MOLECULAR MARKERS,

NATURAL HISTORY
AND EVOLUTION
MOLECULAR MARKERS,
NATURAL BISTORY
AND EVOLUTION

JOHN C. AVISE

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

Copyright C> 1994 by Springer-Seienee+Business Media Dordreeht
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Avise. John C.
Molecular markers, natural hislory & evolution/John C Avise.
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ISBN 978-0-412-03781-8 ISBN 978-1-4615-2381-9 (eBook)
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 Contents
Preface
Part I. Background
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3
WHY EMPLOY MOLECULAR GENETIC MARKERS? ......... 5
Molecular Data Are Genetic . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Molecular Methods Open the Entire Biological World for Genetic
Scrutiny. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 6
Molecular Methods Access a Nearly Unlimited Pool of Genetic
Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Molecular Data Can Distinguish Homology from Analogy ....... 8
Molecular Data Provide Common Yardsticks for Measuring
Divergence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Molecular Approaches Facilitate Mechanistic Appraisals of
Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Molecular Approaches Are Challenging and Exciting .......... 12
WHY NOT EMPLOY MOLECULAR GENETIC MARKERS? ..... 15

2. History of Molecular Phylogenetics .................. 16

DEBATES AND DIVERSIONS FROM MOLECULAR
SYSTEMATICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Empiricism and the Classical-Balance Debate ............... 17
Classical Versus Balance Views of Genome Structure ........ 17
Molecular Input to the Debate . . . . . . . . . . . . . . . . . . . . . . . . . 20
Research Preoccupations of the Protein-Electrophoretic
Revolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Empirical Approaches to the Variability/Fitness Conundrum .... 23
Genetic Theory and the Neutralist-Selectionist Debate .......... 26
Systematic Philosophy and the Phenetic-Cladistic Debate . ....... 34
Phylogenetic Data and the Molecule-Morphology Debate ........ 39
MOLECULAR PHYLOGENETICS . . . . . . . . . . . . . . . . . . . . . . . . 40
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

3. Molecular Tools .................................. 44

PROTEIN ASSAYS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Protein Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Principles and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

v
vi Contents

Protein Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 47
Principles and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . .. 47
Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
DNA ASSAYS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
DNA-DNA Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 53
Principles and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . .. 53
Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Restriction Analyses ................................ 57
Animal Mitochondrial DNA . . . . . . . . . . . . . . . . . . . . . . . . . 60
Plant Mitochondrial and Chloroplast DNA . . . . . . . . . . . . . . . 68
Single-copy Nuclear DNA . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Ribosomal RNA Genes and other Middle-repetitive
Gene Families ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 75
Minisatellite Sequences and DNA Fingerprinting ........... 78
DNA Sequencing and the Polymerase Chain Reaction ......... 82
Principles and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . .. 82
Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
REFERENCES TO LABORATORY PROTOCOLS ............ 90
SUMMARy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

4. Interpretive Tools ................................ 92

CATEGORICAL SUBDIVISIONS OF MOLECULAR GENETIC
DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Protein versus DNA Information . . . . . . . . . . . . . . . . . . . . . . .. 93
Qualitative versus Distance Data ....................... 93
Detached versus Connectible Information. . . . . . . . . . . . . . . . .. 97
Single-Locus versus Multilocus Data .. . . . . . . . . . . . . . . . . . .. 97
Utility of Data Along the Phylogenetic Hierarchy ............ 98
MOLECULAR CLOCKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
General Concepts . ................................. 100
Clock Calibrations and Controversies .................... 103
Absolute and Relative Rate Comparisons .................. 106
Closing Thoughts About Molecular Clocks ................. 108
PROCEDURES FOR PHYLOGENY RECONSTRUCTION ....... 109
Distance Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
UPGMA Cluster Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Fitch-Margoliash Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Neighbor-Joining Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Distance Wagner Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Comparison of Distance Matrix Methods . . . . . . . . . . . . . . . . 119
 Contents vii

Character-State Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Hennigian Cladistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Maximum Parsimony . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Conclusions About Phylogenetic Procedures . ............... 124
GENE TREES VERSUS SPECIES TREES . . . . . . . . . . . . . . . . . . 126
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Part II. Applications

5. Individuality and Parentage ....................... 141
GENETIC IDENTITY VERSUS NON-IDENTITY ............. 141
Human Forensics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Ramets and Genets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Background and Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Spatial Distributions of Clones . . . . . . . . . . . . . . . . . . . . . . . 150
Ages of Clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Genetic Mosaics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Clonal Reproduction in Microorganisms .................. 160
Gender Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
PARENTAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
Paternity Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Multiple Paternity in Animals .. . . . . . . . . . . . . . . . . . . . . . . 172
Paternity in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Sperm and Pollen Competition, Precedence ............... 182
Maternity Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

6. Kinship and Intraspecific Phylogeny ................ 190

CLOSE KINSHIP AND FAMILY STRUCTURE .............. 190
Eusocial Colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Noneusocial Colonies and Groups . ...................... 199
Kin Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
GEOGRAPHIC POPULATION STRUCTURE AND
GENE FLOW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Autogamous Mating Systems .......................... 210
Gametic and Zygotic Dispersal . ........................ 213
Pollen and Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Marine Gametes and Larvae . . . . . . . . . . . . . . . . . . . . . . . . . 215
Direct Estimates of Dispersal Distances ................. 221
Vagility, Philopatry, and Dispersal Scale . ................. 222
Physical Dispersal Barriers . . . . . . . . . . . . . . . . . . . . . ..... 223
Philopatry to Natal Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Vlll Contents

Gender-biased Dispersal and Gene Flow . . . . . . . . . . . . . . . . . 227

Non-neutrality of Some Molecular Markers . . . . . . . . . . . . . . . 230
Historical Demographic Events . . . . . . . . . . . . . . . . . . . . . . . 232
PHYLOGEOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Case Histories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
The Origin of Green Turtles on Ascension Island . . . . . . . . . . . 236
Migratory Histories of Monarch Butterflies . . . . . . . . . . . . . . . 236
Interbrood Switching in Periodical Cicadas . . . . . . . . . . . . . . . 237
Natural Selection and Biogeographic History in the Killifish ... 238
Human Populations-Out of Africa? . . . . . . . . . . . . . . . . . . . 238
Genealogical Concordance and the Sharp-tailed Sparrow . . . . . . 241
Phylogeography of a Regional Fauna .. . . . . . . . . . . . . . . . . . 242
General Conclusions About Intraspecific Phylogeography ....... 246
MICROTEMPORAL PHYLOGENY . . . . . . . . . . . . . . . . . . . . . . 248
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

7. Speciation and Hybridization ...................... 252

THE SPECIATION PROCESS . . . . . . . . . . . . . . . . . . . . . . . . . . 257
How Much Genetic Change Accompanies Speciation? ......... 257
Traditional Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Molecular Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Do Speciations Entail Severe Population Bottlenecks? ......... 264
Are Speciation Rates and Divergence Rates Correlated? ....... 267
Can Speciation Occur Sympatrically? .................... 269
Flocks of Fishes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Host- or Habitat-Switching in Insects . . . . . . . . . . . . . . . . . . . 272
Can Related Species be Diagnosed Reliably? ............... 274
Should a Phylogenetic-Species Concept Replace the BSC? ...... 278
HYBRIDIZATION AND INTROGRESSION . . . . . . . . . . . . . . . . . 280
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
Frequencies and Geographic Settings of Hybridization ....... 280
Case History Involving Treefrogs . . . . . . . . . . . . . . . . . . . . . . 284
Hybrid Zone Asymmetries . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Differential Introgression Across a Hybrid Zone ........... 287
Haldane's Rule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Differential Mating Behaviors . . . . . . . . . . . . . . . . . . . . . . . . 291
Differential Gametic Exchange . . . . . . . . . . . . . . . . . . . . . . . 293
Reticulate Evolution Evidenced by cpDNA Phylogenies ...... 295
Speciation by Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
 Contents ix

Diploid or Homoploid Speciation . . . . . . . . . . . . . . . . . . . . . . 297

Origins of Unisexual Biotypes . . . . . . . . . . . . . . . . . . . . . . . . 299
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

8. Species Phylogenies and Macroevolution ............. 306

RATIONALES FOR PHYLOGENY ESTIMATION ............ 307
Phylogenetic Mapping of Organismal Traits . . . . . . . . . . . . . . . . 307
Anatomical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Behavioral, Physiological, and Life History Features ........ 315
Biogeographic Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Vicariance Versus Dispersal . . . . . . . . . . . . . . . . . . . . . . . . . 321
Common Ancestry Versus Convergence . . . . . . . . . . . . . . . . . 326
Recent Islands, Ancient Inhabitants . . . . . . . . . . . . . . . . . . . . 328
Academic Pursuit of Genealogical Roots .................. 329
SPECIAL APPROACHES TO PHYLOGENY ESTIMATION ..... 331
DNA-DNA Hybridization and Avian Systematics . ............ 331
Mitochondrial DNA and the Higher Systematics of Animals ..... 334
Eccentric mtDNA Markers . . . . . . . . . . . . . . . . . . . . . . . . . . 334
mtDNA Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
Chloroplast DNA and the Higher Systematics of Plants ........ 337
Eccentric cpDNA Markers . . . . . . . . . . . . . . . . . . . . . . . . . . 337
cpDNA Restriction Sites and Sequences . . . . . . . . . . . . . . . . . 339
Slowly Evolving Gene Sequences and Deep Phylogenetic
Branches ................... . . . . . . . . . . . . . . . . . . . 341
PROSPECTUS FOR A GLOBAL PHYLOGENY .............. 350
SPECIAL TOPICS IN MOLECULAR PHYLOGENETICS ....... 352
Horizontal Gene Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
Phylogeny of Retroviruses and Transposable Elements ......... 354
Molecular Paleontology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

9. Conservation Genetics ............................ 361

ISSUES OF HETEROZYGOSITY . . . . . . . . . . . . . . . . . . . . . . . . 362
Molecular Heterozygosity in Rare and Threatened Species ...... 362
Does Reduced Molecular Heterozygosity Matter? ............ 366
ISSUES OF PHYLOGENY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
Parentage and Kinship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Population Structure and Intraspecific Phylogeny ............ 372
Genetic Partitions Within Rare and Threatened Species ....... 373
Stock Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
Conclusions About Intraspecific Phylogeography ........... 380
x Contents

Speciation and Conservation Biology . .................... 382

Recognition of Endangered Species .................... 382
Molecular Forensics and Law Enforcement ............... 386
Hybridization and Introgression . . . . . . . . . . . . . . . . . . . . . . . . 388
Biological Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Legal Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
Species Phylogenies and Macroevolution .................. 393
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397

Literature Cited .................................... 399

Index to Taxonomic Genera .......................... 501
General Index ...................................... 507
 Preface

I never cease to marvel that the DNA and protein markers magically appearing
on laboratory gels and autoradiographs can reveal so many otherwise hidden facets
about the world of nature. Can individual plants sometimes exist as genetic
mosaics derived from multiple zygotes? Is reproduction in unicellular eukaryotes
predominantly sexual or clonal? In the absence of recombinationally derived
genetic variability, how long do evolutionary lineages persist in parthenogenetic
all-female "species"? Do social groups within various species of insects, fishes,
and other organisms consist of close relatives whose behavior might have evolved
under the influence of kin selection? How often does intraspecific brood parasitism
occur in birds, whereby females "dump" eggs in the nests offoster parents? When
distinctive morphotypes exist among related fishes within a lake, do these reflect
species' differences (as opposed to developmental switches within a species), and,
if so, did the species flock arise in situ? Do migratory marine turtles return to natal
sites for nesting? How often has carnivory evolved among plants? What are the
evolutionary origins of mitochondrial DNA and chloroplast DNA within eukary-
otic cells? Are transposable elements the phylogenetic precursors of retroviruses,
or vice versa? Can plate tectonics and continental drift account for the presence
of large flightless birds in Australia, South America, and Africa? Have demo-
graphic bottlenecks diminished genetic variability in some populations to the
extent that they can no longer adapt to environmental challenges? How distinct
genetically are the taxa currently afforded protection under the Endangered Spe-
cies Act? These are but a small sample of the diverse problems addressed and
answered (at least provisionally) through use of molecular genetic markers.
This treatment of molecular natural history and evolution is written at a level
appropriate for the advanced undergraduate or graduate student, or for scientists
in fields such as ecology, genetics, ethology, molecular biology, population
biology, and conservation biology who may wish a readable introduction or
refresher to the burgeoning application of molecular markers to problems in their
disciplines. I hope to have captured and conveyed the genuine excitement that
can be brought to such fields when genetic attributes with known patterns of
inheritance are applied to organismal-Ievel issues. I also hope to have provided
a wellspring of research ideas for those entering the field. My goal has been to
present material in a manner that is technically straightforward, without sacri-

xi
xii Preface

ficing the richness of underlying concepts and biological applications. For the
reader, the only necessary prerequisites are an introductory knowledge of genet-
ics and an acute interest in the natural biological world.
Why is a treatment of this topic necessary when numerous excellent texts in
molecular evolution already are available? Recent publications in the field have
focused on (a) proteins and DNA as primary objects of interest from a molecular
perspective; (b) broad conceptual issues regarding patterns and processes of
molecular evolution; (c) statistical or mathematical aspects of population genet-
ics theory; (d) procedures of molecular data analysis; or (e) descriptions of
laboratory methods. Some books have approached more closely what is at-
tempted here, but are restricted either to topic, laboratory method, or taxonomic
group. Hoelzel and Dover (1991a) and Hoelzel (1992) have produced fine in-
troductions to molecular ecology, but to my knowledge, no extended available
treatment explicitly addresses the multifaceted biological applications for mo-
lecular genetic markers from the perspectives of population biology, natural
history, and organismal phylogeny.
The field of molecular evolution is at a stage where reflection on the past 30
years may provide useful historical perspective, as well as a springboard to the
future. The mid-1960s witnessed an explosion of interest in molecular techniques
with the seminal introduction of protein electrophoretic approaches to popUlation
genetics and evolutionary biology. In the late 1970s, attention shifted to methods
of DNA analysis, primarily through restriction enzymes, and in the 1980s,
mitochondrial DNA analyses and DNA fingerprinting approaches gained im-
mense popUlarity. Recently, the introduction ofPCR-mediated DNA sequencing
has brought the field close to a final technical fruition by providing the first ready
access to the "ultimate" genetic data, nucleotide sequences themselves. None-
theless, it would be a mistake to conclude that direct sequence information
invariably provides the preferred or most accessible pool of genetic markers for
all biological applications. Several alternative assays retain tremendous power
and utility and, because of ease, cost, amount of genetic information accessed,
or simplicity of data interpretation, continue to be the techniques of choice for
many evolutionary problems. Ecologists, as well as molecular geneticists, some-
times are unaware of the arguments for and against various molecular genetic
approaches, and one major goal of this book is to clarify these positions.
In scientific advance, timing and context are all-important. Imagine for sake of
argument that DNA sequencing methods had been widely employed for the past
30 years and that only recently had protein-electrophoretic approaches been
introduced. No doubt a headlong rush into allozyme techniques would ensue, on
justifiable rationales that (a) the methods are cost-effective and technically sim-
ple, (b) the variants revealed reflect independent Mendelian polymorphisms at
several loci scattered around the genome (rather than as linked polymorphisms in
a single stretch of DNA), and (c) the amino acid replacement substitutions
 Preface Xlll

uncovered by protein electrophoresis (as opposed to the silent base changes often
revealed in DNA assays) might bring molecular evolutionists closer to the real
"stuff" of adaptive evolution. To carry the argument farther, suppose that mo-
lecular genetic methods had been employed throughout the last century but that
an entrepreneurial scientist finally ventured into the world of nature and discov-
ered organismal phenotypes and behaviors. Finally, the interface of gene prod-
ucts with the environment would have been revealed! Imagine the sense of
excitement and the research prospects!
These fanciful scenarios are raised to emphasize a point-molecular ap-
proaches carry immense popularity at the present time, but they nonetheless
provide but one of many avenues toward the goal of understanding the natural
histories and evolutionary biologies of organisms. Studies of morphology, ecol-
ogy, and behavior undeniably have shaped the great majority of our perceptions
about the natural world. Molecular approaches are especially exciting at this
point in time because they open new empirical windows and novel insights on
these traditional subjects.
In this book, I have attempted to identify and highlight select case histories
where molecular methods have made significant contributions to natural history
and evolutionary biology. The treatment is not intended to be exhaustive, be-
cause literally thousands of studies have utilized genetic markers. Rather, I have
tried to choose classic, innovative, or otherwise interesting examples illustrative
of the best that molecular methods have to offer. The literature is represented
through 1992. Overall, I have attempted to retain a balanced taxonomic perspec-
tive that includes examples from plants, animals, and microbes, and indeed I
hope that common threads will be evident that tie together the similar classes of
biological questions that frequently apply to such disparate organisms.
This book is organized into two parts. Part I provides introductory material
and background: the rationale for molecular approaches in natural history and
evolution (Chapter 1); the history of molecular phylogenetics (Chapter 2); out-
lines of various laboratory methods and the nature of the genetic data each
. molecular method provides (Chapter 3); and descriptions of some of the inter-
pretive tools of the trade, including molecular clock concepts and phylogenetic
methods as applied to molecular data (Chapter 4).
Part n departs significantly from previous books in molecular evolution by
emphasizing empirical examples of significant biological applications for mo-
lecular genetic markers. Topics are arranged along a phylogenetic hierarchy
ranging from micro- to macro-evolutionary: the assessment of genetic identity!
nonidentity and parentage (Chapter 5); kinship and intraspecific phylogeny
(Chapter 6); speciation, hybridization, and introgression (Chapter 7); and assess-
ment of the deeper phylogenetic structures in the evolutionary tree of life (Chap-
ter 8). A concluding chapter deals with the relevance of molecular studies to
conservation biology and to the preservation of genetic diversity (Chapter 9).
xiv Preface

I would like to dedicate this book to my current and fonner graduate students
and research technicians, without whom most of our work would not have been
possible, nor nearly as much fun: Charles Aquadro, Marty Ball, Eldredge Ber-
mingham, Brian Bowen, Robert Chapman, Michael Douglas, Matt Hare, Steve
Karl, Lou Kessler, Trip Lamb, Joe Neigel, Bill Nelson, Jay Parker, John Patton,
Joe Quattro, Carol Reeb, Nancy Saunders, Kim Scribner, DeEtte Walker, and
Kurt Wollenberg. Recent graduate students were forced to read early drafts of
several chapters, and they responded by providing numerous helpful criticisms.
Drs. Fred Allendorf, Jim Hamrick and Linda Maxson read drafts of the entire
book and suggested numerous improvements. Excellent criticisms of individual
sections or chapters were kindly provided by Drs. Jeff Palmer, David Stock,
Gary Olsen, and Carl Woese. Of course, I remain responsible for any errors or
omissions. I am also indebted to my own advisors in graduate school-Robert K.
Selander, Michael H. Smith, and Francisco J. Ayala-for getting me started; and
to my wonderful colleagues at the University of Georgia-Wyatt Anderson, Jim
Hamrick, Jonathan Arnold, Marjorie Asmussen, John McDonald, Mike Arnold,
and others-for keeping me going. Much of this book was written during a
sabbatical supported by the Sloan Foundation, and I am indebted to my host
Denny Powers for providing a wonderful working environment at the Hopkins
Marine Station of Stanford University. Over the years, my laboratory has been
supported by grants primarily from the National Science Foundation and the
National Geographic Society.
I especially want to dedicate this book to my wife Joan, with whom I have
collaborated to put into practice the fitness concepts that we evolutionary biol-
ogists so often discuss. The result was the light of my life, Jennifer. Finally, I
want to thank my parents, Dean and Edith, for unwavering support.
 Part I

Background
 1

Introduction

The stream of heredity makes phylogeny; in a sense, it is

phylogeny. Complete genetic analysis would provide the
most priceless data for the mapping of this stream.

G.G. Simpson, 1945

This book is about the study of phylogeny and natural history, late 20th-century
style. Scientists now routinely utilize the genetic infonnation in biological mac-
romolecules-proteins and DNA-to address numerous aspects of the behav-
iors, life histories, and evolutionary relationships of organisms. When used to
best effect, molecular data are integrated with infonnation from such fields as
ethology, field ecology, comparative morphology, systematics, and paleontol-
ogy. These time-honored biological disciplines remain highly active today, but
each has been enriched if not rejuvenated by contact with the relatively young but
burgeoning field of molecular evolution.
Interest in molecular evolution can center in either of two areas: (a) charac-
terization of the molecular basis of variation in particular genetic systems or (b)
application of genetic data to questions in natural history and organismal evo-
lution. The latter constitutes the primary focus of this book. However, molecular
and organismal issues are intertwined. Knowledge of such molecular-level prop-
erties as the mode of genetic transmission and the nature of mutational differ-
ences underlying a polymorphism are critical to proper interpretation of molec-
ular markers in a population context; and, patterns of variation and divergence in
genetic markers can be highly infonnative regarding evolutionary forces imping-
ing on molecular evolution.

3
4 Introduction

In the not-too-distant future, it might become possible to isolate and charac-

terize directly the genes contributing to variation in particular phenotypic fea-
tures and adaptations. Such molecular-level analyses will revolutionize the study
of phenotypic variability and its maintenance. But there is much more to evo-
lution, and many topics in organismal biology already can be addressed critically
by examining variation in "randomly chosen" DNAs or proteins-in other
words, by employing molecular genetic markers. These subjects include organ-
ismal forensics, mating systems, population structure, gene flow, speciation,
hybridization, and systematics, to name a few.
Phylogeny is evolutionary history-the topology of the proverbial tree of life.
All organisms share certain features (most notably, nucleic acids as hereditary
material) that suggest a single or monophyletic origin on earth, some three to
four billion years ago. The pattern of phylogeny involves successive branching
from this trunk of life, with organisms alive today representing tips of the twigs
on the currently outermost branches. A complete picture of phylogeny requires
knowledge of both the branching order (cladogenetic splitting of lineages) and of
the branch lengths (anagenetic changes within lineages through time). The pos-
sibility of occasional genetic transfer between branches (reticulate evolution),
perhaps mediated by interspecific hybridization, viral conveyance, or establish-
ment of endosymbiotic associations among genomes, also must be considered.
Most studies that utilize molecular markers can be viewed as attempts to
estimate phylogeny, broadly defined, at one or another hierarchical stage of
evolutionary divergence (Fig. 1.1). Phylogenetic relationships thus can be as-
sessed at levels ranging from extreme micro- to macro-evolutionary: (a) genetic
identity versus nonidentity, as in clonally-reproducing organisms; (b) parentage
(maternity and paternity); (c) extended kinship within a local group or popula-
tion; (d) differentiation among geographic populations and subspecies; (e) dif-
ferentiation among reproductively isolated species; and (f) phylogenetic structure
at intermediate and great evolutionary depths in the tree of life. Different types
of molecular assay provide genetic information ideally suited to different subsets
of this hierarchy, and a continuing challenge is to develop and utilize molecular
methods appropriate for a particular biological problem at hand.
It is also appropriate to orient this book around phylogeny because of the
central importance of historical events in evolutionary biology. As noted by
Hillis and Bull (1991), "Virtually all comparative studies of biological variation
among species depend on a phylogenetic framework for interpretation." If
Dobzhansky's (1973) famous dictum is correct, that "Nothing in biology makes
sense except in the light of evolution," then it might be appended that "much in
evolution makes more sense in the light of phylogeny. " Brooks and McLennan
(1991) provide an eloquent and extended treatment of the general role of phy-
logenetic analysis in ecology and ethology, as do Eldredge and Cracraft (1980)
and Harvey and Pagel (1991). Molecular approaches will contribute to these
 Introduction 5

•__.rO-

.......
..-_.,-
.......
....

~
-"-.

,...............

. L -_ _ _ _ _ _ _ _ _ _ ~ .L-_ _ _ _ _ _ _ _ _ _ ~

., .,
:c
.
II)
c::
" ~
II)

iii

..
.c E
:<: E pedigree
E
.
D-
E

macro- micro-

PHYLOGENY
Figure 1.1. The hierarchical nature of phylogenetic assessment (after Avise et aL.
1987a).

phylogenetic enterprises because with evolutionary relationships properly sorted

out at the molecular level, unprecedented opportunities emerge for understanding
the evolutionary origins and histories of other organismal attributes.

WHY EMPLOY MOLECULAR GENETIC MARKERS?

In describing organismal relationships, the approaches previously available

have rested on the data of comparative morphology, physiology, and other
assayable phenotypic features. Molecular systematists also employ the compar-
ative method, but the comparisons involve direct or indirect information on
nucleic acid and protein sequences. Why are molecular data of special signifi-
cance to phylogeny estimation?

Molecular Data are Genetic

This simple truism is of overriding significance. Because phylogeny is "the

stream of heredity," only genetically transmitted traits are informative to phy-
logeny estimation. Not only are protein and DNA features heritable, but most
6 Introduction

molecular assays reveal variable character states whose particular genetic bases
and modes of transmission can be specified explicitly. Thus, from knowledge of
the amount and nature of genetic information assayed, statements of relative
confidence can be placed on molecular-based phylogenetic conclusions.
This situation contrasts with the insecure knowledge concerning the genetic
bases of most conventional characters employed in organismal systematics (Bar-
low, 1961; Boag, 1987). Seldom can the genes and alleles controlling particular
morphological, physiological, or behavioral traits be specified, and, indeed,
some such traits conventionally utilized in taxonomy may be influenced by
nongenetic factors. For example, a significant fraction of the variance in mor-
phometric features used to describe named subspecies of the red-winged black-
bird (Agelaius phoeniceus) proved to be due to environmental rearing conditions:
from eggs experimentally transplanted from one geographic population to an-
other, the resulting progeny converged significantly on some of the morpholog-
ical features of their foster parents (James, 1983). Developmental or phenotypic
plasticity, whereby form is influenced directly by environment, is especially
pronounced in many plants (Clausen et aI., 1940). Of course, such ecotypic
variation can be misleading if interpreted as providing genetic or systematic
characters.

Molecular Methods Open the Entire Biological World for Genetic Scrutiny

Prior to the introduction of molecular approaches, genetic studies mostly were

confined to the small handful of species that could be crossed under controlled
laboratory conditions, such as Mendel's pea plants, the bacterium Escherichia
coli and its phages, the corn Zea mays, the fruit fly Drosophila melanogaster.
and the house mouse Mus musculus. From transmission patterns across gener-
ations, the genetic bases of assayable morphological or physiological traits in
these species were deduced, but such analyses could hardly be expected to
capture the full flavor of genetic diversity across the earth's broader biota (nor the
richness of diversity among genetic elements within a genome). By contrast,
various molecular assays provide direct structural evidence regarding essentially
any genes or their protein products and can be applied to the genetics of any
creatures, microbes to whales.

Molecular Methods Access a Nearly Unlimited Pool of Genetic Variability

Genomes are enormous in information content. For example, a typical mam-

malian genome consists of some three thousand million nucleotide pairs, in a
composite linear sequence roughly 100 times longer than the total string of letter
characters in an 18-volume World Book encyclopedia. Like an encyclopedia,
each genome is a repository of information, coding not only the proteins and
 Introduction 7

other cellular machinery of life but also retaining within its nucleotide sequence
a record of evolutionary relationships to other genomes. Bacterial genomes range
in size from about 0.6 X 106 to 13.2 X 106 base pairs (bp); protist genomes
from 23 X 106 to 686 X 109 bp; and genomes of multicellular fungi, plants and
animals from 8.8 X 106 to more than 300 x 109 bp (Cavalier-Smith, 1985; Li
and Graur, 1991; Sparrow et al., 1972). Molecular assays involve sampling,
often more or less at random, from such vast informational pools.
Genetic variability within most species is also tremendous. Consider, for
example, the human gene pool, which in comparison to many other species is
unexceptional if not low with regard to magnitude of variation (Li and Sadler,
1991). Although genome-wide estimates of nucleotide diversity still are some-
what uncertain, a conservative estimate is that homologous pairs of randomly
drawn human DNA sequences may differ at 0.03% or more of nucleotide posi-
tions, on average (Ewens et al., 1981; Li and Sadler, 1992). The human genome
sequencing project, perhaps to be completed within the next decade, promises to
provide the complete three-billion-bp nucleotide sequence of one genome equiv-
alent. Thus, if a second human genome were to be sequenced, it would likely
differ from the first at nearly one million nucleotide sites! Nucleotide diversities
in some other species have been estimated at 0.5-2.0% (Stephan and Langley,
1992), such that an average pair of chromosome sets drawn from such popula-
tions likely would differ at several million nucleotide positions.
Obtaining complete DNA sequences from more than a few model organisms
will remain impractical for the foreseeable future [the first complete sequence of
a single entire chromosome from any organism, the 315 kb chromosome III of
the yeast Saccharomyces cerevisiae, was obtained in 1992 (Oliver et al., 1992)].
Complete genomic sequences will also remain quite unnecessary for purposes of
providing genetic markers in population biology and evolution because available
methods already can uncover ample variability for even the finest of diagnostic
requirements. For example, over 2000 human DNA polymorphisms revealed by
restriction endonuclease analysis had been cataloged by 1990 (Stephens et al.,
1990a) and the list is growing rapidly (Weissenbach et al., 1992); nearly 100
human protein and blood group polymorphisms had been surveyed by that time
among the major human races (Nei and Livshits, 1990). Assume for the sake of
an extremely conservative argument that only 200 polymorphisms were present
in the human genome, each with the minimum possible two alleles. Rules of
Mendelian heredity show that the potential number of different human genotypes
would then be an astronomical 3200 , or roughly 10, 000, 000, 000, 000, 000,
000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,
000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000. The total number of
people estimated to be alive in the year 2000 is 6,000,000,000, and the total
number of humans who have ever lived will be roughly 13,000,000,000 by then.
Thus, the potential number of distinct human genotypes vastly exceeds the
8 Introduction

number of individuals who will ever inhabit the earth, and no humans past,
present, or future (barring identical twins) are likely to be the same genetically.
In human forensic practice, assays of even modest numbers of highly allelic
Mendelian polymorphisms have proved sufficient to provide individual-specific
DNA fingerprints that have withstood jurisprudential scrutiny (Chapter 5).

Molecular Data Can Distinguish Homology from Analogy

The central problem of phylogenetics always has been to distinguish the com-
ponent of biological similarity due to descent from common ancestry (homology)
from that due to convergence from different ancestors (analogy). Evolutionary
classifications should reflect only true homologies, records of genealogical an-
cestry. But shared morphologies and behaviors, which until recently provided
the major guides to classification, sometimes can evolve independently in unre-
lated species in response to common environmental challenges. For example,
Old World and New World vultures share numerous adaptations for carrion-
feeding existence (e. g., soaring food-search behavior, featherless face and head,
powerful hooked bill) which formerly were thought to be indicative of a close
evolutionary relationship and resulted in their taxonomic placement with other
diurnal raptors (Falconiformes). However, recent data from DNA-DNA hybrid-
ization (as well as detailed reappraisals of morphology) showed conclusively that
New World vultures are related more closely to storks (formerly Ciconiiformes)
and have independently acquired adaptations for the carrion-feeding life-style
(Sibley and Ahlquist, 1990).
In referring to DNA hybridization methods, the paleontologist Gould (1985)
wrote "I do not fully understand why we are not proclaiming the message from
the housetops . . . We finally have a method that can sort homology from
analogy. " Gould was referring to the fact that when species are assayed with
respect to hundreds or thousands of molecular characteristics, any widespread
and intricate similarities that appear are unlikely to have arisen by convergent
evolution and, therefore, must reflect true phylogenetic descent. This is not to
say that particular molecular characters, such as individual nucleotide sites, are
free from homoplasy (convergences, parallelisms, or evolutionary reversals that
muddy the historical record). Indeed, some molecular characteristics considered
individually may be especially prone to homoplasy, due to a small number of
interconvertible character states and to a sometimes rapid rate of change among
them (e.g., each nucleotide position is characterized by only four assumable
states-adenine, guanine, thymine, and cytosine). Other molecular features,
such as duplications, deletions, or rearrangements of genetic material, may be
relatively rare events for which hypotheses of monophyly can sometimes be
justified. In generating molecular-based phylogenetic hypotheses, the nature of
the underlying data must be taken into account.
 Introduction 9

Molecular Data Provide Common Yardsticks for Measuring Divergence

A singularly important aspect of molecular data is that they allow direct

comparisons of relative levels of genetic differentiation among essentially any
groups of organisms (Wheelis et al., 1992). Suppose, for example, that one
wished to quantify evolutionary differentiation within a taxonomic family or
genus of fishes as compared to that within a taxonomic counterpart of birds. The
kinds of morphological features traditionally employed in fish systematics (e.g.,
number of lateral line scales, fin rays, gill rakers, and position of the swim
bladder) clearly would be of little utility for direct comparison with avian sys-
tematic characters (e.g., plumage features, structure of the syrinx, or arrange-
ment of toes on the feet), and perhaps for such reasons, traditional systematic
practices and standards have developed separately among ichthyologists, orni-
thologists, and other organismal specialists. However, birds and fishes (as well
as other animals, plants, and microbes) do share numerous molecular traits, such
as genes and enzymes involved in central metabolic pathways for the respiration
and synthesis of carbohydrates, fats, amino acids, and nucleic acids.
To introduce the concept of common yardsticks in molecular evolution, con-
sider Figure 1.2 which summarizes reported levels of genetic divergence among
congeneric species representing five vertebrate classes, as measured by the com-
mon denominator of protein electrophoretic divergence among similar suites of
enzymes. One unanticipated generality to emerge is that by this criterion, avian
congeners typically exhibit far less genetic differentiation than do many reptilian
or amphibian species within a genus, despite considerable anatomical differen-
tiation among birds (Wyles et aI., 1983). Perhaps avian congeners are evolu-
tionarily younger than many nonavian congeners, or perhaps avian proteins
evolve more slowly on average than those of amphibians, for example (Avise,
1983a; Avise and Aquadro, 1982). In either event, these comparative genetic
results raise exciting evolutionary issues that were not evident from morpholog-
ical comparisons alone.
In another example of this "common yardstick" perspective, King and Wil-
son (1975) reviewed evidence that the assayed proteins and nucleic acids of
humans and chimpanzees are only about as divergent as are those of morpho-
logically similar sibling species of fruit flies and rodents, and much less diver-
gent than those of typical amphibian congeners. They suggested that the tradi-
tional placement of humans and chimps in different taxonomic families
(Hominidae and Pongidae) might have stemmed from morphological differences
attributable to changes at a few regulatory genes acting during ontogeny. An
alternative possibility is that the perceived morphological distinctiveness of hu-
mans from chimps and other primates may have been exaggerated for reasons of
anthropocentric bias. In a fascinating paper entitled "Frog perspective on the
morphological difference between humans and chimpanzees," Cherry et al.
10 Introduction

• I
Plethodon (325)
Hyla (21) rn
• . z

.
, Lltorla (120)
•• • Hydromantes (10) '"
iii

J '"i:
Rana (21)
I •i SCaphlOpUS (10) IL
" Tarlcha (3) :::E

]
• -I BlpeS (3)
+ _ - -_ _ -4----,----1•..._-+---4----;--." Anolls (72) rn
• .; Lacerta (3) ~
+ Uma (3)
~
IL
W
..... Crotaphytus (6) a:

-------,---1.1-'--+---i--..,.' Hotropls (1081)

• Lepomls (45)
Ii Menldla (10)
., Etheostoma (3) rn
1,',: :,'. Bathygoblus (3)
Coregonus (6)
w
::t:
rn
io--e--+- Thoburnla (3) Ii:
, .-----. , Hypentellum (3)
..... : Campostoma (3)
i ++-1 Cyprlnodon (10)

---tIt-.----c--f--.' Cercocebus (6)

• Geomys (10)
---'--~--+-".--'-'--+---+. i Peromyscus (190)
I:

i
Laslurus (15)
• Dlpodomys (55)
.;.- i Macaca (15)
:,.... ':, Spermophllus (3)
Heotoma (3)
, . . . : Thomomys (10)
. . ' paplo (6)

,~,---iII'II------i---· , VIreo (10)

.. Ammodramus (3)
•• .' Vermlvora (6)
; .. ' Parus (3)
i----....: Anas (45)
.. ; Toxostoma (3)
. .; Zonotrlchla (3)
rn
c
. .: Aythya (10) a:
.......... Dendrolca (66) iii
I: Anser (3)
!,',i Catharus (6)
A A A " .., Geosplza (15)
/ V V V--i------i-------1f------T---t-----+---1
1.2 1.0 0.8 0.6 0.4 0.2 0.0
3.0 2.0

GENETIC DISTANCE (ALLOZYMES)

Figure 1.2. A comparative genetic yardstick for vertebrate genera. Shown are means and
ranges of molecular differentiation, plotted on a common scale, among congeneric species
within each offive vertebrate classes (after Avise and Aquadro, 1982). Genetic distances
are in units of codon substitutions per locus, as assessed by multilocus protein electro-
phoresis. In parentheses are numbers of pairwise species comparisons for each genus.
 Introduction 11

(1978) employed the anatomical traits normally used to discriminate among frogs
(eye-nostril distance, forearm length, toe length, etc.) to quantify the human-
chimpanzee morphological separation. By these criteria, morphological diver-
gence between the two primates was large even by frog standards (whereas the
molecular divergence was not), a result interpreted as consistent with the pos-
tulate that morphological and molecular evolution can proceed at independent
rates. It is ironic, but understandable, that this rare attempt to utilize a compar-
ative yardstick approach in morphological evolution came from a research lab-
oratory normally devoted to molecular genetics, where such comparisons come
quite naturally.
Thus, one challenging question posed by molecular approaches is whether a
genetic basis for taxonomy can be developed that for the first time will provide
a universal standard for comparing all forms of life (Chapter 8). This is not to
imply that the overall magnitude of genetic divergence between taxa is neces-
sarily the only or even the best guide to phylogenetic relationships within groups
(Chapters 2 and 4), but it certainly is a novel and important consideration for
intergroup comparisons that simply falls outside the framework of traditional
systematic practice.

Molecular Approaches Facilitate Mechanistic Appraisals of Evolution

Although traditional assessments of phenotype have been immensely infor-

mative in identifying spontaneous visible mutations and in elucidating the action
of evolutionary forces including natural selection, the mechanistic (i.e., molec-
ular) underpinnings of such attributes remained unknown. Modern methods such
as DNA sequencing provide previously inaccessible information about the fun-
damental molecular basis of evolutionary change. For example, various mor-
phological and physiological mutations in Drosophila now have been character-
ized at the molecular level and have proved attributable to specifiable point
mutations in the coding regions of single-copy genes, mutations in flanking and
nonflanking regulatory domains, insertions of transposable elements, and to
"homeotic" mutations in special classes of repetitive segmentation loci (Lam-
bert et al., 1988; Lewin, 1990). A growing enterprise in quantitative genetics
involves use of DNA markers to physically map loci for polygenic traits (phe-
notypic characteristics influenced by variation at multiple genes) (e.g., Lander
and Botstein, 1989; B. Martin et al., 1989). Scientists are still a long way from
the goal of relating all phenotypic differences to particular DNA changes, but
increasing numbers of genetic disorders in humans and a few other species are
yielding to detailed molecular-level characterizations (e.g., Randall, 1991;
Stine, 1989; Wallace et aI., 1988).
Although these kinds of mechanistic appraisals fall somewhat outside the
subject matter of this book, data of relevance to molecular biology frequently arise
12 Introduction

as a direct or indirect by-product of molecular phylogenetic analyses. For ex-

ample, through phylogenetic reconstructions based on restriction-site maps for the
alcohol dehydrogenase locus in Drosophila melanogaster, researchers have dis-
covered that a mutation conferring a higher capacity to utilize and/or detoxify
environmental alcohols arose recently in evolution, perhaps within the past few
thousand to one million years (Aquadro et aI., 1986; Stephens and Nei, 1985).
From sequence analyses of introns at "t-Ioci" on chromosome 17 in house mice,
particular chromosomal inversions influencing embryonic development were es-
timated to have originated about three million years ago, and to have accumulated
recessive lethality factors that have been spread worldwide within the last 800,000
years (Morita et aI., 1992). Detailed molecular analysis of an esterase locus in
mosquitos has shown that the global distribution of an insecticide-resistance
attribute most likely resulted from the migrational spread of a single mutation,
rather than a high mutation rate to independent resistance alleles (Raymond et ai. ,
1991); a similar molecular-based conclusion was reached for the global spread of
a methicillin-resistance gene in the pathogenic bacterium Staphylococcus aureus
(Kreiswirth, 1993). Much interest in human medicine now centers on the use of
molecular markers to assess whether particular genetic disorders (such as phe-
nylketonuria in Yemenite Jews, Huntington's chorea in Afrikaners, or the fragile
X syndrome) are of monophyletic or polyphyletic mutational origin (Avigad et aI.,
1990; Diamond and Rotter, 1987; Hayden et al., 1980; Richards et al., 1992). In
general, the mapping of phenotypic traits onto phylogenies estimated from mo-
lecular markers is revolutionizing understanding of the evolutionary origins of
numerous organismal features (Chapter 8).

Molecular Approaches are Challenging and Exciting

Another appeal of molecular phylogenetics is the sheer intellectual challenge

provided by this new discipline. Many of the discoveries in molecular biology
clearly affect the practice of molecular phylogenetic assessment, and some rel-
evant molecular-level phenomena taken for granted today were undreamed of
even a few years ago. For example, nucleotide sequences among loci belonging
to some multigene families often evolve in concert within a species, and thereby
remain relatively homogeneous. This process of concerted evolution (Zimmer et
aI., 1980), first noted by Brown et ai. (1972), apparently is due to the homog-
enizing effects of unequal crossing over among tandem repeats and to gene
conversion events even among unlinked loci (Arnheim, 1983; Dover, 1982;
Ohta, 1980, 1984; Smithies and Powers, 1986). Thus, the genes in multilocus
families may not provide the independent bits of phylogenetic information for-
merly assumed (Ohno, 1970). To the extent that sequences of multicopy genes
within a species evolve in concert subsequent to the duplications from which they
 Introduction 13

arose, the usual distinction between orthology (sequence similarity tracing to a

speciation event) versus paralogy (sequence similarity tracing to a gene dupli-
cation) loses some relevancy (Fig. 1.3).
Another example of the changing perspectives on phylogeny prompted by
molecular methods involves the introduction of mitochondrial (mt) DNA meth-
ods to population biology in the late 1970s. Prior to that time, most biologists
viewed intraspecific evolution primarily as a process of shifting allele frequen-
cies, a perspective that fit well with the traditional language and framework of
population genetics but that failed to accommodate adequately the phylogenetic
component of population history (Avise et al., 1987a; Wilson et al., 1985). By
providing the first accessible data on "gene genealogies" at the within-species
level, mtDNA methods have forged an empirical and conceptual bridge between

... ancestral
gene

... ance~tral
~__~~__~~~~__~~~~ species

extant species 1 extant species 2

Figure 1.3. Possible allelic relationships within a multigene family. The two circles
indicate gene duplication events from an ancestral locus, producing three extant genes a,
b, and c. The three ellipses represent allelic separations, leading to the extant alleles aI'
b l , and C I in species 1 and ~, b2, and c2 in species 2. Genetic comparisons between aI'
and a2' b l and b2, or c l and c2 are orthologous, whereas all other comparisons (e.g.,
between a l and b2 , a l and c 2' or a l and bl) are paralogous. In the absence of concerted
evolution, orthologous similarities should stem to times near the speciation event,
whereas paralogous similarities date to the relevant gene duplication events (which could
vastly predate the speciation times of the organisms compared). However, under strong
concerted evolution, all or portions of aI' b l , and C I would appear related more closely
to one another than to their respective allelic counterparts in species 2.
14 Introduction

Table 1.1. Eleven unorthodox perspectives on evolution prompted by genetic findings

on animal mitochondrial DNA.a

1. Asexual Transmission (Chapter 3)

Cytoplasmic genes within sexually-reproducing species can exhibit uniparental,
nonrecombining transmission.
2. Population Hierarchy
Populations of mtDNA molecules inhabit each somatic and germ cell lineage
within the individuals comprising organismal populations (Birky et al., 1989).
3. Non-universal Code (Chapter 8)
MtDNA genetic codes sometimes differ from one another, and from the nuclear
codes formerly thought to be universal.
4. Conserved Function, Rapid Evolution (Chapters 3 and 4)
Considerations in addition to level of functional constraint must be required to
explain the rapid pace of animal mtDNA evolution.
5. Lack of Mobile Elements, Introns, Repetitive DNA
Genes with selfish motives gain no fitness advantage by becoming repetitive
within an asexually-transmitted genome (Hickey, 1982).
6. Endosymbiotic Origins (Chapter 8)
Eukaryotic organisms are genetic mosaics containing nuclear and organellar
genomes with formerly independent evolutionary histories.
7. Intergenomic Conflicts of Interest
Mutations with sex-limited detrimental effects on male fitness may accumulate
disproportionately in maternally-transmitted genomes and lead to conflicts of
interest between nuclear and cytoplasmic genes.
8. Intergenomic Cooperation
Interactions between products of mitochondrial and nuclear genes also lead to
expectations of functional coevolution.
9. Matriarchal Phylogeny (Chapter 6)
Mutational differences among mtDNA haplotypes record the phylogenetic histories
of female lineages within and among species.
10. Gene Trees Versus Organismal Phylogenies (Chapters 4, 6, and 7)
Organismal pedigrees contain gene genealogies that can differ greatly from locus
to locus.
11. Degenerative Diseases
Genetic defects in mitochondrial oxidative phosphorylation provide a new
paradigm for the study of aging and degenerative disease (Wallace, 1992).
"For elaboration, see the chapters or references listed, and also Avise (1991a).
 Introduction 15

the nominally rather separate disciplines of population genetics (micro-evolu-

tion) and traditional phylogenetics (mostly macro-evolutionary study) (Avise,
1989a). Indeed, a current intellectual challenge is how to come to grips with the
true meaning of "organismal phylogeny," which in some sense must be viewed
as an emergent property of the multitudinous genealogies for independent loci
whose surviving alleles have trickled through an animal or plant pedigree under
the vagaries of Mendelian genetic transmission (Chapters 4, 6, and 7). Overall,
phylogenetic studies on mtDNA have stimulated a wide variety of novel per-
spectives on evolution (Table 1.1); similar claims could be made for some other
molecular systems as well.

WHY NOT EMPLOY MOLECULAR GENETIC MARKERS?

Against these advantages of molecular genetic methods appear to stand only

two major detractions: (a) considerable training normally is required of practi-
tioners and (b) the monetary cost is high (but also quite variable across methods)
relative to the standards of nonmolecular systematics (Weatherhead and Mont-
gomerie, 1991). However, a fact sometimes overlooked is that the great majority
of molecular-level assessments merely support earlier phylogenetic hypotheses
based on morphology (e.g., Avise, 1974; Sibley and Ahlquist, 1990). Thus, a
complete molecular reanalysis of the biological world is unnecessary. Molecular
markers are used most intelligently when they address controversial areas or
when they are employed to analyze problems in natural history and evolution that
have proven beyond the purview of traditional nonmolecular observation.
 2

History of Molecular
Phylogenetics

The field . . . -molecular evolution-does not have clas-

sification as its primary objective. relevant though molec-
ular data may be to that end.

R.K. Selander, 1982

From its inception in the latter half of this century, the field of molecular evo-
lution has been dominated by a series of fundamental controversies about the
nature and evolutionary significance of genetic variation. Underlying these de-
bates (discussed later) were exciting conceptual issues that understandably cap-
tured the attention of molecular evolutionists. However, to a considerable degree
these controversies also directed attention away from what often were perceived
as more mundane applications of molecules as genetic markers. Thus, prior to
the mid-1980s, with a few notable exceptions, most applications of molecular
markers in areas of natural history or phylogenetic estimation were viewed as
ancillary by-products of research programs whose main goals were to uncover
genetic mechanisms or elucidate broad evolutionary processes. Only in the last
few years has molecular phylogenetics per se begun to assume its appropriate
status as an essential scientific discipline, both empirically rich and conceptually
challenging. What were these broad controversies in evolutionary biology that so
dominated attention, relegating molecular phylogenetics to the back burner?
These historical debates are the primary subject of this chapter.

16
 History of Molecular Phylogenetics 17

DEBATES AND DIVERSIONS FROM MOLECULAR SYSTEMATICS

Empiricism and the Classical-Balance Debate

CLASSICAL VERSUS BALANCE VIEWS OF GENOME STRUCTURE

One long-standing controversy concerned the magnitude of genetic variability

characterizing animal and plant genomes. Evolution has been defined as a change
in genetic composition of populations through time (Dobzhansky, 1937). Ge-
netic variation is prerequisite. Understandably, a central challenge for empirical
population genetics always has been to measure genetic variability, under the
rationale that such quantification would enlighten understanding of the operation
of natural selection, drift, or other evolutionary forces. Unfortunately, the vari-
ation routinely observable at the phenotypic level seldom can be characterized in
population genetic terms (e.g., the numbers of variable loci and the alleles
responsible). This problem of empirical insufficiency plagued population genet-
ics throughout the first half of this century, as evidenced by the development of
diametrically opposed scientific opinions about the magnitude of genetic vari-
ability in nature. Advocates of the "classical school" maintained that genetic
variability in most species was low, such that con specific individuals were ho-
mozygous for the same "wild-type" allele at nearly all genetic loci. Proponents
of the "balance view" maintained that genetic variation was high, such that most
loci were polymorphic and individuals typically heterozygous at a large fraction
of genes (Fig. 2.1).
Numerous corollaries and ramifications stem from these opposing schools of
thought (Lewontin, 1974). Under the classical view, natural selection was seen
as a purifying agent, cleansing the genome of inevitable mutational variation.
Deleterious recessive alleles in heterozygotes might temporarily escape elimina-
tion, but were prevented from reaching high frequencies in populations because
of their negative fitness consequences when homozygous. The classicists did not
deny adaptive evolution but felt that the process was attributable to rare, selec-
tively advantageous mutations that quickly swept through a species to become
the new wild-type alleles. Because little variability existed to be shuffled into
new multilocus allelic combinations, recombination was viewed as a relatively
insignificant process. Furthermore, any genetic differences discovered between
populations or species must be of profound importance (because of the low
within-population component of variation). Central to the classical school was
the concept of genetic load (see Wallace, 1970, 1991)-the idea that genetic
variability produced a heavy burden of diminished fitness, which in the extreme
might even cause population extinction. This perception of genetic variation as
a curse was summarized forcefully by Muller (1950), who predicted, from ge-
18 History of Molecular Phylogenetics

A 1 B 1 C 1 0 1 E 2 F 1 G 1 H 1 I 1 J 1 K 1 L 1 M1 N 1 ° 1

CLASSICAL

A1 B 2 C S D4 E 2 F S G 2 H 1 1 2 J 7K3L4MSN20S

BALANCE (AND NEOCLASSICAL)

Figure 2.1. Classical versus balance views of genome structure. Shown for each case are
two homologous chromosomes with genes A-O exhibiting alleles indicated by subscripts.

netic load calculations, that only 1 locus in 1000 would prove to be heterozygous
in a typical human individual.
On the contrary, the balance school viewed natural selection as favoring
genetic polymorphisms through balancing mechanisms such as fitness superiority
ofheterozygotes (Dobzhansky, 1955), variation in genotypic fitness among hab-
itats, or frequency-dependent fitness advantage (Ayala and Campbell, 1974).
Genetic variability was thought to be both ubiquitous and adaptively relevant.
Deleterious alleles were not ruled out, but these were held in check by natural
selection and contributed little to heterozygosity. Because of the high variability
predicted for sexually reproducing species, no allele could properly be termed
wild-type. Genetic recombination, therefore, assumed a greater significance than
de novo mutation in producing interindividual fitness variation from one gener-
ation to the next. Furthermore, genetic differences among populations were
perhaps of less import because of the large within-population component of
overall variability. How much genetic variation was predicted under the balance
view? Wallace (1958) raised a proposal that seemed extreme at the time, but not
at all unreasonable today: "the proportion of heterozygosis among gene loci of
representative individuals of a population tends towards 100 percent."
The balance hypothesis gained indirect support from several lines of evidence:
(a) extensive phenotypic variation in most natural populations, which in a few
well-studied species often proved to have a genetic basis and to be of adaptive
relevance (e.g., Ford, 1964); (b) a genetic basis for many naturally occurring
morphological variants and fitness characters in populations that could be ma-
 --
0~
30

vertebrates
>-
(.)
20
(648 species)
t:
Q)
::J 10
C'"
........
Q)

--
0~
15
invertebrates
(370 species)
>-
(.)
10

t:
Q)
::J 5
C'"
........
Q)

--
~
o
18

plants
(785 species)
12

o
o .10 .20 .30 .40 >0.40

heterozygosity (H)
Figure 2.2. Empirical heterozygosity estimates from allozymes. Shown are
frequency distributions of mean H per species derived from protein-electro-
phoretic surveys conducted on 1803 species of vertebrate and invertebrate
animals (after Ward et al., 1992) and plants [data originally tabulated for a
review by Hamrick and Godt (1989) and kindly provided by J. HamrickJ. An
average of more than 20 loci was scored per study.
20 History of Molecular Phylogenetics

nipulated experimentally (e.g., by inbreeding, or by use of "common garden"

experiments in which the fraction of phenotypic differentiation attributable to
genetic variation could be estimated by controlling for environmental differ-
ences); and (c) the genetic response to artificial selection exhibited by many
domesticated animals and plants for numerous traits (see the review in Ayala,
1982a). However, none of these or related observations allowed direct answers
to the fundamental question: what fraction of genes is heterozygous in an indi-
vidual and polymorphic in a population? An answer requires that variability be
assessed at multiple independent loci, chosen without bias with respect to mag-
nitude of variability. But this requirement introduces a Catch-22 for any genetic
appraisal based on traditional Mendelian genetic approaches: Genes underlying
a phenotypic trait can be identified only when they carry segregating polymor-
phisms. In other words, because genetic assignments for phenotypic features
were inferred from the segregation patterns of allelic variants, invariant loci
escaped detection and no accumulation of such data could provide an uncolored
estimate of overall genetic variability. Other means were required to screen
genetic variation more directly, and in a manner that allowed assay of an unbi-
ased sample of polymorphic and monomorphic loci.

MOLECULAR INPUT TO THE DEBATE

A fundamental breakthrough occurred in 1966, when independent research

laboratories published the first estimates of genetic variability based on multilo-
cus protein electrophoresis (Harris, 1966; 10hnson et al., 1966; Lewontin and
Hubby, 1966). This method involves separation of non-denatured proteins by net
charge under influence of an electric current, followed by application of his-
tochemical stains to reveal the enzyme or other protein products of particular,
specifiable genes (Chapter 3). Because proteins are revealed whether or not they
vary, this approach provided the first valid attempt to obtain an unbiased estimate
of genome variability at a reasonable number (usually 20-50) of genetic loci.
The empirical results were clear-fruit fly and human genomes harbored a
wealth of variation, with 30% or more of assayed genes polymorphic, and
roughly 10% of loci heterozygous in a typical individual (Box 2.1). Over the
next two decades, multilocus electrophoretic surveys conducted on hundreds of
plant and animal species revealed levels of genetic variation that normally were
high but also variable among species (Fig. 2.2; see the reviews in Hamrick and
Godt, 1989; Nevo, 1978; Powell, 1975; Ward et al., 1992).
Protein electrophoretic techniques were not entirely new in 1966-indeed,
crude methods had been available for nearly 30 years (see Brewer, 1970).
Rather, the scientific impact of the landmark electrophoretic surveys lay prima-
rily in the manner in which methods and data from the field of molecular biology
were applied for the first time to long-standing issues in the previously separate
 History of Molecular Phylogenetics 21

Box 1.1. Measures of Genedc Variability wIttIln a Population

For multilocus protein electrophoretic data, one useful measure of genetic diversity
is population heterozygosity (H), defined as the mean percentage of loci heterozygous
per individual (or equivalently, the mean percentage of individuals heterozygous per
locus). Estimates of H can be obtained by direct count from a raw data matrix, the
body of which consists of observed diploid genotypes as in the following hypothetical
example involving eight allozyme loci (A-H) scored in each of five individuals:

Locus (j)

Individual (i) A B C D E F G H h;

1 aa aa aa aa ab aa aa aa 0.125
2 bb ab ab ab bb aa ab aa 0.500
3 cc ac be bd dd ad cd be 0.750
4 aa aa aa aa aa aa aa aa 0.000
5 cc cc cc cc cc cc cc cc 0.000
hj 0.0 0.4 0.4 0.4 0.2 0.2 0.4 0.2 H = 0.275

Here, diploid genotypes are indicated by lowercase letters (each letter representing an
electrophoretic allele), and heterozygotes have been underlined. In this example, 11 of
40 assayed genotypes are heterozygous (H = 0.275). Equivalently, H may be inter-
preted as the mean of the row or column totals, which represent direct-count heterozy-
gosities for single individuals (h;) or single loci (h), respectively. Heterozygosities
also may be estimated from observed frequencies of alleles (rather than genotypes),
assuming the population is in Hardy-Weinberg proportions. Thus hj = 1 - Iq/,
where qk is the frequency of the kth allele. Other common measures of population
variability for allozyme data are the mean number of alleles per locus and the per-
centage of polymorphic loci (P), which is 0.6 in the above example. To avoid an
expected positive correlation between P and sample size, a locus usually is considered
polymorphic only if the frequency of the most common allele falls below an arbitrary
cut-off, typically 0.99 or 0.95. In most protein-electrophoretic surveys, estimates of H
occur in the range of 0.0-0.2 (Fig. 2.2) and estimates of P range from 0.0 to 0.80.
For DNA-level data (which typically involve restriction sites or sequences along
a particular stretch of DNA that can be thought of as a "locus"), a useful statistic
summarizing heterozygosity at the nucleotide level is nucleotide diversity (Nei and Li,
1979; Nei and Tajima, 1981), or the mean sequence divergence between haplotypes
(alleles): P = I/;~p;j' where!; and~ are the frequencies of the ith andjth haplotypes
in the population, and Pij is the sequence divergence between these haplotypes. An-
other informative measure is haplotype diversity: h = 1 - I/;2. This measure is a
DNA-level analogue of the h for protein-electrophoretic data because its calculation
entails no assessment of the magnitude of genetic divergence between the alleles
involved. Depending on the loci and species surveyed, nucleotide diversities within a
population typically fall in the range 0.0005-0.020 (Stephan and Langley, 1992), and
haplotype diversities sometimes approach 1.0 for rapidly evolving genomes such as
animal mtDNA (Avise et aI., 1989).
22 History of Molecular Phylogenetics

discipline of population genetics. After the mid-1960s, contacts between molec-

ular and population genetics were only to expand, and at an ever-faster pace.
The data and conceptual orientations of the early protein electrophoretic (al-
lozyme) studies were to exert overriding influence on research goals in popula-
tion genetics for the next quarter century. Although this work provided important
information on genetic variation in natural populations, it also inhibited a stron-
ger molecular phylogenetic perspective in at least two ways. First, allozyme
methods revealed for each locus allelic products (electromorphs) that represent
qualitative multistate characters, the phylogenetic order of which cannot safely
be inferred from the observable property, band mobility on the gel. Much of
traditional population genetic theory, built around the seminal works of Fisher
(1930), Haldane (1932), and Wright (1931), can be couched in the language of
the expected frequency dynamics of such phylogenetically unordered alleles
under the separate or joint evolutionary forces of natural selection, genetic drift,
gene flow, mutation, and recombination. Protein electrophoretic approaches pro-
duced data that could be interpreted within the perspectives and language of
traditional population genetics theory, but the net effect was to channel thought
away from the phylogenetic perspectives which increasingly characterized other
areas of evolutionary biology. Thus, 20 years after the protein-electrophoretic
revolution began, Lewontin (1985) concluded, "population genetics is concep-
tually the study of gene lineages, [but] until now, the data to study such lineages
have not really been available."
Second, by focusing on issues of genetic variation per se (rather than on the
possible phylogenetic content of the molecular information), the seminal protein
electrophoretic surveys launched an intensive and still-continuing quest to refine
and interpret empirical estimates of molecular variability. The field of empirical
population genetics, thus, became preoccupied with several nonphylogenetic
questions, as described next.

RESEARCH PREOCCUPATIONS OF THE PROTEIN-ELECTROPHORETIC REVOLUTION

(a) How much protein variation remained hidden beyond the resolving power
of conventional gel electrophoresis? To address this question of cryptic poly-
morphism, two general experimental protocols were followed. In "backward
experiments" (Selander and Whittam, 1983), protein variants of known amino
acid sequence were electrophoresed to determine the proportion of known alleles
detectable (Ramshaw et al., 1979). In "forward experiments," assay conditions
were varied in attempts to discriminate additional alleles within the electromorph
classes identified in the original tests. Such assays involved the use of varied
buffers or other electrophoretic running conditions, "gel-sieving" through
acrylamide matrices of differing pore size, thermostability analyses, and mis-
cellaneous other biochemical techniques. These approaches frequently uncov-
 History of Molecular Phylogenetics 23

ered hidden protein variants (particularly at loci that were polymorphic in the
initial assays), and left the general impression that the original electrophoretic
methods had revealed only the tip of the genetic variability iceberg (Aquadro and
Avise, 1982a, 1982b; Ayala, 1982b; Bernstein et al., 1973; Bonhomme and
Selander, 1978; Coyne, 1982; Johnson, 1976a, 1977; McDowell and Prakash,
1976; Milkman, 1976; Prakash, 1977). Unfortunately, data from some of these
biochemical methods were difficult to interpret because the particular genetic
bases of the polymorphisms seldom were verifiable directly from the assays.
Primarily for this reason, none of these "refined" methods proved widely useful
as a source of genetic markers for population biology.
(b) How representative of other genes were the variability estimates derived
from protein electrophoretic loci? Because the availability (and cost) of his-
tochemical stains were deciding criteria for inclusion of proteins in most elec-
trophoretic surveys, dehydrogenases and other enzymes of the glycolytic path-
way and citric acid cycle were represented disproportionately. An initial concern
was whether variability at the loci encoding these proteins might be misrepre-
sentative of other protein-coding genes. For a brief time in the late 1970s and
early 1980s, attention was directed to the assay of abundant membrane-associ-
ated, ribosomal, and other structural proteins revealed by nonspecific protein
stains, using newly introduced two-dimensional gel techniques (which separate
proteins on the basis of charge by isoelectric focusing in the first dimension, and
then by molecular weight in the second dimension) (O'Farrell, 1975). Results
from several species indicated somewhat lower heterozygosities than had been
estimated from the original protein electrophoretic surveys (Aquadro and Avise,
1981; Leigh Brown and Langley, 1979; Racine and Langley, 1980; Smith et al.,
1980). However, any lingering thoughts that genomes might lack appreciable
variation were dispelled conclusively with the explosion of data on restriction
fragment-length polymorphisms (RFLPs) and DNA sequences that already had
begun to revolutionize the field by the early 1980s. One legacy of the genomic
variability debate is that an important branch of population genetics still focuses
on global estimates of genomic variation per se as a means of attempting to
understand the evolutionary forces governing genome structure (Gillespie, 1987;
Kimura, 1991; Kreitman, 1987; Li, 1978; Ohta and Tachida, 1990).
(c) How was genetic variability related to organismal fitness? Considering the
electrophoretic results in the historical context of the debate between the classical
and balance schools, it is hardly surprising that many population geneticists
turned empirical research efforts to the problem of how natural selection might
maintain so much protein polymorphism. As described next, allozyme research-
ers attacked this problem in several ways.
EMPIRICAL ApPROACHES TO THE VARIABILITy/FITNESS CONUNDRUM

In the "muItilocus" approach, searches were launched for correlations be-

tween mean overall heterozygosity and organismallife history attributes or fit-
24 History of Molecular Phylogenetics

ness components. One widely addressed issue was whether protein variability
might be correlated with environmental heterogeneity (Hedrick, 1986; Levene,
1953; Soule and Stewart, 1970). Some intriguing associations were reported. For
example, Nevo and Shaw (1972) attributed low heterozygosity in burrowing
mole rats to selection for homozygosity in the supposedly constant and narrow
subterranean niche. Selander and Kaufman (1973a) suggested that genic het-
erozygosity generally was highest in small, immobile animals that perceive
environments as coarse-grained patches of alternative habitat (Levins, 1968) and
lowest in large mobile animals that perceive environments as fine grained. Smith
and Fujio (1982) concluded that heterozygosities in marine fishes were correlated
positively with degree of habitat specialization. Powell and Taylor (1979) sum-
marized evidence that ~nvironmental heterogeneity in conjunction with habitat
choice contributed to genotypic diversity, whereas Valentine and Ayala (1974)
favored an environmental selection model consistent with an observed correla-
tion in marine invertebrates between low genetic variability and temporal trophic
resource stability (Ayala et al., 1975a; Valentine, 1976). Two influential studies
using experimental cages of fruit flies reported significantly higher heterozygos-
ities in populations maintained under variable as opposed to uniform environ-
mental regimes (McDonald and Ayala, 1974; Powell, 1971).
On the other hand, Sage and Wolff (1986) suggested that differing levels of
genic heterozygosity in large mammals were attributable not to varying envi-
ronmental selection pressures per se, but rather to environment-dependent pop-
ulation histories and effects of genetic drift-species in glaciated regions tended
to have lower variabilities than their counterparts in temperate and tropical re-
gions, purportedly due to population bottlenecks accompanying serial recoloni-
zations of northern latitudes following retreats of the Pleistocene glaciers. In
general, unless species are normally at genetic equilibrium (which seems un-
likely), the extant standing crop of genetic variation must be a function both of
the genetic diversity originally available to a species (its phylogenetic legacy)
and of additional processes such as selection, gene flow, and the mating system
that govern how that available variation is partitioned within and among popu-
lations.
Positive correlations also were noted between genetic variability and particular
life history attributes, such as short generation time, small maximum body size,
and small egg size in bony fishes (Mitton and Lewis, 1989; but see Waples,
1991), and high fecundity, outcrossing mode of reproduction, pollination by
wind, and long generation time in plants (Hamrick et al., 1979). Among con-
specific organisms, correlations were reported between individual heterozygosity
and a variety of phenotypic characters presumably associated with fitness (see the
review in Mitton, 1993): exploratory behavior in mice (Garten, 1977); antler
characteristics in deer (Scribner and Smith, 1990); shell shape in blue mussels
(Mitton and Koehn, 1985); growth rate in fishes, salamanders, oysters, trees and
 History of Molecular Phylogenetics 25

other species (Ferguson, 1992; Garton et al., 1984; Koehn et al., 1988; Ledig et
al., 1983; Mitton and Grant, 1984; Pierce and Mitton, 1982; Singh and Zouros,
1978); herbivory resistance in pines (Mopper et al., 1991); disease resistance in
trout (Ferguson and Drahushchak, 1990); and developmental stability in many
species, as supposedly evidenced by lower phenotypic variance between indi-
viduals (Lerner, 1954; Zink et al., 1985) or by lower "fluctuating asymmetry"
(the difference between bilateral features) within individuals (Allendorf and
Leary, 1986; Leary et al., 1985; Palmer and Strobeck, 1986; Van Valen, 1962).
Of course many of these physiological and developmental characteristics likely
are interrelated.
Another aspect of the multilocus approach involved searches for molecular or
metabolic features correlated with heterozygosity. Among the examined factors
arguably associated with genic variability were the following: molecular size of
the enzyme (Eanes and Koehn, 1978a); quaternary structure (Sole-Cava and
Thorpe, 1989; Ward, 1977; Zouros, 1976); level of intragenic recombination
(Koehn and Eanes, 1976); physiological role in regulating flux through metabolic
pathways (Johnson, 1976b); enzymatic action on intracellular versus extracellu-
lar substrates (Ayala and Powell, 1972a; Gillespie and Langley, 1974; Kojima et
al., 1970); and others (reviews in Koehn and Eanes, 1978; Selander, 1976).
Several difficulties accompanied attempts to interpret such multilocus associ-
ations, beyond the obvious point that correlation by itself cannot prove causality.
First, there likely is a reporting bias in favor of positive correlations, and the
number of variables that can be examined essentially is limitless. Second, mean
heterozygosity as estimated from a small number of protein loci may not accu-
rately rank-order specimens within a population with respect to genome-wide
variability (Chakraborty, 1981; Mitton and Pierce, 1980) [unless, perhaps, in-
dividuals vary dramatically along an outbred-inbred continuum, due to demo-
graphic cycles, fine demic structure, or mating behaviors (Mitton, 1993; Scrib-
ner, 1991; Smith et al., 1975; Smouse, 1986)]. This point led some authors to
conclude that associations of individual heterozygosity with fitness were attrib-
utable not to differing levels of genome-wide variation, but rather to physiolog-
ical advantages stemming from heterozygosity at the particular glucose-metab-
olizing or other enzymes under survey [or perhaps to tightly linked genes in the
chromosomal blocks that they mark (Koehn et aI., 1983; Mitton and Grant,
1984)]. Third, several of the associations with heterozygosity listed above in-
volved weak trends for which exceptions could readily be cited, or alternative
explanations advanced. For example, high genetic variability characterizes some
species inhabiting proverbially "stable" environments such as the deep sea, and
caves or other subterranean settings, and low genetic variability certainly can
result from demographic population contractions in any environment (Avise and
Selander, 1972).
Ward et al. (1992) recently updated the correlational approach to the study of
26 History of Molecular Phylogenetics

allozyme heterozygosity. From a literature survey of over 1000 animal species,

they concluded that approximately 21-34% of the variance in mean protein
heterozygosity could be attributed to taxonomic effects (e.g., fishes tend to have
low H values, amphibians the highest such values), and 41-52% of the variance
was due to protein effects (including subunit size, subunit number, enzyme
function, etc.). No comparable summary appears to be available for plants.
Nonetheless, with regard to postulated molecular or physiological correlates of
multilocus heterozygosity, the conclusion reached in 1976 by Selander still
stands: ". . . notwithstanding the immense amount of effort expended in sur-
veying variation in organisms in the last decade, the sample sizes of loci are
generally inadequate for satisfactory analyses of the variation: molecular heter-
ogeneity is too great." The numerous correlations involving heterozygosity
listed above remain intriguing, but largely unexplained.
Frustration with such multilocus approaches to assessing natural selection's
role in maintaining genetic variability led other researchers to the "vertical" or
"single-locus" approach, wherein particular polymorphisms were studied at
multiple levels ranging from biochemistry, physiology, and developmental ex-
pression to transmission patterns, population dynamics and ecological associa-
tions (Clarke, 1975; Koehn and Hilbish, 1987; McDonald, 1983). Intensive
studies of several such model systems all uncovered convincing evidence for
differences between allozyme genotypes upon which selection probably operates
(Table 2.1). Unfortunately, relatively few polymorphisms have been analyzed so
intensively. Furthermore, most of the genes studied had been identified a priori
as likely candidates for natural selection, and thus the polymorphisms analyzed
by the vertical approach probably comprise a biased sample with regard to the
issue of selective maintenance.

Genetic Theory and the Neutralist-Selectionist Debate

The discovery of extensive molecular variation did not clinch the case for the
philosophical perspective on genetic variation embodied in the balance school of
thought, but instead stimulated development of an alternative explanation for
molecular genetic variability that was to assume a prominent role in population
genetics to the present time. Under the strict neutral mutation theory, alternative
alleles confirm no differential fitness effects on their bearers. As summarized by
Kimura (1991), "the great majority of evolutionary mutant substitutions at the
molecular level are caused by random fixation, through sampling drift, of se-
lectively neutral (i.e., selectively equivalent) mutants under continued mutation
pressure. " As applied to intraspecific molecular variability, neutrality theory
predicts that polymorphisms are maintained by a balance between mutational
input and random allelic extinction by genetic drift. Neutralists did not deny the
existence of high molecular variability, but rather questioned its relevance to
Table 2.1 Examples of major research programs on allozyme polymorphisms that employed the "vertical" approachQ

Protein Organism Evidence Introductory references

Alcohol Drosophila Kinetic differences between isozymes associated Aquadro et al., 1986;
dehydrogenase with differences in survivorship, Clarke, 1975; van
developmental time, and environment Delden, 1982.
a-glycerophosphate Drosophila Kinetic differences correlated with flight Miller et al., 1975;
dehydrogenase metabolism, power output, and environmental O'Brien and
temperature MacIntyre, 1972;
Oakeshott et al., 1982.
Carboxylesterase Drosophila Differences in enzyme activity associated with Gilbert and Richmond,
reproduction 1982; Richmond et al.,
1980.
Glucose-6 phosphate Drosophila Differences in metabolic flux associated with Barnes and Laurie-
dehydrogenase and differences in fitness Ahlberg, 1986;
6-phosphogluconate Cavener and Clegg,
dehydrogenase 1981; Hughes and
Lucchesi, 1977.
Glucosephosphate Colias butterflies Kinetic differences correlated with mating Watt, 1977; Watt et al.,
isomerase success and survivorship 1983, 1985.
Glutamate pyruvate Tigriopus copepods Enzyme activity differences associated with Burton and Feldman,
transaminase differential responses to hyperosmotic stress 1983.
Lactate dehydrogenase Fundulus fishes Differences in kinetic and other biochemical DiMichele et al., 1986,
properties associated with differences in 1991; Place and
metabolism and fitness Powers, 1979, 1984.
Leucine Mytilus mollusks Enzyme activity differences associated with Hilbish and Koehn, 1985;
aminopeptidase osmoregulation and fitness Hilbish et al., 1982;
Koehn and Immennan,
1981.
~n each case, kinetic differences demonstrated between allelic products have suggested that the polymorphisms may be maintained
by natural selection.
28 History of MoLecuLar PhyLogenetics

organismal fitness. Because neutralists and classicists share the position that
balancing selection plays little role in maintaining molecular polymorphism and
that most selection is directional or "purifying" against deleterious alleles,
neutrality theory also has been referred to as the neoclassical theory (Lewontin,
1974).
Several points should be made clear at the outset. First, neutralists do not
suggest that most genes or allelic products are dispensable (of course they are
not). Rather, they propose that different alleles are functionally equivalent such
that organismal fitness is not a function of the particular genotypes possessed.
Sec~d, neutralists do not deny that many de novo mutations are deleterious and
eliminated by purifying selection. Rather, the focus is on the supposed neutrality
of segregating polymorphisms that escape selective elimination. Indeed, one
cornerstone of neutrality theory is that nucleotide positions or genic regions that
are functionally less constrained are those most likely to harbor neutral variation
and to exhibit the most rapid pace of allelic substitution. Third, neutralists do not
challenge the Darwinian mode of adaptive evolution for organismal morpholo-
gies and behaviors [although some important recent extensions of the neutrality
theory do propose a significant role for genetic drift in organismal evolution as
well (Kimura, 1990)]. Rather, neutrality theory developed in response to the
intellectual challenge provided by the unexpectedly high levels of molecular
variability observed.
Neutrality concepts were introduced in the late 1960s (Kimura, 1968a,
1968b), and gained immediate widespread attention due in part to a paper by
King and Jukes (1969) provocatively titled: "Non-Darwinian evolution: random
fixation of selectively neutral mutations." Indeed, the theory did challenge a
prevailing approach of naively extending to molecular biology the neo-Darwin-
ian views on the adaptive significance of nearly all organismal differences (see
Gould and Lewontin, 1979). It is quite remarkable that within a decade, and
continuing today, neutrality theory gained sufficient acceptance to be viewed
widely as molecular evolution's gigantic "null hypothesis"-the simplest and
most straightforward way to interpret molecular variability, and the hypothesis
whose predictions were to be falsified before alternative proposals involving
balancing selection could be entertained seriously. This is not to say that the
selectionist-neutralist debate is fully resolved.
The neutrality school has strong roots in the quantitative tradition of theoret-
ical population genetics developed earlier in the century (Fisher, 1930; Haldane,
1932; Wright, 1931). An elegant and elaborate theory predicts the amount of
genetic variability within a given population as a function of mutation rate, gene
flow (where applicable), and population size (Kimura and Ohta, 1971). Con-
spicuously absent from the calculations are selection coefficients, because alleles
are assumed to be neutral. Under strict neutrality theory, molecular variability is
a function of the neutral mutation rate and the evolutionary effective population
 History of Molecular Phylogenetics 29

Box 2.2 Effective Population Size

Not all individuals in a population contribute gametes to the next generation with
equal probability. This has led to the concept of effective population size (Ne), orig-
inally due to Wright (1931). The effective number of individuals refers to the size of
an idealized population that would have the same genetic properties (such as the
intergeneration variance in allele frequencies due to chance sampling error) as that
observed for the real population. Usually, N" is much smaller than N (the census size)
for one or more of the following reasons:
(a) Separate sexes: In organisms with separate sexes, one gender may be more
common than the other. Let N m and Nf be the census numbers of males and females
in such a population. Then the effective population size due to this disparity alone is
Ne = 4N"/vJ(Nm + Nf ). UnlessNm = Nf , this equation shows thatN" is less than the
total census count (Nm + Nf ).
(b) Fluctuations in population size: Most populations in nature probably fluctuate
greatly in size, due to diseases, changes in habitat quality, predation, etc. The effective
population size due to such fluctuations is equal to the harmonic mean of the breeding
population sizes across generations. A harmonic mean is a function of the mean of
reciprocals, or in this case N" = n/[I(lIN)l. where N; is the population size in the ith
generation and n is the number of generations. A harmonic mean is closer to the
smaller rather than to the larger of a series of numbers being averaged, so N" can be
much lower than most population censuses. A severe reduction in population size is
called a "population bottleneck" and can greatly depress evolutionary N e •
(c) Combination of separate sexes and fluctuating population size: If census pop-
ulation sizes of males and females are known across multiple generations, the joint
effects of the above factors on Ne can be determined. For each generation, the census
sizes of males and females are converted to an effective size for that generation, as in
(a). Then the equation in (b) is employed to take the harmonic mean of the single-
generation estimates.
(d) Variation in progeny numbers: Even in a nonfluctuating population with equal
numbers of males and females, some individuals may leave many more progeny than
others, creating a large variance across families. Only when offspring numbers follow
a Poisson distribution with mean (and hence variance) of 2.0 per parent, does Ne =
N. In more realistic situations, where the variance often exceeds the mean, N" is
smaller than the census breeding population size (Crow, 1954). Hedgecock et al.
(1992) have argued that organisms with extremely high fecundities are particularly
prone to gross disparities between Ne and N due to high variability in fertility across
individuals; empirically, such disparities indeed have been reported for several aquatic
and marine species such as shellfish (Hedgecock and Sly, 1990).
(e) Other factors: Various other factors also can reduce Nt! relative to N. For
example, in a species composed of many subpopulations each of which is subject to
periodic extinction and recolonization, the species as a whole will have a far lower Ne
than might have been predicted had only composite census sizes for particular gen-
erations been available (Maruyama and Kimura, 1980).
30 History of Molecular Phylogenetics

size, Ne (Box 2.2). For example, the heterozygosity expected for electrophoret-
ically detectable alleles at equilibrium between mutation and genetic drift is
given by
(2.1)
where J.L is the per locus per generation mutation rate to neutral alleles (Ohta and
Kimura, 1973). Figure 2.3 plots this expected relationship between H and Ne for
reasonable neutral mutation rates and also shows the range of allozyme heterozy-
gosities empirically observed for numerous animal species with indicated pop-
ulation census sizes. Such comparisons should deal with species effective sizes
because the theory involves equilibrium expectations over long-term evolution.

1 .0

.-
In 0.8
o
C)
- 9
~ Jl = 10
N 0.6
o... ~
CD
~
0.4
CD
.c
C 0.2
as
CD
== 0.0
2 5 8 11

log [N]
10
Figure 2.3. Predicted relationship between species effective population size
and protein-electrophoretic heterozygosity under neutrality theory. Expecta-
tions for two neutral mutation rates (J.L) are presented. Also shown (shaded
area) are observed heterozygosities for numerous animal species as a function
of current-day population size (N. shown logarithmically as powers of 10)
(after SouIe, 1976).
 History of Molecular Phylogenetics 31

Except perhaps for some of the least abundant species, observed values of H
have proved to be much lower than neutrality theory predicts. This conclusion
generally holds even when mutations are assumed to be mildly deleterious (Nei,
1983). One likely explanation for the relative paucity of genetic variation is that
long-term effective population sizes for most species are vastly smaller than
might otherwise be supposed from current-day census sizes.
This raises a remarkable irony about the neutralist-selectionist debate that
stems from historical precedents of the classicist-balance controversy. When
protein variation was uncovered in the seminal electrophoretic surveys, selec-
tionists interpreted the observations as consistent with the balance view and
sought (as described earlier) to discover the selective forces responsible for such
extensive polymorphism. At the same time, neutralists were facing a dilemma of
how to account for the paucity of polymorphism relative to neutrality expecta-
tions, given mutation rates and population sizes thought to characterize most
species. The dearth of variation from the neutralist perspective extends to the
level of some DNA sequences as well (Box 2.3). For example, with regard to
mitochondrial DNA alleles (which are maternally inherited), the expected mean
time to common ancestry under neutrality theory is approximately
(2.2)
where G is the number of generations and Nf{e) is the effective population size of
females (Avise et al., 1988). Figure 2.4 plots values of Nf{e) for a number of
vertebrate and invertebrate species as estimated from observed mtDNA haplo-
type distances (Avise, 1992), using conventional evolutionary rate calibrations
for the mtDNA molecule (Brown et al., 1979). Most observed values fall orders
of magnitude below theoretical expectations based on neutrality theory and
present-day census population sizes (Nt values). In other words, despite exten-
sive genetic heterogeneity, mtDNA diversity typically is much lower than neu-
trality theory predicts. Either mtDNA evolution is slower than generally believed
or evolutionary effective population sizes are vastly lower than are present-day
population sizes for most species. In summarizing these types of observations,
Nei and Graur (1984) concluded that" ... polymorphism is actually much lower
than the neutral expectation and that if the bottleneck effect is not sufficient for
explaining the observed level, the type of selection to be considered is not
diversity-enhancing selection but diversity-reducing selection." The irony of this
neutralist perspective in the history of the classical-balance debate still is not
appreciated widely by many of the proponents of balancing selection.
Another important aspect of the neutral mutation theory concerns predictions
about molecular evolutionary rate. Two aspects of rate must be distinguished
carefully. With regard to shifts in frequencies of preexisting alleles. the rate of
neutral evolution can be greater in small populations. Genetic drift refers to
random changes in allele frequency due to sampling variation of gametes from
32 History of Molecular Phylogenetics

Box 2.3. Mean TImes to Shared AlleUc Ancestry

Another way to formulate neutrality theory regarding the association between ge-
netic variability and population size is through consideration of the expected frequency
distribution of times to common ancestry among alleles. Imagine an idealized popu-
lation with nonoverlapping generations and large constant size N. Suppose further that
in each generation, individuals contribute to a gamete pool from which 2N nuclear
gametes are drawn at random (effectively with replacement) to produce individuals of
the next generation. The probability that two gametes carry copies of the same allele
from the prior generation is lI2N. This is also the probability that the time to common
ancestry of two alleles is one generation ago (G = I). The probability that a pair of
alleles is not identical from the prior generation is 1 - 1I2N. Thus, the probability that
these latter alleles trace to an identical copy two generations ago is (l - l/2N)(1I2N).
From an extension of such reasoning, the probability that two randomly chosen alleles
derive from a common ancestral allele that existed G generations ago is
fiG) = (1I2N)(l - lI2N)G - 1, or approximately (l/2N)e-(G - 1)/2N.

These equations give the probability distribution of times to common ancestry in terms
of the number of generations (Tajima, 1983). The distribution is geometric, with mean
approximately 2N. The mean time to shared haplotype ancestry for mtDNA genes can
be derived similarly (Avise et al., 1988), but is only one-fourth as large as for nuclear
genes, the difference being attributable to a twofold effect due to the haploid trans-
mission of mtDNA and another twofold effect due to mtDNA's normal pattern of
uniparental transmission through females. The above theory assumes that times to
common ancestry for allelic pairs are independent. Therefore, in interpreting empirical
data for any particular species against these expectations, caution must be exercised
because the history of lineage coalescense within a real population imposes a severe
correlation on the pairwise comparisons (Ball et al., 1990; Felsenstein, 1992; Hudson,
1990; Slatkin and Hudson, 1991).

generation to generation and is a special case of the more general phenomenon

of sampling error, which is inversely related to sample size. However, with
regard to the origin and substitution of new alleles, the rate of neutral evolution
is independent of population size and depends only on the mutation rate to
neutral alleles.
This latter conclusion can be demonstrated as follows. In a diploid population
of size N, there are 2N allelic copies of each nuclear gene. In time, the descen-
dents of only one of these copies will remain (i.e., is destined for fixation). The
chance that any newly arisen neutral mutation will undergo random fixation is
simply V2N. On the other hand, the probability that a new neutral mutation arises
in a population is 2NfJ" where fJ, is again the mutation rate to neutral alleles. It
follows that the rate of fixation of new neutral mutations is the product of the
origination rate of mutations and their probabilities of fixation once present, or
2NfJ, x V2N = fJ,. In other words, the rate of substitution in evolution under strict
 History of Molecular Phylogenetics 33

•
•
•
(estimated •
from mtDNA
variation) 1 04
•
•
•
2
10
Hi
present population size
Figure 2.4. Relationship between current-day census population size and evo-
lutionary effective population size, as estimated from empirical mtDNA nucle-
otide diversities for several marine species in the southeastern United States
(after Avise, 1992). Both axes are in logarithmic scale.

neutrality equals the rate of mutation to neutral alleles. This simple conclusion is
the theoretical basis for the neutrality prediction that biological macromolecules
can provide standard "molecular clocks" (Chapter 4), irrespective of population
size.
The selection-neutrality controversy has dominated both the theoretical and
empirical sides of population genetics for the last 25 years (see the reviews in
Avise, 1977a; Ayala, 1976a; Lewontin, 1974, 1991; Nei and Koehn, 1983), and
the debate is not yet settled, for at least two major reasons. First, both selectionist
and neutralist theories are immensely powerful constructs in the sense of being
capable of explaining nearly any set of observations by appropriate alteration of
parameters and assumptions. Because of the multitudinous ways in which natural
selection can operate, falsification of all selectionist scenarios for a given data set
is nearly impossible (indeed this was a primary motivation for the development
of a quantitative neutrality theory that specifies expectations explicitly). But
neutrality theory also can yield a nearly limitless array of predictions by varying
34 History of Molecular Phylogenetics

parameters that are notoriously difficult to measure in real populations [Ne , IJ.,
and selection intensities against alleles that may be slightly deleterious or
"nearly neutral" (Ohta, 1992a)]. Furthermore, many apparent departures from
neutrality expectations might be due to unknown historical factors that no doubt
remove populations and species from the equilibrium conditions that commonly
are assumed in most neutral models.
A second probable reason why the selection-neutrality debate continues with-
out final resolution is the difficulty of defining exactly what is meant by natural
selection. For example, is the phenomenon of "meiotic drive" (whereby certain
alleles appear to "cheat" during meiosis by distorting Mendelian segregation
ratios in their favor) to be viewed as a form of natural selection at the gametic
level? In general, are "selfish genes" (Dawkins, 1989) that compete for trans-
mission within an organismal lineage to be interpreted as evolving under the
influence of natural selection? Holmquist (1989) argues that the molecules inside
a cell form an interacting community rather like an ecosystem. This "molecular
interplay" is quite different from what traditionally has been meant by natural
selection at the organismallevel (Ohta, 1992b). Perhaps the concept of natural
selection should be broadened to encompass evolutionary effects at hierarchical
levels both below (Dawkins, 1989) and above (Gould, 1980) the level of dif-
ferential fitness among individuals.
In any event, the final answer likely lies somewhere between the polarized
neutralist and selectionist camps. Certainly some loci are under strong balancing
or other forms of natural selection, whereas the segregating alleles at many other
loci must have negligible differential influence on organismal fitness. Knowledge
about the evolutionary forces governing the dynamics of molecular markers
probably is much more important for some applications (e.g., phylogeny recon-
struction) than it is for others (e.g., clonal identification and parentage assess-
ment).

Systematic Philosophy and the Phenetic-Cladistic Debate

Another area of evolutionary research that diverted attention from the appli-
cation of molecules as genetic markers centered around conflicting philosophical
approaches to systematics. In the 1960s, a heated debate developed (between the
pheneticists and the cladists) that was to dominate attention in the field of sys-
tematics for more than two decades. The philosophical differences involved,
relevant though they may be to the field of molecular evolution, initially centered
around interpretations of morphologic and other traditional systematic characters.
Until the mid-l900s, the science of classifying organisms involved the oper-
ational approach of comparative morphological assessment, introduced in crude
form by Carl Linnaeus two centuries earlier (Linnaeus, 1759). Typically, spe-
cialists devoted years of study to a particular group such as birds or beetles, and
 History of Molecular Phylogenetics 35

on the basis of accumulated experience and gestalt classified their creatures into
a hierarchical taxonomy. This approach contributed greatly to a cataloging of the
tremendous diversity of the natural world and resulted in most of the biological
classifications still followed today. Potential difficulties of this approach
stemmed from the lack of unifying or standardized classification methods (either
conceptual or operational) with the following consequences: (a) the centering of
systematic authority within a small number of researchers for each taxonomic
group; (b) the lack of formalized procedures for corroboration or refutation of a
proposed classification; (c) the absence of a uniform measure by which classi-
fications for different taxonomic groups might meaningfully be compared; and
(d) the lack of a clear philosophical orientation on precisely which aspects of
evolution were reflected in a particular classification.
Explicit concern with these shortcomings of traditional systematic practice
prompted the rise of numerical taxonomy, or the phenetic approach to systematics
(Sokal and Sneath, 1963). Pheneticists proposed that organisms should be grouped
and classified according to overall similarity (or its converse, distance), as mea-
sured by defined rules preferably using as many organismal traits as possible.
Among the principles guiding numerical taxonomy are the following (Sneath and
Sokal, 1973): (a) the best classifications usually result from the analyses based on
the largest numbers of characters; (b) at least at the outset, every character is to
be afforded equal weight; (c) classifications are based on quantitative measures
of overall (phenetic) similarity or distance between the taxa (operational taxo-
nomic units or OTUs) under comparison; and (d) patterns of character correlations
can be used to recognize distinct taxa and to draw systematic inferences under
certain assumptions about evolutionary pathways and mechanisms. Operation-
ally, numerical taxonomy involves the application of quantitative methods for
estimating phenetic similarity, examining character correlations, and grouping
OTUs. Philosophically, "numerical taxonomy aims to develop methods that are
objective, explicit, and repeatable .... " (Sneath and Sokal, 1973).
The development of numerical taxonomy provided a valuable service to sci-
ence by opening for scrutiny traditional practices in systematics that had been
needlessly opaque. Nonetheless, pheneticists were attacked on several fronts,
most notably by cladists who proposed an alternative philosophy and protocol for
phylogeny reconstruction and classification (Eldredge and Cracraft, 1980). Un-
der tenets of the cladistic school, organismal relationships cannot be deduced
from overall similarity, but are reflected in a particular subset of similarity
attributable to synapomorphic or shared-derived traits (Box 2.4; Fig. 2.5). Cla-
dists focus almost exclusively on assessing the branch-splitting component of
evolutionary trees (cladogenesis) rather than branch lengths (accumulated change
within lineages, or anagenesis). The ultimate goal is to develop organismal
classifications based on correctly inferred cladogenetic histories.
As sometimes practiced, cladistic approaches themselves are not entirely im-
Box 2A. Oadlsdc TennlnololY and Concepts
The following definitions are relevant to cladistic-phenetic discussions:
I. Classes of organismal resemblance:
a. Phenetic similarity-overall resemblance between organisms.
b. Patristic similarity-the component of overall similarity due to shared ancestry.
c. Homoplastic similarity (homoplasy)-the component of overall similarity due to
convergence from unrelated ancestors. [The term homoplasy also is used fre-
quently to describe the "extra steps" implied in a phylogenetic network beyond
those that distinguish taxa in the raw data matrix. In this usage, homoplasy may
arise from convergence, parallelism, or evolutionary reversals in character states
(Chapter 4)].
II. Classes of character state used to characterize organismal resemblance:
a. Plesiomorph--an ancestral character state (one present in the common ancestor
of the taxa under study).
b. Symplesiomorph-an ancestral character state shared by two or more descen-
dant taxa.
c. Apomorph-a derived or newly evolved character state, not present in the
common ancestor of the taxa under study.
d. Synapomorph--a derived character state shared by two or more descendant taxa.
e. Autapomorph--a character state unique to a single taxon.
III. Other relevant definitions
a. Monophyletic group or clade-an evolutionary assemblage that includes a com-
mon ancestor and all of its descendents.
b. Polyphyletic group-an artificial assemblage derived from two or more distinct
ancestors.
c. Paraphyletic group-an artificial assemblage that includes a common ancestor
and some but not all of its descendents.
d. Outgroup-a taxon phylogenetically outside the clade of interest.
e. Sister taxa-taxa stemming from the same node in a phylogeny.
Phenetic resemblance may be due to patristic and/or homoplastic similarity. Patristic
similarity may arise from symplesiomorphic and/or synapomorphic character states.
Cladists attempt to distinguish between symplesiomorphic and synapomorphic simi-
larity and to identify clades on the basis of synapomorphs only (Fig. 2.5). Pheneticists
usually make no such attempts to distinguish sources of resemblance. Phylogenetic
reconstructions based on either cladistic or phenetic principles can be compromised by
extensive homoplasy.
Because cladists must distinguish symplesiomorphs from synapomorphs, much ef-
fort is devoted to the elucidation of evolutionary "polarities" (derived versus ancestral
conditions) of character states. The following are among the criteria that have been
used to suggest primitiveness for a character:
a. Presence in fossils
b. Commonness among an array of taxa
c. Early appearance in ontogeny
d. Presence in an outgroup
Criterion (d) is most widely employed now, as the others have proved misleading or
incorrect in many instances (Stevens, 1980).
 History of Molecular Phylogenetics 37

CHARACTERS AND STATES

x y z

Figure 2.5. Philosophical rationale underlying Hennigian cladistic attempts to distin-

guish sources of similarity. Shown is the true (but unknown to the researcher) phylogeny
for taxa A-F and the distribution of observed binary states for characters X, Y, and Z.
Suppose that taxon A is known to be an 0UtgroUp for B-F, whose phylogeny is to be
reconstructed. Character states XI' YI' and Zp possessed by various taxa and the out-
group, are symplesiomorphs (shared ancestral states), and hence identify no clades. In
particular, Yl and ZI could be positively misleading in amalgamating ingroup members
(C-F and B, C, F, respectively) had their ancestral status gone unrecognized. Character
state Y2 defines no multitaxon clade because it is an autapomorph, and Z2 could be
misleading as a putative clade marker because it evolved in parallel (convergent) fashion
in taxa D and E. Only x 2 is a valid synapomorph in this example, correctly identifying
the true clade composed of taxa B-D.

mune from criticism. For example, a widely held belief is that "one true syn-
apomorphy is enough to define a unique genealogical relationship" (Wiley,
1981). Although incorrect [in part because of the distinction between phyloge-
nies for particular genes and organismal pedigrees (Chapter 4)], this perception
sometimes has led to dogged advocacy for putative clades that receive support
from only a few presumptive but favored synapomorphs. Thus, unless many
characters are assayed (as advocated under the phenetic school), there is a po-
tential danger in cladistics of the kinds of authoritarianism that plagued system-
atics earlier in the century and that prompted the original rise of numerical
taxonomy.
Indeed, there seems little justification for the rancor of the cladistic attack on
phenetics, for at least two reasons: (a) cladism owes a deep debt to numerical
taxonomy for having first opened new discussions and viewpoints on traditional
systematic practices, and (b) curiously, cladistic methods as applied to large data
38 History of Molecular Phylogenetics

sets can come rather close to the procedures of numerical taxonomy (because for
such data, character conflicts in clade delineation almost inevitably arise, thereby
requiring some form of numerical tallying of putative synapomorphs). Thus,
numerical cladistics and numerical phenetics are not as distinct operationally as
they might at first appear, although cladistic methods do attempt to distinguish
among sources of similarity and thereby account for specified character state
distributions in terms of phylogenetic history.
The original "bible" of the cladistic school, published in 1950 by the German
entomologist Willi Hennig, was translated to the English version Phylogenetic
Systematics in 1966. In the last two decades, cladistic methods based on Hen-
nig's insights have revolutionized systematic practice as applied to traditional
taxonomic characters. Cladograms generated for many taxonomic groups now
summarize phylogenetic reconstructions based on hypotheses concerning the
polarities (ancestral versus derived conditions), transformations, and temporal
orders of appearance of various morphological, physiological, or behavioral
character states. Thus, one major strength of cladistic approaches involves the
formulation of explicit, potentially testable hypotheses for the origin of particular
character states (Buth, 1984; Patton and Avise, 1983). The cladistic school also
has given rise to the important "vicariance" subdiscipline of biogeography
(Chapter 8).
Few researchers now dispute the philosophical pillar of the cladistic school-
that shared-derived traits are a solid basis for clade delineation. Rather, most
questions center on operational issues: How reliably can synapomorphies be
identified? What kinds of characters are best suited? How are character conflicts
resolved when putative clades identified by different presumptive synapomorphs
disagree? How is a phylogeny to be translated into a classification?
The relatively simple principles of Hennigian cladistics have attracted much
devotion and prompted strident claims concerning the theory and practice of
systematics. Future historians of science no doubt will address reasons for the
heated polarization between the phenetic and cladistic camps [see Hull (1988) for
an early perspective], but at least two important ramifications for molecular
evolution have stemmed from this controversy. First, the debate itself unques-
tionably gave a renewed energy to morphology-based systematics. This devel-
opment came at a time when some traditional systematists may have felt threat-
ened by the rise and increasing dominance of molecular biology. One
unfortunate and unnecessary consequence of this timing is that molecular and
morphological approaches to systematics sometimes have been viewed in oppo-
sition, a perception with no valid basis. Second, the cladistic-phenetic war,
although waged primarily in the context of morphology-based systematics, oc-
casionally spilled over such that molecular phylogenetics also was caught in the
crossfires. For example, strict Hennigian approaches cannot be applied to raw
data consisting solely of numerical distance values between taxa, and as a result
 History of Molecular Phylogenetics 39

some cladists automatically discredited all such infonnation derived from the
important immunological and nucleic acid hybridization methods of molecular
biology (Chapter 3). Attacks also were mounted against the widespread practice
of summarizing molecular data using the UPGMA algorithm of numerical tax-
onomy, despite the fact that an important assumption of this phenetic procedure
(constant evolutionary rate across phenogram branches) appeared to mesh well
with the considerable independent evidence of a clocklike behavior for many
biological macromolecules (Chapter 4). Furthermore, many molecular data,
even those in the form of qualitative character states such as protein electro-
morphs or DNA sequences, are not particularly well suited for strict Hennigian
cladistic analysis, in part because of the high risk of homoplasy at the level of
individual electromorph or nucleotide character states (Straney, 1981).
Apparent conflicts among characters in clade delineation arise primarily from
homoplasy (Box 2.4). To minimize the number of ad hoc hypotheses required to
resolve character conflicts along a phylogeny, principles of "maximum parsi-
mony" are employed widely today (Felsenstein, 1983; Sober, 1983). As applied
to phylogenetic inference, parsimony algorithms operate by estimating evolu-
tionary trees of minimum total length (i.e., trees that minimize the number of
evolutionary transformations among character states required to explain a given
data set). Although notions of parsimony have long been a part of general
biological reasoning, developments in cladistic philosophy provided an impor-
tant historical step in the further elaboration of parsimony approaches in phy-
logeny reconstruction. Thus, a close intellectual connection exists between nu-
merical cladistics and numerical parsimony, both of which strive to resolve data
conflicts in the simplest possible ways consistent with particular assumptions
about the evolutionary process (Swofford and Olsen, 1990). Further discussion
of maximum parsimony and other phylogenetic methods as applied to molecular
data will be deferred to Chapter 4.

Phylogenetic Data and the Molecule-Morphology Debate

Overlapping to some degree with the phenetics-cladistics debate was another

controversy about whether molecules or morphologies provide a better guide to
phylogeny reconstruction. Particularly in the 1960s and early 1970s (in the early
years of protein electrophoresis), many systematists trained in traditional organ-
ismal disciplines understandably viewed the new molecular data with consider-
able incredulity. As the field of molecular evolution grew, this overt skepticism
softened somewhat to a fundamental disquietude over the relative merits of
molecular versus morphological infonnation in systematics. Nonetheless, the
underlying tone of antagonism between molecular and morphological approaches
persisted, and remains today in many circles. The question too often has been
"Which of these data bases is superior?"
40 History of Molecular Phylogenetics

Perhaps we recently have entered a more mature era in systematic relations

with the recognition that molecular and morphological data can be reciprocally
informative, and indeed require each other's services (Hillis, 1987). For exam-
ple, a new enterprise in molecular evolution may be termed "phylogenetic
character mapping" (Chapter 8). This involves plotting the taxonomic distribu-
tion of morphological (or other) characters along a molecular-inferred phylog-
eny, the intent being to uncover the evolutionary histories of organismal at-
tributes. Of course, phylogenetic mapping also may be conducted in the reverse
direction; that is, by plotting the distributions of molecular characters along a
morphology-inferred phylogeny. For example, several instances of horizontal
gene transfer between otherwise unrelated organisms have been revealed under
the compelling logic of this general approach (Chapter 8). Such enlightened
analyses that attempt to capitalize on the comparative information content of
multiple classes of data should lead eventually to a more contented marriage
between molecular- and morphology-based systematics. Under this developing
perspective, the interplay between alternative lines of evidence becomes of
greater interest and significance than does either data source considered alone.

MOLECULAR PHYLOGENETICS

While these grand controversies of evolutionary biology were being played

out, other researchers adopted a more pragmatic approach of simply applying
protein and DNA markers to resolvable problems in natural history and evolu-
tion. Beginning as a subsidiary endeavor in molecular evolution, this previously
neglected perspective has grown steadily and now occupies a position of central
prominence, as this book will attest.
Advances in molecular systematics have proceeded as a series of waves, each
initiated by the development of a new laboratory method. A typical pattern is as
follows. A laboratory technique is introduced, and a flurry of evaluative activity
follows. Methods that fail to meet advance billing (e.g., for reasons of technical
difficulty, poor repeatability, or ambiguity in genetic data) are abandoned. Ap-
proaches that survive the initial screening are then incorporated first into research
programs addressing the general conceptual issues of evolutionary biology de-
scribed above. For example, concerns invariably arise about whether natural
selection plays a role in maintenance of molecular polymorphisms revealed by
the new technique under consideration, and numerous tests (involving observa-
tional or experimental data evaluated against the predictions of neutrality theory)
are applied. Discussion also ensues about methods of analysis most appropriate
for the new class of molecular data. In the meantime, genetic markers provided
by each method are applied to particular problems in natural history or evolution
where their use appears appropriate. Success in such endeavors stimulates further
 History of Molecular Phylogenetics 41

interest, and the more utilitarian of the molecular approaches wash over the field.
Eventually, usually after a period of several years, the enthusiasm crests, and a
new wave of interest in ~other method may begin to take shape. Typically, the
earlier methods are not abandoned, 'but merely become incorporated into the
growing pool of molecular techniques that find continued application in studies
of natural history and evolution.
The first molecular approach employed widely in the field was protein elec-
trophoresis as applied to allozyme and isozyme systems. "Allozymes" are
protein variants of a genetic locus that behave in straightforward Mendelian
fashion and, hence, are interpretable as simple allelic products of a gene.
"Isozymes" are a broader class encompassing all protein variants observed on
electrophoretic gels, including heteromeric products of multiple loci, posttrans-
lational variants, and other protein alterations (Chapter 3). Allozyme methods
were introduced in the mid-1960s, and for the next 10 years dominated molecular
systematics (see the reviews in Avise, 1974, 1983b; Buth, 1984; Gottlieb, 1977;
Whitt, 1983, 1987). Today, protein electrophoresis remains a popular method for
generation of molecular markers.
The next widely employed technique in molecular systematics involved anal-
yses of "restriction fragment-length polymorpbisms" (RFLPs) in DNA. For
both technical and conceptual reasons, mitochondrial (mt) DNA received a great
bulk of the early attention. Mitochondrial approaches dominated molecular sys-
tematics during the late 1970s and 1980s (Avise et aI., 1979a, 1979b; Brown and
Wright, 1979; Brown et aI., 1979; reviews in Avise, 1986, 1991a; Avise and
Lansman, 1983; Birley and Croft, 1986; Harrison, 1989; Moritz et aI., 1987;
Palmer, 1990; Wilson et aI., 1985), much as had allozyme studies a decade
earlier, and strong interest in mtDNA markers continues today. In the middle and
late 1980s, another wave of excitement attended RFLP analyses as applied to
hypervariable nuclear DNA regions, in a class of procedures that because of
diagnostic power became known as "DNA fingerprinting" methods (Burke,
1989; Hill, 1987; Jeffreys et aI., 1985a,b, 1988a; Kirby, 1990).
The current wave of excitement taking shape in molecular phylogenetics be-
gan with the introduction of the polymerase chain reaction (PCR) for in vitro
amplification of specific DNA fragments (Erlich and Arnheim, 1992; Erlich et
aI., 1991; Mullis, 1990; Mullis et aI., 1986; Saiki et aI., 1988; White et aI.,
1989). When coupled with the further development of amplification primers
(Kocher et aI., 1989), improved laboratory methods for sequence determination
(Innis et aI., 1988; Ruano et aI., 1990; Scharf et aI., 1986; Wrischnik et aI.,
1987), and development of appropriate methods for interpretation of haplotype
data (Clark, 1990; Stephens et aI., 1990b), PCR-based approaches permit in-
creased direct access to the phylogenetic information content of DNA sequences
from both nuclear and cytoplasmic genes. Furthermore, because the PCR can
amplify particular DNA segments from tiny amounts of starting tissue [or even
42 History of Molecular Phylogenetics

Box 2.5. Abbreviated ChronololY of Some Significant Historical

Developments In the AppUcatlon of Molecular Markers (for an
extended hiStory of genetic discoveries, see King and Stansfield,
1990).
1944 Avery, MacLeod, and McCarty provide experimental evidence that DNA and
not protein is the genetic material.
1953 Watson and Crick propose a molecular model for the structure of DNA.
1955 Smithies uses starch-gel electrophoresis to identify protein polymorphisms.
1963 Margoliash determines amino acid sequences for cytochrome c in severhl taxa
and generates the first phylogenetic tree for a specific gene product:
1966 Several independent researchers (see text) use electrophoretic methods and
histochemical enzyme stains to assess levels of genetic variability in ani-
mal populations and humans.
1967 Sarich and Wilson provide an early application of protein immunological
methods and discover a more recent shared ancestry for human and great
apes than previously suspected.
1968 Kimura proposes the neutral theory of molecular evolution. Meselson and
Yuan isolate and characterize the first specific restriction enzyme. Britten
and Kohne use DNA hybridization methods to characterize animal ge-
nomes.
1971 Publication of the first periodical devoted explicitly to molecular evolution
(Journal of Molecular Evolution).
1975 Southern describes a method for transferral of DNA fragments to nitrocellu-
lose filters, hybridization to radioactive probes, and detection of fragments
by autoradiography.
1977 Maxam and Gilbert, and Sanger, Nicklen, and Coulson describe laboratory
methods for DNA sequencing.
1978 Maniatis and colleagues develop a procedure for gene isolation which involves
construction and screening of cloned libraries of eukaryotic DNA.
1979 Avise, Lansman, and Shade, and Brown, George, and Wilson introduce
mtDNA approaches to analyses of natural animal populations.
1981 Palmer and colleagues begin an important series of papers utilizing cpDN A for
phylogenetic reconstructions in plants.
1985 Jeffreys, Wilson, and Thein develop the DNA fingerprinting technique and
point out its potential for forensic science. Saiki and six colleagues report
the enzymatic amplification of DNA using the polymerase chain reaction.
1989 Kocher and six colleagues report the discovery of conserved PCR primers that
can be employed to amplify mtDNA segments from many species.
1992 Proliferation of periodicals devoted to evolutionary applications for molecular
markers, for example, Molecular Ecology (Blackwell); Molecular Phylo-
genetics and Evolution (Academic Press); Molecular Marine Biology and
Biotechnology (Blackwell).
 History of Molecular Phylogenetics 43

from some well preserved fossils (Chapter 8)], it has extended molecular appli-
cations to a much wider biological arena (Arnheim et al., 1990).
In addition to these major approaches in molecular systematics, other powerful
but less widespread methods have added significant contributions. Particularly
important among these have been immunological comparisons of proteins, which
provided some of the initial evidence for molecular clocks (Benjamin et al.,
1984; Goodman, 1963; Sarich and Wilson, 1966, 1967; Wilson et al., 1977) and
are still employed today (Maxson and Maxson, 1986, 1990), and DNA-DNA
hybridization methods, which have been available for many years (Britten and
Kohne, 1968; Doty et al., 1960) and have had special impact in the systematics
of certain groups such as birds (Sibley and Ahlquist, 1990), insects (Caccone and
Powell, 1987; Caccone et al., 1988a,b) and hominoid primates (Caccone and
Powell, 1989; Sibley and Ahlquist, 1987).
Given the burgeoning interest today in molecular phylogenetics and molecular
ecology, it is useful to remain cognizant of the remarkably shallow history of
these scientific disciplines. An abbreviated chronology of some significant de-
velopments in the application of molecular markers is summarized in Box 2.5.

SUMMARY
1. The study of evolution from a molecular perspective is a fairly recent
enterprise, dating in substantive form only to the latter half of the 20th
century.
2. Several major controversies have dominated attention in molecular evolu-
tion and related fields. These include the classical-balance debate on the
magnitude of genetic variation, the selection-neutrality debate on the adap-
tive significance of molecular variation, the phenetic-cladistic debate on
procedures for interpreting molecular or other data in a systematics con-
text, and the relative phylogenetic utility of molecular versus morphologic
characters. The latter controversies include issues that are particularly rel-
evant to some phylogenetic applications for molecular data.
3. To a considerable extent, these important debates diverted attention from
some of the more utilitarian applications for protein and DNA markers in
natural history and evolution. Only recently has a focus of primary research
effort shifted to this latter arena.
4. Several waves of excitement in molecular evolution have followed intro-
duction of new laboratory techniques. Among the most influential methods
have been protein electrophoresis in the late 1960s and 1970s, RFLP anal-
yses of mtDNA in the late 1970s and 1980s, DNA fingerprinting in the
mid-to-Iate 1980s, and PCR-mediated DNA sequencing in the 1990s.
 3

Molecular Tools

Perhaps nowhere has the power of the scientific method

been more brilliantly demonstrated than in the develop-
ment of procedures for the study of the chemistry of life.

M.O. Dayhoff and R.Y. Eck. 1968

There exists a wide variety of laboratory assays for revealing molecular genetic
markers. Detailed laboratory protocols can be found in the papers and manuals
listed at the end of this chapter, but in practice there is no substitute for hands-on
training "at the bench" under the guidance of an experienced practitioner.
Therefore, this chapter will merely outline the procedural steps of various mo-
lecular methods and will emphasize instead the nature of genetic information
produced by each of the several molecular techniques that has had major impact
in ecological and evolutionary studies.

PROTEIN ASSAYS

Protein Immunology

PRINCIPLES AND PROCEDURES

Immunological methods rely on the antigenic properties of proteins (Fig. 3.1).

When a protein from species A is injected into a suitable host such as a rabbit,
this antigen elicits production of antibodies with high specificity for antigenic

44
 Molecular Tools 45

1) Purify protein from

reference species 2) Inject into rabbit

3) Collect antibodies 4) Prepare extract from

test species

~ +
>-
yA-<
+ -- ---- --
- -
.>....

+ - -
5) Mix antigens and
complement

- ---
antibody
>-
~+ yA-<
.>....

6) Measure amount of
+ --
--
bound complement

+ ----
Figure 3.1. General protocol for immunological comparisons of proteins by rnicrocom-
plement fixation (after Wilson, 1985).

sites on the injected protein. In laboratory assays, heterologous antigen (a related

protein from species B) also is tested for its capacity to elicit a reaction from this
antibody. Cross-reactivity with the heterologous antigen can be quantified and
compared to results of the control reaction involving the original ("homolo-
gous") antigen. The difference in antigen-antibody reactivities in tests involving
homologous versus heterologous antigens provides a measure of the genetic
relationship (usually expressed as immunological distance or ID units) between
46 Molecular Tools

these proteins from species A and B. A fundamental assumption underlying all

immunological approaches is that level of antibody-antigen "recognition" pro-
vides a quantitative guide to the genetic similarity of the antigenic proteins
involved.
The first immunological assays performed in a phylogenetic context (Nuttall,
1904) involved precipitin tests, whereby antibodies against unpurified proteins
were mixed with antigens in solution, and the resultant antibody-antigen pre-
cipitate was measured. More recently, these and other crude immunological tests
have been supplanted by microcomplement fixation (MCF) methods, in which
antibodies against purified proteins form the basis for the refined estimates of
antigen-antibody cross-reactivity. An outline of the procedure is as follows (see
Fig. 3.1). A protein such as albumin is purified from a reference species, using
standard biochemical protocols. The highly purified protein is injected several
times into a rabbit, over a 3-month period. One week after the last injection,
rabbit antiserum is collected and titered (standardized to a given level of reac-
tivity under specified MCF conditions). The antiserum is stable and may be
stored frozen indefinitely. When a heterologous MCF is to be performed, the
antiserum is mixed with varying concentrations of antigen from the diluted
plasma (purified protein is not required) of a test species. Included in the reaction
mixtures is "complement," a group of proteins normally found in vertebrate
serum that becomes trapped in the developing latices of the antigen-antibody
reaction. The amount of complement "fixed" in the reaction is a function of the
cross-reactivity between antibody and antigen. In spectrophotometric assays,
levels of complement remaining unbound are monitored as a function of antigen
concentration, and the level of immunological response thereby is quantified.

DATA

The data from MCF or other immunological techniques consist of a quanti-

tative measure of divergence (immunological distance or ID) between related
forms of a protein carried by the two or more species. The cross-reactivity
registered in immunological tests depends on the affinity and specificity of the
antibodies to the antigen, properties which are functions of the immunization
protocol as well as the genetic relationships between these molecules. Thus,
standardization in techniques is essential. High-affinity antibodies as monitored
by MCF are capable of detecting single amino acid replacements within the
antigenic portions of challenging proteins.
The MCF assay in effect counts the number of amino acid replacements
responsible for differences between the antigenic sites of related (preferably
orthologous) protein forms. The usual protein of choice in vertebrate studies has
been albumin for the following reasons: It is monomeric and encoded by a single
gene; it is abundant, Ubiquitous, and easily purified; it has a large number
 Molecular Tools 47

(25-50) of major antigenic sites (Benjamin et al., 1984), at which amino acid
substitutions translate into antigenic differences detectable in the MCF assay;
and, it evolves at a rate appropriate for studies of intermediate taxonomic levels
such as subgenera, genera, or families (the usual categories of application for
MCF approaches). Maxson and Maxson (1986) present evidence that albumin ID
as measured by MCF is a linear estimator of amino acid replacement differences
(see also Prager and Wilson, 1993). Other proteins used in MCF assays have
included lysozyme, ovalbumin, and transferrin in vertebrates (Leone, 1964;
Prager and Wilson, 1976; Wright, 1974), glycerophosphate dehydrogenase, acid
phosphatase, and larval proteins in invertebrates (Beverley and Wilson, 1985;
Collier and MacIntyre, 1977; MacIntyre et al., 1978), and alkaline phosphatase
in bacteria (Cocks and Wilson, 1972; additional references in Maxson and Max-
son, 1990).
For a complete assessment of taxa, MCF assays require that antibodies be
produced from each species under consideration. Otherwise, only a subset of
cross-reactivities between species pairs can be attempted, leading to missing
elements in the pairwise ID matrix (the basis for phylogeny reconstruction).
Generating and testing antisera for moderate or large numbers of species is
time-consuming, but offers the additional advantage of permitting tests of reci-
procity (anti-A vs. B and anti-B vs. A). Differences between reciprocal out-
comes provide a measure of experimental error in the MCF procedure (Maxson
and Wilson, 1975).

Protein Electrophoresis

PRINCIPLES AND PROCEDURES

This method takes advantage of the fact that nondenatured proteins with dif-
ferent net charge migrate at different rates through starch or acrylamide gels (or
other supporting media such as cellulose acetate strips) to which an electric
current is applied (Fig. 3.2). The charge characteristics stem primarily from the
three amino acids with positive side chains (lysine, arginine, and histidine) and
the two with negative side chains (aspartic acid and glutamic acid). The net
charge of a protein, which varies with the pH of the running condition, deter-
mines the protein's movement toward the anode (positive pole) or cathode (neg-
ative pole) in the gel. Protein size and shape also can interact with pore size in
the electrophoretic matrix to influence migrational properties.
Because of low cost, safety, and ease of use, gels made from hydrolyzed
potato starch are employed most widely. The "starch gel electrophoresis" (SGE)
procedure begins with the extraction of water-soluble proteins from a particular
source (leaves, roots, liver, heart, blood, skeletal muscle, etc.). The extract from
each individual is soaked onto a paper wick, and 20 or more such wicks are
48 Molecular Tools

......
.. ,..
...... -
~

~~o
1) Dissect tissues 3) Centrifuge, collect
supernatant
2) Homogenize

power supply

0
---
-- --
--- -
-
---- --.
---------
5) Stain gel slice 6) Score population

4) Electrophorese

Figure 3.2. General protocol for protein-electrophoretic surveys (see text).

placed side by side along a slit (the origin) cut into the gel. The gel is placed in
a buffer tray connected to an electrical power supply, and electrophoresis pro-
ceeds over several hours. The gel then is removed, sliced horizontally, and the
wafer-thin slices incubated with histochemical stains specific for the enzymes
under assay. Each stain contains a commercially available substrate for the
enzyme, necessary cofactors, and an oxidized salt (usually nitro-blue tetra-
zolium, NBT). For example, the staining solution for lactate dehydrogenase
(LOH) includes lactic acid (the substrate), nicotinamide adenine dinucleotide
(NAO, the cofactor), phenazine methosulfate (PMS, an intermediary catalyst),
and NBT. At the position(s) in the gel to which LOH from the study organism
has migrated, a reaction is catalyzed whereby lactic acid is oxidized to pyruvic
acid and the salt is reduced to a blue precipitate visible to the naked eye as a
discrete band. The band profile is the "zymogram" pattern for the enzyme, and
usually can be interpreted in simple genetic terms.
Histochemical stains single out the products of particular genes from among
the thousands of other undetected proteins also migrating through a gel. Their
development (Hunter and Markert, 1957), coupled with improvements in elec-
trophoretic procedures and media, eliminated need for laborious protein purifi-
cation procedures that continue to preclude direct amino acid sequencing from
most population applications. Recipes and ingredients for more than 100 his-
tochemical stains are available widely. Not all enzymes resolve well for a given
taxon, however, and a typical multilocus SGE survey involves successful assay
 Molecular Tools 49

of about 10-30 enzymes, perhaps encoded by 15-50 genes (some enzymes are
encoded by multiple loci). Often, hundreds or even thousands of individuals are
analyzed.
For example, one starch gel carrying extracts from 25 individuals can be
sectioned into about 5 replicate slices and each slice incubated with a different
stain. Twenty such gels per day might be run in an active laboratory. Thus, in a
single day, a total of 2500 genotypes (25 individuals x 20 gels x 5 enzyme
stains) could reasonably be scored. This is a conservative estimate because many
stains reveal genotypes for two, three, or four gene copies whose products
catalyze essentially the same reaction. Such masses of genetic data are incredible
by the standards of premolecular genetics, where the elucidation of even a small
handful of Mendelian loci and genotypes in a few individuals required breeding
studies conducted over multiple generations. Indeed, within a short time of the
onset of the allozyme revolution in the mid-1960s, vastly more genotypic data
from natural populations were gathered than in all the 100 years since Gregor
Mendel.

DATA

Zymogram patterns normally are interpretable in terms of Mendelian geno-

types at particular loci. The Mendelian bases of observed polymorphisms may be
verified in several ways. First, experimental crosses may be conducted to provide
relatively direct appraisals of the genetics underlying zymogram phenotypes. For
example, a cross between two presumptive homozygotes exhibiting different
bands on a gel should produce uniformly heterozygous progeny with predictable
zymogram appearance (see following text and Fig. 3.3), and backcrosses of such
progeny to either parent should produce heterozygotes and appropriate homozy-
gotes in approximately equal frequency. Similar' 'family tests," often conducted
with plants, circumvent the need to conduct crosses by addressing whether an
array of progeny exhibits banding patterns consistent with Mendelian expecta-
tions given the genotype of the known mother. In early protein electrophoretic
surveys, such direct experimental validation of zymogram variation was com-
monplace, but as experience with the simple genetic bases of the banding pat-
terns for commonly used enzyme systems accumulated, further corroborations
through breeding or family studies became less critical. Another approach to
verifying the Mendelian basis of zymogram variation involves population genetic
considerations, whereby frequencies of electrophoretic types are compared
against Hardy-Weinberg expectations for neutral alleles. For populations that are
outcrossed, and free from pronounced microspatial subdivision, frequencies of
suspected genotypes almost invariably have proved to be close to those predicted
from observed electromorph frequencies, thus providing strong support for the
Mendelian nature of the variants.
50 Molecular Tools

Third, molecular considerations can help document the Mendelian basis of

zymogram variations (Fig. 3.3). Most enzymes surveyed electrophoretically
have a known quaternary structure. For example, phosphoglucomutase (PGM) is
a monomer, composed of a single polypeptide subunit with catalytic activity.
Thus, homozygotes at a PGM gene show one band on gels, and heterozygotes
show a simple two-band pattern, one band produced by each of the two PGM
alleles. An example of a dimer is glucosephosphate isomerase (GPI), whose
catalytic activity requires the joining of two polypeptide subunits. The zymo-
gram for GPI heterozygotes-a three-band gel profile in which the middle band
is approximately twice the intensity of the flanking bands-reflects random
dimeric associations between the polypeptides produced by the two alleles.
Purine-nucleoside phosphorylase (PNP) is an example of a trimer, and LDH is a
tetramer, such that heterozygotes for these loci normally exhibit four-band and
five-band zymograms, respectively, with characteristic band intensities (Fig.
3.3).
Most observed allozyme variants probably are attributable to nucleotide sub-
stitutions causing replacements of the charged amino acids, although direct mo-
lecular evidence for this conclusion seldom is available. In any event, because

heterozygotes
, ,

-
AA

- - -
AB
a
AB
aa
AB

- -•
aaa

aab
AB
- aaaa
aaab
BB

--
• ab aabb

-
_ abb

-
abbb
b - bb - bbb bbbb
Figure 3.3. Examples of single-locus zymogram patterns. Lowercase letters indi-
cate polypeptide subunits produced by alleles A or B; uppercase letters indicate
diploid genotypes. Shown in the four central lanes (columns) are the expected
zymogram patterns for a heterozygous individual when the enzyme in question is
(from left to right) monomeric, dimeric, trimeric, or tetrameric, respectively. For
example, with a tetrameric protein system, a heterozygote typically would exhibit
five bands on the gel with the following subunit compositions: aaaa, aaab, aabb,
abbb, and bbbb. If the two alleles produce similar polypeptide concentrations and
subunit assembly is random, then intensities of the five respective bands should
appear in the ratio 1 : 4 : 6 : 4 : 1.
 Molecular Tools 51

particular DNA mutational profiles underlying electromorph differences nor-

mally remain unknown, the multiple allozyme alleles at any locus must be
viewed as qualitative multistate traits, the phylogenetic order of which cannot be
safely inferred from the observable property, electrophoretic mobility. Allozyme
data normally consist, then, of the accumulation of such genotypic information
from a number ofloci, typically unlinked and scattered about the nuclear genome
(Pasdar et al., 1984; Shows, 1983; Wheat et al., 1973). Apart from their many
uses as single-locus Mendelian markers in such areas as parentage assessment
and gene flow estimation, allozyme frequencies observed at multiple loci also
can be employed to compute quantitative measures of genetic distance (Chapter
4) between the populations, species, or other taxa under comparison. Such
genetic distances represent a composite numerical summary of all allozyme data
included in the survey.
Protein electrophoretic assays also reveal various aspects of gene expression
that may be under genetic control. Because gene expression patterns themselves
are products of the evolutionary process, they too can be informative as phylo-
genetic markers. For example, among 26 orders and more than 40 assayed
families of living birds, only the woodpeckers (Picidae), honeyguides (Indica-
toridae), barbets (Capitonidae) and toucans (Rhamphastidae) consistently exhibit
a unique three-band zymogram for malate dehydrogenase (MOH), perhaps at-
tributable to a gene duplication event (Avise and Aquadro, 1987). The taxo-
nomic distribution of this fortuitously discovered MDH pattern helped settle a
long-standing debate about whether these birds, which superficially look so
different, are indeed allied phylogenetically as their traditional placement within
Piciformes would suggest. However, apart from identifying this single clade, the
MDH marker was of no further utility for avian phylogenetic assessment [quite
in contrast to the conventional class of information on allozymic allele frequen-
cies, where a more comprehensive picture of piciform relationships has emerged
(Lanyon and Zink, 1987)]. Thus, the rarity of idiosyncratic allozyme characters
is both a phylogenetic blessing and a curse-rarity suggests monophyly and
thereby implies that special weight be given to eccentric molecular features, but
it also means that the applicability of such markers is limited.
On the other hand, not all idiosyncratic patterns of gene expression have
proved to be informative phylogenetically. For example, Mindell and Sites
(1987) assayed tissue expression patterns for 30 presumptive isozyme loci in
representatives of two avian orders (Charadriiformes and Passeriformes) and
observed numerous inconsistencies with the accepted taxonomy. For these sys-
tems, they concluded that' 'widespread homoplasy, opposite polarities and lim-
ited predictive capability for the isozyme tissue expression patterns suggest that
most may be more useful in studies of gene regulation than in higher level
taxonomy." Clearly, caution must be exercized in the utilization of gene ex-
pression patterns as phylogenetic markers.
52 Molecular Tools

Many enzymes are encoded by two or more loci that arose through gene
duplications via polyploidy, aneuploidy, or regional intrachromosomal duplica-
tion (Buth, 1983; MacIntyre, 1976; Ohno, 1970). Their zymogram patterns
usually are predictable (and interpretable) from rules governing polypeptide
assembly into functional enzymes with known quaternary structure. From such
evidence, Gottlieb (1988) and Soltis et ai. (1987) have identified several dupli-
cations of isozyme-encoding nuclear genes among various species of diploid
Clarkia plants. These duplications individually appeared to be rare and, hence,
of potential phylogenetic relevance. On the other hand, three complications
dealing ultimately with establishment of gene homologies served to temper en-
thusiasm somewhat (Sytsma and Smith, 1992). First, the convergent origin of a
duplicate gene in independent lineages remains a possibility. Second, postdu-
plication silencing of either member of a duplicate pair may occur, perhaps on
separate occasions in different lineages. Third, the presence of duplicate loci
might represent a plesiomorphic condition at the hierarchical level of the taxon-
omy examined. The latter two possibilities indeed have been documented in the
Clarkia studies (Gottlieb, 1988).
The protein products of duplicated genes frequently have diverged in structure
and regulatory control since the duplication event and may show striking onto-
genetic changes or tissue specificities of potential relevance to phylogenetic
assessment. Thus, whereas most vertebrates express a single GPI gene, bony fish
express two unlinked GPI loci, one predominantly in skeletal muscle and the
other in liver (Avise and Kitto, 1973; Whitt et al., 1976). All vertebrates (with
the exception of lamprey fishes) have muscle- and heart-specific LDH expression
involving the "A" and "B" genes (Markert et al., 1975). As gauged by zy-
mogram patterns, the assembly of heterotetramers between the LDH loci some-
times is nonrandom, presumably due to taxon-specific genetic regulatory influ-
ences (Murphy, 1988; Sites et al., 1986). Some birds (doves) and mammals have
an additional LDH enzyme (produced by the "e" locus) expressed only in the
primary spermatocytes (Blanco and Zinkham, 1963; Matson, 1989; Zinkham et
aI., 1969); also, bony fish carry a third gene for LDH, expressed in a variety of
tissues in primitive species and predominantly in eye or liver of advanced teleosts
(Horowitz and Whitt, 1972; Markert and Faulhaber, 1965; Shaklee et al., 1973;
Whitt et al., 1975). It is doubtful that this third LDH locus in fish is orthologous
to those in either the doves or mammals (Fisher et al., 1980; Quattro et al.,
1993), but in any event, these LDH loci clearly have evolved distinctive regu-
latory profiles in different taxonomic groups. Other multilocus protein systems
studied extensively with regard to patterns of gene expression and phylogeny
include malate dehydrogenase, glycerol-3-phosphate dehydrogenase, creatine
kinase (Buth et aI., 1985; Fisher and Whitt, 1978; Fisher et al., 1980; Philipp et
al., 1983), and the globin superfamily of oxygen-carrying molecules (Dayhoff,
1972; Doolittle, 1987).
 Molecular Tools 53

Duplicate genes also are subject to evolutionary silencing or loss, and these
patterns can be infonnative phylogenetically. For example, Ferris and Whitt
(1978, 1979) utilized patterns of enzyme loss and change of gene expression to
reconstruct the phylogeny of the catostomid suckers, a group of freshwater fishes
that underwent a polyploidization event some 50 million years ago and subse-
quently became "diploidized" at approximately 50% of assayed structural
genes. The rationale for these reconstructions was as follows. Immediately fol-
lowing the polyploidization event, all loci in the sucker genome must have been
duplicated, and the "primitive" condition from that point forward became pres-
ence of each duplicate gene. As mutations accumulated, some genes lost ex-
pression (and become pseudogenes), whereas other duplicate copies diverged in
structure and function (Ferris and Whitt, 1977). These processes presumably
were nearly irreversible (however, see Buth, 1979, 1982), such that taxa sharing
possession of the derived states (loss or alteration of gene expression) likely
belonged to Hennigian clades (assuming that the same losses of gene expression
had not occurred independently in separate evolutionary lineages).

DNA ASSAYS

DNA-DNA Hybridization

PRINCIPLES AND PROCEDURES

This method (see Fig. 3.4) relies on the double-stranded nature of duplex DNA,
and the fact that paired nucleotides on the two complementary strands are held
together by hydrogen bonds (two coupling each adenine-thymine base pair, and
three coupling each guanine-cytosine). These hydrogen bonds are the weakest
links in DNA, so that when native DNA is boiled in solution, the duplexes
dissociate or "melt" into single strands but otherwise remain structurally intact.
As the melted sample is cooled, strands collide by chance, and those with
complementary nucleotide sequence reassociate into double-stranded molecules
as their respective bases pair and refonn hydrogen bonds. A rapidly reassociating
component represents repetitive DNA (because homologous strands are in high
number and collide most frequently). This fraction is removed. The remaining
fraction represents single- or low-copy sequences in the genome. Such single-copy
DNA strands are then mixed under conditions where duplex fonnation occurs. The
mixtures may involve DNA strands from a single sample or species (in which case
homoduplexes are produced) or the mixtures may involve strands from two species
(fonning heteroduplexes). The culminating step in the hybridization protocol
involves characterizing the thennal stabilities of these homoduplexes and het-
eroduplexes by gradually raising temperatures and monitoring the course of
54 Molecular Tools

molecular dissociation to single strands. The thennal stability exhibited by any

duplex depends largely on the similarity of nucleotide sequences in its two strands,
because only properly paired bases are hydrogen-bonded. The measured differ-
ence in thennal stability between homoduplexes and heteroduplexes provides a
quantitative estimate of the genetic divergence between the two species.
Further details of the DNA hybridization process are as follows. First, DNA

1) Extract DNA 2) Shear DNA to = 500 bp fragments

repeated
sequences

cool and incubate

3) Prepare single-copy
DNA
•
~ _.ingle.copy
~.equenc.s

4) Radioactively label

5) Prepare hybrid

6) Determine melting
heat by 2.5 degree. heat by 2.5 degree.
temperature of hybrid • •
DNA by assaying ~
radioactivity in '{f\fV
bottom vials -. 1\IV"
Figure 3.4. General protocol for DNA-DNA hybridization (after Sibley and Ahlquist,
1986).
 Molecular Tools 55

is extracted from the nucleus of cells, separated from RNA and proteins, and
physically sheared into fragments averaging 500 nucleotides in length (to reduce
viscosity and to permit subsequent fractionation of repetitive from single-copy
DNA). The sheared fragments are boiled, cooled, and their reassociation kinetics
employed [as first described by Britten et al. (1974)] to remove most of the
repetitive fraction. This is accomplished by incubating the DNA in solution at
about 50°C for a short time, such that the repetitive sequences preferentially
anneal and most single-copy sequences remain unpaired. This solution is passed
through a hydroxyapatite column, which binds double-stranded DNA only. The
single-stranded DNA which passes through the column is labeled with radioac-
tive iodine (and becomes known as the tracer) and is mixed with a much larger
amount of unlabeled DNA (the driver) from the same or a different species. This
mixture is incubated at 60°C for several days to form hybrid DNA molecules that
have one labeled and one unlabeled strand. The sample is then placed on a
hydroxyapatite column, and gradually heated in a water bath at 2.5°C increments
over a 6O-95°C range. At each temperature increment, additional duplexes that
have melted (a function of degree of base-pair mismatching) are washed from the
column into a vial. Counts of radioactivity in the vials record the amount of
duplex DNA that melted at the various temperatures.

DATA

The raw data from DNA-DNA hybridization consist of "thermal elution

profiles" (Fig. 3.5) that summarize the observed percentages of dissociated,
single-stranded DNA as a function of melting temperature. From such cumula-
tive melting curves, various statistics can be generated that provide quantitative
estimates of the degree of base-pair mismatch between the DNAs under com-
parison (e.g., Britten, 1986; Sarich et al., 1989; Sheldon and Bledsoe, 1989).
Considerable debate (beyond the scope of current discussion) has centered on
which of these distance measures is to be preferred, but in practice all tend to be
highly correlated (Kirsch et al., 1990). One such distance measure is based on
Tm' defined as the interpolated temperature at which 50% of the hybrid molecules
that were formed remain in duplex condition. The differences in Tm values
between homoduplex and heteroduplex melting profiles (IlTm) constitute the
"raw" distance data used in phylogenetic reconstructions based on the DNA
hybridization approach.
The relationship between IlTm and percent base-pair mismatch is thought to be
linear (Britten et al., 1974; Caccone et al., 1988b; Kohne, 1970), but the exact
conversion between the two is uncertain. Several studies have examined the
relationship by studying the thermal stability properties of synthetic oligonucle-
otides or other sequences of known base composition (Bautz and Bautz, 1964;
Hutton and Wetmur, 1973; Laird et al., 1969; Springer et al., 1992) and have
 56 Molecular Tools

Figure 3.5. Thennal elution profiles from

A DNA-DNA hybridization. Shown are cu-
mulative melting curves for the single-

-
~
100 copy fraction of nDNA in some flightless
0 ratite birds (after Sibley and Ahlquist,
80
1990). (A) Homoduplex DNA of the emu
.-
CO
U 60
(Dromaius novaehollandiae) (closed
0 squares) and heteroduplexes between that
t/)
t/) 40 species and the southern cassowary (Ca-
suarius casuarius) (open squares),
0 20 greater rhea (Rhea americana) (open cir-
~
0 cles) , ostrich (Struthio camelus) (closed
65 70 75 80 85 90 95 triangles) , and domestic fowl (Gallus
gallus-a nonratite outgroup) (closed cir-
Temperature (OC) cles). In these comparisons, the cas-
sowary appears genetically closest to the
emu, followed in order by the rhea, os-
B trich, and domestic fowl. (B) Homodu-
plex DNA of the ostrich (closed squares)
and the heteroduplex with the greater

-
100
~
rhea (open squares). Note that although
.-
0
80 the melting curves involving the rhea and
CO
ostrich are nearly identical when corn-
U 60
0 pared against the emu (panel A), this does
t/) not necessarily imply that these latter spe-
t/) 40
cies are genetically close to one an-
0 20 other-they could differ from the emu in
~
0 different phylogenetic directions. Indeed,
0
60 65 70 75 80 85 90 95
differences between melting curves in
panel B indicate a large genetic distance
Temperature (OC) between the rhea and ostrich.

reported that incremental aTm values of 1°C correspond roughly to 0.7-1.7%

base-pair mismatch. As a working rule of thumb, a aTm of 1°C often is equated
to 1.0% base mispairing (see Britten, 1986; Koop et aI., 1986). However,
Powell et al. (1986) and Caccone et al. (1988b) obtained somewhat higher
conversion ratios (aTm of 1°C equaling 1.5-2.0% base-pair mismatch) in their
studies of cloned DNAs of known sequence.
Because DNA hybridization data involve an averaging of genetic differences
across a large fraction of the genome (primarily the single-copy portion), they
sometimes have been promoted as the strongest available source of phylogenetic
information (Sibley and Ahlquist, 1990). Thus, it is somewhat surprising that
these methods have not been employed more widely by molecular systematists.
DNA-DNA hybridization approaches have had tremendous impact in molecular
 Molecular Tools 57

genetics by revealing important aspects of genomic structure-amounts of re-

petitive DNA, lengths of repeated sequences, and interspersion patterns among
repetitive and low-copy sequences (e.g., Britten and Kohne, 1968). With regard
to phylogenetic applications, reservations expressed about the DNA hybridiza-
tion approach include the fact that the raw data consist solely of distance values
(rather than directly observed molecular character states) and that the influences
of factors affecting the kinetics of hybridization (such as differences in base
composition, DNA fragment size, and genome size) are incompletely under-
stood. Some of these factors nonetheless are partially controlled or standardized
in most DNA hybridization studies. For example, effects of base compositional
differences (numbers of A-T versus C-G pairs) among sequences can be ame-
liorated by use of chaotropic solvents (Werman et al., 1990) and comparisons
can be confined to particular organismal groups (such as birds) where compli-
cations arising from confounding variables such as pronounced differences in
genome structure or organization should be minimized.
The development of automated thermal elution devices (such as the
"DNAnalyzer" of Sibley and Ahlquist, 1981) greatly expedited the process of
gathering DNA hybridization data. Indeed, the honor for the largest number of
species yet included in any molecular systematic survey no doubt belongs to
Sibley and Ahlquist (1990), who have conducted some 27,000 DNA-DNA
hybridizations involving about 1700 avian species (only about 350 of these
species provided DNA used as tracer, however, so that only a small subset of all
possible pairwise comparisons was accomplished).

Restriction Analyses

The discovery of restriction endonucleases (Linn and Arber, 1968; Meselson

and Yuan, 1968) revolutionized molecular biology. Type II restriction enzymes
(Kessler, 1987) cleave duplex DNA at particular oligonucleotide sequences,
usually either four, five, or six base pairs in length. For example, EcoRI (named
after the bacterium Escherichia coli from which it was isolated) acts like a
precise scalpel to cut double-stranded DNA wherever the nonmethylated 5'-
GAATTC-3' sequence occurs. Several hundred such enzymes, most with dif-
ferent recognition sequences, have been isolated and characterized from various
bacterial strains (Roberts, 1984; examples in Box 3.1). In a bacterium, these
enzymes protect against invasion by foreign DNA (host DNA is protected by
bacterial-specific methylation systems). In a molecular genetics laboratory, re-
striction enzymes find wide application in assays of DNA restriction fragment-
length polymorphisms (RFLPs).
All RFLP analyses involve cutting (restricting) DNA with one or more endo-
nucleases, separating the resulting fragments according to molecular weight by
gel electrophoresis, and visualizing the size-sorted fragments. Differences
58 Molecular Tools

Box 3.1. Recognition Sequences and Predse Cutting Sites (*) In

Double-Stranded DNA for Some of the Indonudeases Commonly
Imployed In Restriction Site Surveys. Py and Pu Indicate
pyrimidines (T or C) and purines (A or G), respectively.

Enzyme Recognition Sequence Enzyme Recognition Sequence

AvaI 5' ... G*Py C G Pu G ... 3' EeoRV 5' ... G A T*A T C ... 3'
3' .. . G Pu G C Py*C . . . 5' 3' ... C T A*T A G ... 5'
BamHI 5' ... G*G A T C C ... 3' MboI 5' ... *G A T C ... 3'
3' ... C C T A G*G ... 5' 3' ... C T A G* ... 5'
Bell 5' .. . T*G ATe A . . . 3' PstI 5' ... C T G C A*G ... 3'
3' . .. ACT A G*T . . . 5' 3' ... G*A C G T C ... 5'
BglII 5' ... A*G A T CT ... 3' Pvull 5' ... C A G*C T G ... 3'
3' .. . T C T A G* A . . . 5' 3' ... G T C*G A C ... 5'
ClaI 5' ... A T*C GAT ... 3' SpeI 5' ... A *C TAG T . . . 3'
3' ... TAG C*T A ... 5' 3' ... T GAT C*A ... 5'
EeoRI 5' ... G*A A T T C ... 3' XbaI 5' ... T*C TAG A ... 3'
3' ... C T T A A*G ... 5' 3' ... A GAT C*T ... 5'

among individuals in these "digestion profiles" may result from base substitu-
tions within cleavage sites, additions or deletions of DNA, or sequence rear-
rangements, with each source of variation producing characteristic banding
changes. Three important and partially interrelated variables in the assays in-
clude electrophoretic media employed, means of fragment visualization, and
choice of DNA to be analyzed. These general considerations will be discussed
first, and methodological details for particular applications will be added later.
The usual electrophoretic media are agarose or acrylamide gels. These form
dense matrices through which larger DNA fragments migrate more slowly than
smaller fragments under the influence of an electric current. At neutral pH, DNA
is negatively charged (due to properties of the sugar-phosphate backbone), and
therefore moves toward the anode of electrophoretic gels at rates determined by
molecular size. Agarose gels (0.6-2.0% agarose) are most useful for separating
DNA fragments in the size range 300-20,000 base pairs (bp) and acrylamide
gels (3.5-20.0%) in the range 10-1000 bp. To facilitate estimation of restriction
fragment lengths in the sample DNAs, molecular size standards (commercially
available) typically are included in each gel.
Visualization of DNA fragments can be accomplished by several means. Some
electrophoretic assays begin with highly purified DNA isolated from particular
sources (such as mitochondria), in which case DNA fragments in the gel are
revealed by chemical or radioactivity "stains." When DNA amounts are high
 Molecular Tools 59

(> 50 ng per gel band), ethidium-bromide provides a convenient chemical agent

for fragment detection. Ethidium-bromide binds to DNA in such a way that
staining intensities are proportional to fragment sizes and digestion profiles ap-
pear stoichiometric. Silver-staining is similar and reportedly provides greater
sensitivity in detecting small DNA quantities [< 100 pg (Guillemette and Lewis,
1983)]. In highly sensitive "end-labeling" procedures, DNA digestion frag-
ments are labeled radioactively with 32p_ or 35S_tagged nucleotides prior to
electrophoretic separation. After the gel has been run, it is vacuum-dried and
overlaid by X-ray film whose development as an autoradiograph reveals posi-
tions to which the DNA fragments migrated. With end-labeling, band intensities
are independent of fragment size (because all fragments have two labeled ends),
and the method, therefore, is useful in revealing smaller fragments when DNA
amounts are limited.
Other RFLP assays begin with DNA of heterogeneous classes (e.g., total
nuclear DNA preparations), with elucidation of DNA fragments from particular
genes accomplished after electrophoresis by the technique of "Southern hybrid-
ization" (Southern, 1975). In this method, all DNA fragments in the gel are
denatured in a basic solution and then transferred as single strands (by capillary
action or electrophoresis) to a nylon or nitrocellulose membrane. The membrane
is incubated with a single-stranded "probe"-DNA previously isolated, puri-
fied, and radioactively labeled-under conditions where any strands in the mem-
brane that are complementary to those of the probe hybridize with it to form
radioactive duplexes. When high-stringency conditions are employed, hybrid-
ization with distantly related or nonhomologous DNA is avoided. Thus, the
probe in effect picks complementary and (ideally) homologous sequences to
itself from among the thousands or millions of undetected fragments that also
have migrated through the gel. These fragments with sequence similarity to the
probe then are visualized by autoradiography of the "Southern blot."
The probe in Southern hybridizations thus identifies the DNA under assay in
a particular study. This probe may constitute, for example, a single gene from a
nuclear or cytoplasmic genome, a noncoding stretch of DNA sequence, or an
entire animal mtDNA. If the probe contains DNA present in multiple copies in
the genome, the Southern blot reveals fragments from all members of the family
to which the probe has hybridized. In some such cases, Southern blots may
reveal highly complex digestion profiles wherein nearly all individuals are dis-
tinguished by their "DNA fingerprints." The probes in Southern hybridizations
may come from DNA highly purified by physical means (e.g., mtDNA isolated
via CsCI gradient centrifugation), or more normally via cloning of particular
genes through biological vectors (Sambrook et al., 1989). For rapidly evolving
sequences, utility of the probe may be confined to assays within or among closely
related species, whereas for slowly evolving sequences the probes may retain
cross-hybridizing utility across broader taxonomic assemblages. The most com-
60 Molecular Tools

mon limiting factor in Southern hybridization studies has been the availability of
suitable probe DNA.
Because different classes of DNA differ dramatically in terms of the nature of
genetic information provided by restriction analysis, as well as in additional
details of isolation procedure, they will be discussed separately in the sections
that follow.

ANIMAL MITOCHONDRIAL DNA

Procedures A crucial initial step in isolation of animal mtDNA is the

efficient separation of cytoplasm (where mitochondria are housed) from cell
nuclei (see Fig. 3.6). Soft tissue such as heart, liver, or ovary is minced, gently
homogenized, and centrifuged at low speed (700 X g) to remove some of the
nuclei and cellular debris. Subsequent centrifugation at higher speed (20,000 x
g) pellets the mitochondria which then are washed and lysed. The next step in the
purification involves CsCl-EtBr gradient centrifugation (160,000 x g) for 48 h
[or less, depending on the speed and type of rotor employed (Carr and Griffith,
1987)]. The mtDNA, which appears as a discreet band in the gradient, is re-
moved by hypodermic needle and separated from remaining contaminants by
dialysis. Purified mtDNA samples can be stored frozen indefinitely. This purified
mtDNA then can be used either as a probe in Southern blots to reveal mtDNA
bands in heterogeneous DNA samples or the purification process can be repeated
for each individual in the genetic survey and the RFLPs elucidated directly by
chemical staining or radioactive end-labeling. A rate-limiting step in mtDNA
analysis by the above protocol involves the lengthy gradient centrifugations.
Various shortcuts for mtDNA isolation are available when large quantities of
mitochondrial-rich cells (such as oocytes) are available, or when only small
amounts of mtDNA are required (Chapman and Powers, 1984; Jones et al.,
1988; Palva and Palva, 1985; Powell and Zuninga, 1983).
Many laboratories working with animal mtDNA employ end-labeling proce-
dures. As typically accomplished, mtDNA samples purified from each individual
are digested with restriction endonucleases, and the resulting fragments are ra-
dioactively tagged and electrophoretically separated. Development of an auto-
radiograph reveals the mtDNA digestion profile for each enzyme. For plant
mitochondrial and chloroplast genomes, which are much larger, the normal
laboratory procedures involve Southern blotting using cloned genes or subsets of
the genome as probe. [One recently discovered complication that could apply to
the Southern blotting approach, but not to end-labeling of purified mtDNA,
occurs when homologues of particular cytoplasmic genes occur also in a cell's
nucleus as a result of intergenomic transfer (M.F. Smith et al., 1992).]
Data The structure and genetic basis of variation in metazoan animal
mtDNA is probably better understood than that of any comparably sized region
 Molecular Tools 61

1) Dissect and homogenize

tissues
2) Centrifuge to pellet
mitochondria

-
mineral oil

protein, SDS

nDNA

glycogen -
-
RNA -

4) Digest mtDNA
3) Purify mtDNA by CsCI-gradient and radioactively
centrifugation; remove mtDNA band end-label

X-RAY
-- -
FILM - -- ---

5) Electrophorese 6) Develop autoradiograph

Figure 3.6. General protocol for mtDNA restriction site analysis by radioactive end-
labeling.

of the nuclear genome (see the reviews in Attardi, 1985; Brown, 1985; Cantatore
and Saccone, 1987; Gray, 1989; Wallace, 1982; Wolstenholme, 1992). With few
exceptions, animal mtDNA is a closed circular molecule, typically 15-20 kilo-
bases (kb) in length, and composed of about 37 genes coding for 22 tRNAs, 2
rRNAs, and 13 mRNAs specifying proteins involved in electron transport and
62 Molecular Tools

oxidative phosphorylation (Wallace, 1986; Fig. 3.7a). A "control region" of

about l-kb initiates replication and transcription. Gene arrangement in mtDNA
appears generally stable, although differences in gene order do distinguish some
higher animal taxa (Desjardins and Morais, 1990; Okimoto et al., 1992; Piiiibo
et aI., 1991; Chapter 8). Nearly the entire mtDNA genome is involved in the
coding function: introns, large families of repetitive DNA, pseudogenes, and
even sizable spacer sequences between genes are rare or lacking.
With regard to the general mode of animal mtDNA evolution, much also is
understood (see the reviews in Avise and Lansman, 1983; Avise et al., 1987a;
Birley and Croft, 1986; Harrison, 1989; Moritz et aI., 1987; Wilson et al.,
1985). Individuals commonly are homoplasmic or nearly so, with a single
mtDNA sequence predominating in all tissues (however, see Bermingham et al.,
1986; Hale and Singh, 1986; Moritz and Brown, 1987, and references therein for
exceptions). The reason for this relative sequence homogeneity within individ-
uals probably involves bottlenecks in mtDNA numbers in intermediate germ cell
generations (Birky et aI., 1989; Chapman et al., 1982; Clark, 1988; Laipis et aI.,
1988; Rand and Harrison, 1986; Solignac et aI., 1984, 1987; Takahata, 1985).
Mitochondrial DNA normally evolves rapidly at the sequence level, no doubt
due in part to a lack of known repair mechanisms for mutations that arise during
replication (Wilson et al., 1985). Some sequences within the control region
evolve with exceptional rapidity and have proved to be of utility in high-reso-
lution analyses of population structure (Stoneking et al., 1991). Although
addition/deletion changes in mtDNA are not rare, most differences between
sequences reflect point mutations, with a strong initial bias for transitions over
transversions (Aquadro and Greenberg, 1983; Brown and Simpson, 1982; Brown
et al., 1982; Greenberg et al., 1983).
Finally, and most importantly for genetic marker purposes, mtDNA is trans-
mitted predominantly through maternal lines in most species (Avise and Vrijen-
hoek, 1987; Dawid and Blackler, 1972; Giles et al., 1980; Gyllensten et aI.,
1985a; Hutchison et al., 1974). Several exceptions to strict maternal inheritance
are known (see Avise, 1991b; Gyllensten et aI., 1991; Kondo et al., 1990), but
even in the few species such as marine mussels (Mytilus) where "paternal
leakage" is relatively common (Zouros et al., 1992), the paternally- and
maternally-derived molecules are not known to recombine genetically in progeny
(Hoeh et al., 1991). Genotypes for mtDNA thus represent nonrecombining char-
acters, asexually transmitted usually via females through the pedigrees of what
otherwise may be sexually reproducing species. Thus, for simplicity, mtDNA
genotypes are referred to as clones or haplotypes, and their inferred evolutionary
interrelationships interpreted as estimates of "matriarchal phylogeny" (Avise et
al., 1979b). From a functional perspective, mtDNA consists of about 37 genes,
but from a phylogenetic perspective the entire mtDNA molecule represents one
nonrecombining genealogical unit with multiple alleles.
 CR

human
3
(a) mtDNA
( == 16.5 kb)

LSC

tobacco
cpDNA
(b)
(==156kb)

SSC
Figure 3.7. Major structural features of animal mitochondrial DNA and plant
chloroplast DNA (the two molecules are not drawn to the same scale). (a) Human
mtDNA, composed of a control region (CR), and genes encoding 2 rRNAs (l2S
and 16S), 22 tRNAs (open circles), and 13 polypeptides. Also shown are the sites
(OrH and OrL ) at which replication is initiated along the complementary DNA
strands. (b) Tobacco (Nicotiana tabacum) cpDNA. composed of large and small
single-copy regions (LSC. SSC), and a large inverted repeat (lR). Also shown is
the position of the rbeL gene, which has figured prominently in nucleotide se-
quencing studies (Chapter 8).
64 Molecular Tools

The raw data in mtDNA restriction surveys consist of fragment length profiles
produced by individual enzymes (Fig. 3.8). Because mtDNA is a closed circle,
the number of linear fragments is equal to the number of restriction sites recog-
nized by the endonuclease. A useful check on gel scoring is provided by the
mtDNA genome size within a given species, to which observed fragment sizes
should sum. This feature also facilitates direct comparisons of digestion profiles
across studies or among different laboratories (a desirable property not shared
fully by allozyme methods, where meaningful comparisons of allelic products
require that known electromorph standards be run in all gels). A typical mtDNA
population survey may involve use of 10-20 different restriction enzymes and
reveal perhaps 50-100 restriction fragments per individual. Because the en-
zymes employed are commonly five- and six-base cutters, this is equivalent to
assaying 250-600 bp of recognition sequence per specimen. The larger mtDNA
population surveys sometimes include many hundreds of individuals.
Differences among mtDNA digestion profiles normally arise from point mu-
tations that create or destroy enzyme recognition sequences. Commonly, the
pattern of interconversion among digestion profiles resulting from restriction site
mutations can be deduced directly from the single-enzyme gel patterns them-

Box 3.2. Presence (+) Versus Absence (0) Restrlcdon Site Matrix.
this example Involves 96 restrlcdon sites represendng 20
different mtDNA haplotypes (a-t) observed among 107
sharp-tailed sparrows, Ammodramus caudacutus (data from
Rising and Avlse, t 993)•
••••••••.• • 00.0 •••••• 0 ••••••••••••••••••• 0000 •••••••••••••••••• 0 •••• 0 •••••• o. 0.0 •••••••••• 0 •••••
••••••••••• 00.0 •••••• 0 •• · •••••••••••••••• 0000 ••••••••••••••••• 00 •••• 0 •••••• 0. 0.0 •••••••••• 0 •••••
•••••••••• • 00.0 •••••• 0 •••••••••••••••••••• 000 •••••••••••••••••• 0 •••• 0 ...... 0.0.0.·.·.· •••• 0 •••••
•••••••••• • 00.0 ...... 0 ••••••••••••••••••• 0.00 .................. 0 •••• 0 ...... 0. 0.0 .......... 0 •••••
.......... • 00.0 .......................... 0000 ................. 00 .... 0 ...... 0. 0.0 ............... .
.... ...... • 00.0 ...... 0 ................... 0000 .................. 0 •••• 0 ........ 0.0 .... • ••••• 0 •••.•
• .......... 00.0 ...... 0 ................... 0000 .................. 0 .......... 00.0.0 .......... 0 •••••
...... .... ·00.0 ...... 0 .................... 0 .... • ... 0 .......... 0 ........... 00·0.0 ..... 00 ... 0 •••••
................... .. 00 ·0 ........... 0 ....................................... 0.0 ............ 0 ................... 0 ...................... 00 .. 0.0 ....... .. 00 ..... 0 ........ .
.......... • 0 .. 0 ...... 0 .................... 0.0 ...... 0 .......... 0 ........... 00.0.0 .... ·00 ... 0 •••••
.......... • 00.0 ...... 0 .................... 0.0 ...... 0 .......... 0 ........... 00 ••• 0 ..... 00 ••• 0 •••••
.......... • 00.0 ...... 0 .................... 0.0 ...... 0 .......... 0 ..... 0 ..... 00· •• 0 ..... 00.·.0 ••• • •
............ 0.00 ..... 0 .................... 0.0 ...... 0 .......... 0 ........... 00 .0.0 ..... 00 ••• 0 •••••
......... .. 00.00 ..... 0 .................... 0.0 •••••• 0 .......... 0 ........... 00 .0.0 .... ·00 ... 0 •••••
......... .. 00.0 ...... 0 .................... 0. 00 ..... 0 .......... 0 ........... 00 .0 ....... 00.0.0 •••••
......... .. 00.0 ...... 0 .................... 0.00 ..... 0· ......... 0 ........... 00. 0 ....... 00 ... 0 •••••
........... 00.0 ...... 0 .................... 0.00 ..... 0 .......... 0 ........... 00 ......... 00.0.0 •••••
• ....... .. ·00 ........ 0 .................... 0.0 ...... 0 .......... 0 ........... 00.0.0 ..... 00 ... 0 •••••
.......... • 00.0 ...... 0 .... 0 ............... 0.0 •••••• 0 .......... 0 ........... 00.0.0 ..... 00 ... 0 •••••
...................... 00 .. 0 ......... 0 ...... 0 ........................... 0. 0 .. +- ....... 0 ................... 0 .................... 00 ..... 0.+ ..... 00 ..... 0 ........ ..
 EcoRI
Figure 3.8. Interpreta-
tion of mtDNA digestion
profiles (from Avise,
1987). Shown is an au-
toradiograph of EcoRI
digests of mtDNA from
B c E
o ---
------------1....-------- ...
18 eels (genus Anguilla).
The seventh lane from

- ...... -
the right is a molecular
size standard, in which
-- -
.._---------- .....•
the darkest band is 1. 6 ~
kb in size, successive
bands above it are ap-
proximately 2, 3, 4, 5,

-
6,7,8, ... kb, and the
band below it is 1.0 kb.

• -
Five EcoRI patterns
(A-E) are evident; their
interrelationships are
summarized in a parsi-
mony network shown _ . . _ "~ _ _ _ _. . . .
under the radiograph
(the arrows indicate the
direction of restriction
site loss, and not neces-
sarily the direction of ev-
olution). For example,
pattern A differs from B A~B~C
by loss of an EcoRI re-
striction site, which con-
verts the two larger frag-
1\
D E
ments in the B profile
(4.6 and 8.0 kb) to the
largest fragment in A (12.6 kb). In turn, C differs from B by gain of an EcoRI site which
converts B's 8.0-kb fragment to C's fragments of sizes 5.1 kb and 2.9 kb. Pattern E
apparently has a "doublet" (two fragments of indistinguishable molecular weight) at
3.1 kb.

selves, using the infonnation from mtDNA fragment sizes (Fig. 3.8). Such
infonnation can be accumulated across restriction enzymes and used to generate
composite mtDNA clonal descriptions. Data also may be recorded as binary
characters and summarized in a presence-absence matrix of restriction sites
across individuals or mtDNA clones (Box 3.2). Studies may go further by
mapping the positions of sites relative to one another or to landmarks on the
66 Molecular Tools

mtDNA genome, using double-digestion or partial-digestion procedures (Fig.

3.9). Normally, however, sites can be mapped only to within a few tens or
hundreds of base pairs of their true locations.
Some mtDNA gel profiles are sufficiently different or complex that pattern
interconversions cannot readily be deduced. One may nonetheless count percent-
ages of shared (and presumably homologous) fragments, but caution is indicated
because, unlike site changes, not all fragment changes are independent. For
example, a point mutation creating a restriction site will result in the correlated
appearance of two smaller fragments with the same total molecular weight as the
one larger fragment lost (Fig. 3.9). If many such changes distinguish digestion
profiles, the evolutionary interconversions are difficult to deduce. (For similar
reasons, digestion profiles produced by restriction enzymes with four-base rec-
ognition sites can be difficult to score-they typically involve 10-20 or more
mtDNA fragments each-whereas five- and six-base cutters normally produce
simple gel patterns with only about 2-10 bands.) Statistical methods that take
into account such correlated fragment changes are available for converting per-
centages of shared fragments to estimates of sequence divergence (e.g., Nei and
Li, 1979; Upholt, 1977). However, because of uncertainties that the molecular
assumptions underlying these conversion formulas are met in any given study,
analyses based on "site" differences are desirable wherever possible.
Additional sources of mtDNA variation stem from occasional differences in
mtDNA size, most often due to variations in copy number of localized tandem
repeats usually in or near the control region of the molecule (Bermingham et al.,
1986; Harrison et al., 1985; Moritz and Brown, 1986, 1987). The larger of these
localized repeat regions (they can range in size from a few base pairs to more
than 1 kb), are readily distinguished from restriction-site changes because they
alter concordantly the sizes of particular restriction fragments across all digestion
profiles (smaller fragment-size differences might be overlooked, however, par-
ticularly when they reside in high-molecular-weight gel bands). It is important to
properly characterize genomic size changes so that they are not misinterpreted as
independent restriction-site changes involving multiple enzymes.
Restriction analyses of animal mtDNA normally are applied at the level of
conspecific popUlations and closely related species, but other features of the
molecule have proved useful in higher-level phylogenetics. From sequencing
studies, it is known that transitions greatly outnumber transversions in the early
stages of mtDNA divergence, but because transversions tend to accumulate in a
ratchet fashion through time, the observed ratio of transitions to transversions
may itself provide a useful dating device (Moritz et aI., 1987). Other potentially
useful phylogenetic features of mtDNA include gene arrangement and gene
content [which are known to differ among some higher animal taxa (Hoffmann
et al., 1992; Hyman et al., 1988; Moritz et al., 1987; Wolstenholme et aI.,
1985»), structures of the tRNA and rRNA genes and their products (Cantatore et
 c 8 c a c a

8 a

Single-enzyme digests Double-enzyme digests

/r-------=---- / -------,
8 b ~ 8+b b+c 8+C
c
,-- - - , ,--- , , , , " ,-------, ,--------,
ABC ABC ABC ABC ABC ABC
16.5--
14.5
12.6
12.0
10.6
8.8 8.8_
7.7
6.5_
6.1
4.5
3.9 3.9
3.2
2.7
2.0
1.2-

Parsimony network: A- • C ...... S

Figure 3.9. Scoring and mapping of mtDNA restriction sites. Shown are three different mtDNA haplotypes (A, B, and C) as evidenced
by digestion patterns produced by three restriction endonucleases (a, b, and c). The restriction maps at the top are unknown at the outset,
but have been deduced from observed gel profiles produced in the single- and double-enzyme digestions. Fragment sizes (in kb) are
indicated. A parsimony network at the bottom summarizes the most parsimonious pathway of evolutionary interconversion between
haplotypes with respect to the sites assayed. Note that, in this case, restriction site changes and the parsimony network (but not the full
restriction-site map) also could have been deduced directly from the single-enzyme digestion profiles.
68 Molecular Tools

al., 1987; Wolstenholme et al., 1987), the genetic codes utilized, modes of
mtDNA replication and transcription, and, as described later, various mtDNA
sequences themselves (Chapter 8).

PLANT MITOCHONDRIAL AND CHLOROPLAST DNA

Several proven features in the evolution of animal mtDNA were completely

unanticipated (Avise, 1991a; Avise and Lansman, 1983). These include the
usual pattern of within-individual homoplasmy (predominance of a single
mtDNA sequence), despite between-individual sequence differences, and the
rapid pace of nucleotide substitution in the face of what would seem to be severe
evolutionary constraints on function as judged by the "genetic economy" of the
molecule (Attardi, 1985). However, once the major evolutionary patterns of
animal mtDNA were revealed, it might be supposed that they would apply to
other cytoplasmic genomes as well. Surprisingly, such has not proved to be the
case.
Plant mtDNA is highly variable in size [200-2400 kb (Palmer, 1985; Pring
and Lonsdale, 1985; Ward et al., 1981)] and typically exists as a collection of
different-sized circles arising from extensive recombinational processes within
individuals that interconvert between a "master" molecule and subgenomic
circles (Hanson and Folkerts, 1992; Palmer and Herbon, 1986; Palmer and
Shields, 1984). Inheritance is usually but not invariably maternal (Birky, 1978;
Forsthoefel et al., 1992). Although plant and animal mtDNAs are similar with
regard to gene content and general function, their evolutionary patterns differ
diametrically (Birky, 1988; Palmer, 1992). For species assayed to date, plant
mtDNA appears to evolve rapidly with respect to gene order, but slowly in
nucleotide sequence [perhaps l00-fold slower than animal mtDNA (Palmer and
Herbon, 1988)]. Reasons for the slow accumulation of point mutations are not
understood, but one ad hoc hypothesis is that plant mitochondria may possess
relatively error-free DNA replication systems, or perhaps highly efficient en-
zymes for repair of DNA damage (Palmer and Herbon, 1988). In any event, the
complicated recombinational features and technical difficulties in laboratory as-
say have conspired to limit the utility of plant mtDNA in molecular systematics.
Chloroplast DNA provides yet another story. cpDNA is transmitted maternally
in most plants (Birky, 1978; Gillham, 1978; Hachtel, 1980), biparentally in
some (e.g., Metzlaff et al., 1981) and paternally in several assayed gymno-
sperms (e.g., Dong et al., 1992; Szmidt et al., 1987; Wagner et al., 1987; see
also Schnabel and Asmussen, 1989). cpDNA sequences are known to move
occasionally to the nucleus (Baldauf and Palmer, 1990; Baldauf et al., 1990;
Gantt et al., 1991), and indeed there is considerable indirect support for past
exchanges among the mtDNA, cpDNA, and nDNA genomes (Fox, 1983; Gel-
lissen et al., 1983; Gray, 1989; Nugent and Palmer, 1991; Stem and Palmer,
 Molecular Tools 69

1984). The molecule varies in size from about 120 to 217 kb in photosynthetic
land plants, due largely to extent of reiteration of a large inverted repeat that
includes genes for the rRNA subunits (Zurawski and Clegg, 1987) (Fig. 3.7b).
With some possible exceptions (Milligan et al., 1989; Wagner et al., 1987), the
rate of cpDNA evolution generally appears slow both in terms of primary nu-
cleotide sequence [silent substitution rates have been estimated at only three to
four times greater than those of plant mtDNA (Wolfe et al., 1987)] and in terms
of gene rearrangement (Curtis and Clegg, 1984; dePamphilis and Palmer, 1989;
Palmer, 1990; Ritland and Clegg, 1987). Because of the large size of cpDNA
and its leisurely pace of evolution, most systematic treatments have involved
restriction site or sequence determinations for particular genes (Clegg et al.,
1986; Palmer, 1987; Palmer et aI., 1988a; Zurawski and Clegg, 1987) or have
monitored the taxonomic distributions of unique cpDNA structural features
across higher-level plant taxa (e.g., Downie and Palmer, 1992; Jansen and
Palmer, 1987; see Chapter 8). Nonetheless, some studies have uncovered con-
siderable intraspecific cpDNA variation as well (D.E. Soltis et al., 1992).

SINGLE-COPY NUCLEAR DNA

Procedures Restriction analyses of single-copy nuclear (sen) DNA tradi-

tionally have relied on Southern blotting procedures, with the probes represent-
ing DNA sequences cloned into a biological vector such as lambda phage or a
bacterial plasmid (Kochert, 1989; Fig. 3.10). Once suitable probes are available,
the general Southern blotting methodology proceeds as summarized in Figure
3.11.
Clones used as probes may represent particular genes of known function or
anonymous gene regions drawn at random from single-copy sequences in a
genomic library (a library is a collection of cloned DNA fragments). Construc-
tion of suitable probes is the most challenging aspect of scnRFLP analysis.
Probes for genes of known function sometimes are derived from "c" (comple-
mentary) DNA-sequences produced by reverse transcription of a particular
messenger RNA. Such probes have been developed for a wide variety of genes
and species, and are exchanged routinely among research laboratories. An al-
ternative approach involves generation of anonymous single-copy probes, as
follows. Total cell DNA is extracted and digested to completion with a restriction
enzyme such as HindIII. Fragments of size 500-5000 bp are isolated by gel
electrophoresis and cloned into a suitable vector, thereby generating a DNA
library. The library then is screened for single-copy sequences by "dot blot"
hybridization (Fig. 3.12), whereby the DNA contents of each clone are hybrid-
ized under controlled conditions with radioactively labeled total cell DNA. The
radioactive signal intensity of the clone dot is a reflection of the genomic copy
number of a particular sequence, such that strong signals identify clones carrying
70 Molecular Tools

1) Extract DNA
-
2) Digest DNA with
restriction enzyme

3) Open bacterial plasm ids by

digestion with same
restriction enzyme

4) Ligate DNA into plasmids

6) Culture transformed bacteria

5) Insert recombinant plasmids
to multiply DNA
into bacteria

Figure 3.10. General protocol for DNA cloning and genomic library construction.
 Molecular Tools 71

- A

- B

1) Dissect and homogenize

tissues
2) A: Extract total cell DNA with phenol/chloroform
B: Digest DNA with restriction enzyme

3) Electrophorese
4) Denature DNA and transfer
to nylon membrane

- --
X·RAY

FILM

6) Develop autoradiograph
5) Add radioactive probe,
hybridize to DNA in membrane

Figure 3.11. General protocol for surveys of single-copy nDNA polymorphisms

through Southern blotting (after Burke, 1989).
72 Molecular Tools

0_-
.-.....-
'.

1) Extract DNA 2) Cut DNA 3) nONA library

o.oo •• oeo

8
GO.OO.OOO
o •• oeooeo
.00000000
oo • • ooe • •
•• oooeooe

4) Determine copy 5) Make primers 6) Extract DNA

number from low-copy clones

, Hindlll
" EcoRI
, EcoRI

--------
= === - ==== --------
-------
= =
----- -
---:---- --------------------
--
7) PCR amplify 8) Screen enzymes 9) Screen population

Figure 3.12. General protocol for surveys of scnDNA polymorphisms through the
use of the polymerase chain reaction.

repetitive DNA and absent or weak signals identify clones containing single- or
low-copy sequences. One important distinction between the use of cDNA and
genomic clones as probes is that the fonner represent processed coding se-
quences for the transcribed gene product, whereas the latter also may include
regions flanking the gene, and introns (noncoding stretches carried within most
genes that are interspersed with the sequences specifying amino acids). Some of
these noncoding sequences may evolve especially rapidly and provide an addi-
tional class of genetic markers.
Recently, alternative methods for generating scnRFLPs have been developed
that take advantage of the polymerase chain reaction (peR; Saperstein and Nick-
 Molecular Tools 73

erson, 1991). For single-copy sequences identified in a nuclear genomic library,

primers for the PCR reaction (described later) are generated and employed to
amplify homologous DNA from each sampled individual. These amplified
DNAs then are digested by restriction enzymes, electrophoresed, and stained
directly (e.g., with EtBr). The process is summarized in Figure 3.12. This
PCR-based method offers several advantages over the Southern blotting ap-
proach (Karl and Avise, 1993): Because only a small amount of DNA is required
for the PCR reaction, assays can be conducted even when tissue sources are
limited; the DNA analyzed is of defined length (i.e., bounded by primers), so
size differences underlying digestion profiles readily can be distinguished from
restriction-site differences, and scoring generally is facilitated; because DNA is
amplified in vitro with the PCR, DNA methylation can be eliminated as a
potential source of fragment variation (amplified DNA is completely unmethy-
lated); and finally, because large amounts of DNA can be produced by ampli-
fication, the PCR-based method eliminates the need for radionucleotides and the
long exposure times (up to 7 days) required to produce a scorable signal by
autoradiography of Southern blots. On the negative side, considerable effort and
expense are entailed in library construction, screening for single-copy sequences,
and generation of primers for the PCR reactions.
Data Single-copy sequences are those that occur with a frequency of one
per haploid genome (as opposed to various classes of repetitive DNA that occur
in multiple copies). All methods for restriction analysis of single-copy DNA are
intended to reveal polymorphisms (nucleotide substitutions, addition/deletions,
inversions, etc.) within such particular stretches of sequence. The raw data are
in many respects analogous to those provided by protein electrophoresis: For
each gene region assayed, diploid individuals can be described as homozygous
or heterozygous for various restriction-site polymorphisms (Fig. 3.13); the Men-
delian nature of the polymorphisms can be verified by breeding studies or by
agreement of allele frequencies with Hardy-Weinberg expectations for popUla-
tions suspected of random mating; genotypic descriptions can be accumulated
across many enzyme/gene combinations; a population may exhibit multiple al-
leles at each "locus"; and the alleles thus identified usually remain unordered
phylogenetic ally (unless multisite haplotype characterizations can be accom-
plished in regions of low recombination-see the following paragraphs and also
the discussion of gene trees in Chapter 4). The major advantage over protein
electrophoresis is that, in principle, a nearly unlimited pool of genetic variants
may be tapped. Thousands of single- or low-copy regions, to each of which
many restriction enzymes can be applied, are present in most organisms (single-
copy DNA constitutes 20-80% of most plant and animal genomes-see the
review in Sibley and Ahlquist, 1990). Also, the assays reveal polymorphisms at
both silent and replacement nucleotide positions (as well as that due to other
sources such as duplications/deletions and insertions of transposable elements).
 , 1: 7
probe
12
9 1
enzyme
enzyme 2

enzyme 1 enzyme 2
010 011 1/1 010 011 1 11
19-
-14-
12-
9-

7 -
5 -
-4-

probe

site
• 5 (5)
A
6 2:)
B
7
• enzyme 3

A 010 011 1 11 010 011 011 1 11 DID Oil 1 11

B DID 010 DID Oil Oil 011 Oil 1 11 1/1 1/1

(cis) (Irans)

-18- 18-

13_
13 -
11 - 11- 11-

7 -
6 6 -
5 - 5 5 -

Figure 3.13. Interpretation of restriction digestion profiles for Men-

delian polymorphisrns in diploid organisms, as assayed by Southern
blotting using scnDNA probes. Shown are gel patterns produced by
each of three enzymes whose restriction site positions are indicated
by circles, triangles, and ovals. Closed symbols indicate nonvariable
restriction sites; open symbols indicate variable sites that are either
present (I) or absent (0) along a given chromosome. Heterozygous
individuals are designated as "011" and respective homozygotes by
"111" and "0/0." In the bottom figure, note that each lane (indi-
vidual) in the gel is characterized by two diploid genotypes, reflect-
ing various combinations of presence versus absence at each of two
adjacent but variable restriction-site positions. Other numbers in the
figure indicate the sizes (in kb) of various restriction fragments.
 Molecular Tools 75

The major disadvantage in comparison to protein electrophoresis is that the

methods are expensive, both in effort and materials. Perhaps for this reason,
rather few applications of scnRFLP approaches in evolutionary biology have as
yet appeared, despite the fact that these methods are widespread in related
research endeavors including the mapping of disease genes and quantitative trait
loci (Botstein et al., 1980; B. Martin et al., 1989; Paterson et al., 1988; Weller
et al., 1988), and in breeding studies and strain verification in domestic species
(Apuya et al., 1988; Beckmann et al., 1986).
Even when DNA sequences assayed by the above procedures are short (i.e.,
0.5-2 kb), particular segments sometimes exhibit two or more linked restriction
polymorphisms (Fig. 3.13). When multiple RFLPs are revealed within a defined
gene region, their phases become of additional interest. Phase refers to the
pattern of association (cis versus trans configuration) among multiple variants
along a chromosome. Suppose, for example, that two linked polymorphisms in
a diploid organism each involve presence (1) versus absence (0) of a restriction
site, such that the four possible states for a nuclear chromosome are 1,1 and 0,0
(cis configurations) and 1,0 and 0,1 (trans configurations). These states are
evident directly from the assayed diploid genotypes of all individuals except
double heterozygotes. In populations where the unambiguous diploid genotypes
reveal a consistent phase for the variable sites (complete disequilibrium), it is
probably safe to assume that the double heterozygotes carry two chromosomes
with these same linkage phases (Karl et al., 1992). But when disequilibrium is
incomplete, the phases of double heterozygotes cannot always be recovered from
the gel digestion patterns (Fig. 3.14). The difficulties of phase determination in
diploids become even greater when more than two linked polymorphisms are
involved. For these reasons (and because of the likelihood of intragenic recom-
bination in the history of the assayed region), complete haplotype determinations
are seldom accomplished from scnRFLP approaches. Thus, unlike mitochondrial
RFLP data, where haplotype phase is obvious and assessment of phylogeny
among alleles routine, evolutionary relationships among scnRFLP alleles of a
particular locus are seldom determined.
RIBOSOMAL RNA GENES AND OTHER MIDDLE-REpETITIVE GENE FAMILIES

When Southern blotting involves the use of a DNA probe that contains ho-
mologies to repetitive genomic sequences, the probe hybridizes to all such se-
quences and thereby simultaneously reveals restriction fragment changes at mul-
tiple members of the gene family. Ribosomal RNA genes in the nuclei of
eukaryotic cells usually exist as tandemly repeated elements, each repeat unit
composed of a highly conserved coding sequence of total length about 6 kb, plus
shorter and more variable noncoding spacer regions (Fig. 3.15). The repeated
rONA modules may occur at one or several chromosomal sites. The copy num-
ber of rONA repeats per genome varies from several hundred in some mammals
and insects to many thousands in plants (Long and Dawid, 1980).
76 Molecular Tools

,
adjacent variable sites
, ,
non-adjacent variable sites
,

• • ]
1 2 3 4 1 5 6 7
16 6 .6
+ 2 + 3 4 "
• (I)
cis

• •
+ 5 6 + 7
1 • • "

•
1 + 5
.6 •
6 7

1
1(1)
5

• "
6 + 7
}"n.

cis
! trans

1+5_ 1+5_
5- 5-
6+7- 6+7-

7- 7-

6- 6
1_

Figure 3.14. Effects of the position of variant restriction sites on haplotype phase
determination in diploid individuals that are double-heterozygotes (after Quinn and
White, 1987). When the variant sites (open circles) for a restriction enzyme are
adjacent (left-hand column), RFLP digestion profiles differ between the cis and trans
phases. When one or more invariant sites (closed circles) intervene between the
variant sites (as in the right column), cis and trans heterozygotes produce identical gel
profiles and, hence, cannot be distinguished.
 Molecular Tools 77

16S 23S 5S

Escherichia coli

18S 5.8S 26S

Vicia taba

18S 5.8S 28S

Homo sapiens

Figure 3.15. Structural features of the rDNA repeat module (drawn to approximate
scale) in E. coli, and in a representative plant and animal (after Appels and
Honeycutt, 1986). Hatched regions indicate loci encoding the "small" (16S and
18S) and "large" (23S, 26S, 28S) subunits of ribosomal RNA, as well as the "5S"
rRNA elements. Black regions indicate internal transcribed spacers, which often
differ in length.

The ready availability of probes for ribosomal RNA genes has prompted a
number of studies of population variation and differentiation in these genetic
regions based on Southern blotting methods (Appels and Dvorak:, 1982; Arnold
et al., 1991; Davis et al., 1990; Rieseberg et al., 1990a,b; Rogers et al., 1986;
Saghai-Maroof et al., 1984; Schaal et al., 1987; Williams et al., 1985). These
studies have revealed RFLP markers that often distinguish closely related spe-
cies, and sometimes conspecific populations. The genetic differences normally
involve varying lengths of the repeat unit due to heterogeneity in size of the
spacer regions, with additional variation occasionally stemming from restriction
site changes in both the coding and spacer segments (Schaal, 1985).
The occurrence of multiple rDNA copies per genome opens the possibility of
intraindividual polymorphism, which indeed has been observed within both
plants and animals. A potential difficulty in interpreting genetic markers pro-
vided by any multigene family involves understanding the degree to which
mechanisms of concerted evolution may have homogenized the repeated DNA
sequences (Arnheim, 1983; Ohta, 1980; Ohta and Dover, 1983), and hence the
extent to which different family members can be viewed as providing indepen-
dent bits of phylogenetic information. Various complications ultimately related
to the problem of distinguishing orthology from paralogy (see Chapter 1) can
arise. For example, Williams et al. (1987) showed that variants for rRNA genes
have a nonrandom distribution among the X and Y chromosomes in Drosophila,
suggesting that within-chromosome homogenizing mechanisms are more effi-
78 Molecular Tools

cient than those working among chromosomes; Arnold et al. (1988) showed that
biased gene conversion has influenced the distributions of sequences for rRNA
genes in a hybrid zone between grasshopper subspecies. Thus, the use of nuclear
rRNA genes (and other repetitive DNA families) as genetic markers in micro-
evolutionary studies could be compromised by the heterogeneity of molecular
processes governing the evolutionary turnover and, hence, genetic relationships
among the repeat elements themselves (Schaal et al., 1991). On the other hand,
Hamby and Zimmer (1992) conclude that "the most remarkable feature of rDNA
is the overall sequence homogeneity among members of the gene family." For
use as simple genetic markers, an ideal situation would be where concerted
evolution was so pronounced that all copies of a repetitive sequence within an
individual were quickly homogenized, and each specimen could thus be char-
acterized by an unambiguous, specifiable genotype. However, such extreme
concerted evolution also would carry the consequence of confining the informa-
tion content of a multigene family to that of a single "locus."
Despite these potential complications, RFLP markers from rDNAs have con-
tributed to studies of geographic popUlation structure and patterns of introgression
in hybrid zones (Arnold et al., 1987; Bakeret al., 1989; Cutler et aI., 1991; Learn
and Schaal, 1987). Far more prominent, however, have been nucleotide sequence
analyses of both nuclear and cytoplasmic ribosomal RNA genes (Hillis and Dixon,
1991; Mindell and Honeycutt, 1990), which have provided much of the molecular
data for phylogenetic reconstructions among deeper branches in the tree of life
(Chapter 8). Naturally, these studies have focused on slowly evolving coding
regions of the rRNA genes. At these macro-evolutionary scales, the fraction of
overall rDNA sequence heterogeneity attributable to intraindividual or intraspe-
cific polymorphism becomes negligible, and safely can be neglected.
Southern blotting procedures have been employed to assess levels of genetic
variability in other kinds of multigene families as well. For example, the major
histocompatibility complex (MHC) is a family of tightly linked homologous loci
that encodes cell surface antigens involved in the immunological response. In
humans, mice, and other mammals, particular MHC genes are known to be
extremely polymorphic, some with scores of alleles (Hedrick et aI., 1991;
Hughes and Nei, 1988, 1989; Klein, 1986). Feline probes homologous to one
class ofMHC loci were employed by Yuhki and O'Brien (1990; see also Winkler
et aI., 1989) to assess molecular variation in African cheetahs and Asiatic lions,
species suspected by other criteria to possess low genome-wide variability due to
historical bottlenecks in population size (Chapter 9). Mammalian MHC probes
have also revealed DNA polymorphisms in birds (Gibbs et al., 1991).
MINISATELLITE SEQUENCES AND DNA FINGERPRINTING

The genetic complexity inherent in repetitive DNA families sometimes can be

turned to advantage in terms of providing individual-specific genetic markers.
 Molecular Tools 79

The tenn "DNA fingerprinting" usually is associated with a molecular approach

introduced by Jeffreys et al. (l985a), in which Southern blot assays of hyper-
variable regions of DNA reveal gel banding profiles that distinguish most or all
individuals (barring monozygotic twins) within a sexually reproducing species.
The DNA probes originally employed by Jeffreys (l987) hybridize to conserved
core sequences (10-15 bp long) scattered in numerous arrays about the human
genome as part of a system of "dispersed tandem repeats" (Fig. 3.16), also
referred to as minisatellite DNA sequences or as VNTR (variable number of
tandem repeat) loci. Each repetitive unit within an array is about 16-64 bp long.
Increases and decreases in the lengths of particular arrays result from changes in
the repeat copy number arising from high rates of unequal crossing-over during
meiosis [indeed, there is speculation that the minisatellite sequences themselves
provide hotspots for recombination (Jarman and Wells, 1989)].
The original Jeffreys' probes were isolated from a myoglobin intron in humans
and applied to problems in human forensics (Dodd, 1985; Gill et al., 1985;
Jeffreys et al., 1985b, 1985c), but these probes also cross-hybridize to reveal
DNA banding profiles in many species including other mammals (Hill, 1987;
Jeffreys and Morton, 1987; Jeffreys et aI., 1987), birds (Brock and White, 1991;
Burke and Bruford, 1987; Hanotte et al., 1992a; Meng et al., 1990), fishes
(Baker et aI., 1992), and even some invertebrates such as corals and snails
(Coffroth et aI., 1992; Jarne et al., 1990, 1992). Other probes for hypervariable
minisatellites have been identified (such as one from the M13 phage) that behave
similarly in providing DNA fingerprints in additional vertebrate taxa (Georges et
aI., 1987; Longmire et al., 1990, 1992; Vassart et aI., 1987), as well as in some
invertebrate animals (Zeh et al., 1992), plants (Rogstad et al., 1988), and mi-
crobes (Ryskov et al., 1988).
The complex gel profiles characteristic of DNA fingerprints appear when an
enzyme is employed that cleaves outside the repeat arrays (Fig. 3.16). From each
homologous chromosome position in an individual, either one or two bands will
be revealed, depending on whether the specimen is homozygous or heterozygous
with respect to number of tandem repeats in the array. The presence of several
such arrays scattered about the genome results in multilocus digestion profiles
typically consisting of some 20 or more scorable bands per individual in the
4-23-kb size range. In an animal population, dozens of alleles characterized by
differing lengths may be segregating at each chromosomal position. The multi-
locus nature of the data and the sheer complexity in single gel profiles provide a
convenient means for establishing genetic identity versus nonidentity, as well as
for assessing parentage [because barring spontaneous de novo mutation (Jeffreys
et aI., 1988b), each band in a individual's DNA fingerprint must derive from
either its biological mother or father].
In multilocus DNA fingerprints, the advantage of genetic complexity for in-
dividual diagnosis becomes a liability in other contexts. Generally, it remains
 chromosomal segments in individual A

1 2

3 4
•• •••• ••••••••••

~
5 6

A B c

gel
autoradiograph

Figure 3.16. DNA fingerprinting from VNTR loci. Shown are six dispersed chromo-
somal segments (on the same or different chromosomes), each of which may harbor
variable numbers of the tandem repeat elements. Solid circles within the repeats
indicate the conserved core sequence to which the probe hybridizes in a Southern blot.
A restriction enzyme that cuts (arrows) outside the repeat regions thus reveals a
complex digestion profile on a gel autoradiograph.
 Molecular Tools 81

unknown which bands in a fingerprint belong to which loci (array), so allelism

is not established. Thus, whether individuals are homozygous or heterozygous at
particular "genes" seldom is ascertained, nor can the allele or genotype fre-
quencies characterizing populations be determined. These problems seriously
compromise attempts to estimate genetic relatedness from the measurable at-
tribute in DNA fingerprints-the fraction of shared marker bands (Lynch, 1988;
but see also Kuhnlein et al., 1990 and Packer et al., 1991)--or to estimate gene
flow or other population parameters employing the usual statistical algorithms
that require straightforward Mendelian markers.
These shortcomings of multilocus DNA fingerprinting methods for applica-
tions other than genetic identity and parentage assessment have prompted devel-
opment of methods for analyzing particular mini satellite regions, one at a time.
These include use of refined DNA probes and more stringent hybridization
conditions in Southern blotting, or PCR-based methods (Horn et al., 1989; see
next section) to reveal genetic variation tied to individual hypervariable loci
(Jeffreys et al., 1988a; Nakamura et al., 1987; Wong et al., 1986, 1987). For
example, under stringent assays, a "3'HVR" probe consisting of tandem 17-bp
repeats hybridizes to a single region on human chromosome 16 downstream from
the a-globin complex (Higgs et al., 1986; Jarman et al., 1986). Each such
assayed VNTR locus produces only one or two bands in a particular DNA
digestion, depending on whether the individual in question is homozygous or
heterozygous for the number of repeats (or in other flanking regions) between
restriction sites. In human populations, several such hypervariable loci exhibit
dozens of alleles, some of which may be difficult to distinguish unambiguously
by gel mobility alone (Devlin et al., 1991). Another recent variant of the
"single-locus" fingerprinting approach circumvents these ambiguities in allelic
identification by characterizing patterns of base substitution (in addition to repeat
copy number) within particular minisatellite loci (Jeffreys et al., 1990, 1991).
The accumulation of genetic information from several hypervariable single-
locus regions combines the advantages of relatively straightforward allelic in-
terpretation (characteristic of the allozyme and scnDNA approaches described
earlier) with the individual-diagnostic power of multilocus DNA fingerprinting.
However, as with allozyme and scnDNA methods (but unlike the situation for
mtDNA) , detailed phylogenetic relationships among the various alleles of a
VNTR locus normally are not assessed. Use of single-locus VNTR assays is the
current method of choice by companies and agencies involved in human forensic
practice (Balazs et al., 1989; Budowle et al., 1991; Chapter 5). Single-locus
VNTR probes also are being developed for other taxa such as birds (Burke et al.,
1991; Hanotte et al., 1991). A current limitation of this approach is that the
probes often prove highly taxon-specific (unlike the multilocus Jeffreys' probes
employed under low-stringency conditions). However, some minisatellite probes
do cross-hybridize under high stringency conditions to highly variable loci in
numerous related species (Hanotte et al., 1992b).
82 Molecular Tools

Recently, another class of tandem repetitive elements referred to as "micro-

satellites" has been discovered in various animals (Fries et al., 1990; Litt and
Luty, 1989; Stallings et al., 1991; Tautz, 1989; Weber and May, 1989) and
plants (Nybom et al., 1992). Each microsatellite locus consists of reiterated short
sequences (particular di-, tri- or tetra-nucleotides) tandemly arrayed (Hamada et
al., 1984), with variations in repeat copy number accounting for a profusion of
distinguishable alleles. In humans, for example, hundreds of microsatellite loci
have been characterized, each typically with several alleles (5-10 or more) that
may be revealed as RFLPs following PCR amplifications using conserved flank-
ing regions (see Valdes et al., 1993). These highly-allelic Mendelian polymor-
phisms [known also as "simple sequence length polymorphisms" or SSLPs
(Schlotterer et al. (1991)] should find widespread application in various areas of
population biology such as gene flow estimation and parentage assessment (Elle-
gren, 1991). Although micro satellite approaches are still in their infancy with
regard to non-human population applications (at the time of this writing), they
evidence the broader point that additional sources for molecular markers likely
will be developed and employed widely in the near future.

DNA Sequencing and the Polymerase Chain Reaction

PRINCIPLES AND PROCEDURES

Two methods for direct sequence determination of purified DNA have been
available for nearly two decades. One approach, introduced by Maxam and Gilbert
(1977, 1980) (Fig. 3.17), relies on chemical cleavage reactions specific to indi-
vidual bases (A, T, C, or G). The ends of a targeted stretch of DNA are
radioactively labeled and the DNA is divided into four subsamples which then are
treated with different chemical reagents that cleave at base-specific positions. For
example, one subsample is treated with dimethyl sulfate and piperidine, which
results in DNA cleavage only at the G positions. Reactions are carried out under
conditions such that only a small, random fraction of sites actually is cleaved in
any molecule, so that the composite digestion contains a collection of fragments
of varying length, each terminated at a G position. Then fragments are separated
electrophoretically in a polyacrylamide gel and visualized by autoradiography.
Parallel reactions specific for the other three bases are carried out and the frag-
ments separated on adjacent lanes of the gel. Thus, the DNA sequence can be read
directly from the ladderlike banding pattern appearing on an autoradiograph (Fig.
3.17).
An alternative sequencing procedure, introduced at the same time by Sanger
et al. (1977), is the usual technique of choice today. This method relies on the
controlled interruption of in vitro DNA replication (Fig. 3.17). Double-stranded
DNA is denatured to single strands, and a short DNA segment (the primer) known
 Molecular Tools 83

Isolate DNA; denature S·, ........... GATCAGGCTTAAGCA ...... -3·

Into single strands

1) Radioactively 1) Anneal primer and radioactively label

end-label

• S·- ........... GATCAGGCTTAAGCA ........... -3·

***~.5·

2) Cleave chemically 2) Add labeled dXTP's, DNA polymerase,

In four treatments and ddXTP in four treatments

o
Cleave at:

1 (T+C 1G
ddCTP ddTTP ddATP ddGTP

0000
-- -- -- --
..
(A+G

GATCA G GA GAT
+--w -vi +-w ~
GATCAG GATC GAT GATCAGG
--w--w _ _ ~

etc. etc. etc. etc. etc. etc. etc. elc.

3) Separate fragments 3) Separate fragments

electrophoretlcally el ectrophoretlcally

~ ~ ~ ~ ~ ~ ~ ~
G A+G T+C C C T A G

A
C G
G C
A T
A T
T A
T A
C G
G C
G C
A T
C G
T A
A T

Maxam-Gilbert approach Sanger approach

Figure 3.17. General protocols for DNA sequencing via the Maxam-Gilbert (left panel)
and Sanger (right panel) approaches (after Hillis et al., 1990).
84 Molecular Tools

to be complementary to a sequence on the target DNA is annealed to the target

sample. This primer/template mixture is divided into four subsamples, each of
which is subjected to a primer extension reaction catalyzed by DNA polymerase.
All four reactions contain the four deoxynucleotides (dA, dC, dG, and dT), plus
a single dideoxynucleotide (ddN, a nucleotide that lacks the 3' -OH group present
in deoxynucleotides). The synthesized DNA strand is made radioactive, either by
labeling the end of the primer or by the incorporation of a labeled deoxynucleotide
during synthesis. DNA sequence extension occurs by attachment of nucleotides
to a free 3' -OH, so that wherever ddNTP has been incorporated into the growing
strand, further strand extension is arrested. The polymerase reaction is carried out
under conditions such that the incorporation of ddNTPs is rare and random. Thus,
different DNA molecules in a subs ample achieve varying lengths before termi-
nation at a particular base. As under the Maxam-Gilbert approach, fragments from
the four subsamples are separated electrophoretic ally in a polyacrylamide gel,
visualized by autoradiography, and the DNA sequence read directly.
In the past, sequencing applications were limited by the availability of puri-
fied, homologous DNA sequences from different organisms. Such sequences had
to be isolated and amplified in vivo by laborious procedures of cloning into
microbial vectors (Fig. 3.10). The recent development of the polymerase chain
reaction (Mullis and Faloona, 1987; Saika et al., 1985, 1988) has changed this
situation dramatically by permitting rapid in vitro DNA amplifications. As a
result, DNA sequencing has grown explosively in the last decade and has be-
come among the most popular of methods for phylogenetic reconstruction
(Miyamoto and Cracraft, 1991).
The PCR technique involves three steps (Fig. 3.18): (a) denaturation of
double-stranded DNA by heating; (b) annealing of extension primers to sites
flanking the region to be amplified; and (c) primer extension, in which strands
complementary to the region between the flanking primers are synthesized under
the influence of a DNA polymerase (Taq) which is thermostable. The double-
stranded products are cycled repeatedly through steps (a)-(c). In each round of
denaturation-annealing-extension, the target sequence is roughly doubled in the
reaction mixture, so that after 20 or more rounds the product assumes over-
whelming preponderance and can be sequenced directly as purified DNA span-
ning the primers. Indeed, the amplification primers for PeR also can be em-
ployed as sequencing primers in the Sanger reactions, allowing a direct coupling
of these approaches. The PCR and sequencing procedures are becoming increas-
ingly automated and can be carried out with commercially-available temperature
cyclers coupled to a sequencing apparatus.
The primers employed to initiate the peR process are short sequences (about
20-30 nucleotides long) that exhibit high sequence similarity (particularly in the
3' end) to regions flanking the target sequence. For example, several such prim-
ers have been identified in animal mtDNA (Box 3.3), which for reasons dis-
 Molecular Tools 85

1) Isolate DNA -:::=:::"iii!i1i!ii!"!!i1i!!ii!"~

cycle 1

2) Denature and anneal primers

3) Primer extension := ,,''''11''''''''''=;;::::::::

,Ii Ii Ii Ii __

cycle 2

4) Denature and anneal primers

-"""II'''''''''''''''''';;::::::::
",,'" -
- -
5) Primer extension
,,11,,"iiiiii'"
:=:::::;iI!", ""dllilili

cycle 3

-
6) Denature and anneal primers

- -II
7) Primer extension
111111" ''''',',',',',:iiiilill

:::::;:; "",,'!iiiii""'_
8) Repeat cycles
Figure 3.18. General protocol of the polymerase chain reaction for amplifying DNA
(after Oste, 1988).
86 Molecular Tools

Box 3.3. Examples of Conserved Primer Sequences Within

Various MItochondrial Genes That Have Proved Useful for peR
AmpUftcadons.
Sequences are given in the 5' to 3' direction; Y indicates either a cytosine (C) or
thymine (T); R either an adenine (A) or guanine (G); and W indicates an A or T.
Several of these primers were developed for fishes, but in many cases they successfully
amplify DNA from other animals as well.

Gene Primer Primer Sequence Reference

Control Ll5926 TCAAAGCTTACACAGTCTTG- Kocher et al., 1989

region TAAACC
HI6498 CCTGAAGTAGGAACCAGATG Meyer et al., 1990
Ll6518 CATCTGGTTCTTTCTTCAGGG- Meyer, 1993
CCAT

12S rRNA Hll09 GTGGGGTATCTAATCCCAGTT Meyer, 1993

Ll091 AAAAAGCTTCAAACTGGGATT- Kocher et al., 1989
AGATACCCCACTAT
H1478 TGACTGCAGAGGGTGAC- Kocher et aI., 1989
GGGGCGGTGTGT

Cytochrome L5950 ACAATCACAAAGAYATYGG Normark et al., 1991

oxidase I H7196 AGAAAATGTTGWGGGAARAA Normark et aI., 1991

ATPase 6 H8517 GGGRACTTTGACTGGTACT Meyer, 1993

L8580 AGCCCCACATACCTAGGTA- Meyer, 1993
TCCC
H8907 GGGGTTCCTTCAGGCAAT- Meyer, 1993
AAATG

Cytochrome L9225 CACCAAGCACACGCATACCA- Meyer, 1993

oxidase III CAT
H9407 AAAGTTCCTGTGGTGTG- Meyer, 1993
CGGGGG

Cytochrome b Ll4841 AAAAAGCTTCCATCCAA- Kocher et aI., 1989

CATCTCAGCATGATGAAA
Ll5020 GCYAAYGGCGCATCCT- Meyer, 1993
TYTTYTT
H15149 AAACTGCAGCCCCTCAGAA- Kocher et al., 1989
TGATATTTGTCCTCA
 Molecular Tools 87

-- -- - -- - - - -
51 52 F1 Examples of F 2 or backcross genotypes
, ,

- - -- -- -- - - --
Figure 3.19. Nature ofRFLP data generated by the "RAPD" procedure. In this approach,
short random primers are employed in PeR reactions to amplify anonymous DNA seg-
ments (in this case, from four unlinked gene regions). The presence of a band indicates
successful amplification; absence indicates unsuccessful amplification, perhaps resulting
from naturally occurring mutations in the primer recognition site. Thus, RAPDs in diploid
organisms usually behave in a dominant/recessive fashion (although some cases of
codominant inheritance are known-e.g., Fritsch and Rieseberg, 1992). As exemplified
in this figure, the RAPD approach may be especially useful in characterizing first-gen-
eration or later-generation hybrids between genetically distinct species, Sl and S2'

cussed later is the molecule toward which the majority of PCR efforts in popu-
lation biology thus far has been directed. The PCR also can be coupled to DNA
assays other than sequencing, for example in the generation of nuclear RFLPs
from the amplified sequences, as in the microsatellite and various other ap-
proaches described earlier. Another PeR-based RFLP approach is the "RAPD"
method (pronounced "rapid," for random amplified polymorphic DNA)
(Williams et aI., 1990). This technique involves screening DNA for interpretable
polymorphisms using short (= 10 bp) primers of arbitrary sequence to amplify
at random from a few anonymous genomic sequences (Welsh et aI., 1991). The
method typically yields polymorphisms with dominance-recessive characteristics
(Fig. 3.19). At the time of this writing, the RAPD approach has not yet been
employed widely in population biology, but it has been promoted strongly by
some authors (see Hadrys et aI., 1992; Hedrick, 1992).

DATA

In a sense, all assay methods short of DNA sequencing can be thought of as

indirect attempts at sequence acquisition. Indeed, the availability of nucleotide
sequence data in principle allows recovery of genetic information at less-detailed
levels (e.g., amino acid sequences, RFLP maps), whereas the converse is not
true (Box 3.4). However, for logistic reasons, DNA sequences (typically 500 bp
or more in length per sample) usually are gathered from only one or a few genes
Box 3A. Nucleodde Sequences In the mtDNA Cytochrome b Gene of Marine Turtle Species (Taxa a-n)
(after Bowen et aI., 1993a).
The entire data set involved more than 500 nucleotide positions per sample (only 63 of which are shown here), which is typical for many
sequencing studies.
1. Nucleotide sequences. Each dash indicates a base identical to that of the reference species, "a."
Taxon Nucleotide Sequence
a ACC GOA ATC TIC TTG GCA ATA CAC TAT TCA CCA GAT Aer TCC erG GCA TIC TCA TCA ATC ATC
b C-A --T --C --C -TC A-A --C --T --T -C-
c C-A --T --C --C -TC A-A --C --T --T -C-
d --T C-A --T --C ·TC A-A --C --T TC-
e --T C-A --T --C -TC A-A --C --T TC-
f --T G-- C-A --T --C -TC A-A --C --T TC-
g --T C-A --T --c -TC --T A-A --C -C-
h C-A --C --C -TC A-A --T --T --T ocr
--T C-A --C --C -TC A-A --T -T --T ocr
--T C-A --C --C -TC A-A --T --T --T ocr
k --T C-A --C --C -TC A-A --T --T --T ocr
C-A --C -TC A-A --T GC-
m --A --C -T- --C -C G-T ocr
n --A --C -T- --C --C G-T ocr
2. Same data coded as purines ("0") versus pyrimidines ("I ").
a 011 000 011 III 110 010 010 101 101 110 110 001 011 111 110 010 111 llO llO Oll Oll
b 0-- --1
c 0-- --1
d 0-. --1 1--
e 0- --1 1--
0-- --1 1--
g 0-- --1
h 0-- --I
0-- --I
0-- --I
k 0-- --I
0-- --I
m --I --I
n --I --I
3. Same data translated into amino acid sequences (by reference to the mitochondrial genetic code).
a thr gly ile phe leu ala met his tyr ser pro asp thr ser leu ala phe ser ser ile ile
b ile met thr
c ile met thr
d ile met ser
e ile met ser
val ile met ser
g ile met thr
h ile met ala
ile met ala
ile met ala
k ile met ala
I ile met ala
m ile val ala
n ile val ala

Any of these coded data sets could be analyzed phylogenetically by appropriate computer programs. Note that when the data are coded as
purines versus pyrimidines, only trans versions would be counted in the resulting phylogenetic estimates; and when the data are coded as
amino acids, only replacement substitutions would be counted. These data codings exemplify a trade-off common to most sequencing
studies: Although these latter two treatments weight heavily for mutational events that are rare and thus less likely to be homoplasious over
short evolutionary time scales (see Chapter 4), much information of potential phylogenetic significance (particularly at lower sequence
divergence levels) can be lost by neglecting the silent transitions. For example, in the data sets above, 20 of the 63 nucleotide positions
shown were variable overall, whereas only four positions displayed transversions and only five represented replacement substitutions.
90 Molecular Tools

in a given study and from relatively small numbers of individuals. Thus, these
data provide high resolution for molecular aspects of sequence differences (e. g. ,
changes in coding versus noncoding regions; transitions versus transversions;
silent versus replacement substitutions in coding regions; base substitutions ver-
sus addition/deletion changes or nucleotide rearrangements, etc.), but at the
expense of sacrificing genetic information from a broad base of loci and indi-
viduals. Contemplated biological applications for DNA sequencing must weigh
such considerations.
There are several other clear and unique advantages to sequencing methods,
particularly when coupled to PCR. The PCR requires as a template only minute
quantities of undegraded DNA, as has been obtained from such unlikely sources
as single feathers (TaberIet and Bouvet, 1991), hairs (Morin et al., 1992; Tab-
erIet and Bouvet, 1992; Vigilant et al., 1989), animal excrement (H6ss et aI.,
1992), museum-preserved material (Higuchi et aI., 1984), and even fossils
(Chapter 8). Amplification of DNA by the PCR is much faster and easier than by
cloning. Finally, sequences that evolve at different rates may be chosen for
analysis (Chapter 4), so that sequencing studies can be tailored to phylogenetic
analyses at any desired level of evolutionary separation.
Potential concerns about PCR-based assays include the following. Because of
the extreme sensitivity of the PCR, sample contamination (e.g., by microbes,
physical handling, or any source of contact with nontarget DNA) poses a serious
difficulty. Degree of fidelity of the PCR amplification has been of some concern
(Dunning et aI., 1988; Ennis et al., 1990; Pfrfrbo and Wilson, 1988; Saiki et aI.,
1988), because any misincorporation of nucleotides in the early rounds of am-
plification will result in an amplified sequence differing at least slightly from the
original template. Low-frequency errors in amplification have been observed,
but because these are normally far less than the incidence of natural nucleotide
site polymorphisms within populations, their effects in most phylogenetic appli-
cations (particularly at higher taxonomic levels) probably are negligible (Kwia-
towski et al., 1991). Finally, most PCR applications (other than RAPDs) require
prior knowledge of sequences flanking the target DNA. This latter obstacle has
diminished considerably as sequence data have accumulated from numerous
genes and species.

REFERENCES TO LABORATORY PROTOCOLS

For the interested reader, the following references provide far more detailed
descriptions of the various laboratory methods.
Protein immunology: Champion et al., 1974; Maxson and Maxson, 1990.
Protein electrophoresis: Harris and Hopkinson, 1976; Murphy et al., 1990;
Selander et al., 1971; Shaw and Prasad, 1970.
 Molecular Tools 91

DNA-DNA hybridization: Sibley and Ahlquist, 1990; Werman et al., 1990.

DNA restriction analysis: Dowling et al., 1990; Hames and Higgins, 1985;
Hoelzel, 1992; Karl and Avise, 1993; Lansman et al., 1981; Quinn and
White, 1987; Sambrook et al., 1989; Watson et al., 1992.
DNA fingerprinting: (single-locus and multilocus): Bruford et al., 1992.
DNA sequencing and the PCR: Hillis et al., 1990; Innis et al., 1990; Palumbi
et aI., 1991; Sanger et al., 1977.

SUMMARY
1. A rich potpourri of laboratory methods exists for revealing genetic mark-
ers. Protein assays with the greatest impact in the fields of population biology and
evolution have been the immunological approach of microcomplement fixation
(MCF) and multilocus starch-gel electrophoresis (SGE). The most influential of
the DNA assay methods have been DNA-DNA hybridization, analyses of re-
striction fragment-length polymorphisms (RFLPs), and nucleotide sequencing.
2. Each of these assay methods is applied most fruitfully to particular
classes of genes or their products: MCF to abundant, purifiable proteins encoded
by single genes, such as albumin; SGE to enzymatic proteins for which specific
histochemical stains are available; DNA-DNA hybridization to the single-copy
fraction of the nuclear genome; RFLP analyses to a variety of genetic systems,
including mitochondrial DNA, chloroplast DNA, single-copy nuclear DNA,
ribosomal RNA genes, and the various categories of repetitive elements under-
lying DNA fingerprints; and nucleotide sequencing to virtually any segments of
DNA, particularly those that can be amplified via the polymerase chain reaction.
3. The multivarious laboratory methods available differ widely in the na-
ture of genetic information provided. The next chapter will attempt to organize
thoughts about how these diverse molecular approaches can be cataloged with
regard to phylogenetic information content.
 4

Interpretive Tools

Thus the hereditary properties of any given organism

could be characterized by a long number written in a
four-digital system.

G. Gamow, 1954

Once molecular data have been gathered, how are they analyzed and interpreted
in a phylogenetic context? The answer, of course, depends on the particular
biological problem addressed and the nature of the molecular information. The
biological settings are nearly as diverse as one's imagination allows. The inter-
pretive tools are those of population genetics and phylogenetics. Some ap-
proaches to molecular data analysis are highly idiosyncratic to a particular bio-
logical problem or data base (examples appear throughout Part II), whereas
others are standard and generalizable. This chapter will introduce some of the
fundamentally important principles and procedures in this latter class of analyt-
ical tools.

CATEGORICAL SUBDIVISIONS OF MOLECULAR GENETIC DATA

One initial way to organize thought about analytical approaches is to subdivide

the diverse types of molecular data into categories for which shared philosophical
approaches to analysis and interpretation may pertain. The following are some of
these partitions (Fig. 4.1).

92
 Interpretive Tools 93

DNA-DNA DNA
HYBRIDIZATION SEQUENCING

RFLPs--
MICROCOMPLEMENT
MULTILOCUS
FIXATION
FINGERPRINTING

RLFPs--
MULTILOCUS MULTIPLE
ALLOZYMES scnDNAs
RFLPs--
mtDNA

Figure 4.1. Alternative ways to "slice the pie" of molecular genetic data classes. The
heavy black line separates protein assays from those dealing directly with DNA. The
heavy gray line divides the methods according to whether the raw data consist of
qualitative character states or distance values only. The stippled slices of the pie
indicate those techniques that normally supply information from only one gene (or
linkage group) at a time, as opposed to the remaining methods that usually access
genetic data from multiple loci either simultaneously (lined slices) or cumulatively
(open slices).

Protein Versus DNA Information

An obvious consideration is whether a molecular technique reveals variation at

the level of proteins or, alternatively, at the level of DNA. Methods in the former
category unmask primarily the genetic changes in coding regions that have
altered amino acid sequence. On the other hand, some nucleic acid techniques
provide access to a far greater panoply of architectural changes in both coding
and noncoding regions of the genome. Thus, informative genetic markers may be
derived from synonymous as well as non synonymous (amino acid altering) nu-
cleotide substitutions in protein-coding sequences, from genetic changes in in-
trons and gene-flanking regions, and from additions and deletions of genetic
material, sequence rearrangements, or other DNA-level features.

Qualitative Versus Distance Data

Some molecular techniques, notably DNA-DNA hybridization and micro-

complement fixation, provide information solely in the form of numerical or
94 Interpretive Tools

quantitative distance values between taxa (see Springer and Krajewski, 1989).
Most other molecular approaches provide raw data in the form of qualitative
character states-electromorphs, restriction sites or fragments, or nucleotide
sequences. The latter can be converted to quantitative estimates of genetic dis-
tance, if so desired, but the converse is not true; that is, qualitative character
states cannot be recovered from distance data alone. This distinction between
qualitative and distance data is important for two fundamental reasons. First,
many biological applications (e.g., forensic identification, parentage determina-
tion, kinship assessment, gene flow estimation, and the characterization of hy-
brids) require qualitative genetic markers, whereas other applications such as
phylogenetic estimation can employ either qualitative or distance data. Second,
several phylogenetic or tree-building algorithms (e.g., parsimony and cladistic
analyses) require qualitative data, whereas other algorithms utilize matrices of
genetic distances among taxa.
The concept of genetic distance is fundamental to molecular systematics. A
genetic distance between two sequences, individuals, or taxa is a quantitative
estimate of how divergent they are genetically. The units of distance depend on
the nature of the molecular information summarized. For example, values of
Nei's (1972) D for protein electrophoretic data are interpreted as the net numbers
of codon substitutions per locus that have accumulated since separation of any
two populations and can be either corrected or uncorrected for presumed multiple
substitutions ("hits") at the same amino acid site. An analogous measure for
DNA restriction or sequence data is p, the estimated number of base substitutions
per nucleotide (or percent sequence divergence if uncorrected for multiple hits).
Genetic distance (~Tm) values from DNA-DNA hybridization have no immedi-
ate molecular interpretation beyond the measured difference in thermal stability
per se, although additional independent information may permit a calibration of
~Tm to magnitude of sequence divergence. Similarly, the immunological dis-
tance (ID) values from microcomplement fixation are, at face value, merely
descriptors of antigen-antibody cross-reactivity (although again, with additional
information they may be calibrated to numbers of amino acid substitutions).
Definitions of some of the standard distance statistics for various types of mo-
lecular genetic data are summarized in Box 4.1.
The converse of genetic distance is "genetic similarity. " Thus, when genetic
distance is low, genetic similarity is high, and vice versa. In the early literature
of molecular evolution, it was customary to refer to "percent homology" be-
tween DNA sequences or other molecular characters under comparison. This
practice now is discouraged. The word homology properly refers to organismal
features (such as genes) that trace to a shared ancestral condition, so that
sequences are either homologous or they are not. (Complications can arise,
however, when a complex gene or genome contains sequence elements of
both homologous and nonhomologous origin). In any event, truly homologous
Box 4.1. lxamples of Genetic Distance Statistics
AIIozymes
For protein-electrophoretic data, the two most commonly employed distance mea-
sures are as follows:
(a) Rogers' (1972) distance. For a given locus with m alleles, let Xi and Yi be the fre-
quencies of the ith allele in populations X and Y, respectively. Rogers' D is defined as
D = [0.5 ~(Xi - Yi)2]0.5, (4.1)
where the summation is over all alleles. When data from more than one gene are
considered, the arithmetic mean of such values across loci provides the overall genetic
distance estimate. Rogers' D can take values between zero and one; Rogers' similarity
S = 1 - D.
(b) Nei's (1972) standard genetic distance (see also Nei, 1978). At any locus, Nei's
"genetic identity" (similarity) is defined as
1 = ~J'i / (Y; ~y/)0.5. (4.2)
For mUltiple loci, the overall similarity or identity is
1 = J xy / (J)(.9°. 5 , (4.3)
where Jx ' Jy , and J xy are the arithmetic means across loci of Lx/, "Ly/, and Lxi Yi'
respectively. Nei's I can range from zero to one. Nei's standard genetic distance then
is calculated as
D = -In I. (4.4)
This D, which can take values between zero and infinity, is interpreted as the mean
number of codon substitutions per locus, corrected for multiple hits.
Many such distance estimates for protein-electrophoretic data have been proposed
(see Nei, 1987) and their relative merits in various applications debated at length.
However, for particular data sets, most distance measures are correlated highly (al-
though they may differ in magnitude, especially at larger values).
DNA restriction fragments or sites
(a) Restriction fragments. Upholt (1977) was the first to derive the relationship
between the proportion of fragments shared in the mtDNA digestion profiles of two
taxa, and an estimate of their nucleotide sequence divergence. Let N x ' Ny, and N xy be
the number of restriction fragments observed in sequences X and Y and shared by X
and Y, respectively. The overall proportion of shared fragments is calculated as
(4.5)
Then the number of base substitutions per nucleotide (or, approximately, the percent-
age of nucleotides substituted) is estimated by
p = 1 - [0.5( -F + {F2 + 8F}0.5)]l!r, (4.6)
where r is the number of base pairs in the enzymes' recognition site. The value of p
must be computed separately for enzymes recognizing four-, five-, and six-base se-
quences, and the final distance value is the average of these estimates (weighted by the
total numbers of fragments produced by the respective enzyme classes). Using a
slightly different approach, Nei and Li (1979) derived a relationship between F andp
essentially identical to that of Upholt.
 (b) Restriction sites. For "site" data, let N x • Ny. and N xy be the number of sites
observed in sequences X and Y and shared by X and Y, respectively. The overall
proportion of shared sites is calculated as
S = 2Nxy 1 (Nx + Ny). (4.7)
Then the number of base substitutions per nucleotide is estimated by
P = -InS 1 r. (4.8)
Values of p again must be calculated separately for enzymes cleaving at four-, five-,
and six-base recognition sites and the overall distance computed as the weighted mean
of these values.
Nucleotide sequences
Only the simplest case will be considered, in which sequences of the same length
can be aligned without ambiguity. Let Zd be the number of nucleotides which differ
between two sequences, and z, be the total number of nucleotides compared. Then the
percent sequence difference is simply
(4.9)
For sequences exhibiting little divergence, p is a close approximation to the accumu-
lated number of nucleotide substitutions per site because no correction is needed for
mUltiple substitutions at a site. Particularly when sequence divergences are larger,
corrections for multiple hits have been suggested. One simple correction was provided
by Jukes and Cantor (1969):
D = -3/410(1 - 4pI3). (4.10)
This equation was derived under the assumption that at any nucleotide position,
substitutions occur with equal probability to any of the remaining nucleotides. Other
corrections have been developed by varying these assumptions, for example by al-
lowing different rates for transitional versus transversional substitutions as in the
widely employed "two-parameter" model of Kimura (1980).
Note that unlike the allozyme genetic distances that are based on population allele
frequencies and, hence, provide distances between populations or species, these latter
estimates apply to sequence divergence between particular genes or alleles. If the se-
quences come from haploid individuals (as is effectively true for uniparentally inherited
cytoplasmic genomes), the calculated values also can be interpreted as between-indi-
vidual distances at that locus. When many such sequences within a population are
assayed, mean genetic distance [or nucleotide diversity (Box 2.1)] then is estimated by
mean p = '£fdj P;j (4.11)
where/; andfj are the frequencies of the ith andjth sequences in the sample, andpu
is the estimated sequence divergence between the ith and jth sequences (Nei, 1987).
If two or more populations have been assayed, estimates of net sequence divergence
(Pcorr) between populations also can be calculated, based on a correction for within-
population polymorphism:
Pcorr = Pxy - O.5fpx + py] (4.12)
where Pxy is the mean genetic distance in pairwise comparisons of individuals between
populations X and Y, and Px and Py are the mean genetic distances among individuals
within these respective populations. This correction assumes that sequence diversity
within the extant populations is representative of the level of variation present in the
common ancestor.
 Interpretive Tools 97

sequences among an array of organisms can exhibit a wide range of genetic

similarities, the values depending in part on how long ago the extant taxa sep-
arated from common ancestors.

Detached versus Connectible Information

Some types of molecular data can be connected readily across studies, whereas
others cannot. Good examples of connectible data are DNA sequences. Once a
nucleotide sequence is available for any gene or species, newly obtained se-
quences can be compared against the original without the need to repeat the
earlier assays. Other kinds of information are less connectible, in the sense that
data from one study may be impossible to link directly with similar data from
others. For example, a !1Tm value (from DNA-DNA hybridization assays) be-
tween the genomes of species A and B is of no immediate service in assessing
the relationship of these species to taxa C and D for which another !1Tm value
may be available. In contrast, DNA sequences from these same four species
could be used to estimate a phylogeny, irrespective of when or where the se-
quences were determined.
The distinction between connectible versus detached data is not the same as
that between qualitative versus distance information. For example, protein elec-
trophoresis provides qualitative genotypic data, but the electromorphs them-
selves are distinguished by gel mobilities relative to one another. Thus, it is
difficult to compare the particular electromorph genotypes reported in one study
with those of another, unless shared standards had been employed in both.
Another point is that a connectibility for data does not necessarily imply an ease
of phylogenetic analysis and, indeed, can create special difficulties due to the
combinational properties of such information. Assays for DNA sequencing, for
example, have become so proficient that "Methods of data acquisition have
vastly outstripped current systems of data management" and "contemporary
algorithms for phylogenetic and evolutionary analysis are not equal to the tasks
presented by sequence data sets now in hand" (Clegg and Zurawski, 1992).
Thus, the phylogenetic opportunities provided by connectible data also pose
profound challenges in the development of appropriate analytic methodologies.

Single-Locus versus M ultilocus Data

As normally applied, some molecular techniques (such as microcomplement

fixation and DNA sequencing) entail the acquisition of data from individual loci,
whereas others (such as DNA-DNA hybridization and multilocus fingerprinting)
inherently access genetic information from multiple gene regions (Fig. 4.1). This
distinction is important because the amount of genetic information assessed
influences the interpretations to be drawn from data. A major advantage of
98 Interpretive Tools

DNA-DNA hybridization approaches is that the estimated !1Tm 's reflect an av-
erage across the thermal stability properties of multitudinous sequence regions;
and the individual-diagnostic power of multilocus DNA fingerprinting stems in
part from independent assortment among the multiple dispersed arrays of repet-
itive elements.
For most applications involving genetic markers, the number of functional
genes assayed is less important than the number of linkage groups represented,
which influences how many independent bits of phylogenetic information are
revealed. For example, animal mtDNA is composed of some 37 functional
genes, all transmitted as a nonrecombining unit primarily through female lines.
Thus, from a phylogenetic perspective, the entire 16-kb mtDNA molecule is only
a single "gene."
Multilocus assay methods can be categorized further into those which assess
information from multiple loci simultaneously (e.g., DNA-DNA hybridization
and multilocus DNA fingerprinting) versus sequentially (multilocus protein elec-
trophoresis and assays of scnDNA RFLPs) (Fig. 4.1). Only the latter normally
provide information interpretable in simple genetic terms (i.e., as Mendelian
genotypes at particular loci). Although the number of genes included in such
allozyme and scnDNA assays typically is small or moderate, even a handful of
interpretable genetic polymorphisms considered in aggregate can provide re-
markable power in applications such as forensics, parentage assessment, gene
flow estimation, and the characterization of hybrids.

Utility of Data Along the Phylogenetic Hierarchy

Another way to slice the molecular-techniques pie is with regard to the level
of evolutionary separation at which the various methods can be applied (Box
4.2). Most assay methods provide an empirical window of opportunity that is
fairly narrow relative to the broad field of potential phylogenetic applications.
For example, microcomplement fixation and DNA-DNA hybridization methods
are suitable for phylogenetic studies at intermediate taxonomic levels, when
species' separations date to approximately 2-100 million years ago (mya). At
shorter divergence times, genetic distances estimated by these methods tend to be
small and not significantly different from zero. At the microevolutionary end of
the continuum, multilocus DNA fingerprinting is highly appropriate for ques-
tions of genetic identity versus nonidentity and parentage. Studies of RFLPs
from mtDNA and scnDNA have been most fruitful at levels of conspecific
populations and closely related species, as have allozyme surveys. Among the
available molecular methods, only DNA sequencing can find application at vir-
tually any taxonomic level. This flexibility stems from the fact that different
DNA sequences evolve at highly different rates (see the next section), such that
studies can be tailored around the choice of appropriate sequence elements.
Box 4.2. Levels of Evolutionary Divergence at Which Various Molecular Genetic Methods Normally
Provide Informative Phyogenetlc Markers (Modified from Hlms and Moritz, 1990).
RFLP Analyses of
Hierarchical Protein Protein DNA-DNA DNA
Level Immunology Electrophoresis Hybridization mtDNA scnDNA VNTR Loci Sequencing

Genetic identityl * * * ** *
nonidentity
Parentage * * ** ** *
Con specific ** ** ** * *
populations
Closely * ** * * * *
related species
Intermediate ** * ** **
taxonomic levels
Deep separations * * **
(>50 mya)

(**)--highly infonnative; (*)--marginally infonnative, but not an ideal approach for reasons of cost-ineffectiveness or other difficulties;
(-)--inappropriate use of method. Not all categorizations are absolute. For example, some isozyme characters such as presence/absence of
duplicate gene products can be useful at higher taxonomic levels.
100 Interpretive Tools

Nonetheless, because of labor and expense, obtaining DNA sequences from

large numbers of individuals and genes in a population context is not particularly
cost-effective, and sequencing studies usually are conducted at intennediate or
higher levels of the phylogenetic hierarchy.
Of course, not only the molecular method but also the amount of genetic
infonnation obtained influence the resolution obtainable in a given application.
For example, restriction-site or sequencing studies of animal mtDNA often in-
volve assay of about 500 nucleotide pairs per individual. At the conventional
sequence divergence rate of 2% per million years (see next section), roughly 1
of 500 base pairs is expected to change after 100,000 years of maternal lineage
separation, thus establishing 100 millenia as an approximate lower limit of
resolving power for de novo mutations with this level of effort. If the entire
16,OOO-bp mtDNA genome could be assayed, the ability to detect a single
nucleotide change would be increased by more than 30-fold, suggesting a lower
limit on resolving power of just a few thousand years. However, these statements
refer only to the accumulation of novel mutations and not to significant shifts in
allelic frequencies of ancestral polymorphisms (which could take place in as little
as a single generation by genetic drift, natural selection, or migration).

MOLECULAR CLOCKS
General Concepts

Zuckerkandl and Pauling (1965) were the first to propose that various proteins
and DNA sequences might evolve at constant rates over time, and thereby
provide internal biological timepieces for dating past evolutionary events. The
concept of a molecular clock fits well with neutrality theory because, as dis-
cussed in Chapter 2, the rate of neutral evolution in genetic sequences is equal
simply to the mutation rate to neutral alleles. However, clock concepts are not
necessarily incompatible with selectionist scenarios: If a large number of assayed
genes was acted on by muItivarious selection processes over long periods of
time, short-tenn fluctuations in selection intensities might tend to average out
and the magnitudes of overall genetic distance between isolated taxa could well
be correlated strongly with the time elapsed since common ancestry.
Few concepts in molecular evolution have been more debated (or abused) than
those concerning molecular clocks. At the outset, three general points must be
understood. First, the debate is not whether molecular clocks behave metronom-
ically, like a working timepiece-they do not. If molecular clocks exist, both
neutralists and selectionists predict at best a "stochastically constant" behavior,
like radioactive decay (Ayala, 1982c; Fitch, 1976). Second, not all molecular
phylogenetic applications hinge critically on the reliability of molecular clocks.
For example, genetic characters likely to be of monophyletic origin (such as gene
 Interpretive Tools 101

duplications or differences in gene arrangement) remain powerful as phyloge-

netic markers, regardless of the rate of evolution at the level of DNA sequence;
and, appraisals of genetic identity and parentage (e.g., by DNA fingerprinting)
hardly depend on a steady pace for DNA sequence evolution. Many tree-building
algorithms based on genetic distance matrices relax assumptions of rate homo-
geneity among lineages (see the next section), and indeed branching orders, in
principle, can be inferred directly from distributions of qualitative character
states using techniques such as cladistic or parsimony analyses that remain valid
irrespective of whether molecules evolve in strictly time-dependent fashion.
Nevertheless, data and concepts pertaining to molecular clocks are of funda-
mental importance in many phylogenetic contexts.
A third point about molecular clocks is that, from an empirical standpoint,
different DNA sequences unquestionably evolve at markedly different rates (see
the reviews in Li and Graur, 1991; Nei, 1987; A.C. Wilson et al., 1987). These
rate heterogeneities are apparent at several levels: (a) across nucleotide positions
within a codon [mean rates for synonymous substitutions normally are several
times higher than those involving nonsynonymous changes in protein-coding
regions (Figs. 4.2a,b)]; (b) among nonhomologous genes within a lineage [non-
synonymous rates can vary by orders of magnitude, as between the slowly
evolving histones and rapidly evolving relaxins (Fig. 4.2a)]; (c) among classes of
DNA within a genome [e.g., introns and pseudogenes evolve more rapidly than
do nondegenerate sites in protein-coding genes (Fig. 4.2c)]; and (d) among
genomes within an organismallineage (e.g., synonymous substitution rates in
cpDNA are several-fold lower than those of plant nuclear genomes, and verte-
brate mtDNA evolves on average about 5-10 times faster than does most single-
copy nuclear DNA). Under neutrality theory (Kimura, 1983), such rate hetero-
geneities across nucleotide sites, genes, and genomes within a phylogenetic
lineage are interpreted to reflect varying intensities of purifying selection asso-
ciated with differing levels of functional constraint on DNA sequences, perhaps
in conjunction with a variation in the underlying rate of mutation to strictly
neutral alleles (Britten, 1986). Ironically, these kinds of extreme rate heteroge-
neities can be highly beneficial for phylogenetic studies, by permitting choice of
appropriate DNA regions geared to the time-scale requirements of a particular
phylogenetic problem (Box 4.2). For example, slowly evolving sequences for
ribosomal RNA genes have been extremely informative in reconstructing deep
branches in the tree of life (Chapter 8), whereas rapidly evolving mtDNA se-
quences have revolutionized phylogenetic studies of animals at the intraspecific
level (Chapter 6). Within a given molecule such as mtDNA, the more slowly
accumulating replacement substitutions and transversions may be most informa-
tive at the levels of species, genera, or families, whereas rapidly accumulating
silent substitutions and transitions often provide numerous markers for the study
of local populations (Bowen et al., 1993a).
102 Interpretive Tools

a) nonsynonymous
rate
~ .(
Ii
...II> .... 10
8 - c
]
iii
c

.. '"
a. II> aI c 0 0
,.., 6 .., C :;; ii ~
c: :;;
" 0
0 0
... 4 •c .£
-;
0

. 0....
.!
I;
.2- .!c
0
.""
~ ~
0 E
-; ~
a. 2
:c .£ E

.
.c
:I 0

b) synonymous rate

..'2
.(
iii
!
.
c c
c
'f
-c
c

0 c :;; :;;
Ii 10 OJ ".
.c ""
~" .!!-
0
...co :c "" 0.
0
~ 8 ...E
.""
0
a. -= .! "0
..
01
,..,
II>
6
c
c:
0 '"0... 4 Figure 4.2. Observed rates of nucleotide
~;; ;;;
a. 2 substitution in various genes and gene re-
.
.c
:I 0 gions (generated from the information in Li
and Graur, 1991). (a) and (b) Nonsynony-
mous and synonymous substitution rates,
respectively, in eight protein-coding genes
c) average rate sequenced from both humans and rodents.
. .!
c(
zQ
The rate calculations are based on the as-
sumption that observed sequence differ-
;;

..
"c0
';;,
" .!
;; ..
~.
E ences accumulated over 80 million years .
(c) Average rates of substitution in differ-
!
c
0
0.
~
. . . .!
c
... ent parts of these and other genes. Nonde-
. ..,.
.! ~
t!
.
Ii ~ ;; DI
generate nucleotide sites are those at which
.....
10 C
c
:; ...
II
e DI
all possible substitutions are nonsynony-
a. 8
...'"
..."
01 0

II
,.., II>
6 mous; at fourfold degenerate sites, all pos-
c:
~
'"0
4 "" sible substitutions are synonymous, and at
~ ;;; twofold degenerate sites, one of three pos-
;; a. 2
sible nucleotide changes is synonymous
.
.c
:I 0 and the other two are nonsynonymous .
 Interpretive Tools 103

Clock Calibrations and Controversies

In discussions of phylogeny reconstruction, a more controversial form of rate

heterogeneity involves possible differences in the evolutionary tempo of homol-
ogous DNA sequences across organismal lineages. For dating cladogenetic
events, approximate uniformity in such evolutionary rates clearly would be de-
sirable (A.C. Wilson et. aI., 1987) and, indeed, numerous "universal" rate
calibrations have been suggested for a variety of genes and assay methods.
For example, a conventional calibration for evolutionary rate of animal
mtDNA [derived from the slope of the linear portion of the divergence curve
(Fig. 4.3A)] is about 2% sequence divergence per million years between pairs of
lineages separated for less than 10 million years, or 1 x 10- 3 substitutions per
site per year per evolutionary line (Brown et aI., 1979). In referring to mtDNA
in higher animal taxa, Wilson et ai. (1985; see also Shields and Wilson, 1987a)
concluded that "no major departures from this rate are known for the molecule
as a whole." However, this conclusion is now controversial for the following
empirical reasons. First, the ratios of mean mtDNAlscnDNA divergence rates
have been shown to differ significantly between animal groups (Caccone et aI.,
1988a; DeSalle et aI., 1987; Powell et aI., 1986; Vawter and Brown, 1986), and
whether the rate heterogeneity is attributable to variation in mtDNA, scnDNA,
or both is unclear. Second, different nucleotide positions and genes within
mtDNA evolve at varying rates within a lineage (Brown et aI., 1982; Gillespie,
1986; Moritz et aI., 1987) and particular mtDNA genes (such as cytochrome
oxidase) reportedly show rate differences as high as fivefold across taxa (Brown
and Simpson, 1982; Crozier et al., 1989). Third, recent studies have reported
significant mean mtDNA rate heterogeneities across lineages of Hawaiian Dro-
sophila (DeSalle and Templeton, 1988), mammalian orders (Hasegawa and
Kishino, 1989), vertebrate classes (Avise et al., 1992a; Bowen et aI., 1993a),
and homeothermic versus heterothermic vertebrates (Kocher et al., 1989). The
argument has been raised that the pace of mtDNA evolution among animal
groups may be linked causally to differences in metabolic rate and/or to the
generation-length and body-size differences with which metabolic rate is nega-
tively correlated (Avise et al., 1992a; A.P. Martin et al., 1992; Thomas and
Beckenbach, 1989). A related suggestion involves the concept of "nucleotide
generation time"-the time elapsed between episodes of DNA replication or
repair. The idea is that nucleotide positions with higher turnover (shorter repli-
cation intervals) are subjected to more mutational opportunities per unit of si-
dereal time; species with higher metabolic rates and briefer organismallife spans
tend to have shorter nucleotide generation times and, therefore, may have higher
absolute rates of molecular evolution (Martin and Palumbi, 1993).
Examples of reported calibrations for other putative molecular clocks are
summarized in Fig. 4.3. Again, controversies surround the validity and univer-
104 Interpretive Tools

.,---r----r-----,.-----" 0 0
gg 60
. . .; mtDNA
..
:s .-...
'"
., 1 50 r---r----,.------,.-------,

~ ~
1 .
100
CDQ)-40 ·•.. 0 80:;:::",_:-
o ..
~~ 20 V··~~~;........ c: §~ 50
rn.:! - ........ 11 60 ~ .:.. E 'C
'if!. '0 ········--~u_ ... _.
o 40 #. E
o 20 40 60 80 15 30 45 60
Divergence time (mya) Divergence time (mya)
A B

0.75
.,... 90

'.-"5! .
c:: C

.. - .''0;"
0.50
c 60

", Z
::>.- 30
E'C
0=..--<"'---'---'-----' 8 16 24 32
o 30 60 90 120
C Divergence time (mya) Divergence time (mya)
D

30r--,---,--r--,
scnDNA .,......., 30

I- 20
hybrids ., c::.,
c:: 20

<l
10
.,::>", .,'">
cr~
10 ribosomal
:.!! .- RNA
o 'C
o~~~-~--'---~
o 15 30 45 60 500 1000 1500 2000
Divergence time (mya) Divergence time (mya)
E F

Figure 4.3. Examples of "clock calibrations" reported for various types of molecular
genetic data. All dates along the abscissa came from fossil or biogeographic evidence. (A)
Solid line: mitochondrial DNA sequence divergence for various mammals (after Brown,
1983). The slope of the linear portion of this curve gives the conventional mtDNA clock
calibration of 2% sequence divergence per million years between recently separated
lineages (note that beyond about 15-20 million years, mtDNA sequence divergence
begins to plateau, presumably as the genome becomes saturated with substitutions at the
variable sites). Hatched line: percentage observed transitions for various mammals (open
squares, after Moritz et aI., 1987) and Drosophila (closed squares, after DeSalle et aI.,
1987). (B) Albumin immunological distances (as estimated by microcomplement fixation)
for various carnivorous mammals and ungulates (after Wilson et aI., 1977). (C) Accu-
mulated codon substitutions in seven proteins (cytochrome c, myoglobin, (1- and l3-he-
moglobin, fibrinopeptides A and B, and insulin) for various mammalian species [after
Langley and Fitch (1974) and Nei (1975)]. The three squares below the solid line involve
primate comparisons. (D) Codon substitutions per locus (Nei's D) based on allozyme
comparisons for carnivores (dots) and primates (squares) (after Wayne et al., 1991a). (E)
!1T values from DNA-DNA hybridizations involving carnivores (dots) and primates
(squares) (after Wayne et al., 1991a). (F) Percent sequence divergence in the 16S ribo-
somal RNA gene for various eubacterial forms: (a) cyanobacteria; (b) chloroplasts; (c)
microaerophiles; (d) mitochondria; (e) obligate aerobes; (f) Photobacterium; (g) Rhizo·
bium and Bradyrhizobium; and (h) Escherichia (after Ochman and Wilson, 1987). The
wide horizontal bars indicate considerable uncertainty about the actual divergence times
from nonmolecular evidence. The slope of the line is drawn arbitrarily to represent an
evolutionary rate of 1% sequence divergence per 50 million years.
 Interpretive Tools 105

sality of such results, particularly as applied across taxonomic groups. The

inherent appeal of molecular clocks appears to have led to some egregious
claims. For example, the early protein-electrophoretic literature conveyed the
strong impression that allozyme distances could be used reliably and accurately
to date speciations; many papers concluded that observed genetic distances were
consistent with the suspected separation times for particular species as gauged by
nonmolecular evidence. But different authors were employing (perhaps unwit-
tingly) allozyme rate calibrations that differed by more than 22-fold from one
another! (see the review in Avise and Aquadro, 1982.) Given such a huge range
of potential "clock" calibrations in the literature, it is difficult to imagine any
observed allozyme distance that could not be accommodated with a given fossil-
based scenario! Ironically, if these researchers individually were correct that a
molecular clock was ticking within each taxonomic group, then collectively no
single allozyme clock could apply across these same taxa.
Ayala (1986) summarized evidence for the erratic behavior of particular pro-
tein clocks across lineages, as did Britten (1986) and Brunk and Olsen (1990) for
the overall rate of scnDNA sequence divergence as gauged by DNA-DNA hy-
bridization. Some investigators have concluded that although mean molecular
rates in the nuclear genome may vary among taxa, they do so in a predictable or,
at least, a consistent fashion. For example, molecular evolution arguably appears
slower in primates than in rodents and appears especially slow in the hominoids
(Goodman et al., 1971; Koop et al., 1989; Li and Tanimura, 1987; Li et al.,
1987; Maeda et aI., 1988; but see also Caccone and Powell, 1989; Easteal, 1991;
Kawamura et al., 1991; Sibley and Ahlquist, 1987). Several of the published
interpretations are similar to those presented earlier for mtDNA. Thus, for avian
and mammalian species, Sibley and colleagues [long-term advocates of the con-
cept of a "uniform rate of DNA evolution" (Sibley and Ahlquist, 1984)] pre-
sented recent evidence that short generation time or increased number of germ-
line cell divisions may be associated with a higher mean rate of scnDNA
sequence evolution (Catzeftis et aI., 1987; Sibley et al., 1988). More generally,
apparent variation in nuclear substitution rates among taxa has been attributed to
differences in generation lengths (Gaut et aI., 1992; Kohne, 1970; Laird et al.,
1969; Li et aI., 1987), numbers of DNA replications in germ-line cells (Wu and
Li, 1985), repair efficiencies during DNA replication (Britten, 1986), or degree
of exposure to mutagens [including the free radicals whose DNA-damaging
effects appear correlated with metabolic rates among species (Adelman et al.,
1988)].
On the other hand, some researchers consistently have maintained that sidereal
time is the best predictor of genetic divergence and that molecular clocks can be
calibrated universally across organismal groups (Wilson, 1985). As stated by
Wilson et ai. (1987): "Molecular evolutionary clocks have ticked at much the
same rate per year in many eubacterial genes as in the nuclear genes of animals
106 Interpretive Tools

and plants." They also propose an intriguing hypothesis for this conclusion,
based on the following assumptions: (a) most nucleotide substitutions involve
neutral mutations; (b) the mutation rate per year is higher in short-generation
organisms; (c) the fraction of mutations that is effectively neutral is lower in
larger populations (because of the greater impact there of deterministic forces
including natural selection); and (d) species with shorter generations tend to have
larger populations. If these speculations hold, a greater mutation rate in short-
generation species might be counterbalanced by a lower fraction of effectively
neutral mutations, such that overall evolutionary rates remain relatively constant
among diverse taxa.

Absolute and Relative Rate Comparisons

In any event, how are molecular rates assessed, and how is it that the heated
debates about rate heterogeneity have continued for so long without final reso-
lution? One difficulty is that molecular-distance measures often become nonlinear
with time at various evolutionary depths (e.g., Figs. 4.3A,D), and some argu-
ments against molecular clocks have stemmed from appraisals involving inap-
propriate regions of divergence curves. Another problem involves the difficulty
of determining confidence limits for genetic-distance estimates (Nei, 1987). Thus,
any distance in Fig. 4.3 is merely a point value with estimation errors of various
types. Another aspect of statistical concern is whether, for a given molecule, the
mean of substitution rates among lineages is equal to the variance, as predicted
under neutrality theory for a Poisson-like process. Several studies have concluded
that the empirical variance in substitution rates slightly exceeds the mean (Langley
and Fitch, 1974; Ohta and Kimura, 1971), and this has been interpreted as
evidence against a uniform clock (Gillespie, 1986, 1988; Takahata, 1988).
Perhaps the most serious difficulty in the calibration of molecular distance
against sidereal time is that firm independent knowledge from fossil or biogeo-
graphic evidence is required also. Unfortunately, such information is insecure or
lacking for most taxa (if this were not true, there would be little motivation for
molecular phylogenetic appraisals!). For example, all separation dates in Fig.
4.3 came from the fossil record and the range in estimates of divergence time is,
in some cases, extremely wide (e.g., Fig. 4.3F). Fossils seldom provide the solid
anchor of separation times that molecular biologists sometimes suppose-pre-
served remains often are scanty and confined to a few phenotypic attributes
whose phylogenetic relevance is suspect. These problems are especially acute for
morphologically simple creatures like bacteria.
Biogeographic evidence also can be difficult to interpret, even in the cleanest
of instances. To cite one example, it is well documented that the Isthmus of
Panama rose about three million years ago and must, therefore, have curtailed
any former gene flow between tropical marine faunas in the eastern Pacific and
western Atlantic oceans. Today, the green turtle (Chelonia mydas) is distributed
 Interpretive Tools 107

circumtropically and shows a distinct break in mtDNA phylogeny that distin-

guishes all assayed populations from the Atlantic versus Pacific (Bowen et al.,
1992; Chapter 6). However, the magnitude of mtDNA sequence divergence
between these clades is only Pcorr = 0.006, a value tenfold lower than expected
under the postulated three million years of separation, provided the mammalian
mtDNA evolutionary rate of 2% sequence divergence per million years applies
(Fig. 4.3A). Perhaps mtDNA evolution in green turtles is slower than in mam-
mals by an order of magnitude (Avise et al., 1992a). Alternatively, turtle
mtDNA might evolve at a more usual pace, but Atlantic and Pacific populations
were in genetic contact more recently (perhaps through dispersal of green turtles
around South America or South Africa during Pleistocene interglacial periods
when climates may have been warmer than now). Similar genetic studies (of
allozymes and mtDNA) have been conducted on several Atlantic-Pacific gemi-
nate species pairs of fishes and sea urchins separated by the Panamanian Isthmus,
with conflicting interpretations drawn regarding possible heterogeneity in evo-
lutionary rates of homologous genes (Bermingham and Lessios, 1993; Grant,
1987; Lessios, 1979, 1981; Vawter et al., 1980).
To circumvent the uncertainties of the species' divergence dates from fossil or
biogeographic evidence, molecular evolutionists also employ "relative-rate"
tests that do not depend on knowledge of absolute divergence times (Sarich and
Wilson, 1973). Each test requires at least two related species (say A and B) and
an outside reference species (C) known to have branched off prior to the sepa-
ration of A and B. The rationale is illustrated in Fig. 4.4a. By definition, the true
evolutionary distance between A and B (dAB) is equal to the sum of their branch
lengths from a common ancestor at point 0 (Le., dOA + daB)' Similarly,
or rearranged, dOA = d Ac - doc (4.13)

and
or rearranged, daB = d BC - doc (4.14)

(a) (b) (c)

o
A B c A B c A B c
Figure 4.4. Rationale (a) and potential difficulties (h,c) for the relative rate test (see text).
108 Interpretive Tools

Subtracting Eq. (4.14) from Eq. (4.13) yields

d OA - dOB = d AC - d BC (4.15)
According to the molecular clock, d OA and dOB should be equal (dOA - dOB
= 0) and, hence, d AC = d BC ' Genetic distances d OA and dOB cannot be mea-
sured empirically, but d AC and d BC can. The relative-rate test asks whether d AC
and dBC as estimated by a particular molecular approach are statistically different
and, hence, incompatible with the molecular clock hypothesis.
Many relative rate tests for molecular data have been conducted (see the review
in Li and Graur, 1991). For example, from their DNA-DNA hybridization
studies, Sibley and Ahlquist (1986) report that "genetic distances between the
outlier and each of the other species ... are always equal, within the limits of
experimental error" and "thousands of such trios of species ... yield the same
result and attest to the uniform average rate of the DNA clock in birds." Rats,
mice, and hamsters also have passed the relative-rate test (Li et aI., 1987; O'hUi-
gin and Li, 1992). On the other hand, failures to pass the test contributed to the
conclusion that nDNA sequences evolve more rapidly in rodents than in primates
(Li et aI., 1987; Wu and Li, 1985) and that certain cpDNA sequences evolve more
rapidly in annual angiosperms than in other plants (Bousquet et al., 1992).
The relative-rate test is not without difficulties, as illustrated in Fig. 4.4. If A
and B separated very recently compared to C (Fig. 4.4b), d AC and d BC may
appear equal in an empirical test even under a highly erratic clock (because the
vast majority of evolution has taken place in the long and shared OC branch).
Conversely, if the separation of A and B was close to that of the common
ancestor with C (Fig. 4.4c), an incorrect assumption about the hypothesized
branching order of the three species might exist, such that d AC and d BC could
appear different even under a perfectly constant clock. Thus, errors both of false
acceptance and false rejection of evolutionary clocks can be envisioned in par-
ticular tests of relative rate.
Closing Thoughts About Molecular Clocks

What can be concluded from the immense effort expended on assessment of

molecular clocks? It is now undeniable that some and probably most molecular
systems evolve at heterogeneous rates across at least some taxa. Thus, if precise
clocks exist, they are local rather than universal. Yet the time elapsed since
common ancestry (whether calibrated in absolute time units or generation lengths
is uncertain) remains perhaps the single best predictor of molecular divergence,
especially when genetic distance is measured across large numbers of loci. What
justifies this bold statement? The evidence is mostly indirect, inconclusive in
individual instances, but compelling cumulatively: (a) the nodes in numerous
molecular phylogenetic trees generally have proved consistent with independent
time estimates, insecure as these may be (e.g., Fig. 4.3); (b) evolutionary rates
 Interpretive Tools 109

for molecules proceed mostly independently of those for morphology and other
phenotypic attributes, where rates can vary wildly under the influence of differing
selection regimes; and (c) given current understanding of the mechanistic basis
of molecular evolution (particularly at the level of DNA sequence), it would be
most surprising if mean genome-wide genetic distances did not increase with time.
In many instances, molecular timepieces with less than full precision none-
theless can provide significant improvement over phylogenetic understanding
gained from nonmolecular data. Consider for example the single-celled mi-
crobes, whose phylogeny was totally unknown prior to the application of mo-
lecular information. Phylogenetic patterns in ribosomal RNA genes and other
loci have revealed stunning genetic relationships and subdivisions among mi-
crobial taxa, including those that early in evolution entered into endosymbiotic
relationships with protoeukaryotic cells (Chapter 8). Molecular clocks keep far
from perfect time, but to dismiss the time-dependent properties of molecular
evolution out of hand would be to deny access to an invaluable and sometimes
sole source of temporal information.

PROCEDURES FOR PHYLOGENY RECONSTRUCTION

Phylogenetic trees are graphical representations consisting of nodes (taxo-

nomic units) and branches (pathways connecting nodes) that summarize the
evolutionary relationships among organisms (Fig. 4.5). In most studies, the

A
B
C

c E
D

F F
L....J
1 DISTANCE
UNIT TIME---~~~

Figure 4.5. Alternative representations of a phylogenetic tree for six extant OTUs.
Left: Unrooted network with scaled branches. Right: Rooted tree with unsealed
branches (the root is the heavy line and the tree is oriented along a temporal axis).
Internal nodes are indicated by black dots. Note that branch angles have no meaning-
branches may be rotated freely about any internal node without materially affecting
tree topology.
110 Interpretive Tools

operational taxonomic units (OTUs) are species or higher taxa, such that the tree
topology can be characterized properly as nonanastomotic. In some cases, the
OTUs also may be well-isolated conspecific populations, individuals, or nonre-
combined alleles of a gene. The external nodes in a phylogenetic tree represent
extant OTUs, and internal nodes are the ancestral units. Peripheral branches lead
to external nodes, and interior branches connect internal nodes. Branch lengths
reflect the number of evolutionary changes along each ancestral-descendant path-
way. If the genetic distance between any two OTUs is equal to the sum of all
branch lengths connecting them, the tree is said to be strictly additive. Depar-
tures from additivity provide one measure of the degree of distortion in phylog-
eny estimation due to homoplasy in the data (or perhaps to improper behavior of
the distance measures or phylogenetic algorithms employed).
Phylogenetic trees may be graphed in several ways (Fig. 4.5). A tree is scaled
when branch lengths are drawn proportional to the numbers of genetic changes;
otherwise, it is unscaled (although branch lengths may be indicated numerically
along the diagram). A tree is rooted when an internal node is specified that
represents the common ancestor of all OTUs under study; otherwise, it is un-
rooted and commonly referred to as a network. A tree is bifurcating when two
immediate descendant lineages come from each node, and multifurcating when
three or more lineages do so.
One important class of applications for molecular data is estimation of phy-
logenetic trees. The process is challenging for several reasons. First, even small
numbers of OTUs can be connected into astronomical numbers of different trees,
only one of which actually is correct. For nOTUs, the number of different
bifurcating rooted trees (NTr ) possible is given by
N Tr = [(2n - 3)!]/[2n - 2(n - 2)!] (4.16)
and the number of bifurcating unrooted trees (NTu) for n ~ 3 is
N Tu = [(2n - 5)!]/[2n - 3(n - 3)!] (4.17)

(Felsenstein, 1978). Thus, the number of possible trees increases rapidly as the
number of taxa increases, and even the small value of n = 10 yields N Tr =
34,459,425 and N Tu = 2,027,025. Many phylogenetic algorithms work by
searching among trees for those that exhibit desirable properties according to
some specified optimality criterion (e.g., shortest total branch length under par-
simony). Unfortunately, when the number of OTUs is more than about a dozen,
it is usually impractical for even the fastest computers to examine all possible
trees, and various truncated search procedures must be implemented. A second
difficulty is that any molecular reconstruction is a function both of the genetic
data themselves and of the distance measures and phylogenetic algorithms ap-
plied to them, and these influences can be difficult to tease apart. Finally, true
phylogeny seldom is known with certainty from independent evidence (however,
 Interpretive Tools 111

Table 4.1. Hypothetical distance matrix for five OTUs (see text)

A B C D E
A 0.08 0.19 0.70 0.65
B 0.17 0.75 0.70
C 0.80 0.60
D 0.12
E

see Atchley and Fitch, 1991), so appraisals of phylogenetic methods normally

rest on indirect evidence. The net result of these difficulties in evaluating alter-
native tree-building approaches is that much scope has existed for argumentation
over which method of phylogenetic reconstruction is "best."
What follows are brief descriptions of some of the algorithms most commonly
employed in molecular phylogenetics. These may be divided usefully into quan-
titative (distance) methods versus qualitative (character-state) approaches
(Avise, 1983b). Extended treatments of these and other phylogenetic procedures
(including "maximum likelihood," which is beyond the current scope) can be
found in Felsenstein (1982, 1988), Sneath and Sokal (1973), Swofford and Olsen
(1990), and references therein.

Distance Approaches

All distance-based approaches begin with an OTU x OTU matrix, the body
of which consists of estimated pairwise genetic distances between taxa. For n
OTUs, there are n(n - 1)/2 pairwise distances (excluding "self" comparisons
along the matrix diagonal). Clearly, because of phylogenetic connections among
taxa, such estimates cannot be treated as independent values from a statistical
perspective. Indeed, the historical interconnections that the distances reflect are
the primary focus. In Table 4.1 is presented a hypothetical distance matrix for
five OTUs (10 pairwise comparisons) that will provide a basis for illustrating
various distance algorithms for tree construction.

UPGMA CLUSTER ANALYSIS (SNEATH AND SOKAL, 1973)

Cluster analyses group OTU s according to overall similarity or distance and are
the simplest methods computationally. The clustering procedure most commonly
employed is the "unweighted pair group method with arithmetic averages"
(UPGMA), which operates as follows. A distance matrix (such as in Table 4.1)
is scanned for the smallest distance element, and the OTUs involved are joined
at an internal node drawn in an appropriate position along a distance axis (Fig.
4.6a). In our example, OTUs A and B are joined first, at distance level d = 0.04
(because the sum of lengths of branches connecting A and B is the observed d =
0.08). This distance element in the matrix is then discarded. The matrix is scanned
112 Interpretive Tools

A
B
C
D
(a)
E

.40 .30 .20 .10 o

GENETIC DISTANCE

A C D
(b)
.05 ).05/09 ;>'
.03 .55 .01 E
B

"
A D

(c) x
1.
y
04

z
;Y
.10 .60

B/.: -~E
Figure 4.6. Evolutionary trees produced by alternative algorithms
applied to the genetic distance matrix in Table 4.1. (a) UPGMA
dendrogram. (b) Scaled neighbor-joining (N-J) network [a Fitch-
Margoliash (F-M) network proved nearly identical to this N-J tree
for these data]. (c) Distance Wagner network, drawn as unsealed
but with distances indicated along branches. The latter network
also exemplifies a common graphical presentation in which inter-
nal nodes are labeled. In this instance, note how rooting along the
Y-Z branch and appropriate rotation of branches can cause the
Wagner, N-J, and F-M networks to resemble closely the UPGMA
dendrogram.
 Interpretive Tools 113

again for the smallest remaining distance, which in this case is d = 0.12 joining
D and E. These OTUs are clustered at level d = 0.06. The next smallest distance
in the matrix is d = 0.17 between C and A. However, A is already part of a
previously formed cluster with B, so C cannot be joined directly to A but rather
must be connected through the A-B internal node. This clustering level is de-
termined by the arithmetic mean of the distances between C and the OTUs in the
previous cluster [d = (0.19 + 0.17)/2 = 0.18]. Thus, C joins the A-B group
at d = 0.09. All that remains is to join the A-B-C cluster with the D-E cluster.
The level of joining is determined from the mean of all pairwise distances between
the OTUs in these clusters [d = (0.70 + 0.65 + 0.75 + 0.70 + 0.80 + 0.60)/6
= 0.70]. Note that the final cycle of clustering in UPGMA exhausts all remaining
distance values in the matrix. The final "tree" (UPGMA figures usually are
referred to as phenograms or dendrograms) is shown in Fig. 4.6a.
Several points about UPGMA should be clarified. In each cycle of the clus-
tering procedure, OTUs or previously formed clusters are grouped according to
the smallest mean distance between the taxa involved (rather than smallest single
distance element in the original matrix). Each OTU contributes equally to the
calculation of these mean distances (hence the term "unweighted"). All extant
OTUs are depicted as "right justified" along the genetic distance axis (Fig.
4.6a), such that the branches from a node are in this respect unscaled. The
dendrogram is rooted implicitly, at the point where the last (i.e., deepest) clus-
ters join. Finally, in some UPGMA presentations, the numerical scale of the
distance axis is "doubled" (i.e., the scale in Fig. 4.6a could be drawn equiva-
lently with respective distances 0,0.20,0.40,0.60,0.80), such that the distance
values refer to the joint (rather than single) branch lengths. The particular con-
vention on distance axes that has been followed in a given study can be checked
easily by reference to the original distance matrix.
The major assumption of UPGMA clustering is the equal rate of evolution
along all dendrogram branches (thUS, any true rate heterogeneity would remain
undetected in a UPGMA reconstruction). Despite this strong assumption,
UPGMA performs unexpectedly well in recovering tree topologies, particularly
branch lengths, in empirical tests using computer simulations (Nei et al., 1983;
Sourdis and Krimbas, 1987; Tateno et al., 1982). This seems to be due to the fact
that genetic-distance estimates are subject to large stochastic error and the dis-
tance-averaging aspects of UPGMA tend to reduce the effects of this error (Nei,
1987). In any event, because UPGMA groups taxa that differ least genetically
(without finer points of consideration), UPGMA is a simple and intuitively
appealing example of the phenetic approach to data summary (Chapter 2).
FITCH-MARGOLIASH METHOD (1967)

The "F-M" procedure was one of the first distance algorithms to relax the
assumption of uniform evolutionary rate, and is still widely employed. The
114 Interpretive Tools

general approach is illustrated most easily by reference to three taxa. Consider

OTUs A-C in Table 4.1, for which we wish to construct the branch lengths x,
y, and z in the following network.

A\
A B c
By definition, dAB = X + y, d AC = X + z, and d BC = Y + z. Although x, y,
and z cannot be observed directly, they may be estimated from the observable
distances among extant OTUs. From Table 4.1, x + y = 0.08; x + z = 0.19;
and y + z = 0.17. Simultaneous solution of these three equations with three
unknowns yields the desired branch lengths, as follows:
+y =
(x 0.08)
plus +z=
(x 0.19)
yields (2x + y + z = 0.27)
minus (y + z = 0.17)
yields 2x = 0.10 or x = 0.05.
The unique solutions are x = 0.05, y = 0.03, and z = 0.14. Note that these
branch lengths produce a perfectly additive tree, but this seldom is true when n
> 3 OTUs are considered. Note also that the branches x and y connecting A and
B to their immediate common ancestor can differ in length (hence the capacity
under F-M to infer rate heterogeneity). As applied to more than three OTUs, the
F-M procedure cycles through this tree-fitting process until all branch lengths are
obtained [see Fitch-Margoliash (1967) or Li and Graur (1991) for details].

NEIGHBOR-JOINING METHOD (SAITOU AND NEI, 1987)

This method (N-J) is related conceptually to cluster analysis, but also allows
for unequal rates of molecular change among branches. It does so by construct-
ing, at each step of the analysis, a transformed distance matrix that has the net
effect of adjusting branch lengths between each pair of nodes on the basis of
mean divergence from all other nodes. For example, below the diagonal in the
top matrix of Box 4.3 is a transformed distance matrix for OTUs A-E, generated
from the original matrix (Table 4.1) as follows. The modified distance between
OTUs A and B (d* = -1.03) equals the observed distance between A and B (d
= 0.08) minus the sum of the distances between each of these taxa and all others
(i.e.,0.08 + 0.19 + 0.70 + 0.65 + 0.08 + 0.17 + 0.75 + 0.70 = 3.32),
the latter quantity divided in this case by 3 (or generally by 2 less than the number
of OTUs in the matrix). Similar calculations fill out the modified distance matrix,
 Interpretive Tools 115

which then is searched for the minimum (most negative) value. In our example,
the smallest transformed distance is between D and E (d* = -1.36), so these are
joined first, through the internal "node 1." The branch lengths joining D and E
to node 1 then are calculated as shown in Box 4.3. Note that unlike UPGMA, but
as under F-M, these lengths can differ. The process continues through n - 1
cycles, with joined extant OTUs replaced by internal nodes as the distance
matrices shrink in size. The final tree for our example is shown in Fig. 4.6b.
Those interested in additional details and formal steps of the operation should
consult Studier and Keppler (1988) or Swofford and Olsen (1990).

DISTANCE WAGNER METHOD (FARRIS, 1972)

The F-M and N-J algorithms attempt to fit the distance matrix to an additive
tree, with the view that empirical distances in the matrix may be either under-
estimates or overestimates of their true values. The net effect is that some
pathways connecting OTUs in the tree are longer, and others shorter, than their
empirical distances in the matrix. For example, in the N-J network (Fig. 4.6b),
the sum of branch lengths connecting C to E (0.65) is greater than the estimated
distance in Table 4.1 (d = 0.60), whereas the sum of branch lengths connecting
C to D (0.75) is smaller than its corresponding empirical value (d = 0.80).
The distance Wagner procedure is similar, but assumes that the observed
distances are lower bounds on true values (as perhaps would be the case if
genetic distances were uncorrected for superimposed evolutionary changes).
Path lengths in Wagner trees equal or exceed corresponding observed distances.
A tree that minimizes the total of all branch lengths under this stipulation satisfies
the optimality criterion under the distance Wagner method, and various algo-
rithms for approaching this task have been proposed (Farris, 1972; Swofford,
1981; Tateno et al., 1982; Waterman et aI., 1977). Thus, searches for trees with
minimum total length under distance Wagner and related procedures bear some
analogy to qualitative parsimony approaches described later.
Under the Farris (1972) algorithm, a distance Wagner tree for our five OTUs
is generated as follows (Box 4.4). First, A and B are joined because they exhibit
the smallest genetic distance in the original matrix (Table 4.1). OTU C is added
next, to an internal node X along the A-B path, and the three branch lengths
[distances d(A, X), deB, X), and d(C, X)] are calculated as shown in Box 4.4b.
The inferred branch lengths d(D, X) and deE, X) also are calculated and stored
for later use. Then, a decision is made about which remaining OTU (D or E) next
joins the network, and to which existing pathway it should be added (A-X, B-X,
or C-X). In our case, E is joined to the C-X branch (through a newly created
internal node Y) because this inferred genetic distance is the smallest among the
possibilities (Box 4.4d). The new branch lengths [d(E, Y), d(C, Y), and d(X,
Y)] are calculated as before. The final step in our case is to join D to the network,
116 Interpretive Tools

Box 4.3. Cydlng Operation of the Neighbor-Joining Algorithm

A B C D E r rl3

A 0.08 0.19 0.70 0.65 1.62 0.54

B -1.03 0.17 0.75 0.70 1.70 0.57
C -0.94 -0.99 0.80 0.60 1.76 0.59
D -0.63 -0.61 -0.58 0.12 2.37 0.79
E -0.58 -0.56 -0.68 -1.36 2.07 0.69

Distance D to node 1 = 0.1212 + (0.79 - 0.69)12 = 0.11

Distance E to node 1 = 0.12 - 0.11 = 0.01

A B C Node 1 r rl2

A 0.08 0.19 0.62 0.89 0.44

B -0.82 0.17 0.66 0.91 0.46
C -0.75 -0.79 0.64 1.00 0.50
Node 1 -0.78 -0.76 -0.82 1.92 0.96

Distance C to node 2 = 0.64/2 + (0.50 - 0.96)12 = 0.09

Distance node 1 to node 2 = 0.64 - 0.09 = 0.55

A B Node 2 r rll

A 0.08 0.08 0.16 0.16

B -0.26 0.10 0.18 0.18
Node 2 -0.26 -0.26 0.18 0.18

Distance A to node 3 = 0.0812 + (0.16 - 0.18)12 = 0.03

Distance node 2 to node 3 = 0.08 - 0.03 = 0.05
B Node 3

B 0.05
Node 3

r is the sum of the observed distances between the OTU of that row and other extant OTUs or
nodes. All values were rounded to two decimal points.
 Interpretive Tools 117

Growing Network

~D
E

6:.L_c_ _ _ _---c:~ 0
E

A C 0
,_L ~
.. 0lil:.:....------<"./
E

A C 0
~~~----------<C'./
)-L ~
B
Box 4.4. Stepwise Operation of the farris Algorithm for
Identifying a Distance Wagner Network from the Data
In Table 4.1.
(a) Join OTUs with smallest observed distance. A B
(b) Add next OTU, create internal node, and calculate branch lengths: .08
d(C, X) = (I12) [d(C, A) + d(C, B) - d(A, B)]
= (112) [0.19 + 0.17 - 0.08] c
= 0.14
d(A, X) = d(A, C) - d(C, X)
= 0.19 - 0.14
.14
= 0.05
d(B, X) = d(B, C) - d(C, X)
= 0.17 - 0.14 A---X---B
= 0.03
.05 .03
(c) Calculate additional branch lengths:
d(D, X) = suP{[d(D, C) - d(C, X)] d(E, X) = suP{[d(E, C) - d(C, X)]
[d(D, A) - d(A, X)] [d(E, A) - d(A, X)]
[d(D, B) - d(B, X)]} [d(E, B) - d(B, X)]}
= suP{[0.80 - 0.14] = suP{[0.6O - 0.14]
[0.70 - 0.05] [0.65 - 0.05]
[0.75 - 0.03]} [0.70 - 0.03]}
= 0.72 = 0.67

(d) Determine OTU to be added next, and branch to which it is joined:

d[D(A. X)] = (1/2) [d(D, A) + d(D, X) - d(A, X)] d[E(A, X») = (112) [d(E, A) + d(E, X) - d(A, X)]
= (112) (0.70 + 0.72 - 0.05) = (112) (0.65 + 0.67 - 0.05)
= 0.685 = 0.635
d[D(B, X)] = (112) [d(D, B) + d(D, X) - d(B, X») d[E(B, X)] = (112) [d(E, B) + d(E, X) - d(B, X)]
= (112) (0.75 + 0.72 - 0.03) = (112) (0.70 + 0.67 - 0.03)
= 0.72 = 0.67
d[D(C, X») = (112) [d(D, C) + d(D, X) - d(C, X») d[E(C, X») = (112) [d(E, C), + d(E, X) - d(C, X»)
= (112) (0.80 + 0.72 - 0.14) = (1/2) (0.60 + 0.67 - 0.14)
= 0.69 = 0.565

(e) Add next OTU, create internal node, and calculate branch lengths:
d(E, Y) = (112) [d(E, X) + d(E, C) - d(C, X)]
.56 .04
= (1/2) [0.67 + 0.60 - 0.14)
= 0.565 E V ---c
d(X, Y) = d(E, X) - d(E, Y)
= 0.67 - 0.565
= 0.105
.10
d(C, Y) = d(C, E) - d(Y, E)
= 0.60 - 0.565
= 0.035 A X- - - B
.05 .03
(f) Continue to cycle through steps c--e until all OTUs are added.

d is an observed or inferred genetic distance; sup indicates choosing the maximum value from a set of numbers.
 Interpretive Tools 119

at a position determined by its smallest inferred distance to existing branches. In

general, the Farris algorithm repeats these tree-construction rules, keeping track
of internal nodes and branch lengths at each stepwise addition of an extant OTU.
The final distance Wagner network for OTUs A-E is presented in Fig. 4.6c.

COMPARISON OF DISTANCE MATRIX METHODS

Much debate has concerned which of these (or other) distance algorithms
produces the "best" tree. One basis for choice is "goodness-of-fit," a measure
of how well the inferred distances in the tree match the empirical distance values
in the original matrix. Branch lengths between OTUs read from the tree (output
distances) can be compared to input distances in the matrix by any of several
suggested statistics, including a "cophenetic correlation" (Sneath and Sokal,
1973), "percent standard deviation" (Fitch and Margoliash, 1972), or an "F"
statistic (Prager and Wilson, 1978). As an example, the latter is defined as

F = 100 ~
S
I Ii - 0i I /
~
S
Ii (4.18)

where for the s pairwise comparisons among OTUs, I and 0 are the input and
output distances, respectively. Smaller values of F indicate better fit. As might
be expected, procedures such as F-M or N-J that explicitly adjust tree branches
to achieve a fit to an additive tree generally outperform methods such as UPGMA
that do not (Avise et aI., 1980; Berlocher, 1981; Prager and Wilson, 1978). On
the other hand, the nature of the distance measure itself (e.g., whether it could,
in principle, yield an additive tree) also can affect the outcome, as can to some
extent the definition of the statistic (such as F) used to assess the agreement
between the tree and data.
A second basis for choice among distance algorithms involves degree of
congruence among trees derived from different sets of data. Because a given
array of species has a single phylogenetic topology along which all characters
have evolved, methods of data summary producing more highly congruent trees
might be judged superior (Farris, 1971). Several measures of congruence among
trees have been suggested (e.g., Farris, 1973; Mickevich, 1978). Unfortunately,
such comparisons seldom have been applied to molecular information because of
the usual absence of multiple independent data sets for a particular taxonomic
group.
A third approach for comparing the performance of phylogenetic algorithms
involves computer simulation of evolutionary change along specified model
trees. From observed genetic distances among extant computer OTUs, phylog-
enies are estimated and an algorithm's performance is evaluated by how well it
120 Interpretive Tools

recovers the known tree (Fiala and Sokal, 1985; Jin and Nei, 1991; Saitou and
Nei, 1987). Potential shortcomings of this approach are (a) the difficulty of
assessing the biological plausibility of assumptions underlying the simulated
evolution of genetic characters through the computer tree and (b) the danger of
circular reasoning when the best phylogenetic algorithm proves to be the one
whose assumptions most closely match those underlying the simulation. All
phylogenetic algorithms involve assumptions (explicit or implicit). A continuing
challenge is to identify and understand the assumptions and to assess their ap-
propriateness for the evolutionary mode of the characters under assay. Note also
that facility with implementing a phylogenetic algorithm, even by hand, is no
guarantee that the underlying assumptions will become apparent (if you do not
believe this, try working through the F-M, distance Wagner, or N-J examples
provided earlier).
A fourth and powerful method for evaluating algorithm performance was
introduced by Hillis et al. (1992). They serially propagated bacteriophage T7 in
the presence of a mutagen, experimentally dividing the culture at various time
intervals such that a known phylogeny was produced. Then the terminal lineages
were assayed for restriction-site maps and the empirical data were used to infer
the evolutionary history by various phylogenetic methods. All five algorithms
employed, which included F-M, N-J, and UPGMA (as well as a qualitative
parsimony approach-see next section), produced the correct branching order of
the known topology, but differed slightly in ability to recover correct branch
lengths. Of course, such direct appraisals of phylogenetic methods for living
organisms will be possible only in a few systems such as T7 where mutation rates
are high and thousands of generations occur each year (see Chapter 6).
Among the distance methods considered, only UPGMA automatically pro-
duces a rooted tree. To root any of the other networks, two procedures may be
followed. First, outgroup OTUs (those known from independent evidence to
have branched off earlier from other taxa under study) can be included in the
empirical analysis, in which case the root is placed between an outgroup and
the node leading to ingroup members. Alternatively, if an approximate uniform
rate of evolution is assumed for long time periods, the network may be rooted at
the midpoint of the longest pathway between any extant OTUs (note that this
assumption is less stringent than that of rate homogeneity in all tree branches).
For example, the longest pathway in the N-J network in Fig. 4.6b is of length
0.76 (between B and D). Placement of the root midway along this path produces
a rooted tree that in this case resembles closely the UPGMA dendrogram (Fig.
4.6a).
Character-Stole Approaches
Some molecular techniques provide discrete character information, such that
a data matrix can be developed that assigns a character state (Xii) to each OTU 0)
 Interpretive Tools 121

for each character (J) (e.g., Boxes 3.2 and 3.4). The types of molecular char-
acters and their states vary with circumstance and must be specified carefully in
each study. For example, in an allozyme survey, the gene for LDH could be
considered one character, with different states being the observed electromorphs;
or the character might be a particular nucleotide position in a DNA sequence,
with possible states A, T, C, or G. Such characters which can assume three or
more states are called multi state traits. Binary characters are those that can
assume only two states. For example, the presence versus absence of a RFLP
restriction site at a particular map location exhausts the two possible states for
that character. In some cases, characters might be defined at a more inclusive
level. For example, the collection of different restriction-site maps produced by
a given endonuclease, or the collection of DNA sequences for a given gene,
might justifiably be considered the multiple states of their respective characters.
Multistate characters may be unordered or ordered. Electromorphs of an al-
lozyme locus are examples of unordered character states because their evolu-
tionary interrelationships cannot be deduced directly from the observed electro-
phoretic mobilities. Similarly, the alternative states at a given nucleotide position
normally are considered unordered because there is no a priori reason to assume
a particular evolutionary pathway for the interconversion among A, T, C, and G.
On the other hand, restriction-site maps for a given enzyme (or DNA sequences
for a given gene) may be treated as ordered multistate characters when their
probable evolutionary transformations have been deduced from reasonable criteria
such as parsimony (Figs. 3.8 and 3.9). However, such character-state phylogenies
are themselves evolutionary inferences (hypotheses) derived from the application
of phylogenetic procedures to information accumulated from lower-level char-
acter-state descriptions (individual restriction sites or nucleotide positions, in
these cases). Thus, character-state matrices for most computer-based phylogenetic
algorithms consist of data coded at these more fundamental levels.
The polarity of character states refers to the direction of evolution and, hence,
is distinct from the concept of character order. Polarized characters are those for
which ancestral and descendant states have been determined. Thus binary char-
acters, and ordered or unordered multistate characters, may be either polarized or
nonpolarized. Use of inferred character polarity is essential in Hennigian cladis-
tic analysis (Fig. 2.5) but is not necessary in all forms of parsimony. What
follows are brief descriptions of the qualitative character-state algorithms most
commonly employed in molecular phylogenetics.

HENNIGIAN CLADISTICS

The philosophy underlying Hennigian cladistics was described in Chapter 2.

The critical feature is that synapomorphic character states must be identified and
that they alone provide the basis for clade identification. If there were no dis-
122 Interpretive Tools

agreements in clade delineation among putative synapomorphs and each character

state was monophyletic, a Hennigian reconstruction should capture the true
evolutionary tree. Molecular features that are likely of monophyletic origin (e.g.,
unique gene arrangements or other idiosyncratic genetic attributes) are especially
well suited for Hennigian cladistic interpretations. Unfortunately, most large
molecular (and nonmolecular) data sets include some conflicts among characters,
no doubt due in part to the possibility of polyphyletic mutational origins of
individual character states (such as the electromorphs of a locus, nucleotides at
a sequence position, or sites in a restriction map). To resolve the apparent
dilemmas in character-state distributions across taxa, parsimony approaches are
employed widely.

MAXIMUM PARSIMONY

A most-parsimonious tree is one that requires the smallest number of evolu-

tionary changes to explain the observed differences among OTUs. Consider
Figure 4.7, which presents a hand-generated estimate of a parsimony network for
10 extant OTUs based on 9 variable characters. (Hypothetical OTUs HYPI and
HYP2, not directly observed, were added arbitrarily to make all branches of unit

Stul- EcoRI*

CHARACTERS
.....
· .....
;; ;; it ue ;;
~ 0
~ ;;
OTUs c g g w ;: Z Go OJ
A c c
B d c $pel
C b c EcoRl
D Pvull
Dr.1I
E c c Avail

·•
H b d b b d
b d b b
b b b b
b d d b b
M d b d b b c

Figure 4.7. Estimate of an unrooted maximum parsimony network (right) based on an

OTU X character matrix (left) for a subset of mtDNA clones observed in green turtles
(after Bowen et aI., 1992). The lowercase letters are mtDNA digestion profiles produced
by nine restriction enzymes (letters adjacent in the alphabet denote profiles that differed
by a single restriction site). Inferred restriction site changes along branches of the network
are indicated.
 Interpretive Tools 123

length in the upper portion of the tree.) Construction of the network could be
initiated by connecting any OTU to its nearest genetic neighbors via reference to
the character-state matrix. For example, haplotype D is one mutation step re-
moved from each of three other haplotypes (A, C, and E) that are two steps
removed from one another. Haplotype B, in turn, is one step from A, two steps
from D, and three steps each from C and E. Thus, the distribution of character
states for OTUs A-E yields the singular most-parsimonious network shown at
the bottom right of Figure 4.7. Note that this portion of the network is strictly
additive.
Generation of the complete network for all 10 extant OTUs illustrates com-
plications that may arise. First, there is a large genetic gap distinguishing OTUs
A-E from the assemblage H, I, J, L, M, so where the branch connecting these
groups should be placed is not obvious initially. Here, D and HYP2 are joined
because they differ by five steps, whereas any other intergroup branches would
involve six steps or more. Second, some genetic character states appear in
different (presumably distantly related) portions of the network. For example,
the character state StuI-d appears in some representatives of both the upper and
lower OTU groups, probably due to polyphyletic origins from StuI-c. Similarly,
the state EcoRI-e appears in OTUs I and J that are not adjacent genetically as
judged by the other assayed characters. These character states contribute to
homoplasy by introducing additional steps along network branches beyond those
differentiating OTUs in the original character-state matrix. Nonetheless, in this
example, the sum of all pairwise output distances in the network (256) is only
slightly greater that the sum of all input distances (250), indicating strong good-
ness-of-fit between tree and data.
In usual practice, computer-based parsimony algorithms operate by searching
alternative trees for minimum total length. Sometimes, many trees of different
topology prove equally parsimonious or require similar numbers of steps. None-
theless, such networks constitute only a small fraction of the vast universe of
potential trees, most of which, therefore, can be eliminated from further con-
sideration.
Actually, parsimony approaches comprise a family of related methods with
varying assumptions about how character-state transformations occur. The most
commonly employed parsimony algorithms and their assumptions are as follows:

a. Wagner parsimony (Farris, 1970; Kluge and Farris, 1969). This approach
(not to be confused with the distance Wagner method) allows free reversibility of
character states in the tree, with changes in either direction equally likely (thus
alternative rootings produce no change in tree length). Characters may be binary
or ordered multistate, although transformations among multistate characters must
occur through intervening states only. The network in Figure 4.7 is based on
Wagner assumptions and includes both binary and ordered multistate characters.
124 Interpretive Tools

Fitch's (1971) modification of the Wagner approach allows direct transforma-

tions among any unordered multi state characters.
b. Dollo parsimony (Farris, 1977). This parsimony approach assumes that an
ancestral condition for each binary or ordered multistate character can be spec-
ified and that each nonancestral character is uniquely derived (multiple rever-
sions to the ancestral condition are allowed). The analysis seeks to minimize total
tree length under these conditions. In principle, Dollo parsimony should be
appropriate when probabilities of change among character states are highly
asymmetric. For example, because a mutation in any nucleotide position of a
restriction site eliminates enzyme recognition, loss of a particular restriction site
might be more likely than its gain (DeBry and Slade, 1985; Templeton, 1983,
1987). However, if derived states are polyphyletic even occasionally, Dollo
parsimony can exhibit pathologic behavior (Swofford and Olsen, 1990).
c. Camin-Sokal (1965) parsimony. This form of parsimony carries the strin-
gent assumption that all evolutionary change is irreversible. Thus, the approach
goes beyond Dollo parsimony by disallowing reversions to the ancestral condi-
tion. The method is not employed widely with genetic data because most mo-
lecular characters probably violate this assumption.
d. Generalized parsimony (Swofford and Olsen, 1990). In effect, all parsi-
mony methods (including Wagner parsimony) make assumptions about the
"costs" of transformation among character states. A generalized parsimony
approach allows flexibility in assignment of these costs among characters, ideally
utilizing independent evidence about the relative frequencies of different kinds of
molecular changes. Understandably, such approaches are expensive computa-
tionally, and usable algorithms still are in their infancy.

Conclusions About Phylogenetic Procedures

For the practicing empiricist, what recommendations can be made concerning

this plethora of phylogenetic algorithms? Of course, it is important to attempt a
match between assumptions of the phylogenetic procedure(s) and the nature of
evolution for the molecular characters under assay. Second, it generally is ad-
visable to attempt multiple methods of data analysis, particularly if these entail
philosophically distinct approaches. For example, both UPGMA clustering (a
distance-based method epitomizing the phenetic philosophy) and Wagner parsi-
mony (a character-state approach more akin to cladistic thought) reasonably can
be applied to many molecular data sets and the results compared. Generally,
most of the significant features in a given data set (e.g., major genetic breaks
reflected as long internodal distances) are revealed irrespective of the phyloge-
netic algorithm employed, whereas problematic differences among trees tend to
involve topology shifts where OTUs or internal nodes are bunched closely.
 Interpretive Tools 125

Third, at least one character-state approach should be included when the data
pennit. Because tree topologies derived from qualitative approaches provide
explicit inferences about character-state distributions along branches, they are
extremely rich in empirical content (sensu Popper, 1968); in other words, they
offer testability and accountability (Avise, 1983b; Baverstock et al., 1979; Patton
and Avise, 1983). Consider again Figure 4.7, where character-state changes are
specified explicitly along branches. Such detailed evolutionary understanding is
not possible with distance analyses alone. Furthennore, if portions of the tree
topology are suspect by external evidence, the problem areas and the characters
responsible can be identified. Perhaps the offensive characters were scored in-
correctly or mistransferred during data analysis. Perhaps they arose polyphylet-
ically (an important observation in its own right), in which case the character states
might be subjected to further examination (sequencing in this case) to assess the
molecular basis of the apparent convergence. The point is that further hypothesis
testing flows readily from explicit accounts of character-state distributions.
Fourth, it is highly desirable to include confidence statements about putative
clades revealed in a phylogenetic reconstruction. One common approach is boot-
strapping (Felsenstein, 1985a; Hedges, 1992), which involves resampling (with
replacement) from the existing data sets and assessing the frequency with which
particular groups or clades appear in trees generated from the resampled data.
Thus, bootstrapping indicates how well various groupings of OTUs are sup-
ported by existing data (though not necessarily how well the available data
represent un sampled genetic characters). Finally, use of outgroups is to be en-
couraged because this allows rooting of the tree and helps to establish character
state polarities.
Because of the large size of most molecular data sets, computer programs are
required to calculate population genetic parameters and distance matrices and to
implement phylogenetic algorithms. The software packages employed most
commonly are PHYLIP (available from Joseph Felsenstein, University of
Washington, Seattle), PAUP (written by David L. Swofford and distributed by
the Illinois Natural History Survey, Champaign), BIOSYS-l (Swofford and
Selander, 1981, also available from the Illinois Natural History Survey),
FREQPARS (Swofford and Berlocher, 1987, available from Swofford at the
Smithsonian Institution, Washington, D.C.), Hennig86 (James S. Farris, Port
Jefferson Station, New York), and MacClade (written by Wayne P. Maddison
and David R. Maddison, and available from Sinauer, Sunderland, MA). Brief
descriptions of these programs can be found in Swofford and Olsen (1990).
Before closing this section, it should be emphasized that all phylogenetic
algorithms discussed earlier are based on two fundamental assumptions: that the
characters are (a) homologous and (b) independent. The concept of independence
warrants elaboration. Characters are independent in a mechanistic sense if
changes in one character occur independently of those in another, such that the
126 Interpretive Tools

character states do not co-vary because of pleiotropic effects in the underlying

mutational process. For example, restriction fragment gains and losses tend to
co-vary across digestion profiles, whereas the responsible site changes do not,
and for this reason it is preferable (when possible) to code RFLP data as pres-
ence/absence of restriction sites rather than fragments (or at least to accommo-
date the covariance of fragments in the phylogenetic analysis). The assumption
of independence, critical to most computational algorithms, is probably valid for
most molecular characters (unlike the situation for many morphological traits)
and indeed is a major strength of multicharacter molecular approaches.
However, there is another sense in which some molecular characters may be
partially nonindependent evolutionarily. When molecular characters are tightly
linked, as is usually the case for restriction sites or sequence data at a particular
locus, molecular states tend to co-vary in transmission across organismal gen-
erations (barring intragenic or interallelic recombination). Some molecular sys-
tems such as animal mtDNA presumably are free of recombination almost en-
tirely. Thus, although such character states are independent in the mechanistic
sense of mutational origins, they may not elucidate independent transmission
pathways through an organismal pedigree. Recognition of this fact has contrib-
uted to the fundamental distinction, now recognized widely in molecular phy-
logenetics, between a "gene tree" and an organismal phylogeny (Avise, 1989a;
Doyle, 1992; Neigel and Avise, 1986; Pamilo and Nei, 1988; Tajima, 1983;
Tateno et al., 1982; Wilson et al., 1985).

GENE TREES VERSUS SPECIES TREES

When OTUs are the alleles of a locus (e.g., haplotype sequences or restriction-
site maps), a reconstructed phylogeny represents a gene tree. Any group of
organisms has a single pedigree that extends back through time as an unbroken
chain of parent-offspring genetic transmission, but due to the nondeterministic
nature of biparental Mendelian heredity in sexually-reproducing species, not all
genes will have trickled through this organismal pedigree in identical fashion.
Thus, gene trees are likely to differ somewhat from one (unlinked) locus to the
next (Ball et aI., 1990). Furthermore, a gene tree may differ in topology from that
of the population tree or species tree through which it has been transmitted [even
in the absence of introgressive hybridization (Chapter 7)], due to an inevitable
process called "stochastic lineage sorting" that may be exemplified as follows.
Consider a single population pedigree through which haplotypes have de-
scended. A simple case conceptually involves mtDNA inherited through female
lines, but the principles apply to haplotypes of particular nuclear genes as well.
As shown in Figure 4.8, some females by chance leave no daughters (those
mtDNA lineages terminate), whereas others produce one or more daughters that
may contribute mtDNA to successive generations. Thus, as an inevitable con-
 Interpretive Tools 127

J EARLIER HISTORY]

1
:
lll ll l
::: :: :

en
z
o
-<C
I-
10
a:
w
z
w
G

20
Figure 4.8. The allelic lineage sorting process within a popUlation. Shown is an
mtDNA gene tree through 20 generations, where each node represents an individual
female, and branches lead to daughters. The tree was generated by assuming a
Poisson distribution of progeny numbers with a mean of one daughter per female
(after Avise, 1987).
128 Interpretive Tools

sequence of differential organismal reproduction, the mtDNA gene tree is con-

tinually "self-pruning"-certain branches are lost as others proliferate. At equi-
librium, the expected frequency distribution of times to common ancestry can be
approximated (Box 2.3). In general, it is unlikely that two or more founding
lineages by chance will survive beyond 4N generations, where N is the popula-
tion size (Fig. 4.9).
Now consider the lineage sorting process extended to two daughter taxa (A
and B) stemming from a common ancestral population. With regard to a gene
tree within these sister populations or species, three phylogenetic categories are
possible (Fig. 4.10): (I) reciprocal monophyly, in which case all alleles within
each sister taxon are genealogically closer to one another than to any heterospe-
cific alleles; (II) polyphyly, where some alleles in each taxon are genealogically
closer to heterospecific alleles than to homospecific alleles; and (III) paraphyly,
in which case all alleles within one daughter taxon are one another's closest
relatives, whereas some alleles in the second taxon are genealogically closer to
heterospecific alleles. The first category (Fig. 4.10, case I) illustrates how the
depths of gene trees can vary even when their branching topologies agree with
the species tree (e.g., the alleles in B trace to an ancient node b whereas the

1.0

0.8
n 10,000
0.6
1,000
~ 65
0.4 10

0.2

0.0
1
generations
Figure 4.9. Probabilities (1T) of survival of two or more founding lineages through time.
Shown are probability curves for populations of various size in which females produce
daughters according to a Poisson distribution with mean l.0 (after Avise et aI., 1984a).
 Interpretive Tools 129

I II III

A B A B A B

Figure 4.10. Three phylogenetic relationships possible between two sister taxa (A and B)
with respect to an allelic genealogy (after Avise et al., 1983). Lowercase letters point out
important ancestral nodes to which extant alleles or haplotypes trace. The solid dark bars
indicate barriers to reproduction (extrinsic or intrinsic). The phylogenetic categories in the
gene tree are as follows: I, reciprocal monophyly; II, polyphyly; III, paraphyly.

alleles in A trace to a recent node a). The latter two categories of relationship
(Fig. 4.10, cases II and III) illustrate how a gene tree also can differ in funda-
mental branching order from a species tree. These discordances arise because
many allelic separations inevitably predate the species split (unless the ancestral
form went through an extreme population bottleneck just prior to speciation).
Figure 4.11 shows diagrammatically how these three categories of phylogenetic
relationship may characterize one-and-the-same pair of sister taxa at different
times following their speciation.
If genetic distances among haplotypes are measured in units of time since
common ancestry, these categories of phylogenetic relationship between sister
taxa (with respect to a gene tree) may be defined formally by the inequalities in
Table 4.2. Neigel and Avise (1986) employed computer simulations to monitor
the phylogenetic status of sister taxa with respect to mtDNA lineages (Fig. 4.12).
Shortly after speciation, the probability is high that sister taxa exhibit a poly-
phyletic gene-tree status. At intermediate times since speciation (typically N-3N
generations, where N is the population size of each sister taxon), probabilities of
poly-, para-, and monophyly are intermediate as well. Only after about 4N
generations do sister taxa appear reciprocally monophyletic with high probabil-
ity. Similar results apply to nuclear genes (Nei, 1987), although the times to
monophyly are extended accordingly because of the expected fourfold larger
effective population sizes for nuclear loci (Box 2.3).
When balancing selection maintains polymorphisms within the species, the
expected times to reciprocal monophyly in a gene tree may be extended consid-
130 Interpretive Tools

SPECIES A SPECIES B
Figure 4.11. The lineage sorting process extended to two sister taxa (after Avise and Ball,
1990). Shown are distributions of allelic lineages at a single gene through an ancestral
population subdivided at time X into two daughter populations or species. With respect to
this gene tree, the sister species are polyphyletic between time levels X and Y (follow
tracings from nodes s, t, u, and v), show paraphyly between levels Y and Z, and are
reciprocally monophyletic beyond level Z.
 Interpretive Tools 131

Table 4.2. Definitions of the phylogenetic status of two sister taxa with respect to a
gene tree they containd

Phylogenetic Phylogenetic Distance

Category Status Relationship

1 A and B monophyletic max d AA < min dAB and

max d BB < min dAB

n A and B polyphyletic max d AA > min dAB and

max dDD > min dAB

illa A paraphyletic with max dAA > min dAB and

respect to B max d DD < min dAB

nib B paraphyletic with max d AA < min dAB and

respect to A max d BB > min dAB
aAfter Neige1 and Avise, 1986. Maximum distances within either taxon (max dAA or max doo) versus
minimum distance between sister taxa (min dAB) are the deciding criteria (see Fig. 4.10 and text).

100

P 50

"""...'"11..."......,

o
o 200 400 600 800

generations
Figure 4.12. Probabilities of reciprocal monophyly (I), polyphyly (II), and paraphyly
(ill) for two sister taxa G generations following a simulated speciation (after Neigel
and Avise, 1986). In each of 100 replicate computer runs, the daughter species were
founded by 20 and 30 individuals respectively and allowed to grow rapidly to
carrying capacity N = 200.
132 Interpretive Tools

erably beyond those expected under the neutral model. Much recent interest has
attended the discovery of two such apparently balanced polymorphisms that have
persisted for millions of years and across several speciation events. These in-
volve the major histocompatibility loci in rodents and primates (Figueroa et aI.,
1988; Lawloret aI., 1988; McConnell et aI., 1988; Takahataand Nei, 1990), and
the self-incompatibility locus in Solanaceae plants (Ioerger et aI., 1990).
Discordances between species-splitting patterns and the topologies of gene
trees also can characterize taxa that separated anciently but whose speciations
occurred close together in time (Fig. 4.13). The same kinds of lineage sorting
processes are responsible; in this case, lineages from the polymorphic ancestral
gene pool that happen to have reached fixation in the descendent taxa may by
chance be those that produce a gene-tree/species-tree discordance (Takahata,

(a) (b)

-----t 1------+

- - - - t 2 - - - -..

A B c A B c
Figure 4.13. Gene genealogies across ancient nodes close in time. Shown are two
topologically distinct gene trees (dark lines) possible within a species tree consisting
of two sister taxa (A and B) and an outgroup (C). In diagram (a), the gene tree and
species tree have the same branching pattern, whereas in (b) the branching topol-
ogies differ. For neutral alleles, the probability of the discordance exemplified by
diagram (b) is given by ¥3e- TJ2N• (Nei, 1987), where T = tl - t2 (t 1 is the time of
the first speciation and t2 is the time of the second speciation), andN" is the effective
population size.
 Interpretive Tools 133

1989; Wu, 1991). In the primate literature, intense debates have concerned
which "true" tree characterizes the phylogeny for human, chimpanzee, and
gorilla, a related triad of species whose branching pattern appears to consist of
two closely spaced nodes like those in Figure 4.13. Many molecular assays have
been applied, but not all agree in outcome (Chapter 8). Perhaps with respect to
gene trees, no single outcome should be expected.
These perspectives stemming from molecular research reveal several points of
qualitative importance to phylogenetics, beyond the immediate fact that gene trees
and species trees can differ in branching topology. First, with regard to relation-
ships in particular molecular (or other) characters, the phylogenetic status of a
given pair of species is itself an evolutionarily dynamic characteristic, with a usual
time course subsequent to speciation being polyphyly~paraphyly~monophyly
(Fig. 4.12). Second, the phylogenetic status of species is a function both of the
pattern of population splitting and of the historical demography within the pop-
ulations involved (Avise et aI., 1984a). Thus, for example, species with a larger
effective population size (Box 2.2) will tend to retain polyphyletic or paraphyletic
status for longer times than will species with smallNe , all else being equal. Third,
in accounting for the appearance of "heterospecific" alleles within a species, it
now is apparent that possibilities involving lineage sorting from an ancestral gene
pool must be considered in addition to the usual scenarios of interspecies transfer
mediated via introgressive hybridization (Chapter 7).
Because of its rapid pace of evolution, "haploid" packaging within most
organisms, and nonrecombining mode of transmission, mtDNA has provided the
vast majority of empirical data suitable for estimating gene trees over micro-
evolutionary time scales. In principle, data from nuclear loci can be exploited
similarly, but two major difficulties arise. First is the technical problem of
isolating individual haplotypes of a locus from diploid organisms. Box 4.5
describes several genetic systems or experimental approaches where this diffi-
culty might be circumvented. A second potential complication, particularly at the
intraspecific level, is intragenic recombination (or gene conversion). Such shuf-
fling of genetic material among alleles, if frequent over the time scales relevant
to a genealogical reconstruction, will obscure the otherwise linear evolutionary
histories of particular haplotypes within a species (Hudson, 1990). Patterns of
nonrandom association (disequilibrium) among tightly linked polymorphic mark-
ers can help to reveal how frequently recombination may have occurred in the
history of a gene region (Clark, 1990; Stephens, 1985). Nonetheless, for secure
recovery of a gene genealogy, attention normally must be confined to DNA
segments with little or no recombination (Box 4.6).
Gene trees and species trees are equally "real" phenomena, merely reflecting
different aspects of the same phylogenetic process. Thus, occasional discrepan-
cies between the two need not be viewed with consternation as sources of
"error" in phylogeny estimation. When a species tree is of primary interest,
Box 4.5. Some Special Genedc Systems and Potendal
Approaches for the lsoladon of DNA Haplotypes.
Approach Rationale Comments Example Reference
mtDNA Most individuals By far, most widespread Avise, 1989a
effectively source for gene-tree
homoplasmic and data
haploid for mtDNA;
nonrecombining
genetic transmission
Sex chromosomes (e.g., Heterogametic sex is Few genes as yet Bishop et al. 1985;
X or Y in mammals, haploid; limited identified or surveyed Vulliamy et al. 1991
Z or W in birds) recombination,
particularly in Y or
W chromosomes
Species with Males are haploid in No known attempts as Hall, 1990
haplo-diploid sex many hymenopteran yet for full gene trees
determination insects
Species with prominent For example, No known attempts as McDermott et al., 1989
haploid phase of life gametophyte stage of yet for full gene trees
cycle mosses is haploid
Species with prominent Endosperm in seeds of Does not apply to
haploid tissue gymnosperms is a angiosperms, where
haploid product endosperm is triploid
(gametophyte) from
the female parent
Haploid species Haploid microorganisms Relatively few attempts Nelson et al., 1991
should be suitable,
provided sexual
reproduction and
recombination are
limited
DNA amplification in Gametes are haploid; In principle, single H. Li et al., 1988;
vitro (PeR) from each molecule gametes or molecules Boehnke et al., 1989;
single gametes, or represents one can be isolated by Ruano et al., 1990
single DNA haplotype serial dilution
molecules
DNA amplification in Cloning passes DNA Relatively laborious Scharf et al., 1986
biological vectors through a bottleneck
of one molecule
Extraction of individual Use of inbred strains, or Methods most readily Aquadro et al., 1986,
chromosomes of controlled crosses available in 1991
producing individuals Drosophila
with chromosomes
identical by descent;
especially powerful
when applied to
genes within
chromosome
inversion systems
where recombination
is limited or absent

When such genetic systems also exhibit limited recombination, they could provide ideal opportunities for
construction of gene trees.
 Interpretive Tools 135

Box 4.6. Intraspecific Gene Trees for Nuclear Locl

Most early attempts to study nuclear gene trees at the intraspecific level involved Dro-
sophila species because experimental crosses can be conducted readily to "extract" indi-
vidual chromosomes from diploid fruit flies taken from nature, thereby generating strains in
which particular chromosomal homologues are "identical by descent" (Box 4.5). Figure
4.14 shows one such genealogy estimated for ADH haplotypes extracted from D. melano-
gaster.
Construction of a gene genealogy requires a history of limited or no recombination
among homologous haplotypes over the time scales of the phylogeny. It has proven difficult
to predict which gene regions are likely to be sufficiently free of such recombination. For
example, similar attempts to construct an intraspecific allelic phylogeny for ADH in a
related fruit fly D. pseudoobscura have been thwarted by an absence of strong non-random
associations among linked restriction sites, apparently due to a history of higher interallelic
exchange (Schaeffer and Miller, 1992; Schaeffer et al., 1987). The xanthine dehydrogenase
region in D. pseudoobscura also shows few nonrandom associations among restriction sites
(Riley et al., 1989), as do several assayed loci other than ADH in D. melanogaster [e.g.,
the notch, white, zeste-tko, and perhaps amylase gene regions (Aguade et al., 1989a;
Langley and Aquadro, 1987; Langley et al., 1988; Schaeffer et al., 1988)]. Many demo-
graphic as well as molecular factors can influence the history of effective recombination
within a gene region. For example, the allelic contents of species that are highly subdivided
spatially should tend to exhibit greater disequilibrium than those of "high gene flow"
species in which the alleles are routinely brought together such that recombination among
them at least is possible. Different chromosomal regions also are known to differ inherently
in recombination rates, with likely consequences extending to several aspects of genome
structure including patterns of intragenic marker associations (Aguade et al., 1989b; Begun
and Aquadro, 1992).
When stretches of DNA with limited recombination can be identified, the gene geneal-
ogies that they imply might be used to map phenotypic mutations (Templeton et al., 1992).
The rationale for this endeavor is that any such mutations linked with the nonrecombining
marker region under study would be embedded within the same evolutionary history that is
represented by the allelic cladogram.
Aquadro et al. (1991) have capitalized on the recombination-suppression properties of
chromosomal inversions in D. pseudoobscura to generate a phylogeny for an amylase gene
(Amy) that is contained within the inverted region of the third chromosome. The reduction
in effective recombination in inversion heterozygotes is dramatic and occurs because crossing
over inside the inverted region normally produces dysfunctional duplication and deficiency
products that are shunted to polar bodies where they fail to participate in zygote formation
(and Drosophila males lack recombination). The significance of recombination suppression
in this system is that the maintenance oflinked and presumably adaptive complexes of genes
within the inverted region is facilitated. A gene tree based on restriction site maps for 28
Amy haplotypes is presented in Figure 4.15, from which the following major conclusions
emerged (Aquadro et al., 1991): (a) restriction-site differences are considerably greater
among the various gene arrangements than among haplotypes within the same gene ar-
rangement; (b) the gene phylogeny based on Amy appears concordant with the inversion
phylogeny generated independently from cytologic considerations; and (c) from application
of a molecular clock, the inversion polymorphism may be about two million years old.
136 Interpretive Tools

fast
1a

fast
10f
=
=
=
=
slow
=
=
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
,.

=
,.

\~ i'-------,
slow slow
1a 2 3 4 5 5f 11a

Figure 4.14. Gene tree for alcohol dehydrogenase haplotypes in Drosophila

melanogaster (after Aquadro et al., 1986 as redrawn by Avise, 1989a). The
haplotype labeled "slow" in the largest box serves as an arbitrary reference
in which the numbers refer to the standard states for 15 restriction sites and
other characters arrayed in the 5' to 3' direction of the ADH gene. Each
genetic variant from the reference is represented by a lowercase letter.
"Slow" and "fast" refer to a replacement substitution (character 10) that
underlies two common protein electromorphs at the ADH locus. The pre-
sumed transformations among character states can be read cumulatively
through the network. For example, haplotype "slow 5f lla" differs from
the reference in genetic states for characters 2, 5, and 11. Lines crossing
branches of the network indicate numbers of character-state changes inferred
along a path. The haplotype at the lower left represents a probable interal-
lelic recombinant between the two haplotypes to which it is connected by
paths. From this phylogeny and additional genetic evidence, it was surmised
that the "fast" allozyme genotype represents a derived condition having
evolved recently from the ancestral "slow" allozyme allele (Aquadro et al.,
1986; Ashburner et al., 1979; Stephens and Nei, 1985).
 TL

2.0 1.5 1.0 0.5 0.0

% nucleotide substitutions per site

Figure 4.15. Molecular and karyotypic phylogenies in Drosophila pseu-

doobscura (see Box 4.6). Above: Gene tree for 28 molecular haplotypes
observed at the amylase (Amy) locus (after Aquadro et al., 1991). Also
indicated are the chromosomal inversion types from which these Amy hap-
lotypes were extracted: ST (Standard); AR (Arrowhead); KL (Klamath-
from D. persimilis); CH (Chiricahua); SC (Santa Cruz); and TL (Treeline).
Below: Cytogenetic phylogeny of these same gene arrangements.
138 Interpretive Tools

gene trees can assist in understanding the population demographies underlying

the speciation process, as well as the species-splitting patterns themselves (Chap-
ters 6 and 7). Of course, for such purposes, it would be desirable to include
information from multiple gene genealogies. Each gene tree also is of inherent
interest because it describes the evolutionary history of genetic changes within a
localized bit of the genome. In studies of the ages and origins of particular
adaptations (or of genetic disorders), such single-locus reconstructions become
the primary foci of attention (Figs. 4.14 and 4.15).

SUMMARY
1. The analytical methods of population genetics and phylogenetics are
applicable to molecular data, but the particular algorithms employed are multi-
various and depend both on the nature of the biological problem and the nature
of the molecular data. Molecular information can be classified into several al-
ternative but partially overlapping categories to which distinctive general meth-
ods of data analysis and interpretation pertain.
2. The concept of molecular clocks has played a major role in molecular
phylogenetics. Nonhomologous sequences are known to evolve at greatly dif-
fering rates, but considerable controversy still exists about the magnitude of the
heterogeneity in rates of homologous sequence evolution among organismal
lineages. Both absolute and relative rate tests have been employed widely in the
assessment of molecular evolutionary tempos.
3. Phylogenetic algorithms may be classified into distance-based versus
character-state approaches. Important examples of the former class include clus-
ter analyses, the Fitch-Margoliash method, neighbor-joining, distance Wagner,
and other algorithms that utilize a matrix of genetic distances between taxa.
Among the character-based methods are Hennigian cladistics and several forms
of parsimony analysis.
4. Important distinctions exist between a gene tree and an organismal phy-
logeny. These two aspects of phylogeny provide different but mutually infor-
mative perspectives on the evolutionary process.
 Part I I

Applications
 5

Individuality and Parentage

With the recognition . . . that the . . . genome is replete

with DNA sequence polymorphisms such as RFLP's, it
was only a small leap to imagine that DNA could, in
principle, provide the ultimate identifier.

E.S. Lander, 1991

Most species of sexually reproducing organisms harbor sufficient variation that

appropriate molecular genetic assays can distinguish one individual from another
with high probability. Furthermore, the transmission of genetic markers across a
single generation, as interpreted against the established rules of Mendelian in-
heritance, provides a powerful means for determining parent-offspring links.
Issues of genetic identity versus nonidentity, and of parentage (maternity and
paternity) fall at the extreme micro-evolutionary end of the phylogenetic con-
tinuum. The classes of genetic markers that have proved most suitable are those
that provide highly variable qualitative character states with known transmission
properties, e.g., allozymes and hypervariable single-locus and multilocus
RFLPs.

GENETIC IDENTITY VERSUS NONIDENTITY

Human Forensics

One of the first legal cases in the United States to admit DNA as evidence,
Pennsylvania v. Pestinikas in 1986, involved use of PeR-based assays to analyze

141
142 Individuality and Parentage

tissue samples from an exhumed corpse (Moody, 1989). The first criminal con-
viction in the United States based in part on DNA evidence came in a 1987 rape
trial (State v. Andrews, Orange County, Florida) and established a legal prece-
dent for the use of "DNA typing" to link a suspect to biological material (e.g.,
blood, semen, or hair follicles) left at a crime scene (Kirby, 1990; Roberts,
1991). By the year 1990, more than 2000 court cases in 49 states had used DNA
evidence in either civil litigations or criminal proceedings (Chakraborty and
Kidd, 1991). As with conventional fingerprinting, DNA typing merely provides
physical evidence that potentially associates victims and suspects with one an-
other or to a crime location and, therefore, must be used in conjunction with
other lines of evidence to establish guilt or innocence. Indeed, the parallels with
traditional fingerprinting appear so strong that at least one legal expert has
predicted that "DNA analysis will be to the end of the 20th century what
fingerprinting was to the 19th" (Melson, 1990, p. 189).
One illustrative example of DNA typing in a homicide case involved a mor-
tuary worker accused of murder and incineration of his estranged wife at a
crematorium in Wichita, Kansas (account in the New York Times, Nov. 21,
1988, as related by Kirby, 1990). Circumstantial independent evidence had
implicated the worker in his wife's death, but he staunchly maintained that she
had not been at the mortuary near the time of her disappearance. However,
bloodstains discovered on the side of the crematorium proved by DNA typing to
match other remaining tissue from the deceased woman. The mortuary worker
was convicted of first-degree homicide and aggravated kidnap. In another un-
usual but illustrative example of the power of DNA typing methods, Hagelberg
et al. (1991) used the PCR to amplify DNA sequences from the 8-year-old
skeletal remains of a murder victim. By comparing microsatellite DNA markers
in the remains with those of the presumptive parents, the victim's identity was
established. Not all forensic applications of DNA typing involve crimes this
macabre, but molecular genetic methods similarly have provided useful physical
evidence in numerous cases of homicide, rape, burglary, assault, hit-and-run
accidents, missing persons, and others.
Early forensic work involved typing various blood groups and serum proteins
that exhibited circumscribed genetic variability and hence provided only limited
evidence on individual identity and uniqueness. The approach now employed
most widely in human forensics utilizes RFLP data accumulated from several
hypervariable VNTR loci, assayed one at a time. Within human populations,
each such locus reveals large numbers of DNA fragments on gels [on the order
of scores or hundreds (Fig. 5.1)] that differ in length due to variations in the
numbers of the small tandem repeat units. Because any DNA fragment size is
measured with some error, the determination of distinct allelic classes from the
quasi-continuous distribution of fragment lengths is not entirely straightforward
(Devlin et al., 1992), and, in practice, various grouping procedures are em-
 Individuality and Parentage 143

160
140

-... -
0
G)
III
C
0
ca
120

100

.rJ
E ...>
G)
80

::s III 60
C .rJ
0 40
20

DNA fragment size (kb)

Figure 5.1. Frequency distribution of restriction fragments (sizes in kb) from the
D2S44 locus in Caucasian samples (after Devlin et ai., 1992). Data from Life-
codes, Inc.

ployed to pool fragments of similar length into allelic classes or "bins," the
widths of which are functions of the magnitude of experimental error in fragment
migration across replicates (Budowle et aI., 1991). Even so, each VNTR locus
employed by forensics laboratories exhibits many distinguishable alleles (usually
10-30), with most being uncommon or rare (Table 5.1). One consequence is that
the great majority of individuals appears heterozygous (Box 5.1), exhibiting two
DNA gel bands. Another consequence is that the probability of a single-locus
genetic match between randomly chosen individuals is low. As illustrated in Box
5.1, genotypic frequencies from several unlinked VNTR loci then are combined
to calculate the multilocus probabilities of observing a given DNA profile in a
random draw from the population.
Several laboratories operated by private companies or governmental units
(notably Cellmark, Lifecodes, and the Federal Bureau of Investigation) routinely
conduct DNA typing using VNTR loci. Provided the relevant tissues samples left
at the crime scene have yielded assayable DNA, two evidential outcomes are
possible: (a) the samples do not match the suspect by DNA typing, in which case
the evidence may be declared exculpatory; or (b) the tissues are declared a
match. In the western judicial tradition where a suspect is considered "innocent
until proven guilty," the latter situation clearly focuses the following question:
What is the likelihood that such a DNA match occurs by chance? At face value,
such probabilities calculated from available VNTR data are infinitesimally small
(3 X 10- 9 in the example in Box 5.1), such that matches usually are interpreted
as establishing genetic identity "beyond reasonable doubt. "
144 Individuality and Parentage

Table 5.1. Frequencies of alleles (bins) at four hypervariable VNTR

loci in Caucasians a

VNTR Locus D 1S7

Binned allele frequencies in
Frequencies in Caucasian samples at VNTR loci
Binned Caucasians Blacks D2S44 Dl7S79 D4S139
Allele (n = 605) (n = 372) (n = 802) (n= 563) (n= 460)
0.004 0.007 0.005 0.010 0.004
2 0.006 0.009 0.003 0.003 0.010
3 0.009 0.011 0.016 0.007 0.006
4 0.012 0.007 0.024 0.004 0.014
5 0.011 0.016 0.046 0.015 0.033
6 0.014 0.020 0.034 0.223 0.024
7 0.010 0.011 0.123 0.199 0.040
8 0.029 0.035 0.106 0.263 0.047
9 0.021 0.023 0.084 0.200 0.054
10 0.014 0.030 0.049 0.029 0.071
II 0.028 0.030 0.083 0.032 0.108
12 0.031 0.026 0.039 0.010 0.190
13 0.046 0.044 0.041 0.006 0.129
14 0.067 0.069 0.039 0.095
15 0.057 0.065 0.087 0.036
16 0.061 0.073 0.089 0.036
17 0.069 0.054 0.075 0.103
18 0.055 0.051 0.022
19 0.060 0.047 0.018
20 0.063 0.063 0.008
21 0.079 0.062 0.008
22 0.077 0.060
23 0.077 0.074
24 0.032 0.017
25 0.019 0.027
26 0.050 0.071
a Also shown for comparison are allele frequencies at the D IS7 locus in a sample from the Black
population. These data were introduced by the Federal Bureau of Investigation to a criminal case in
Athens, Georgia, May, 1991, and are part of a larger data base that included frequencies from
additional VNTR loci in Caucasians, Blacks, and Hispanics.
 Individuality and Parentage 145

Box 5.1. Probabilities of Single-locus and Multllocus DNA Profiles

Based on tile VNTR Data for Caucasians In Table 5.1.
(a) Probability that an individual is heterozygous at the DIS7 locus:
h = 1 - I[(0.004)2 + (0.006)2 + ... + (0.050)2] = 0.945.
(b) Examples of probabilities of particular allelic combinations at individual loci:
5/22 heterozygote at DIS7: 0.011 x 0.077 x 2.0 = 0.001694
6/8 heterozygote at D2S44: 0.034 x 0.106 x 2.0 = 0.007208
519 heterozygote at D17S79: 0.015 x 0.200 x 2.0 = 0.006000
12/12 homozygote at D4S139: 0.190 x 0.190 = 0.036100
(c) Probability of the multilocus DNA profile in (b):
0.001694 x 0.007208 x 0.006000 x 0.036100 = 3 x 10- 9 .
(d) Relevance: Suppose a crime suspect exhibited the multilocus genotype shown in
(b). Then if assumptions of the model are met, the probability of a match with
a randomly drawn genotype from the available Caucasian sample is about I in
333 million.
These calculations assume random associations of alleles within and among loci (Hardy-Wein-
berg equilibrium and gametic phase eqUilibrium, respectively).

However, these conclusions are based on particular assumptions whose va-

lidity has been questioned. Indeed, recent criticisms raised within the scientific
community have placed DNA forensic procedures themselves on trial (Lander,
1989; Lewontin and Hartl, 1991). One class of problems involves technical
matters relating to allelic discrimination, handling of samples, and the repeat-
ability of laboratory tests. Such difficulties can be alleviated by the adoption of
tighter standards for quality control, as recommended recently by a panel of the
NRC (National Research Council, 1992). Another contentious issue involves the
probability calculations for genotypic matches. The calculations exemplified in
Box 5.1 involve the potentially erroneous assumption that genotype frequencies
across loci are independent. One likely source of nonindependence is population
subdivision. Human populations are not entirely homogeneous, but rather exhibit
genetic substructure that is expected to produce allelic correlations due to non-
random mating and historical separations. (For example, alleles for blond hair
and blue eyes, though present at loci that are independent in a physiological and
transmission sense, nonetheless are highly correlated in humans due to racial and
populational histories.) On the other hand, in statistical analyses of the empirical
data bases for the VNTR loci employed in human forensics (taken from the FBI
and Lifecodes), few significant genetic correlations within or among loci have
been found (Risch and Devlin, 1992; Weir, 1992).
146 Individuality and Parentage

The effect of population substructure on forensic conclusions is a matter of

degree (Nichols and Balding, 1991). Consider an extreme example in which a
suspect belongs to a small, inbred community that differs dramatically in allele
frequency from the North American Caucasian population. Use of the Caucasian
data base (Table 5.1) as a reference for the calculation of genotypic probabilities
clearly would be inappropriate, and the direction of error could work against the
defendant. Thus, the likelihood of a genotypic match between the suspect and
another member of the local community who actually may have committed the
crime is much greater than the probability of a match within a broader popula-
tion. To circumvent this problem, each relevant human "subgroup" might be
specified separately and the appropriate probability calculations based accord-
ingly for each case. Unfortunately, such extensive genetic characterization is
infeasible logistically, even if the appropriate subgroups somehow could be
identified. Such concerns led Lewontin and Hartl (1991) to conclude that current
applications of VNTR typing have serious flaws as forensic evidence. Morton
(1992) responded that the Lewontin-Hartl (1991) objections to genotypic prob-
ability calculations are themselves "absurdly" conservative in favor of the de-
fense, and Chakraborty and Kidd (1991) conclude that "If DNA evidence is
excluded from courtroom applications, the prospect of convicting true criminals,
as well as exonerating the falsely accused, will be substantially diminished. "
The degree of human population substructure should not be overstated. In
terms of allozyme and blood-group polymorphisms, Lewontin (1972) earlier had
argued that more than 90% of overall genetic diversity in humans occurred
within (rather that between) races and concluded that "our perception of rela-
tively large differences between human races and subgroups . . . is indeed a
biased perception, and that, based on randomly chosen genetic differences,
human races and populations are remarkably similar to each other .... " Such
conclusions apply to at least some VNTR loci as well (Balazs et aI., 1989), as
illustrated by the similar spectra of allele frequencies at D1S7 in the Caucasian
versus Black populations (Table 5.1). On the other hand, whereas frequencies of
most allelic classes at VNTR loci have proved similar across ethnic groups,
allelic frequencies at some such loci do differentiate among certain human pop-
ulations (Balazs et al., 1992; Krane et al., 1992).
A variety of conservative calculation procedures can be followed that tend to
diminish bias against the defense. These include the use of wider bins for group-
ing DNA fragments and employment of observed rather than expected frequen-
cies of single-locus genotypes in the reference population (to circumvent as-
sumptions of Hardy-Weinberg equilibrium). In one case in Columbus, Georgia
(Georgia v. Caldwell), calculations based on such modifications increased the
probability of a genetic match by 1OO-fold: from 4 x 10- 8 to 4 X 10- 6 (the
suspect nonetheless was convicted). The NRC report (1992) proposed another
ingenious solution, highly conservative in favor of the defense, known as the
 Individuality and Parentage 147

"ceiling principle." They recommend that allele frequencies be estimated at all

marker loci in 15-20 human populations representing a diversity of ethnic
groups. For each allele, its highest frequency in any population or 0.05, which-
ever is higher, should then be employed for estimating the expected genotypic
frequencies against which to evaluate the genotypic profile of the suspect. This
method overestimates the expected frequency of genotypes in the reference data
base, but the effect is such that any errors introduced are in the direction of
decreasing the chances that an innocent suspect is convicted.
Fortunately, the current controversy over DNA forensic practice likely will
become moot in the near future with the adoption of tighter standards for quality
control, development of refined laboratory techniques for distinguishing alleles
(e.g., Jeffreys et al., 1991), inclusion of data from additional hypervariable loci
and more populations, and adoption of the conservative NRC guidelines for
calculating probabilities of a genotypic match.

Ramets and Genets

BACKGROUND AND CONCEPTS

Many and perhaps most species of plants and invertebrate animals reproduce
facultatively by either sexual or asexual (clonal) means (Jackson et al., 1985).
For example, the quaking aspen (Populus tremuloides) can produce sexual seeds
but also proliferates vegetatively via buds that sprout from the roots of a mature
tree. Death of the mother stem then may result in the physical disconnection of
clonemates. Clonal proliferation in other plants may involve runners, stolons,
rhizomes, bulbs, root or stem suckers, plant fragments, or even asexual (apomic-
tic) seeds that combine the advantages of cloning with wide dispersal potential
(Cook, 1980). The latter may arise when a nonmeiotic cell in the ovarian wall
initiates seed formation or when failure of a reduction division in a germ cell
lineage produces eggs with a full complement of chromosomes from the maternal
parent. In many corals such as the staghorn (Acropora cervicornis), a colony
consists of numerous asexually-derived polyps that are genetically identical to
one another and to the sexually-produced planula larvae from which they arose.
The polyps are housed jointly in a secreted calcareous skeleton that occasionally
breaks, thereby producing "daughter" colonies that are genetically identical but
physically disjunct. Some coral species also produce dispersive asexual larvae
(Stoddart, 1983a). Various mechanisms of asexual proliferation, including clonal
production of larval-like propagules, somatic fragmentation, parthenogenesis, or
production of multiple individuals by division of an early embryo or zygote
(polyembryony), are a normal part of the natural histories of many invertebrate
and some vertebrate animals (Blackwelder and Shepherd, 1981; Jackson, 1986).
148 Individuality and Parentage

As phrased by Harper (1985), "It is the nature of many plant and animal growth
fonns that the organism dies in bits and continues growth as separated parts."
In such species with clonal reproduction, challenging questions arise, such as:
What constitutes an individual? What are the units of selection (Buss, 1983,
1985)? Harper (1977) defined the genetic individual or "genet" to include all
entities (however physically organized) that have descended from a single sex-
ually-produced zygote and, hence, that are genotypically identical to one another
(barring mutation). By contrast, a "ramet" is an individual in a physical or
functional sense-a physiologically or morphologically coherent module having
arisen through clonal replication. Thus, a genet may consist of many modular
ramets, asexually derived. Many evolutionary interpretations of field data hinge
critically on the correct distinction of clonemates from nonclonemates. For ex-
ample, secure genetic knowledge of which ramets ultimately derive from the
same zygote is necessary for drawing proper inferences about (a) sex ratios
within sexual-asexual populations, (b) magnitudes and patterns of effective gene
flow, (c) degrees of outcrossing and the mating system, (d) extents of interclonal
competition, and (e) the evolutionary ages of clones (Cook, 1983, 1985).
In many cases, the breeding system of a species is unknown but asexual
reproduction is suspected. Molecular genetic markers can help settle the issue.
For example, some populations of the mustard plant (Arabis holboellii) have
characteristics suggestive of apomictic capabilities (reproduction without fertil-
ization), including the appearance of pollen and embryos with unreduced chro-
mosome number. Roy and Rieseberg (1989) confinned the occurrence of
apomixis in Arabis by showing that assayed siblings were genetically identical to
their respective parent plant at several polymorphic allozyme loci. Many marine
benthic algae release spores into the water column, but whether these are sexual
or asexual propagules remained uncertain. For one such species (Enteromorpha
linza), Innes and Yarish (1984) employed allozyme markers to document clonal
spore production. Using DNA fingerprint assays, Nybom and Schaal (1990)
detected a large number of DNA fingerprint genotypes in a population of the
predominantly sexually-reproducing black raspberry (Rubus occidentalis),
whereas a related species suspected of frequent asexual reproduction (the black-
berry, R. pensylvanicus) exhibited far fewer genotypes. On the other hand, use
of allozyme markers documented sexual reproduction in the free living amoeba
Naegleria lovaniensis (Pernin et al., 1992) and in the fungal pathogen Crumenu-
lopsis sororia (Ennos and Swales, 1987).
Many marine invertebrates brood their young, and it is of interest to know
whether these larvae are the products of sexual or asexual reproduction. Using
allozyme assays, Black and Johnson (1979) showed that brooded young of the
intertidal anemone Actinia tenebrosa were genetically identical to their parents,
indicating asexual reproduction. Similarly, Ayre and Resing (1986) documented
asexual reproduction for two coral species (Tubastraea diaphana and T. coc-
 Individuality and Parentage 149

cinea). On the other hand, in two other coral species assayed allozymically
(Acropora palifera and Seriatopora hystrix), nonparental genotypes were de-
tected in the majority of the larval broods, thus indicating reproduction by sexual
means. Clearly, brooded larvae can be produced both sexually and asexually in
various invertebrate species (Ayre and Resing, 1986).
In many animal populations, the first suggestion of parthenogenesis [whereby
progeny develop directly from an unfertilized female gamete (Soumalainen et
ai., 1976; see Fig. 5.3)] often comes from the indirect evidence of a strongly
female-biased sex ratio in nature. Clonal reproduction then may be confirmed
with genetic markers-true ameiotic parthenogens derived from a single female
are genetically uniform, barring postformational mutations (Hebert and Ward,
1972), and clonal parthenogenetic populations that arose through recent hybrid-
ization exhibit "fixed heterozygosity" at loci distinguishing the parental species
(Des sauer and Cole, 1986). For example, Echelle and Mosier (1981) used al-
lozyme evidence to confirm that a population of silverside fishes (in the Menidia
clarkhubbsi complex) reproduces by clonal means, as did Dawley (1992) for two
killifish populations that proved to have arisen through crosses between the
sexual species Fundulus heteroclitus and F. diaphanus. In the parthenogenetic
aphids Myzus persicae and Sitobion avenae, DNA fingerprint assays revealed
characteristic genetic signatures confirming suspected modes of clonal reproduc-
tion (Carvalho et al., 1991). As discussed later, molecular markers similarly
have substantiated clonal reproduction in many other invertebrates, fishes, am-
phibians, and reptiles.
The hallmark of clonal reproduction is the stable transmission of genotypes
across generations, without the shuffling effects of genetic recombination (the
only source of variation, therefore, being mutation). Thus, the term "clonal
reproduction" sometimes is used also to describe genetic transmission in strictly
self-fertilizing hermaphroditic organisms, where the intense inbreeding that char-
acterizes this reproductive mode could have resulted in near homozygosity at
most loci. Although such organisms may retain meiosis and syngamy (union of
gametes, in this case from a single parent), genetic segregation and recombina-
tion in effect are suppressed once homozygosity through inbreeding is achieved.
For example, the floridian population of the cyprinodontid fish Rivulus mar-
moratus [the only known example of a hermaphroditic vertebrate animal with
internal self-fertilization (Harrington, 1961)] exists in nature as highly homozy-
gous "clones," as gauged by intraclonal acceptance of fin grafts [indicating near
identity at histocompatibility loci (Harrington and Kallman, 1968; Kallman and
Harrington, 1964)] and by complete homozygosity at 31 genes whose protein
products were assayed electrophoretically (Vrijenhoek, 1985). Nonetheless, re-
cent DNA fingerprinting studies (B.J. Turner et al., 1990, 1992) revealed con-
siderable genetic variation that had remained undetected in the earlier assays.
This last result highlights a cautionary note that applies to studies of "clonal
150 Individuality and Parentage

diversity" in any organism-the absolute number of clones recognized can de-

pend on the discriminatory power of the assay as well as the reproductive biology
and evolutionary history of a species. Thus, it is not sufficient to be concerned
merely with the number of identifiable clones; the evolutionary relationships
among the distinguishable genotypes and the biological processes that generated
these genetic differences also must be considered.

SPATIAL DISTRIBUTIONS OF CLONES

Clearly, the geographic distribution of genotypes in sexual-asexual species

will be influenced by the relative frequencies of sexual versus clonal reproduc-
tion and the dispersive characteristics of propagules produced by these respective
reproductive modes. Early attempts to illuminate these population genetic struc-
tures utilized indirect, phenotypic criteria for clonal identifications, e.g., distri-
butions of morphological attributes or phenologies in plants (Barnes, 1966),
agonistic behaviors among sea anemones (Sebens, 1984), and histocompatibility
responses (acceptance versus rejection of tissue grafts) in marine invertebrates
and unisexual vertebrates (Cuellar, 1984; Neigel and Avise, 1983a; Schultz,
1969). Recent attempts to assign ramets to genets and to map the spatial distri-
butions of clones have involved more direct molecular genetic assays.
Plants and Fungi Maddox et al. (1989) used allozyme genotypes at four
polymorphic loci to map the microspatial distributions of goldenrod (Solidago
altissima) clones in fields of various ages. Different allozyme genotypes almost
certainly reflect sexual recruitment (via seeds) into the population, whereas the
multiple ramets of a clone reflect asexual proliferation via rhizomes. Patterns of
dispersion of goldenrod genotypes differed among the fields: Clones were local-
ized in the youngest plots (e.g., Fig. 5.2), whereas older fields exhibited higher
spatial intermixtures of clones and fewer remaining rhizomic connections among
ramets. Apparently, colonization of a field by sexually produced seeds is fol-
lowed by ramet proliferation and eventual spatial mixing of clones over micro-
geographic scales.
Other plant species may have wider clonal distributions (Ell strand and Roose,
1987; Silander, 1985). Clonal growth in the bromeliad Aechmea magdalenae
results in the spread of ramets over distances of several meters, as judged by
distributions of multilocus allozyme genotypes (Murawski and Hamrick, 1990).
Using similar allozyme approaches, Hermanutz et al. (1989) mapped genets in
the arctic dwarf birch (Betula glandulosa), a sexual species that at its northern
limit also reproduces by a process called "vegetative layering" whereby pros-
trate branches beneath the moss layer produce new ramets vegetatively. Single
clones were dispersed over areas of at least 50 m2 (the approximate sizes of the
assayed plots). In the columnar cactus Lophocereus schottii, dispersal of asexual
propagules can take place via detached stem pieces that on occasion may be
 Individuality and Parentage 151

® 0

Figure 5.2. Microspatial map (total area 0.75 m2 ) of allozymically identified clones in
Solidago altissima (after Maddox et aI., 1989). Circles denote current living ramets and
numbers indicate different electrophoretic genotypes. Larger ellipses encompass the prob-
able ramets belonging to each genet.

washed downstream by floodwaters. Parker and Hamrick (1992) found that

although most genetically identified clonemates in this species were spatially
aggregated at scales of a few meters, some widely separated individuals (> 70
m apart) had identical allozyme genotypes that might reflect such instances of
longer-distance clonal dispersal. Dispersal of asexual genotypes over large dis-
tances also has been suggested for other riparian species such as willows (Dou-
glas, 1989).
A form of apomictic reproduction in plants that could result in unusually
widespread dispersion of clones is agamospermy, the formation of unreduced
spores, seeds, or embryos by asexual processes. In the marine alga Enteromor-
pha linza, which can produce asexual spores that disperse in the water column,
152 Individuality and Parentage

particular clones (allozymically identified) were distributed over the entire sur-
vey transect of more than 150 km of shoreline (Innes, 1987). Within each of two
obligate agamospermous populations of dandelions (Taraxacum sp.), all indi-
viduals proved genetically identical at 15 allozyme loci, whereas related sexual
populations were highly diverse genetically (Hughes and Richards, 1988). How-
ever, other apomictic dandelion populations showed considerable genetic vari-
ation and coexistence of multiple clones (Ford, 1985; Ford and Richards, 1985).
Only limited genetic information exists on the broader geographic distribution of
genets within these or other agamospermous taxa (Bayer, 1989; Hughes and
Richards, 1989). One potential complication in interpreting the clonal structures
of ancient and widespread "agamospecies" is in distinguishing sexually-derived
genetic variation from that which may have arisen via postformational mutations
(Brookfield, 1992; see beyond).
According to Cook (1980), perhaps the record for size and age of a plant clone
involves the quaking aspen. Based on a distinctive morphological appearance
and spatial arrangement, one suspected genet appeared to be represented by more
than 47,000 ramets (covering 107 acres) that may trace to a single seed deposited
several thousand years ago at the close of the Wisconsin glaciation (Kemperman
and Barnes, 1976). On the other hand, looks may be deceiving. In allozyme
surveys of other quaking aspen populations, Cheliak and Patel (1984) found that
several "clones" provisionally identified by morphology actually were com-
posed of several distinct electrophoretic genotypes that probably had arisen
through recombination (and hence sexual reproduction). They concluded that
environmental influences on phenotypic appearance in aspens invalidate mor-
phological appraisals as a reliable guide to clone identification. The recent doc-
umentation of extensive genetic variation in quaking aspens as revealed by DNA
fingerprinting raises the hope that clonal identifications and distributions in this
species soon may become definitive (Rogstad et al., 1991).
Genetic documentation is available for the honey mushroom, Armillaria bul-
bosa. in which one genetic clone identified by mtDNA and nuclear RAPD
markers has been claimed as being among the largest and oldest of organisms on
Earth (M.L. Smith et al., 1990, 1992). This pathogenic fungus of tree roots in
mixed hardwood forests can spread vegetatively by cordlike aggregations of
hyphae that weave across the forest floor. Molecular genetic markers revealed
that one presumably interconnected clone of A. bulbosa in northern Michigan
had spread across 37 acres, may weigh in aggregate more than 90,000 kg (about
the size of an adult blue whale) and is perhaps some 1500 years old. A second
and smaller clone nearby covered a mere 5 acres! Recently, a report has appeared
of an even larger fungal clone (in Armillaria ostoyae) covering 1500 acres in
Washington State (Anonymous, 1992), although genetic confirmation in this
case appears to be lacking.
Invertebrate Animals Most seastars (Asteroidea, Echinodermata) can re-
 Individuality and Parentage 153

produce asexually by fission, whereby detached arms regenerate new bodies. To

determine the relative importance of fissiparity versus sexual reproduction in the
seastar Coscinasterias calamaria, Johnson and Threlfall (1987) employed allo-
zyme markers in conjunction with laboratory and field observations. Results
indicated that on local scales, clonal reproduction predominated such that many
individuals within 50 m of one another were clonemates. The importance of
sexual recruitment in this species was documented as well, through the obser-
vation of distinct clonal genotypes characterizing different regions of the study
area in western Australia. Similarly, colonies of the hard coral Pavona cactus
can proliferate by fragmentation, and an allozyme survey revealed the signifi-
cance of this asexual process in distributing clonemates over distances of up to
nearly 100 m along reefs in eastern Australia (Ayre and Willis, 1988). In the
coral Pociliopora damicornis, which produces dispersive planulae larvae by
asexual means, most clonal genotypes were clustered spatially, but nonetheless
particular clones as revealed in protein assays reportedly occurred over distances
of up to several kilometers (Stoddart, 1984a, 1984b). On the other hand, allo-
zyme surveys of the intertidal sea anemone Oulactis muscosa (a species that can
reproduce by fission) revealed a population genetic structure consistent with
recruitment almost exclusively by sexual reproduction (Hunt and Ayre, 1989). In
the sea anemone Actinia tenebrosa (a species that can produce brooded young
asexually), particular clonal genotypes as identified by allozyme assays were
distributed over hundreds of meters of shoreline in Australia (Ayre, 1984). In yet
another species of sea anemone (Metridium senile), allozyme surveys of various
populations in northeastern North America indicated marked differences among
sites in the frequencies with which sexual versus clonal recruitment had taken
place (Hoffman, 1986).
Much discussion has concerned the reliability of histocompatibility bioassays
as a guide to clonal distinctions in marine invertebrates. Within many coral and
sponge species, artificial grafts between colony branches exhibit either an "ac-
ceptance" or "rejection" reaction. Considerable indirect evidence suggests that
these respective responses signal clonal identity versus nonidentity, at least in
some species (e.g., Hildemann et al., 1977; Neigel and Avise, 1983b; review in
Avise and Neigel, 1984). However, protein electrophoretic studies conducted to
date have not fully corroborated this possibility (Curtis et al., 1982; Neigel and
Avise, 1985; Resing and Ayre, 1985; however, see Hunter, 1985). Thus, occa-
sional instances of graft acceptance between colonies differing in allozyme gen-
otype have been reported, as have instances of graft rejection between colonies
identical in genotype at a small number of moderately polymorphic allozyme loci
(Table 5.2). Nonetheless, because of the limited genetic data as well as technical
difficulties with both the field and laboratory procedures (Grosberg, 1988; Neigel
and Avise, 1985), much more attention should be devoted to the issue of how
reliably histocompatibility responses reveal clonal makeup in various marine
154 Individuality and Parentage

Table 5.2. Tissue graft response and multilocus allozyme genotype

(three polymorphic loci) in 65 assayed pairs of sponges within a local
population of Niphates erecta (after Neigel and Avise, 1985).a

Tissue Graft Response

Allozyme
Genotype Acceptance Rejection

Same D 5
(26) (12)
Different 5 32
(21) (35)

aShown in parentheses are similar results obtained for 94 assayed pairs within the
coral species Montipora dilatata and M. verrucosa. where a single polymorphic
allozyme locus was monitored (after Heyward and Stoddart, 1985). For both the
corals and sponges, the associations between tissue graft response and clonal
identity as revealed by allozyme genotype were highly significant statistically.

invertebrate species. Other traditional guides to presumed clonal identity, such as

patterns of aggression among sea anemonies, and morphotypic appearances
among colonies of corals and sponges also have been called into question from
reappraisals based on molecular genetic markers (e.g., Ayre, 1982; Ayre and
Willis, 1988; Solt~-Cava and Thorpe, 1986).
Because of their power and sensitivity, various DNA fingerprinting ap-
proaches should find wide application in studies of clonal population structure in
marine invertebrates [provided that the frequency of sexual reproduction is con-
siderably greater than the minisatellite mutation rate (Brookfield, 1992)], How-
ever, few empirical examples thus far are available. Coffroth et al. (1992)
utilized Jeffrey's and related minisatellite probes to study DNA fingerprints and
clonal structure in a gorgonian coral (Plexaura sp. A) that reproduces by frag-
mentation as well as by sexual production of dispersive larvae. Among 73
assayed colonies on 7 reefs in Panama, 29 different genotypes were identified by
these molecular methods. The only cases of identical DNA fingerprints involved
colonies located on the same reef, usually in close proximity. On two reefs, both
DNA fingerprinting and histocompatibility assays were conducted, and these
approaches revealed similar numbers of clones-17 and 13, respectively. Also
worthy of mention were experimental controls demonstrating that (a) multiple
samples from a single colony produced identical DNA fragment profiles and (b)
zooxanthellae symbiotic to the corals were not the source of the DNA bands
scored. Based on this experience with Plexaura, DNA fingerprinting would
appear to hold much promise for further genetic analyses of corals and other
invertebrates capable of clonal reproduction.
Many invertebrates can proliferate clonally by parthenogenesis. For example,
17 of 33 North American species in the earthworm family Lumbricidae exhibit
 Individuality and Parentage 155

a parthenogenetic reproductive mode that apparently evolved from an ancestral

hermaphroditic condition (Jaenike and Selander, 1979). In one such species
(Octolasion tyrtaeum), Jaenike et al. (1980) used allozyme markers to identify
eight distinct clones, two of which were widespread and common in diverse soil
types over the several thousand square kilometers of study area in the eastern
United States. Thus, some asexual lineages apparently can occupy broad niches
and achieve tremendous ecological success, at least over the short term. Many
freshwater gastropods (snails) likewise reproduce parthenogenetically (larne and
Delay, 1991). Allozyme studies of the polyploid parthenogen Thiara balonnen-
sis in Australia revealed that local populations generally consist of one clone
only, with genetic distance among clones correlated with geographic distance
(Stoddart, 1983b). This pattern of variation was postulated to result from the
gradual evolution of new clones by mutational processes (as opposed to occa-
sional sexual reproduction) in conjunction with geographic separations.
A converse example of high clonal diversity over microgeographic scales was
encountered in another species that exhibits obligate parthenogenesis throughout
much of its range, the cladoceran Daphnia pulex (Hebert and Crease, 1980).
Initial surveys revealed a total of 22 allozymic clones in 11 populations, with up
to 7 genotypes coexisting in a single lake. Subsequent studies of D. pulex using
mtDNA and allozyme markers revealed many additional clones and also dem-
onstrated that obligate parthenogenesis had a polyphyletic origin from facultative
parthenogenesis within this species (Crease et ai., 1989; Hebert et ai., 1989). In
a related species D. magna, tens to hundreds of clones sometimes coexist within
a pond (Hebert, 1974a, 1974b; Hebert and Ward, 1976). Where D. magna
occurs in temporary habitats, it is a cyclic parthenogen: Drought-resistant sexual
eggs (requiring fertilization) are produced each year, and these zygotes reestab-
lish populations that then are maintained by two or three generations of clonal
parthenogenesis until the pond again dries up. In this case, clonal coexistence
might only be short term because new recombinationally-derived clones enter the
population each year from sexual eggs. Interestingly, D. magna in permanent
habitats reproduces by continued parthenogenesis and tends to exhibit fewer
clonal types (Hebert, 1974c).

AGES OF CLONES

A common belief is that populations of clonally reproducing organisms must

have short evolutionary life spans, due to an accumulation of deleterious muta-
tions and gene combinations that cannot be purged in the absence of recombi-
nation [a phenomenon referred to as "Muller's ratchet" (Muller, 1964)], or to a
presumed lack of sufficient recombination ally-derived genotypic diversity to
allow adaptive responses to environmental challenges (Darlington, 1939; Felsen-
stein, 1974; Maynard Smith, 1978; Williams, 1975). However, the evidence
156 Individuality and Parentage

cited above suggests that some clonal lineages in invertebrates and plants can
achieve wide distributions and enjoy at least moderate-term ecological success.
Remarkably, about 70 vertebrate "species" also are known to reproduce by
clonal or quasi-clonal means (Dawley and Bogart, 1989) (the term "biotype" is
preferred for such forms because traditional species concepts hardly apply).
These biotypes typically consist solely of females that propagate by partheno-
genesis or related reproductive modes (Fig. 5.3). Essentially all unisexual ver-
tebrates arose through hybridization between related sexual species, and this
aspect of their evolutionary histories will be deferred to Chapter 7. Here we
consider the ages of particular vertebrate unisexual lineages, as inferred from
recent molecular assays primarily involving mtDNA.
Two conceptual approaches to assessment of vertebrate clonal ages have been
attempted from molecular data. The first involves estimation of the genetic
distance between a unisexual and its closest sexual relative. In a review of 24

PARTHENOGENESIS GYNOGENESIS HYBRIDOGENESIS

Figure 5.3. Three known modes of unisexual reproduction in vertebrates (after

Avise et al., 1992c). In parthenogenesis, the female's nuclear genome is trans-
mitted intact to the egg, which then develops into an offspring genetically identical
to the mother; in gynogenesis, the process is the same except that sperm from a
related bisexual species is required to stimulate egg development; and in hybri-
dogenesis, an ancestral genome from the maternal line is transmitted to the egg
without recombination, whereas paternally-derived chromosomes are discarded
premeiotically, only to be replaced each generation through fertilization by sperm
from a related sexual species.
 Individuality and Parentage 157

unisexual vertebrate lineages that have been so compared (Avise et al., 1992c),
13 proved indistinguishable in mtDNA assays from an extant genotype in the
related sexual taxon, indicating a very recent evolutionary separation; 5 addi-
tional lineages differed from nearest assayed bisexual taxon at sequence diver-
gence estimates of less than 1%, suggesting times of origin within the last
500,000 years (Fig. 5.4). A few unisexual haplotypes did show greater sequence
differences from related sexual forms, and these translate into literal estimates of
evolutionary durations of perhaps a few million years. However, a serious res-
ervation about such estimates is that closer relatives within the sexual progenitor
may have become extinct after unisexual separation, or otherwise remained
unsampled in the collections, such that unisexual ages could be grossly overes-
timated by this approach. Indeed, because of the low mtDNA lineage diversity
observed within most unisexual taxa relative to their sexual cognates (Fig. 5.4),
most authors have concluded that unisexuals arose very recently, even when
genetically close mtDNA lineages were not observed among the sexual relatives
sampled (e.g., Vyas et aI., 1990).
One recent example in which an ancient clonal age was promulgated involves
gynogenetic mole salamanders in the genus Ambystoma. From comparisons of
mtDNA sequences in the unisexuals versus extant sexual relatives, Hedges et al.
(1992a) and Spolsky et al. (1992) estimated evolutionary durations for the gy-
nogens of about four to five million years. However, one reservation about the
relevance of this conclusion to clonal persistence arguments is that these sala-
manders are unusual among the vertebrate unisexuals in that their evolution may
not be strictly clonal-other molecular data suggest that they continually acquire
nuclear DNA from sexual species, presumably via occasional incorporation of
sperm into the egg. If so, the antiquity of these "clonal" salamanders applies
strictly only to the mtDNA lineages that they contain.
A second approach to the estimation of clonal ages involves assessing the
scope of genetic variability within unisexual clades that by independent evidence
are of monophyletic (single-hybridization) origin. This method avoids confound-
ing postformational processes indicative of an old lineage with genetic diversity
that may have arisen from multiple hybrid origins. Quattro et al. (1992a) exam-
ined mtDNA and allozyme variability within a hybridogenetic clade of fishes in
northwestern Mexico (Poeciliopsis monacha-occidentalis) that by independent
zoogeographic evidence and tissue-graft analyses was of monophyletic origin.
The genetic data confirmed the monophyly of the clade and also documented
considerable genetic diversity within it, including the accumulation of several
mitochondrial and allozymic mutations in the monacha portion of the genome
(which comes from the female parent). From the magnitude of this diversity, the
authors estimated that the unisexual clade was more than 100,000 generations
old. One reservation about the relevance of this conclusion to clonal persistence
arguments is that because of the hybridogenetic reproductive mode of these
 time (my)
0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8
16

-en
(U
:::J
12
><
CI)

.-
en
r::::
8

.
:::J
4
0
r::::
0
0.0 0.8 1.6 2.4 3.2 4.0 4.8 5.6

sequence divergence (%)

-
~

en
a..
CI)
60

bisexuals
.-
> 40
"0
- (')

-
CI) ,....
0
20
.- -><
"0
0
CI) 0
CJ
:::J
r:::: 20 unisexuals

Figure 5.4. mtDNA results for unisexual vertebrates (after Avise et aI., 1992c).
Top: Frequency distribution of the smallest genetic distances observed between
mtDNA genotypes in unisexual clades and those in the closest assayed sexual
relatives (also shown are the associated evolutionary ages of the unisexuals based on
the conventional mtDNA clock calibration of 2% sequence divergence per million
years between lineages). Bottom: Nucleotide diversities in 13 sexual species and
their respective unisexual derivatives, arranged in rank order from left to right by the
magnitude of variation within the bisexuals.
 Individuality and Parentage 159

Poeciliopsis fishes (Fig. 5.3), inheritance only of the maternal component of the
lineage is strictly clonal [the somatic complement of each generation includes
new genetic input from the paternal side (P. occidentalis), such that the overall
genetic system ofthe hybridogenetic unisexuals is referred to as "hemiclonal"].
In any event, Maynard Smith (1992) argues that in evolutionary terms,
100,000 years "is but an evening gone" and for this reason results for Poecil-
iopsis do not contradict the conventional wisdom that organismal clones are
short-lived. Regardless of one's perspectives about whether such time scales are
"long" or "short" in the context of clonal persistence debates, molecular data
have provided the first critical information regarding the evolutionary durations
of vertebrate lineages lacking recombination.

Genetic Mosaics

Occasionally, what appears to be a single ramet actually represents two or

more genets that have fused into one morphologically (and perhaps physiolog-
ically) integrated module. A case in point involves strangler figs (Ficus spp.).
These trees often begin growth when bird- or mammal-deposited seeds germinate
in the humus-filled crotches of host trees. Shoots grow upward and roots down-
ward around the host bole. The roots eventually cross and fuse to form a unified
woody sheath surrounding the host, which then may die such that only the fig
tree remains. Thompson et al. (1991) showed by allozyme analyses that indi-
vidual fig trees often were genetic mosaics consisting of multiple genotypes.
Thirteen of the 14 sampled trees showed detectable genetic differences among
branches, such that at least 45 genetic individuals altogether were represented.
Presumably, the mosaicism is attributable to postgermination fusions among
roots that trace to multiple seeds deposited in the host tree.
Fusions among ramets also are common in many marine invertebrates includ-
ing corals, sponges, bryozoans, and hydroids (Grosberg, 1988; Jackson, 1985).
These somatic fusions involve postlarvae more often than mature colonies, and
in some cases the larvae may be asexual products of a single genet. However, in
other situations the fusing larvae are known or suspected to be sexually-produced
siblings, thus generating genetic mosaics. Aggregation and fusion among kin
may be facilitated by cosettlement of larvae with limited dispersal, but evidence
suggests that cellular recognition systems also are involved, and these may
reflect the same highly polymorphic histocompatibility loci that influence graft
acceptance versus rejection responses in mature colonies (e.g., Grosberg and
Quinn, 1986). Unfortunately, molecular genetic markers as yet have not been
widely capitalized on to address the frequency or pattern of mosaicism in colo-
nial marine invertebrates.
The correct identification of genetic mosaics is important in several evolution-
ary regards. For example, outcross pollinations or fertilizations could occur
160 Individuality and Parentage

within a mosaic' 'individual," thus influencing the genotypes of progeny and the
perceived mating system of a plant or animal species. If mosaics are common in
nature, the number of genets in a population could be seriously underestimated
by a mere census of the number of ramets, with consequences extending to any
evolutionary parameters that are influenced by effective population size (such as
the expected magnitude of genetic drift). The occurrence of genetic mosaics also
raises important issues regarding the degree of physiological and functional
integration of composite individuals.
In a quite different sense, the cells of all eukaryotic organisms can be viewed
as genetic mosaics containing nuclear and organellar genomes with formerly
independent evolutionary histories. Mitochondrial and plastid genomes almost
certainly represent the descendents of bacteria that early in evolution entered into
endosymbiotic relationships with protoeukaryotic host cells bearing precursors of
the nuclear genome (Margulis, 1970). As elaborated in Chapter 8, the most
compelling evidence for the mosaic nature of the eukaryotic cell comes from
phylogenetic assessments of genetic markers provided by slowly evolving genes.

Clonal Reproduction in Microorganisms

Eukaryotic protozoans such as the agents of malaria, sleeping sickness, Cha-

gas' disease, and leishmaniases infect more than 10% of the world's human
population and account for tens of millions of deaths every year. Sexual repro-
duction often is assumed for these parasites (most of which are diploid) because
recombination among strains has been observed in the laboratory. Nonetheless,
recent data from molecular markers have demonstrated that several parasitic
protozoans in nature can and often do reproduce by clonal means (Tibayrenc et
al., 1990, 1991a). For example, analyses of scnRFLPs revealed that globally
distributed virulent strains of the protozoan Toxoplasma gondii are nearly ho-
mogeneous genetically, likely because of clonal reproduction and a single evo-
lutionary origin from nonvirulent strains that exhibit moderate polymorphism
and are capable of sexual reproduction (Sibley and Boothroyd, 1992). Such
findings are of medical importance because they can influence the strategies for
diagnosis of the disease agents, as well as for developing vaccines and curative
drugs (Tibayrenc et al., 1991b).
Among other protozoans, the case for clonal reproduction in nature is partic-
ularly strong for Trypanosoma cruzi, the agent of Chagas' disease. Genetic
studies of T. cruzi isolated from humans, insect vectors, and mammals sampled
throughout the geographic distribution of the disease in South and Central Amer-
ica revealed several population genetic signatures characteristic of predominant
clonal reproduction (Tibayrenc et al., 1986; Tibayrenc and Ayala, 1987, 1988;
Zhang et al., 1988). These include fixed heterozygosity and other evidence for
 Individuality and Parentage 161

an absence of segregation genotypes at individual loci, an overrepresentation of

identical multilocus allozyme genotypes that also were geographically wide-
spread, and a correlation in clonal identification between independent sets of
genetic markers (Boxes 5.2 and 5.3). Some of the clones proved remarkably
divergent from one another, a finding that now should be taken into account by
the medical community, which traditionally has tended to view this and other
such protozoan species as undifferentiated quasi-panmictic entities. Similar lines
of molecular genetic evidence for clonality are available also for several other
parasitic protozoans (Table 5.3).
In the cereal rust Puccinia striiformis, Newton et al. (1985) used nuclear-
encoded allozymes and cytoplasmically-transmitted RNAs (from mycoviruses
inside the rust cells) to assay numerous accessions of this fungus from around the
world. Representatives of one geographically widespread group of wheat-attack-
ing forms (P.s. tritici) proved completely uniform by these assays, whereas some
other related fungal species showed much higher levels of genetic variability. P.
striiformis has no known sexual stage, so the presumed clonal transmission

Table 5.3. Population genetic evidence for c10nality in various parasitic protozoans
that are agents of human disease (after Tibayrenc et aI., 1991 b). a

Criterion
Evidence
Organism (a) (b) (c) (d) for Clonalityb

Entamoeba histolytica 0 0 + ND Moderate

Giardia sp. 0 0 + + Moderate
Leishmania major ND + + ND Strong
L. donovanilinJantum ND + + ND Strong
L. tropica + + + ND Strong
L. Old World ND + + ND Strong
Naegleria sp. + ND + ND Weak
Plasmodium Jalciparum 0 0 + ND Weak
Trichomonad sp. 0 0 + ND Weak
Trypanosoma brucei s.l. + + + + Strong
T. congolense + + + ND Moderate
T. cruzi + + + + Strong
"The listed criteria for clonality are described in Box 5.2. +, the criterion is satisfied; ND, not done;
0, data not available because the test is ploidy-level dependent (most taxa appear diploid, although
Plasmodium is haploid and the ploidy levels of Entamoeba, Giardia, and Trichomonad remain
uncertain).
bOverall weight of available evidence, which includes also sample sizes of assayed strains and
numbers of scored polymorphic loci.
162 Individuality and Parentage

probably contributes to the genetic results. However, further comparative studies

of pathogenic fungi with different life cycles will be required before final con-
clusions are drawn.
Bacteria too have been the subject of molecular genetic analyses to assess
reproductive mode in nature. Following the discovery in the 1940s of genetic
recombination mediated by conjugative transfer, transformation, and transduc-
tion in laboratory strains of Escherichia coli, the notion developed that extensive
genetic exchange probably characterized most bacterial taxa (Hedges, 1972).
However, until recently most research on the systematics, epidemiology, and
pathogenicity of bacteria involved physiologic, serologic, or other phenotypic
characteristics that seldom could be tied to specifiable alleles or loci that would
permit reliable elucidation of population genetic structure and reproductive mode
(Selander et al., 1987a). This situation changed beginning with allozyme studies
first conducted by Milkman (1973, 1975), and since then a wealth of protein and
DNA evidence has accumulated, suggesting that clonal reproduction predomi-
nates in many bacterial forms in nature. Because bacterial prokaryotes normally
are haploid, the lines of evidence for clonality consist primarily of criteria (c) and
(d) in Box 5.2.
For example, early work with E. coli revealed that despite high allozymic
variation (H == 0.50, an order of magnitude greater than values for most higher
eukaryotes), the number of distinctive multilocus genotypes was limited se-
verely. Furthermore, identical or closely similar electrophoretic types were ob-
served among some samples from geographically widespread, unassociated hosts
and persisted over periods as long as 100 years (Ochman and Selander, 1984;
Selander and Levin, 1980; Whittam et al., 1983a, 1983b). This and subsequent
molecular research demonstrating strong nonrandom allelic associations across
loci [gametic phase disequilibrium (Box 5.3)] has led to the view that chromo-
somal genetic transmission in E. coli is fundamentally clonal in nature, with
rather infrequent exchange of genes by means of recombination. [Two qualifi-
cations to this conclusion should be stressed: (a) plasmid or other extrachromo-
somal elements of adaptive relevance can be exchanged commonly among E.
coli (and other bacterial) strains (Hartl and Dykhuizen, 1984; Valdes and Pinero,
1992) and (b) occasional exchanges involving chromosome segments apparently
do occur also, producing some limited degree of reticulation among otherwise
isolated chromosomal lineages and leading to the appearance of mosaic chro-
mosomes whose segments trace to different E. coli ancestors (Milkman and
Bridges, 1990; Milkman and Stoltzfus, 1988»).
In any event, many ramifications stem from the new paradigm regarding the
prevalence of clonal inheritance in E. coli. For example, the multiple clones
occurring within an individual host probably represent successive invasions of
multiple founding genotypes (Caugant et aI., 1981) rather than products of
recombination among a small number of pioneering strains as formerly was
 Individuality and Parentage 163

Box 5.2. Populadon Genedc Criteria Strongly Suuesdve of Oonal

Reproducdon In Diploid Populadons (modified from nbayrenc et
aI.,1991b)
Comments and Caveats
(with Regard to Eliminating the
Inferences Possibility of Frequent
(Observations) Sexual Reproduction)

Absence of meiotic segregation at single marker loci

(a) Fixed heterozygosity (most Observation also incompatible with
or all individuals appear self-fertilization; must consider
heterozygous) possibility of mis-scoring due to
gene duplication or polyploidy

(b) Significant deficit in Missing heterozygotes also

frequencies of some consistent with self-fertilization;
expected diploid genotypes; must exclude effects of population
other deviations from subdivision, assortative mating,
Hardy-Weinberg selection, etc.
equilibrium
Absence of recombination among multiple marker loci
(c) Overrepresented, Should consider possible effects of
widespread identical selection or population subdivision;
genotypes; significant deficit must take into account low expected
of expected recombinant frequencies of multilocus genotypes
genotypes; nonrandom when allelic variation is high
associations of alleles
among loci [gametic phase
disequilibrium (Box 5.3)]
(d) Correlation between Should consider possible effects of
independent sets of genetic population subdivision or correlated
markers selection pressures
164 Individuality and Parentage

Box 5.3. Gametic Phase DlsequUlbrtum

Gametic phase disequilibrium is the nonrandom association between alleles of dif-
ferent loci. By "nonrandom" is meant that the multilocus combinations of alleles
depart significantly from expectations based on products of the single-locus allelic
frequencies. Consider the simplest possible case, involving two loci (A and B) each
with two alleles (A I' A2 and B I' B 2) whose frequencies are PI' P2' q l ' and q2'
respectively. Four di-Iocus gametic genotypes (or haplotypes) are possible:

Alleles at Locus A

Alleles at
Locus B

If alleles are associated at random in haplotypes (gametic phase equilibrium), the

expected frequencies of these di-Iocus genotypes are Plql' PIQ2' P2QI' and P2Q2' One
convenient quantitative measure of a departure from this expectation is given by the
gametic phase disequilibrium parameter, defined as

where P 11 and P 22 are the observed frequencies of haplotypes in the "coupling"

phase, and P 12 and P 21 are observed frequencies in the "repulsion" phase. It can be
shown that in a large random mating population, any initial disequilibrium [D(O)]
among neutral alleles will tend to decay toward zero (provided that c *- 0) according
to the equation

D(G) = (l-c)G D(O),

where D(G) is the disequilibrium remaining at generation G, and c is the probability

of a recombination event between the two loci each generation (or the "recombination
fraction"). Thus, for unlinked loci (c = 0.5), disequilibrium decays by one-half each
generation. Rates of disequilibrium decay slow as the recombination fraction de-
creases. When the loci examined involve one nuclear gene and one cytoplasmic gene,
the analogous nonrandom gametic associations are referred to as "cytonuclear dise-
quilibria" (Asmussen et aI., 1987). Because nuclear and cytoplasmic genes are un-
linked, any initial disequilibrium between such loci is expected to decay monotonically
to zero by one-half per generation, in a random mating population.
Gametic phase disequilibrium can arise from any historical or contemporary process
that has restricted recombination among loci, such as physical linkage of genes on a
chromosome, or other factors that can generate nonrandom associations among the
alleles of unlinked loci as well, including population subdivision, founder effects, a
 Individuality and Parentage 165

self-fertilization mating system (Chapter 6), or selection favoring particular multilocus

allelic combinations (Lewontin, 1988). Genomes or portions thereof characterized by
a rarity or absence of recombination may be viewed as linked "supergenes" with
regard to evolutionary dynamics. One likely consequence involves genetic hitchhik-
ing, whereby alleles that are neutral mechanistically nonetheless may spread through
a population because of chance association with an allele favored by natural selection
at another locus. Such hitchhiking on rare favorable mutations could lead to periodic
selection that has the net effect of purging population genetic variability in nonrecom-
bining systems (Levin, 1981). The selective sweeps involved in periodic selection may
account in part for the observation that genetic variability in mtDNA (Chapter 2) and
in bacterial species (Milkman, 1973) is vastly lower than might have been expected
given suspected mutation rates to neutral alleles and apparent population sizes. Such
selective sweeps may account also for the observation of greatly reduced nucleotide
diversity in chromosomal regions of eukaryotic nuclear genomes that are characterized
by low recombination rates (Begun and Aquadro, 1992).

supposed; widespread dispersal of strains (even on a worldwide scale)

apparently can take place; prospects for identifying the phylogenetic origins of
phenotypically characterized strains of E. coli and its relatives [such as Shigella
(Whittam et al., 1983b)] are enhanced when the confounding effects of frequent
recombination can be neglected; and, in general, the dynamics of bacterial
lineages may prove to be governed to a considerable degree by evolutionary
factors including "periodic selection" (sequential replacement by clones of
higher fitness, with net effect of purging population genetic variability) that, in
principle, apply with special force to any low-recombination genetic system
(Box 5.3).
Similar evidence for the prevalence of clonal chromosomal inheritance in
other bacterial species including pathogenic forms is leading to a fundamental
reassessment of bacterial taxonomy and epidemiology (Selander and Musser,
1990). The following are a few examples (Selander et al., 1987b). Based on the
distinctiveness of sets of clones as identified by protein electrophoresis,
Legionella consists of two undescribed species masquerading as L. pneumophila
(Selander et aI., 1985). Some of the more than 50 electrophoretic types (ETs)
are distributed worldwide, and the same clone may cause both Legionnaires'
disease and Pontiac fever. In Bordetella, a pathogen responsible for human
whooping cough and a variety of respiratory diseases in animals, numerous
clones and several genetically distinct species proved to exhibit strong host
specificities (Musser et aI., 1987). For example, clone ET-l of B.
bronchiseptica is a pig specialist, ET-6 a dog specialist, and the named species
B. parapertussis and B. pertussis are other clonal forms of B. bronchiseptica
that have become highly specialized as human pathogens. In Haemophilus
166 Individuality and Parentage

injluenzae, certain clones are distributed worldwide, and one distinctive clonal
group (ET-91-94) causes meningitis and septicemia in human neonates.
Another clone (ET-l) is known to have increased greatly in frequency in the
United States between 1939 and 1954 and now causes about 30-40% of the
invasive disease (Musser et al., 1985). Other Haemophilus clonal groups exhibit
no close associations with particular disease conditions (Musser et al., 1985,
1986). Unusually high clonal variation characterizes Neisseria meningitidis, but
only a few among the hundreds of multilocus genotypes appear to have been
responsible for most major epidemics worldwide over the past 60 years. For
example, one epidemic disease that started in Norway in the mid-1970s and
spread through Europe is caused by a group of clones in the ET-5 complex that
bears little genetic relationship to other electrophoretic types (Caugant et al.,
1986). Another severe epidemic that appeared in Cuba in the late 1970s also is
caused by ET-5 complex clones. These same strains apparently were brought to
Miami via Cuban refugees and initiated an outbreak in Florida in 1980-1981.
For the bacterium Staphylococcus aureus, among the dozens of electrophoretic
types isolated from two continents, a single clone accounted for 88% of the
cases of urogenital toxic shock syndrome in women (Musser et al., 1990). All
of these population genetic studies of clonal structure exemplify the power of
molecular markers in addressing problems in bacterial evolution that are of
diagnostic and epidemiologic relevance.
On the other hand, not all bacterial taxa have proved to be predominantly
clonal. Based on a great diversity of allozyme and RFLP genotypes observed in
a wild population of Bacillus subtilis, Istock et al. (1992) concluded that recom-
bination must be frequent relative to binary fission. This species is known to have
a proclivity for spontaneous transformation, whereby DNA is exchanged by
direct cell to cell contact. Such transformations presumably underlie the recom-
binational events that generated the high genotypic diversity reported.
Results for B. subtilis notwithstanding, available population genetic data in-
dicate that recombination events within many bacterial species are far too rare to
produce random allelic associations. Nonetheless, even for these species occa-
sional chromosomal recombinations are by no means ruled out, and these could
have important consequences over evolutionary time scales. Direct genetic ev-
idence for chromosomal recombination does exist for E. coli and a growing list
of bacterial taxa (DuBose et al., 1988; Selander et al., 1991). Thus, a particular
E. coli strain, for example, could have its chromosomes derived in bits and
pieces from multiple ancestors, the degree of mosaicism depending on the an-
cestors' remoteness, and the historical number of recombinational events in-
volved (also an inverse function of closeness of physical linkage of the genes
(Hartl and Dykhuizen, 1984). If recombination does generate, even occasion-
ally, selectively advantageous genotypes which then are propagated by clonal
 Individuality and Parentage 167

means, such events could have an important impact on bacterial adaptation and
evolution over the long term.

Gender Determination

Ascertainment of an individual's sex can prove difficult in many situations, for

example, in early life history stages, in species with little dimorphism in sec-
ondary sexual characters, or in species with internal gonads (such as birds). Yet,
knowledge of sexual identity is critical in ethological studies, sex ratio estima-
tion, management of matings among captive animals, and other areas of popu-
lation biology. In some groups such as many turtles and reptiles, sex is influ-
enced by the temperature at which eggs are incubated (Bull, 1980), but in most
taxa gender has a clear genetic basis. For these latter species, assay of molecular
markers linked to sex-determining genes or chromosomes can, in principle,
provide a powerful means of gender identification.
Quinn et al. (1990) isolated a piece of DNA homologous to the W (female-
specific) chromosome of the snow goose (Chen caerulescens) and used this
molecular probe to determine the sex of more than 150 birds from which blood
samples had been taken. Similarly, Griffiths and Holland (1990) isolated a
W-specific DNA probe for the herring gull (Larus argentatus), as did Sinclair et
al. (1990) for a Y (male-specific) chromosome in humans. Using DNA finger-
printing assays, Millar et al. (1992) found W-specific bands in the brown skua
(Catharacta lonnbergi) and used these gender markers to document significantly
different sex ratios in adult birds versus chicks. It remains to be seen how widely
these or similar probes can be employed in gender examination across broader
taxonomic arrays. One potential complication already has been documented-
occasional cross-homology between the Z and W chromosomes of birds (and the
X and Y chromosomes of mammals), due perhaps to occasional transposition or
recombination events between these sex chromosomes or to sequence conserva-
tion from an ancestral pair of homologues (Burgoyne, 1986; Ohno, 1967; Page
et al., 1982, 1984). Nonetheless, Sinclair et al. (1990) discovered a Y-specific
probe that could be used in Southern blot analyses to determine sex in a wide
range of mammals.
A remarkable application of sex-specific probes to gender identification in
nature involved humpback whales (Megaptera novaeangliae), which like other
baleen whales lack any obvious secondary sexual characteristics that distinguish
males from females. A human Y-chromosome clone was employed successfully
as a hybridization probe in RFLP analyses to determine the gender of 72 free-
ranging whales from which skin biopsies had been collected by a special dart (C.S.
Baker et al., 1991). The sex of yet another individual was identified by assays of
DNA extracted from sloughed skin collected from the whale's swimming path!
168 Individuality and Parentage

PARENTAGE
Procedures for genetic assessment of parentage are similar in principle to those
used to assess genetic identity/nonidentity, with the added complication that the
rules of Mendelian transmission genetics must be taken into account when com-
paring the genotypes of sexually-produced progeny against those of putative
parents. Parentage analyses typically address some version ofthe following ques-
tion: Are the adults who are associated behaviorally or spatially with particular
young the true biological parents of the offspring in question? If the answer proves
to be no, a genetic "exclusion" has been achieved. Whether the actual mothers
and fathers also can be specified depends on the size and genetic composition of
the pool of candidate parents and on the level of genetic variability monitored.
Sometimes one biological parent is known from independent evidence and the
problem simplifies to one of paternity (or maternity) exclusion or inclusion. In
other cases, neither parent is known with certainty prior to the molecular study.
Knowledge of biological parentage often is important in behavioral and evo-
lutionary studies. For example, matings are difficult to observe directly in nature
for many species, but reproductive behaviors and patterns of gene flow (Chapter
6) nonetheless can be deduced from molecular information on maternity and
paternity. Proper interpretations of behavioral interactions between presumed
family members depend on knowledge of genetic ties, including parentage. Even
when matings can readily be observed, questions of genetic parentage remain of
interest. Thus, in many birds and mammals, copulations are known to occur
outside the socially bonded pair, but the extent to which these result in illegit-
imate young has been uncertain and constitutes a major deficiency in the under-
standing of sexual selection and the evolution of mating systems (Gyllensten et
al., 1990; Mock, 1983; Trivers, 1972). By revealing genetic parentage, molec-
ular data provide more direct assessments of realized reproductive success and,
thus, at least partially circumvent the danger of equating mating prowess or other
components of the reproductive process with actual gene transfer across gener-
ations. Finally, knowledge of biological parentage is critical for correct inter-
pretations of the transmission genetics or heritabilities of any morphological
characters as deduced from field data on presumed parent-offspring associations
(Alatalo et al., 1984).
Parentage analyses utilize the cumulative information from multiple polymor-
phic loci assayed either individually (e.g., allozymes, scnRFLPs) or jointly
(multilocus DNA fingerprinting). General interpretive procedures may be intro-
duced by the following examples:
(a) Maternity and paternity both uncertain, exclusions attempted. T.W.
Quinn et al. (1987, 1989) compared goslings within each of several broods of the
snow goose (Chen caerulescens) against their adult male and female nest atten-
dants (putative parents) using genetic markers from multiple single-copy nuclear
 Individuality and Parentage 169

Box 5.4. Diploid Genotypes (at 14 Nudear RfLP Lod) of Nest

Attendants and GosUngs In Three FamlUes of the Snow Goose
(after T.W. Quinn et at., 1987).
As elaborated in the text, families 1 and 3 show strong genetic evidence of nonatten-
dant parentage for some goslings.
RFLP Locus
A B C D E F G H I J K L M N

Family 1
male attendant 2,2 2,2 2,3 1,2 1,1 1,1 1,4 2,2 1,2 1,2 1,2 1,2 2,2 2,2
female attendant 2,2 2,2 2,2 1,1 1,1 1,1 1,3 1,2 2,2 1,1 1,1 1,2 1,2 1,2
gosling 1 2,2 2,2 2,2 1,2 1,1 1,1 1,1 1,2 1,2 1,1 1,2 1,1 1,2 1,2
gosling 2 2,2 2,2 2,2 1,1 1,1 1,1 3,4 2,2 2,2 1,2 1,2 1,2 2,2 2,2
gosling 3 2,2 2,2 2,2 1,2 1,1 1,1 1,3 2,2 1,2 1,1 1,1 2,2 2,2 2,2
gosling 4 2,3 a 2,2 I,I d 1,1 1,1 1,1 I,2a 1,2 1,2 1,1 1,1 1,1 1,lc I,Ic
Family 3
male attendant 1,2 2,2 2,4 1,1 1,1 1,1 1,1 1,2 1,1 1,1 1,1 1,2 1,1 1,2
female attendant 2,2 2,2 1,2 1,1 2,2 1,1 1,2 1,2 2,2 1,1 1,1 1,1 2,2 1,2
gosling 9 2,2 2,2 1,2 1,1 1,2 1,1 1,1 2,2 1,2 1,2a 1,1 1,1 1,2 1,2
gosling 10 2,2 2,2 2,4 1,1 1,2 1,1 1,2 1,1 1,2 1,1 1,1 1,1 2,2c1,1
gosling 11 1,2 2,2 2,4 1,1 I,l b 1,1 1,1 1,2 2,2c 1,1 1,1 1,1 W b
1,1
gosling 12 2,3a2,2 2,2 1,1 W b 1,1 2,2c 1,1 1,2 1,1 1,1 1,1 I,l b
2,2
gosling 13 1,2 2,2 2,2 1,1 1,2 1,1 1,2 2,2 1,2 1,1 1,1 1,2 1,2 1,2
Family 4
male attendant 2,2 2,2 3,3 1,2 1,1 1,1 1,1 1,2 1,2 1,1 1,1 1,1 1,1 1,2
female attendant 2,2 1,2 1,1 1,1 1,1 1,1 1,1 2,2 1,1 1,1 1,2 1,2 1,2 1,2
gosling 14 2,2 1,2 1,3 1,2 1,1 1,1 1,1 1,2 1,2 1,1 1,2 1,1 1,2 1,2
gosling 15 2,2 1,2 1,3 1,2 1,1 1,1 1, I 1,2 1,2 1,1 1,1 1,2 1,1 1,2
gosling 16 2,2 2,2 1,3 1,2 1,1 1,1 1,1 2,2 1,2 1,1 1,2 1,1 1,2 1,2
gosling 17 2,2 2,2 1,3 1,2 1,1 1,1 1,1 2,2 1,2 1,1 1,1 1,2 1,1 2,2

aExciudes one unspecified parent; bexcludes putative mother; cexcludes putative father; dexcludes
both putative parents

RFLP loci. Prom their data (Box 5.4), the following observations and deductions
were made. Two goslings in family 3 (numbers 11 and 12) proved to be ho-
mozygous at some loci (E and M) for alleles not present in the female attendant.
Such cases excluded the putative mother and were interpreted to reveal instances
of intraspecific brood parasitism (IBP), whereby other females (not assayed)
must have contributed eggs to the nest. Other goslings (e.g., number 10 in family
3) proved to be homozygous (locus M) for alleles not present in the male
attendant. Such cases excluded the putative father and were interpreted to reveal
likely instances of extra-pair fertilization (EPP) by other males in the population.
170 Individuality and Parentage

Some heterozygous loci (e.g., J in gosling 9, family 3) exhibited one allele not
observed in either nest attendant and a second allele present in both attendants.
Such loci exclude one of the putative parents, but do not alone determine which
attendant is disallowed. Finally, some loci (e.g., C in gosling 4, family 1) were
homozygous for alleles not observed in either putative parent, thus excluding
both. Overall, the genetic data confirmed suspicions from field observations that
IBP (and probably EPF) are relatively common in snow geese populations (see
also Lank et al., 1989).
(b) Maternity known, paternity to be decided among a few candidate males.
Burke et al. (1989) applied multilocus DNA fingerprinting assays to the dunnock
sparrow (Prunella modularis), a species with a variable mating system tending
toward polyandry in which two males sometimes mate with and defend the
territory of a single female. In the DNA fingerprints, paternally-derived bands in
progeny were identified as those that could not have been inherited from the
known mother. Then, the true father was determined by comparing bands from
the fingerprints of candidate sires against these paternal alleles in progeny. For
example, Figure 5.5 shows DNA fingerprints from one known mother (M), her
four offspring (D-G), and two candidate sires (P", and P~). In this family, the
genetic data demonstrate that progeny G was sired by Pa' whereas D, E, and F
were fathered by P~. Thus, individual broods of polyandrous dunnock females
indeed can be multiply-sired.
(c) One parent or two? Many plants and invertebrate animals are hermaph-
roditic, i.e., an individual produces both male and female gametes. Such indi-
viduals might self-fertilize (in which case offspring have a single parent) or
matings may be facultative or compulsory with other individuals (thus producing
two-parent progeny). From wild-caught females whose mating habits are in
question, genetic examination of progeny often can reveal whether some progeny
carry alleles not present in the mother and, hence, derive from outcross fertili-
zations. Furthermore, comparisons of genotypic frequencies in a natural popu-
lation against Hardy-Weinberg expectations can aid in deciding whether cross-
fertilization or self-fertilization predominates (because the latter is a most intense
form of inbreeding, whose continuance leads to pronounced deficits in heterozy-
gote frequency and the eventual appearance of completely homozygous strains).
In examples of these approaches, allozyme data were employed to show that
cross-fertilization is the prevailing mode of reproduction in several species of
hermaphroditic freshwater snails in the genera Bulinus and Biomphalaria (Roll-
inson, 1986; Vrijenhoek and Graven, 1992; Woodruff et aI., 1985), that inter-
mediate levels of self-fertilization characterize the Florida tree snail Liguus fas-
ciatus (Hillis, 1989) and the coral Goniastrea favulus (Stoddart et aI., 1988), and
that self-fertilization predominates in populations of the sea anemone Epiactis
prolifera (Bucklin et aI., 1984). In similar allozymic studies of 19 species of
 Individuality and Parentage 171

offspring

kb M RP 'D E F G' ~
10 1- -I

Figure 5.5. Parentage analyses by multi-locus DNA fingerprinting.

Shown is a gel designed to assess whether offspring D-G with known
mother M were sired by Pot' P13 , or both (after Burke et ai., 1989).
Boxes encompass paternally derived bands in progeny that permitted
choice between the two candidate sires. See text for further expla-
nation.

terrestrial slugs in the families Limacidae and Arionidae, the majority of taxa
were shown to be predominant outcrossers (Foltz et al., 1982, 1984).
[As mentioned earlier, another form of uniparental reproduction is partheno-
genesis, which experimentally can be distinguished from self-fertilization by
examination of the diploid genotypes among the offspring of a heterozygous
parent. Fixed heterozygosity among progeny is inconsistent with expectations of
Mendelian segregation under self-fertilization but is a hallmark of ameiotic par-
thenogenetic reproduction. Using such allozymic evidence, Hoffman (1983)
documented that a laboratory population of one slug species formerly suspected
of self-fertilization (Deroceras laeve) actually reproduced by parthenogenesis.
172 Individuality and Parentage

Further discussion of parentage in the context of parthenogenetic reproduction

will be deferred to Chapter 7.]

Paternity Analysis

In species with internal fertilization, or where close physical or behavioral

associations connect mothers with offspring, maternity may be evident, whereas
paternity is uncertain. Such is often the case in humans, where indeed the bulk
of effort by molecular forensic laboratories is devoted to paternity assessment
(Lewis and Cruse, 1992). In many species, establishment of paternity is com-
plicated further by the fact that females may store viable sperm for considerable
periods of time. Many insects possess specialized sperm storage organs (sper-
mathecae). In lizards, female reproductive tracts may retain viable sperm for as
long as two months, and in some snakes for perhaps five to seven years (Devine,
1984). Some avian and mammalian females also may store sperm for periods of
days to weeks (Birkhead and M011er, 1992; Parker, 1984), although in general
both the life spans of sperm and the durations of sperm storage by females are
considerably longer in birds than in mammals (Gomendio and Roldan, 1993).

MULTIPLE PATERNITY IN ANIMALS

It might be supposed that male-specific DNA sequences (e.g., on the Y chro-

mosome in mammals and insects) would be of special use in paternity assess-
ment. Indeed, Williams and Strobeck (1986) reported mUltiple paternity for the
male progeny of some individual Drosophila melanogaster females, as gauged
by the observed diversity in RFLP patterns for ribosomal RNA gene markers that
were known to be confined to the Y chromosome. However, few such case
histories utilizing male-specific chromosomes as yet are available, no doubt for
two reasons: only a limited number of polymorphisms thus far has been uncov-
ered on these sex chromosomes and, in any event, paternity assignments for
female progeny could not be accomplished by this approach.
However, many paternity studies have utilized protein-electrophoretic or au-
tosomal DNA markers. By applying these molecular assays to single-female
litters, broods, or clutches, concurrent multiple paternity has been documented
for a wide variety of species in nature. Notable examples have come from mice
(Birdsall and Nash, 1973; Xia and Millar, 1991), shrews (Tegelstrom et al.,
1991), prairie dogs (Hoogland and Foltz, 1982), snakes (Gibson and Falls,
1975), marine turtles (Harry and Briscoe, 1988), salamanders (Tilley and Haus-
man, 1976), fishes (Chesser et al., 1984; Simonsen and Frydenberg, 1972;
Travis et al., 1990), insects (Milkman and Zeitler, 1974; Parker, 1970), spiders
(Martyniuk and Jaenike, 1982), isopods (Heath et al., 1990; Sassaman, 1978),
 Individuality and Parentage 173

limpets and other snails (Gaffney and McGee, 1992; Mulvey and Vrijenhoek,
1981), and lobsters (Nelson and Hedgecock, 1977).
Several of these studies involved socially polygynous species and thus con-
firmed suspicions that multiple copulations or inseminations could indeed result
in mUltiple successful fertilizations of a progeny cohort. For example, female
Belding's ground squirrels are known to mate with several different males, and
allozymic results established that an estimated 78% oflitters were multiply-sired,
usually by two or three males (Hanken and Sherman, 1981). In a similar study
of the willow leaf beetle (Plagiodera versicolora) , more than 50% of wild caught
females produced egg clutches expressing multiple sires (McCauley and O'Don-
nell, 1984) and in Drosophila pseudoobscura, single-female broods were esti-
mated to have been fathered by 1.7 different males on average (Cobbs, 1977).
On the other hand, not all molecular genetic analyses have uncovered evidence
for multiple paternity. For example, using allozyme assays, Foltz (1981) dem-
onstrated a high degree of monogamy in the oldfield mouse Peromyscus polion-
otus; and in a DNA fingerprinting study, all 99 assayed offspring from more than
25 families of the California mouse (P. californicus) resulted from matings
within individual male-female pairs [a finding that according to Ribble (1991)
provided "the first convincing demonstration of exclusive monogamy in a mam-
mal" (but see also Kleiman, 1977)].
Of greater surprise were genetic observations that single broods in some sup-
posedly monogamous species frequently contain illegitimate progeny resulting
from copulations involving one or more parents other than the care-givers. Per-
haps nowhere has such evidence for cuckoldry been discussed more widely than
in the Passeriformes (perching birds), which as a group traditionally had been
considered among the most monogamous of organisms (Gill, 1990). Gowaty and
Karlin (1984) were among the first to report genetic evidence for multiple pa-
ternity in a purportedly monogamous bird (the eastern bluebird, Sialia sialis), a
species now estimated from genetic data to produce between 8% and 35% ille-
gitimate young (Gowaty and Bridges, 1991a). Many additional examples of
multiple concurrent parentage in birds since have come to light (Table 5.4), with
illegitimate young often in high frequency. For example, an allozyme survey of
the indigo bunting (Passerina cyanea) established that at least 37 of 257 off-
spring (14%) carried genotypes incompatible with the putative father (Westneat,
1987). Statistical corrections based on detection probabilities (because only a
few polymorphic protein markers were employed) raised the estimated frequency
of EPFs in indigo buntings to between 27% and 42%. These latter estimates
agree with a DNA fingerprinting survey of another bunting population, where 22
of 63 nestlings (35%) were shown to have resulted from extra-pair fertilizations
(Westneat, 1990). Not all genetic reappraisals of passeriform species have pro-
duced evidence for cuckoldry; among 176 young from 32 families of warblers in
the genus Phylloscopus, no illegitimate young were found using sensitive single-
Table 5.4. Examples of concurrent multiple paternity or maternity in avian species as revealed in studies that employed
molecular genetic markers.

Observed % Observed Corrected EPF,

No. No. Broods with % % IBP,
Broods Progeny Non-Kin Non-Kin Non-Kin or
Species Assayed Assayed Progenya Progen~ Progenyb Assay Bothe Referenced

Indigo bunting 98 257 24 14 27-42 Allozymes EPF Westneat, 1987

(Passerina cyanea)
Indigo bunting 25 63 48 35 35 DNA EPF Westneat, 1990
(Passerina cyanea) fingerprints
White-fronted bee-eater 65 97 8 7 9-12 Allozymes Both Wrege and
(Merops bullockoides) Ernlen, 1987
Cliff swallow 105 349 21 10 24 Allozymes Both? Brown and
(Hirundo pyrrhonota) Brown, 1988
Eastern kingbird 19 60 47 30 39-53 Allozymes IBP, Both? McKitrick,
(Tyrannus tyrannus) 1990
House wren 18 97 22 6 31 Allozymes IBP, Both? Price et aI.,
(Troglodytes aedon) 1989
Red-winged blackbird 36 111 45 28 28 DNA EPF Gibbs et al.,
(Agelaius phoeniceus) fingerprints 1990
Field sparrow 17 52 41 19 Allozymes Both Petter et aI.,
(Spizella pusilla) 1990
aDefined here as progeny for which at least one of the putative parents is excluded by genetic evidence.
bCorrection based on empirical detection probabilities for the polymorphic loci assayed [by procedures such as those detailed in Westneat et aI. (1987)].
'EPF: extra-pair fertilization; IBP: intraspecific brood parasitism.

dAdditional examples may be found in Birkhead et aI. (1990), Bollinger and Gavin (1991), Evarts and Williams (1987), Gavin and Bollinger (1985), Gelter
and Tegelstrom (1992), Joste et al. (1985), Lifjeld et aI. (1991), Morton et aI. (1990), Mumme et aI. (1985), Seutin et al. (1991), Sherman and Morton (1988),
and Wetton et al. (1987, 1992).
 Individuality and Parentage 175

locus and multilocus VNTR assays (Gyllensten et al., 1990). Nonetheless, the
unexpectedly high frequency of extra-pair fertilizations reported for many birds
is prompting a fundamental reassessment of avian mating systems (Westneat et
aI., 1990).
Studies of multiple parentage are most informative when combined with be-
havioral or life history observations. For example, in eastern bluebirds the fre-
quency of "nondirectly descendant nestlings" (NDDN) proved to be signifi-
cantly greater for (a) males in their first breeding season rather than older males,
(b) males paired with females who more frequently were off territory during their
fertile periods than for males with sedentary mates, and (c) males that stayed
closer to females and thus exhibited what might be interpreted as mate-guarding
behavior (a counter intuitive result, unless it is supposed that males can sense a
propensity for cheating by certain females and attempt to monitor them accord-
ingly). Gowaty and Bridges (1991b) interpreted some of these observed trends as
consistent with the postulate that female bluebirds actively pursue extra-pair
fertilizations (rather than acting as passive or coerced on-territory recipients of
EPF-seeking males, as might be assumed under traditional mating system the-
ory). Whether any fitness advantages accrue to EPF-seeking females remains
unknown, although two conventional lines of speculation are that greater genetic
diversity among progeny might be selectively advantageous, and/or that extra-
pair copulations provide "fertilization insurance."
In other avian genetic studies integrated with demographic or behavioral ob-
servations, Morton et al. (1990) discovered through DNA fingerprinting that
older males in the colonial-nesting purple martin (Progne subis) achieve far
higher fecundities than do younger males, a result attributed to forced copula-
tions by older males that result in age-biased extra-pair fertilizations. In a similar
DNA fingerprinting study of polygynous red-winged blackbirds (Agelaius phoe-
niceus), Gibbs et ai. (1990) found that the proportion of illegitimate chicks is
significantly greater in marshes with higher male densities and that the cuckold-
ing males often are territorial neighbors. In a study of barn swallows (Hirundo
rustica), DNA fingerprints revealed that notwithstanding a pairing preference of
females for males with longer and more symmetrical tail streamers (M~ller,
1992), such males did not receive the expected fitness advantage because of a
significantly increased susceptibility to cuckoldry (H.G. Smith et aI., 1991). The
authors interpreted this result as indicative that longer-tailed males are hampered
in ability to guard mates and that this provides an evolutionary counterbalance to
the sexual selection for longer tails via female choice. In a detailed behavioral
and DNA-fingerprinting analysis of a population of blue tits (Parus caeruleus) in
Belgium, Kempenaers et ai. (1992) found that mate-guarding by males was
relatively ineffective in protecting paternity: "Attractive" males (those with
many visits from neighboring females) actually suffered less loss in paternity
(fewer extra-pair young in their own nests) than did "unattractive" males. These
176 Individuality and Parentage

males also were larger and survived better. Results were interpreted as support-
ive of a "genetic quality hypothesis," whereby females somehow assess male
quality and mate preferentially with superior males, regardless of other social or
populational considerations.
In some organisms such as the striped-backed wren (Camphylorhynchus
nuchalis), young remain in natal groups and appear to assist adult kin in rearing
new broods. Under sociobiological theory, postponement of dispersal and breed-
ing to assist in the rearing of others' progeny may be favored by natural selection
if the helpers' contributions to production of close kin exceeds expected repro-
ductive success had they dispersed (Brown, 1987; Hamilton, 1964). Studies
based on DNA fingerprinting by Rabenold et al. (1990) demonstrated another,
more direct avenue by which the fitness of helpers was enhanced. Some of the
behaviorally dominant males in wren social groups were found to share paternity
with auxiliary males previously thought to be nonreproductive. Thus, such re-
production by subordinate males may further help to explain their long tenure as
"helpers at the nest." In contrast, among females the only reproductives proved
to be dominant individuals, a result interpreted as consistent with the observed
aggressiveness of young female wrens in competition for breeding sites outside
the natal group.
Troops of rhesus macaque monkeys (Macaca mulatta) are characterized by
strong dominance hierarchies (both genders) whose behavioral underpinnings are
postulated to have evolved in response to selective pressures favoring high-
ranking individuals. Do males of higher social rank truly exhibit higher fitness
through greater access to receptive females (Dewsbury, 1982)? In a landmark
genetic study of primates, Duvall et al. (1976) applied allozyme analyses in
conjunction with behavioral observations to a captive group of rhesus macaques
and discovered that only 7 of 29 offspring (24%) produced over the 2 years of the
study actually were fathered by the at (top-ranking) male. Even low- and mid-
ranking adolescent monkeys sired several offspring and, therefore, clearly had
access to ovulating females. In a similar study of another rhesus troop, Curie-
Cohen et al. (1983) found that over an 8-year period the dominant male sired
only 13-32% of the offspring even though he participated in 67% of the ob-
served copulations, and that the second ranking male sired 30-48% of the
offspring despite participation in only 14% of the observed matings. Working
with six groups of the same species, Smith (1981) found that reproductive
success as determined by genetic markers was significantly correlated with rank,
although the pattern was such that changes in rank position appeared to follow
rather than precede changes in reproductive success. Examples of further genetic
work on rhesus monkeys include observations that a reproductive advantage is
enjoyed by the sons of high-ranking mothers, at least prior to their dispersal from
their natal groups (Smith and Smith, 1988), and that males and females of similar
social rank tend not to produce disproportionate numbers of progeny (Small and
 Individuality and Parentage 177

Smith, 1982). The overall picture to emerge from these and other studies is that
social dominance or even copulation frequency within rhesus troops are at best
imprecise predictors of breeding success of males [although the possibility re-
mains that lifetime reproductive fitness of socially dominant males might be
higher due to increased probabilities of survival for their offspring, rather than
increased reproductive activity per se (Bernstein, 1976)].
Rhesus macaques typically have large troops and are seasonal breeders, with
females exhibiting more or less synchronized fertile cycles during a well-defined
period. Stern and Smith (1984) speculated that in such cases it is more difficult
for males to monopolize females than in social systems where troop size is
smaller and estrus is dispersed temporally. A related macaque species (M. fas-
cicularis) breeds nonseasonally and was employed as a test of this hypothesis.
However, genetic paternity analyses of 44 M. fascicularis offspring born over a
28-month period revealed no evidence to support a positive association between
male social rank and number of offspring sired (Shively and Smith, 1985).
A different outcome was uncovered in studies of the red howler monkey
(Alouatta seniculus), where in none of nine surveyed troops could genetic evi-
dence exclude the dominant male as the father of offspring conceived during his
tenure (Pope, 1990). In both single-male and multimale harems, only the top-
ranking male was observed to mount females, and genetic evidence supported
the idea that dominant-male social status confers reproductive success. Red
howler troops are small and spatially cohesive, and this may facilitate behavioral
monitoring by the a male. Pope (1990) further suggested that females may avoid
mating with subordinate males to avoid infanticide, because infants conceived
during successful and attempted status changes by males frequently are killed.
Preliminary studies of molecular paternity also have been conducted on other
primates including baboons, marmosets, lemurs, guenons, mandrills, gorillas,
chimpanzees, and several monkeys and other macaque species (deRuiter, 1992;
R.D. Martin et al., 1992). In comparing results, two complications have been as
follows: DNA fingerprinting methods have worked better for some species than
others (for technical reasons) and levels of genetic variability have proved to
differ among populations and species (T.R. Turner et al., 1992). Nonetheless,
results for the red howler monkey notwithstanding, an emerging generality is that
social status and observed copulation frequency often are poor guides to male
reproductive success. Thus, for many primate species, these traditional methods
of fitness estimation appear to be grossly inadequate predictors of successful
progeny production.

PATERNITY IN PLANTS

In plants, fatherhood results from the spread of pollen, as mediated for ex-
ample by insect pollinators or wind. The same types of molecular genetic anal-
178 Individuality and Parentage

yses as described above can be applied to questions of pollen source (Adams et

al., 1992; Devlin and Ellstrand, 1990). The task again is simplified when the
mother is known (e.g., as the bearer of the seeds in question), but can remain
difficult when there is a large pool of potential pollen donors. Paternity may be
addressed with regard to the multiple seeds within a fruit and/or with regard to
the entire seed set of a maternal parent.
Many plant species are monoecious (hermaphroditic). Not all such plants can
self-fertilize, however, for several reasons: (a) male and female flowers of an
individual may mature at different times or be spatially separated on the plant,
(b) the stamens and stigma within a perfect flower (a flower possessing both
male and female parts) may be positioned such that mechanical pollen transfer is
unlikely, or (c) self-incompatibility genes may be present. These sterility genes
are known to carry multiple alleles that appear to have been selected to prevent
possible deleterious effects of the intense inbreeding that self-fertilization entails.
(Operationally, when the maternal parent and a pollen grain share the same allele
at a self-sterility locus, sporophytic tissue discriminates against gametophytic
tissue, for example by inhibiting growth of the pollen tube down the style.)
Nevertheless, self-fertilization apparently has evolved independently from the
outcrossing mode on numerous occasions (Stebbins, 1970; Wyatt, 1988), prob-
ably at least 150 times in the Onagraceae alone (Raven, 1979).
Thus, one of the first genetic questions regarding a monoecious species
concerns the frequency with which self-fertilization (as opposed to outcrossing)
takes place. When the female parent is known, the problem simplifies to one of
paternity assessment, with the issue in this case being how often the individual
plant that mothered an array of progeny or seeds can be excluded as the father of
those genotypes. For example, any offspring that exhibits an allele not present
in its known mother must have arisen through an outcross event (barring
mutation). Of course, the power to detect outcrossing depends on the number
of polymorphic marker loci monitored and the number and frequencies of
alleles. Several statistical models have been developed to quantitatively estimate
rates of selfing versus outcrossing (s and t, respectively, where s + t = 1) from
genotypic information at one or more loci (Brown and Allard, 1970; Ennos and
Clegg, 1982; Ritland and Jain, 1981; Schoen, 1988; Shaw et al., 1981). For
example, the widely employed "mixed-mating" model assumes that the mat-
ing process can be divided into two distinct components: self-fertilization and
random mating (i.e., random independent draws of pollen from the total pop-
ulation of plants) (Brown, 1989; Clegg, 1980). This model may be especially
appropriate for wind-pollinated species. A variant of this model that is probably
more applicable to insect-pollinated species assumes that outcross events within
a family are correlated because they may involve successive pollen draws from
a single male parent (Schoen and Clegg, 1984). In reality, mean genetic re-
latedness within a progeny array often may fall somewhere between full sibs and
 Individuality and Parentage 179

half sibs, and furthennore the sibship composition no doubt varies from array to
array.
From allozymes and other genetic markers, these "mating system parame-
ters" (s and t) have been estimated empirically for numerous populations and
species of monoecious plants. In an early summary of the literature by Lande and
Schemske (1985), the overall frequency distribution of outcrossing rates proved
to be bimodal, with most species either predominantly selfing or outcrossing
(Fig. 5.6). These authors interpreted the bimodality in mating system as consis-
tent with a scenario in which outcrossing is selected for in historically large
species with substantial inbreeding depression, whereas selfing is favored in
species where pollinator failure or population bottlenecks have greatly reduced
the level of inbreeding depression (via prior purging of deleterious recessive
alleles). Empirical evidence does exist for a high variance among plant species
in degree of inbreeding depression, with outcrossers typically exhibiting the
greatest reductions in fitness under inbreeding (Schemske and Lande, 1985).
Nevertheless, few monoecious plant species are "fixed" for either pure out-
crossing or pure selfing, and different populations within some species show
tremendous variation along the selfing-outcrossing continuum (Fig. 5.6). Fur-
thennore, the bimodality of mating systems noted by Schemske and Lande
(1985) may reflect a bias due to the disproportionate representation of selfing
grasses and outcrossing trees in the early literature (Aide, 1986).
In monoecious species where outcrossing has been established, or in any
dioecious species, the next genetic question likely to arise is: "Which plants
were the pollen donors for particular outcrossed offspring?" As illustrated in
Box 5.5, molecular genetic markers again can supply the answer. The approach
involves comparing the diploid genotype of each seed or progeny with that of its
known mother, and thereby deducing (by subtraction) the haploid genotype of
the fertilizing pollen. Then candidate fathers are screened for diploid genotype,
and paternity excluded for those males whose genotypes could not match the
deduced pollen contribution to the progeny. Sometimes all males except the true
father can be excluded. When multiple candidates remain, statistical procedures
exist for assigning "fractional paternity" based on the probabilities of being the
father. In the first large-scale application of these approaches, Ellstrand (1984)
employed six highly polymorphic allozyme loci to establish paternity for 246
seeds from 9 maternal plants within a closed population of the wild radish,
Raphanus sativus. Multiple paternity was found for all assayed progeny arrays
from a maternal plant and for at least 85% of all fruits, with the minimum
paternal donor number averaging 2.27. The wild radish is a self-incompatible,
insect-pollinated species. Subsequent work established that most multiply-sired
fruits resulted from the simultaneous deposition by a single insect vector of
pollen from several plants ["pollen carryover" (Marshall and Ellstrand, 1985)]
and that a considerable fraction of the seed paternity for some plants (up to 44%)
 25
en
.-
CI)
(.) 20
CI)
C.
en 15
"I-
0
10. 10
CI)
.c
E 5
::::J
C

o- 0.2- 0.4- 0.6- 0.8-

0.2 0.4 0.6 0.8 1.0

outcrossing rate
0 0.2 0.4 0.6 0.8 1.0

A
B ..
• • •••••••
.. .....
-- ..
••
• •• •
••
••
C ... •
0 I I. I

E • • • • •• •
Figure 5.6. Outcrossing rates in plants estimated by molecular markers. Top:
Frequency distribution of mean outcrossing rate as estimated from allozyme
markers for 55 monoecious plant species (after Schemske and Lande, 1985).
Hatched bars are animal-pollinated species, shaded bars are wind-pollinated
species (Aide, 1986). Bottom: Interpopulational variation in outcrossing rate in
each of five plant species: (A) Lupinus succulentus, (B) L. nanus, (C) Clarkia
exilis, (D) C. tembloriensis, and (E) Gilia achilleifolia (after Schemske and
Lande, 1985). Solid circles are population means and horizontal lines represent
observed ranges within each species.
 Individuality and Parentage 181

Box 5.5. IIlustradon of Paternity Assignment for five Progeny

from a Known Mother [taken from a much larger aUozyme data
set In Ulstrand (1984)].
The body of the table consists of observed diploid genotypes at each of six loci in the
wild radish.
Allozyme Locus
LAP PGI PGMI PGM2 6PGD lDH

Potential
Fathers
A 2,2 1,2 2,3 2,2 3,3 1,1
B 2,2 2,3 1,3 1,1 1,3 1,1
C 1,2 1,2 1,3 1,1 1,2 1,2
D 1,5 1,1 1,2 1,3 1,3 1,1
E 2,3 2,2 1,2 1,2 1,1 1,3
F 2,2 1,3 2,2 1,2 1,3 1,1
G 1,1 1,2 1,2 1,2 3,3 1,1
H 1,1 1,2 1,2 1,2 1,3 2,2
1,2 1,1 1,1 1,2 1,3 1,1
J 1,2 2,3 1,2 1,2 3,3 1,2
K 2,2 1,2 1,3 2,2 3,3 1,1
L 1,2 1,1 1,1 2,3 1,3 1,1
M 2,5 1,1 1,2 2,3 1,2 1,3
N 1,1 1,1 1,2 1,1 1,1 1,1
0 1,3 1,2 1,2 1,2 3,3 1,1

Known
Mother
Z 1,2 1,1 I, I 1,2 1,3 1,1

Deduced Paternal
Gamete Assignment
Offspring
P 2,2 1,2 1,3 1,2 1,1 1,2 223-12 C
Q 2,2 1,2 1,3 1,2 2,3 1,1 223-21 C
R 1,2 1,2 1,3 1,1 1,2 1,1 -23121 C
S 1,2 1,1 1,2 2,3 1,1 1,3 -12313 M
T 2,2 1,1 1,1 1,3 2,3 1,3 211323 M
182 Individuality and Parentage

derived from immigrant pollen from populations at least 100 m away (Ell strand
and Marshall, 1985).
In a similar allozymic study of a small forest herb ChalrUlelirium [uteum,
Meagher (1986) established paternity likelihoods for 575 seeds with known
mothers. The distribution of intermate (pollen-flow) distances indicated that more
nearby fertilizations had taken place than expected on the basis of random mating,
but nonetheless some mating pairs were separated from one another by more than
30 m. A follow-up study of established seedlings (where maternity was unknown)
confirmed this pollen-dispersal profile and also demonstrated that pollen and seed
dispersal distances were quite similar (Meagher and Thompson, 1987). Surpris-
ingly, no obvious relationship was found in this species between the size of the
male plant (indicative of reproductive effort) and the paternal success (Meagher,
1991). Hamrick and Murawski (1990) conducted similar genetic paternity anal-
yses on several tropical woody species and showed that (a) a significant proportion
of the pollen received by individuals came from relatively few pollen donors, (b)
many matings (30-50%) appeared to take place between nearest-neighbors, and
(c) some matings (10-25%) nonetheless did involve long-distance pollen flow
(greater than 1 km). The overall breeding structure to emerge from the latter study
appeared to have two components: a leptokurtic pattern of pollen dispersal within
populations, superimposed on a more even distribution of "background" pollen
originating from outside the population.
Results from genetic determinations of pollen source sometimes can assume
important economic and management ramifications for crop species. For example,
commercial seed orchards provide a significant fraction of the zygotes used to
establish pine plantations in the southeastern United States. One such seed orchard
for the loblolly pine (Pinus taeda) in South Carolina was composed of grafted
ramets of 50 loblolly clones that had been chosen and maintained for phenotyp-
ically desirable traits. Using multilocus allozyme markers, Friedman and Adams
(1985) discovered that at least 36% of the seeds from this orchard had been
fertilized by outside pollen, despite a surrounding lOO-m-wide buffer zone po-
sitioned explicitly to prevent such genetic contamination by nonselected males.
In an experimental population of cultivated cucumbers (Cucurbita pepo), Kirk-
patrick and Wilson (1988) showed that approximately 5% of progeny had been
fathered by a wild cucumber relative (C. texana). The authors suggested that such
weed-crop genetic exchange may have occurred over thousands of years and
provided an important source of genetic variability for selected cultivars.

SPERM AND POLLEN COMPETITION, PRECEDENCE

The widespread occurrence of polygyny and the multiple paternity of progeny

arrays means that sperm from two or more males commonly must be placed in
direct competition for fertilization of eggs within a female's reproductive cycle.
 Individuality and Parentage 183

Several morphological characteristics and reproductive behaviors of males have

been interpreted as adaptations to meet the genetic challenges resulting from this
supposed competition with another male's sperm (Parker, 1970). For example,
in many worms, insects, spiders, snakes, and mammals, the male secretes a plug
that serves temporarily as a "chastity belt" to block the female's reproductive
tract from subsequent inseminations. Other widespread male behaviors that have
been interpreted as providing paternity assurance in the face of potential sperm
competition include prolonged copulation (up to one week in some butterflies),
multiple copUlations with the same female, and postcopulatory guarding (Parker,
1984). From a female's perspective, mechanisms to prevent sperm competition
are not necessarily desirable, and this may lead to intersexual conflicts of inter-
est. Among the suggested advantages accruing to females from sperm competi-
tion and mUltiple paternity are an enhanced opportunity to acquire the "best"
available male genotypes, fertility insurance, and an increase in the genetic
diversity among progeny (Knowlton and Greenwell, 1984). Recent evidence also
suggests that the reproductive tracts of some females may playa more active role
than previously supposed in post-copulatory choice among sperm for fertiliza-
tions (Birkhead and M~ller, 1993).
From the progeny arrays of multiply-inseminated females, molecular markers
can be employed to determine which among the competing males' sperm have
achieved the fertilizations. Is the first-mating male at a reproductive advantage,
or does the last-mating male achieve the highest fertilization success? Or is there
no mating-order effect, the probability of fertilization instead merely being pro-
portional to the numbers of sperm inseminated by multiple males (the "raffle"
scenario)? These questions have been addressed using genetic markers for nu-
merous animal species (see the reviews in Birkhead and M~ner, 1992; Smith,
1984). In insects, it is frequently the case [but not always (Laidlaw and Page,
1984)] that the last male to mate with a female sires most of the offspring (Fig.
5.7). For example, in the bushcricket Poecilimon veluchianus, Achmann et al.
(1992) showed by DNA fingerprinting that the last-mated male achieves more

III 15
Q)
III

-...
1"11
U 10
o
Q)
.tJ 5
E 1-----1r--- Figure 5.7. Sperm competition in insects.
::J
c: o I Shown is the frequency distribution of the
proportion of eggs fertilized by the last male
0.0 0 . 25 0.5 0.75 1.0
to mate with mUltiply inseminated females
% eggs fertilized for a variety of species, as determined by
by last-mating male genetic markers (after Parker, 1984).
184 Individuality and Parentage

than 90% of the fertilizations. Mating in this species involves transfer of a large
spermatophore to a female, who often copulates with several males and then may
eat some spermatophores after copulation. The genetic findings appeared to
eliminate the possibility that nourishment gained by a female from the spermato-
phore "gift" of an early-mating male reflects a paternal investment strategy
enhancing that male's fitness.
The term "sperm displacement" conventionally has been employed to de-
scribe the enhanced reproductive success exhibited by last-mating males. In
some insects such as the locust (Locusta migratoria), an active "sperm flushing"
process indeed has been observed that probably contributes to the phenomenon
(Parker, 1984); in the dunnock sparrow, males before copulating peck at the
cloaca of a female, apparently causing her to eject sperm from previous matings
(Birkhead and M~ller, 1992). However, in most cases the mechanisms of sperm
displacement either remain unknown, or appear less active. In Drosophila, for
example, there is no known evidence for any substance in the male's ejaculate
that incapacitates the sperm of competitor males. In chickens and ducks, semen
from different inseminations is stored in separate layers in the female, with the
most recent contribution remaining on top and, therefore, perhaps most likely to
fertilize the next available egg (McKinney et al., 1984). For such cases, more
neutral terms such as "sperm predominance" (Gromko et al., 1984) or "sperm
precedence" are to be preferred.
In some birds, both raffle competition and sperm precedence are known to
operate, but over different time scales. If inseminations occur more than about 4
hours apart, then last-male sperm precedence tends to operate, but a sperm raffle
characterizes the process when two males inseminate a female in rapid succes-
sion (Birkhead and M~ller, 1992).
In a few insects (Fig. 5.7) and other animal species, first-mating males appear
to have the fertilization advantage. For example, in the intertidal copepod Ti-
griopus californicus, allozyme studies showed that virtually all of a female's
progeny are fathered by her first mate (Burton, 1985). In this species, a male
often clasps a female for a period of several days before her sexual maturation.
In light of the genetic observations, Burton (1985) interpreted this prolonged
clasping behavior by males as a precopulatory mate-guarding strategy to assure
that a potential mate has not been inseminated previously.
In the relatively asocial ground squirrel Spermophilus tridecemlineatus, syn-
chronously breeding females are scattered spatially at low densities. As a con-
sequence of this natural history, the mating system probably conforms to what
has been labeled "scramble-competition polygyny," and indeed, behavioral
observations suggest that the strongest phenotypic correlate of male mating suc-
cess is male mobility during the breeding season, with the attendant increased
likelihood of encountering females in estrus (Schwagmeyer, 1988). Through
use of allozyme markers, Foltz and Schwagmeyer (1989) discovered that in wild
 Individuality and Parentage 185

populations of this species, the first male to copulate with a multiply-mated

female sires the majority (on average 75%) of the resulting progeny. Results
were interpreted to indicate that the mating advantage for first males during
precopulatory scramble competition also translates into a genetic advantage dur-
ing the ensuing postcopulatory sperm competition.
A remarkable example of first-male fertilization advantage recently was re-
ported for the spotted sandpiper, Actitis macularia. In this polyandrous avian
species with strong tendencies for behavioral sex-role reversal, territorial females
pair with, defend, and lay clutches for several males. Molecular studies based on
DNA fingerprinting showed that males pairing early in the mating season cuck-
old their females' later mates by means of sperm storage in the females' repro-
ductive tracts (Oring et al., 1992). Thus, not only does an early-pairing male
have a greater confidence of paternity, but he thereby also appropriates the
reproductive efforts of subsequent males toward enhancement of his own fitness!
An intriguing hypothesis of "sperm sharing" has been advanced for some
species of hermaphroditic freshwater snails (Monteiro et al., 1984). Under this
suggestion, a snail might pass on the sperm from a previous mate to another
partner, such that the transmitting individual acts mechanically as a male but
achieves no genetic contribution to progeny. However, an empirical test of this
hypothesis based on allozyme markers failed to support the sperm-sharing hy-
pothesis (Rollinson et al., 1989). Instead, hermaphroditic snails proved capable
of passing on their own sperm while still producing eggs fertilized by sperm
received from an earlier mating.
In plants, opportunities also exist for competition among male gametes from
different donors, as, for example, via differing rates of pollen tube growth
through stigmatic tissue toward the egg (Snow, 1990). Using allozyme markers
to establish paternity, Marshall and Ellstrand (1985) demonstrated that most of
the seeds in multiply-sired fruits of the wild radish (Raphanus sativus) resulted
from the first in a series of sequential pollen donors. Further study revealed that
microgametophyte competition among several pollen donors appeared more pro-
nounced than among male gametophytes produced from a single pollen source
(Marshall and Ellstrand, 1986). In the morning glory Ipomoea purpurea, similar
allozymic analyses also revealed a strong fertilization advantage for first-polli-
nating males, even when pollen donations from a second source occurred im-
mediately after the first (Epperson and Clegg, 1987). In paternity studies of yet
another herbaceous plant Hibiscus moscheutos, allozyme markers revealed that
individuals with fast-growing pollen tubes sired a disproportionate number of
seeds following mixed experimental pollinations (Snow and Spria, 1991).
Maternity AlUllysis
Maternity also is sometimes in question. Indeed, one of the first applications
of DNA fingerprinting involved human maternity assessment in an immigration
186 Individuality and Parentage

dispute (Jeffreys et aI., 1985c). The case concerned a Ghanaian boy born in the
United Kingdom who left to join his father and subsequently returned alone to be
reunited with his mother. However, there was suspicion that a substitution had
occurred, either for an unrelated boy or for a son of one of the mother's several
sisters living in Ghana. At request of the family's solicitor, a DNA-fingerprint
anal ysis was conducted of the boy, his putative mother, and several of the child's
undisputed siblings (the task was complicated because the boy's father was
uncertain). The first step involved deducing paternal-specific bands in the boy's
DNA fingerprint. These were DNA fragments present in at least one ofthe siblings
but absent from the mother. The second step involved subtracting these paternal-
specific bands from the boy's DNA fingerprint. All 40 remaining fragments
matched those present in the woman, indicating that she indeed was the child's
biological mother. These genetic data were provided to the immigration author-
ities who then dropped objections and granted the boy residence in the United
Kingdom.
Tamarin et al. (1983) introduced an ingenious method for maternity assign-
ment in small mammals that, when used in conjunction with genetic markers,
offers special promise for assessing parentage in natural populations. The ap-
proach involves injection of pregnant or lactating females with unique combi-
nations of gamma-emitting radionuclides (e.g., 58CO, 85Sr, 65Zn) that transfer to
progeny via the placenta or the mother's milk. The isotopic profiles of the young
are determined spectrophotometrically and are matched against those of prospec-
tive mothers to establish maternity (a caveat could apply to the analysis if
mothers sometimes nurse offspring other than their own). Sheridan and Tamarin
(1986) combined this method of maternity assignment with protein-electro-
phoretic analyses to assess parentage in 40 offspring from a natural popUlation of
meadow voles (Microtus pennsylvanicus). Knowledge of maternity facilitated
the paternity analyses and led to the conclusion that about 38% of the adult males
in the population bred successfully in the surveyed time period, fathering at most
two litters each.
It might be supposed that rapidly-evolving mtDNA molecules would provide
ideal genetic markers of maternity, and indeed they do with regard to extended
female lineages within a species (Chapter 6). However, although mtDNA has
proved sufficiently variable within some species to provide "fingerprints" that
distinguish most individuals under conventional RFLP assays (A vise et al.,
1989), no explicit applications of mtDNA to questions of single-generation
maternity seem to have been published (however, see Kessler and Avise, 1985).
In practice, the multilocus genotypic variability generated by recombination
among nuclear genes far surpasses that anticipated even for the rapidly mutat-
ing but nonrecombining mtDNA. Thus, genetic approaches that employ mul-
tilocus allozyme or nuclear DNA-fingerprinting assays have provided most of
 Individuality and Parentage 187

the opportunities for maternity assessment, as illustrated by the following

examples.
Each spring, pregnant females of the Mexican free-tailed bat (Tadarida bra-
siliensis) migrate to caves in the American Southwest and form colonies, often
of several million individuals. Most females produce single pups that within
hours of birth are deposited on the cave ceilings or walls in dense creches.
Lactating females return to the creches and nurse pups twice each day. Tradi-
tional thought was that nursing must be indiscriminate such that the mothers act
"as one large dairy herd delivering milk passively to the first aggressive cus-
tomers" (Davis et al., 1962). However, McCracken (1984) challenged this view
with protein-electrophoretic evidence indicating that nursing was selective along
genetic lines. This conclusion stemmed from comparing observed allozyme gen-
otypes in female-pup nursing pairs with the expected frequencies of such geno-
typic combinations if nursing were random. A highly significant deficit of ma-
ternal genetic exclusions (relative to expectations from population genotype
frequencies) indicated selective nursing by females of their own pups (or at least
of pups with similar genotypes). McCracken estimated that only 17% of the
assayed females were nursing pups that could not be their offspring.
VandeBerg et al. (1990) employed protein-electrophoretic markers to validate
pedigrees in captive squirrel monkeys (genus Saimiri). Among 89 progeny for
which parentage had been inferred from behavioral observations, assignments
for 7 individuals proved incorrect, and retrospective examination of colony
records in conjunction with further genetic typing permitted a correction of the
pedigree records. Five of the errors had involved cases of mistaken paternity,
but, interestingly, two involved mistaken maternity. These latter cases appar-
ently were the consequence of infant swapping between dams shortly after birth,
an "allomaternal" behavior that previously had gone unrecognized.
More commonly, questions about maternity arise in oviparous animals such as
birds, fishes, and insects, where prolonged care of eggs outside the female's
body opens possibilities for intraspecific brood parasitism or other means of egg
or progeny mixing. In birds, traditional methods for inferring IBP include mon-
itoring nests for supernormal clutch sizes, noticing the appearance of eggs de-
posited outside the normal laying sequence of the resident female, or detecting
intraclutch differences in the physical appearance of eggs in those species where
interclutch differences in egg patterning are pronounced. Recent approaches
have involved more direct maternity assessment via molecular-genetic markers.
The finding of frequent intraspecific brood parasitism in snow geese (Box 5.4)
already has been mentioned, and additional examples of IBP in avian species are
presented in Table 5.4. For example, in wild zebra finches (Taeniopygia guttata) ,
a DNA-fingerprint examination of 92 offspring from 25 families revealed that
about 11% of offspring and 36% of broods resulted from IBP and that the
188 Individuality and Parentage

average number of parasitic eggs per clutch was greater than 1.0 (Birkhead et al.,
1990). In house wrens (Troglodytes aedon), a similar genetic study based on
allozymes led to the conclusion that as many as 30% of chicks were produced by
females other than the nest attendant (Price et al., 1989). Such unexpectedly high
frequencies of IBP in avian species as revealed by molecular markers (and by
more traditional approaches) have rekindled great interest in this remarkable
"egg-dumping" behavior by females (Petrie and Mf/Jller, 1991).

SUMMARY

1. Qualitative markers from highly polymorphic molecular systems provide

powerful tools for assessing genetic identity versus nonidentity, and parentage
(maternity and paternity).
2. In human forensics, genetic typing by RFLP assays of VNTR loci pro-
vides the late-20th century analogue of traditional fingerprinting and has found
wide application in civil litigations and criminal cases. Conservative procedures
for calculating probabilities of a genotypic match can serve to ameliorate any
potential biases against the defense.
3. For plant and animal species that are known or suspected of reproducing
by asexual (clonal) as well as sexual means, a variety of qualitative molecular
markers has been used to assess the reproductive system, to describe the spatial
distributions of particular genets (all clonal descendants from a single zygote),
and to estimate the ages of clonal lineages. Some clones have proved to be
unexpectedly old.
4. In several microorganisms including various bacteria and eukaryotic
protozoans, molecular markers have revealed unexpectedly strong proclivities
for clonal reproduction. These findings are of medical as well as academic
interest because they may influence strategies for diagnosis of disease agents and
for development of vaccines and curative drugs.
5. Molecular markers have found application in identifying genetic mosaics
in nature and in determining gender, where these features may not be obvious
from external phenotypes alone.
6. Molecular assessments of genetic parentage involve linking an offspring
to its biological mother and/or father when the parents are uncertain from other
evidence. Methods of analysis are influenced by whether paternity, maternity, or
both are uncertain and by the size of the pool of candidate parents.
7. Single-female clutches or broods in many species, including those for-
merly thought to be monogamous, often have proved through molecular analysis
to consist of multiply-sired young. The widespread occurrence of multiple pa-
 Individuality and Parentage 189

ternity raises the possibility of spenn or pollen competition, a phenomenon

studied extensively through molecular markers.
8. Multiple maternity of broods or clutches also has been documented by
molecular markers in several species. In birds, for example, this results from the
previously under-appreciated phenomenon of intraspecific brood parasitism, or
"egg-dumping. "
9. Molecular studies of maternity and paternity are most infonnative and
exciting when coupled with observations on behavior or natural history and
interpreted within the context of behavioral~cologic theory.
 6

Kinship and
Intraspecific Phylogeny

. . . community of descent is the hidden bond which natu-

ralists have been unconsciously seeking.

c. Darwin, 1859

Clonal identity and parentage (Chapter 5) are extreme examples of close kinship,
but now we shall be concerned with genetic relatedness within and among
broader groups of extended kin. Questions of genetic relatedness arise in virtu-
ally all discussions of social species where particular morphologies and behav-
iors might have evolved as predicted under theories of kin selection and inclusive
fitness (Box 6.1). Interest in kinship also arises for any species whose popula-
tions are structured spatially, perhaps along family lines. At increasingly greater
depths in time, all conspecific individuals are related genealogically through an
extended pedigree that constitutes the intraspecific phylogeny of a species.

CLOSE KINSHIP AND FAMILY STRUCTURE

Procedures for the molecular-genetic assessment of close kinship also require

qualitative genetic markers with known transmission properties, such as allo-
zymes or scnRFLPs. However, unlike the straightforward situation in paternity
and maternity assessment, where the single genetic pathway connecting an off-
spring with each of its biological parents permits explicit predictions about a

190
Box 6.1. Genetic Relatedness Within Groups, Induslve fitness,
and Kin Selection
Genetic relatedness. In discussions of close kinship, it is useful to have available a
quantitative measure of genetic relatedness (r) between individuals. An intuitive interpre-
tation of a coefficient of relatedness is provided by an answer to the following question:
What is the probability that an allele carried by the focal individual also is possessed by the
relative in question? Or in other words, what is the expected proportion of alleles shared
between the genomes of these individuals? In normal diploid species, r = 112 for full
siblings and parent-offspring pairs (Fig. 6.1), r = 114 for half-sibs or for an individual with
his uncles, aunts, grandparents, and grandchildren, r = 118 for first cousins, r = 0 for
nonrelatives, etc. Generally, for any known pedigree, values of r can be determined directly
by such pathway analyses (Cannings and Thompson, 1981; Michod and Anderson, 1979).
In the more usual situations in nature where pedigrees are unknown, several statistical
methods have been developed and refined for estimating mean coefficients of relatedness
among group members from polymorphic genetic markers such as those provided by
allozymes (Crozier et al., 1984; Pamilo and Crozier, 1982; Queller and Goodnight, 1989).
For example, Parnilo (l984a) derived an estimate for r that can be expressed in terms of
heterozygosities observed at a locus (hobs,m) and those expected under Hardy-Weinberg
equilibrium (hexp,m) within a colony m with N m individuals, in comparison to the heterozy-
gosities observed (Hobs ) and expected (Hexp) within the broader population composed of c
colonies:

Hexp - (I/c)~ hexp,m - (1/C)~[(IINm - 1)] [hexp,m - (1/2) hobs,m]

r = (6.1)
Hexp - (1/2) Hobs

This coefficient of relatedness may be interpreted as a genotypic correlation among group

members in a subdivided population [see Parnilo (1984a) for derivations and discussion].
Inclusive fitness and kin selection. Classic genetic fitness usually is defined as the
average direct reproductive success of an individual possessing a specified genotype in
comparison to that of other individuals in the population. Inclusive fitness, which entails a
broader view of the transmission of genotypes across generations, incorporates both the
individual's personal or classic fitness as well as the probability that the individual's
genotype in question may be passed on through relatives. These latter transmission prob-
abilities are influenced by the coefficients of relatedness involved. Concepts of inclusive
fitness have been advanced as an explanation for the evolution of •• self-sacrificial" behav-
iors, wherein alleles influencing such altruism may have spread in populations under the
influence of kin selection. According to Hamilton's (1964) rule, a behavior is favored by
selection whenever
(6.2)
where .iw, is the change the behavior causes in the individual's fitness, .iWy is the change
the behavior causes in the relative's fitness, and ryx is the genetic relatedness of the
individuals involved. In general, under Hamilton's rule, an allele will tend to increase in
frequency if the ratio of the cost C that it entails (loss in expected personal reproduction
through self-sacrificial behavior) to the benefit B that it receives (through increased repro-
duction of relatives) is less than r:

CIB < r. (6.3)

Under the proverbial example of altruistic behavior, an individual's alleles WOUld, thus,
tend to increase in frequency if personal fitness was sacrificed for a comparable gain in
personal fitness by more than two full sibs, four half-sibs, or eight first cousins!
192 Kinship and Intraspecific Phylogeny

circumscribed set of expected genotypes in these individuals, most extended

kinship assessments are complicated by the fact that numerous alternative trans-
mission pathways potentially link more distant relatives. Thus, in genetic studies
of broader kinship, particularly those based on small numbers of loci, the focus
typically shifts from attempts to enumerate relationships among particular indi-
viduals (but see later section on noneusocial colonies and groups and also Queller
and Goodnight, 1989) to a concern with probability patterns of mean genetic
relatedness within groups.
The general types of concepts and reasoning that are involved in kinship
assessment may be introduced qualitatively by the following example (from
Avise and Shapiro, 1986). Juveniles of the serranid reef fish Anthias squami-
pinnis occur in social aggregations ranging in size from a few individuals to more
than 100. Although eggs and larvae of this species are pelagic, drifting in the
open ocean, Shapiro (1983) raised the intriguing hypothesis that juvenile cohorts
might consist of close genetic relatives (predominantly siblings from a single
spawn) that had stayed together through the pelagic phase and settled jointly. If
true, kin selection would have to be considered as a factor potentially influencing
behaviors within the social groups and, furthermore, marine biologists would
have to reevaluate the conventional wisdom that the products of separate spawns
are mixed thoroughly during the pelagic phase. To critically test the Shapiro
hypothesis, genotypes were surveyed at each of three polymorphic allozyme loci
in eight discrete social cohorts of juvenile A. squamipinnis from a single reef in
the Red Sea. Allelic frequencies are presented in Box 6.2.
The hypothesis of close genetic affinity within A. squamipinnis cohorts yields
several genetic predictions that were not borne out by the allozyme data. For
example, if progeny within a cohort were full sibs, they should (a) exhibit at
most four alleles at a locus (but instead several cohorts displayed more than four
alleles at the Pgm and Sod loci), (b) exhibit alleles in frequencies consistent with
transmission primarily from only two parents (but instead allele frequencies
departed dramatically from these expectations, and the best goodnesses of fit to
the empirical data required a minimum of 11-16 equally contributing parents per
cohort), (c) share any rare alleles that were observed in the study (but instead all
rare alleles present in more than one individual were distributed across cohorts),
(d) often exhibit strong heterozygote excess relative to expectations from within-
cohort allele frequencies, as, for example, in a mating between alternate ho-
mozygotes or between a homozygote and a heterozygote (but instead no signif-
icant excesses were revealed), and (e) exhibit genotypic relatedness values [r
(Box 6.1)] close to 0.5 (but instead estimates of r were invariably near zero). In
addition, if A. squamipinnis cohorts existed primarily as full-sib assemblages,
there should be (f) large between-cohort variances in allele frequency (but ob-
served variances were close to zero), and (g) large differences among cohorts in
 Kinship and Intraspecific Phylogeny 193

Box 6.2. Observed Allele frequendes at Three AIIozyme loci In

Dpt low Cohorts of the Reef nsh Anthlas squamlplnnJs (after
Avlse and Shapiro, 1986).
Cohort

Gene Allele A B C D E F G H Total

Ldh 2n = 124 140 128 158 152 140 134 106 1,082
a 0.613 0.679 0.680 0.620 0.605 0.643 0.649 0.613 0.638
b 0.379 0.314 0.320 0.380 0.395 0.350 0.351 0.377 0.358
c 0.008 0.007 0.010 0.003
d 0.007 0.001
Pgm 2n= 124 140 128 162 156 140 162 106 1,118
a 0.944 0.936 0.937 0.938 0.904 0.957 0.926 0.943 0.935
b 0.048 0.043 0.016 0.050 0.070 0.022 0.049 0.028 0.042
c 0.088 0.021 0.031 0.012 0.026 0.021 0.025 0.019 0.020
d 0.008 0.001
e 0.008 0.010 0.002
Sod 2n = 124 140 128 162 156 140 162 106 1,118
a 0.758 0.707 0.734 0.729 0.679 0.722 0.698 0.792 0.723
b 0.113 0.150 0.180 0.148 0.211 0.164 0.191 0.142 0.164
c 0.089 0.100 0.070 0.080 0.071 0.057 0.087 0.047 0.076
d 0.040 0.036 0.008 0.037 0.026 0.036 0.012 0.019 0.027
e 0.008 0.013 0.021 0.012 0.007
f 0.007 0.001
g 0.006 0.001
Within-cohort genetic relatedness based on the application of Eq. (6.1) to the three loci yields
estimates ofr = -0.01,0.01, and 0.02, hence providing strong evidence against the proposition
that cohorts consist of close kin (see text).

single-locus and multilocus genotypic frequencies (but no such differences were

observed). Overall, these several lines of reasoning applied to the genetic data
demonstrated that the assayed cohorts of A. squamipinnis do not consist exclu-
sively or predominantly of close genetic relatives, but rather represent nearly
random samples of progeny from many matings.
In general, from such molecular data, estimates of the average genetic relat-
edness within groups can be quantified by approaches such as outlined in Box
6.1. Estimated values of r then may be compared against theoretical expectations
for organisms of known pedigree relationship, and thereby used as a genealogical
backdrop against which to evaluate hypotheses regarding social behaviors and
the possible influence of kin selection.
194 Kinship and Intraspecific Phylogeny

Eusocial Colonies

Species considered eusocial consist of groups of individuals possessing the

following traits (Wilson, 1971): cooperation in the caring for young; reproduc-
tive division of labor, with more-or-Iess sterile individuals working on behalf of
the reproductives; and overlapping generations of colony workers. Eusociality
long has intrigued biologists because its evolution entails a remarkable transition
to sterility of worker individuals-a most extreme fonn of reproductive altruism.
The epitome of eusociality has been reached in colonial hymenopteran insects,
including nearly all ants and the highly social bees and wasps, where sterile
workers (who are all females) provide exaggerated examples of "helpers at the
nest. "
Hamilton (1964) first proposed that the evolution of such extreme reproductive
altruism might have been facilitated by the altered and asymmetric genetic re-
lationship among parents and siblings stemming from haplo-diploid sex deter-
mination. In these hymenopteran species, males develop from unfertilized eggs
and hence are haploid for a single set of chromosomes inherited from their
mother. Females develop from fertilized eggs and are diploid. Thus, daughters
of a monogamous female share three-quarters of their alleles [all alleles from
their father and half from their mother (Fig. 6.IB)], a situation that contrasts with
expected relationships in nonnal diploid species where full sibs share only one-
half of their alleles. Thus, according to Hamilton's insight, an ancestral hy-
menopteran with any behavioral predisposition toward rearing sisters, even at the
risk of producing fewer young, might have increased her inclusive fitness by
adopting this behavioral strategy. In other words, if the genetic ties within a
generation are closer that those between generations, an individual might profit
under the currency of inclusive fitness by investing in a parent's reproductive
success rather than her own.
However, this prediction weakens considerably when the reproductive females
(queens) are multiply mated (polyandry, in this context) and/or when multiple
queens lay eggs in a nest (polygyny) because mean genetic relatedness among
colony workers then is lowered dramatically [by a magnitude depending on the
numbers of parents, their genetic relationships to one another, and their contri-
butions to progeny (Fig. 6.1; Wade, 1982; Wilson, 1971)]. Empirical surveys
using allozyme markers have been conducted on a variety of social hymenopter-
ans to assess mean within-colony genetic relatedness (Table 6.1). For some
species (such as Rhytidoponera ants), average relatedness among colony work-
ers, indeed, has proved to be near the value of r = 0.75 expected when a
singly-inseminated queen founds a monogynous nest. However, genetic relat-
edness estimates for other species have proved to be significantly lower, often far
below the critical threshold of r = 0.50 where the original supposed genetic
advantage of reproductive altruism by workers would seem to be absent. Such
 NORMAL DIPLOID
(A) r: female to mother, 0.50
female to sister, 0.50

HAPLO-DIPLOID (single father)

(8) r: female to mother, 0.50
female to sister, 0.75

HAPLO-DIPLOID (>1 father)

r: female to mother, 0.50
female to sister, 0.25-0.75

DIPLOID (inbred/outbred cycle)

(C) r: female to mother, 0.50
female to sister, =1.0

Figure 6.l. Transmission genetics through single-generation pedigrees.

Shown are expected patterns under rules of (A) normal diploid inheritance,
(B) haplo-diploidy as in eusocial hymenopteran insects (with and without
multiple insemination of queens), and (C) diploidy under an extreme inbreed-
ing-outbreeding cycle (see text). Circles (females) and squares (males) bi-
sected by vertical diameter lines represent diploid individuals; males without
such lines are haploid. Also shown are mean coefficients of genetic related-
ness (r) expected among females under these various reproductive modes. In
the case of haplo-diploidy with more than one father (polyandry), values of r
depend on the number of fathers and their proportionate contributions to the
worker population of a colony. For example, if all of n males contribute
equally, then the average coefficient of genetic relatedness becomes r =
0.5[0.5 + lin]; in general, mean r = 0.5[0.5 + Lf?]' where /; is the
proportionate contribution by the ith male. Reproduction by multiple queens
within a colony (polygyny) would serve further to lower the mean r.
196 Kinship and Intraspecific Phylogeny

Table 6.1. Examples of coefficients of genetic relatedness (r) estimated among

females within colonies of various eusocial hymenopteran insects. All estimates stem
from protein-electrophoretic analyses. a

Comments on
colonies
Species Comparison r and queens b Authority

ANTS
Rhytidoponera Workers 0.76 Monogynous, Ward, 1983
chalybaea (A) monoandrous
Rhytidoponera Workers 0.70 Monogynous, Ward,1983
confusa (A) monoandrous
Nothomyrmecia Workers 0.17 Occasionally Ward and Taylor,
macrops polygynous 1981
Myrmecia pilosula Workers 0.17 Polygynous Craig and
Crozier, 1979
Myrmica rubra Workers 0.02-0.54 Polygynous Pearson, 1983
Solenopsis invicta Workers 0.01-0.08 Polygynous, queens Ross and
singly-mated Fletcher, 1985
Formica aquilonia Workers 0.09 Polygynous Pamilo, 1982
(Espoo)
Formica polyctena Workers 0.19-0.30 Polygynous Pamilo, 1982
Formica sanguinea Workers 0.31-0.42 Polygnous, queens Pamilo and
multiply-mated Varvio-Aho,
1979
Formica Workers 0.33 Polygynous, queens Pamilo, 1981,
transkaucasica singly-mated 1982
WASPS
Agelaia multipicta Workers 0.27 Polygynous West-Eberhard,
1990
Parachartergus Femalesc 0.11 Probably polygynous Queller et al.,
colobopterus 1988
Polybia Femalesc 0.34 Polygynous Queller et al.,
occidentalis 1988
Polybia sericea Femalesc 0.28 Polygynous Queller et al.,
1988
Cerceris antipodes Femalesc 0.25-0.64 Polygynous McCorquodale,
1988
Microstigmus Femalesc 0.60-0.70 Monogynous or Ross and
comes polygynous, Matthews,
queens often 1989a, 1989b
singly-mated
BEES
Apis mellifera Workers 0.25-0.34 Highly polyandrous Laidlaw and
Page, 1984

a Additional examples may be found in Holldobler and Wilson (1990) and Ross and Carpenter
(1991).

hJn most cases, the mating system is not well known.

'Reproductive and nonreproductive females not distinguished.
 Kinship and Intraspecific Phylogeny 197

genetic results, coupled with field observations, have revealed that queens often
are multiply-inseminated, and furthennore that polygyny is common (see the
reviews in Cole, 1983; H6lldobler and Wilson, 1990; Ross and Carpenter,
1991). These findings have raised a conundrum about the evolution of insect
eusociality: Because cogenerational individuals within a nest are less closely
related to one another than they would be to their own sons or daughters, "if kin
selection is a powerful force, what prevents evolution from leading to a more
competitive state in which the workers (who have ovaries) try to take over
reproduction?" (H6lldobler and Wilson, 1990).
One possibility is that a colony is divided into cliques of closely related
individuals who are able to distinguish close kin from more distant kin, and
direct altruistic behaviors accordingly. Experiments indicate that kin discrimi-
nation is possible for some hymenopteran species but not others (H6lldobler and
Wilson, 1990). For example, worker honey bees (Apis mellifera) are able to
"assess" their relatedness to other individuals and preferentially rear queens
from larvae that are related most closely (Visscher, 1986), whereas workers of
the ant Rhytidoponera confusa appear to lack such abilities (Crosland, 1988).
Another suggestion is that the high levels of polygyny and polyandry observed
represent derived behaviors rather than the ancestral conditions under which
eusociality evolved. Under this hypothesis, eusociality tends to arise through kin
selection when populations are highly structured along family lines, whereas
subsequent maintenance and elaboration into advanced eusociality can occur
even when within-colony relatedness decreases due to polygyny and polyandry.
Eusocial colonies once fonned may operate so smoothly and successfully that the
inclusive fitness of workers remains higher than if workers became egg-layers,
such that reversion to a less eusocial condition simply is infeasible. In ants, it is
difficult to test the hypothesis that polygyny and polyandry are derived condi-
tions because most species are strongly eusocial. In the primitively eusocial bee
Lasioglossum zephyrum, an empirical molecular genetic estimate of r = 0.7 is
sufficiently high to indicate that kin selection could operate in this species (Cro-
zier et aI., 1987). However, in several species of primitively eusocial polistine
wasps, r values within colonies sometimes have proved to be moderate or low
(Strassmann et aI., 1989). Although it is not certain that these species provide
valid representations of the ancestral behavioral condition, the findings do dem-
onstrate that low within-colony relatedness is not confined to the advanced hy-
menopteran societies.
Finally, various ecological-genetic hypotheses have been advanced to explain
the conundrum involving low within-colony r. For example, genetic variability
among nestmates might diminish susceptibility to infectious parasites (Shykoff
and Schmid-Hempel, 1991a, 1991b) or promote broader tolerance to variable
environments; caste determination might have a partial genetic basis for which
polyandry or polygyny conceivably allow fuller expression (Crozier and Page,
198 Kinship and Intraspecific Phylogeny

1985); or, perhaps, collaborating queens fare proportionately better than indi-
vidual queens in competition for limited nest sites (Herbers, 1986). Under this
latter hypothesis, inclusive fitness concepts could remain in partial effect if such
cofoundresses are genetic relatives, as sometimes (but not always) appears to be
the case: In various wasp, bee, and ant species, molecular-genetic appraisals of
cofounding queens have revealed mean relatedness values ranging from r = 0.0
- 0.7 (Metcalf and Whitt, 1977; Ross and Fletcher, 1985; Schwartz, 1987; Stille
et al., 1991; Strassmann et al., 1989).
In any event, the altered coefficients of genetic relatedness stemming from
haplo-diploidy cannot provide a universal or complete explanation for the evo-
lution of eusociality because (a) other haploid-diploid arthropod species outside
the Hymenoptera (e.g. some mites, thrip insects, and beetles) do not exhibit
eusociality and (b) some diploid species (notably termites) do. In discussing the
evolution of termite eusociality, Syren and Luykx (1977) and Lacy (1980) noted
that several termite species possess sex-linked multichromosome translocation
complexes that serve to elevate genetic relatedness both between sisters and
between brothers. However, the low genetic relatedness between male and fe-
male siblings under these translocation systems remains difficult to accommodate
with the evolution of termite eusociality (Andersson, 1984; Leinaas, 1983).
Another proposed model that could influence inclusive fitness by altering genetic
relatedness within and among groups involves cyclic inbreeding-outbreeding
(Bartz, 1979; see also Pamilo, 1984b and Williams and Williams, 1957). When
male and female mates are unrelated but are each the product of intense inbreed-
ing, their offspring can be nearly identical genetically but only 50% like either
parent (Fig. 6.1C). When such conditions hold, any behavioral predispositions
of siblings to stay together and to assist parents in rearing the young might be
favored for the same reasons of inclusive fitness as set forth above for the
haplo-diploid hymenopterans (however, see Crozier and Luykx, 1985). Termites
possess several natural history features that favor close social interactions and
might set the stage for such a breeding cycle, such as living in protected and
contained nests conducive to multigenerational inbreeding and passing symbiotic
intestinal flagellates from old to young individuals by anal feeding (an arrange-
ment that necessitates at least some degree of social behavior!) (Wilson, 1971).
A remarkable mammalian analogue of the eusocial system of termites has been
discovered in the naked mole-rat, Heterocephalus glaber (Jarvis, 1981; Sherman
et al., 1991). Brood care and other duties in this colonial underground rodent are
performed cooperatively by mostly nonreproductive workers or helpers, who
represent the young from previous litters and who assist the queen in rearing
progeny that are fathered by a few select males within the burrow system. Are
colony mates especially close genetically, such that kin selection might plausibly
account for the extreme reproductive selflessness displayed by subordinate indi-
viduals? Using DNA-fingerprint assays, Reeve et al. (1990) observed remark-
 Kinship and Intraspecific Phylogeny 199

ably high probabilities (0.88-0.99) of DNA band-sharing within colonies (sim-

ilar to estimated levels of band-sharing in DNA fingerprints of highly inbred
mice or monozygotic twins in cows and humans). From these molecular data,
they estimated a mean coefficient of genetic relatedness significantly greater than
0.50 (r = 0.81 ± 0.10) and concluded that more than 80% of the matings within
a colony likely were among siblings or between parents and offspring. Results
appear consistent with intense within-colony inbreeding and, hence, leave open
the possibility of an influencing role for kinship and inclusive fitness in the
evolution of H. glaber eusociality. Nonetheless, behavioral and life history
characteristics no doubt are important as well, as are phylogenetic constraints, as
indicated by the differences among the eight African mole-rat species in the
degree to which colonial and eusocial behavior is exhibited (Allard and Honey-
cutt, 1992; Honeycutt, 1992).

NoneusocW Colonies and Groups

Most group-living species exhibit far less social organization and subdivision
of labor than do the eusocial hymenopterans and mole-rats, but genetic related-
ness among group members remains of interest. For example, eastern tent cat-
erpillars (Malacosoma americanum) are characterized by cooperative nest (tent)
building, as well as cooperative foraging along pheromonal trails. The adult
moths of this diploid species lay egg masses in trees, from which the first-instar
larvae emerge to feed on young leaves at the twig tips. Later, caterpillars move
to centralized locations to initiate tent construction. In a temporal genetic study
using allozymes, Costa and Ross (1993) found that the mean genetic relatedness
within colonies of newly-emerged larvae (from a single egg mass) was r = 0.49,
not significantly different from the expected value of r = 0.50 for full siblings.
However, relatedness values declined over the next eight weeks, to r = 0.38 (or
to r = 0.25 if colonies on single-tent trees were disregarded). The temporal
reduction in intracolony relatedness represents an erosion of the initial simple
family structure, apparently due to the exchange of individuals among colonies
of a tree when foragers encounter pheromonal trails of nonsiblings. Results
indicate that foreigners (nonsiblings) are not overtly discriminated against by
these caterpillars, but rather can be accepted into a colony.
Day-roosting colonies of the bat Phyllostomus hastatus in Trinidad are sub-
divided into compact clusters of adult females that remain highly stable over
several years and are attended by a single adult male who from allozyme evi-
dence fathers most of the babies born to females within the harem (McCracken
and Bradbury, 1981). The stable groups of adult females are the fundamental
units of social structure in this species and it has been hypothesized that they
arise from active cooperative interactions among females on shared foraging
grounds. Are the harems composed of matrilineally related individuals such that
200 Kinship and Intraspecific Phylogeny

kin selection should be considered as a possible factor influencing social or

cooperative behavior? Using allozyme assays, McCracken and Bradbury (1977 ,
1981) demonstrated that females within a harem are unrelated and represent
random samples from the total adult population. Combined with field observa-
tions, results indicate that juveniles are not recruited into parental social units and
that contemporary kin selection, therefore, cannot explain the maintenance of
behavioral cohesiveness in these highly social mammals.
In some other mammalian species, females comprising a group or colony are
close relatives. For example, black-tailed prairie dogs (Cynomys ludovicianus)
live in social groups called coteries that typically consist of one or two adult males
born in foreign coteries and of several adult females and young that are closely
related due to a strong tendency by females to remain within the natal coterie for
life (Hoogland and Foltz, 1982). Genetic analyses (based on pedigree and al-
lozyme data) verified that despite the matrilineal population structure, colonies are
outbred due to coterie-switching by males and the avoidance of male-daughter
matings (Foltz and Hoogland, 1981, 1983; see also Chesser, 1983). Several other
ground-dwelling squirrels (in the family Sciuridae) also have varying degrees of
social organization built around matrilineal kinship (Michener, 1983). Evidence
on kinship in many other group-living animal species is reviewed by Wilson
(1975). Traditionally, such genealogical understanding has come from difficult
and labor-intensive field observations of mating and dispersal (Fletcher and
Michener, 1987), but as in the black-tailed prairie dog, in several cases molecular
markers have been employed as additional sources of information.
For example, DNA-fingerprinting assays have been applied to prides of Af-
rican lions that from prior field observations were thought to exist as matriarchal
groups (Gilbert et aI., 1991; Packer et aI., 1991). A lion pride typically contains
two to nine adult females, their dependent young, and a coalition of two to six
adult males that have joined the pride from elsewhere. Incoming males collab-
orate to evict resident males and often kill the dependent young from the prior
coalition. Based on observed proportions of bands shared in multilocus DNA
fingerprints of nearly 200 animals, it was concluded that female companions
within prides always are related closely, male coalition partners either are related
closely (particularly within larger coalitions) or are unrelated (in some of the
smaller coalitions involving only two to three males), and mating partners usu-
ally are unrelated (Fig. 6.2). Furthermore, fingerprinting analyses of parentage
revealed that resident males fathered all cubs conceived during their tenure and
that the variance in male reproductive success increased greatly as coalition size
increased. From these observations, the authors concluded that males only act as
nonreproductive "helpers" when coalitions are composed of close relatives.
Several points should be made about these important genetic studies. First,
unlike most analyses cited earlier that estimated mean relatedness within groups
based on genotype frequencies at particular allozyme or other loci, the multilocus
 Kinship and Intraspecific Phylogeny 201

40
(a)
30

20

10

20
>-
(.)
(b)
15
C
CD
:s 10

-
C"
...
G) 5

60
o partners born in same pride
(C) _ born in different prides
45 ~ partners of unknown origin
~ males from neighboring pride
30

15

0
30 40 50 60 70 80 90 100

bands shared (%)

Figure 6.2. Frequency distributions of mini satellite band-shar-
ing in Serengeti lions (after Packer et al., 1991). Band-sharings
are indicated between (a) females born in the same versus dif-
ferent prides, (b) male coalition partners known to have been
born in the same versus different (or in some cases unknown)
prides, and (c) coalition males and resident females.
202 Kinship and Intraspecific Phylogeny

DNA-fingerprinting approaches allowed assessments of genetic relatedness

among particular pairs of individuals. Second, the extent of minisatellite band-
sharing initially was calibrated against known or suspected kinship among lions
for which long-term field observations and pedigrees were available. Third, such
calibrations between band-sharing and r proved to be nonlinear and also differed
between the two lion populations studied (in the Serengeti National Park and the
Ngorongoro Crater). Fourth, the large variances in levels of band-sharing among
individuals with a given known r meant that for lions of unknown relationship,
only the most general of kinship classes (nonrelatives, intermediate relatives, or
very close kin) could be inferred. Overall, notwithstanding the considerable
genetic insights gained on lion prides, these studies also indicate that the mul-
tilocus fingerprinting approach has important limitations for precise estimation of
r between individuals and normally will require independent background infor-
mation and population-specific calibration for proper interpretation.
One reason that DNA-fingerprint calibrations may differ among populations,
and "unrelated" individuals exhibit varying levels of background band-sharing,
could involve earlier populational histories including possible bottlenecks and
inbreeding. An extreme example appears to involve the dwarf fox Urocyon
littoralis, which has colonized several Channel Islands off Southern California
within the last 20,000 years. All assayed foxes from one of the smallest and most
isolated islands, San Nicolas, exhibited identical bands in multilocus DNA fin-
gerprints, and several other island populations also showed greatly enhanced
levels of band-sharing (75-95%) relative to foxes from different islands (16-
56%) and relative to values typifying outbred populations of many other verte-
brate species (10-30%) (Gilbert et aI., 1990). Patterns of band-sharing between
islands also produced an estimated phylogeny that agreed well with archaeozo-
ological and geological records concerning the history of island colonization for
this species. Results indicate that multilocus DNA fingerprinting approaches can
in some cases provide useful information on genetic relationships within and
among small, isolated, and potentially inbred populations. Nonetheless, attempts
to utilize these multilocus fingerprint assays for assessing relationships in larger,
outbred populations have met with little success, primarily because the band
variability is simply too complex (Baker et al., 1992; Prodohl et aI., 1992).
Caution also is indicated because van Pijlen et al. (1991) have found that dif-
ferent DNA-fingerprint probes sometimes can produce grossly different esti-
mates of band-sharing from the same array of populations.
Most cetacean species (whales and dolphins) live in social groups called pods,
and these have been the subject of several genetic studies using DNA finger-
printing and other molecular methods (Hoelzel, 1991a). In the long-finned pilot
whale (Globicephala melas), social groups typically consist of 50-200 animals
whose strong herding instincts have been exploited by native peoples to drive
entire pods into shallow bays for mass slaughter. Preliminary DNA-fingerprint
 Kinship and Intraspecific Phylogeny 203

analyses of tissue samples from several such Faroe Island harvests revealed that
adult males are not related closely to adult females within a pod, and furthermore
that nearly 90% of fetuses could not have been fathered by a resident male (Amos
et al., 1991a, 1991b). From these and additional behavioral observations, the
authors conclude that social groups in the pilot whale are built around matrilineal
kinship, with considerable interpod genetic exchange mediated by males. Ma-
trilineal relationships within other cetacean species have been examined more
directly by mtDNA analyses. For example, among pods of killer whales (Orcinus
orca) near Vancouver Island, British Columbia, two distinct mtDNA types
have been observed that in preliminary assays appear to correspond to a long-
recognized behavioral distinction between sympatric groups with fish-hunting
versus mammal-hunting social traditions (Hoelzel, 1991b; Hoelzel and Dover,
1991b). On the other hand, in similar mtDNA analyses of groups of spinner
dolphins (Stenella longirostris) , no clustering of matrilines within specific schools
or neighboring morphotypes was detected, suggesting significant and recent
genetic interchange (Dizon et al., 1991).
Questions concerning kinship within groups also can arise in plants. The
whitebark pine (Pinus albicaulis) often displays a multistem form, and the stems
within a clump have proved to be genetically distinct individuals that are allo-
zymically more similar to one another than to individuals in other clumps (Fur-
nier et aI., 1987). This family structure appears to be a direct result of the
seed-caching behavior of the primary dispersal agent for the whitebark pine
seeds-the Clark's nutcracker bird. Limber pines (Pinus fiexilis) also frequently
exhibit a multiple-trunk growth form that may result either from damage to
leader shoots of a single genetic individual or to growth from mUltiple zygotes
(seeds) deposited in caches by birds. From allozymic analyses, nearly 20% ofthe
multitrunk clusters included two to four genetically different individuals and the
mean genetic relatedness within these clusters was estimated to be r = 0.19, or
slightly less than expected for half-sibs (Schuster and Mitton, 1991). The authors
note that such grouping of related individuals offers a potential for interactions
such as kin selection or sib competition, factors seldom considered for plants.

Kin Recognition

The occurrence in many species of groups of close kin raises additional ques-
tions about whether individuals can somehow assess their genetic relatedness to
others, and perhaps adjust competitive, cooperative, altruistic, or other behaviors
accordingly (Waldman, 1988; Wilson, 1987). In studying such issues, ethologists
monitor behavioral interactions among organisms exhibiting varying levels of
genetic relatedness. The great majority of such analyses has employed study
organisms whose relatedness was known or suspected from direct field obser-
vations or from pedigree records in captive settings (Fletcher and Michener, 1987;
204 Kinship and Intraspecific Phylogeny

Hepper, 1991). However, in a few cases, molecular genetic markers have assisted
with the relatedness assignments. For example, in the clusters of limber pines
described above, natural fusions or grafts among woody tissues from different
trunks commonly are observed. From allozyme data, Schuster and Mitton (1991)
discovered that fused trees were related significantly more closely than trees that
were unfused. Whether such fusions are adaptively advantageous for the interac-
tants (e.g., though translocation of water and nutrients, or added physical stability)
and, hence, may have evolved through kin selection, remains an open issue.
In a free-living population of Belding's ground squirrels (Spermophilus bel-
dingi) in California, Holmes and Sherman (1982) employed protein-electro-
phoretic techniques to distinguish full siblings from maternal half-sibs (resulting
from multiple mating). Subsequent behavioral monitoring indicated that full
sisters fought significantly less often and aided each other more than did half-
sisters. Such nepotism (favoritism shown kin) must require an ability by ground
squirrels to judge relatedness. Additional experiments indicated that the proxi-
mate cues by which this is accomplished in S. beldingi appear to involve physical
association during rearing, as well as "phenotypic matching," whereby an in-
dividual behaves as if it had compared phenotypic traits (genetically determined)
against itself or a nestmate template (Holmes and Sherman, 1982).
Another postulated advantage to kin recognition involves behavioral avoidance
of close inbreeding (Hoogland, 1982). Like many amphibians, the American toad
(Bufo americanus) exhibits site fidelity to natal ponds for breeding and, thus,
individuals are likely to encounter siblings as potential mates (Waldman, 1991).
Can siblings recognize close kin and avoid incestuous mating? Waldman et al.
(1992) monitored mtDNA genotypes in 86 amplexed pairs of toads and found
significantly fewer matings between possible siblings (haplotypes shared) than
expected from the haplotype frequencies in the local population. From this
preliminary genetic evidence, the authors suggest that •• siblings recognize and
avoid mating with one another. " They further suggested that the proximate cues
employed might include advertisement vocalizations by males because the re-
semblance among male calls proved to be positively correlated with genetic
relatedness as assessed by band similarities in nuclear DNA fingerprints. Thus,
females potentially could employ male vocalizations (and/or other genetically-
based clues such as odors) in kinship assessment.

GEOGRAPIDC POPULATION STRUCTURE AND GENE FLOW

Populations of nearly all species, social or otherwise, exhibit at least some

degree of genetic differentiation among geographic locales (Ehrlich and Raven,
1969), if for no other reason than because siblings usually tend to begin life
spatially near one another and their parents, and mating partners seldom repre-
 Kinship and Intraspecific Phylogeny 205

sent random draws from across the geographic range of a species (Turner et al.,
1982). In an influential early study of microgeographic population structure
based on allozymes, Selander (1970) demonstrated fine-scale spatial clustering
of genotypes of house mice (Mus musculus) within and among barns on the same
farm, apparently due to tribal family structure and genetic drift in small popu-
lations. Such genetic structure sometimes exists even in the most improbable of
settings. For example, mosquitofish (Gambusia affinis and G. holbrooki) are
abundant and highly dispersive, yet large conspecific samples revealed statisti-
cally significant differences in allozyme allele frequencies over a few hundred
meters of shoreline in a stream or reservoir (Kennedy et al., 1985, 1986); also,
significant temporal variation at a locale was observed over periods of a few
weeks to years (McClenaghan et al., 1985). Across broader geographic scales,
populations can show additional differentiation due to spatial habitat structure,
isolation by distance, or other factors. Thus, further genetic structure in mosqui-
tofish, hierarchically arranged, characterized populations across ponds and
streams within a local area, reservoirs within a river drainage, drainages within
a region, and regional collections of drainages that house deep genetic differ-
ences associated with species-level separations probably dating to the Pleistocene
(Scribner and Avise, 1993a; M.H. Smith et aI., 1989; Wooten et al., 1988).
Molecular analyses of geographic population structure similarly have been con-
ducted on hundreds of animal species at a variety of spatial and temporal scales.
Populations of most plant species also vary in genetic composition, sometimes
over microspatial areas of a few kilometers or even meters (Levin, 1979). For
example, large populations of the wild wheat Triticum dicoccoides show pro-
nounced differences in genetic structure over distances of less than 5 km, due, in
part, to limited gene flow and to a self-fertilization reproductive mode (Golen-
berg, 1989). The grasses Agrostis tenuis and Anthoxanthum odoratum show
sharp clinal variation in several genetically based characters across the meter-
wide ecotones between pastures and lead-zinc mines, as a result of strong dis-
ruptive selection for heavy-metal tolerance and flowering time (Antonovics and
Bradshaw, 1970; McNeilly and Antonovics, 1968). In many species, effective
gene flow via pollen and seed dispersal is sufficiently limited that estimates of
neighborhood size (the population within which mating is random) commonly
include less than a few hundred individuals, occupying areas less than 50 m2 (Bos
et al., 1986; Calahan and Gliddon, 1985; Fenster, 1991; Levin and Kerster,
1971, 1974; Smyth and Hamrick, 1987). As with animal populations, additional
genetic structure normally is to be expected over increasing spatial scales.
A continuing challenge is to describe population genetic architectures within
species (Box 6.3) and to identify and order the evolutionary forces responsible in
particular instances. Broadly speaking, these forces involve migration or gene
flow (Box 6.4), random genetic drift, various modes of natural selection, mu-
tational divergence, and the opportunity for genetic recombination mediated by
206 Kinship and Intraspecific Phylogeny

Box 6.3. Descriptive Statistics for Population Structure

Several approaches to the statistical description of population structure are available
(Sokal and Oden, 1978a, 1978b; Weir, 1990). For simplicity, we will confine attention
to "F-statistics" because these have been employed most widely. Wright (1951)
introduced a method of describing the genetic population structures of diploid organ-
isms in terms of three F-statistics or allelic correlations, FIS ' FIT' and FST' whose
theoretical interrelationships can be written as

(6.4)
Wright defined F IS as the correlation between homologous alleles within individuals
with reference to the local population, and FIT as the corresponding allelic correlation
with reference to the total population. FIS and FIT are often called fixation indices (FI).
Their estimated values also may be interpreted as describing departures from the
expected Hardy-Weinberg genotypic frequencies within local populations and within
the total population, respectively (Nei, 1973, 1977):
(6.5)
where hobs and hexp are the observed and expected frequencies of heterozygotes at a
locus (Box 2.1). Thus, positive values for fixation indices indicate positive correla-
tions among uniting gametes (heterozygote deficits), likely due to local inbreeding
(FIS)' or local inbreeding plus population subdivision (FIT).
F ST may also be interpreted as the variance (Vp) of allele frequencies among pop-
ulations, standardized relative to the maximum value possible given the observed
mean allele frequency (ji):

FST = V/fi (1 - p). (6.6)

This statistic is used commonly as a measure of population subdivision, and as shown
in Box 6.4 provides a convenient approach for estimating interpopulational gene flow
in models that assume selective neutrality. The reader should be alerted to the many
nuances (beyond the scope of current discussion) involved in calculating F-statistics
from empirical data, such as how to handle the difficulties of multiple alleles at a
locus, multiple loci, and various sources of sampling error within and among subpop-
ulations (Weir and Cockerham, 1984).

the mating system. Finer considerations require a partitioning of these general

categories into biological factors relevant for each group of organisms. For
example, numerous ecological and life history factors are predicted to influence
plant population structures (Table 6.2), and in a comparative summary of more
than 150 empirical studies of 124 plant taxa based on allozymes, Loveless and
Hamrick (1984) found that interpopulational genetic differences indeed generally
were consistent with predictions based on a plant's breeding system, floral mor-
phology, life cycle, timing of reproduction, and successional stage (see also
 Kinship and Intraspecific Phylogeny 207

Box 6A. Genetic Exchange Among Populations

Gene flow is the transfer of genetic material between populations resulting from
movements of individuals or their gametes. Usually, gene flow is expressed as a
migration rate m, defined as the proportion of alleles in a population each generation
that is of migrant origin. Gene flow is notoriously difficult to monitor directly, but
commonly is inferred from spatial distributions of genetic markers by several statistical
approaches. Most of these approaches are based on equilibrium expectations derived
from theoretical models of population structure under neutrality theory, e.g., the
"island model" wherein a species is assumed to be subdivided into populations
(demes or islands) of equal size N, all of which exchange alleles with equal proba-
bilities, or the "stepping-stone" model wherein only adjacent demes exchange alleles.
Allelic frequencies in finite populations are influenced by random genetic drift also,
which is a function of effective population size (Box 2.2). The influences of drift and
gene flow are difficult to tease apart and, thus, most statistical procedures as applied
to spatial genetic information permit estimates only of the product Nm, which can be
interpreted as the absolute number of individuals exchanged between populations per
generation. Also, Nm is of particular interest because under neutrality theory, at
equilibrium the level of divergence among populations is a function of the numbers of
migrants rather than the proportions of individuals exchanged. The most common
approaches to Nm estimation are as follows:
(a) From F -statistics. Wright (1951) showed that for neutral alleles in an island
model, equilibrium expectations are:

FST == 1 / (1 + 4Nm) or Nm == (1 - F ST ) / 4FsT . (6.7)

Nei (1973) defined a related measure of between-population heterogeneity (gene di-

versity or GST ) that bears the same relationship to Nm and also is employed widely.
Takahata and Palumbi (1985) suggested modifications of these basic statistics for
extranuclear haploid genomes such as mtDNA, and Lynch and Crease (1990) proposed
an analogue of the F ST or GST indices (NST) that is applicable to any data at the
nucleotide level.
(b) From private alleles. Private alleles are those found only in one population.
Slatkin (1985a) showed by computer simulations of a variety of artificial populations
that the logarithm of the average frequency of private alleles [p( I) 1is related approx-
imately linearly to the logarithm of Nm:

In [P(1)] = -0.505 In (Nm) - 2.440 (6.8)

This result proved insensitive to most changes in parameters of the model, except that
a correction for Nm due to differences in the mean number of individuals sampled per
population was recommended (see Barton and Slatkin, 1986). The basic rationale
underlying Slatkin's (l985a) method is that private alleles are likely to attain high
frequency only when Nm is low. In practice, when sufficient genetic information is
available, the F ST and private allele methods are expected to yield comparable esti-
mates of gene flow under a wide variety of population conditions (Slatkin and Barton,
1989).
208 Kinship and Intraspecific Phylogeny

(c) From allelic phylogenies. Unlike the two approaches described above that
can be applied to phylogenetically unordered alleles (such as those provided by allo-
zymes), this method requires knowledge of the phylogeny of nonrecombining seg-
ments of DNA (such as mtDNA haplotypes). Given the correct gene tree and knowl-
edge of the geographic populations in which the allelic clades are found, a parsimony
criterion is applied to estimate the minimum number of migration events consistent
with the phylogeny. Slatkin and Maddison (1989) show that the distribution of this
minimum number is a simple function of Nm, which therefore can be estimated from
empirical data by comparison to tabulated results from their computer-simulated pop-
ulations.
None of these procedures should be interpreted as providing a precise estimate of
genetic exchange among demes; rather, each offers a qualitative (albeit numerical)
guideline as to whether populations likely experience high, moderate, or greatly re-
stricted gene flow. Roughly speaking, the average exchange of one individual per
generation (Nm == 1) between populations, irrespective of deme size, is marginally
sufficient in theory to prevent dramatic genetic differentiation by genetic drift alone
(Allendorf, 1983). The value Nm = 1 corresponds to mean FST = 0.20 [Eq. (6.7)]
or p(l) == 0.085 [Eq. (6.8)]. Thus, as a rule of thumb, "high-gene-flow" species are
expected to exhibit lower estimates of F ST and p(1) than these respective values,
whereas "low-gene-flow" species should display much higher values. Additional
discussion of these procedures for gene flow estimation may be found in Hudson et al.
(1992) and Slatkin (1985b, 1987).
Neigel et al. (1991) introduced a philosophically different approach to gene flow
analysis that yields an estimate of single-generation dispersal distance (rather than
Nm). The method is based on expected spatial distributions of lineages of various
evolutionary age in a gene tree, assuming an evolutionary clock for the molecule and
assuming that dispersal of lineages has occurred via a multigeneration "random-walk"
process from specifiable centers of origin for each clade. As applied to an empirical
mtDNA gene tree for continent-wide populations of the deer mouse Peromyscus man-
iculatus (Lansman et al., 1983), this method yielded estimates of single-generation
dispersal (== 200 m) that agree well with direct mark-recapture data for this species.
This approach should be most suitable for low-dispersal species and for rapidly-
mutating, nonrecombining genetic markers such as mtDNA. In such situations, mu-
tations that delineate new descendent lineages may be dispersed at rates sufficiently
low to prevent the attainment of an equilibrium between genetic drift and gene flow
that many of the earlier models assume.
Table 6.2. Examples of ecological and life history factors and their predicted
influencesa on the population genetic structures of plants (after Loveless
and Hamrick, 1984).
Genetic Genetic Genetic
Heterozygosity Structure Structure
Within Within Among
Factor Populations Populations Populations

Breeding System
Self-fertilizing Low High High
Mixed mating Moderate Moderate Moderate
Outcrossing High Low Low
Floral Morphology
Monoecious Depends on % selfing Depends on % selfing Depends on % selfing
Dioecious High Low Low
Reproductive Mode
Apomictic Depends on other Depends on other Potentially high
factors factors
Sexual Potentially high Depends on other Depends on other
factors factors
Pollination Mechanism
Sedentary animal Potentially low Potentially high High
Dispersive animal High Low Low
Wind High Low Low
Seed Dispersal
Limited 11 Potentially high High
Long range High Low Low
Seed Dormancy
Absent Depends on other Depends on other Depends on other
factors factors factors
Present Increases potential Reduces potential Reduces potential
Phenology
Asynchronous No prediction Increases potential Increases potential
Synchronous No prediction Reduces potential Reduces potential
Life Form
Annual Reduced?? Increases potential Increases potential
Long-lived Increased ?? Reduces potential Reduces potential
Timing of Reproduction
Monocarpicb No prediction Increases potential Increases potential
Polycarpic No prediction Reduces potential Reduces potential
Successional Stage
Early ?? Depends on other Increases potential
factors
Late ?? Depends on other Reduces potential
factors
Geographic Range
Narrow endemic Low Low High
Widespread Potentially High Depends on other Depends on other
factors factors
Population Size, Density
High High Depends on other Depends on other
factors factors
Low Low Depends on other Depends on other
factors factors

"Predictions remain qualified because categories may be interrelated and other confounding vari-
ables may pertain.

~aving only one fruiting period during the life cycle.

210 Kinship and Intraspecific Phylogeny

Hamrick and Godt, 1989). Similarly, a comparative summary of the allozyme

literature for more than 300 animal species (Table 6.3) led Ward et al. (1992) to
conclude that mobility is an important factor influencing the apparent magnitude
of population structure. Thus, many insects and birds, which tend to be vagile
organisms, show significantly lower levels of mean population structure than do
relatively sedentary creatures such as some amphibians (Table 6.3). In the sec-
tions that follow, a few illustrative cases are highlighted that address how par-
ticular demographic or other factors may impinge on molecular population ge-
netic structure. Where possible, attempts will be made to draw parallels between
results for taxonomically distinct organisms, such as plants and animals.

Autogamous Mating Systems

In a classic series of studies employing allozyme markers, Allard and col-

leagues documented the dramatic influence that the mating system can assume,
in conjunction with natural selection, in shaping the multilocus genetic archi-
tecture of plant species. The slender wild oat (A vena barbata) is a predominantly
self-fertilizing species, first introduced from its native range in the Mediterranean
to California during the Spanish period some 400 years ago, and more exten-
sively during the Mission period some 200-250 years ago. Within California, it

Table 6.3. A comparative summary of population structuresG for 321 animal species
surveyed by multilocus protein electrophoresis (after Ward et al., 1992).

Population Differences8 No. of

Taxonomic Group (with Standard Errors) Species

Vertebrates
Mammals 0.242 ± 0.030 57
Birds 0.076 ± 0.020 16
Reptiles 0.258 ± 0.050 22
Amphibians 0.315 ± 0.040 33
Fishes 0.135 ± 0.040 79
TOTAL 0.202 ± 0.015 207
Invertebrates
Insects 0.097 ± 0.Q15 46
Crustaceans 0.169 ± 0.061 19
Mollusks 0.263 ± 0.036 44
Others 0.060 ± 0.021 5
TOTAL 0.171 ± 0.020 114
aShown are the proportions of total genetic variation within species due to genetic differences between
geographic populations, as reflected in the "coefficient of gene differentiation": (HT - Hs) I H T' where
Hs and HT are the mean heterozygosities estimated within local populations and within the entire
species, respectively (Nei, 1973).
 Kinship and Intraspecific Phylogeny 211

has since achieved a remarkable population-genetic structure characterized by a

great predominance of two apparently coadapted multilocus gametic types (Al-
lard et al., 1972; Clegg and Allard, 1972). One genotype, "1,2,2,2,I,B,H"
(where the numbers and letters refer to alleles at each of five allozyme loci and
two morphology genes), is characteristic of the semiarid grasslands and oak
savannahs bordering the central Sacramento-San Joaquin Valley, whereas the
complementary gametic type ("2,1,1,1,2,b,h") is more common in the strip of
coastal ranges and the higher foothills of the Sierra Nevada mountains.
These associations between genotype and the environment (notably xeric versus
mesic soils) also are maintained over microgeographic scales in transitional
(ecotonal) areas (Hamrick and Allard, 1972), notwithstanding the continued
production of recombinant genotypes through occasional outcrossing [the esti-
mated outcrossing rate is t = 0.02 (Clegg and Allard, 1973)]. The authors
conclude that genetic variability in A. barbata in California has been internally
organized and spatially structured within just 400 years by intense natural selection
operating in conjunction with severe constraints on recombination afforded by the
mating system (Allard, 1975). These studies added great empirical force to
theoretical arguments that any factors such as linkage or inbreeding that tend to
restrict intergenic recombination can facilitate the operation of natural selection
in moulding coadapted multilocus gene complexes (Box 6.5; Clegg et al., 1972;
Jain and Allard, 1966).
"Autogamy" refers to the process of self-fertilization or self-pollination. In a
review of the literature on spatial genetic variation in plants, Heywood (1991)
concluded that autogamous species often display remarkable levels of local ge-
netic differentiation that far surpass those of predominantly outcrossing species.
For example, in the annual plant Plectritis brachystemon that is highly autoga-
mous (outcrossing rate 2%), most of the allozyme diversity proved to be inter-
populational; whereas in the sympatric congener P. congesta that is predominantly
allogamous (outcrossing rate 70%), most of the allozymic diversity is intrapop-
ulational (Layton and Ganders, 1984). Another conclusion was that the spatial
genetic architectures observed for predominantly autogamous species frequently
are similar to those of taxa that reproduce by a mixture of apomixis (Chapter 5)
and sexual outcrossing (Heywood, 1991). Especially when such species occur in
low-density situations, the effects of genetic drift, selection on multilocus gen-
otypes, and the restricted recombination engendered by the mating system, all can
collaborate to produce striking spatial variation in genotypic distributions.
Analogous population genetic analyses have been conducted on a hermaph-
roditic land snail that also reproduces by facultative self-fertilization (Rumina
decollata), and these studies have demonstrated the potential for adaptive con-
vergence in the genetic architectures of animals and plants (Selander and
Kaufman, 1975a; Selander et al., 1974). This gastropod, native to the Mediter-
ranean region, was introduced to the eastern United States before 1822 and
212 Kinship and Intraspecific Phylogeny

Box 6.5. Self-Fertllizadon, Restricted Genedc Recomblnadon, and

Muldlocus Organlzadon
Self-fertilization is the most extreme fonn of inbreeding. One consequence of pre-
dominant selting is a severe restriction on inter-genic recombination, and an associated
enhanced opportunity for the maintenance of coadapted gene complexes, as can be
illustrated qualitatively as follows. Assume that at each of four di-allelic loci, the
haploid genotypic combinations 1,1, 1,1 and 2,2,2,2 (numbers refer to the allele at
each locus) confer high viability on their diploid bearers and that the other 14 multi-
locus haplotypes (l ,2, 1, 1; 2,1,2,1; etc.) lead to adaptively inferior individuals. During
the course of a generation, the favored genotypes will tend to increase in frequency
under natural selection from early to late stages in the life cycle. However, under
random outcrossing, recombination at gametogenesis will tend to undo the effects of
selection in maintaining the favored multigene complexes across generations. This
process of decay in gametic phase disequilibrium (D) can be inhibited if the loci are
physically linked on a chromosome because intergenic recombination then may be
restricted (Box. 5.3). Inbreeding similarly retards the rate at which D converges to
zero, by limiting effective recombination. For two loci under a model of mixed self-
mating and random mating, this rate is given by
1 - 0.5 {0.5 (1 + A + s) + [(0.5 (1 + A + S»2 - 2SA]0.5}, (6.9)
where s is the selting probability and A is the amount of linkage (for A = 0, the
recombination fraction is c = 0.5) (Weir and Cockerham, 1973). For example, with
s = 0.98 and A = 0.00 (or with s = 0.00 and A = 0.98), the rate of decay of D is
1.0% per generation. The point is that linkage and selting can be seen to enter this
equation similarly and, therefore, they retard disequilibrium decay in similar fashion.
One important difference between the effects of linkage versus inbreeding in re-
stricting recombination is that the fonner acts locally among physically linked loci,
whereas the latter acts globally, restricting recombination between all loci whether on
the same or different chromosomes. Thus, in theory, inbreeding in conjunction with
selection could serve to organize entire genomes into integrated multilocus systems
(Allard, 1975).

subsequently has spread across much of North America. In its native range, R.
decollata exists as a complex of inbred monogenic or mildly polygenic strains
characterized by different suites of allozymic and morphological markers. Two
strains ("light" and "dark") predominate in intensively surveyed areas in south-
ern France and show fixed differences at 13 of 26 allozyme loci. The dark form
typically occupies protected mesic environments such as under logs or rocks,
whereas the light form is associated with open xeric habitat. Occasional out-
crossing between these strains releases extensive recombinational variation that
otherwise is expressed as between-strain genetic differences. Nevertheless, the
two forms tend to retain their separate identities and habitat correlations in nature,
 Kinship and Intraspecific Phylogeny 213

suggesting that, as in Avena barbata, strong multilocus associations and pro-

nounced population genetic structures probably stem from natural selection [as
well as stochastic population factors (Selander, 1975)] operating in conjunction
with the self-fertilizing breeding system that reduces effective recombination
(Selander and Hudson, 1976). As a further note of interest, some inbred strains
of R. decollata apparently have achieved extensive geographic distributions and
high population numbers in the absence of appreciable genetic variation; all
assayed populations in North America are allozymically identical and, thus,
probably derive from a single strain introduced from Europe (Selander and
Kaufman, 1973b).

Gametic and Zygotic Dispersal

POLLEN AND SEEDS

In outcrossing plants, mobile male gametes (pollen) are transferred to sedentary

eggs in the female flower by wind or by animal vectors such as insects, birds, or
mammals, some of which may be capable of moving long distances. The zygotes
(seeds) produced then are dispersed from the maternal parent either by gravity,
wind (sometimes assisted by a winged or plumose seed condition), or by animals
(via seed attachment to the vector's body or passage through the digestive system).
To what extent do varying dispersal mechanisms for gametes and seeds influence
gene flow regimes and genetic structures of plant populations?
Most tropical trees outcross predominantly, with pollination and seed dispersal
usually animal-mediated (Hamrick and Murawski, 1990). Using allozyme data,
Hamrick and Loveless (1989) compared the population genetic structures in
several outcrossing species of trees and shrubs in Panama. The authors rank-
ordered 14 species with respect to a priori predictions about expected interpop-
ulational genetic structure based primarily on considerations of pollen and seed
dispersal mechanisms and found that the empirical estimates of gene flow [Nm
(Box 6.4)] were correlated significantly with this ranking (Fig. 6.3). They con-
cluded that geographic population structure appeared greater in species with
weak-flying pollinators and anemic means of seed dispersal. The few wind-
pollinated species examined also had relatively strong population structures,
consistent with earlier suggestions that wind may not be a particularly effective
agent for pollen flow in the low-density populations of conspecifics in tropical
forests. Nonetheless, the correlations between reproductive biology and genetic
structure were moderate at best, explaining far less than 50% of the overall
variance in outcomes. Furthermore, over the spatial scales of several kilometers
monitored, all species exhibited Nm > 1.0. This raises the more fundamental
point that gene flow appears moderate to high in most populations of these
tropical forest trees.
214 Kinship and Intraspecific Phylogeny

12

10
• •
~ 8

- •
'1:J
CD 6
C\J
• •• • •
E
; 4 •
(II
CD
2 • •• ••
TROPICAL TREES
0 Figure 6.3. Relationship be-
2 4 6 8 10 12 14 tween gene flow and life history
mediated dispersal potential.
predicted rank order of
Top: Fourteen species of trop-
population structure
ical trees and shrubs (data from
Hamrick and Loveless, 1989);
Bottom: Nine species of marine
shore fishes (Waples, 1987).
> 30 • • • • • Species are plotted in rank order
according to the predicted mag-
nitude of popUlation structure
14 • based on suspected dispersal ca-
pabilities of pollen and seeds
12
~ (plants) or larvae (fishes). Gene
flow (Nm) values were esti-

-
10
'1:J mated from allozyme data using
CD
C\J 8
• Wright's FST approach (Box
E 6.4). The Spearman's coeffi-
Ui cients of correlation between
•
6
CD
N m and predicted rank were r =
4 MARINE FISHES 0.68 (P < O.Ol,plants)andr =
0.88 (P < 0.01, fishes). Cau-
2 tion is indicated, however, be-
0 • cause Nm values estimated from
Slatkin's private-allele ap-
2 3 4 5 6 7 8 9
proach for these same species
predicted rank order of were not significantly correlated
population structure with dispersal rank.
 Kinship and Intraspecific Phylogeny 215

Temperate trees, more of which are wind pollinated, also commonly yield
moderate to high estimates of gene flow. For example, among populations of
pitch pine (Pinus rigida) throughout the species' range in the eastern United
States, mean FST across allozyme loci was only 0.024 (Guries and Ledig, 1982),
a value associated with Nm = 10.2. Other wind-pollinated pines that exhibit low
among-population differentiation across broad and continuous ranges include P.
banksiana [Nm = 6.7 (Dancik and Yeh, 1983)], P. contorta [Nm = 8.1
(Wheeler and Guries, 1982)], and P. ponderosa [Nm = 16.4 (Hamrick et al.,
1989)]. On the other hand, pine species whose populations are distributed as
scattered isolates tend to show far greater spatial structure, as might be expected,
and this is reflected in lower estimates of gene flow: P. torreyana, Nm = 0
(Ledig and Conkle, 1983); P. halapensis, Nm = 0.6 (Scheller et al., 1985); and
P. muricata, Nm = 1.0 (Millar, 1983).
Hamrick et al. (1992) reviewed published allozyme data on conspecific pop-
ulation structures within 322 woody plant species. The overall mean estimate for
the interpopulational component of genetic diversity was GST = 0.085 (Nm =
2.7). However, only 16% of the heterogeneity in genetic structure across species
could be accounted for by differences in seven life history and ecological traits
considered. The authors conclude that other influences, including the specific
and idiosyncratic evolutionary histories of species, must have played important
roles in determining how genetic diversity is partitioned. Most woody species are
long-lived and self-fertilization is rare. Another review of the broader allozyme
literature for 449 herbaceous and woody plants permitted breeding system and
life form to be added to the list oflife history influences considered (Hamrick and
Godt, 1989). Across this wider taxonomic scale of comparison, the most im-
portant predictors of the magnitude of interpopulational structure were the selfing
versus outcrossing breeding system and annual versus perennial life form. On
average, 146 annual species showed GST = 0.357 (Nm = 0.45) and 78 selfing
species showed GST = 0.510 (Nm = 0.24). Thus, plant species that live for
short periods and self-fertilize tend to exhibit far greater spatial structure than
those with contrasting features.

MARINE GAMETES AND LARVAE

Many marine invertebrates and fishes shed gametes and larvae into the water
column, in analogous fashion to the atmospheric release of pollen and seed by
many land plants. The planktonic duration varies widely for different species.
For example, larvae of the sessile polychaete Spirorbis borealis remain free-
living for a few hours at most and are competent to settle immediately on release
from their parents, whereas larvae in another polychaete genus Phyllocha-
etopterus can delay settlement and metamorphosis in excess of one year (Schel-
tema, 1986). More usually, the planktonic larval duration of benthic marine
216 Kinship and Intraspecific Phylogeny

invertebrates lasts from a few days to weeks. Among marine fishes, larvae
commonly remain in the plankton for weeks or months (e.g., Victor, 1986), but
the remarkable leptocephalus larvae of some eels may remain pelagic for three
years or more (Castle, 1984). On the other hand, other marine invertebrates and
fishes produce nonplanktotrophic eggs or larvae. These less-dispersive
propagules may be demersal or may be brooded by parents [e.g., in oral cavities
(marine catfishes), abdominal pouches (pipefishes and seahorses), or other stor-
age sites (many invertebrates)].
Are these widely varying potentials for gene flow via eggs and larvae corre-
lated with population genetic structures in marine species? Some evidence sug-
gests that they are. Thus, for invertebrates, several genetic studies have reported
a correspondence between increased potential for larval dispersal and diminished
genetic differentiation among geographic popUlations (Berger, 1973; Crisp,
1978; Gooch, 1975; Liu et al., 1991). For example, the larval-brooding snail
Littorina saxatilis exhibited greater interpopulational variation in allozymic and
morphologic characters than did a congeneric free-spawning species L. littorea
(Janson, 1987). In sea urchins of the genus Heliocidaris. one species (H. tuber-
culata) with a several-week planktonic larval stage showed little differentiation
in mtDNA genotypes between populations separated by 1000 km of open ocean,
whereas populations of a congener (H. erythrogramma) with only a tbree- to
four-day planktonic larval duration were strongly partitioned over comparable
geographic scales (McMillan et al., 1992) . Among the vertebrates, Ehrlich
(1975) noted that Pacific damselfishes with pelagic larvae show allozyme uni-
formity over huge areas, whereas the one assayed species that lacks a pelagic
larval phase (Acanthochromis polyacanthus) was highly structured genetically.
Waples (1987) assessed allozyme differentiation in several species of marine
shore fishes sampled along the same geographic transect in the eastern Pacific
and reported a strong negative correlation with dispersal capability inferred from
planktonic larval durations (Fig. 6.3). The species with the lowest potential for
dispersal (a livebearer with no pelagic larval stage-Embiotocajacksoni) exhib-
ited the highest spatial genetic structure, whereas a species with the highest
dispersal potential (a fish associated with drifting kelp and characterized by an
extended larval duration-Medialuna californiensis) exhibited no detectable spa-
tial genetic differentiation. Such results appear generally consistent also with the
long-standing observation that marine species with dispersive larvae show a
greater tendency to colonize oceanic islands and to exhibit broader geographic
ranges than those with sedentary larvae (Jablonski, 1986; Thorson, 1961; how-
ever, see Thresher and Brothers, 1985 for counterexamples).
Population genetic structures in North Atlantic eels have attracted particular
interest because of the extraordinary catadromous life histories of these species.
Juvenile eels (Anguilla rostrata in the Americas and A. anguilla in Europe)
inhabit coastal and inland waters, but during sexual maturation migrate to the
 Kinship and Intraspecific Phylogeny 217

Sargasso Sea (western tropical mid-Atlantic Ocean) where spawning takes place.
Conventional wisdom (reviewed by Williams and Koehn, 1984) is that the pe-
lagic larvae produced from this suspected mass spawn disperse passively to
continental margins via ocean currents, perhaps settling at locales randomly
oriented with respect to the homesteads of their parents. If mating is indeed
panmictic, and larval dispersal passive, all continental populations could repre-
sent random draws from a single gene pool and lack spatial structure accord-
ingly. To a first approximation, genetic data for A. rostrata collected throughout
eastern North America appear consistent with this scenario. Thus, Williams et al.
(1973) and Koehn and Williams (1978) observed only very mild spatial structure
at polymorphic allozyme loci (which they took as evidence for clinal selection in
a panmictic species), and Avise et al. (1986) detected no geographic structure in
mtDNA with the available sample sizes. However, American and European eels
proved to be clearly distinct genetically, confirming the much-debated presence
of at least two largely independent gene pools in the North Atlantic (Avise et al.,
1986). Further genetic analysis also revealed a low-frequency presence of hy-
brids between A. rostrata and A. anguilla in Iceland, an island longitudinally
intermediate to North America and Europe (A vise et al., 1990b). This unex-
pected appearance of hybrid eels in Iceland, thousands of kilometers from where
the zygotes presumably were produced in the Sargasso Sea, raises the intriguing
possibility of hybrid intermediacy in larval migratory behavior.
In general, a long-duration planktotrophic larval stage certainly affords the
opportunity for extensive gene flow, and this potential appears to have been
realized in many marine species as evidenced by a near absence of genetic
(allozyme or mtDNA) differentiation over vast areas: for example, among pop-
ulations of the sea urchin (Strongylocentrotus purpuratus) along the 2500-km
west coast of North America (Palumbi and Wilson, 1990); among popUlations of
the red rock lobster (Jasus edwardsii) across 4600 km of Australasian habitat
(Ovenden et al., 1992); within each of six species of reef fishes from locales
1000 km apart in the Caribbean (Lacson, 1992); among red drum populations
from the Atlantic and Gulf of Mexico seaboards in the southeastern United States
(Bohlmeyer and Gold, 1991; Gold and Richardson, 1991); among damselfish
(Stegastes Jasciolatus) populations throughout the 2500-km length of the Hawai-
ian archipelago (Shaklee, 1984); among milkfish (Chanos chanos) populations
from localities up to 10,000 km apart in the Pacific (Winans, 1980); between
populations of tuna (Katsuwonus pelamis and Thunnus alalunga) in the Atlantic
versus Pacific Oceans (Graves et al., 1984; Graves and Dizon, 1989); and among
global populations of the orange roughy fish (Hoplostethus atlanticus) (Smith,
1986). Extensive movement of adults no doubt also contributes to the geographic
uniformity in some of these fishes.
On the other hand, several other marine species with pelagic larvae do exhibit
dramatic population differentiation over microgeographic or macrogeographic
218 Kinship and Intraspecific Phylogeny

scales (Avise, 1987; Burton, 1983, 1986; Hedgecock, 1986). For example,
rock-pool inhabiting populations of the copepod Tigriopus californicus show
strong genetic (allozymic) differentiation both regionally and locally, notwith-
standing a natural history that includes free-swimming adult and larval forms
(Burton et aI., 1979; Burton and Feldman, 1981). Populations of the American
lobster Homarus americanus, whose pelagic larval stage lasts two to eight weeks,
show genetic differences between the Atlantic Ocean and the Gulf of St. Lawrence
in the northeastern United States (Tracey et aI., 1975). In the horseshoe crab
Limulus polyphemus, a dramatic genetic distinction in mtDNA exists between
continuously-distributed adult populations along the Gulf of Mexico and Atlantic
coasts in the southeastern United States (Saunders et aI., 1986), despite the
presence in this species of a trilobite larval stage that is "specialized for dispersal"
(Rudloe, 1979).
Significant but ephemeral genetic patchiness also characterizes local popUla-
tions of an intertidal limpet (Siphonaria sp.) and a sea urchin (Echinometra
mathaei) in western Australia (Johnson and Black, 1982; Watts et al., 1990),
despite macro geographic similarities in allele frequencies and the presence of
potentially dispersive planktonic larval stages. Such "chaotic patchiness" was
attributed to idiosyncrasies among local sites in histories of larval recruitment.
From a compilation of such examples, Burton (1983) concluded that although
invertebrate species with planktotrophic larvae tend to show less spatial hetero-
geneity than those with nonmotile larvae, "previously described relationships
between length of planktonic larval life and the geographic boundaries of pan-
mictic populations are not strongly supported. In particular, substantial differ-
entiation has been observed in several species that appear to have high dispersal
capabilities. "
There are several reasons why high dispersal potential of gametes or larvae
may not always translate into spatial population genetic homogeneity and the
attendant high estimates of gene flow (Hedgecock, 1986). First, actual levels of
gene flow may be lower than presumed because of physical impediments to
larval dispersal. Thus, the influences of particular oceanic currents in New En-
gland and in the southeastern United States may account in part for the genetic
differences reported between regional populations of the American lobster and
the horseshoe crab, respectively (see above). Second, larvae may not always be
the passive dispersal propagules commonly assumed, but rather may adopt more
active migrational behaviors and settlement choices in some species. Crisp
(1976) and Woodin (1986) review the available evidence for discriminatory
larval settlement in benthic intertidal and infaunal marine invertebrates, respec-
tively. Some larvae fall far short of their dispersal potential. For example, in the
shrimp Alphaeus immaculatus that has an extended free-swimming larval stage
but whose adults live symbiotically with sea anemones, a detectable proportion
of successful recruits settles on anemone colonies within a few meters of the
parents (Knowlton and Keller, 1986).
 Kinship and Intraspecific Phylogeny 219

Diversifying natural selection operating on particular loci via differential sur-

vival or mating success also could convey a false impression of low gene flow
among populations. For example, in the blue mussel Mytilus edulis, allele fre-
quencies at a leucine amino-peptidase (Lap) allozyme locus are significantly
heterogeneous spatially, but strongly correlated with environmental salinity.
Physiological and biochemical studies indicate that the alleles involved function
differentially in relieving osmotic stress in varying salinity environments, via
their influence on the free amino acid pools and volumes of cells (Hilbish et al.,
1982). Thus, frequencies of the non-neutral Lap alleles probably say more about
environmental conditions than about the gene flow regime of the species (Boyer,
1974; Koehn, 1978; Theisen, 1978). At other polymorphic allozyme loci, mussel
populations exhibit large, moderate, or small interpopulational variances in al-
lelic frequencies (Koehn et al., 1976), such that estimates of gene flow under
assumptions of neutrality differ considerably across genes.
A particularly sobering example of how different genetic markers can some-
times yield contradictory pictures of gene flow involves populations of the Amer-
ican oyster (Crassostrea virginica) from the Gulf of Mexico and Atlantic coasts
of the southeastern United States. Surveys of polymorphic allozymes revealed a
near uniformity of allele frequencies throughout this range (Fig. 6.4), a result that
understandably was attributed to high interpopulational gene flow resulting from
"the rather long planktonic stage of larval development, since this species has the
ability to disperse zygotes over great distances when facilitated by tidal cycles and
oceanic currents" (Buroker, 1983). However, mtDNA genotypes revealed a
dramatic genetic "break," involving cumulative and nearly fixed mutational
differences that cleanly distinguished most Atlantic from Gulf oyster populations
(Reeb and Avise, 1990). Subsequent surveys of nuclear RFLPs tended to support
the dramatic Atlantic/Gulf mtDNA dichotomy (Karl and Avise, 1992; Fig. 6.4)
and, thus, appear to eliminate possible differences in dispersal of male gametes
versus female gametes as a potential explanation for the contrasting allozymic and
mitochondrial population structures. One remaining possibility is that several of
the allozyme loci may be under uniform balancing selection, and thus do not
record the population subdivision that seems evidenced so clearly by mtDNA and
by some of the nuclear RFLP distributions (Karl and Avise, 1992). This sug-
gestion appears consistent also with the long-standing observation that multilocus
allozyme heterozygosities in mollusks are associated strongly with presumed
fitness components such as metabolic efficiency and growth rate (Garton et al.,
1984; Zouros et al., 1980). Whether this explanation or its converse is correct (that
allozymes faithfully register high gene flow in oysters, but mtDNA and some
scnRFLPs differ in the Atlantic versus Gulf because of diversifying selection), the
conclusion remains that natural selection must have acted on at least some of the
genetic markers. This finding underlines the need for caution in inferring pop-
ulation structure and gene flow from any single gene or perhaps even class of
marker loci.
 1.0

>- 0.8 - '----. .~. 0----..

~
(.)
c:
CI)
:s 0.6

-
c::r
...
CI)

0.4
CI)
CI) allozyme loci
0.2
as

0.0

MA sc GA FL FL FL FL FL LA

coastline location

1.0
mtDNA and
0.8 scnDNA loci

0.6

0.4
CI)
CI)
0.2
as

0.0

MA sc GA FL FL FL FL FL LA

coastline location
Figure 6.4. Allele frequencies in oyster populations. Shown are frequen-
cies of the most common alleles at five polymorphic allozyme loci (top)
and five DNA loci (bottom) along a coastline transect running from
Massachusetts through South Carolina, Georgia, Florida, and Louisiana
(after Karl and Avise, 1992). The allozyme loci are Est1, Lap1, 6Pgd,
Pgi, and Pgm (data from Buroker, 1983); loci assayed at the DNA level
are mtDNA (heavy line; from Reeb and Avise, 1990) and each of four
anonymous single-copy nuclear genes.
 Kinship and Intraspecific Phylogeny 221

DIRECT EsTIMATES OF DISPERSAL DISTANCES

In some cases, molecular markers that are rare or unique have been employed
to monitor gene dispersal from known point sources over one or a few generations.
Such "genetic-branding" approaches (Ferris and Berg, 1986) are similar in con-
cept to traditional labeling and tracking studies based on nonheritable markers
[such as physical tags, fluorescent dyes, radiotracers, morphological characters,
parasite loads, etc. (see the review in Levin, 1990)], with the added benefit that ac-
tual gene movement may, in some cases, be monitored over multiple generations.
For example, Burton and Swisher (1984) introduced copepods (Tigriopus
californicus) carrying rare allozyme alleles into several natural tide-pool popu-
lations on rocky California headlands. There was an initial decline in the fre-
quencies of the introduced alleles, but within six weeks the alleles had spread to
nonrecipient pools on the same outcrop, and within eight months all subpopu-
lations on an outcrop were nearly homogeneous in allelic frequencies, indicating
rather extensive interpool gene flow over these small spatial scales. Similarly,
Dillon (1988) introduced freshwater snails (Goniobasis proxima) carrying unique
allozyme alleles into isolated streams in the southern Appalachians of Virginia.
Few introductions were successful (2 of 12), but among those that were, intro-
duced alleles spread at a rate of about 15-20 m per year upstream and only 5-10
m per year downstream. Thus, one of the peculiar natural history features con-
firmed for this species is its behavioral tendency to migrate by crawling against
the current.
Gametic dispersal can be investigated in a similar fashion. Grosberg (1991)
monitored the effective dispersal of sperm by placing allozymically marked
colonies of the marine ascidian Botryllus schlosseri onto the pilings of a harbor
dock and later assaying brooded embryos within nearby natural colonies for the
presence of the introduced allele (signaling fertilization by dispersed sperm).
From the rapid decline in fertilization success observed beyond about 50 cm,
Grosberg concluded that effective sperm dispersal in this species is extremely
limited. Similarly, Schaal (1980) monitored effective pollen flow in the Texas
bluebonnet (Lupinus texensis) by assaying for heterozygotes among the FI prog-
eny in an experimental population where an allozymically marked pollen donor
was surrounded by plants of alternate genotype. Schaal found that gene dispersal
via pollen was quite restricted (a few meters), with most gene movement being
to neighboring plants. Nonetheless, this movement of pollen genes was signif-
icantly greater than would have been inferred from interplant flight distances by
the pollinators (bees), apparently because of "pollen carryover" wherein some
fraction of the pollen a bee deposits on a stigma comes from flowers visited
before the most recent. Such studies of allelic dispersal that involve screening of
progeny arrays for introduced genetic markers are allied closely to the parentage
analyses discussed in Chapter 5.
222 Kinship and Intraspecific Phylogeny

Nonetheless, several limitations attend attempts to monitor dispersal directly

by genetic markers. First is the problem of generating or finding unique alleles
that with assurance are not found also in the natural population to which the
markers are introduced. Second is the possibility that the markers themselves, or
perhaps the selective breeding programs often used in their generation, may
impair or otherwise modify the dispersal capabilities of the propagules in com-
parison to the natural state. A related possibility is that the dynamics of the
introduced markers might be influenced primarily by natural selection acting on
the markers or their genetic backgrounds rather than by the dispersal and gene
flow regimes that usually are of primary interest in such studies. Finally, the
greatest problem is the infeasibility of monitoring long-distance movement of
potentially vagile markers, due to large spatial scales and the inevitable effects
of dilution from the point source (Jones et aI., 1981). Such undetected long-
distance gene flow, even if rare, could have a significant homogenizing influence
on the genetic structure of a species.

Vagility, Philopatry, and Dispersal Scale

Particularly in animals with nondispersive gametes and larvae, adult vagility

in conjunction with habitat "grain" [the geographic scale at which organisms
"perceive" environmental patchiness (Levins, 1968)] no doubt assume major
roles influencing gene flow and population structure. To a land snail such as
Helix aspersa, parking lots and streets may be formidable barriers to movement,
and colonies within and among city blocks indeed do exhibit significant popu-
lation structure as registered by allozymes (Selander and Kaufman, 1975b). At
the other extreme, some turtles are oceanic mariners that swim many thousands
of kilometers during their lifetimes; surveys of scnRFLPs in green turtles (Che-
lonia mydas) indicate population genetic structures throughout entire ocean ba-
sins comparable in magnitude to those among Helix populations on adjacent city
blocks within a town [mean FST == 0.13 (Karl et aI., 1992)].
Although the spatial scales of potential gene flow clearly are influenced by the
mobilities of animals, apparent popUlation genetic structure is not inextricably tied
to an organism's vagility for several reasons, including (a) the presence of physical
or ecological barriers that overmatch dispersal capability, (b) behavioral consid-
erations such as habitat choice, or philopatry (site faithfulness) with regard to
reproduction, (c) gender-biased patterns of dispersal and gene flow (applying
particularly to genetic markers with sex-limited transmission), (d) influences of
natural selection on particular genetic markers (or on loci linked to them), or (e)
historical demographic events that have removed populations from equilibrium
expectations between gene flow and genetic drift. These will be illustrated in turn.
 Kinship and Intraspecific Phylogeny 223

PHYSICAL DISPERSAL BARRIERS

Bluegill sunfish (Lepomis macrochirus) are active and mobile swimmers abun-
dant throughout their freshwater range in North America. An allozyme survey of
2560 specimens divided equally among 64 localities (8 sites per reservoir, 4
reservoirs in each of 2 adjacent river drainages) revealed that nearly 90% of the
overall variance in allele frequency occurred between reservoirs of a drainage
(Avise and Felley, 1979). Among locales within reservoirs (which ranged in size
to more than 100,000 acres), allele frequencies seldom were significantly het-
erogeneous. Some of the reservoirs surveyed were geographically proximate, but
intervening dams no doubt constitute formidable barriers to bluegill movement.
Clearly, the subdivided structure of the physical environment has imposed a
corresponding genetic structure on these otherwise highly mobile freshwater
fishes.
Gyllensten (1985) reviewed allozyme literature on population structures for 19
species of fishes characterized according to habitat and life-style: strictly fresh-
water, anadromous (migrating from saltwater to freshwater to spawn), and ma-
rine. The average proportion of total intraspecific gene diversity allocated be-
tween locales increased considerably from the marine species and anadromous
forms (1.6% and 3.7%, respectively) to the freshwater taxa (29.4%). Thus, the
differences in the distribution of genetic variability coincided generally with
qualitative differences in the occurrence of obvious geographic barriers to mi-
gration. Nevertheless, some marine and anadromous fish do exhibit levels of
population structure across their ranges that are more or less comparable to those
of some freshwater fish species (Avise et al., 1987b; Bowen and Avise, 1990).
In a flightless waterstrider (Aquarius remigis) that migrates by rowing on water
surfaces, an allozyme survey by Preziosi and Fairbairn (1992) revealed that
populations from different streams of a watershed are highly structured (FST =
0.46), whereas samples within a stream are undifferentiated (FST = 0.009). By
contrast, another waterstrider with functional wings (Limnoporus canaliculatus)
exhibited nearly homogeneous allele frequencies throughout several Atlantic
seaboard states (lera, 1981), suggesting that the differences in dispersal capacity
of these two Hemipteran species exert an important influence on their population
genetic structures. On the other hand, a comparison of population structures in
five species of carabid ground beetles revealed no correlation with degree of
flight-wing development (ranging from vestigial to fully winged), but a positive
correlation was noted between F ST values and the elevations of collecting sites
(Liebherr, 1988). Perhaps the greater fragmentation of highland habitats was a
more important factor than inherent dispersal capability in influencing magnitudes
of genetic structure in these beetles, or perhaps different populational histories
were involved.
224 Kinship and Intraspecific Phylogeny

Numerous other species likewise occupy discontinuous habitats and, depend-

ing on dispersal capabilities, may show significant population genetic structure
related to environmental patchiness. To troglobitic (obligate cave dwelling)
crickets in the genera Hadenoecus and Euhadenoecus, different cave systems
represent isolated pockets of habitat, and these species tend to exhibit greater
allozymic population structure than do their epigean (surface dwelling) counter-
parts (Caccone and Sbordoni, 1987). To Peromyscus mice on islands, ocean
channels severely constrain dispersal, such that small island populations often
show diminished within-population variability and exaggerated between-island
genetic differences relative to their mainland counterparts (Ashley and Wills,
1987, 1989; Avise et aI., 1974; Selander et aI., 1971). To any habitat specialist,
suitable environments may be scattered. In the beetle Collops georgianus, which
is endemic to granitic outcrops in the southeastern United States, differentiation
among distant outcrops appeared pronounced (FST = 0.19) relative to the spatial
genetic heterogeneity exhibited over microgeographic scales (FST = 0.01)
(King, 1987). These same granitic outcrops also serve as rather isolated island
habitats for endemic plant populations, where allozyme data have indicated
severe restrictions on gene flow (Wyatt et aI., 1992).

PHILOPATRY TO NATAL SITE

Each reproductive season, many marine turtles migrate hundreds or thousands

of kilometers from foraging grounds to particular nesting locales, where eggs are
deposited on sandy beaches. For example, female green turtles (Chelonia mydas)
that nest on Ascension (a small isolated island on the mid-Atlantic oceanic ridge)
otherwise inhabit feeding grounds along the coast of Brazil, some 2000 km
distant. From repeated captures of physically tagged adults, it long has been
known that green turtles exhibit strong nest-site fidelity, i.e., Ascension females
nest there and nowhere else, Costa Rican and Venezuelan nesters are faithful to
their respective rookeries, and so on. What had remained unknown is whether
the site to which a female is fidelic as an adult is also her natal rookery. If female
"natal homing" prevails, rookeries within an ocean basin should exhibit clear
genetic differences from one another with regard to maternally-transmitted ge-
netic traits (such as mtDNA), even if appreciable interrookery exchange of
nuclear genes occurs via the mating system and male-mediated gene flow (Karl
et al., 1992). In recent surveys of mtDNA from green turtle rookeries around the
world (Bowen et al., 1992; Meylan et aI., 1990), it was discovered that (a) a
fundamental phylogenetic split distinguishes all specimens in the Atlantic-Med-
iterranean from those in the Indo-Pacific Oceans and (b) additional genetic sub-
structure characterizes popUlations within each ocean basin, as evidenced by
fixed or nearly fixed genotypic differences between most rookeries (Fig. 6.5).
Evidently, female green turtles have a strong propensity for natal homing. Re-
 Kinship and Intraspecific Phylogeny 225

ATLANTIC -
MEDITERRANEAN
INDIAN- ,r----------------------~,

PACIFIC ~
~~
~~

~::

... ......
···•• ...•••
~~
,r------------------------~, ~

~
AVES (-),

COSTA RICA( ... ),

·.
POLYNESIA ( 0 )

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POLYNESIA ( 0),
FLORIDA(-)
JAPAN ( 0),

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JAPAN ( 0) AVES (-)
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sequence divergence (%)

Figure 6.5. UPGMA dendrogram summarizing relationships among 226 sampled nests of
the green turtle (after Bowen et aI., 1992). To conserve space, the sequence divergence
(P) axes on the bottom are presented as mirror images centered around the root leading to
two distinct clonal assemblages (Atlantic-Mediterranean versus Indian-Pacific ocean ba-
sins) that also were evident in qualitative parsimony analyses of the same data.
226 Kinship and Intraspecific Phylogeny

suIts exemplify how populations of a highly mobile species nonetheless can be

structured dramatically, due in this instance both to geographic constraints (phys-
ical separations between oceans) as well as to a philopatric reproductive behavior
(natal homing within oceans).
Whales too are impressive oceanic travelers, normally moving many thou-
sands of kilometers seasonally. Baker et al. (1990) analyzed mtDNA from skin
biopsies of 84 humpback whales (Megaptera novaeangliae) , and found clear
haplotype differences between groups previously reported to show distinct pat-
terns of migration between summer feeding grounds in subpolar or temperate
environs and winter breeding areas in the tropics. The authors interpreted this
spatial segregation of genotypes, that occurred within as well as between ocean
basins, "to be the consequence of maternally directed fidelity to migratory
destinations," again illustrating the importance of site-fidelic behavior on pop-
ulation structure in a highly mobile animal.
Salmon are active and powerful swimmers, but also are notorious for sus-
pected natal-homing propensity. In anadromous forms of these species, parr
(juveniles) spawned in freshwater streams migrate to sea before eventually re-
turning as adults to natal streams to complete the life cycle. Several allozyme and
mtDNA studies of both Atlantic and Pacific species have revealed significant
genetic differences among spawning popUlations at various spatial scales (Bill-
ington and Hebert, 1991; Ferguson, 1989; Gyllensten and Wilson, 1987a; Ry-
man, 1983; Stahl, 1987). For example, Atlantic salmon (Salmo salar) from
North America and Europe could be distinguished cleanly in genotypic compo-
sition (Bermingham et aI., 1991; Davidson et aI., 1989). However, on the
smaller spatial scales of nearby drainages, genetic differences at most loci tend
to involve allele frequency shifts that are less pronounced (e.g., G.M. Wilson et
al., 1987). Presumably, the lack of greater local differentiation may be due to
occasional "mistakes" in natal homing that provide gene flow between other-
wise isolated spawning areas, as well as to the fact that most high-latitude
streams currently utilized by salmon must have been colonized within the last
few thousand years (following glacial retreats) and, hence, the populations no
doubt are tightly connected in a historical sense.
Birds present a special enigma with regard to magnitude and pattern of pop-
ulation structure. On the one hand, most birds have high dispersal potential
(because of flight, and often a migratory propensity), and many species have
broad geographic distributions, suggesting that gene flow may be high and pop-
ulation differentiation minimal. On the other hand, many species also exhibit
strong tendencies for nest-site philopatry and show obvious geographic variation
in body size, song, or plumage (often leading to recognition of subspecies)
suggesting that interpopulational gene flow may be low. Barrowclough (1983)
reviewed the allozyme literature on geographic variation among populations
within 57 vertebrate species. In comparison to most nonavian vertebrates, the
 Kinship and Intraspecific Phylogeny 227

surveyed birds (primarily temperate zone passerines) exhibited only minor geo-
graphic differentiation (mean FST = 0.02, compared to mean FST estimates of
0.11, 0.23, 0.30, and 0.38 for fishes, mammals, reptiles, and amphibians,
respectively). Results were interpreted as "consistent with a presumed generally
greater vagility in birds than in animals such as salamanders and rodents."
Barrowclough (1983) also noted, however, that the results might reflect differ-
ences in the ages of populations.
Subsequent research employing the more discriminatory mtDNA molecule
(Avise and Zink, 1988) has revealed that avian species exhibit a wide variety of
population genetic structures (see the review in Avise and Ball, 1991). Some
species such as the red-winged blackbird (Agelaius phoeniceus) and downy
woodpecker (Picoides pubescens) show little differentiation in terms of mtDNA
phylogeny across the entire North American continent (Ball et al., 1988; Ball and
Avise, 1992), whereas others such as the seaside sparrow (Ammodramus mari-
timus), Canada goose (Branta canadensis), and fox sparrow (Passerella iliaca)
show deep divisions in intraspecific mtDNA phylogeny that tend to align with
geographic or morphologic partitions (Avise and Nelson, 1989; Van Wagner and
Baker, 1990; Zink, 1991). Most of these studies involved restriction site analyses
of total mtDNA. Recently, Wenink et al. (1993) employed sequences from the
hypervariable mtDNA control region to demonstrate significant regional popu-
lation structure in the dunlin (Calidris alpina) , a long-distance migrant shorebird
with Holarctic nesting distribution. Thus, high-resolution molecular assays likely
will reveal significant geographic structure in many avian species, perhaps even
over relatively small spatial scales [as has been demonstrated for example in the
song sparrow Melospiza melodia (Hare and Shields, 1992; Zink, 1991)].

GENDER-BIASED DISPERSAL AND GENE FLOW

The degree of faithfulness to natal site or to social group often is gender-

dependent. For example, as described earlier, lion prides normally consist of
matrilineally related females and most interpride movement is by males (Packer
et aI., 1991; Schaller, 1972). Conversely, Florida scrub jays (Aphelocoma co-
erulescens) live in extended family groups built around patrilineal kinship-the
young often remain at the nest as helpers, accession of territories is patrilineal,
and most dispersal is by females (Woolfenden and Fitzpatrick, 1984). In general,
most mammalian species with asymmetric philopatry exhibit male-biased dis-
persal, whereas most such avian species exhibit female-biased dispersal (Green-
wood, 1980; Greenwood and Harvey, 1982). One likely consequence of such
asymmetric dispersal is that a species may exhibit qualitatively different patterns
of geographic population structure at genes with biparental transmission (most
nuclear loci) versus those at which transmission occurs through only one sex
(e.g., mtDNA, the Y chromosome of mammals and insects, or the W chromo-
228 Kinship and Intraspecific Phylogeny

some of birds). Although several Y- or W-linked genes have been identified in

various mammalian and avian species, respectively (Casanova et al., 1985; Ellis
et al., 1990; Page et aI., 1985; Rabenold et al., 1991; Rasheed et al., 1991;
Whisenant et aI., 1991), few as yet have proven sufficiently variable to be of
particular phylogenetic utility at the intraspecific level (Maynard Smith, 1990;
but see Bishop et al., 1985). Thus, most molecular studies of gender-biased
dispersal have relied on data from mtDNA in conjunction with those from al-
lozymes or nuclear RFLPs.
A case in point involves macaque (Macaca) monkeys, where mirror-image
patterns of geographic variation have been reported in nuclear-encoded allozymes
versus mitochondrial DNA. Male macaques typically leave their natal group
before sexual maturity, whereas females remain for life. Melnick and Hoelzer
(1992) reviewed the literature on molecular variation in several macaque species
(M.fascicularis, M. mulatta, M. nemestrina, andM. sinica) and reported that the
observed patterns of geographic population structure appear consistent with these
gender-specific behaviors. For example, in the nuclear genome of M. mulatta,
only 9% of the total intraspecific diversity proved attributable to variation among
geographic locales, whereas 91 % of the overall diversity in mtDNA occurred
between populations. Thus, the population genetic structures registered by the two
genomes are "very different from one another ... (and) ... intimately linked
to the asymmetrical dispersal patterns of males and females and the maternal
inheritance of mtDNA" (Melnick and Hoelzer, 1992).
Another example of distinctive genetic signatures resulting from gender-based
differences in behavior may involve the green turtle (Chelonia mydas). As already
mentioned, most rookeries within an ocean basin are strongly isolated with regard
to mtDNA lineages (mean inferred Nm == 0.3), indicating a strong propensity for
natal homing by females (Bowen et aI., 1992). However, these same rookeries
are somewhat less differentiated at assayed scnRFLPs (mean Nm = 1.7; Karl et
aI., 1992), perhaps because of occasional male-mediated gene flow operating
through the mating system. Green turtles are known to mate at sea, often on
feeding grounds or other locales spatially removed from the nesting sites. Thus,
interrookery matings may provide an avenue for the exchange of nuclear genes
that largely is closed to mtDNA because of female natal philopatry.
The lesser snow goose (Chen caerulescens) provides an exception to the
prevalent pattern of male-biased philopatry in birds. In this species, like many
other migratory waterfowl, pair formation occurs on wintering grounds where
considerable mixing of birds from different nesting areas can take place. Yet a
mated pair normally returns to the female's natal or prior nesting area. Among
all avian species for which direct banding returns are available (Cooke et al.,
1975), according to Greenwood (1980) "The lesser snow goose is the best
documented example of male biased natal and breeding dispersal. . . ." This
natural history pattern suggests considerable intercolony gene flow mediated by
 Kinship and Intraspecific Phylogeny 229

males, an expectation consistent with results of both allozymic (Cooke et al.,

1988) and scnRFLP studies (Quinn, 1988; Quinn and White, 1987). The behavior
also suggests that colonies should be isolated with regard to matriarchal lineages,
but surprisingly this has not proved to be the case. In a mtDNA survey of 160 geese
from colonies across their breeding range (from Wrangel Island, Russia to Baffin
Island in the eastern Canadian Arctic), no significant differences were observed
in the spatial frequencies of two major mtDNA clades, a result strongly indica-
tive of considerable population connectedness and gene flow involving females
(Avise et aI., 1992b; see also Quinn, 1992). One likelihood is that the entire
current range of the snow goose was colonized recently from expansion out of
Pleistocene refugia where the separation between the two mtDNA clades may have
been initiated. A related possibility is that of ongoing gene flow, either via
occasional lapses in philopatry by individual females (a phenomenon indeed
documented from banding returns) or by episodic pulses of gene flow during
periods of colony perturbation (also suspected from field observations). Whatever
the process, snow goose colonies clearly have been in recent matrilineal contact,
notwithstanding the propensity for natal philopatry by females.
Important object lessons are to be gained from these comparisons of banding
and genetic data for snow geese. First, direct behavioral or marking studies on
contemporary populations can, in some cases, provide a misleading picture of
the geographic distributions of genetic traits because they fail to access the
important evolutionary aspects of population connectedness revealed in the
genes. Second, and conversely, geographic distributions of genetic markers can,
in some cases, provide a misleading picture of contemporary dispersal and gene
flow because they retain a record of evolutionary events and demographic pa-
rameters that may differ from those of the present. Thus, a full appreciation of
the geographic population structure of a species requires an integration of evo-
lutionary (genetic) and contemporary (behavioral) perspectives.
Africanized bees, or "killer bees" of the popular press, are aggressive fonns
of Apis melli/era that have spread rapidly in the New World following the
introduction of African honeybees into Brazil in the late 1950s. The mode of
spread and composition of colonies have been much debated, and at least two
hypotheses were prevalent. First, perhaps queens are relatively sedentary, and
most of the geographic expansion in aggressive behavior results from gene flow
mediated by drones. Under this hypothesis, these males might travel consider-
able distances and mate with the more docile honeybees of European ancestry
that fonnerly comprised the domesticated hives in the Americas. Alternatively,
perhaps the gene flow results from colony swarming, a mechanism of maternal
migration whereby a queen and some of the workers leave a hive and fly else-
where to establish a new colony. Under this scenario, hybridization with the
domesticated European fonns is not necessarily required. Two independent re-
search groups (Hall and Muralidharan, 1989; D.R. Smith et al., 1989) applied
230 Kinship and Intraspecific Phylogeny

mtDNA assays to Africanized honeybees (mostly feral hives) in the neotropics

and showed that these colonies usually carry African-type as opposed to Euro-
pean-type mtDNA. These results document the spread of African honeybees as
continuous matrilines (the swarming hypothesis), thus eliminating the possibility
that drones alone are responsible for the gene flow (see also Hall and Smith,
1991). Subsequent studies based on nuclear RFLPs and allozymes showed that
African and European honeybees also hybridize at least occasionally in the
neotropics and that this has led to introgression of nuclear genes as a part of the
Africanization process, although to an argued degree (Hall, 1990; Lobo et aI.,
1989; Rinderer et aI., 1991; Sheppard et aI., 1991).
NON-NEUTRALITY OF SOME MOLECULAR MARKERS

Lewontin and Krakauer (1973) pointed out that one expected signature of
natural selection on genetic markers is the appearance of significant heteroge-
neity across loci in the variances of allele frequencies among geographic popu-
lations. Because genetic drift, gene flow, and the breeding structure of a species
should, in principle, affect all loci in a similar fashion, different population
genetic structures across loci might indicate either that (a) allele frequencies at
geographically variable loci are under diversifying selection (despite high gene
flow as evidenced by geographically uniform genes) or (b) allele frequencies at
geographically uniform loci are under stabilizing or equilibrium selection (de-
spite low gene flow as evidenced by heterogeneous allele frequencies at geo-
graphically variable loci). Lewontin and Krakauer applied this reasoning to
suggest that natural selection acted on at least some human-blood-group poly-
morphisms (Cavalli-Sforza, 1966), which on a global scale showed interpopu-
lational allele frequency variances spanning a wide range (from FST = 0.03 to
FST = 0.38)! The "Lewontin-Krakauer" test subsequently was criticized on the
grounds that their proposed statistical methods seriously underestimated the vari-
ances in gene frequencies expected under the null (neutral) theory (Nei and
Maruyama, 1975; Robertson, 1975; see also Lewontin and Krakauer, 1975).
Nevertheless, it remains true that different loci within a species can sometimes
paint very different pictures of population structure and gene flow when inter-
preted under models of selective neutrality.
An extreme example involves the deer mouse Peromyscus maniculatus. In
allozyme surveys of populations from across North America, F ST values at six
polymorphic loci ranged from 0.04 (inferred Nm = 6) to 0.38 (Nm = 0.4)
(Avise et aI., 1979c). Particularly noteworthy was the observation that popula-
tions from central Mexico to northern Canada, and from the east to the west
coasts of the United States, invariably exhibited the same two electromorphs at
the aspartate aminotransferase (Got-lor Aat-I) locus, and in roughly similar
frequencies (FST = 0.05). Subsequent molecular screening by varied electro-
phoretic techniques and other discriminatory protein assays failed to reveal any
 Kinship and Intraspecific Phylogeny 231

appreciable "hidden variation" within these two Aat-l electromorph classes

(Aquadro and Avise, 1982b). Yet this relative geographic homogeneity at Aat-l
contrasts sharply with the extreme geographic heterogeneity exhibited by this
species in morphology, ecology, karyotype, and mtDNA sequence (Blair, 1950;
Baker, 1968; Bowers et aI., 1973; Lansman et al., 1983). In particular, the
number of acrocentric chromosomes ranges from 4 to 20 across populations
(Bowers et aI., 1973), and regional populations often show deep divisions in a
mtDNA genealogy involving cumulative and fixed mutational differences (Lans-
man et al., 1983). It is difficult to escape the conclusion that Aat-l provides a
serious underestimate of the magnitude of geographic population structure in this
species, perhaps because geographically uniform selection balances Aat-l allele
frequencies despite the severe historical and contemporary restrictions on gene
flow that appear to be registered by many other genetic traits.
The converse of this situation may apply to a well-studied allozyme polymor-
phism in Drosophila melanogaster for alcohol dehydrogenase (Adh), an enzyme
whose main biochemical function is to catabolize ethanol that is abundant in
fermented fruits in the fruit flies' natural environment. Several studies have
shown that the AdhF allele has significantly higher enzymatic activity than Adhs ,
but is less heat resistant, and that these and other biochemical and physiological
attributes can translate into fitness differences between Adh genotypes in partic-
ular experimental regimes (Sampsell and Sims, 1982; van Delden, 1982). In
natural populations, frequencies of these two Adh alleles sometimes vary locally
[e.g., inside vs. outside wine cellars! (Hickey and McLean, 1980)], and also
show strong latitudinal clines with AdhF more common with increasing latitude
in both the northern and southern hemispheres (Oakeshott et aI., 1982). Such
evidence for environmental selection on Adh implies that prima facie conclusions
about gene flow based on this polymorphism alone could be misleadingly low.
From numerous other genetic traits, Singh and Rhomberg (1987) conclude that
gene flow in D. melanogaster indeed is moderately high even on continental
scales (Nm == 1-3), but overall that natural selection in combination with mi-
gration [as well as population history (Hale and Singh, 1991)] must be consid-
ered in studies of geographic variation in molecular markers.
Based on similar arguments from comparative geographic patterns, natural
selection has been implicated for various molecular polymorphisms in other
species as well (e.g., Ayala et aI., 1974). When coupled with further lines of
evidence for balancing or other forms of selection acting on particular protein
systems (Chapter 2), as well as on some DNA characters (e.g., Hughes and Nei,
1988; Kreitman, 1991; MacRae and Anderson, 1988; Nei and Hughes, 1991), it
becomes clear that interpretations of geographic population structure under the
assumption of strict neutrality are made with some peril. At the very least,
conclusions about the forces shaping popUlation structure in any species should
be based on information from multiple independent loci.
232 Kinship and Intraspecific Phylogeny

HISTORICAL DEMOGRAPHIC EVENTS

Because of mathematical tractability, many theoretical models in population

genetics yield only the equilibrium expectations between counteracting evolu-
tionary forces [e. g., the diversifying influence of genetic drift in small populations
versus the homogenizing influence of gene flow under an island or stepping-stone
model (Box 6.4)]. Seldom is it feasible to consider formally the idiosyncratic
histories of particular species or to treat nonequilibrium situations. Yet the de-
mographic histories and phylogenies of real species are highly idiosyncratic and
likely to produce nonequilibrium population structures. Hence, interpretation of
empirical genetic structures of natural populations is especially challenging.
For example, in comparative analyses of three anadromous species of fish along
the same coastline transect in the southeastern United States, Bowen and Avise
(1990) were led to consider several historical demographic and zoogeographic
factors that might have produced the observed differences in geographic popu-
lation structure as registered by mtDNA. All species showed significant haplotype
differences between the Atlantic and Gulf of Mexico, but the magnitude and
pattern of the genetic variation differed greatly among the taxa. The black sea bass
(Centropristis striata) showed little within-region polymorphism and a clear
phylogenetic distinction between Atlantic and Gulf popUlations; menhaden
(Brevoortia tyrannus andB. patronus) showed extensive within-region polymor-
phism and a paraphyletic (Fig. 4.10) genealogical relationship between the At-
lantic and Gulf; and sturgeon (Acipenser oxyrhynchus) exhibited extremely low
mtDNA variation both within regions and overall. From the mtDNA variabilities
observed, estimates of evolutionary effective popUlation sizes (Chapter 2) proved
to vary by more than four orders of magnitude, from Nf{e) = 50 (Gulf of Mexico
sturgeon) to Nf{e) = 800,000 (Atlantic menhaden), and to be rank-order correlated
with current-day census sizes. These differences in Nf{e) , which presumably reflect
widely different demographic histories of the three species, may help to explain
some of the phylogenetic features, including the clean distinction between Atlantic
and Gulf forms ofthe sea bass versus the paraphyletic pattern in menhaden. From
prior population genetic analyses of several other maritime species (see upcoming
section on phylogeography), Atlantic versus Gulf populations commonly exhibit
genetic differences that appear to trace to Pleistocene events. All else being equal,
smaller populations isolated by such events should evolve to a status of reciprocal
monophyly in a gene tree more rapidly than large populations (Chapter 4).
However, historical separations between the Atlantic and Gulf cannot explain all
features of the population genetic structure in these three fishes. Thus, for the
menhaden and sturgeon (but not the sea bass), recent gene flow between the
Atlantic and Gulf also was implicated strongly by the shared presence of a few
nearly identical mtDNA haplotypes (that would not likely have remained unaltered
over long evolutionary periods).
 Kinship and Intraspecific Phylogeny 233

Whether these particular inferences are correct or not, they serve to introduce
some of the historical demographic considerations and nonequilibrium environ-
mental conditions that must have impacted the genetic structures of real popu-
lations. In interpreting empirical data on population structures, a challenge in-
volves deciding how far to pursue idiosyncratic demographic explanations,
particularly because these can seldom be tested critically in controlled or repli-
cated settings (however, see Fos et al., 1990; Scribner and Avise, 1993b; Wade
and McCauley, 1984) and because alternative scenarios might account for the
data. Nonetheless, cognizance of the limitations of equilibrium theory, and of the
potential impact of historical demographic factors on population-genetic struc-
tures, must represent steps toward greater realism.

PHYLOGEOGRAPHY
Background

The introduction of mtDNA data to population genetics in the late 1970s

prompted a revolutionary shift in attitude toward such historical, phylogenetic
perspectives on intraspecific population structure. Because of the maternal, non-
recombining mode of mtDNA inheritance and rapid evolution in mtDNA se-
quence, the molecule often provides multiple alleles or haplotypes that can be
ordered phylogenetically within a species, yielding intraspecific phylogenies
(gene genealogies) interpretable as a matriarchal component of the organismal
pedigree. MtDNA transmission in animal species constitutes the female analogue
of male surname transmission in many human societies (Avise, 1989b}--both
sons and daughters inherit their mother's mtDNA genotype, which only daughters
normally transmit to the next generation. Thus, mtDNA lineages reflect muta-
tionally interrelatable "female family names" of a species, and their historical
dynamics can be interpreted according to the types of theoretical models long used
by human demographers to analyze surname distributions (Lasker, 1985; Lotka,
1931; Chapter 4). Furthermore, mtDNA clones and clades within many species
have proved to be geographically localized. Such observations prompted intro-
duction ofthe word "phylogeography" (Avise et al., 1987a), which refers to the
study of the principles and processes governing the geographic distributions of
genealogical lineages, including those at the intraspecific level.
One of the first phylogeographic applications of mtDNA data still serves as a
useful introduction to the types of population structures frequently revealed. The
pocket gopher (Geomys pinetis) is a fossorial rodent that inhabits a three-state
area in the southeastern United States. Analysis of 87 individuals from across
this range by 6 restriction enzymes revealed 23 different mtDNA genotypes
whose phylogenetic relationships and distributions are presented in Figure 6.6.
Clearly, most mtDNA haplotypes in these gophers are localized geographically,
234 Kinship and Intraspecific Phylogeny

Figure 6.6. mtDNA phylogeny for 87 pocket gophers (after Avise et aI., 1979b).
Lowercase letters represent different mtDNA genotypes, which are connected by
branches in a parsimony network that is superimposed over the geographic sources of
the collections in Alabama, Georgia, and Florida. Slashes across network branches
reflect the numbers of inferred mutational steps along a pathway. Heavier lines en-
compass two distinctive mtDNA clades that differ by at least nine mutational steps.
 Kinship and Intraspecific Phylogeny 235

appearing only at one or a few adjacent collection sites. Furthermore, genetically

related clones tended to be geographically contiguous or overlapping, and a
major gap in the matriarchal phylogeny exhibited a strong geographic orientation
cleanly distinguishing eastern from western populations.
Population subdivision characterized by localized genealogical structure
and/or significant mtDNA phylogenetic gaps across a species' range subse-
quently have been reported in a wide variety of animal species: mammals ranging
from voles and mice to some whales (Carr et al., 1986; Cronin et al., 1991a;
Cronin, 1992; MacNeil and Strobeck, 1987; Plante et al., 1989; Prinsloo and
Robinson, 1992; Riddle and Honeycutt, 1990; Wada et al., 1991); birds ranging
from sparrows to geese (Avise and Nelson, 1989; Shields and Wilson, 1987b;
Van Wagner and Baker, 1990; Zink, 1991); reptiles ranging from gekkos to
tortoises (Densmore et al., 1989a; Lamb and Avise, 1992; Lamb et al., 1989;
Moritz, 1991); amphibians (Wallis and Arntzen, 1989); freshwater and some
marine fishes (Avise, 1987; Bermingham and Avise, 1986; Crosetti et al., 1993);
insects (Hale and Singh, 1987, 1991; Harrison et al., 1987); snails (Murray et
al., 1991); horseshoe crabs (Saunders et al., 1986); and many others (reviews in
Aviseetal., 1987a;Moritzetal., 1987; Wilson et aI., 1985). On the other hand,
several species have proved to exhibit little or no mtDNA phylogeographic
structure across broad ranges; for example, some large mammals such as black
bears (Cronin et aI., 1991b) and coyotes (Lehman and Wayne, 1991; Lehman et
al., 1991); some birds (Ball et aI., 1988; Tegelstrom, 1987a); several marine
invertebrates and fishes (Arnason et al., 1992; Avise, 1987; Ovenden et al.,
1992; Palumbi and Wilson, 1990); and the nematode Ostertagia ostertagi [a
parasite of cattle that may have been spread widely by transport of livestock
(Blouin et al., 1992)]. In general, differences in organismal mobility and in
environmental fragmentation appear to exert important influences on patterns of
mtDNA phylogeographic structure. Thus, low-vagility pocket gophers and tor-
toises exhibit pronounced spatial-genetic structure, as do many freshwater fishes
across isolated drainages, whereas mobile species that occupy more or less
continuously suitable habitat-e.g., some birds, marine fishes, and large terres-
trial mammals-often exhibit less spatial differentiation.
Presumably, the localization of related mtDNA clones in most species reflects
limited contemporary gene flow (at least via females), and the deeper genetic
breaks sometimes observed may evidence longer-term historical population sep-
arations. Several authors have discussed the philosophical distinctions between
contemporary gene flow versus historical connectedness between populations in
a genealogical sense (Avise, 1989a; Larson et al., 1984; Slatkin, 1987). What
follows are a few illustrations of how mtDNA analyses have added a phyloge-
netic dimension to perspectives on intraspecific population structure.
236 Kinship and Intraspecific Phylogeny

Case Histories
THE ORIGIN OF GREEN TURTLES ON ASCENSION ISLAND

This major rookery for green turtles (Chelonia mydas) is a tiny (8 km in

diameter) island situated on the mid-Atlantic ridge, halfway between Brazil and
Liberia. From direct tagging studies, it is known that the females that nest on
Ascension otherwise inhabit shallow-water feeding pastures along the South
American coastline. Thus, for each nesting episode (every two to three years for
an individual), females embark on a 5000-km migration to Ascension Island and
back, in a several-month odyssey involving navigational and exertional feats that
nearly defy human comprehension. How might Ascension turtles have established
such an unlikely migratory circuit, particularly since suitable nesting beaches
along the coast of South America are utilized by other green turtles? Carr and
Coleman (1974) proposed an historical biogeographic scenario involving plate
tectonics and natal homing. Under their hypothesis, the ancestors of Ascension
Island green turtles nested on islands adjacent to South America in the late
Cretaceous, soon after the opening of the equatorial Atlantic Ocean. Over the past
70 million years, these volcanic islands have been displaced from South America
by sea-floor spreading (at a rate of about 2 cm per year). A population-specific
instinct to migrate to the present-day Ascension Island thus was proposed to have
evolved over tens of millions of years of genetic isolation (at least with regard to
matriarchal lineages) from other green turtle rookeries in the Atlantic.
Bowen et al. (1989) critically tested the Carr-Coleman hypothesis by com-
paring mtDNA genotypes of Ascension Island nesters with those of other green
turtles. They found fixed or nearly fixed mtDNA differences between many
Atlantic rookeries, consistent with severe restrictions on contemporary interrook-
ery gene flow by females and, thus, with the natal homing aspects of the Carr-
Coleman hypothesis. However, the magnitude of sequence divergence of the
Ascension rookery from others in the Atlantic [p < 0.002 (Fig. 6.5)] indicates
a very recent separation, within at most the last 100,000 years (under a conven-
tional vertebrate mtDNA clock) or 800,000 years [under a slower mtDNA clock
calibration proposed for turtles (A vise et al., 1992a)]. Indeed, these are upper
estimates of divergence times because in available assays the Ascension mtDNA
haplotype proved indistinguishable from that in a geographically proximate Bra-
zilian rookery (Bowen et al., 1992). In any event, the genetic results clearly are
incompatible with the Carr-Coleman scenario--colonization of Ascension Is-
land, or at least extensive matriarchal gene flow into the population, has been
evolutionarily recent.
MIGRATORY HISTORIES OF MONARCH BUTTERFLIES

Each year, monarch butterflies (Dana us p/exippus) from across North America
undertake massive migrations that culminate in huge overwintering aggrega-
 Kinship and Intraspecific Phylogeny 237

tions. Monarchs from east of the Rocky Mountains migrate to refugia in the
Transvolcanic Range of Central Mexico, and those from west of the Rockies
winter along the central California coast. In terms of allozymes, Eanes and
Koehn (1978b) first noted that monarchs from across the eastern range showed
very low geographic differentiation when compared to other species, and a recent
survey based on mtDNA restriction sites was unable to document any genetic
differences between populations east versus west of the rockies (Brower and
Boyce, 1991). Although it is impossible to prove a null hypothesis (in this case
that there are no genetic differences between eastern and western populations),
the results do suggest that historical contact between the two areas has been
extensive and recent. The monarch is the only temperate representative of an
otherwise exclusively tropical subfamily of the Nymphalidae, so on these
grounds too it appears plausible that the current migrational phenomenon may
represent a post-Pleistocene colonization of North America from one or a few
nearly homogeneous ancestral sources.

INTERBROOD SWITCHING IN PERIODICAL CICADAS

Species of Magicicada have an unusual life cycle in which larvae remain in the
ground for long periods, only to emerge en masse and mate at periodic intervals
(e.g., every 17 years in M. septendecim, and every 13 years in M. tredecim).
Each year class is referred to as a brood. Life-cycle length commonly is assumed
to be a genetically fixed species-specific trait, and differences in emergent pe-
riodicity supposedly impose severe temporal reproductive isolation between
broods (13 and 17 year broods should co-emerge only once every 13 x 17 =
221 years!). Alternatively however, periodical cicadas might switch between
life-cycle lengths in response to genetic or environmental factors (Lloyd and
White, 1976; Lloyd et al., 1983). Some such switches must have occurred
evolutionarily to account for the origins of broods with different periodicities.
Martin and Simon (1988, 1990) observed an anomalous distribution of
mtDNA, allozyme, and morphological markers in M. septendecim and M.
tredecim in the central and eastern United States; in all data sets, 13-year cicadas
from a large region of their northern range proved more similar to 17-year
cicadas than to 13-year cicadas collected elsewhere in the same year. This
distribution of phylogenetic markers, in conjunction with geographical informa-
tion and other aspects of the data, were interpreted to evidence an historical
switch in maturation time, in which a large number of 17-year cicadas underwent
a 4-year acceleration in development to become 13-year cicadas, thus isolating
the new population temporally from the parent brood. Furthermore, in this case,
the developmental acceleration led to an overlap in emergence time with preex-
isting 13-year broods, thus opening new opportunities for gene flow between
former temporal isolates.
238 Kinship and intraspecific Phylogeny

NATURAL SELECTION AND BIOGEOGRAPHIC HISTORY IN THE KILLIFISH

In Fundulus heteroclitus, two common alleles at a lactate dehydrogenase

(LDH) nuclear gene exhibit a pronounced clinal shift in frequency along the east
coast of the United States (Fig. 6.7). Detailed laboratory studies have revealed
kinetic and biochemical differences between these LDH alleles that predict sig-
nificant differences among individuals in metabolism, oxygen transport, swim-
ming performance, developmental rate, and relative fitness (see the review in
Powers et al., 1991a). The nature of these differences is such that latitudinal
shifts in environmental temperature have been posited as directly responsible for
the clinal allelic structure (Mitton and Koehn, 1975; Powers et al., 1986). Does
adaptation to local conditions provide the entire story for the genetic architecture
of these killifish populations?
Gonzalez-Villasenor and Powers (1990) surveyed populations along the same
coastal transect with regard to restriction site variation in mtDNA and demon-
strated a pronounced phylogenetic subdivision of F. heteroclitus into northern
versus southern units. Quite likely, these populations were isolated during the
Pleistocene, and now hybridize secondarily along the mid-Atlantic coast in such
a way as to contribute to the clinal structure observed in LDH and in some other
nuclear genes. This example demonstrates how phylogenetic and selective mech-
anisms need not be opposing influences, but may in some cases act in concert to
achieve an observed population structure (Powers et al., 1991b). Although the
differences in LDH allele frequency are mediated to a significant degree by
environmental selection, the historical context in which this selection has taken
place adds an important dimension to our understanding of the contemporary
population genetic architecture.

HUMAN POPULATIONs---OUT OF AFRICA?

Not surprisingly, a great deal of attention has been directed toward estimating
and interpreting mtDNA phylogeny within Homo sapiens (e.g., Ballinger et al.,
1992; Cann et al., 1984; Denaro et al., 1981; DiRienzo and Wilson, 1991;
Excoffier, 1990; Johnson et al., 1983; Merriwether et al., 1991; Stoneking et aI.,
1986; Torroni et aI., 1992; Ward et aI., 1991; Whittam et aI., 1986). However,
two publications stand out as having had major historical and conceptual impact.
First, Brown (1980) provided an early glimpse of global mtDNA diversity by
assaying mtDNA RFLPs from 21 humans of diverse racial and geographic ori-
gin. Genetic differentiation was rather limited, with mean sequence divergence
estimated at only p = 0.0036. Using standard mtDNA clock calibrations, Brown
(1980) concluded that this level of mtDNA sequence heterogeneity "could have
been generated from a single mating pair that existed 180-360 X 103 years ago,
suggesting the possibility that present-day humans evolved from a small mito-
 Kinship and Intraspecific Phylogeny 239

1.0
CD

-ca
CD 0.8 Fundulus
heteroclitus
..a
m 0.6
•
J:
C 0.4
...I
.
-...
C" 0.2
CD

0.0
44 42 40 38 36 34 32 30

degrees north latitude

) northern
populations

southern
populations

2.0 1.0 0.0

% sequence divergence
(mtDNA)
Figure 6.7. Molecular geographic patterns in the killifish (after
Powers et al., 1991a). Top: Latitudinal cline in population frequen-
cies of the nuclear "b" allele of a lactate dehydrogenase polymor-
phism. Bottom: Phenogram of mtDNA haplotypes in these same
populations, showing the major phylogenetic distinction between
genotypes characteristic of northern versus southern populations.
240 Kinship and Intraspecific Phylogeny

chondrially monomorphic population that existed at that time." The magnitudes

of these estimated sequence divergences and separation times have changed
relatively little with the addition in subsequent studies of many more samples as
well as direct mtDNA sequence information (e.g., Hasegawa and Horai, 1991;
Vigilant et al., 1991). Thus, the overall mtDNA picture for humans remains one
of fairly shallow lineage separations, at least relative to conspecific populations
of many other species similarly assayed. [In this regard, the mtDNA results also
parallel findings from nuclear genomes that human populations and races are
remarkably similar in molecular composition, notwithstanding obvious pheno-
typic differences in traits such as hair texture and skin color (Nei and Livshits,
1990; Nei and Roychoudhury, 1982). For example, in an early summary of the
protein electrophoretic literature, Nei (1985) concluded that "the net gene dif-
ferences between the three races of man, Caucasoid, Negroid, and Mongoloid,
are much smaller than the differences between individuals of the same races, but
this small amount of gene differences corresponds to a divergence time of 50,000
to 100,000 years."]
In any event, the provocative conclusion of the Brown (1980) study was that
the coalescence of extant human mtDNA lineages might trace to a single female
(dubbed "Eve" by the popular press) within the last few hundred thousand
years, and furthermore that this indicated a severe bottleneck in absolute human
numbers (the "Garden of Eden" scenario). This latter aspect of the scenario
soon was challenged with results of models and computer simulations of popu-
lation lineage sorting as a function of popUlation demography (Chapter 4). From
such a theory on gene genealogies, Avise et al. (1984a) concluded that" 'Eve'
could have belonged to a population of many thousands or tens of thousands of
females, the remainder of whom left no descendants to the present day, due
simply to the stochastic lineage extinction associated with reproduction." In
other words, the observation that the genealogy of mtDNA (or any other locus)
coalesces does not necessarily imply an extreme bottleneck in absolute popula-
tion numbers (see also Hartl and Clark, 1989, p. 90). Latorre et al. (1986) were
more blunt: Even if it is true that all extant mtDNA lineages do trace to a single
female some 200,000 years ago, "the Mother Eve hypothesis is ... fallacious."
Recent analyses of allelic genealogies at nuclear loci have bolstered these con-
clusions. Thus, as summarized by Takahata (1993), "Genetic variation at most
loci examined in human populations indicates that the (effective) population size
has been == 104 for the past I Myr ... (and) that the population size has never
dropped to a few individuals, even in a single generation."
A second influential study extended the mtDNA survey to 147 humans from
around the world and produced a parsimony tree whose root traced to the African
continent (Cann et al., 1987). The findings led to the "out of Africa" hypoth-
esis, whereby human maternal lineages originated in Africa and spread within
the last few hundred thousand years to the rest of the world, replacing those of
 Kinship and Intraspecific Phylogeny 241

other archaic populations. This conclusion also has proved highly controversial.
One criticism has come from some paleontologists who on the basis of fossil or
other evidence favor a multiregional origin for humans that far predates the
apparent time scale of the mtDNA spread (e.g., Wolpoff, 1989; Wolpoff et al.,
1984; however, see also Stringer and Andrews, 1988; Wilson et aI., 1991).
Another criticism has come from some geneticists who found that the postulated
African root of the molecular phylogeny was not strongly supported (nor refuted)
when additional tree-building analyses were applied to the mtDNA data (Hedges
et aI., 1992c; Maddison, 1991; Templeton, 1992). [Evidence remains unchal-
lenged that extant populations in Africa house the highest level of mtDNA
polymorphism and this has provided a second argument for an African mtDNA
root (Cann et al. 1987).] Perhaps the truth lies somewhere between the extreme
versions of the out-of-Africa and the multiregional models. Thus, a third possible
scenario is that humans with modern anatomical features appeared first in Africa
and then spread throughout the world, not completely replacing archaic popu-
lations but rather interbreeding with them to some extent (Li and Sadler, 1992).
If so, the gene genealogies for some fraction of nuclear loci might well retain
branches tracing to early roots on continents other than Africa.
In any event, most of the recent discussions of human origins based explicitly
on gene trees have involved only the matrilineal component of our genetic
history, as recorded by mtDNA. At first thought, it might be supposed that
knowledge of the analogous patrilineal component (i.e., the transmission path-
way of the Y chromosome) would complete the story by revealing an "Adam"
["the father of us all" (Gibbons, 1991)], but this is fallacious. The vast majority
of our genetic heritage involves nuclear loci whose alleles trace through both
sexes during human evolutionary history. In theory, such nuclear genealogies
can, of course, differ from that of mtDNA as well as from one gene to the next.
As yet, few such nuclear genealogies have been estimated empirically, but
analyses of haplotypes at the loci encoding apolipoprotein and B-globin also
were interpreted to support an African origin for Homo sapiens sapiens, with
later dispersal to Europe and the Pacific (Rapacz et aI., 1991; Wainscoat et al.,
1986; however, see Giles and Ambrose, 1986). On the other hand, recent anal-
yses of the apolipoprotein C-II gene led Xiong et al. (1991) to suggest an Asian
origin for these sequences. Nonetheless, because of the limited amount of ge-
nealogical data currently available for nuclear loci and the great potential for
such information gain through additional sequencing, at this time only one aspect
of human origins seems absolutely certain-that interest and debate will continue
(Goldman and Barton, 1992).
GENEALOGICAL CONCORDANCE AND THE SHARP-TAILED SPARROW

Because gene trees within an organismal phylogeny can differ considerably

from locus to locus (Chapter 4), under what circumstances might the lineages
242 Kinship and Intraspecific Phylogeny

within gene genealogies faithfully reveal significant phylogenetic subdivisions

within conspecific pedigrees? One initial consideration is whether a particular
gene tree receives statistical support by criteria such as bootstrapping. Given that
a clade in a gene tree has been verified, the next consideration is whether
concordant genealogical support exists at other, independent loci. Ideally, an
examination of intraspecific phylogenies (of the sort provided by mtDNA) for
each of several unlinked nuclear genes would be desirable. In the absence of such
extensive genealogical data, surrogate genetic information must be employed.
For example, a recent molecular analysis of sharp-tailed sparrows (Ammodra-
mus caudacutus) uncovered a highly significant mtDNA distinction that divides
the species into two phylogeographic groups: a "northern" assemblage currently
inhabiting the interior prairies of the United States and Canada, the Hudson Bay
lowlands, the St. Lawrence River Valley, and the Canadian maritime provinces
to Maine; and a "southern" group that occurs along the Atlantic coast from
southern Maine to Virginia (Rising and Avise, 1993). Populations belonging to
these two mtDNA clades proved to be recognizable concordantly by multivariate
analyses of morphological attributes, as well as by behavioral distinctions in-
cluding song and flight displays (Greenlaw, 1993; Montagna, 1942). Such con-
cordance among independent lines of evidence suggests strongly that, in this
case, the split in the mtDNA gene tree also reflects a phylogenetic distinction in
the organismal phylogeny (likely tracing to Pleistocene population separations).
This example is of further interest because the subdivision of the sharp-tailed
sparrow into two distinctive phylogenetic units does not coincide with the ex-
isting subspecific designations for the complex, nor with the present-day geo-
graphic subdivisions upon which this taxonomy in part is based.

PHYLOGEOGRAPHY OF A REGIONAL FAUNA

Another potential class of genealogical concordance involves shared phylo-

geographic patterns across unrelated taxa, presumably due to similar historical
influences on intraspecific genetic architectures. A remarkable case in point
involves the maritime and freshwater faunas of the southeastern United States,
where a total of 19 species has been compared for phylogeographic patterning in
mitochondrial (and in some cases nuclear) markers (see the review in Avise,
1992). Among 10 estuarine species or species complexes surveyed throughout
most of their respective distributions [including creatures as diverse as the horse-
shoe crab (Limulus polyphemus), American oyster (Crassostrea virginica), black
sea bass (Centropristis striata), diamondback terrapin (Malaclemys terrapin),
and seaside sparrow (Ammodramus maritimus)], popUlations of at least five and
probably eight species evidence a fundamental mtDNA phylogenetic break be-
tween the Atlantic and Gulf of Mexico shorelines (Figs. 6.8 and 6.9). The
striking concordance in geographic patterns (though not always in the magni-
 Kinship and Intraspecific Phylogeny 243

Black Sea B~'7) Atl.

8) Gulf
I I I I I I I

:q"
3.0 2.5 2.0 1~ 1.0 0.5 0.0

American
Oyster
Seaside Sparrow ) Atl.

14] Gulf

I I I I I I I
3.0 2.5 2.0 1.5 1.0 0.5 0.0

Gulf

4:) AH.

"]~ Gulf

I I I I I

3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.0 2.5 2.0 1.5 1.0 0.5 0.0
Sequence Divergence (%) Sequence Divergence (%)

Figure 6.8. Relationships among mtDNA haplotypes in four maritime species

of the southeastern United States (from Avise, 1992). Numbers of individuals
belonging to various mtDNA clones are indicated to the right, whereas ter-
minal branches without numbers were represented by single assayed individ-
uals. Note that all UPGMA phenograms are plotted on the same scale of
estimated mtDNA sequence divergence.

tudes of separation) strongly suggests an overriding influence of shared historical

biogeographic forces. Most likely, ancestral forms of the various taxa were
separated during the Pleistocene into disjunct Atlantic and Gulf isolates (perhaps
via changes in sea level and the associated alterations in estuarine habitat), with
subsequent dispersal and contemporary ecological influences leading to the
present-day distributions of these clades. The different depths in the mtDNA
gene trees might reflect heterogeneity in molecular evolutionary rates across taxa
and/or isolations that date to different Pleistocene glacial or interglacial episodes.
The Gulf Stream, a major oceanic current that flows out of the Gulf of Mexico
and hugs the southern Florida coastline, is most likely an important contempo-
rary influence on gene flow, perhaps facilitating in some species the inferred
"leakage" of Gulf mtDNA haplotypes into southeastern Florida (Fig. 6.9). The
244 Kinship and Intraspecific Phylogeny

(J

Horseshoe American Seaside

Crab Oyster Sparrow

(J (J
(J

Diamondback Black
Terrapin Sea Bass

Figure 6.9. Geographic distributions ofmtDNA genotypes within each of

six maritime taxa (from Avise, 1992). Shown are pie diagrams summa-
rizing frequencies of the two fundamental clades in populations of each
species.

zone of demarcation between conspecific populations near Cape Canaveral (in

eastern Florida) is also a long-recognized boundary between marine zoogeo-
graphic provinces, as identified by a more traditional type of zoogeographic
evidence-concentrations of species' distributional limits (Briggs, 1958, 1974).
Similar historical processes may be evidenced by these intraspecific and inter-
specific phenomena; for example, if either of the distinctive conspecific units
recognized in the mtDNA analyses had become extinct, current northern or
southern range limits of the extant sister taxon likely would occur along eastern
Florida.
A similar pronounced pattern of intraspecific mtDNA genealogical concor-
dance across taxa characterizes freshwater fishes in the southeastern United
States (Avise, 1992; Bermingham and Avise, 1986; Scribner and Avise, 1993a).
Within each of six species or species complexes examined genetically, ranging
from the bowfin (Amia calva) to mosquitofish (Gambusia affinislholbrooki) and
spotted sunfish (Lepomis punctatus), a sharp phylogenetic subdivision distin-
guishes populations in eastern (primarily Atlantic coast) from western (primarily
Gulf coast) drainages (Figs. 6.10 and 6.11). The geographic dividing line differs
 Kinship and Intraspecific Phylogeny 245

Bowfin
j eastern

J western

6.0 6.0 4.0 2.0 0.0

eastern

1
1
western

6.0 6.0 4.0 2.0 0.0

j
Warmouth Sunfish
eastern

western

J
6.0 6.0 4.0 2.0 0.0

Redear Sunfish
) eastern Figure 6.10. Relationships among mtDNA
haplotypes in four freshwater fish species of
J western the southeastern United States [after Ber-
mingham and Avise (1986) as summarized
6.0 6.0 4.0 2.0 0.0 by Avise, 1992]. Note that all UPGMA
Sequence Divergence (%) phenograms are plotted on the same scale of
estimated mtDNA sequence divergence.

somewhat from one species to the next (as do the magnitudes of genetic dis-
tance), but considering the number and variety of species examined, shared
aspects of the phylogeographic patterns remain striking. As in the maritime
realm, these concordant features presumably reflect shared elements in biogeo-
graphic histories, likely involving Pleistocene refugia in eastern and western
drainages with subsequent dispersal routes influenced by subsequent drainage
interconnections. The general zone of demarcation between the eastern and
western freshwater populations also corresponds well to a contemporary bound-
ary between piscine zoogeographic provinces as identified by concentrations of
species' distributional limits (Swift et al., 1985).
It remains to be seen how often multispecies assemblages within other regional
246 Kinship and Intraspecific Phylogeny

Warmouth Sunfish Redear Sunfish Spotted Sunfish

Bowfin Bluegill Sunfish Mosquitofish

Figure 6.11. Geographic distributions of mtDNA genotypes within each of six fresh-
water fish species (from Avise, 1992). Shown are pie diagrams summarizing fre-
quencies of the two fundamental clades in populations of each species.

biotas will prove to exhibit such pronounced patterns of phylogeographic con-

cordance. In the American southwest, populations of the desert tortoise (Xero-
bates agassizi) east versus west of the Colorado River exhibit pronounced
mtDNA differentiation that was interpreted provisionally as stemming from the
isolating effects of historical marine incursions in the area (Lamb et al., 1989).
However, subsequent mtDNA analyses of two other reptile species with similar
ranges (the desert iguana Dipsosaurus dorsalis and the chuckwalla Sauromalus
obesus) failed to reveal any phylogeographic concordance with the desert tortoise
pattern (Lamb et al., 1992). Each species displays a unique signature of genetic
differentiation, but there is no evidence across species for the overriding impact
of any singular biogeographic factor.

General Conclusions About IntraspecifIC Phylogeography

Experience with the intraspecific mtDNA architectures of numerous species has

led to the formulation of several phylogeographic hypotheses and their corollaries
(Box 6.6). Whether or not these particular hypotheses are confirmed with addi-
tional data from mtDNA and nDNA genealogies, it can be argued that a more
explicit concern with the historical aspects of population structure should assume
a place in evolutionary study at least commensurate with that of' 'ecogeography,"
a long-established discipline that focuses on selection-mediated trends in geo-
graphic distributions of organismal attributes. For example, Bergmann's eco-
 Kinship and Intraspecific Phylogeny 247

Box 6.6. PhylOleolraphlc Hypotheses Derived from Studies

of mtDNA Gene Genealosles Within Spedes (after Avlse
et aI., • 987a)
I. Most species are composed of geographic populations whose members occupy
different branches of an intraspecific, phylogenetic tree (pedigree).
n. Species with limited or "shallow" phylogeographic population structure have
life histories conducive to dispersal and have occupied ranges free of long-standing
impediments to gene flow.
m. Monophyletic groups distinguished by large phylogenetic gaps usually arise
from long-term extrinsic (biogeographic) barriers to gene flow.
Corollaries
(a) As time since isolation increases, the degree of phylogeographic concordance
across separate gene genealogies increases.
(b) The geographic placements of phylogenetic gaps may tend to be concordant
across species.
(c) Phylogenetic gaps within species tend to be geographically concordant with
boundaries between traditionally-recognized biogeographic provinces.

geographic rule notes the tendency in homeotherms for larger body sizes at higher
latitudes, presumably a surface/volume adaptation for heat conservation in colder
climates; Allen's rule notes a latitudinal trend in lengths of limbs, perhaps because
shorter extremities serve similarly to conserve heat in cold climates; Gloger's
ecogeographic rule notes a tendency for populations in humid areas to be more
heavily pigmented, perhaps a manifestation of selection for background-matching
related to predation and competition. Emphasis on these and related ecogeo-
graphic trends perhaps reflects the prior dominance of an "adaptationist para-
digm" (Gould and Lewontin, 1979) for viewing intraspecific evolution, but this
perspective should now be balanced with the additional insight to be gained from
a phylogeographic perspective. The proposed place for phylogeography within the
broader discipline of biogeography is summarized in Figure 6.12.
A gene genealogy within any species represents but one realization of the
process of lineage sorting through an organismal pedigree and, hence, must be
interpreted with caution as an indicator of overall genomic history. In other
words, in the absence of concordant support from the phylogenies of independent
loci, significant partitions in a gene tree cannot necessarily be assumed to evi-
dence fundamental historical subdivisions at the population level (Chapter 4;
Avise and Ball, 1990). However, additional support for long-standing population
subdivisions may take any of several forms, including (a) a correspondence
between significant phylogenetic subdivisions in a gene tree and the boundaries
between historical biogeographic provinces as evidenced by nonmolecular data,
(b) a concordance between subdivisions in a gene genealogy and subdivisions
248 Kinship and Intraspecific Phylogeny

Biogeography
Ecogeography Phylogeography

e.g., Dispersal Vicariance

Bergmann's rule
Allen's rule
Gloger's rule

Figure 6.12. The proposed status of "phylogeography" within the

broader discipline of biogeographic study. An explicit concern with
phylogenetic aspects of intraspecific population structure should
help to broaden and enrich the selectionist perspectives of "eco-
geography" and encompass the realization that both past demo-
graphic conditions (induding dispersal and gene flow), as well as
historical environmental disjunctions [vicariance (see Chapter 8»),
no doubt have played influencing roles in generating present-day
lineage distributions.

registered by other attributes such as morphology or behavior (as in the sharp-

tailed sparrow), or (c) geographic concordance in significant genealogical par-
titions among the populations of several independent species (as for the fresh-
water and maritime faunas of the southeastern United States).
Thus, in interpreting gene phylogenies, a distinction should be drawn between
generalized conclusions about the magnitude of population structure and gene
flow, and the evidence for particular historical population separations. In species
whose populations exhibit contemporary isolation by distance in the absence of
long-standing biogeographic barriers to dispersal, phylogenies of independent
genes each may reveal significant population subdivision, yet exhibit little con-
cordance in the particular population units identified. Such results imply gener-
alized contemporary restrictions on gene flow, but the particular populations
distinguished by any gene may be of little phylogenetic consequence. In contrast,
when populations or metapopulations are recognized concordantly by multiple
lines of molecular or other genetic evidence, significant historical separations in
the organismal pedigree are implicated.

MICROTEMWORALPHYLOGENY
Most DNA sequences evolve far too slowly to permit the direct temporal
monitoring of significant phylogenetic changes in populations over yearly or
 Kinship and Intraspecific Phylogeny 249

decade-long time scales. One valiant attempt to describe such changes involved
the comparison of mtDNA sequences in extant and museum-preserved samples
of the Panamint kangaroo rat (Dipodomys panamintinus) taken from the same
locales in California (Thomas et aI., 1990). The museum samples were dried
skins prepared in 1911, 1917, and 1937, from which a mtDNA segment was
amplified by the PCR. The results showed a temporal stability in genotype
composition over this century, with relationships among three geographic pop-
ulations being the same for the modern as for the museum collections. However,
even if genetic changes had been observed, presumably most would have in-
volved shifts in frequencies of preexisting ancestral haplotypes (as can occur
rapidly under genetic drift or shifting family structure, for example) rather than
the evolutionary accumulation of de novo mutations over such a short time scale.
Molecular systems which apparently do evolve rapidly enough to permit the
monitoring of short-term mutational changes involve RNA viruses including
HIV, the human immunodeficiency retrovirus that produces AIDS (acquired
immune deficiency syndrome). The mean rate of synonymous nucleotide sub-
stitution for the HIV genome is approximately 10 x 10- 3 per site per year
(W.-H. Li et aI., 1988), or about 1,000,000 times greater than typical rates in the
nuclear genomes of most higher organisms (Chapter 2). Presumably, a rapid
pace of change in particular regions of these RNAs (along with the possibility of
inters train recombination) underlies the observed rapid rates of change in viral
pathogenicity and antigenicity (Coffin et aI., 1986; Gallo, 1987). Another con-
sequence of the rapid mutational process is that HIV s from different individuals
[and sometimes within the same individual through time (Holmes et aI., 1992)]
usually are found to be genetically distinct but related, and such heterogeneity
has found epidemiologic and forensic applications. For example, in sequence
comparisons of a HIV from a dentist, 7 of his infected patients, and 35 other HIV
carriers from the local geographic area, it was shown that the viruses from the
dentist and 5 of his patients were related closely (Ou et al., 1992). These
molecular markers provided the first genetic confirmation for transmission of
HIV from an infected health care worker to clients.
The human AIDS virus appears to have originated in Africa, perhaps within
the past few decades following a postulated transfer from wild monkeys (Dia-
mond, 1992) and, subsequently, has infected millions of people worldwide.
Studies of HIV nucleotide sequences have played an important role in docu-
menting the histories and patterns of migration of the virus (Desai et al., 1986;
Gallo, 1987; Yokayama and Gojobori, 1987). For example, W.-H. Li et ai.
(1988) analyzed the sequences from 15 HIV isolates from the United States,
Haiti, and Zaire to generate the estimated phylogeny reproduced in Figure 6.13.
Results are consistent with the hypothesized African ancestry of HIV, its sub-
sequent spread to Haiti, and later to the United States. Most remarkable about
these phylogenetic reconstructions are the micro-time-scales involved. From the
250 Kinship and Intraspecific Phylogeny

United
States

~'--------l
J""Ii
] Zo;"

o 2 4 6 8 10 12

% sequence divergence from root

Figure 6.13. Phylogenetic relationships among 15 my isolates based

on nucleotide sequence data (after W.-H. Li et al., 1988). Note the
micro-time-scales as reflected by recent dates for various nodes in the
tree.

synonymous substitution rates in HIV, the inferred separation date of the New
World from the African isolates was 1969, and the separation date of the United
States from the Haitian isolates was 1977!

SUMMARY
1. All conspecific individuals are related genealogically through an ex-
tended pedigree that constitutes the intraspecific phylogeny of a species.
2. Molecular approaches for assessment of kinship within a species nor-
mally require highly-polymorphic qualitative markers with known transmission
patterns. However, because of the complexity of potential transmission pathways
between relatives and the indeterminate nature of Mendelian inheritance, exact
kinship relations between particular pairs of individuals seldom can be estab-
lished, and analyses normally are confined to assessment of mean genetic relat-
edness within groups.
3. In eusocial species such as many haplo-diploid hymenopterans, mean
intracolony relatedness values sometimes have proved to be high, but numerous
exceptions. present an enigma for sociobiological theories on the evolution of
reproductive altruism.
4. Within noneusocial groups, a variety of mean genetic relatedness values
 Kinship and Intraspecific Phylogeny 251

has been observed using molecular markers, and these often appear interpretable
in terms of the suspected behaviors and natural histories of the species assayed.
Genetic markers also have helped to address questions regarding mechanisms
and genetic consequences of kin recognition.
5. Most populations are genetically structured geographically. These ge-
netic architectures have been revealed for numerous species using molecular
markers and appear to be influenced by a variety of factors including the nature
of the mating system and the gene flow regime. These, in turn, are influenced by
the species-specific dispersal capabilities of gametes and zygotes and by the
vagilities as well as philopatries of postzygotic life-history stages.
6. The genetic structure of any species also has been influenced by histor-
ical biogeographic and demographic factors. In large measure, historical per-
spectives on population genetic structure were stimulated by the relatively un-
ambiguous genealogical reconstructions made possible by development of assays
for nonrecombining mtDNA haplotypes. A new discipline termed' 'phylogeog-
raphy" has enriched biogeographic study and provides a balance to traditional
ecogeographic perspectives.
7. Some viruses evolve so rapidly that genetic changes can be observed
directly over time scales of years or decades. Molecular phylogenetic appraisals
of the HIV virus have revealed details of its origin and spread within this century.
 7

Speciation and Hybridization

Without gene flow. it is inevitable that there will

be speciation.

M.H. Wolpoff. 1989

With regard to sexually-reproducing organisms, several "species concepts"

have been advanced (Box 7.1). Most of these entail the perception of conspecific
populations as a field for gene recombination-in other words, as an extended
reproductive community within which genetic exchange potentially takes place.
For example, under the popular "biological species concept" (BSC) champi-
oned by Dobzhansky (1937), species are characterized as "groups of actually or
potentially interbreeding natural populations which are reproductively isolated
from other such groups" (Mayr, 1963). Many authors have expressed sentiments
on the BSC similar to those of Ayala (1976b): "among cladogenetic processes,
the most decisive one is speciation-the process by which one species splits into
two or more ... Species are, therefore, independent evolutionary units. Adap-
tive changes occurring in an individual or population may be extended to all
members of the species by natural selection; they cannot, however, be passed on
to different species." Thus, under the BSC and related concepts, species are
perceived as biological and evolutionary entities that are far more meaningful
and less arbitrary than other taxonomic categories such as subspecies, genera, or
orders (Dobzhansky, 1970). Nonetheless, several complications can attend the
application of BSC principles.
One of these difficulties involves the discretionary judgments required about
the specific status of related extant forms in allopatry (as well as of extant forms

252
 Speciation and Hybridization 253

Box 7.1. lxamples of Various Spedes Concepts and Definitions

1. Biological species concept (BSC) (Dobzhansky, 1937): "Species are systems of
populations: the gene exchange between these systems is limited or prevented by a
reproductive isolating mechanism or perhaps by a combination of several such mech-
anisms. "
Comment: Unquestionably the most influential species concept, and one that re-
mains popular today.
2. Evolutionary species concept (ESC) (Simpson, 1951): "a lineage (ancestral-
descendant sequence of populations) evolving separately from others and with its own
unitary evolutionary role and tendencies."
Comment: Applicable both to living and extinct groups, and to sexual and asexual
organisms. However, this concept is vague operationally in what is meant by "uni-
tary evolutionary role and tendencies."
3. Recognition species concept (RSC) (Paterson, 1985): the most inclusive population
of biparental organisms which share a common fertilization system.
Comment: Similar to the BSC in viewing conspecific populations as a field for gene
recombination. However, this concept shifts attention from isolating mechanisms as
barriers to gene exchange, to a focus instead on the positive function of these
mechanisms in facilitating reproduction among members of a species. Although
reproductive barriers can indeed arise as a by-product of speciation, they are not
viewed as an active part of the speciation process.
4. Cohesion species concept (CSC) (Templeton, 1989): "the most inclusive population
of individuals having the potential for cohesion through intrinsic cohesion mecha-
nisms. "
Comment: Attempts to incorporate strengths of the BSC, ESC, and RSC, and avoid
their weaknesses. The major classes of cohesion mechanisms are genetic exchange-
ability (factors that define the limits of spread of new genetic variants through gene
flow) and demographic exchangeability (factors that define the fundamental niche
and the limits of spread of new genetic variants through genetic drift and natural
selection) .
5. Phylogenetic species concept (PSC) (Cracraft, 1983): a monophyletic group com-
posed of "the smallest diagnosable cluster of individual organisms within which there
is a parental pattern of ancestry and descent. "
Comment: Explicitly avoids all reference to reproductive isolation and focuses in-
stead on phylogenetic histories of populations. A serious problem involves how
monophyly is to be recognized and how to distinguish histories of traits (e.g., gene
trees) from histories of organisms (pedigrees).
6. Concordance principles (CP) (Avise and Ball, 1990): a suggested means of recog-
nizing species by the evidence of concordant phylogenetic partitions at multiple in-
dependent genetic attributes.
Comment: Attempts to incorporate strengths of the BSC and PSC and avoid their
weaknesses. This approach accepts the basic premise of the BSC, with the under-
standing that the reproductive barriers are to be interpreted as intrinsic as opposed
to extrinsic (purely geographic) factors. When phylogenetic concordance is exhib-
ited across genetic characters solely because of extrinsic barriers to reproduction,
subspecies status is suggested.
254 Speciation and Hybridization

to their evolutionary ancestors). Inevitably, reproductive isolating barriers [RIBs

(Box 7.2)] develop between geographically separated popUlations as a nonadap-
tive by-product of genomic divergence, but the time scales involved and the
magnitudes of differentiation are matters for study in particular instances. The
"acid test" for biological species status-whether the populations retain sepa-
rate identities in sympatry-often has not been carried out in nature. A second
practical difficulty involves the issue of how much genetic exchange disqualifies
populations from status as separate biological species. Thus, the study of spe-
ciation conceptually links the topic of gene flow (Chapter 6) with that of hy-
bridization and introgression. Under the BSC, there are no black-and-white
solutions to either of these two difficulties because evolutionary divergence and
speciation normally are gradual processes, and because levels of genetic ex-
change can vary along a continuum (Dobzhansky, 1976).
Another challenge in applying the BSC involves the need to distinguish the
evolutionary origins of RIBs from their genetic consequences. Normally, repro-
ductive barriers under the BSC are considered intrinsic biological factors rather
than purely extrinsic limits to reproduction resulting from geographic separation
alone. However, this distinction blurs when syntopic popUlations (those occu-
pying the same macrohabitat) are isolated via preferences for different micro-
habitats, particularly when these habitat proclivities are coupled with differences
in mate choice (Diehl and Bush, 1989). In such situations, one substantive as
well as semantic issue is whether speciation occurred sympatrically versus allo-
patrically followed by secondary range overlap. Another issue is whether certain
types of RIBs arise in direct response to selection pressures favoring homotypic
matings (for example) or whether they reflect nonselected by-products of ge-
nomic differentiation that occurred for other reasons (Box 7.2).
Associated with the speciation process, under any definition, is the conversion
of genetic variability within a species to between-species genetic differences.
However, because RIBs retain primacy in demarcating species under the BSC,
no arbitrary magnitude of molecular genetic divergence can provide an infallible
metric to establish specific status, particularly among allopatric forms. Further-
more, as noted by Patton and Smith (1989), almost "all mechanisms of speciation
that are currently advocated by evolutionary biologists . . . will result in para-
phyletic taxa as long as reproductive isolation forms the basis for species defi-
nition. " How then may molecular markers inform speciation studies? First,
molecular patterns might provide distinctive genetic signatures relatable to de-
mographic events during speciation or to the geographic settings in which spe-
ciation took place (Neigel and Avise, 1986; Templeton, 1980a; Fig. 7.1). Second,
estimates of genetic differentiation between populations at various stages of RIB
acquisition might be useful in assessing temporal aspects of the speciation process
(Coyne, 1992). Finally, molecular markers can prove invaluable for assessing the
magnitude and pattern of genetic exchange among related forms, and thereby can
contribute to an understanding of the intensity and nature of RIBs.
 Speciation and Hybridization 255

Box 7.2. Oasslftcatlon of Reproductive Isolating Barriers (RIBs)

1. Prezygotic Barriers
(a) Ecological or habitat isolation: Populations occupy different habitats in
the same general region, and most matings take place within these mi-
crohabitat types.
(b) Temporal isolation: Matings take place at different times, e.g. seasonally
or diurnally.
(c) Ethological isolation: Individuals from different populations meet but do
not mate.
(d) Mechanical isolation: Interpopulational matings occur but no transfer of
male gametes takes place.
(e) Gametic mortality or incompatibility: Transfer of male gametes occurs
but eggs are not fertilized.
2. Postzygotic Barriers
(a) F 1 inviability: F 1 hybrids have reduced viability.
(b) F 1 sterility: F 1 hybrids have reduced fertility.
(c) Hybrid breakdown: F 2 , backcross, or later-generation hybrids have re-
duced viability or fertility.
One rationale for distinguishing between prezygotic and postzygotic RIBs is that in
principle only the former are selectable directly. Under the "reinforcement" scenario
of Dobzhansky (1940; Blair, 1955), natural selection can act to superimpose prezy-
gotic RIBs over any preexisting postzygotic RIBs (that may have arisen, for example,
in former allopatry). As stated by Dobzhansky [1951, as quoted from Butlin (1989)],
"Assume that incipient species, A and B, are in contact in a certain territory. Muta-
tions arise in either or both species which make their carriers less likely to mate with
the other species. The nonmutant individuals of A which cross to B will produce a
progeny which is adaptively inferior to the pure species. Since the mutants breed only
or mostly within the species, their progeny will be adaptively superior to that of the
non-mutants. Consequently, natural selection will favour the spread and establishment
of the mutant condition."
Notwithstanding its conceptual appeal, Dobzhanksy's suggestion has proven diffi-
cult to verify observationally or experimentally (see the review in Butlin, 1989).
Koopman (1950) and Thoday and Gibson (1962) provide widely-quoted examples of
selective reinforcement of prezygotic RIBs, but other such experimental studies have
produced equivocal outcomes (e.g., Spiess and Wilke, 1984).
(a) vq
abc d e f

abc d e f

(b)
'«)7
abc d e f

(c)
'«)7
abc d e f' f

(d)
~
abc d e f' f

(e)
~
Figure 7.1. Five models of speciation and corresponding gene
trees (after Harrison, 1991). Shown are distributions of alleles
in the two daughter species. For simplicity, each population is
represented as monomorphic, and the gene genealogy in each
case is «(a,b)(c»«d)(e,f»). In reality, most populations are
likely to be polymorphic and, hence upon separation, are ex-
pected to evolve through intermediate states of polyphyly and
paraphyly in the gene tree (Chapter 4). The five models are (a)
speciation by geographic subdivision with the physical partition
congruent with an existing phylogenetic discontinuity; (b) spe-
ciation by subdivision with the partition not congruent with an
existing phylogenetic discontinuity; (c) speciation in a periph-
eral population; (d) speciation via colonization of a new habitat
by propagule(s) from a single population; and (e) local sympat-
ric speciation. In the allelic phylogenies, solid versus hatched
lines represent lineages occurring in the two respective daughter
species.
 Speciation and Hybridization 257

THE SPECIATION PROCESS

What follows are examples of some questions in speciation theory that have
been readdressed through use of molecular genetic markers.

How Much Genetic Change Accompanies Speciation?

TRADITIONAL PERSPECTIVES

One long-standing view of species differences, stated clearly by Morgan early

in this century (1919), is that species differ from each other "not by a single
Mendelian difference, but by a number of small differences. " A counterproposal
frequently expressed was that new species or even genera might arise by single
mutations of a special kind-"macromutations" or "systemic mutations"
(deVries, 1910; Goldschmidt, 1940}--that suddenly transform one kind of or-
ganism into another. Although such suggestions for saltational speciation prob-
ably are untenable in their original formulation, more recent theories have
stressed plausible routes by which species might arise rapidly, perhaps in some
cases with minimal molecular genetic divergence overall. Examples of such
avenues to saltational speciation in plants and animals are summarized in Box
7.3. But apart from these "special cases" (which nonetheless may be quite
widespread), can species arise quickly and with little genetic alteration?
One class of argumentation for sudden speciation comes from paleontology.
Based on a reinterpretation of "gaps" in the fossil record, Eldredge and Gould
(1972) proposed that diagrams of the "tree of life," in which divergence is
plotted on one axis and time on the other, are better represented as "rectangular"
branching patterns (Stanley, 1975) reflecting evolution through "punctuated
equilibria." According to this view, a new species arises rapidly and, once
formed, represents a well-buffered homeostatic system, resistant to within-
lineage change (anagenesis) until speciation again is triggered, perhaps by an
alteration in ontogenetic (developmental) pattern (Gould, 1977). A second class
of argumentation for sudden speciation has come from molecular genetics: The
molecular events responsible might involve changes in gene regulation perhaps
mediated by relatively few control elements that could have a highly dispropor-
tionate influence on organismal evolution, including speciation (Britten and Da-
vidson, 1969, 1971; Krieber and Rose, 1986; McDonald, 1989, 1990; Rose and
Doolittle, 1983; Wilson, 1976). A third class of argumentation involves demo-
graphic and population-genetic considerations. For example, Mayr (1954) sug-
gested that "founder effects" in small, geographically isolated populations
might produce "genetic revolutions" leading to new species; Carson (1968)
advanced a "founder-flush" model in which rapid population expansion and
relaxed selection following a severe founder event might facilitate the appear-
258 Speciation and Hybridization

Box 7.3. Sudden Spedadon

There are several known or suspected pathways to rapid speciation that entail little
or no change in genetic composition at the allelic level (beyond rearrangements of
genetic variation from the ancestral forms). These include the following:
(a) Polyploidization. The origin of stable polyploids usually is associated with
hybridization between populations or species differing in chromosome constitution. If
the hybrid is sterile only because its parental chromosomes are too dissimilar to pair
properly during meiosis, this difficulty is removed by a doubling of chromosomes
producing a polyploid hybrid. Furthermore, such a polyploid species spontaneously
exhibits reproductive isolation from its progenitors because any cross with the parental
species produces progeny with unbalanced chromosome sets (e.g., a cross between a
tetraploid and a diploid progenitor produces mostly sterile triploids).
Examples: The treefrog Hyla versicolor is a tetraploid that on the basis of allozyme
and immunological comparisons arose recently from hybridization between distinct
eastern and western populations of its cryptic diploid relative H. chrysoscelis (Maxson
et aI., 1977; Ralin, 1976). However, polyploidy is relatively uncommon in animals
and confined primarily to forms that reproduce asexually (see the section on hybrid-
ization in this chapter). On the other hand, recent estimates indicate that 70-80% of
angiosperm plant species may be of polyploid origin (Lewis, 1980), and molecular
genetic data often provide definitive evidence identifying the ancestral parental spe-
cies. For example, allozyme analyses established that the tetraploid goatsbeard species
Tragopogon mirus and T. miscellus arose recently from crosses between the diploids
T. dubius and T. porrifolius, and T. dubius and T. pratensis, respectively (Roose and
Gottlieb, 1976). These allopolyploids (polyploids arising from combinations of ge-
netically distinct chromosome sets) expressed additively all examined allozyme alleles
inherited from their respective progenitors. In several cases, similar molecular genetic
analyses involving allozymes or cpDNA have demonstrated that particular polyploid
forms are of polyphyletic (multihybridization in this case) origin, e.g., Plagiomnium
bryophytes (Wyatt et aI., 1988), Asplenium ferns (Werth et al., 1985), Glycine soy-
beans (Doyle et al., 1990), Heuchera alumroots (Soltis et aI., 1989) and Senecio
composites (Ashton and Abbott, 1992). New autopolyploid taxa (polyploids that arise
by the multiplication of one basic set of chromosomes) also have been described
through molecular assays (Rieseberg and Doyle, 1989). An especially ingenious ap-
plication of genetic markers involved documentation of a complicated cytological
pathway leading to a new tetraploid species of fern, Asplenium plenum. Using protein
electrophoresis, Gastony (1986) showed that A. plenum must have arisen through a
cross between triploid A. curtissii (which had produced an unreduced spore) and a
diploid A. abscissum (which had produced a normal haploid spore). The nearly-sterile
triploid A. curtissii itself was shown to have arisen through a cross between a tetraploid
species A. verecundum and diploid A. abscissum.
(b) Chromosomal rearrangements. Closely related taxa differing in a variety of
structural chromosomal features-including translocations, inversions, or chromo-
some numbers-may exhibit reproductive isolation for at least two reasons. First, the
structural differences themselves may cause difficulties in chromosomal pairing and
proper disjunction during meiosis in hybrids, resulting in partial or complete sterility.
Second, the gene rearrangements may otherwise diminish fitness in hybrid classes
through disruptions of gene expression patterns resulting from position effects.
 Speciation and Hybridization 259

Examples: White (1978a) compiled evidence for animals that chromosomal rear-
rangements commonly are involved in the speciation process (see also Sites and
Moritz, 1987), as did Grant (1981) for plants. When chromosomal rearrangements
have conferred reproductive isolation recently, allelic differentiation between the de-
scendant species may yet be minimal. Three examples in which the magnitude of
allozymic differences between chromosomally differentiated forms are reported to be
about the same as those among populations within a species involve subterranean
Thomomys and Spalax rodents (Nevo and Shaw, 1972; Nevo et al., 1974; Patton and
Smith, 1981) and Sceloporus lizards (Sites and Greenbaum, 1983). In some of these
cases, however, the chromosomal differences do not provide complete barriers to
reproduction.
(c) Changes in the mating system. Many plant species exhibit self-incompatibility
whereby pollen fail to fertilize ova from the same individual. The mechanisms may
involve alleles at a self-incompatibility locus known to be highly polymorphic within
some species (Ioerger et al., 1990) or to a physical barrier such as a difference in the
lengths of styles and stamens (heterostyly) that inhibits self-pollination. A switch in
mating system [for example, from self-incompatibility to self-compatibility (autog-
amy) as mediated by a change from heterostyly to homostyly] could, in principle,
precipitate a rapid "speciation" event with little overall change in genic composition.
Other alterations of the breeding system such as the timing of reproduction similarly
might generate reproductive isolation rapidly.
Examples: In many plant groups, closely related taxa exhibit contrasting reproduc-
tive modes suggesting that "the evolution of floral syndromes, and their influence on
mating patterns, is intimately associated with the development of reproductive isola-
tion and speciation" (Barrett, 1989). For example, self-compatible Stephanomeria
malheurensis apparently arose from a self-incompatible progenitor S. exigua ssp.
coronaria and also differs from it by chromosomal rearrangements which are the
principle cause of the hybrid sterility (Stebbins, 1989). As judged by high allozymic
similarities (Gottlieb, 1973b), the process took place recently such that the derivative
species "was extracted from the repertoire of genetic polymorphisms already present
in the progenitor" (Gottlieb, 1981). Such evolution of self-fertilization probably fa-
vors the establishment of chromosomal rearrangements contributing to reproductive
isolation of the selfing derivative (Barrett, 1989).

ance and survival of novel recombinants leading to a new species; and, Templeton
(1980b) introduced a "transilience model" in which speciation involves a rapid
shift to a new adaptive peak under conditions where a founder event causes a
rapid but temporary accumulation of inbreeding without severe depletion of
genetic variability [see Carson and Templeton (1984) for comparisons of these
models, and Provine (1989) for the history of concepts]. The generality of such
rapid-speciation scenarios has proved difficult to document because speciation no
doubt is a highly eclectic process and because many of the proposed genetic and
demographic events likely occur also at the populational level without producing
new species.
260 Speciation and Hybridization

On the other hand, many authors have viewed the speciation process (though
not necessarily its products) as a rather unexceptional continuation of the mi-
croevolutionary processes generating geographic population structure, with the
added factor of the evolutionary acquisition of intrinsic reproductive isolation (see
the reviews in Barton and Charlesworth, 1984; Charlesworth et al., 1982). This
view was termed "phyletic gradualism" by Eldredge and Gould (1972). How-
ever, Wright (1931) and some others who interpret speciation mostly as a con-
tinuation of microevolution (Provine, 1986) nonetheless emphasize how episodic
shifts in evolution can result from genetic drift (in conjunction with selection) in
facilitating rapid leaps across adaptive peaks. Thus, the crucial distinction is not
whether evolutionary change is gradual versus episodic, but whether speciation
as a process is somehow decoupled from processes of intraspecific population
differentiation [as Gould (1980) suggested]. As an approach to addressing these
issues, many assessments have been made of the magnitude and pattern of genetic
differentiation associated with species formation.
Traditional nonmolecular approaches to quantifying genetic divergence be-
tween closely related species involve study of phenotypic traits among the later-
generation progeny of hybrid crosses. One method is to measure the variance
among F 2 hybrids for particuiar behavioral or morphological characters. Fre-
quently, it was observed that such variances greatly exceed those in the parental
popUlations and in the FJ's, and that few F2's fall into the parental classes
(DeWinter, 1992; Lamb and Avise, 1987; Rick and Smith, 1953). Such results
appear attributable to recombinationally derived variation and indicate that for
these assayed characters the parental species must differ in multiple genes, each
with small effect [although only the minimum number of such polygenes actually
can be estimated by this approach (Lande, 1981)]. Another traditional method of
assessing the genetic differences between species involves chromosomal map-
ping of RIB genes through searches for consistent patterns of co segregation in
experimental backcross progeny (see the review in Charlesworth et al., 1987).
For example, in sibling species of Drosophila, partial hybrid sterility and invi-
ability have proven attributable to differences at several (usually anonymous)
loci on each chromosome (Dobzhansky, 1970, 1974; Orr, 1987, 1992), with
X-linked genes typically having the greatest effects (Coyne and Orr, 1989a).
However, there are at least two serious limitations to these classical Mendelian
approaches. First, they can be applied only to hybridizable taxa. Second, pat-
terns of allelic assortment can be inferred only for loci distinguishing the parental
species-genes identical in the parents escape detection. But to determine the
proportion of genes distinguishing species, both divergent and nondivergent loci
must be monitored. Thus, following the introduction of allozyme methods in the
mid-1960s, many researchers reexamined the issue of genetic differentiation
during speciation, under the rationale that direct molecular assays permitted, for
the first time, an examination of a large sample of gene products unbiased with
 Speciation and Hybridization 261

regard to the divergence issue (see the reviews in Avise, 1976; Ayala, 1975;
Gottlieb, 1977).

MOLECULAR EVIDENCE

The most extensive allozyme survey of genetic differentiation accompanying

RIB acquisition involved the Drosophila willistoni complex (Ayala et al.,
1975b), which includes populations at several stages ofthe speciation process as
gauged by reproductive relationships and geographic distributions. This complex
is distributed widely in northern South America, Central America, and the Ca-
ribbean, and provides a paradigm example of gradual speciation involving
(a) geographic populations that are fully compatible reproductively, (b) different
"subspecies" that are allopatric and exhibit incipient reproductive isolation
in the form of postzygotic RIBs (hybrid male sterility in laboratory crosses),
(c) "semispecies" that overlap in geographic distribution and show both postzy-
gotic RIBs and prezygotic RIBs (homotypic mating preferences), the latter pre-
sumably having evolved under the influence of natural selection after sympatry
was achieved between formerly separated subspecies (Box 7.2), (d) sibling spe-
cies that show complete reproductive isolation but remain nearly identical mor-
phologically, and (e) nonsibling species that diverged earlier. Frequency distri-
butions of genetic similarities across 36 allozyme loci are summarized in Figure
7.2. Between subspecies (or semispecies), nearly 15% of these genes showed
substantial or fixed allelic frequency differences involving detected replacement
substitutions. Results were interpreted to indicate that "a substantial degree of
genetic differentiation occurs during the first stage of speciation" (Ayala et al.,
1975b). The subsequent allozyme literature on 119 pairs of closely related
Drosophila taxa was reviewed by Coyne and Orr (1989b). These taxa provided
a cross section of populations at various stages of the speciation process, as
defined by geographic distributions and experimentally determined levels of
prezygotic and postzygotic reproductive isolation. Results demonstrated that
large genetic distances often are associated with various levels of partial repro-
ductive isolation (Table 7.1).
Other noteworthy early studies demonstrating moderate to large allozymic
distances between populations at various stages of speciation involved Lepomis
sunfishes (Fig. 7.2), Peromyscus mice (Zimmerman et al., 1978), and Helian-
thus sunflowers (Wain, 1983). In a sense, these and the Drosophila studies
merely affirm what already was emphasized in Chapter 6-that considerable
genetic differentiation among geographic populations can accumulate prior to the
completion of intrinsic reproductive isolation.
Among the vertebrates, perhaps the current record for magnitude of genetic
differentiation prior to the completion of speciation involves the salamander
Ensatina eschscholtzii. This complex of morphologically differentiated subspe-
262 Speciation and Hybridization

Drosophila Lepomis
willistoni sunfish
complex
100

I = 0.97 I = 0.98 geographic

50
populations

90

I = 0.83 I = 0.84 subspecies

50
or
semispecies

80
sibling
= 0.43 I = 0.54
'u 50
I
or
!! nonsibling
<f.
species
0.5 0 0.5
genetic sImilarity genetic sImilarity

Figure 7.2. Distributions of allozyme loci with respect to genetic

similarity (Nei' s 1972 measure) in comparisons among populations
at various stages of evolutionary divergence. Shown are results from
Drosophila willistoni complex fruit flies (after Ayala et al., 1975b)
and Lepomis sunfish (after Avise and Smith, 1977). For the fruit
flies, some categories such as "subspecies" and "semispecies" that
were treated separately in the original paper are grouped here, with
equal weighting.
 Speciation and Hybridization 263

Table 7.1. Means and standard errors of genetic distance (Nei's D, allozymes)
characterizing Drosophila taxa at indicated levels of prezygotic and postzygotic
reproductive isolationa

Reproductive Mean Genetic Distance (SE)

Isolation No. of
Index Comparisons Prezygotic Postzygotic

0.00 13 0.122 (0.046) 0.138 (0.058)

0.25 8 0.370 (0.078) 0.251 (0.083)
0.50 21 0.257 (0.080) 0.249 (0.032)
0.75 29 0.578 (0.098) 0.722 (0.198)
1.00 13 0.523 (0.089) 0.991 (0.127)

1 _ (frequency heterotypic matings) .

aPrezygotic isolation index:
(frequency homotypic matings)
Postzygotic isolation index: A measure of hybrid inviability and hybrid sterility,
scaled from zero to one.
Source: After Coyne and Orr (l989b)

cies encircles the Central Valley of California in ring-like fashion, with adjacent
populations along the chain normally capable of genetic exchange. However, at
the southern end of the distribution, populations stemming from coastal areas and
the interior overlap sympatrically with limited or no hybridization and thus appear
to have achieved the status of biological species in this area (Wake et al., 1986).
Remarkably, various populations in this ring complex show profound allozymic
distances, often greater than D = 0.5 [far greater than values for many congeneric
vertebrate species (Fig. 1.2)] (Wake and Yanev, 1986). Huge genetic distances
(p > 0.12) are apparent in the mtDNA genomes as well (Moritz et al., 1992).
Wake et al. (1989) interpret the results to evidence "several stages of speciation
in what appears to be a continuous process of gradual allopatric, adaptive di-
vergence." If genetic distances provide even a crude measure of evolutionary
time, speciation in this salamander complex must be extremely slow.
On the other hand, by these same allozymic criteria, other recognized species
of animals and plants are characterized by much smaller genetic distances, often
within the range of values more normally associated with conspecific popula-
tions, i.e., D less than about 0.05 (Fig. 1.2). Examples can be found within
herbaceous plants (Ganders, 1989; Witter and Carr, 1988), insects (Harrison,
1979; Simon, 1979), snakes (Gartside et al., 1977), mammals (Apfelbaum and
Reig, 1989; Hafner et al., 1987), birds (Thorpe, 1982), and many others. Pre-
sumably, the presence of few or no differentiating electromorphs between related
biological species indicates that insufficient time has elapsed for the accumula-
tion of greater de novo mutational differences in these assays. Indeed, this
264 Speciation and Hybridization

time-dependent aspect of allozyme divergence has proved useful in reassessing

speciation dates in several instances. For example, two minnow species in Cal-
ifornia that had been placed in different genera (Hesperoleucus and Lavinia)
proved to exhibit an allozyme distance of only D == 0.05, suggesting a far more
recent separation than had been implied from their generic assignments (A vise et
aI., 1975). In the plant genera Clarkia, Lycopersicon, Erythronium, and Gaura,
particular progenitor-derivative species pairs with low allozymic distances now
are interpreted to be of relatively recent origin (Gottlieb, 1974; Gottlieb and Pilz,
1976; Pleasants and Wendel, 1989; Rick et al., 1976). Conversely, the self-
pollinating plant Clarkia franciscana was thought to have evolved recently by
rapid reorganization of chromosomes from the morphologically similar C. rubi-
cunda, but has proved to be totally divergent from that species at 75% of
allozyme loci, indicating that the phylogenetic separation of the two species
occurred much longer ago than formerly supposed (Gottlieb, 1973a).
Apart from allozyme methods, few molecular techniques have been brought to
bear explicitly on the issue of magnitude of genetic differentiation during spe-
ciation (Harrison, 1991). Multilocus DNA fingerprinting approaches are too
sensitive, and DNA-DNA hybridization methods are generally too insensitive
(however, see Caccone et al., 1987), for addressing these meso-evolutionary
issues. Single-locus sequencing or RFLP approaches applied to mtDNA or
scnDNA may offer the proper window of resolution, but normally provide only
individual gene genealogies that in theory (Chapter 4) and practice (see next
section) may differ from one locus to another and from the composite species
phylogeny. Although it will be interesting to reexamine "genetic differentiation
during speciation" using more refined molecular approaches, any enhanced res-
olution of species differences might well be matched by increased resolution of
populational differences. Thus, in any future studies using refined genetic mark-
ers, a significant challenge again will be to obtain a suitable frame of reference
by extensive sampling of intraspecific variation and by assessment of the mag-
nitude and pattern of interlocus variation.

Do Speciations Entail Severe Population Bottlenecks?

All sudden modes of speciation described in Box 7.3 no doubt are initiated by
very small numbers of individuals who first acquire the relevant chromosomal or
reproductive alterations. Apart from such situations, do founder events underlie
speciations in many other animal and plant groups? If so, significant shifts in
frequencies of ancestral polymorphisms might be entailed, but at the outset
probably little de novo sequence divergence will have accumulated. A severe and
prolonged population bottleneck accompanying speciation should also greatly
diminish genetic variability in the neospecies.
 Speciation and Hybridization 265

A remarkable radiation of drosophilid flies has occurred in the Hawaiian

archipelago, with about 800 species endemic to the islands (compared to about
2000 species in the remainder of the world) (Carson and Kaneshiro, 1976;
Wheeler, 1986). Founder-induced speciation models have figured prominently in
discussions of the prolific speciation among Hawaiian Drosophila (Giddings et
al., 1989), where species formation is postulated to follow the colonization of
new islands perhaps by one or a small number of gravid females. However,
molecular genetic data are equivocal on these scenarios. Some sister species such
as D. silvestris and D. heteroneura do indeed exhibit high allozymic similarities
suggestive of recent speciation (Sene and Carson, 1977). On the other hand,
many recently derived Hawaiian species appear no less variable allozymically
than do typical continental Drosophila, a result used by Barton and Charlesworth
(1984) to dispute founder events, but defended by Carson and Templeton (1984)
as consistent with the founder-flush and transilience models of speciation. The
Hawaiian endemics D. silvestris and D. heteroneura also show relatively high
genotypic and nucleotide diversities in mtONA (OeSalle et al., 1986a, 1986b),
further suggesting to Barton (1989) that founder-induced speciations were not
involved.
Ovenden and White (1990) surveyed both allozyme and mtONA variation in
the Australian fish Galaxias truttaceus, a landlocked form of which constitutes
an incipient species separated from coastal ancestors within the last 3000-7000
years (based on geologic evidence). Among the total of 58 mtDNA haplotypes
observed in coastal populations, only two characterized the landlocked forms.
Heterozygosities at allozyme loci nonetheless were nearly identical in the fresh-
water and coastal populations. The genetic results were interpreted to indicate
that a severe but transitory population bottleneck accompanied the speciational
transition to lacustrine habitat (because in principle, such bottlenecks might
affect genotypic diversity of mtDNA more greatly than that of nDNA).
In terms of gene genealogy, a founder-induced speciation should initially
produce a paraphyletic relationship between the ancestral and descendant species
(Fig. 7.1; Chapter 4). Indeed, several examples of paraphyly for mtDNA or
scnONA gene trees recently have been reported for related species (Powell,
1991). For example, in terms of mtDNA phylogeny, the deer mouse (Peromys-
cus maniculatus), which occupies most of North America, exhibits a paraphyl-
etic relationship to the old-field mouse (P. polionotus) (Fig. 7.3), a species
confined to the southeastern United States. Similarly, the mallard duck (Anas
platyrhynchos) , with broad Holarctic distribution, appears paraphyletic in
mtDNA genealogy to the American black duck (Anas rubripes), which inhabits
eastern North America only (Fig. 7.3). However, the mere appearance of gene-
alogical paraphyly is insufficient for concluding that founder-induced speciations
necessarily were involved for these or other species, for several reasons. First,
paraphyly is expected even when a derivative, geographically restricted species
266 Speciation and Hybridization

) rubripes
Peromyscus Anas
polionotus

1P"""'."'"

rubripes

maniculatus

20 16 12 8 1.0 0.8 0.6 0.4 0.2 0.0

mutation steps % sequence divergence

Figure 7.3. Empirical examples of paraphyletic relationships in the mtDNA gene

genealogies of closely related species. Left: Peromyscus mice (after Avise et aI.,
1983); right: Anas ducks (after Avise et aI., 1990a).

arises via gradual allopatric divergence in the absence of population bottlenecks

(Fig. 7.1 c). Second, under most geographic modes of speciation entailing mod-
erate or large populations, paraphyly in gene trees is an anticipated stage tem-
porally intermediate to polyphyly and reciprocal monophyly (Chapter 4). Fi-
nally, the appearance of paraphy ly also could result from secondary introgressive
hybridization that has transferred some allelic lineages from one species to an-
other (see section on hybridization and introgression later in this chapter).
The influences of founder events on the magnitude of genic variability within
species, and on the pattern of genetic differentiation among species, are in theory
also functions both of the size and duration of the population bottleneck and the
subsequent rate of population growth when variation recovers (Nei et aI., 1975).
All else being equal, mtDNA haplotypes might register founder effects more
clearly than autosomal nuclear loci because of their expected fourfold lower
effective population size (Wilson et al., 1985). However, in any given instance
much stochasticity must exist with regard to which particular lineages happen to
survive a bottleneck and thereby contribute to genetic diversity during popUlation
recovery. Thus, population genetic inferences about the demographic events ac-
companying past speciations can be complicated and are confounded further by
 Speciation and Hybridization 267

the possibility that nonspeciational bottlenecks can either predate or postdate

erection of the RIBS themselves.

Are Speciation Rates and Divergence Rates Correlated?

One intriguing possibility is that speciation events themselves greatly accel-

erate evolutionary differentiation within clades. If so, the magnitude of diver-
gence between extant species should be proportional to the numbers of speciation
events in their evolutionary histories rather than to the sidereal time elapsed since
the species last shared a common ancestor. With regard to morphological diver-
gence, this, indeed, is a logical consequence of the original model of punctuated
equilibrium (Eldredge and Gould, 1972), which proposes a general stasis for
organismallineages except during speciation events. To test this possibility at the
genetic level, Avise and Ayala (1975) introduced a conceptual approach that
involves comparing mean genetic distances within clades (phylads) that are of
similar evolutionary age but have experienced different rates of speciation. If
genetic divergence is proportional to time, mean distances should be similar
among extant members of rapidly speciating (speciose) and slowly speciating
(depauperate) phylads. On the other hand, if genetic divergence is a function of
the number of speciation events, mean genetic distance among extant forms
should be much greater in the speciose clade (Fig. 7.4).
One set of empirical tests utilizing this approach involved the speciose North
American minnows (approximately 200 species, more than 100 within Notropis
alone) versus the relatively depauperate sunfish (approximately 30 species, 11
within Lepomis). From fossil evidence, these groups appear to be of similar
evolutionary age on the continent (Miocene), and an important assumption un-
derlying the test is that the greater number of minnow species is a consequence
of a more rapid pace of speciation (rather than a lower rate of species extinction,
for example). Pairwise comparisons of more than 80 species based on multilocus
protein electrophoretic methods revealed that mean genetic distances within the
minnows and sunfish were similar (Avise, 1977b; Avise and Ayala, 1976): for
example, the mean genetic distance among 47 assayed species of Notropis was
D = 0.62, and among 10 species of Lepomis was D = 0.63 (thus, DRIDp ~
1.0). Furthermore, the minnow and sunfish species had comparable levels of
mean allozymic heterozygosity (H = 0.052 and H = 0.049, respectively),
suggesting that the apparent differences in rates of speciation were not attribut-
able to differing levels of intraspecific genetic variability available for conversion
to between-species differences (Avise, 1977c). Overall, the results for minnows
and sunfish were interpreted as consistent with phyletic gradualism and incon-
sistent with punctuated equilibrium, at least with regard to allozyme evolution.
Gould and Eldredge (1977) justifiably questioned the relevance of these find-
ings to the broader punctuated equilibrium/phyletic gradualism debate. They
268 Speciation and Hybridization

time
distance matrix for phylad P:
4 3 2 1 o A B C 0

A 2 4 4
I
I B 4 4

C 3 3 2
I 0 3 3
I
R
-
I phyletic gradualism:
I Dp = 2 0 16 = 3. 3 3
DR = 392/120= 3.27
I DR 1 Dp = 0.98
I
punctuated equilibrium:
Dp 14/6 = 2.33
A
B DR 664/120 = 5.53
C DR 1 Dp = 2.37
o
Figure 7.4. Explanation of models underlying the test for whether rates of genetic
divergence and speciation are correlated (after Avise and Ayala, 1975). Rand P are
species-rich and species-poor phyJads, respectively, of comparable evolutionary age.
The distance matrix in the upper right applies to phylad P (the larger matrix for R is not
presented) and shows expected distances between species pairs when differentiation is
time dependent (phyletic gradualism, above diagonal) versus speciation dependent
(punctuated equilibrium, below diagonal). In the lower right are shown expected ratios
of mean distances (D values) for extant species within R and P under these competing
models. Under phyletic gradualism, DR/Dp == 1.0, whereas under punctuated equilib-
rium, DR/Dp ;s> 1.0.

pointed out that the controversy refers primarily to patterns of morphological

divergence, whereas allozymes might merely be neutral molecular characters
and, hence, irrelevant to the discussion. Douglas and Avise (1982) therefore
reexamined these fishes in terms of quantitative morphological differences and
showed by multivariate character analyses that the magnitude of phenotypic
divergence was approximately the same in the rapidly speciating minnows and
the slowly speciating sunfish, a result again inconsistent with some of the pre-
dictions of punctuated equilibrium.
Mayden (1986) also criticized the original genetic tests on minnows and sun-
fish, on grounds that the groups examined may not be monophyletic and that
 Speciation and Hybridization 269

their ages were poorly known from fossils. However, if the minnows do not
constitute a clade, they should be older than assumed and, therefore, display
even larger distances under the time-divergence model (and group ages are
irrelevant to predictions of punctuated equilibrium). Nonetheless, several reser-
vations do remain about the validity and generality of this case study. For
example, the minnow and sunfish taxa originally were recognized on the basis of
qualitative morphological appraisals, so the quantitative reassessment by Doug-
las and Avise (1982) likely includes an element of "circular" reasoning in this
test of "rectangular" evolution!
In any event, considering the importance of whether most change in evolution
arises via anagenesis or cladogenesis, it is surprising that few other tests of this
sort involving neontological data (those from extant species) have been at-
tempted (Lemen and Freeman, 1981, 1989; Ricklefs, 1980). Recently, Mindell
et al. (1990) summarized the literature on allozyme genetic distances within 111
vertebrate genera and reported a positive correlation between genetic divergence
and species richness that they attributed to the accelerating influence of specia-
tion on molecular differentiation. However, among taxa this diverse, one cannot
be certain that many other uncontrolled variables do not also correlate with and
possibly influence molecular rates.

Can Speciation Occur Sympatrically?

A long-standing controversy in evolutionary biology concerns how often new

species arise sympatrically, most likely under the influences of diversifying
natural selection on resource utilization (Levine, 1953; Maynard Smith, 1966;
Tauber and Tauber, 1989). Some biologists question the occurrence of sympatric
speciation (e.g., Felsenstein, 1981; Futuyma and Mayer, 1980; Mayr, 1963),
whereas others perceive it as a prevalent mode at least in some groups such as
insects (e.g., Bush, 1975; Kondrashov and Mina, 1986). This debate will not be
rehashed here; instead, a few examples will be presented in which molecular
methods have shed some light on evolutionary relationships in settings where
sympatric speciation had been suspected.

FLOCKS OF FISHES

Within each of several isolated lakes or drainages scattered around the world,
closely related fishes comprise "species flocks" (Echelle and Kornfield, 1984)
that might have arisen through sympatric speciations. The numbers of named
"species" in such flocks range from just a few as in salmonid complexes in
high-latitude lakes of the Northern Hemisphere to more than 500 for cichlid
fishes in the rift-valley lakes of east Africa (Fryer and Iles, 1972; Greenwood,
1981).
270 Speciation and Hybridization

For such flocks, a primary question is whether the differences among sympatric
morphotypes do indeed reflect the presence of distinct species, or alternatively
whether some or all of the differences might reflect phenotypic plasticity within
species [perhaps due to developmental switches triggered by environmental con-
ditions such as diet (Meyer, 1987)]. Polymorphic nuclear markers such as those
provided by allozymes are well suited for addressing this issue. For example,
among Cichfasoma fishes in an isolated basin near Coahuila Mexico, three trophic
morphs described as separate species are present: a snail-eating form with mo-
lariform (crushing) teeth and a short gut; a detritus- or algae-eating form with
papilliform teeth and a long gut; and a fish-eating form with fusiform body. At
27 monomorphic and polymorphic allozyme loci, these morphs proved indistin-
guishable genetically (Kornfield and Koehn, 1975; Kornfield et al., 1982; Sage
and Selander, 1975), a result which led Sage and Selander (1975) to conclude that
"trophic radiation in the Cuatro Cienegas cichlids has been achieved through
ecological polymorphism rather than speciation. " Subsequent studies confirmed
this conclusion-different morphotypes among progeny within a brood can be
generated by altering the rearing conditions (Meyer, 1987).
Another reported example of pronounced trophic polymorphism within a spe-
cies involves stream-dwelling fishes in the genus llyodon. In certain rivers in
Mexico, two sharply dichotomous trophic forms formerly considered distinct
species were shown to be indistinguishable at several polymorphic allozyme loci,
with pooled genotypic frequencies in agreement with Hardy-Weinberg expecta-
tions for single random-mating populations (Grudzien and Turner, 1984; Turner
and Grosse, 1980). "Conspecificity of the trophic types appears to be the most
straightforward interpretation of the data" (Turner and Grosse, 1980).
In many trout and salmon, coexisting forms often exhibit contrasting life
histories: nonanadromous individuals spend their entire lives in freshwater,
whereas anadromous individuals hatched in the same streams or lakes migrate to
sea before returning to freshwater to spawn. In addition, some landlocked pop-
ulations (those that lack present-day access to the ocean) include both stream-
resident and lake-migratory individuals. A long-standing question is whether
these coexisting life history types represent genetically separate populations
(i.e., species). Protein-electrophoretic studies of several such fish complexes-
rainbow trout Safmo gairdneri (now Oncorhynchus mykiss) (Allendorf and Utter,
1979), cutthroat trout O. clarki (Campton and Utter, 1987), brown trout S. sutta
(Hindar et al., 1991), Atlantic salmon S. safar (Stahl, 1987), and sockeye
salmon O. nerka (Foote et al., 1989)-have revealed that freshwater-resident
forms often are genetically close to or indistinguishable from anadromous pop-
ulations in the same regions, whereas by these same assays, populations from
geographically separate spawning localities frequently show highly significant
differentiation (Ferguson, 1989; Ryman, 1983; see Chapter 6). On the other
hand, several other studies on Salmoniformes have reported mild but significant
 Speciation and Hybridization 271

molecular genetic differences among nearby or sympatric forms that differ in

lifestyle (Baby et al., 1991; Birtet al., 1991; Krueger and May, 1987; Skaala and
Nrevdal, 1989; Vuorinen and Berg, 1989). In most of these latter cases, it has
remained unclear whether the genetic differences were attributable to extrinsic
environmental structure per se, or to innate behavioral preferences for different
microhabitats or spawning times.
Whether or not these nonanadromous fishes at a given locale are fully isolated
reproductively from their anadromous counterparts at the present time, the small
genetic distances normally involved, and the polyphyletic appearance of partic-
ular life history patterns suggest that such isolations might well be ephemeral
evolutionarily. Switches between life-styles must have occurred commonly in
the past, and any contemporary phylogenetic separations are probably of recent
origin (as indicated also by the fact that most of the locales under consideration
were covered by glacial ice as recently as 10,000 years ago). Furthermore,
rearing and tagging studies of brown trout have shown that freshwater-resident
individuals can develop from anadromous parents, and vice versa (Skrochowska,
1969), indicating a considerable element of phenotypic plasticity in life-style. In
this case, a possibility is that the freshwater-resident behavior is associated with
a slow growth rate of parr (Hindar et al., 1991), which itself probably is influ-
enced by both genetic and environmental factors.
Molecular analyses of several other putative fish species flocks have revealed
significant differences in allozymes or mtDNA among strictly sympatric forms
and, thus, confirmed the suspected status of separate biological species. Examples
include representatives of the atherinids of central Mexico (Echelle and Echelle,
1984), cyprinodontids of eastern Mexico (Humphries, 1984), coregonids of
Canada and the northern United States (Kirkpatrick and Selander, 1979), certain
of the cichlids in the African rift-valley lakes (Sage et al., 1984; Sturmbauer and
Meyer, 1992), and the now-extinct cyprinids of Lake Lanao in the Philippines
(Kornfield and Carpenter, 1984). However, despite significant allelic differences
at particular loci, in several of these cases the overall genetic distances among
species remained small (Nei's D < 0.05 in the allozyme studies), suggesting that
the evolutionary separations were recent.
A follow-up question for such validated species flocks is whether the specia-
tions took place sYmpatrically, within the lake or drainage basin. In the Allegash
basin of eastern Canada and northern Maine, coexisting dwarf and normal-
sized lake whitefish (Coregonus clupeaformis) represent independent gene pools,
albeit at a small overall level of genetic distance (Kirkpatrick and Selander,
1979). Might this represent a case of sympatric speciation (perhaps involving
changes at a few loci regulating rate of maturation and time of spawning)? If so,
populations of dwarf and normal-sized fish within the Allegash should be one
another's closest relatives (i.e., sister taxa), whereas if speciation was allopatric
and sympatry achieved secondarily, closer genealogical ties might exist with
272 Speciation and Hybridization

populations outside this drainage. Bernatchez and Dodson (1990) discovered

from RFLP analyses of mtDNA that the Allegash populations probably represent
a secondary overlap of two monophyletic groups that evolved allopatrically in
separate refugia during the last (Wisconsin) Pleistocene glaciation. The evidence
was that nearby western populations outside the Allegash belong to one mtDNA
clade, eastern populations to another, and only in the Allegash basin do the two
clades overlap and appear alternately fixed in the dwarf and normal-sized forms.
(A competing hypothesis-that the Allegash basin was the original, continuously
occupied homeland for whitefish and still retains ancestral polymorphisms that
have been fixed elsewhere-appears unlikely because this drainage was glaciated
entirely during the Wisconsin.) An extension of the mtDNA survey to popula-
tions from Alaska to Labrador revealed additional mtDNA clones and clades
whose distributions appear interpretable in terms of the suspected Pleistocene
glacial histories of these drainages (Bernatchez and Dodson, 1991).
A different phylogenetic outcome was revealed in mtDNA analyses of the
African rift-valley cichlids in lakes Victoria and Malawi. Comparisons of some
800 nucleotide positions in the cytochrome b gene, two transfer RNA genes,
and a normally highly variable portion of the control region revealed almost
no differentiation among species within Lake Victoria, yet a large genetic
distinction (> 50 base substitutions) from assayed fishes in Lake Malawi
(Meyer et al., 1990). Results strongly support a recent monophyletic origin for
these cichlid fishes within lakes and conflict diametrically with a taxonomic
reappraisal based on morphology in which Greenwood (1980) had concluded
that "the overall picture is one of a super-flock comprised of several lineages
whose members cut across the boundaries imposed by the present-day lake
shores." The genetic results leave open the possibility of sympatric speciation
in these African cichlids and raise a number of additional questions such as how
so many species can arise so rapidly and coexist ecologically within a lake
(Avise, 1990).
Another example of the variety of genetic outcomes observed among fish
species flocks involves the sculpin (Cottus) radiation in Lake Baikal in Russia.
Grachev et al. (1992) recently reported rather extensive mtDNA sequence dif-
ferences among three species, indicating that by whatever pathway speciation in
this flock took place, "the cottoid fish of Lake Baikal are much more ancient
than cichlid fish of the great lakes of Africa. "

HOST- OR HABITAT-SWITCHING IN INSECTS

Changes in host utilization by phytophagous and zoophagous parasites might,

in principle, give rise quickly to new species reproductively isolated from their
sympatric progenitors (Bush, 1975). If such speciations took place recently, the
genetic footprints again should involve a paraphyletic relationship for each ge-
netically similar progenitor/derivative parasite pair and probably a reduction of
 Speciation and Hybridization 273

variation in the neospecies. Furthermore, if such speciations were common in the

past, the phylogenies of parasites and their hosts could be conspicuously decou-
pled, the host-shifts, in effect, producing reticulations of parasite lineages across
host lineages (e.g., Baverstock et al., 1985). However, caution is indicated
because a lack of congruence between the phylogenies of parasite and host also
could result from asynchronized speciations and lineage sorting processes, such
that surviving lineages in the parasite trace to phylogenetic splits either predating
or postdating nodes in the host phylogeny (Fig. 7.5). Such patterns of parasite

cospeciation of
host and parasite

b
asynchrony of
host/parasite speciations
and lineage sorting

c
cospeciation, with
occasional host-switching

Figure 7.5. Possible relationships between the species' phylogeny of

a parasite (inner branches) and that of its host taxon (thick branches).
Black dots denote parasite speciations. (a) Cospeciation, wherein
host and parasite phylogenies are perfectly concordant; (b) asynchro-
nous speciation, wherein nonconcordant lineage sorting characterizes
host and parasite lineages; (c) host-switching by the parasite.
274 Speciation and Hybridization

lineage sorting within a host phylogeny bear some analogies to similar processes
that can distinguish a gene tree from a species tree (Chapter 4) and also are rem-
iniscent of Fitch's (1970) distinction between orthology and paralogy (Chapter 1).
One of the classic examples of host-switching involves frugivorous Rhagoletis
flies, where several host-specific forms such as the "apple race" and the "haw-
thorne race" of R. polmonella are postulated to be in the process of sympatric
speciation currently (Bush, 1969). Available genetic data are not inconsistent
with this possibility. In allozymic composition, collections from the apple and
hawthorne forms in local sympatry show small but significant differentiation
(Feder et aI., 1988; McPheron et aI., 1988), and such differences between paired
samples extend across the eastern United States and Canada (Federet al., 1990a,
1990b). Furthermore, Berlocher and Bush (1982) concluded from allozymic data
that the phylogeny for many Rhagoletis flies and relatives differs from that of the
hosts, a result at least consistent with host-switching aspects of the sympatric
speciation scenario. Of course, such consistencies do not necessarily eliminate
all competing scenarios.

Can Related Species Be Diagnosed Reliably?

Regardless of origin, molecular differences between species can be of great

utility in diagnosing closely related forms, even where morphological or other
traditional markers have failed or are ambiguous. For example, despite a near
identity in morphological appearance, several sibling species of Drosophila
proved readily separable with a battery of allozyme markers (Ayala and Powell,
1972b; Ayala et aI., 1970; Hubby and Throckmorton, 1968; Fig. 7.2), as did
sibling species of Trachyphloeus weevils (Jermiin et aI., 1991) and Chthamalus
barnacles (Hedgecock, 1979). Mitochondrial sequences in morphologically sim-
ilar Penaeus shrimp show huge genetic differences, greater than those distin-
guishing some orders of mammals (Palumbi and Benzie, 1991). A polychaete
worm (Capitella capitata) widely used as an indicator of marine pollution and
environmental disturbance was thought to be a single cosmopolitan species, but
allozyme analyses indicate the presence of at least six sibling species (Grassle
and Grassle, 1976). In corals assigned to "Montastraea annularis," enormous
phenotypic variation of questionable genetic basis proved to involve at least three
unnamed species, as judged by significant differences in allozyrnic composition
among sympatric morphotypes (Knowlton et al., 1992).
Among the vertebrates also, numerous problematic species have been diag-
nosed using molecular characters. Morphologically cryptic fish species have
been revealed through isozymic or other molecular studies (see the reviews in
Powers, 1991; Shaklee, 1983; Shaklee et aI., 1982). For example, several
closely related species of tuna (Thunnus) can be distinguished by allozyme and
mtDNA markers (Bartlett and Davidson, 1991; Dotson and Graves, 1984; Sharp
 Speciation and Hybridization 275

and Pirages, 1978). In frogs of the genus Gastrotheca, immunological and

protein electrophoretic evidence revealed that at least six species in two different
groups formerly were masquerading under the name G. riobambae (Duellman
and Hillis, 1987; Scanlan et aI., 1980). Remarkable examples of morphological
stasis in the face of extensive speciation involve lungless salamanders in the
family Plethodontidae, where multiple fixed allozymic differences are the rule
rather than the exception among populations formerly thought to be con specific
(Larson, 1984, 1989). [For example, the slimy salamander in the eastern United
States had been considered a single species (Plethodon glutinosus), but dramatic
differences in allozymes and albumin (Highton, 1984; Maha et al., 1989), in-
cluding those among sympatric populations, revealed at least 16 groups that
appear to have achieved the species or semispecies level of divergence (Highton
et aI., 1989).] Among avian taxa, sibling species in the flycatcher genera Em-
pidonax and Contopus are difficult to distinguish morphologically, but often
exhibit diagnostic allozyme alleles (Zink and Johnson, 1984). In general, birds
are among the best known of organisms at the species level, but a new species
of skrike (Lanarius liberatus) recently was discovered that differs from conge-
ners by large mtDNA distances and represents the first avian species described
solely on the basis of molecular data (Hughes, 1992; E.F.G. Smith et aI., 1991).
In many species, adults are distinguishable morphologically, whereas their
juveniles or larvae are not. By comparing molecular characters in unknown
larvae against known adults, species assignments often can be made. In exam-
ples of this approach, researchers have employed protein electrophoresis or DNA
restriction mapping to identify the larvae of a variety of marine organisms,
including oysters (Hu et aI., 1992), white perch and striped bass (Morgan, 1975;
Sidell et aI., 1978), other basses (Graves et aI., 1989), flounders (Smith and
Benson, 1980), tunas (Graves et al., 1988), and snappers (Smith and Crossland,
1977). In the near future, PCR-based methods should prove of particular utility
in identifying small larval forms or eggs. Silberman and Walsh (1992) used the
PCR to amplify nuclear 28S rRNA genes from phyllosome larvae of three spe-
cies of spiny lobsters (Panulirus argus, P. guttatus, and P. laevicauda). The
amplified products then were subjected to restriction digests that revealed
species-diagnostic banding patterns. In an interesting problem of larval identi-
fication involving sea cucumbers (Echinodermata), Olson et al. (1991) used
PCR-based sequences from the 16S ribosomal RNA gene in mtDNA to assign a
collection of bright red pentacula larvae to Cucumaria frondosa. This assignment
came as a surprise-from the coloration and other morphological considerations,
the larvae had been assumed to belong to a distantly related sea cucumber,
Psolus fabricii.
Economic or medical exigencies often require the correct identification of
sibling taxa, and molecular markers can play an important role. For example, the
two principle mosquito vectors for malaria in Africa, Anopheles gambiae and A.
276 Speciation and Hybridization

arabiensis, differ in behavior and preferred habitat but are indistinguishable

morphologically. Finnerty and Collins (1988) isolated a diagnostic RFLP probe
from rDNA that can be used to assay single dried adults, thus extending earlier
species-discriminating capabilities for the gambiae complex based on allozymes
(Miles, 1978). In the eastern United States, another Anopheles complex (for-
merly considered a single species A. quadrimaculatus) has proved from molec-
ular and cytological evidence to consist of at least four morphologically cryptic
species. Numerous molecular markers [including those from allozymes (Narang
et al., 1989a, 1989b, 1989c), rRNA genes (Mitchell et al., 1993), and mtDNA
(Kim and Narang, 1990)] show concordant genetic partitions that agree also with
reproductive boundaries as revealed through experimental crosses. The genetic
differences indeed are large, such that many molecular characters are diagnostic,
and dichotomous keys to species identification in Anopheles can be constructed
(Table 7.2). In another mosquito genus (Aedes), similar work based on allo-
zymes was used to discriminate morphologically cryptic and sometimes sym-
patric forms (Munstermann, 1988), including behavioral types within the Aedes
aegypti complex that is the primary vector to humans of the viruses for dengue
fever and yellow fever (Powell et aI., 1980; Tabachnick and Powell, 1978;
Tabachnick et aI., 1979).
Further development of PCR-based methods for DNA amplification likely will
revolutionize abilities to discriminate among microbes and other small organ-
isms. For example, Wimpee et al. (1991) employed two highly conserved re-
gions of the luxA gene as PCR primers to prepare species-specific probes from
four major groups of marine luminous bacteria. The probes subsequently were
used to identify various bacterial species from field isolates. Schmidt et al.
(1991) examined the species composition represented in bulk genomic DNA
isolated from picoplankton collected in the central Pacific Ocean. The approach
involved cloning the mixed population of DNA into phage, screening these
clones for presence of the 16S rRNA genes, and subsequent nucleotide sequenc-
ing of these isolates. The data were used to establish the identities of the pico-
plankton by comparison with an established information base of rRNA gene
sequences. This method allowed identification of 15 bacterial sequences that
could be related to cyanobacteria and proteobacteria. Similar molecular ap-
proaches have been employed to characterize the phylogenetic positions of pre-
viously unknown microbial taxa from the Sargasso Sea (Giovannoni et al.,
1990). With the accumulation of larger data bases, such approaches might also
allow much finer taxonomic assignments (Weller and Ward, 1989; Weller et aI.,
1991). For example, Fell et al. (1992) used rRNA gene sequences from 117
species representing 23 genera of basidiomycetous yeasts to identify and char-
acterize marine collections of these micro-eukaryotes.
Zooxanthellae are unicellular algae that occur as endosymbionts in many
species of marine invertebrates. Due to a paucity of informative morphological
 Speciation and Hybridization 277

Table 7.2. Diagnostic allozyme loci (A) and dichotomous biochemical key (B) to four
sibling species in the Anopheles quadrimaculatus complex of mosquito species. a

A. Diagnostic Loci for Species Pairs

A:B A:C A:D B:C B:D C:D
Idh-I Acon-l Acon-l Acon-l Acon-l Got-l
Idh-2 Idh-2 Idh-2 Idh-l Idh-l Had-l
Est-2 Had-l Got-l Had-l Got-l Had-3
Est-5 Had-3 Got-2 Had-3 Got-2 Pep-4
Est-7 Pep-2 Pep-2 Got-2 Pep-2 Pgi-l
Had-l Got-2 Pep-4 Pep-2 Pep-4 Me-l
6Pgd-l Pgi-l Me-l Pgi-l Me-l Est-2
Est-2 Mpi-l Est-4 Est-2 Mpi-l
Est-6 Est-5 Est-7
Mpi-l Est-6 Mpi-l
6Pgd-l Est-7
Xdh-3 Mpi-l
Ao-l Xdh-3
B. Biochemical Key
1. Mpi-l slow (62 allele, rarely with 52 as heterozygote) . . . . . . . . . . species D
Mpi-l faster (78 or greater) . . . . . . . . . . . . . . . . . . . . . . . . . . . . go to 2
2. Idh-l slow (86) and Idh-2 fast (162) . . . . . . . . . . . . . . . . . . . . . . species B
Idh-l faster (~ 100, sometimes with 86 as heterozygote);
Idh-2 fast or slower (100, 132, 162) . . . . . . . . . . . . . . . . . . . . . go to 3
3. Had-3 slow (45); Pgi-l slow (95) . . . . . . . . . . . . . . . . . . . . . . . species C
Had-3 faster (100, sometimes with 45 as heterozygote);
Pgi-l faster (100, rarely with 95 as heterozygote) . . . . . . . . . . . . species A
"The diagnostic loci provide correct identification with probability greater than 99%. In the key
shown (one of many that could be generated), the numbers indicate electromorph mobilities relative
to a standard strain.
Source: After Narang et al. (1989b).

characters, most zooxanthellae have been placed provisionally in the genus Sym-
biodinium, but the numbers of species are unknown as are their evolutionary
relationships and possible host-specificities. Rowan and Powers (1991) assayed
RFLPs and sequences in the small-subunit nuclear ribosomal RNA gene from
zooxanthellae isolated from 22 host taxa and found several genetically distinct
forms whose estimated phylogenetic relationships bore little resemblance to the
taxonomies of their hosts. For example, some genetically similar zooxanthellae
were isolated from hosts of ordinal or greater taxonomic distance, such as anem-
ones, corals, and gorgonians; some dissimilar zooxanthellae were isolated from
congeneric hosts. Overall, results suggest that many zooxanthellae species exist
and that the symbioses can arise by a shuffling of symbionts among even unre-
278 Speciation and Hybridization

lated host taxa. The conclusion that Symbiodinium contains many species was
supported further by the finding that genetic diversity in ribosomal RNA gene
sequences within this genus is comparable to that among different orders of
nonsymbiotic dinoflagellates (Rowan and Powers, 1992)!
Not surprisingly, such molecular-based identifications of microbial taxa also
have found applications in more applied areas, including medicine and the food
industry. For example, Regnery et al. (1991) employed PCR-amplified DNA
sequences from the citrate synthase and the 190-kDa antigen genes as substrates
for RFLP assays that proved to differentiate among various rickettsial species
causing spotted fever; Salama et al. (1991) employed subspecies-specific rRNA
gene probes to identify Lactococcus lactis cremoris, a bacterium whose few
available strains are relied on by the dairy industry for the manufacture of
cheddar cheese free of fennented and fruity flavors.

Should a Phylogenetic Species Concept Replace the BSC?

For more than 50 years, the biological species concept (BSC) has been the
major theoretical framework orienting research on the origins of biological di-
versity. Recently, a serious challenge has come from some systematists who
argue that the BSC lacks a sufficient phylogenetic perspective and, hence, pro-
vides an inappropriate guide to the origins and products of evolutionary diver-
sification (de Queiroz and Donoghue, 1988; Donoghue, 1985; Eldredge and
Cracraft, 1980; Mishler and Donoghue, 1982; Nelson and Platnick, 1981). Many
critics of the BSC argue that' 'reproductive isolation should not be part of species
concepts" (McKitrick and Zink, 1988). This has led to a call for replacement of
the BSC with a "phylogenetic-species concept" (PSC-Box 7.1) under which a
species has been defined as a monophyletic group composed of "the smallest
diagnosable cluster of individual organisms within which there is a parental
pattern of ancestry and descent" (Cracraft, 1983). Because molecular data pro-
vide unprecedented power for phylogeny estimation, it might be supposed that
molecular evolutionists would be strong advocates for the PSC, but this has not
necessarily been the case.
One fundamental difficulty with existing PSC proposals concerns the nature of
evidence required to justifiably diagnose a monophyletic group warranting spe-
cies recognition. Molecular technologies have made it abundantly clear that
multitudinous derived traits often can be employed to subdivide named species
into diagnosable subunits (Chapter 6). Indeed, most individuals within sexually-
reproducing species can be distinguished from one another with high-resolution
assays such as DNA fingerprinting (Chapter 5). If each individual is genetically
unique, then the grouping of individuals into phylogenetic "species" requires
that distinctions below some arbitrary threshold be ignored. The evolutionary
significance of any such threshold surely must be questionable. For these and
 Speciation and Hybridization 279

other reasons, Avise and Ball (1990) suggest that if a broader framework of the
PSC is to contribute to a significant advance in systematic practice (and they
believe that it can), a shift from issues of diagnostics to those of magnitudes and
patterns of phylogenetic differentiation, and of the historical and reproductive
reasons for such patterns, will be required. Toward this end, they suggest how
principles of "genealogical concordance" might be employed to combine de-
sirable elements of the PSC and the BSC.
Within any organismal pedigree, allelic phylogenies can differ greatly from
locus to locus (Ball et al., 1990), due to the inevitable Mendelian vagaries of
meiotic segregation and syngamy and to the reproductive successes of individ-
uals through which the alleles happen to have been transmitted. An array of
individuals phylogenetically grouped by one locus may differ from an array of
individuals grouped by another locus, unless some overriding evolutionary
forces may have concordantly shaped the phylogenetic structures of the inde-
pendent genes. One such force expected to generate genealogical concordance
across loci is intrinsic reproductive isolation (the focal point of the BSC).
Through time, due to processes of lineage turnover, populations isolated from
one another by intrinsic RIBS (i.e., biological species) inevitably tend to evolve
toward a status of reciprocal monophyly with respect to particular gene geneal-
ogies (Chapter 4); furthermore, through time, the genealogical tracings of inde-
pendent loci almost inevitably sort in such a way as to partition these isolated
populations concordantly (Avise and Ball, 1990). Thus, genealogical concor-
dance per se becomes a deciding criterion by which to recognize significant
evolutionary partitions at the level of organismal phylogeny.
However, genealogical concordance across loci also can arise from extrinsic
barriers to reproduction, as among populations geographically isolated for suf-
ficient lengths of time [relative to effective population size (Chapter 4)]. As
emphasized in Chapter 6, dramatic phylogenetic partitions commonly are ob-
served among populations considered conspecific under the BSC. It might be
argued that such populations also warrant formal taxonomic recognition, on the
grounds that they represent significant biotic partitions of relevance in such areas
as biogeographic reconstruction (Chapter 6) and conservation biology (Chapter 9).
From consideration of these and other factors, Avise and Ball (1990) sug-
gested the following guidelines for biological taxonomy, based on applications
of genealogical concordance principles. The biological and taxonomic category
"species" should continue to refer to groups of actually or potentially inter-
breeding populations isolated by intrinsic RIBS from other such groups. In other
words, a retention of the philosophical framework of the BSC is warranted, in no
small part because reproductive barriers generate significant (concordant) gene-
alogic partitions at multiple loci in an organismal phylogeny. At the intraspecific
level, "subspecies" warranting formal recognition then may be defined as
groups of actually or potentially interbreeding populations phylogenetic ally dis-
280 Speciation and Hybridization

tinguishable from, but reproductively compatible with, other such groups. Im-
portantly, the evidence for phylogenetic distinction must come, in principle,
from concordant genetic partitions across multiple, independent, genetic-based
traits (be they molecular or phenotypic). This phylogenetic approach to tww-
nomic recognition represents a novel and direct outgrowth of molecular-based
perspectives on the evolutionary process.

HYBRIDIZATION AND INTROGRESSION

The term "hybridization" is as challenging to define as is "speciation," and

for the same reasons. In the early literature of systematics, a hybrid was defined
as an offspring resulting from a cross between species, whereas "intergrade"
was reserved for any product of a cross between distinctive conspecific popula-
tions or SUbspecies. But as we have seen, this distinction can be rather arbitrary,
and in the more recent literature hybridization often is employed in a broad sense
to include crosses between genetically "differentiated" forms regardless of their
current taxonomic status. Introgression refers to the movement of genes between
species (or between well-marked genetic popUlations) mediated by backcrossing.

Background

FREQUENCIES AND GEOGRAPHIC SETTINGS OF HYBRIDIZATION

Hybridization and introgression are common phenomena in many plant and

animal groups. More than 20 years ago, Knoblock (1972) compiled a list of 23 ,675
reported instances of interspecific or intergeneric plant hybridization, despite the
availability of detailed studies on only a small fraction of the botanical world.
Introgression is more challenging to assess, but Rieseberg and Wendel (1993)
provided a compilation of 155 "noteworthy" cases of plant introgression, many
of which include molecular documentation. Similarly, hybridization and intro-
gression have been noted involving numerous animal taxa (Harrison, 1993). For
example, Schwartz (1972, 1981) compiled a list of 3759 references dealing with
natural and artificial hybridization in fishes, many cases of which have been
verified and characterized further using molecular markers (Campton, 1987;
Verspoor and Hammar, 1991). Among the vertebrates, fishes appear most prone
to hybridization, but the phenomenon is widespread.
The frequency of hybridization and the extent of introgression both can vary
along a continuum, and molecular markers are invaluable for assessing where a
given situation falls. At one extreme, hybridization may be rare and confined
primarily to production of F J hybrids, a situation likely to arise when such
progeny have greatly reduced viability or fertility. For example, the two largest
 Speciation and Hybridization 281

mammalian species, the blue whale (Balaenoptera musculus) and fin whale (B.
physalus), normally are distinct but also are suspected of occasional hybridiza-
tion. Recent molecular analyses of three anomalous specimens by nuclear and
mitochondrial DNA revealed that these individuals were indeed interspecific
hybrids resulting from crosses that had taken place in both directions with respect
to sex; one hybrid female also carried a backcross fetus with a blue whale father
(Arnason et aI., 1991; Spilliaert et aI., 1991). In milkweed bugs (Oncopeltus),
genetically divergent species that are capable of producing hybrids under labo-
ratory conditions failed to show significant allozymic exchange in nature due to
strong prezygotic and postzygotic RIBs (Leslie and Dingle, 1983). At the other
extreme, hybridization can be extensive and the hybridizing taxa may merge
completely into one panmictic gene pool. This situation is exemplified by a local
hybrid swarm between two genetically well-marked subspecies of bluegill sun-
fish (Lepomis macrochirus macrochirus and L.m. purpurescens). These subspe-
cies inhabit different regions of the southeastern United States, but overlap in
parts of Georgia and the Carolinas where hybridization takes place (Avise and
Smith, 1974). In one well-characterized hybrid population, mtDNA and allo-
zyme markers normally diagnostic for the parental subspecies proved to be
shuffled thoroughly: Gametic phase disequilibria (Box 5.3) involving nuclear
and cytonuclear allelic associations all were negligible (Asmussen et al., 1987;
Avise et al., 1984b).
In many taxonomic groups, organisms separated for long periods of evolu-
tionary time nonetheless may retain the anatomical and physiological capacity
for hybrid production. Using microcomplement fixation to assay the albumins of
50 pairs of frog species known to be capable of generating viable hybrids,
Wilson et al. (1974a) demonstrated a mean immunological distance of 36 units,
which corresponds to an average separation time of about 21 million years under
a conventional albumin clock (Prager and Wilson, 1975; Wilson et al., 1977).
From similar molecular assays, hybridizable species-pairs of birds were esti-
mated to have last shared common ancestors on average 20 mya, whereas hy-
bridizable pairs of mammals separated on average only 2 my a (Fig. 7.6). From
these dramatic differences, it was concluded that birds and frogs have lost the
potential for interspecific hybridization slowly in comparison to mammals, per-
haps because of a slower pace of chromosomal evolution and/or a hypothesized
lower rate of change in regulatory genes (Prager and Wilson, 1975; Wilson et aI.,
1974a, 1974b). In any event, some organisms clearly retain a potential for
hybridization over extremely long evolutionary periods. How often such poten-
tial is realized in nature is, of course, another issue, and one that also can be
addressed powerfully through molecular approaches.
Frequently, hybridization follows human-mediated transplantations. In the
early 1980s, the pupfish Cyprinodon variegatus was introduced to the Pecos
River in Texas where it then hybridized with an endemic species C. pecosensis.
282 Speciation and Hybridization

20
CIl
'-
C'CS
c..
I
10

CIl
Q)
0
()
Q)
c.. 20
CIl
Q)
-
..c
10

C'CS
N
.- 0
"0
'- 20
..c
~
..c: I mammals I
10

0
C 0
0 32 64 96
immunological distance
(albumin)
Figure 7.6. Immunological distances between vertebrate species
capable of producing viable hybrids [data from Prager and Wilson
(1975) and Wilson et al. (1974a)]. Data are shown for more than
100 species-pairs, many of which appear quite old. Albumin
clocks have been calibrated at about 1.7 immunological distance
(ID) units per million years in frogs and mammals (Prager and
Wilson, 1975) and about 0.6 ID units per million years in birds
(Prager et aI., 1974).
 Speciation and Hybridization 283

Protein-electrophoretic data revealed that within five years, panmictic admix-

tures of the two pupfishes occupied approximately 430 river-kilometers, or
roughly one-half the historic range of the endemic species (Echelle and Connor,
1989; Echelle et al., 1987). In the land snail genus Cerion, Bahamian individuals
of C. casablancae introduced in 1915 into the range of C. incanum on Bahia
Honda Key, Florida, have been hybridizing with the latter during this century.
Analyses of allozymes (and morphology) revealed that these snails now are
panmictic, that no pure C. casablancae remain, and that there has been a 30%
reduction in frequency of the introduced genome (Woodruff and Gould, 1987).
Other examples of extensive introgression following artificial transplantations
involve trout species in the western United States, where repeated introductions
of millions of hatchery-reared rainbow trout into endemic cutthroat trout habi-
tats, and of cutthroat populations from one locale to another, have resulted in
extensive introgressive hybridization well documented by molecular markers
(Allendorf and Leary, 1988; Busack and Gall, 1981; Forbes and Allendorf,
1991; Gyllensten et al., 1985b; Leary et aI., 1984).
Natural hybridization may occur sporadically between broadly sympatric spe-
cies or be confined to particular geographic contact areas. Hybrid zones, which
often appear linear (Hewitt, 1989) or mosaic (Harrison and Rand, 1989; Rand and
Harrison, 1989), are regions in which genetically distinct populations meet and
produce progeny of mixed ancestry (Barton and Hewitt, 1989; Harrison, 1990).
Most hybrid zones represent a secondary overlap between formerly allopatric or
parapatric taxa. [However, the usual evidence for secondary contact, concordance
in character clines across the zone, could in theory also be a primary outcome of
intense diversifying selection within a single continuously-distributed population
(Endler, 1977).] If secondary hybrid zones are not ephemeral, their persistence
might be explained by the hypothesis of "bounded hybrid superiority" wherein
hybrids have superior fitness in areas of presumed ecological transition, or by the
"dynamic-equilibrium" hypothesis wherein hybrid zone maintenance is achieved
through a balance between dispersal of parental types into the zone and hybrid
inferiority (Moore and Buchanan, 1985).
In any event, hybrid zones are marvelous settings to apply molecular markers
for several reasons (Hewitt, 1988). First, the populations or species involved are
genetically differentiated (by definition), such that multiple markers normally
can be uncovered for characterizing the hybrid gene pool. Second, because true
hybrid zones involve amalgamations of independently evolved genomes, exag-
gerated effects of intergenomic interactions might be anticipated, thus magnify-
ing the impact of such processes as recombination and selection, and perhaps
making these evolutionary forces easier to study. Third, various sexual asym-
metries frequently are involved in hybrid zones, and powerful means now exist
for dissecting these factors by utilizing joint data from cytoplasmic and nuclear
markers.
284 Speciation and Hybridization

CASE HISTORY INVOLVING TREEFROGS

To introduce the power of molecular genetics in hybridization analysis, con-

sider an example involving the cytonuclear dissection of an introgressed popu-
lation of tree frogs in ponds near Auburn, Alabama. Hyla cinerea andH. gratiosa
are distinctive species distributed widely and sympatrically throughout the south-
eastern United States. From morphological evidence, they are known to hybrid-
ize at least sporadically, and at the Auburn site extensive introgressive hybrid-
ization has continued for more than 25 years since its initial discovery. One
reason for particular interest in this population stems from behavioral observa-
tions suggesting the potential for a sexual bias in the direction of interspecific
matings. During the breeding season, H. gratiosa males call from the water
surface, whereas H. cinerea males call from perches along the shoreline (Fig.
7.7). In the evenings, gravid females of both species approach the ponds from
surrounding woods and become amplexed. Thus, the hypothesis was raised that
most interspecific matings might involve H. cinerea males with H. gratiosa
females, rather than the converse: H. gratiosa females presumably must "hop
the gauntlet" of H. cinerea males before reaching conspecific pairing partners!
Lamb and Avise (1986) employed five species-diagnostic allozyme loci plus
mtDNA to characterize 305 individuals from this hybrid population. The allo-
zyme loci were chosen because they exhibit fixed allelic differences between the
species, thus allowing provisional assignment of each assayed individual to one
of the following six categories: pure H. cinerea, pure H. gratiosa, F 1 hybrid,
progeny from a backcross to H. cinerea, backcross to H. gratiosa, or later-
generation hybrid. For example, a F1 hybrid should be heterozygous at all
marker loci, and a H. cinerea backcross progeny likely would appear heterozy-
gous at some loci and homozygous for H. cinerea alleles at others. [With
multiple diagnostic markers, the probabilities of misclassifying an individual into
an alternative category are low and can be calculated readily from Mendelian
considerations; for example, a true first-generation H. cinerea backcross progeny
would be mistaken for a pure H. cinerea with probability k = (0.5t, where n
is the number of fixed marker loci, so that in this case k = 0.031.] The mtDNA
genotypes then allowed assignment of the female (and hence male) parent for
each allozymically characterized specimen.
The molecular data revealed a striking genetic architecture for the hybrid
population that generally proved consistent with suspected mating behaviors of
the parental species (Table 7.3; Fig. 7.7). Thus, all 20 F1 hybrids carried "gra-
tiosa" -type mtDNA, indicating that they had H. gratiosa mothers. Furthennore,
52 of 53 individuals identified as backcross progeny to H. gratiosa possessed
"gratiosa"-type mtDNA (as predicted, because their mothers were either F1 's or
pure H. gratiosa). Furthennore, among the progeny of backcrosses to H. ci-
nerea, individuals carrying either "gratiosa" -type or "cinerea" -type mtDNA
 Speciation and Hybridization 285

@
® @ ®
@

®
@J (£)
®
@ @ ®

gratiosa cinerea

all 9

backcrosses backcrosses
to gratlosa to cinerea

Figure 7.7. Biological setting of the hybrid Hyla population (see

text). Top: Diagrammatic aerial view of an Auburn pond, with
calling male treefrogs denoted by c (H. cinerea) and g (H. gra-
tiosa). Bottom: Expected pedigree involved in production of
F 1 hybrids and various backcross classes, under the behavior-
motivated assumption that hybridizations primarily entail matings
of male H. cinerea with female H. gratiosa. Each letter (c or g) in
the pedigree refers to the species origin of the mtDNA genotype;
circles and squares indicate females and males, respectively.
286 Speciation and Hybridization

Table 7.3. Genetic architecture of a hybrid population involving the tree frogs Hyla
cinerea and H. gratiosa (after Lamb and Avise, 1986).a
"gratiosa" mtDNA "cinerea" mtDNA
Allozyme
Category Observed Expected Observed Expected

Pure H. gratiosa 103 o

Pure H. cinerea o 60
FI hybrid 20 20 o o
H. cinerea backcross 22 29 36 29
H. gratiosa backcross 52 53 1 o
Later-generation hybrids 9 Some b 2 Some b

"For each category of frogs as identified allozymically, numbers of observed individuals with
indicated mtDNA genotypes are presented. Also shown are these expected numbers based on the
behaviorally motivated hypothesis that interspecific crosses are in the direction H. cinerea male X H.
gratiosa female (see text), and that to a given backcross category, FI hybrids of both sexes (who thus
have H. gratiosa mtDNA) have contributed equally.
~oth "cinerea" and "gratiosa" mtDNA genotypes are expected among later-generation hybrids,
but relative frequencies are dependent on additional factors and are, thus, hard to predict.

were both well represented (also as predicted, because the mtDNA genome
transmitted in a given mating would depend on whether the F J hybrid parent was
a male or female-Fig. 7.7). Nevertheless, the asymmetric mating pattern alone
probably cannot explain all aspects of the data, because individuals with pure H.
cinerea and H. gratiosa genotypes remained present in high frequency (Table
7.3). Additional factors at work may involve selection against hybrids and/or
continued migration of the parent species into the area. In formal models that
allowed variation in parental immigration rates and included tendencies for pos-
itive assortative mating between conspecifics, Asmussen et al. (1989) found an
excellent fit to the empirical cytonuclear data when, at equilibrium, about 32%
of the inhabitants of the hybrid zone were pure-species immigrants each gener-
ation. Of course, other unexplored scenarios (e.g., involving selection against
hybrids) also might explain the data.
How much of this pronounced genetic structure in the Hyla population would
have been uncovered from a traditional morphological assessment alone? Lamb
aild Avise (1987) applied multivariate analyses to numerous phenotypic charac-
ters in the same Hyla individuals and compared results against those obtained
from the molecular genetic assessments. Although the pure H. gratiosa and H.
cinerea specimens (as classified by molecular genotype) could be distinguished
cleanly by discriminate analyses of morphological traits, various hybrid classes
proved less recognizable. For example, in terms of morphology 18% of true F J 's
were indistinguishable from pure parental species. Other percentages of misclas-
sification by morphology were as follows: 27% of backcrosses in either direction
 Speciation and Hybridization 287

were not distinguished from F l ' s; 50% of gratiosa backcross progeny were
misidentified as pure H. gratiosa; and 56% of cinerea backcross progeny were
misidentified as pure H. cinerea. By contrast, the expected levels of misclassi-
fication based on the surveyed molecular genotypes were always less than 4%
(based on straightforward Mendelian considerations). Furthermore, the pro-
nounced asymmetry in the mating behavior that apparently exerted exceptional
influence on the genetic architecture of the hybrid population would have re-
mained completely undetected by morphological assessment alone.
Numerous molecular genetic analyses of hybridization and speciation have
appeared in the past two decades, and thorough recent reviews can be found in
Arnold (1992), Barton and Hewitt (1985), and Harrison (1993), among others.
A few select examples will be presented here to illustrate the diversity of issues
addressed.

Hybrid Zone Asymmetries

Various types of genetic asymmetries frequently attend hybridization and in-

trogression processes. Some of the asymmetries can stem from differential com-
patibilities of various alleles when placed in heterologous genetic backgrounds
via introgression and may be revealed by comparisons among multiple indepen-
dent molecular markers. Other asymmetries can stem from different behaviors or
fitnesses between the sexes and may be revealed by comparisons of cytoplasmic
and nuclear genomes.

DIFFERENTIAL INTROGRESSION ACROSS A HYBRID ZoNE

A classic case of differential introgression across a well-defined hybrid zone

involves the house mice Mus musculus and M. domesticus. These forms, pre-
viously considered subspecies, meet and hybridize along a narrow line that
extends throughout central Europe (Fig. 7.8). From a molecular genetics per-
spective, this situation first was studied by Selander et al. (1969), who docu-
mented pronounced divergence between the taxa (fixed or nearly fixed allelic
differences at 6 of 40 allozyme loci). Subsequent genetic analysis of nearly 2700
mice from the contact zone across Denmark revealed the following (Hunt and
Selander, 1973): (a) free interbreeding within the hybrid zone (as indicated by
agreement of genotype frequencies with random-mating expectations); (b) an
asymmetry of introgression adjacent to the zone, with extensive introgression of
some of the domesticus alleles into musculus but little gene movement in the
other direction; and (c) a marked increase in the width of the zone in western
Denmark, compared to the east where 90% of the transition in genic character
occurred over a distance of only 20 km. Furthermore, different slopes and
patterns of allelic clines across loci were observed and interpreted as evidence for
288 Speciation and Hybridization

Figure 7.8. Distributions of two

commensal species of house mice
in central Europe. The heavy line
indicates the position of a hybrid
zone at the contact between Mus
musculus (to the north and east, in-
cluding Scandinavia) and M. do-
mesticus (to the south and west).
Open and closed circles indicate
mtDNA genotypes normally char-

••
acteristic of M. domesticus and M .
musculus, respectively, and the ar-
row indicates the postulated route
domesticus of colonization of Scandinavia by
female M. domesticus (Gyllensten
o
•
and Wilson, 1987b). Note that the
distributions of the mtDNA geno-
types are strikingly discordant with
the ranges of the two species as
defined by morphology and nu-
clear genes.

different selective values for alleles as determined in part by the internal genetic
environment. In other words, "selection against introgression of the genes
studied (or the chromosomal segments that they mark) is presumed to involve
reduced fitness in backcross generations caused by disruption of co-adapted
parental gene complexes" (Hunt and Selander, 1973). Another molecular study
of the 20-km-wide hybrid zone in southern Germany revealed that about 98% of
the mice had backcross genotypes. Furthermore, these hybrids were unusually
susceptible to parasitic pinworms, other nematodes, and tapeworms, leading to
the conclusion that their reduced fitnesses "act as a genetic sink, interfering with
the flow of genes between the two species" (Sage et al., 1986). Subsequent
study of Y-chromosome allelic frequencies (based on Southern-blot assays using
diagnostic Y-specific probes) through portions of the hybrid zone in Bulgaria and
Denmark revealed sharp clines indicative of severe restrictions on Y-introgres-
sion as well (Vanlerberghe et al., 1986).
Throughout most of Europe, M. musculus and M. domesticus also differ
clearly in mtDNA composition (mean p == 0.05; Ferris et al., 1983a,b). How-
ever, an unexpected pattern emerged in parts of Scandinavia, where mice that by
the evidence of nDNA and morphology appear to be M. musculus nevertheless
carried M. domesticus-type mtDNA exclusively (Fig. 7.8). Gyllensten and Wil-
 Speciation and Hybridization 289

son (1987b) proposed that the "foreign" mtDNA originated from a small num-
ber of female M. domesticus that colonized Sweden from a southern source,
perhaps in association with the spread of farming from northern Germany to
Sweden some 4000 years ago. Continued backcrossing to M. musculus males
might thereby have introduced mtDNA from M. domesticus into populations that
retain a predominant M. musculus nuclear genetic background. Further study of
the hybrid zone in central Denmark revealed that mtDNA genotypes in northern
Danish populations, though also characteristic of M. domesticus, nonetheless
differ detectably from present-day M. domesticus mtDNA in southern Denmark,
a finding that adds support to the Gyllensten-Wilson scenario by eliminating the
possibility that contemporary introgression alone can account for the mtDNA
transfer (Vanlerberghe et al., 1988).
In some hybrid zones, patterns of variation for cytoplasmic and nuclear mark-
ers are highly concordant (Baker et aI., 1989; Nelson et al., 1987; Szymura et
al., 1985), but in several other instances pronounced discordances across loci (or
between molecular and morphological data) appear to reflect differing historical
patterns of introgression (Table 7.4). One phenomenon frequently reported is
that of cytoplasmic "capture," wherein the mtDNA or cpDNA genotypes nor-
mally characteristic of one species sometimes occur against a predominant nu-
clear background of another species, presumably because of current or past
introgressive hybridization. One speculation has been that mtDNA tends to in-
trogress more readily than nuclear DNA because genes contributing to repro-
ductive isolation might be housed primarily in the nucleus (Barton and Jones,
1983). However, even if true, many additional factors governing introgression
must be involved. The following sections provide examples of how asymmetries
stemming from different behaviors and/or fitnesses of males and females can lead
to patterns of differential exchange for nuclear and cytoplasmic loci.

HALDANE'S RULE

An empirical generality first noticed by Haldane (1922) is that "When in the

F I offspring of two different animal races one sex is absent, rare, or sterile, that
sex is the heterozygous [heterogametic] sex." Thus, in species with heteroga-
metic females (such as birds and butterflies), female hybrids more often show
decreased fitness, whereas in species with heterogametic males (such as fruit flies
and mammals), male hybrids tend to show more severe reductions in viability or
fertility (Coyne and Orr, 1989a).
Tegelstrom and Gelter (1990) compared nuclear and cytoplasmic markers in a
contact zone between two hybridizing flycatcher species in Europe, Ficedula
albicollis and F. hypoleuca. Allozyme differentiation was negligible, due per-
haps to extensive ongoing introgression of nuclear genes via male hybrids [which
from DNA fingerprint analyses of parentage are known to be fertile (Gelter et al.,
290 Speciation and Hybridization

Table 7.4. Examples of mtDNA and cpDNA "capture" reportedly due to introgressive
hybridization between related species of animals and plants, respectively.o

Genus Common Name Reference

Animal mtDNA
Caledia Grasshoppers Marchant, 1988
Clethrionomys Voles Tegelstrom et al., 1988
Drosophila Fruit flies Solignac and Monnerot, 1986
Gryllus Crickets Harrison et aI., 1987
Hyla Treefrogs Lamb and Avise, 1986
Mus House mice Ferris et aI., 1983a
Notropis Minnows Dowling et al., 1989;
Dowling and Hoeh, 1991
Odocoileus Deer Carr et aI., 1986
Rana Frogs Spolskyand Uzzell, 1984,
1986
Plant cpDNA
Argyroxiphium Silverswords Baldwin et al., 1990
Brassica Cabbages and allies Palmer et aI., 1983
Dubautia Silvers words Baldwin et al., 1990
Gossypium Cotton Wendel et aI., 1991;
Wendel and Albert, 1992
Helianthus Sunflowers Rieseberg et aI., 1990b
Heuchera Heucheras Soltis et aI., 1991
Persea Avacados Fumier et aI., 1990
Pisum Peas Palmer et al., 1985
Populus Poplars Smith and Sytsma, 1990
Quercus Oaks Whittemore and Schaal, 1991
Salix Willows Brunsfeld et al., 1992
Tellima (herb. perennial) Soltis et al., 1991
Zea Teosintes, maize Doebley, 1989
OSee text for discussion, and consult Harrison (1989) and Rieseberg and Soltis (1991) for additional
examples.

1992)]. However, mtDNA divergence between these avian species remained

high, a result interpreted by the authors to evidence a lack of interspecific
cytoplasmic genetic exchange (the heterogametic hybrid females appear sterile,
in accord with Haldane's rule). However, samples from outside the zone of
sympatry were not compared, so the geographic extent of nuclear introgression
remains unknown (and indeed, alternatives to the biased introgression scenario
might be entertained). In voles (Clethrionomys), interspecific crosses result in
fertile female but sterile male offspring (Tegelstrom et al., 1988). In northern
Scandinavia, an observed discordance between species boundary and mtDNA
phylogeny led to the conclusion that mtDNA stemming from C. rutilus has
 Speciation and Hybridization 291

passed the species boundary and now is found in C. glareolus. perhaps as a result
of a limited hybridization episode perhaps dating to the postglacial colonization
of the region some 10,000 years ago (Tegelstrom, 1987b). Thus, this scenario is
very similar to that for the unidirectional introgression of mtDNA in the Scan-
dinavian Mus populations discussed above.
In Drosophila hybrids, the heterogametic males frequently show partial or
complete sterility (sometimes in one direction of a cross only), whereas the
homogametic females nonnally remain fertile. Male sterility has proved to be
polygenic, with responsible loci mapped (using chromosomal or molecular
markers) to the X and Y chromosomes and to various auto somes (Dobzhansky,
1974; Vigneault and Zouros, 1986). This asymmetry of hybrid fertility has been
invoked to explain the apparent ease with which mtDNA appears to cross some
species' boundaries (because the fertile females leave open an avenue for inter-
specific cytoplasmic exchange). For example, in D. mauritiana. a high propor-
tion of individuals carries a mtDNA genotype also found in nearby populations
of D. simulans. and these observations were interpreted to indicate a recent
introgression of D. simulans mtDNA into the Mauritiana population (Solignac
and Monnerot, 1986). This hypothesis subsequently gained support from popu-
lation cage studies in which the predicted takeover by D. simulans mtDNA was
documented experimentally over a few generations of introgressive hybridization
(Aubert and Solignac, 1990). Powell (1983) reported a similar situation where
suspected hybridization was postulated to account for a greater mtDNA similar-
ity between populations of D. persimilis and D. pseudoobscura in sympatry than
in allopatry [however, see Powell (1991) for possible reinterpretations]. On the
other hand, DeSalle and Giddings (1986) report that the mtDNA phylogeny for
several closely related species of Hawaiian Drosophila matches the suspected
species phylogeny quite well, despite a postulated historical introgression that
has complicated phylogenetic reconstructions based on nuclear genes.

DIFFERENTIAL MATING BEHAVIORS

The Hyla example discussed earlier provides a powerful illustration of how a

behavioral asymmetry has influenced the genetic architecture of a hybrid zone.
Another example involves a contact zone in France between the hybridizing newts
Triturus cristatus and T. marmoratus (Arntzen and Wallis, 1991). Allozymes
again were employed to characterize the hybrid status of individuals, and mtDNA
genotypes were used to identify the female parents. All F 1 hybrids possessed the
T. cristatus-type mtDNA, perhaps due to a strong asymmetry in mate choice.
Furthennore, an observed absence of introgression of mtDNA in areas where T.
cristatus replaced T. marmoratus is consistent with this interpretation.
Sunfish in the genus Lepomis are renowned for propensity to hybridize, both
in artificial pond situations and in nature. As stated by Breder (1936), "There is
292 Speciation and Hybridization

probably no group of fishes, North American at least, in which there would seem
to be a concatenation of reproductive and other events so well arranged as to lead
to extensive hybridizing; i.e., the species are numerous; there is less geographic
separation than usual; spawning occurs at about the same temperature threshold;
spawning sites are limited and similar for most species; nests are exchanged
among species." From the observation of diminished hybrid fertility in both
sexes, Hubbs and Hubbs (1933; see also Hubbs, 1955) concluded that natural
hybridization probably was limited to the F 1 generation, but later work with
experimental populations revealed that "a number of different kinds of hybrid
sunfishes ... are not sterile, are fully capable of producing abundant F2 and F3
generations, and can be successfully backcrossed to parent species and even
outcrossed to nonparental species" (Childers, 1967). Nonetheless, the 11 rec-
ognized species within the genus normally exhibit large genetic distances at
protein-coding loci (Fig. 7.2) and, for the most part, retain distinctive morpho-
logical identities throughout their respective ranges. Thus, questions remain
about the degree of Lepomis hybridization in nature and whether introgression
has played a significant role in the evolution of the group. Avise and Saunders
(1984) characterized a total of 277 sunfish from two locations in north Georgia
for species-diagnostic allozymes and mtDNA. The genetic data revealed the
following: (a) a low frequency (5%) of interspecific hybrids, all of which ap-
peared to be F l ' s; (b) the involvement of five sympatric Lepomis species in the
production of these hybrids; and (c) no evidence for introgression in these study
locales. With respect to the current discussion, two additional points were of
interest: There was a strong tendency for the hybridizations to take place between
parental species differing greatly in abundance, and a tendency for the rare
species in the hybrid cross to provide the female parent. Although conclusions
are tentative because the number of hybrids discovered was small, the data
suggest a density-dependent mating pattern in which the absence of conspecific
pairing partners and spawning stimuli for females of rarer species might be
important factors in increasing the likelihood of interspecific hybridization.
Another molecular study of hybridization in sunfish involved bluegills (Lepomis
macrochirus) and pumpkinseeds (L. gibbosus) in a lake in southern Canada.
Among 44 hybrids examined by allozymes and mtDNA, all appeared to be Fl'S,
and to have had pumpkinseed mothers (Konkle and Philipp, 1992). From prior
studies, male bluegill are known to exhibit two alternative life history strategies
with respect to reproduction: a "parental pathway," in which sexual maturation
is delayed until about eight years of age, at which time the male builds and defends
a nest, courts and spawns with females, and guards eggs and young after fertil-
ization; and a "precocial pathway, " in which a male matures at about two years
of age, fails to build a nest or court females, and instead "steals" fertilizations
from conspecific parental males through sneaky behavior or female mimicry
(Gross, 1979; Gross and Charnov, 1980). The highly asymmetrical genetic results
 Speciation and Hybridization 293

in the hybrid population studied are consistent with the postulate that precocial
male bluegills may also be cuckolding heterospecific pumpkinseed males, and at
a higher frequency than is true for crosses in the converse direction.

DIFFERENTIAL GAMETIC EXCHANGE

Particularly in plants, a pronounced decoupling of male and female components

of gene flow across species is possible due to the two distinct avenues for genetic
movement-pollen and seeds (Arnold, 1992; Paige et al., 1991). When genetic
transfer is mediated solely by pollen, maternally inherited genetic markers (e.g.,
cpDNA in most angiosperms) will not introgress. On the other hand, immigration
of seeds into a foreign population might lead to the introgression of both nuclear
and cytoplasmic genes when the resulting plants are fertilized by pollen from
resident individuals. Clearly, alternative modes of gene transfer can differentially
influence cytonuclear associations (Asmussen and Schnabel, 1991).
Arnold et al. (1990a, 1990b, 1991, 1992) have employed molecular markers
from allozymes, nONA, and cpDNA to document asymmetrical gene flow be-
tween the Louisiana irises Iris fulva and I. hexagona, part of a species complex
representing a classic example of plant hybridization and introgression (Ander-
son, 1949). At particular locales, nuclear alleles that normally are species-
diagnostic have been shuffled into a variety of recombinant genotypes, confirm-
ing suspicions from morphological analyses that introgressive hybridization has
occurred. Furthermore, many individuals of hybrid ancestry (as gauged by nu-
clear genes) nonetheless retain the cpDNA of I. hexagona (Fig. 7.9), suggesting
that they are the products of pollen transfer from I. fulva onto I. hexagona
flowers. Thus, introgression of nONA markers has occurred in the absence of
cpDNA introgression. Results appear consistent with the natural history of these
species, which includes pollination by highly mobile bumblebees and an absence
of special adaptations for seed dispersal.
Might long-distance pollen movement lead to the exceptionally wide areas of
introgression postulated for some plant species complexes? In the southeastern
United States, three parapatric species of buckeye trees (Aesculus sylvatica, A.
jlava, and A. pavia) appear to be engaged in introgression over a broad region at
least 200 km wide, as inferred from patterns of morphology, geographic distri-
bution, and meiotic irregularities associated with decreased gerrninability of
pollen from putative interspecific hybrids. Allozyme data gathered for these
species also are consistent with the introgression scenario, and further raise the
possibility that long distance movement of genes beyond the morphology-
recognized hybrid area may have taken place (dePamphilis and Wyatt, 1990).
For example, one of the hybrid zones appears highly asymmetrical, with alleles
characteristic of coastal plain A. pavia also found in Piedmont populations where
A. sylvatica normally occurs. The authors hypothesized that an important polli-
294 Speciation and Hybridization

nuclear genes cpDNA

I bayou I I bayou
0
I
C!! ~
~
o 0
e% aCO
=~l' CbOcPt
~ CO
~
~o
83
00
I road I I road
0
I
~

LJ
<t
<t<t

0 LJ
• ·0
0
10 m 10 m

••
() 0
()
a
Figure 7.9. Asymmetrical introgression between Iris fulva and I. hexagona likely
resulting from pollen flow (after Arnold, 1992). Each circle represents a single plant.
Left: Relative proportion of I. fulva (shaded) and I. hexagona (unshaded) nuclear
markers. Right: Similar representation for maternally-transmitted chloroplast DNA
markers. Note, in particular, the population between the road and bayou, where
multilocus nuclear genotypes suggest the presence of advanced generation hybrids or
backcrosses, despite the apparent absence of seed dispersal that would be registered
by cpDNA from I. fulva in this area.

nation agent for buckeyes, the ruby-throated hummingbird, may have effected
long-distance pollen flow during its northward spring migrations, thus account-
ing for both the width and asymmetry of the hybrid zone (dePamphilis and
Wyatt, 1989).
Once pollen have arrived on a heterospecific style, additional challenges await
that might lead to asymmetric barriers to successful hybridization. For example,
in interspecific crosses among Eucalyptus species, E. nitens pollen tubes grow
slowly and never reach full length in the larger E. globulus styles, whereas E.
globulus pollen tubes grow rapidly in E. nitens styles and enter the ovary (Gore
et al., 1990). Hybridization between several species of Eucalyptus occurs rather
commonly in nature (Griffin et at, 1988), and unilateral cross-incompatibility
might play an important role in structuring cytonuclear associations. A variety of
other selective mechanisms operating at prezygotic or postzygotic stages also
could lead to asymmetrical introgression in Eucalyptus species and elsewhere
(Gore et al., 1990; Potts and Reid, 1985).
 Speciation and Hybridization 295

One such postzygotic mechanism is "cytoplasmic male sterility" (CMS), a

widespread phenomenon in plants (Edwardson, 1970) involving pollen abortion
in hybrid progeny due to an interaction of cytoplasmically-transmitted (usually
mtDNA) mutations with a foreign nuclear background (Hanson, 1991). Male
sterility in first-generation hybrids or backcross progeny could have the effect of
attenuating the introgression of nuclear genes, while nonetheless leaving open an
avenue for cytoplasmic transfer via fertile females. CMS is known to occur, for
example, in hybrids between some of the Helianthus species that have figured
prominently in discussions of intertaxon exchange of cpDNA, as described next.

RETICULATE EVOLUTION EVIDENCED BY cpDNA PHYLOGENIES

Owing to a strong propensity for hybridization in many plant taxa, botanists

have been particularly concerned with the possibility of widespread reticulate
evolution (Grant, 1981; Stebbins, 1950), wherein phylogenies of some groups
might be characterized as anastomotic or "netlike," rather than strictly dichot-
omous and branching. Recent molecular analyses based on comparisons of
nDNA and cpDNA have uncovered considerable evidence for this phenomenon.
In Helianthus sunflowers, gross incongruities between phylogenies estimated
from morphological and chromosomal variation, experimental crossing success,
and various molecular markers have led to the conclusion that both recent and
relatively ancient episodes of intertaxon gene exchange have produced a retic-
ulate pattern of relationships among these species (Rieseberg, 1991; Rieseberg et
aI., 1988, 1991). For example, the cpDNA-based phylogeny for numerous He-
lianthus taxa shown in Figure 7.10 contrasts significantly at the indicated posi-
tions with suspected relationships based on morphological characters and on the
nuclear genes for ribosomal RNA. Thus, each of five species (H. anomalus, H.
annuus, H. debilis, H. neglectus, and H. petiolaris) possesses two or more
highly distinct cpDNA genotypes otherwise characteristic of different phyloge-
netic groups within the genus. Furthermore, some species appear to have cap-
tured the cytoplasm of other species on multiple occasions. For example, H.
petiolaris has acquired the cytoplasm of H. annuus at least three times, as
evidenced by additional information on geographic locations of the introgressed
populations and by the particular H. annuus cpDNA genotypes exhibited (Riese-
berg and Soltis, 1991).
As with animal mtDNA, the cpDNA molecule appears especially helpful (as
an adjunct to nuclear analyses) in revealing such cases of reticulation because its
clonal transmission allows particular ancestral sources to be identified without
the complication of recombination (including that at the intragenic scale) that can
lead to a mosaic ancestry for the nuclear genome. In addition, there may be
several biological factors including CMS that facilitate cytoplasmic (relative to
nuclear) exchange across species (see earlier in this Chapter). Rieseberg and
296 Speciation and Hybridization
porteri
anomalus *
annuus
650/0 ~------------- paradoxus *
petiolaris
debilis
neglectus
neglectus
anomalus*
71%
deserticola *
petiolaris
76%
debilis
69% niveus
bolanderi
debilis
100% awmwmml annuus
argophyl/us
debilis
praecox

Figure 7.10. Evidence for cytoplasmic introgression

in Helianthus sunflowers (after Rieseberg and Soltis,
1991). Shown is a most-parsimonious tree (Wagner
network) based on cpDNA data and indicating areas
of gross discrepancy (hatched branches) with a mor-
phological classification. These discrepancies were
interpreted to be the result of interspecies transfer of
cpDNA mediated by hybridization. Numbers indicate
levels of bootstrap support for putative clades. Aster-
isks specify taxa that are stabilized hybrid derivatives.

Soltis (1991) reviewed the rapidly growing literature on instances of interspecific

cpDNA "capture" attributable to introgressive hybridization (Table 7.4) and
concluded that reticulate evolution is indeed a widespread phenomenon in plants.
Such reticulations can seriously compromise phylogeny reconstructions based on
small numbers of characters or those generated under the assumption that phy-
logenies are strictly branching and hierarchical. Furthermore, no taxonomic zone
may be completely safe from the phylogenetic consequences of reticulate evo-
lution, because gene transfer" ... between major evolutionary lines during early
stages of their divergence has the potential to impact cpDNA phylogenetic re-
construction at all taxonomic levels .... " (Rieseberg and Soltis, 1991).
In summarizing this general section, it is now abundantly clear that hybridiz-
 Speciation and Hybridization 297

ation phenomena are best analyzed through multiple lines of evidence involving
numerous molecular (and other) markers. Barriers to reproduction between close-
ly related taxa seldom are absolute and, in addition, often appear differentially
"semipermeable" to cytoplasmic and various nuclear alleles. Thus, a rich and
varied fabric of gene genealogies (seldom evident from traditional morphological
assessment alone) characterizes many hybrid contacts, revealing varying degrees
of reticulation among the phylogenetic branches connecting related species.

Speciation by Hybridization

Can differentiated genomes brought together through hybridization sometimes

produce a new species? One such mechanism already has been described (Box
7.3)--allopolyploidization. From surveys of molecular markers, other hybrid-
ization-mediated routes to speciation have been revealed as well (Abbott, 1992).

DIPLOID OR HOMOPLOID SPECIATION

In the plant literature, a traditional proposal has been that species isolated by
a chromosomal sterility barrier might give rise via hybridization to new fertile
diploid species that are at least partially isolated reproductively from both parents
(Grant, 1963; Stebbins, 1950). Such "recombinational speciation" (Grant,
1981) has been tested under artificial conditions by experimental synthesis of
new hybrid species (see the review in Rieseberg et al., 1990a), but questions
remained as to the prevalence of this speciational mode in nature. Numerous
candidates for hybrid species status were identified from early studies of mor-
phology, ecology, and geographic distributions of plant taxa in nature, but final
confirmation of hybridity awaited the application of molecular markers.
Stephanomeria diegensis is a diploid annual plant native to southern Califor-
nia, purportedly derived following stabilization of hybrid segregants from a
natural cross between two divergent diploid relatives (S. exigua and S. virgata)
with the same chromosome number. Gallez and Gottlieb (1982) demonstrated
that S. diegensis indeed displays an additive profile of the allozyme alleles of its
presumed relatives, a finding consistent with the plants' intermediate morphol-
ogy and karyotype and, thus, supportive of the postulated hybrid origin. Simi-
larly, analyses of allozymes and other nuclear markers in the putative hybrid Iris
nelsoni indicate that this species possesses a combination of nuclear genes char-
acteristic of three species that thus appear involved in its formation-I. fulva. I.
hexagona, and I. brevicaudis (Arnold et aI., 1990b, 1991). However, not all
molecular genetic reappraisals have confirmed the suspected hybrid origins of
problematic plant taxa. The diploid annual Lasthenia burkei proved not to pos-
sess a combination of allozyme alleles present in L. conjugens and L. jremontii,
298 Speciation and Hybridization

thus disputing earlier hypotheses that L. burkei is a stabilized hybrid derivative

of the latter two species (Crawford and Ornduff, 1989).
Rieseberg et al. (1990a) noted that, by hard criteria, apparent additivity of
alleles in the nuclear genome is insufficient to fully confirm the hybrid origin of
a diploid species because a nonexcluded alternative is that the taxon in question
might be ancestral to its putative parents. One solution to this problem is to use
additional markers (such as those from cpDNA) to establish the polarity of
relationships. By adopting this philosophy as applied to allozymes and cpDNA
genotypes in diploid sunflowers, Rieseberg et al. (1990a) concluded that two
problematic taxa, appropriately named Helianthus paradoxus and H. neglectus,
had dissimilar pathways of origin: The former is indeed a hybrid species having
arisen from H. annuus and H. petiolaris, whereas the latter appears to be a recent
nonhybrid derivative of H. petiolaris.
Somewhat less attention has been devoted to the possibility of homoploid
speciation via hybridization in animals. On the basis of morphological and al-
lozyme data, Highton et aI. (1989) suggested that the salamander Plethodon
teyahalee had a hybrid origin as a result of interbreeding between white-spotted
P. glutinosus and red-legged P. jordani. Among fishes, several examples of
stabilized hybrid forms have been suspected from morphological or distributional
considerations. For example, the "zuni sucker" in the Little Colorado drainage
of the western United States was suggested to be a hybridization-derived inter-
mediate between Catostomus discobolus of the Colorado drainage and C. ple-
beius of the Rio Grande; and the "white shiner" in the Roanoke and adjacent
drainages of the eastern United States was proposed to have arisen from hybrid-
ization between nearby Luxilus cornutus and L. cerasinus. However, molecular
reevaluations of these forms either did not support the hybrid origin scenario [in
the case of the zuni sucker (Crabtree and Buth, 1987)] or were equivocal [in the
case of the white shiner (Meagher and Dowling, 1991)] due to the difficulty of
discriminating among the possibilities of ancestral polymorphism, convergence,
and past hybridization to account for the observed allozymic distributions.
Molecular reappraisals have provided support for the postulated hybrid origin
of another recognized fish species. On the basis of an intermediate morphology,
Gila seminuda in the Virgin River of the western United States had been pro-
posed as a hybrid derivative between the roundtail chub (Gila robusta) and
bony tail chub (Gila elegans). In terms of allozymes, G. seminuda proved to be
polymorphic for alleles at two allozyme loci otherwise diagnostic for the putative
parental taxa; as judged by mtDNA, the matriarchal lineage retained by G.
seminuda derives from G. elegans (DeMarais et aI., 1992). As noted by the
authors, such stabilized hybrid derivatives might be relatively common in some
groups of fishes, but remain unrecognized due to a lack of detailed molecular
studies. As also noted by the authors, the formal taxonomic status of such
introgressed forms likely will remain a point of contention, particularly when the
 Speciation and Hybridization 299

introgressed population currently is isolated from its parental species by extrinsic

(geographic) barriers to reproduction. Should the hybrid form be considered a
distinct population, subspecies, or species? This question is not merely aca-
demic, but also is relevant to the implementation of conservation programs
(Chapter 9).

ORIGINS OF UNISEXUAL BIOTYPES

Unisexual biotypes that reproduce by parthenogenesis, gynogenesis, or hybri-

dogenesis (Fig. 5.3) are not biological species in the usual sense applied to
sexually reproducing taxa, but nonetheless they are isolated genetically from
their sexual relatives (as well as from other unisexual lineages) and typically are
afforded formal taxonomic recognition. Among the vertebrates, essentially all of
the 70 known unisexual taxa arose through hybridizations between related sexual
species. In a number of studies, molecular markers have been highly informative
in revealing the mode of origin and parentage of these all-female biotypes.
In most cases, the particular bisexual progenitors of various unisexual verte-
brates had been suspected from earlier comparisons of morphology, karyotype,
geographic range, or other information, but molecular surveys of allozymes and
mtDNA have confirmed the suspected hybrid origins in several instances, and for
the first time revealed the directions of the original crosses. For example, the
gynogenetic live-bearing fish Poecilia formosa in northeastern Mexico exhibits
nearly fixed heterozygosity at numerous protein and allozyme loci that distin-
guish or are polymorphic in P. Latipinna and P. mexicana (Abramoff et al., 1968;
Balsano et at., 1972; Turner, 1982), thus confirming evidence from morphology
and geography that P. formosa arose via hybridization between these sexual
species. The gynogen P. formosa also carries the mtDNA of P. mexicana, which
is highly divergent (p == 0.07) from that of P. Latipinna, indicating that the
direction of the formational hybrid cross(es) was P. mexicana female x P.
Latipinna male (Avise et al., 1991). To date, similar molecular inspections have
allowed unambiguous determination of the sexual progenitors for more than 25
unisexual biotypes (Table 7.5).
A few unisexuals carry genomic contributions from more than two sexual
ancestors. The gynogenetic fish Poeciliopsis monacha-Lucida-viriosa, for exam-
ple, includes P. viriosa nuclear genes apparently introgressed as a result of
occasional matings of P. monacha-Lucida females with P. viriosa males rather
than with males of their usual sexual host P. Lucida (Vrijenhoek and Schultz,
1974). As judged by allozymes and other evidence, several triploid parthenoge-
netic lizards in the genus Cnemidophorus also carry genes from three sexual
progenitors (Des sauer and Cole, 1989; Good and Wright, 1984), likely as a result
of multiple hybridization events involving these species.
In many cases, molecular analyses have further pinpointed the geographic and
300 Speciation and Hybridization

Table 7.5. Examples of species parentage determined for unisexual vertebrates (for an
extended list, see A vise et aI., 1992c). a

Bisexual Parental Species

Ploidy Reprod.
Unisexual Biotype Level Mode b Male Female Ref.c

Cnemidophorus lizards
uniparens 3n P burti inornatus (2) a
tesseLatus 2n P septemvittatus marmoratus b
veLox 3n P inornatus (2) burti or costatus c
laredoensis 2n P sexlineatus guLaris d
Heteronotia lizards
binoei (widespread
form) 3n P binoei sp. "CA6" e
Menidia fish
clarkhubbsi complex 2n G beryllina peninsulae f
Phoxinus fish
eos-neogaeus 2n,3n G eos neogaeus g
Poecilia fish
formosa 2n G Latipinna mexicana h
Poeciliopsis fish
monacha-Lucida 2n H Lucida monacha
monacha-
occidentalis 2n H occidentalis monacha

"The bisexual parental species were identified from comparisons of allozymes, morphology, kary-
otype, geographic ranges, or other information, and the female parent was identified by mtDNA
comparisons in the references indicated.
bp = parthenogenetic; G = gynogenetic; H = hybridogenetic (see Fig. 5.3).

"References: (a) Densmore et aI., 1989a; (b) Brown and Wright, 1979; Densmore et aI., 1989b;
(c) Moritz et aI., 1989; (d) Wright et aI., 1983; (e) Moritz, 1991; (f) A.A. Echelle et aI., 1989;
(g) Goddard et aI., 1989; (h) Avise et aI., 1991; (i) Quattro et aI., 1991; (j) Quattro et aI., 1992a.

genetic source of particular unisexuals. For example, from mtDNA comparisons,

the matriarchal components of nine unisexual biotypes in the sexlineatus group
of Cnemidophorus lizards all appear to stem from females within one of the four
nominate geographic subspecies of C. inornatus-C. i. arizonae (Densmore et al.,
1989a); the maternal ancestry of five triploid unisexual strains in the Poeciliopsis
monacha-lucida fish complex trace phylogenetically to an extant bisexual P.
monacha from the Rio Fuerte in northwestern Mexico (Quattro et al., 1992b).
Typically, the bisexual relatives of unisexuals have proved highly distinct in
mtDNA genotype, whereas mtDNAs of the unisexuals are closely related to or
indistinguishable from those of only one of the sexual progenitors. Thus, an
emerging generalization is that most extant unisexual biotypes originated through
 Speciation and Hybridization 301

asymmetrical hybridization events, occurring in one direction only (e.g., A fe-

male x B male versus B female x A male). Whether this reflects some asym-
metrical mechanistic constraints on the origin of unisexuals, or merely the sur-
vival of a limited subset of lineages from crosses in both directions, generally
remains unclear. However, in the case of the Poeciliopsis hybridogens, labora-
tory crosses of P. monacha females x P. Lucida males sometimes result in the
spontaneous production of viable hybridogenetic lineages, whereas the recipro-
cal matings do not (Schultz, 1973). This direction of cross is consistent with the
molecular-inferred origins of natural hybridogenetic strains, all of which possess
P. monacha-type mtDNA (Quattro et aI., 1991). Furthermore, these extant nat-
ural hybridogens have arisen multiple times through separate hybridization
events, as gauged by their links to several different branches in the mtDNA
phylogeny of their maternal ancestor P. monacha (Fig. 7.11).
One exception to such straightforward hybrid origins involves the hybridoge-
netic frog Rana escuLenta of Europe, in which individuals exhibit mtDNA geno-

e···
Figure 7. 11. Relationships among mill NA haplotypes in the
sexual fish Poeciliopsis monacha (open circles) and its unisex-
ual derivative P. monacha-lucida (shaded circles) (after Quat-
tro et aI., 1991). Slashes represent inferred mutations along the
parsimony network; dotted lines indicate alternative network
pathways.
302 Speciation and Hybridization

types nonnally characteristic of either R. lessonae or R. ridibunda (Spolsky and

Uzzell, 1986). The hybridogen R. esculenta is unique among the assayed "asex-
ual" biotypes in consisting of high frequencies of both males and females. From
behavioral considerations, the initial hybridizations producing R. esculenta were
postulated to involve male R. lessonae x female R. ridibunda. Once the hybri-
dogen was fonned, occasional matings of male R. esculenta with female R.
lessonae secondarily may have introduced R. lessonae-type mtDNA into R.
esculenta. Furthennore, females belonging to such R. esculenta lineages appear
to have served as a natural bridge for the interspecific transfer of R. lessonae
mtDNA into certain R. ridibunda populations via matings with R. ridibunda
males (Spolsky and Uzzell, 1984). Such crosses apparently produced "R. ridi-
bunda" frogs with nonnal nuclear genomes (because the R. lessonae chromo-
somes are excluded during meiosis), but with R. lessonae-type mtDNA.
Another complex scenario surrounds the hypothesized maternal ancestry of the
triploid salamander Ambystoma 2-laterale-jeffersonianum, which by allozyme
evidence contains nuclear genomes of the bisexual species A. laterale and A.
jeffersonianum, but reportedly carries mtDNA from A. texanum (Kraus and
Miyamoto, 1990). These authors favor an explanation in which an original A.
laterale-texanum hybrid female produced an ovum with primarily A. laterale
nuclear chromosomes, but the female-determining sex chromosome (W) and the
mtDNA of A. texanum. When fertilized by a male A. laterale, female progeny
with two A. laterale nuclear genomes and the mtDNA of A. texanum would
result. Subsequent hybridization with male A. jeffersonianum could then pro-
duce the observed A. 2-laterale-jeffersonianum biotypes carrying A. texanum
mtDNA. Although this scenario remains highly speCUlative, its mere feasibility
suggests that distinct reticulate histories could characterize different genomic
elements in some hybridogenetic taxa.
Another question about unisexual vertebrates addressed by molecular markers
concerns mechanistic modes of polyploid fonnation. More than 60% of known
unisexual biotypes are polyploid (Vrijenhoek et al., 1989) and two competing
hypotheses have been advanced to account for the origins of these fonns (Fig.
7.12). Under the "primary hybrid origin" hypothesis, a disruption of meiotic
processes in an F I interspecific hybrid leads to the production of unreduced diploid
gametes, whose subsequent fertilization by spenn leads to a triploid condition
(Schultz, 1969). Alternatively, under the "spontaneous origin" hypothesis, par-
thenogenetic triploids might have arisen when unreduced oocytes from a diploid
nonhybrid were fertilized by spenn from a second bisexual species (Cuellar, 1974,
1977). As diagrammed in Figure 7.12, joint comparisons of mitochondrial and
nuclear markers pennit an empirical test of these competing possibilities. If a
unisexual biotype arose spontaneously from sexual ancestors and hybridization
was involved only secondarily, the paired homospecific nuclear genomes should
derive from the maternal parent, and thus should be coupled with mtDNA derived
 Speciation and Hybridization 303

primary hybrid origin spontaneous origin

/~--------------------------------~\ /r--------------~\

genome addition genome duplication

Figure 7.12. Competing scenarios for the origin of triploid unisexual taxa (from
Avise et al., 1992c; see text). Each uppercase letter represents one nuclear gene set
(A or B) from the respective parental species, and the lowercase letters in boxes
similarly refer to the maternally transmitted mtDNA genomes. Smaller ovals indicate
sperm and eggs, respectively, the latter being unreduced where indicated by stars. In
the genome duplication scenario, this suppression of reduction occurs during an
equational division such that the AB hybrid produces AA (or BB) ova.

from the same species. Conversely, under a model of primary hybrid origin, the
paired homospecific nuclear genomes could be coupled with the mtDNA type from
either of the sexual ancestors, depending on additional details by which the nuclear
genome was duplicated or added (see below).
Cytonuclear genetic analyses for several unisexual taxa have provided support
for the "primary hybrid origin" hypothesis. For example, the triploid parthe-
nogen Cnemidophorus jlagellicaudus possesses the mtDNA of C. inornatus but
two homospecific nuclear genomes from C. burti, and a similar type of cytonu-
clear pattern was observed for 8 of 10 parthenogenetic Cnemidophorus biotypes
examined (Densmore et al., 1989a; Moritz et al., 1989). Similarly, the triploid
gynogenetic fish Poeciliopsis monacha-2 lucida possesses the mtDNA of P.
monacha but two nuclear genomes from P. lucida (Quattro et al., 1992b). Thus,
these results appear to refute the "spontaneous origin" scenario (unless the
diploid nonhybrid that produced unreduced gametes was a male, in which case
all bets are off!).
304 Speciation and Hybridization

Assuming correctness of the primary hybrid origin scenario, two further cy-
togenetic pathways to triploidy can be distinguished (Fig. 7.12). Under the
"genomic addition" scenario (Schultz, 1969), interspecific Fl hybrids produce
unreduced ova (AB) that on backcrossing to one of the sexual ancestors leads to
allotriploid biotypes AAB or ABB. Under the "genomic duplication" scenario
(Cimino, 1972» suppression of an equational division in an F 1 hybrid could
produce unreduced AA or BB ova, which following a backcross to species A or
B would produce AAB or ABB offspring (autopolyploid AAA or BBB progeny
could also result from this process, but no self-sustaining populations of au-
topolyploid unisexual vertebrates are known). An important distinction between
these pathways involves the predicted level of heterozygosity in the homo specific
nuclear genomes. Heterozygosity should be extremely low under the genome
duplication pathway (the only variation being derived from postformational mu-
tations), whereas normal heterozygosity is predicted under the genomic addition
pathway. At least one test of these scenarios is available-in triploid Poeciliopsis
gynogens, all assayed strains proved to be heterozygous for homospecific nuclear
markers at one or more allozyme loci, a result which effectively excludes the
genome duplication hypothesis for these fishes (Quattro et al., 1992b).
Recently, molecular markers contributed to the discovery of the first instance
in nature of "androgenesis" in an animal species. Androgenesis is a sort of male
analogue of gynogenesis (Fig. 5.3), involving the development of an individual
solely under the influence of its paternally derived chromosome set (i.e., in the
absence of instructions from the mother's genetic material). The process had
been demonstrated experimentally in the laboratory (see Giorgi, 1992) and was
uncovered recently in nature as well. Using protein electrophoresis in conjunc-
tion with chromosomal markers, Mantovani and Scali (1992; see also Mantovani
et al., 1991) first identified two hybridogenetic strains of stick insects in Italy
(Bacillus rossius-grandii benazzii and B. rossius-grandii grandii) which had
arisen from hybridization between B. rossius females and two subspecies of B.
grandii males. The males of these hybridogenetic strains are infertile, whereas
females can reproduce both by hybridogenesis or gynogenesis. The most sur-
prising discovery, however, was that when a female B. rossius-grandii benassii
is fertilized by a B. rossius male, up to 20% of the offspring have the nuclear
genetic makeup solely of the father. Thus, these individuals are androgenetic.
They also have proved to be diploid (through duplication of the male chromo-
somes or by fusion of two sperm) and fertile.
Overall, the detailed understanding of the evolutionary genetics of "unisex-
ual" taxa provided by molecular markers was unimaginable even a short time
ago. As recently as 1978, in referring to a hybrid-derived parthenogenetic grass-
hopper, a leading student of the speciation process lamented that "we are never
likely to know which species was the female parent" (White, 1978b). Since
then, parentage determinations for taxa with parthenogenetic or related modes of
 Speciation and Hybridization 305

reproduction have become routine, and indeed are viewed as but a starting point
for more refined genetic analyses of evolutionary origins and pathways.

SUMMARY
1. Various speciation patterns are expected to leave characteristic phylo-
genetic signatures on the genomes of recently separated species. By revealing
such signatures, molecular markers have prompted reexamination of long-
standing issues in speciation theory, including: How much genetic change ac-
companies speciation? Do most speciations entail severe population bottlenecks?
Are rates of speciation correlated with rates of genetic evolution? Have specia-
tions occurred in sympatry? These and related questions have been answered for
several studied groups.
2. Molecular markers have pragmatic utility in distinguishing closely re-
lated taxa, including sibling species that may have gone unrecognized by non-
molecular appraisals. Molecular diagnoses sometimes involve sibling species of
medical or economic importance.
3. Molecular approaches have enriched the traditional biological species
concept (BSC) by adding a phylogenetic perspective to discussions of population
relationships. The distinction between gene trees and species trees has led to the
development and elaboration of principles of genealogical concordance for the
recognition of subspecies and species.
4. Molecular markers provide powerful means for identifying hybrid or-
ganisms and for characterizing patterns of introgression. Degrees of hybridiza-
tion and introgression have proved to vary along a continuum, from instances of
sporadic production of F l' S to extensive introgression leading to genetic merging
of formerly separate taxa.
5. Through joint examination of multiple nuclear loci and cytoplasmic
genes, several sources of genetic asymmetry in hybrid zones have been recog-
nized. These differential patterns of introgression can be due to interlocus vari-
ation in selection intensity against alleles on heterologous genetic backgrounds,
Haldane's rule whereby gender-specific fitness differences characterize hybrid
organisms, differential mating behaviors of the hybridizing taxa, or other sources
of differential gametic exchange.
6. Phylogenetic studies of cpDNA suggest that reticulate evolution has
been common in many plant groups.
7. Hybrid origins for several recognized taxa have been revealed through
molecular markers. Particularly informative have been genetic characterizations of
both the parentage and the cytological pathways leading to production of
hybridization-derived unisexual biotypes.
 8

Species Phylogenies
and Macroevolution

All the organic beings which have ever lived on this

Earth may be descended from some one primordial form.

c. Darwin, 1859
. . . study of the gene at the most fundamental level will
soon tell us more about the phylogenetic relationships of
organisms than we have managed to learn in all the 173
years since Lamarck.

R.K. Selander, 1982

We proceed now to the provenance traditionally equated with molecular phylo-

genetics-estimation of evolutionary relationships among species and higher
taxa. After reproductive barriers have been erected and the speciation process
completed, molecular characters continue to evolve in a more or less time-
dependent fashion (Chapter 4), such that the overall genetic distance between
species under study provides a compelling guide to the general magnitude of
evolutionary time since their common ancestry. Furthermore, many qualitative
molecular markers considered individually or in combination can provide pow-
erful characters for clade delineation. In assessing relationships among species,
a variety of molecular techniques has been brought to bear, including protein
electrophoresis, immunological assays, DNA restriction analysis, DNA-DNA
hybridization, and others. In recent years, nucleotide sequencing has become
widespread and currently is revolutionizing the field. Collectively, these methods
have been applied to phylogenetic estimation in many hundreds of taxonomic
306
 Species Phylogenies and Macroevolution 307

groups, at evolutionary depths ranging from closely related congeners to the

deepest branches in the tree of life. No attempt will be made here to summarize
this vast literature exhaustively. Rather, the purpose of this chapter is to illustrate
by chosen examples some of the wide variety of problems and approaches in
supraspecific phylogeny tackled through molecular markers.

RATIONALES FOR PHYLOGENY ESTIMATION

Phylogenetic Mapping of Organismal Traits
Phylogenetic hypotheses (explicit or implicit) Qnderlie virtually all conclu-
sions in comparative evolution (Harvey and Purvis, 1991). For example, the
inference that powered flight in mammals is a derived rather than ancestral
condition stems ultimately from the restricted phylogenetic position of bats (Chi-
roptera) within a group (Mammalia) whose ancestral forms unquestionably were
terrestrial. More challenging in this case is whether wings and flight evolved
once or more than once (convergently) in bat evolution, a contentious issue
whose resolution depends on whether Chiroptera is a monophyletic or polyphy-
letic assemblage (see below and Fig. 8.1).
An important task in evolutionary biology is to understand adaptations. Un-
fortunately, direct experimental analyses often are not possible, so evolutionists
rely heavily on the comparative method to infer underlying evolutionary pro-
cesses. A significant complication in this general approach is the potential lack

megabats

(a) microbats

primates

megabats

(b) primates

microbats
Figure 8.1. Competing hypotheses concerning the phylogenetic rela-
tionships of microchiropteran bats, megachiropteran bats, and primates
(simplified from R.J. Baker et al., 1991). Slashes across tree branches
indicate hypothesized origins of powered flight. Molecular data provide
significant support for scenario (a).
308 Species Phylogenies and Macroevolution

of statistical independence among data points due to phylogenetic legacy (Felsen-

stein, 1985b; Richman and Price, 1992). Thus, in comparative phylogenetic
analyses, if it is erroneously assumed that each taxon constitutes an independent
unit, the degrees of freedom in a statistical test could be grossly overestimated.
For example, many passerine birds tend to have more or less monogamous mating
systems (however, see Chapter 5), whereas large herbivorous mammals usually
are highly polygynous; how many of the examples of these contrasting mating
systems are to be interpreted as independent adaptive responses to different eco-
logical circumstances faced by birds and mammals (as opposed to the mere reten-
tions of these respective ancestral conditions across the evolutionary radiations of
avian and mammalian species)? By seeking independent evolutionary origins of
organismal characteristics through phylogenetic analyses, conclusions from com-
parative studies can be placed on a firmer empirical and statistical grounding.
In other words, suppose that species' phylogenies were known with certainty.
Taxonomic distributions of morphological, behavioral, or life history characters
superimposed on these phylogenetic trees should then illuminate the evolutionary
origin(s) and directions of change in each organismal feature. "Phylogenetic
character mapping" involves the matching of particular traits with their associ-
ated species on a cladogram, with the purpose of revealing evolutionary patterns
in those traits. Clearly, the phylogeny or cladogram itself must be estimated
using data that are different from and independent of the attributes to be mapped.
With the advent of molecular approaches, such independent appraisals of phy-
logeny have become commonplace.
ANATOMICAL FEATURES

Because bats are such unusual and distinctive creatures, it might seem unlikely
that they could have evolved multiple times from independent ancestors. None-
theless, on the basis of newly-observed neuroanatomical features, a "diphyletic
hypothesis" was proposed in which megabats (the large "flying foxes," subor-
der Megachiroptera) are closer phylogenetically to primates than to microbats
(typical bats, Microchiroptera) (Pettigrew, 1986, 1991). If so, mammalian wings
and powered flight must have evolved from nonflying ancestral conditions on at
least two separate occasions: once in an early ancestor of the Microchiroptera,
and again in a separate line leading to the Megachiroptera after their ancestors
separated from the Primates (Fig. 8.1). On the contrary, under the traditional
monophyletic view, microbats and megabats are one another's closest relatives.
Thus, a clear dilemma exists: either the neuroanatomical characters shared by
primates and megabats are homoplasious (show similarity due to convergence or
reversal) or the shared features of wing structure and powered flight are ho-
moplasious in megabats and microbats.
Recent molecular analyses have led to a rejection of the "flying primate"
hypothesis for megabats. Nucleotide sequences from the 12S ribosomal RNA
gene and the cytochrome oxidase gene in mitochondria (Adkins and Honeycutt,
 Species Phylogenies and Macroevolution 309

1991; Bennett et al., 1988; Mindell et al., 1991), and from the €-globin gene in
the nucleus (Bailey et aI., 1992), all support a monophyletic scenario for Chi-
roptera. For example, 39 derived nucleotide sequence changes observed at the
€-globin locus were shared uniquely by microbats and megabats, whereas only
two or three such changes were shared by megabats with primates; and at the 12S
locus, about 10 more synapomorphies united megabats and microbats than was
true for less parsimonious, alternative tree topologies. Thus, from molecular
appraisals of phylogeny, the anatomical features associated with powered mam-
malian flight appear to be of a single evolutionary origin.
The king crabs of Alaska (genera Lithodes and Paralithodes) are well-known
decapod crustaceans that look like typical "crabs" (apart from extraordinarily
large size), with a strongly calcified exoskeleton and a greatly reduced abdomen
that is folded up under the body. By contrast, hermit crabs (some 800 species in
more than 80 genera) have a long, decalcified abdomen that the animals coil into
adopted gastropod shells which provide the hermits with a protective home. Thus,
at least superficially, the morphology of hermit crabs is quite different from that
of king crabs, being intermediate between true crabs and the other major groups
of decapod crustaceans-lobsters and shrimps. Nonetheless, morphologists long
have suspected a close genealogical tie between hermit crabs and king crabs, for
the following reasons (Gould, 1992): The abdomen of king crabs, though reduced,
is asymmetrical as in hermit crabs; one or two pairs of legs are reduced greatly
in the king crabs and hermit crabs, respectively, whereas all 10 legs are developed
fully in typical crabs; larval forms of the two groups are remarkably alike; and
carcinization (the evolution of crablike features) appears to have been a recurring
theme in hermit crab evolution under ecological circumstances where the shells
of gastropod snails are of limited availability (as for example in the deep sea).
Recent molecular data from a ribosomal RNA gene in mtDNA appear to clinch
the case for a close genetic link between hermit and king crabs and, in addition,
allow placement of a provisional time scale on the evolutionary separation (Fig.
8.2). From phylogenetic analyses of DNA sequences of 12 species of hermit and
king crabs, plus an outgroup the brine shrimp (Artemia salina), the king crabs
appear to have branched off from a restricted genealogical subset of hermit crabs
and, indeed, are nested within the hermit crab genus Pagurus. Furthermore, this
split from hermit ancestors was estimated to have occurred about 13 to 25 million
years ago, thus placing an upper bound on the time transpired during the evolu-
tionary loss of shell-living habit and the complete carcinization of king crabs (Cun-
ningham et al., 1992). Evolutionary changes in the timing of organismal devel-
opment (heterochrony) likely account for these dramatic morphological shifts.
Living cetaceans traditionally have been divided into two distinctive suborders,
the Odontoceti (echolocating toothed whales and dolphins) and the Mysticeti
(filter-feeding baleen whales). Surprisingly, recent sequence analyses of mito-
chondrial ribosomal RNA genes have suggested that the carnivorous sperm whales
are related more closely to baleen whales than to other toothed whales (Fig. 8.3),
310 Species Phylogenies and Macroevolution

100
p

p
p
hermit crabs
y
97

) king crabs
100 •
L_....!8~8~========~) "left-handed"
hermit crabs

L...-_ _ _ _ _ _ _ _ _ _ _ _ brine shrimp

Figure 8.2. Molecular phylogenetic tree for brine shrimp (Artemia), 2 species of king
crabs (genera Lithodes and Paralithodes), and 10 species of hermit crabs (after Cun-
ningham et aI., 1992). The letter P indicates species in the genus Pagurus. Both
distance-based and parsimony methods applied to mtDNA ribosomal RNA gene se-
quences produced essentially the same phylogeny. Numbers indicate percentages of
bootstrap support for the various nodes in the parsimony analysis. From molecular
clock considerations and extrapolation downward from nodes dated by independent
fossil and geographic evidence, the phylogenetic separation of king crabs from hermit
crabs was estimated at about 13-25 mya.

such that the evolutionary relationship between Mysticeti and Odontoceti is one
of paraphyly rather than reciprocal monophyly. This suggested phylogeny for the
two whale groups, if correct, leads to a parsimonious inference that baleen whales
probably lost the echolocation capability secondarily [alternatively, echolocation
would have to have been gained independently by sperm whales and other toothed
cetaceans (Milinkovitch et aI., 1993)]. In other regards, the phylogeny estimated
from mtDNA sequences is consistent with conventional wisdom about relation-
ships within Cetacea, thus providing increased confidence that the mtDNA se-
quences are informative phylogenetic ally . Important areas of agreement between
molecular and traditional systematic data include an inferred monophyletic status
for each of the following: beaked whales (Ziphiidae), baleen whales (Balaenop-
teridae), sperm whales (Physeteroidea), dolphins (Delphinidae), and porpoises
(Phocoenidae) (Fig. 8.3).
Molecular data also have led to a reassessment of the phylogenetic position of
cetaceans within mammals. The transition from a terrestrial to a fully aquatic
life-style required a gross remodeling of several biological systems and has led
to specialized cetacean anatomies, physiologies, and behaviors that have com-
 Species Phylogenies and Macroevolution 311

) porpoises

dolphins
Odontocetl

) sperm
whales
~
)
~ ~
baleen
Mystlceti

I
whales

) beaked
whales
~ Odontocetl

other
mammals

Figure 8.3. Molecular phylogenetic tree for 16 cetacean species based on rRNA
gene sequences in mtDNA (after Milinkovitch et al., 1993). Levels of bootstrap
support were near 100% for most (but not all) of the putative clades identified
by parsimony analyses and neighbor-joining, including the hypothesized rela-
tionship of sperm whales to baleen whales which was supported at about the
90% level of confidence.

plicated attempts to establish genealogical ties to other mammalian groups. Early

paleontological data suggested that condylarths (archaic ungulates, now extinct)
might provide the ancestral link between extant cetaceans and the modern un-
gulates (hoof-bearing mammals), two prominent orders of which are Perisso-
dactyla (odd-toed forms, including horses, tapirs, and rhinoceroses) and Artio-
dactyla (even-toed forms, including pigs, camels, and deer). A variety of
molecular data are either consistent with or positively supportive of the view that
cetaceans indeed are related more closely to ungulates than to other mammals-
these include information from protein sequences (Goodman et al., 1985;
Miyamoto and Goodman, 1986), mtDNA sequences (Irwin et aI., 1991; Milink-
ovitch et al., 1993; Southern et al., 1988), and DNA-DNA hybridization (Milink-
ovitch, 1992). Among the extant ungulates, recent molecular as well as paleon-
tological evidence suggest a close association of cetaceans with Artiodactyla
(Milinkovitch et al., 1993; Novacek, 1992).
Earlier in this century, the startling discovery of a living coelacanth (a member
of an otherwise extinct subset of crossopterygian fishes) created hope that this
"living fossil" would provide a long-sought missing link morphologically close
312 Species Phylogenies and Macroevolution

to the ancestral form of tetrapods (land vertebrates). However, even with extant
material available, systematists have disagreed on the exact placement of the
evolutionary root for tetrapods along the candidate phylogenetic branches that
interconnect the land vertebrates, ray-finned fishes (Actinopterygii), and lobe-
finned fishes (which in addition to the coelacanth include extant lungfishes).
Three plausible resolutions have been suggested (Fig. 8.4): (a) lungfishes as the
sister group to coelacanths plus tetrapods; (b) tetrapods as the sister group to
lungfishes plus coelacanths; and (c) coelacanths as the sister group to lungfishes
plus tetrapods. A recent analysis based on mtDNA sequences (slowly-evolving
positions at the cytb and 12S rRNA loci) provided initial support for phylogenetic
arrangement (c) (Meyer and Dolven, 1992; Meyer and Wilson, 1990). Onto this
molecular phylogenetic backdrop, numerous morphological features then were
mapped (Table 8.1). Through this approach, 14 head and body traits were pos-
tulated to have undergone a single change on the lungfish-tetrapod stem. These
include acquisition of internal nostrils and controlled access to the trachea
through a glottis (both of which facilitate the tetrapod mode of feeding while
breathing), a division of the heart auricle leading to separation of oxygenated

tetrapods

coelacanths
(a)
lungfish

ray-finned fishes

lungfish

coelacanths
(b)
tetrapods

ray-finned fishes

tetrapods

lungfish
(c)
coelacanths

ray-finned fishes

Figure 8.4. Alternative hypotheses for the phylogenetic

root of tetrapods (after Meyer and Wilson, 1990). These
authors interpreted molecular data as favoring scenario (c).
 Species Phylogenies and Macroevolution 313

Table 8. 1. Phylogenetic mapping of 22 morphological traits along the

molecular-inferred tree (Fig. 8.4c) for tetrapods, lobe-finned, and ray-finned fishes.

Presence ( +) Versus Absence (-) in

Ray-Finned
Trait Lungfish Tetrapods Coelacanth Fish

Single Hypothesized Origin

1. Internal nostrils + +
2. Palate fused with + +
neurocranium
3. Glottis + +
4. Pharyngobranchial gill + +
arches
5. Autopalatine bone + +
6. Depressor mandibulae + +
muscle
7. Free hyomandibular + +
bone
8. Ethmoid sensory canal + +
9. Saccus vasculosus of + +
pituitary
10. Pars nervosa of pituitary + +
11. Truncus anteriosus of + +
heart
12. Divided auricle of heart + +
13. Limbs with >4 + +
mesomeres
14. Pelvic girdles joined + +
Parallelisms or Reversals
1. Labial pit and muscular + +
lip-fold
2. Electroreceptors mostly + +
on snout
3. Septum dividing brain + +
hemispheres
4. Thickened dorsal + +
thalamus
5. Maxilla bone + +
6. Short dentary bone + +
7. Glenoid convex + +
8. Endolymphatic + +
commissure
Source: After Meyer and Wilson (1990).
314 Species Phylogenies and Macroevolution

from unoxygenated blood, the fusion of pelvic girdles, and the incorporation of
more mesomeres into the limbs. By contrast, the distributions of eight other
morphological traits along the molecular-inferred phylogeny called for more
complex explanations involving parallelisms or reversals along the tree.
This study is among the most ambitious and explicit of available attempts to
phylogenetic ally map large numbers of organismal characters. However, the
conclusions also have been called into question, primarily on the grounds that the
molecular phylogeny may not be well supported. For example, Gorr et al. (1991)
interpreted hemoglobin sequences as indicative that the coelacanth rather than
the lungfish was the closest living relative of tetrapods, and reanalyses of these
same data by Stock and Swofford (1991) and Sharp et al. (1991) suggested that
the phylogenetic placement of the coelacanth simply was inconclusive. Further
phylogenetic analyses of cytb and other mitochondrial genes (Normark et aI.,
1991), and of 18S rRNA sequences (Stock et aI., 1991), likewise found incon-
clusive support for the postulated lungfish-tetrapod clade. Whether the phyloge-
netic arrangement proposed by Meyer and Wilson ultimately proves correct
remains to be seen, but, in any event, the important take-home message is that
phylogenetic character mapping depends critically on a correct tree topology. If
the lungfish and tetrapods do not constitute a clade, phylogenetic scenarios for
many of the characters listed in Table 8.1 would change dramatically.
Since first described in 1869, the giant panda of China (Ailuropoda melano-
leuca) has been a phylogenetic enigma. It certainly looks like a bear (family
Ursidae), but also has many traits that are not at all bearlike: flattened teeth and
various other adaptations associated with a bamboo diet; an opposable "thumb";
lack of hibernation; a bleating voice like a sheep; and a karyotype consisting of
21 pairs of chromosomes that appears superficially unlike that of bears with 37
chromosome pairs. In terms of chromosomal numbers, the giant panda more
closely resembles the red panda (Ailurus julgens, 22 chromosome pairs), a very
different-looking species also from China that traditionally had been considered
a member of the raccoon family Procyonidae. Over 40 morphological treatises
on the subject of giant panda ancestry have reached no consensus on whether A.
melanoleuca is allied more closely to bears, raccoons, or to neither (O'Brien,
1987). However, recent molecular appraisals appear to have solved the mystery,
showing that the giant panda stemmed (==20 mya) from an early offshoot of the
lineage leading to modern Ursidae [whereas the ancestor of the red panda sep-
arated (==30 mya) from a different line leading perhaps to the modern Pro-
cyonidae or other carnivores (Fig. 8.5; O'Brien et aI., 1985a)).
This study illustrates two general points. First, molecular phylogenies are
most convincing when supported concordantly by multiple lines of evidence.
The molecular assays applied to pandas and relatives included multilocus protein
electrophoresis (Goldman et al., 1989), protein-immunological methods (Sarich,
1973), and DNA-DNA hybridization, and these several data sets converged on
 Species Phylogenies and Macroevolution 315

six
a bear
species

A
b spectacled
bear

c giant
panda
lJd
~
d
raccoon
B

~
e red
panda

40 30 20 10 0

millions of years ago

Figure 8.5. Consensus molecular phylogeny showing the genealogical position of the
giant panda relative to the taxonomic families of bears (Ursidae, node A) and raccoons
(Procyonidae, node B) (after O'Brien, 1987). Lowercase letters represent suggested sub-
family designations.

the consensus phylogeny depicted in Figure 8.5. A second point is that consensus
molecular phylogenies can be infonnative as a backdrop for interpreting the
evolutionary histories of problematic molecular or cellular features, just as they
are for morphological traits. In this case, the consensus molecular phylogeny
prompted a reexamination of the bizarre karyotype of the giant panda, using
refined methods that reveal details of chromosomal banding patterns. Results
showed conclusively that previously noted differences between the gross kary-
otypes of giant pandas and bears (42 versus 74 chromosomes, respectively) were
superficial, attributable to simple centromeric fusions in the line leading to the
giant panda (O'Brien et aI., 1985a).

BEHAVIORAL, PHYSIOLOGICAL, AND LIFE-HISTORY FEATURES

Molecular-based phylogenies also can provide useful backdrop for the recovery
of the evolutionary histories of other classes of organismal characteristics. An
available case in point involves the phenomenon of interspecific brood parasitism
in birds (the laying of eggs by individuals of one species into nests of another
316 Species Phylogenies and Macroevolution

species). Most New World blackbirds (Icterinae) exhibit conventional parental

life-styles, but several cowbirds in this subfamily display varying degrees of
specialization for brood parasitism: M olothrus badius takes over the nests of other
species but rears its own young; M. rufoaxillaris specializes on a single host
species; M. aeneus and Scaphidura oryzivora parasitize confamilial genera only;
and M. ater and M. bonariensis are generalists, utilizing a wide assortment of host
taxa as foster parents. Recent parsimony analyses applied to sequence data from
an 852-bp region of the cytochrome b gene in mtDNA revealed that among 26
species of Icterinae examined, all of the brood parasites (listed above) formed a
monophyletic group (Lanyon, 1992). Furthermore, the two generalist taxa (M.
ater and M. bonariensis) proved to constitute a molecular clade whose sister taxon
(M. aeneus) is a brood parasite with intermediate host specificity. These results
led to the conclusion that brood parasitism probably evolved a single time within
Icterinae, and that the generalized form of brood parasitism is the derived con-
dition. This finding is important because it appears to conflict with a prevailing
hypothesis that as hosts develop defense mechanisms against brood parasitism,
parasites might evolve toward specialization on fewer host taxa.
Another example in which the direction of evolutionary change in behavior
was inferred from molecular phylogenetic analysis involves Lasioglossum sweat
bees of the subgenus Evylaeus, a hymenopteran group that contains both solitary
and eusocial species exhibiting a wide range of nest architectures. Packer (1991)
employed multilocus allozyme approaches to estimate a cladogram for eight Old
World species, and then mapped a variety of behavioral characteristics onto the
molecular phylogeny (Fig. 8.6). Among the major conclusions drawn from this
exercise in phylogenetic mapping were the following: most Evylaeus species
share sociality by descent from a eusocial common ancestor; multiple-foundress
associations are a derived condition within Evylaeus; and an architectural trait
featuring an extended opening of brood cells during development probably orig-
inated twice independently among the species considered.
Endothermy, the ability to maintain elevated body temperature by metabolic
means, is rare among fishes, the only examples having been documented within
large oceanic teleosts of the suborder Scombroidei (including tunas, mackerels,
and billfishes). Did endothermy evolve once or multiple times within this as-
semblage? A recent mapping of this physiological adaptation on a phylogeny
estimated from the cytochrome b sequences in mtDNA suggests that endothermy
arose at least three times independently within the Scombroidei (Fig. 8.7). These
convergent appearances of endothermy have involved diverse physiological and
morphological pathways (Block et al., 1993).
Tunicates (ascidians or sea squirts) have been thought to be primitive chor-
dates, due largely to the presence a well-organized notochord in their tadpole-
type larvae. Recent phylogenetic analyses based on sequences from rRNA genes
(Field et al., 1988) and on molecular features of muscle actins (Kusakabe et al.,
 Species Phylogenies and Macroevolution 317

4
malachurum

lineare

laticeps

calceatum

albipes
3 5 7 9
marginatum
4
pauxillum

fulvlcorne

1) eusociallty
2) nonoverlapplng caste sizes
3) less than 1% workers mate
4) more than one worker brood per year
5) perennial societies
6) multiple foundress nests
7) nests without cavity
8) lateroid present
9) brood cells open during juvenile development

Figure 8.6. Allozyme-based c\adogram for sweat bees,

onto which have been mapped the various behavioral
evolutionary changes listed (after Packer, 1991). Solid
bars crossing branches indicate traits inferred to be of
monophyletic origin within the group; open bars indi-
cate traits with suggested multiple origins.

1992) tend to support this view by placing the ascidians somewhat closer to the
vertebrates than to the invertebrates. Among the Ascidiacea (of which about
2300 species are known), two distinctive reproductive/developmental modes are
exhibited: some (solitary ascidians) live as individuals, whereas others (colonial
ascidians) form colonies and can propagate asexually by budding, strobilation, or
regeneration. Under an orthodox classification, based primarily on morpholog-
ical features of the branchial sac and gonad, ascidians have been divided into two
taxonomic orders (Enterogona and Pleurogona), irrespective of whether the life-
style is solitary or colonial. If this view is correct, and reflective of phylogeny,
then one or both life history modes probably arose multiple times in evolution.
Alternatively, the distinctive life-styles themselves might register a fundamental
318 Species Phylogenies and Macroevolution

/stiophorus p/stypterus
Mskaira nigricans
Tetrspturus s/bidus
Tetrapturus sudsx
Te trap turus angustirostris
A Tetrap/urus be/one
Makaira indica
Xiphias g/adius
a/a/ungs
s/bacares
Thunnus maccoyii
c Thunnus thynnus

Euthynnus slletters/us
Auxis /hazard
Sarda chiliensis

Gss/erochisms me/ampus
Scomber jsponicus

Ruvettus pretiosus
Scomberomorus cavslla
Scomberomorus maculats
Trichiurus lepturus
Sphyraena sphyraena
Coryphaena equiselis
Serranidae

Figure 8.7. Phylogenetic mapping of endothermy among

marine fishes in the suborder Scombroidei (after Block et
al., 1993). The phylogeny was estimated from nucleotide
sequences of a portion of the cytochrome b gene in
mtDNA. Letters indicate three separate origins of en-
dothermy, each with a different physiological basis:
A-modification of the superior rectus muscle into a ther-
mogenic organ; B-modification of the lateral rectus mus-
cle into a thermogenic organ; and C-use of vascular
countercurrent heat exchangers in the muscle, viscera, and
brain.
 Species Phylogenies and Macroevolution 319

phylogenetic split, such that the branchial sac and gonadal characters would be
homoplasious. To test these possibilities, Wada et al. (1992) sequenced portions
of the 18S rRNA gene in 8 diverse species of ascidians representing both solitary
and colonial forms. Phylogenetic analyses of these sequences grouped the as-
cidians into two distinct lineages that agreed with traditional ordinal assign-
ments. Thus, the solitary and colonial life-styles likely evolved independently
after a true phylogenetic split between the Enterogona and Pleurogona.
Of course, plants also have morphologies and behaviors that can be subjected
to phylogenetic mapping using molecular markers. Androdioecy (in which male
and hermaphroditic flowers occur on separate plants) traditionally has been
viewed as an intermediate step in the evolution of dioecy from hermaphroditism.
However, in the tree genus Datiscaceae which contains three dioecious and one
androdioecious species, androdioecy (rather than dioecy) has been postulated to
represent the derived condition. To test this hypothesis, Rieseberg et ai. (1992)
utilized restriction sites from PCR-amplified cpDNA fragments to construct mo-
lecular phylogenies for 14 species (Datiscaceae and 10 outgroups) against which
to interpret the taxonomic distributions of reproductive traits. Results supported
the contention that the androdioecious species (Datisca glomerata) occupies a
derived position relative to dioecious members of the family and, hence, that
androdioecy evolved from dioecy in this plant family.
Carnivory in flowering plants involves a large suite of morphological and
physiological features associated with the attraction, retention, trapping, killing,
and digestion of animals, and absorption of their products. On this basis, it might
be supposed that the evolutionary acquisition of a carnivorous habit would be ex-
tremely difficult and, hence, rare. Nonetheless, mapping of this life-style against
a molecular-based estimate of phylogeny for more than 70 taxonomic plant
families [from nucleotide sequences of the rbcL gene (see section on chloroplast
DNA later in this chapter)] has revealed that carnivory undoubtedly is polyphyl-
etic. Furthermore, "flypaper traps" appeared to have had at least five separate
origins among dicotyledons, and "pitcher traps" had at least three independent
evolutionary origins (Albert et aI., 1992). However, some of the more detailed
components of carnivory did appear to be clustered phylogenetic ally , such that
the overall syndrome displayed a mixture of homologous and analogous elements.
As stated by the authors, "form is not a reliable indictor of phylogenetic rela-
tionships among carnivorous plants at highly inclusive levels (such as trapping
mechanism), whereas it appears to be at less inclusive ones (such as glandular
anatomy)." Figure 8.8 provides a schematic view of how the polygenic deter-
minants of complex phenotypic features might wax and wane along phylogenetic
branches, occasionally crossing thresholds where the more inclusive syndromes
are exhibited.
Even the simplest of eukaryotic organisms also have provided subject material
for phylogenetic character mapping (Knoll, 1992). Molecular data, particularly
320 Species Phylogenies and Macroevolution

A B

Figure 8.8. Concept of gradients and thresholds in the phylogeny of quantitative traits .
Consider a polygenic trait whose level of expression is a function of appropriate alleles at
numerous unlinked loci (as is no doubt true for many phenotypic attributes). The diagram
shows how genetic changes at these loci can produce gradual evolutionary shifts in the
level of expression (intensity of shading) of the final trait. Furthermore, different suites of
underlying alleles may be responsible for a given level of trait expression (as indicated by
various patterns of barring and stippling) . Suppose now that the phenotypic trait requires
some threshold number of alleles for expression. For example, assume that black in the
diagram is above the required threshold (indicating "presence" of the trait) and non-black
is below the threshold (indicating trait "absence"). "Trait presence" would then have
arisen polyphyletically (at positions A-E), due to shifts in levels of polygenic support.
Thus, a quantitative trait could have a complex mixture of both homologous and ho-
moplasious elements.

from ribosomal RNA genes and some conservative protein-coding loci, have
suggested that a basal split in eukaryotic evolution was between a group dubbed
the Archaezoa (see Cavalier-Smith, 1991) and all other eukaryotes (induding
most protists) . All examined members of the Archaezoa lack mitochondria,
peroxisomes (membrane-bound organelles containing enzymes that metabolize
hydrogen peroxide), Golgi apparatus (a cell organelle that sequesters substances
synthesized by the membraneous endoplasmic reticulum), and spliceosomal in-
trons, all features that also are lacking in noneukaryotes. From such evidence, it
now seems likely that ancestral eukaryote(s) may have been far simpler in cel-
lular and molecular organization than are most extant eukaryotic forms.
 Species Phylogenies and Macroevolution 321

Before leaving this general section, it should again be emphasized that the
phylogenies derived from molecular markers, like those from other systematic
characters, represent estimates (rather than definitive documentations) of phy-
logeny. Thus, the conclusions stemming from phylogenetic character mapping
must be viewed as provisional evolutionary hypotheses, subject to further eval-
uation. Nonetheless, the continued exploration of the comparative evolutionary
distributions of different classes of attributes (molecular and organismal) holds
great promise.

Biogeographic Assessment
A second rationale for phylogeny estimation is in biogeographic reconstruc-
tion. Just as phylogeographic relationships among conspecific populations have
been revealed through molecular analyses (Chapter 6), so too have phylogeo-
graphic relationships among species and higher taxa.

VICARIANCE VERSUS DISPERSAL

The potential geographic range of any taxon, of course, is limited by the

suitability of environmental conditions. Within that zone of ecological tolerance,
the realized geographic distribution is influenced additionally by historical fac-
tors. The "success" of numerous human-mediated introductions of plants and
animals around the world is testimony to the fact that not all habitats suitable for
a species are occupied naturally (or, perhaps, that humans have so disturbed
native habitats that introductions can succeed). Thus, whether a species occurs in
a particular region is a function also of historical demographic factors and dis-
persal patterns, which themselves are influenced by the proximity and spatial
relationships of the environments potentially habitable. The study of such his-
torical factors is the focus of molecular phylogeography.
In cases where related taxa show disjunct geographic distributions, two com-
peting hypotheses often are advanced to account for the spatial arrangements (Box
8. 1). Under "dispersalist" scenarios, such a taxonomic group came to occupy its
current range through active or passive dispersal across a preexisting geographic
or ecologic barrier. Alternatively, under "vicariance" scenarios, the more or less
continuous ranges of ancestral forms may have been split by particular vicariant
geographic events, e.g., the rise of a mountain range that sundered lowland taxa,
a continental breakup that partitioned populations of terrestrial organisms, or
subdivision of a body of water that split aquatic or marine populations. Both
theories subscribe to the proposition that speciation is predominantly allopatric.
However, an important prediction of vicariance biogeography (not shared by
dispersalist scenarios) is that the cladogram for a group of related species should
match the historical "area-cladogram" of environments occupied (Box 8. 1).
Most tests of particular vicariance scenarios have involved phylogenetic as-
sessments using traditional nonmolecular characters (e. g. , Cracraft, 1986), but in
322 Species Phylogenies and Macroevolution

Box 8 ••• Vicariance Versus Dlspersallst Biogeography

Disjunct distributions of related taxa pose special challenges for biogeographic
interpretation. Traditionally, disjunct ranges were interpreted to evidence organismal
dispersal across preexisting geographic or ecological barriers, usually from a biogeo-
graphic "center of origin" where a given taxonomic group presumably originated
(Darlington, 1957, 1965). Dispersalist explanations sometimes became quite strained,
however, as for example in accounting for the global distributions of relatively sed-
entary creatures such as the large flightless birds (ostriches, rheas, emus, and their
presumed relatives). In recent decades, vicariance biogeographers have challenged the
dispersalist paradigm by proposing that disjunct distributions also can result from
environmentally-mediated separations of widespread ancestral forms, without the need
to invoke long-distance or improbable dispersal events ["sweepstakes" dispersal
(Simpson, 1940)].
Vicariance biogeography as a formal discipline (Humphries and Parenti, 1986;
Nelson and Platnick, 1981; Nelson and Rosen, 1981; Rosen, 1978) grew out of several
developments in the 1960s and 1970s, prominent among which were the following
(Wiley, 1988): (a) a growing appreciation from the study of plate tectonics and other
geological processes that the earth's features were not fixed, but rather showed tre-
mendous historical dynamism; and (b) the growth of cladistics (Chapter 2), which
armed researchers with new conceptual outlooks and analytical tools for recovering the
historical, phylogenetic component of evolution.
Under strict vicariance hypotheses (in contrast to dispersalist expectations), cladistic
relationships among related disjunct taxa should mirror faithfully the historical rela-
tionships among the geographic regions occupied. Indeed, one major goal of vicari-
ance biogeography is to reveal relationships among the units of real estate themselves
[e.g., "Is Cuba more closely related to Hispaniola than to Jamaica?" (Wiley, 1988)],
through comparative searches for congruent patterns ( •• generalized tracks") in organ-
ismal phylogeography.
geographic phylogeny:

species phylogeny:
 Species Phylogenies and Macroevolution 323

recent years molecular methods have played an increasingly important role. For
example, Bermingham et al. (1992) used data from mtDNA restriction sites to test
a vicariance model for the evolution of North American birds in the black-throated
green warbler complex. It had been proposed that episodic glacial advances during
the Pleistocene repeatedly fragmented the ranges of forest-dwelling birds into
eastern and western populations, in such a way that subsequent speciations
produced a series of western endemics each linked phylogenetically to the wide-
spread eastern form (Dendroica virens in this case), but at different evolutionary
depths (Mengel, 1964). However, molecular data appeared inconsistent with
certain cladistic aspects of this scenario, suggesting instead that some of the
endemic western warblers in the black-throated green complex budded off from
one another (perhaps via intermontane isolations) rather than directly from D.
virens. Nonetheless, other aspects of Mengel's scenario were supported by the
molecular data, including the basal branching of D. nigrescens from the D. virens
lineage and a probable Pleistocene origin for several avian species-pairs.
A similar Pleistocene scenario constituted the conventional wisdom for anuran
evolution in southwestern Australia, where several western endemics were hy-
pothesized to have arisen following multiple invasions from eastern source
stocks. However, microcomplement fixation studies of albumin evolution in
more than 20 species of frogs representing 6 genera revealed several discrepan-
cies with phylogenetic predictions of this model and led to a rejection of the
multiple-invasion scenario in favor of a model that includes speciation events
within southwestern Australia (Maxson and Roberts, 1984; Roberts and Maxson,
1985). Furthermore, by molecular evidence, many of these speciation events
appeared to predate the Pleistocene significantly.
Allozyme data have been used to test competing vicariance versus dispersalist
models for the trans-Pacific distributions of many tropical marine shore fishes
(Rosenblatt and Waples, 1986). More than 50 such taxa occur on both sides of
this ocean basin, despite 5000 km separating the closest islands of the central
versus eastern Pacific. One possibility is that recent long-distance dispersal
(probably west to east, via free-swimming adults or planktonic larvae utilizing
equatorial currents) connects these trans-Pacific fishes. Alternatively, as had
been proposed for certain invertebrate taxa with similar distributions, these fish
populations in the eastern Pacific might conceivably represent vicariant relicts of
former worldwide ancestors in the Tethyan Sea (an ocean body that separated
Laurasia from Gondwana following the breakup of the supercontinent Pangaea
during the Mesozoic) (McCoy and Heck, 1976). The molecular results proved
consistent with the hypothesis of recent or ongoing dispersal from the west
Pacific. Thus, in each of 12 comparisons attempted, allozyme distances between
trans-Pacific forms were small and markedly lower than genetic distances ob-
served between amphi-American sister taxa that were separated into Atlantic and
Pacific units by the Isthmus of Panama, which arose only 3 mya.
324 Species Phylogenies and Macroevolution

Thus, one important advantage of molecular approaches is that temporal issues

(based on molecular clock considerations) can be examined in addition to cla-
distic assessments per se. In another example, Hedges et al. (1992b) utilized
immunological distances in albumins to assess the evolutionary times of sepa-
ration between a wide variety of amphibians and reptiles in the Caribbean region.
Geologic evidence indicates that the Greater Antilles islands were formed in
close proximity to North and South America about 110-130 mya, but under the
influence of plate tectonics began separating from the mainlands by the late
Cretaceous (80 mya). It had been proposed previously that much of the present
West Indian terrestrial biota reflects these ancient proto-Antillean vicariant sep-
arations. Alternatively, postvicariant overwater dispersal might account for the
presence of related taxa on the various islands. The molecular data for 38 pairs
of terrestrial vertebrates proved inconsistent with the hypothesis of ancient vi-
cariance, in two regards (Fig. 8.9): (a) in comparisons involving particular island
pairs or an island versus the mainland, genetic distances between independent
pairs of taxa showed a large variance, suggesting widely differing times of
colonization as might be expected under a dispersalist scenario; (b) nearly all
estimates of divergence time (assuming an albumin evolutionary clock as cali-
brated from independent data) postdated considerably the vicariant geographic
events hypothesized to have isolated the respective populations. Many additional
phylogeographic details also have been revealed through these and other protein
and DNA comparisons of the Caribbean herpetofauna (Burnell and Hedges,
1990; Hedges, 1989; Hedges et aI., 1991). For example, the six species of Anolis
lizards currently on Jamaica apparently represent a monophyletic group that
radiated mostly during the Pliocene from a colonizer that arrived in the mid-
Miocene, about 14 my a (Hedges and Burnell, 1990).
A remarkable instance of long-distance historical dispersal documented by
molecular markers involves two species of annual plant, the diploid Senecio
flavus of the Sabaro-Arabian and Namibian deserts in Africa and the tetraploid S.
mohavensis of the Mojave and Sonoran deserts of North America. Using mul-
tilocus allozyme assays, Liston et al. (1989) showed that these two species
indeed are remarkably similar genetically (Nei's I ~ 0.95). The genetic results
not only affirm the traditional congeneric status of these taxa (based on gross
morphology), but also imply strongly that recent intercontinental dispersal must
account for their highly disjunct distributions. How this' 'sweepstakes dispersal"
(Box 8.1) occurred remains a mystery, but one possibility is that the sticky seeds
were transported by migrating or lost birds.
Not all molecular appraisals have cast doubt on vicariance scenarios. One
long-standing evolutionary enigma concerns how the large flightless birds (the
"ratites," which include the ostriches of Africa, rheas of South America, and the
emus, cassowaries, and kiwis of Australia, New Guinea, and New Zealand,
respectively) came to possess such a wide distribution in the Southern Hemi-
 Species Phylogenies and Macroevolution 325

asteroid
impact

p roto-A n t i lie sl
Geologic from mainland Cuba/Hsp/PR
separation
-L
West Indies/mainland
• •• ••• • •• • •
Cuba/Jamaica
• • • ••

- --
Jamaica/H ispan io la • • ••
Cuba/Hispaniola
•
Hispaniola/Puerto Rico
•
I I I I I I

90 60 30 o
millions of years ago

(albumin immunological distance)

Figure 8.9. Empirical molecular tests of dispersalist versus vicariance hypotheses

for the origins of terrestrial vertebrates in the Caribbean region (after Hedges et al.,
1992b). Shown are immunological distances in albumin proteins, and the associated
time scales under an evolutionary clock, between various island and mainland
species. Shaded areas indicate dates of faunal separation predicted under vicariance
scenarios based on geologic events.

sphere. One possibility is that these birds are not linked phylogenetically, their
similarities in morphology being attributable instead to convergent evolution
from unrelated ancestors on separate continents. Alternatively, under a vicari-
ance model, lineage separations eventually leading to the extant ratites may
have stemmed from the Mesozoic breakup of Gondwana and subsequent drift of
the southern continents via plate tectonics. This vicariance model carries at least
two testable predictions: ratites should be monophyletic; and the separations
among extant species should be ancient (e.g., beginning> 70 mya). Remark-
ably, available molecular data support these predictions. Using immunological
distances in transferrins, Prager et al. (1976) concluded that the ratites indeed
are monophyletic and that the molecular-based estimates of separation times
are congruent with the suspected dates of particular continental separations. Data
from DNA-DNA hybridizations (e.g., Fig. 3.5) (Sibley and Ahlquist, 1981,
1990), and from amino acid sequences of an a-crystallin eye lens protein (Stapel
326 Species Phylogenies and Macroevolution

et aI., 1984), similarly indicate that the living ratites are both monophyletic and
old. For example, ostriches and rheas appeared to be sister taxa that separated
about 80 mya, a date generally consistent with the opening of the South Atlantic
Ocean between Africa and South America (Sibley and Ahlquist, 1981).
Except for purposes of organizing thought, it is probably unwise to dichotomize
dispersalist versus vicariance scenarios too strongly, because both factors prob-
ably have played a role in many instances. For example, although the global
phylogeography of the ratite birds has been interpreted as consistent with vicar-
iance explanations, recent molecular findings also suggest a role for dispersal in
the colonization of New Zealand. Both kiwis and the now-extinct moas inhabited
New Zealand during the Pleistocene, and conventional wisdom is that these groups
shared a common ancestor on the island. However, a phylogeny estimated from
12S rRNA gene sequences suggests that the kiwis are related more closely to
Australian and African ratites than to the moas and, thus, that New Zealand
probably was colonized at least twice by ratite ancestors (Cooper et aI., 1992).

COMMON ANCESTRY VERSUS CONVERGENCE

In phylogenetic mapping, a general goal is to distinguish whether shared

organismal features arose through common ancestry or through convergent ev-
olution from unrelated ancestors. These issues often come into particularly sharp
focus in a geographic context. For example, various marsupial mammals in
Australia bear at least superficial resemblance in behavioral or morphological
features to particular placental mammals elsewhere in the world. These ecolog-
ical similarities (e.g., between the Tasmanian wolf and placental carnivores, and
between some bandicoots and placental rabbits) are "known" to reflect conver-
gent evolution because the marsupials retain other detailed and distinctive evo-
lutionary signatures interpreted as conclusive evidence of common ancestry
(such as a marsupium or pouch). Furthermore, the suspected monophyly of the
marsupials (almost all of which occur in the Southern Hemisphere) makes geo-
graphic sense. In particular, Australia was isolated from other landmasses during
the early and middle Tertiary period (from about 30-60 mya) , during which time
a remarkable marsupial radiation took place that filled many ecological niches on
that continent that elsewhere in the world were occupied by "ecological equiv-
alents" among the placentals. Many temporal and cladistic details of this phy-
logenetic radiation of Australian marsupials have been worked out using molec-
ular methods including protein immunology (Baverstock et al., 1987; Kirsch,
1977) and DNA-DNA hybridization (Kirsch et al., 1990; Springer and Kirsch,
1989, 1991; Springer et al., 1990).
One of the most remarkable and provocative scenarios to emerge in all of
molecular phylogenetics concerns a similar in situ adaptive radiation recently
proposed for Australian songbirds. Most of the birds of Australia were discov-
ered and named after European ornithologists already had classified other ele-
 Species Phylogenies and Macroevolution 327

ments of the world's avifauna. Many of the Passeriformes ("perching birds") in

Australia appeared to fit neatly into taxonomic categories previously established,
e.g., Australian warbler-like birds into Sylviinae (true warblers), Australian
flycatchers into Muscicapidae (Afro-Eurasian flycatchers), treecreepers into
Certhiidae (Eurasian-American creepers), and sitellas into Sittidae (Holarctic
nuthatches). Nonetheless, evidence from DNA-DNA hybridization has sug-
gested that these traditional taxonomies are incorrect when interpreted as phy-
logenetic arrangements. Instead, many of the Australian songbirds may stem
from a common ancestor on the continent (Sibley, 1991; Sibley and Ahlquist,
1986). From molecular comparisons of many hundreds of avian species, Sibley
and Ahlquist (1990) conclude that the songbirds of the world should be divided
(phylogenetically) into two groups-tbe suggested suborder Passerida whose
members appear to have evolved in Africa, Eurasia, and North America, and the
Corvida which originated in Australia. The latter includes numerous species
thought previously to be unrelated to one another (Fig. 8.10). If this fascinating

reduced melting
temperature (0 C)
20 10 o
CORVOIDEA: AUllralian robins, lo,,·runners, pseudo-babblers,
sltellas, whistlers, fantails, monarchs, shrikes, butcherbirds.
Australian vangas, woodswallows, birds of paradis., crows, magpies, jay.,

root ~ ~______ bush-Shrike., helmet shrikes, Old World orioles, cuckoo-shrikes

MELIPHAGOIDEA: falr-y wrens, honeysater., pardalotes,

thronbills, scrUb-wrens

MENUROIDEA: AUllralian Ireecreepers, scrub-birds,

Iyreblrds, bowerbirds

MUSCICAPOIDEA: waxwln"s, dlpperl, starlings, mockingbirds,

thrushes, Old World chats and lIycatchers

SYLVIOIDEA: wrens, gnate.lehers. nuthatches, titmice,

swallOWS, klngle's, bulbul., Old World warbl.fs, babbler.,
-
~
whlte-ey ••

no( ______ PASSEROIDEA: larks, plpitl, .unblrdl, flowerpecker.,

weaverbirds, sparrows, New World orlol.s, wood warble,.,
Australian blackbirds. tanager., carduellne and emberlzlne finches
root
other paa.erlne birds

90 60 30 o
millions of years ago

Figure 8.10, Phylogeny of the perching birds (order Passerifonnes) based on DNA-DNA
hybridization data (after Sibley and Ahlquist, 1986). Two major groups of the suborder
Passeres were postulated: one tracing to an Australian root (some of whose members such
as crows and jays secondarily radiated elsewhere in the world) and the other tracing to a
non-Australian origin.
328 Species Phylogenies and Macroevolution

proposal for a monophyletic origin of Corvida indeed is correct, then the phy-
logeographic history of a major segment of the Australian avifauna parallels that
of the native Australian mammals.

RECENT ISLANDS, ANCIENT INHABITANTS

Another spectacular evolutionary radiation studied through molecular markers

involves representatives of the Drosophilidae flies of the Hawaiian Islands, home
to an estimated 800+ species (a remarkable count, because the entire archipel-
ago accounts for only 0.01 % of the earth's land area). These flies traditionally
are divided into two recognizable groups, the "drosophiloids" and the "scap-
tomyzoids," that have been postulated to derive from only one or perhaps two
founder populations of unknown continental source (Throckmorton, 1975). The
oldest of the major, present-day Hawaiian Islands dates by geological evidence
to only 5 mya. Was the incredible proliferation of drosophiline species on the
Hawaiian Islands truly accomplished within such a short evolutionary time span?
From immunological comparisons of larval hemolymph proteins, Beverley
and Wilson (1985) provided the first extensive molecular evidence that some of
the lineage separations in Hawaiian drosophilines vastly predate the volcanic
emergences of the present-day islands. According to their molecular clock cal-
ibrations, the Hawaiian Drosophilidae stem from a colonist that landed on the
archipelago about 42 mya. To explain the paradox, they note that the current
Hawaiian Islands are merely the latest in a series of former islands that date back
over 70 million years, the remnants of which remain today as eroded seamounts
or low atolls to the northwest of the current chain. Thomas and Hunt (1991) and
DeSalle (1992a, 1992b) have reexamined this issue using DNA sequences from
the alcohol dehydrogenase nuclear locus and mtDNA, respectively, and their
findings strongly support the contention that Drosophila inhibited the Hawaiian
chain well before 5 my a (although estimates of the exact divergence times differ
somewhat). Thus, according to molecular evidence, many speciation events
probably occurred on islands no longer in existence (as flies island-hopped to
newly arisen terrain), such that the drosophiline radiation was accomplished over
a considerably longer temporal framework than suspected previously. Extensive
molecular and other data on particular Drosophila subgroups (such as the' 'pic-
ture-winged" flies) demonstrate that at least some speciations also have taken
place recently on the present-day Hawaiian Islands (e.g., Carson, 1976, 1992).
Analogous questions apply to the biota of another volcanic archipelago that
has figured prominently in evolutionary studies-the Galapagos Islands. All of
the present-day Galapagos are less than three million years old, and some re-
searchers have considered this to represent the maximum age over which the
exuberant evolution on the island chain must have taken place (Hickman and
Lipps, 1985). Indeed, the small genetic distances among several species of
 Species Phylogenies and Macroevolution 329

Darwin's finches (Geospizinae) are consistent with the hypothesis of a very

recent radiation within this avian clade, perhaps within the last one million years
(Polans, 1983; Yang and Patton, 1981). On the other hand, much larger genetic
distances have been found between the marine iguana (Amblyrhynchus cristatus)
and the two land iguanas of the Galapagos (Conolophus pal/idus and C. sub-
cristatus), suggesting separation dates of perhaps 15-20 my a (Wyles and Sarich,
1983). One possibility is that these genera stem from different (but unknown)
ancestral stocks on the South American mainland [such scenarios also have been
advanced to account for large genetic distances observed among some other
native Galapagos lizards and rodents (Lopez et al., 1992; Patton and Hafner,
1983; Wright, 1983)]. However, another possibility is that speciations took place
on former islands before they sank beneath the ocean surface. For this reason,
considerable excitement attended the recent geological discovery of drowned
islands downstream from the Galapagos volcanic hotspot (Christie et al., 1992).
These geological observations and radiometric data indicate that islands have
been present over the Galapagos area for at least nine million years, and perhaps
much longer. Whether this temporal extension of available habitat actually ac-
counts for ancient speciation events in the Galapagos iguanas or its other biotas
remains to be determined.

Academic Pursuit of Genealogical Roots

In truth, most studies in molecular phylogeny probably are initiated out of

sheer intellectual curiosity about the ancestry of a particular group. Most evo-
lutionists have a favorite taxon (be it fishes or fungi), the phylogenetic under-
standing of which can become an obsession. Although it would be presumptuous
here to choose particular examples in molecular systematics as being of special
inherent interest, one case history stands out as worthy of discussion-the phy-
logenetic position of humans relative to the great apes and other primates. Prob-
ably no other topic in molecular evolution has attracted so much interest, or
controversy.
Traditionally, Bomo sapiens has been placed alone (in terms of extant species)
in the Hominidae, a taxonomic family belonging to the superfamily Hominoidea
which also includes the Asiatic apes [gibbons (Bylobates), siamangs (Sympha-
langus) , and orangutans (Pongo)] and the African apes [gorillas (Gorilla) and
chimpanzees (Pan)]. Closest relatives outside the Hominoidea are the Old World
monkeys (Cercopithecoidea). Within Hominoidea, conventional (but not univer-
sal) wisdom has been that humans' closest living relatives are the great apes of
Africa (Pongidae). Beyond these points, agreement usually ended.
Prior to the availability of molecular data, a common paleontological scenario
was that the line leading to humans split from a line leading to gorillas and
chimpanzees about 15-30 my a (see Patterson, 1987). In 1967, a stunning report
330 Species Phylogenies and Macroevolution

by Sarich and Wilson based on immunological studies of albumin challenged this

belief in two major respects (Fig. 8.11). First, the molecular data were inter-
preted to indicate that the phylogenetic split leading to Homo occurred much
more recently than formerly supposed-only about 5 mya. Second, the molec-
ular data suggested that the African apes might not form a distinct clade-rather,
chimpanzees, gorillas, and humans constituted a close-knit but unresolved phy-
logenetic "trichotomy." Nearly 30 years and dozens of molecular studies later,
the two major conclusions of Sarich and Wilson both have been vindicated. It
now is accepted widely that the human lineage separated from apes about 4-8
mya, and that humans, chimpanzees, and gorillas are related more or less equi-
distantly to one another. These conclusions represent an impressive consensus of
information from immunological assays, protein electrophoresis, amino acid
sequencing, DNA-DNA hybridization, DNA restriction-site analyses, and nu-
cleotide sequencing from mtDNA and several nuclear genes and noncoding
regions (e.g., Bruce and Ayala, 1979; Caccone and Powell, 1989; Goodman et
al., 1990; Hasegawa, 1990; Miyamoto and Goodman, 1990; Nei and Tajima,
1985; Sibley et aI., 1990; Williams and Goodman, 1989; and references therein).

,
OLIGOCENE MIOCENE : PLIOCENE : +-:-- PLEISTOCENE

gibbon
siamang
orangutan

,:~,
human
chimpanzee
gorilla
L.....-_....;..._ _ _ _ _ _ _
Old World
~--....,:__:

monkeys

30 20 10 o
time (millions of years)

Figure 8.11. One of the first molecular-based estimates of the phylogenetic position
of Homo sapiens within the primates (after Sarich and Wilson, 1967; see also
Goodman, 1962). This phylogeny, based on immunological distances in albumins,
revolutionized thought about human origins (see text), and its major features have
been confirmed with much additional molecular evidence.
 Species Phylogenies and Macroevolution 331

Earlier competing scenarios based on morphological and paleontological evi-

dence also have been reinterpreted to accommodate (at least partially!) this
overwhelming molecular evidence (Andrews, 1987; Pilbeam, 1984).
In recent years, most attention has focused on resolving the human--chimp-
gorilla trichotomy, and this has included discussions of the suitability of various
classes of molecular data as well as methods of statistical analysis (e.g., Kishino
and Hasegawa, 1989; Nei et aI., 1985; Saitou and Nei, 1986; Templeton, 1983).
Although the issue is not yet settled to everyone's satisfaction, a developing
consensus favors a human-chimpanzee clade as a sister group to the gorilla
(Hasegawa, 1990; Horai et aI., 1992; Li and Graur, 1991; Sibley and Ahlquist,
1984; Williams and Goodman, 1989). In any event, the major point is that
molecular data have revealed just how tightly the phylogenetic ties bind humans
to our primate relatives. The traditional taxonomic placement of Homo sapiens
within the monotypic Hominidae probably says much more about anthropocen-
tric bias than it does about objective phylogenetic reality.

SPECIAL APPROACHES TO PHYLOGENY ESTIMATION

Another way to organize thought about the available plethora of molecular

phylogenetic studies is to focus on the principle methodologies employed. This
section will highlight additional major approaches that have been or promise to
be of special importance in phylogeny estimation.

DNA-DNA Hybridization and Avian Systematics

At the time of this writing, the clear record for number of published taxa
assayed in a molecular phylogenetic study belongs to Charles Sibley and Jon
Ahlquist, who over a 12-year period applied DNA-DNA hybridization methods
to more than 1700 avian species representing all but 3 of the 171 taxonomic
families of birds conventionally recognized. The result was an extended estimate
of avian phylogeny [a printed version of which spans 42 pages in their summary
tome (Sibley and Ahlquist, 1990)] that in ornithological circles has become
known simply as "the Tapestry. " Some examples of conclusions derived from
their work already have been cited (on ratite birds and on the Australian avi-
fauna, this chapter; New World and Old World vultures, Chapter 1). In general,
most of their molecular-based phylogenetic conclusions agree quite well with
traditional ornithological thought, thus providing considerable confidence in the
approach. However, many problematic and contentious results also emerged
(Table 8.2), and the Tapestry and its underlying data have by no means been met
with universal approval (e.g., Cracraft, 1992; Cracraft and Mindell, 1989; Sarich
etal., 1989).
332 Species Phylogenies and Macroevolution

Table 8.2. Examples of surprising and provocative conclusions from DNA-DNA

hybridization studies of birds.

Example Explanation

I. Barbets and toucans Traditional view: African and South American barbets a
monophyletic group within Piciformes
Molecular view: New World barbets and toucans are
related more closely to one another than either is to
Old World barbets
2. Hoatzin Traditional view: a specialized form that has been a
complete taxonomic puzzle
Molecular view: a highly modified cuckoo related to
roadrunners and anis (Cuculiformes)
3. Owls Traditional view: allied either to diurnal birds of prey
(Falconiformes) or to "nightjars" (Caprimulgiformes)
Molecular view: related to nightjars, and not to diurnal
birds of prey
4. Totipalmate swimmers Traditional view: fully-webbed toes and other shared
features indicative of monophyly for Pelecaniformes
(pelicans, boobies, gannets, cormorants, anhingas,
frigatebirds, and tropicbirds)
Molecular view: Pelecaniformes likely a polyphyletic
assemblage
5. Grebes Traditional view: closely related to loons (Gaviiformes)
Molecular view: not related closely to loons, and indeed
have no close living relatives
6. Sandgrouse Traditional view: related perhaps to pigeons
(Columbiformes), plovers (Charadriiformes), or
chickens (Galliformes)
Molecular view: closest allies among the Charadriiformes
7. Vultures Traditional view: New World and Old World vultures (in
Falconiformes) closely related
Molecular view: New World vultures related more closely
to storks (Ciconiiformes) than to Old World vultures
8. Shoebill Traditional view: related to storks or herons
(Ciconiiformes)
Molecular view: related more closely to pelicans
(Pelecaniformes)
9. Starlings Traditional view: related closely to crows (Corvidae)
Molecular view: a sister group to mockingbirds
(Mimidae), unrelated to crows
Source: Sibley and Ahlquist (1990).
 Species Phylogenies and Macroevolution 333

Whether the more provocative of the Sibley-Ahlquist conclusions are proved

correct remains to be determined by comparative analyses using additional mo-
lecular (and other) appraisals. In any event, two important points regarding this
immense effort warrant attention here. First, this study is among the most
broadly based of molecular attempts to capitalize explicitly on the "common
yardstick" rationale in phylogenetic reconstruction (Chapter 1). Sibley and Ahl-
quist promulgated a molecular metric (I1T, that presumably is related to evolu-
tionary time) that could be applied as a standardized measure of the magnitude
of evolutionary separation among any avian (or other) taxa, be they humming-
birds or ostriches and rheas. Second, they boldly advocated a direct translation
of these metrics into a formal taxonomy that is intended to encompass the
concept of "categorical equivalency" (Table 8.3). For example, they suggest
that species be classified at the family level when they exhibit a I1T in the range
9-11°C, and at the subordinallevel when a I1Tof 18-20°C is observed. Whether
or not the particular details of this translation are adopted widely, the Sibley-
Ahlquist study remains a monumental and pioneering empirical effort toward the
revolutionary goal of a universal systematics-an aspiration that is one of the
ultimate promises of molecular phylogenetics.

Table 8.3. Suggested levels of genetic divergence (as measured by DNA-DNA

hybridization) to be associated with indicated levels of taxonomic recognition in birds.

Taxonomic Category Ending llTsrJl Range Example

Class 31-33 Aves

Subclass -ornithes 29-31 Neornithes
Infraclass -aves 27-29 Neoaves
Parvclass -ae 24.5-27 Galloanserae
Superorder -morphae 22-24.5 Anserimorphae
Order -iformes 20-22 Anseriformes
Suborder -i 18-20
Infraorder -ides 15.5-18 Anserides
Parvorder -ida 13-15.5
Superfamily -oidea 11-13
Family -idae 9-11 Anatidae
Subfamily -inae 7-9 Anatinae
Tribe -ini 4.5-7 Anatini
Subtribe -ina 2.2-4.5
Congeneric spp. 0-2.2 Anas (puddle ducks)
Source: After Sibley et al. (1988).
334 Species Phylogenies and Macroevolution

Mitochondrial DNA and the Higher Systematics of Animals

In addition to its utility as an intraspecific, micro-evolutionary marker (Chap-

ter 6), mtDNA also is employed widely as an informative guide to phylogenetic
relationships among higher animal taxa. For these latter purposes, unique struc-
tural alterations in the molecule, or sequence regions that evolve more slowly
than average, are monitored.

EcCENTRIC MTDNA MARKERS

With respect to the intended property of providing a numerical "average" of

genetic distances over a large but unspecified number of (single-copy) genes, the
DNA-DNA hybridization studies discussed earlier represent one end of a philo-
sophical continuum in molecular systematics. Near the other end of this contin-
uum are analyses that focus on idiosyncratic molecular traits which, because of
supposed singularities of origin, should be of special significance in a cladistic
sense. One promising suite of such characters involves gene orders and other
unusual compositional properties of mtDNA.
The same ensemble of about 37 genes comprises the mtDNA of most animal
species, but gene orders sometimes differ. For example, identical mtDNA gene
arrangements are displayed by assayed placental mammals, amphibians, and
fishes, but these differ from the gene alignment in quail by a transposition
involving five loci (Desjardins and Morais, 1991). Also, three assayed marsu-
pials have rearranged tRNA clusters in mtDNA relative to the common verte-
brate theme (PiHibo et al., 1991). More comprehensive surveys of additional
avian and marsupial taxa should reveal precisely where these apparently unique
and derived transpositions arose and, hence, the clades that they delineate.
Distinctive mtDNA gene arrangements recently have been employed as phy-
logenetic markers in the Echinodermata, an invertebrate phylum traditionally
divided into five classes: Asteroidea (sea stars), Echinoidea (sea urchins), Oph-
iuroidea (brittle stars), Holothuroidea (sea cucumbers), and Crinoidea (crinoids).
From fossil evidence, these classes are believed to have separated from one
another early in the Paleozoic (some 450-550 mya), but the exact phylogenetic
branching sequence remained controversial (Smith, 1992). Recent studies of
mtDNA gene order provided an additional clue: ophiuroids and asteroids have a
similar mtDNA gene arrangement that contrasts sharply with the gene order
common to echinoids and holothuroids (crinoids were not assayed) (Jacobs et aI.,
1988,1989; M.J. Smith et al. , 1989, 1990, 1993). Outgroupcomparisons (against
vertebrates) suggest that the asteroid-ophiuroid condition is synapomorphic and,
therefore, defines a clade.
Rearrangements in mtDNA gene order also have been employed successfully
as phylogenetic markers among Suillus and related mushroom genera (Bruns and
Palmer, 1989; Bruns et aI., 1989). More comprehensive surveys of mtDN A gene
 Species Phylogenies and Macroevolution 335

order across metazoan phyla currently are underway (e.g., Okimoto et al., 1992).
Rare mtDNA structural rearrangements appear to hold special phylogenetic prom-
ise (Sankoff et al., 1992), as, for example, in characterizing higher invertebrate
taxa where evolutionary relationships heretofore have been highly conjectural.
Ascertainment of the phylogenetic relationships among the taxonomic classes
of Cnidaria (corals, anemones, jellyfishes, and allies) has been a classic problem
in invertebrate zoology, due to a paucity of morphological characters that are
independent of the dramatic differences in the lifecycles of various forms. The
recent discovery that some cnidarians possess native mtDNA in linear (rather than
circular) form has prompted use of this structural feature as a phylogenetic marker.
All surveyed members of the classes Cubozoa, Scyphozoa, and Hydrozoa proved
to possess linear mtDNA, whereas all Anthozoa, Ctenophora (the supposed
outgroup), and most other surveyed metazoan phyla have circular mtDNA (Bridge
et al., 1992). Thus, the derived (linear) condition appears to define a clade, and
if so, a mapping of life-cycle characters on this rooted phylogeny implies that the
benthic polyp stage (rather than the pelagic medussa stage) probably came first
in cnidarian evolution (Bridge et al., 1992).
Several other kinds of genetic novelties (e.g., differences in secondary struc-
tures of tRNAs, sizes of rRNA genes, and peculiar features of the control region)
also distinguish the mtDNA genomes of various metazoan groups (Wolstenholme,
1992). One particularly intriguing class of potential phylogenetic markers in-
volves different patterns of codon assignment in protein translation. Following the
initial discovery of modified codon assignments in mammalian mtDNA (Barrell
et aI., 1979), it was postulated that "drift" in codon usage away from the "uni-
versal" code of most nuclear genomes had occurred during mtDNA evolution.
Wolstenholme (1992) has summarized available information on mtDNA codon
assignments across 19 metazoan animals representing six phyla and superimposed
the differences on a suspected phylogenetic tree (Fig. 8.12). Several of the
differences appeared to be informative phylogenetically. For example, in the
mtDNAs of all assayed invertebrate phyla (with the exception of Cnidaria), AGA
and AGG specify serine, whereas in vertebrate mitochondria they cause chain
termination. In the Cnidaria, as in the universal nuclear code, these same codons
specify arginine. Thus, from the shared-derived codon conditions, vertebrates
appear to constitute a clade, as do the assayed invertebrates exclusive of Cnidaria.
On the other hand, some evidence for evolutionary convergence in codon
assignments also was uncovered (Fig. 8.12). The best example involved the
nucleotide triplet AAA, which in the Echinodermata and Platyhelminthes (phyla
otherwise thought to be unrelated) appears to specify asparagine, rather than
lysine as in other metazoan mtDNAs and in the universal code. If these unusual
codon assignments for asparagine are confirmed (the evidence is not yet conclu-
sive), the derived condition probably arose twice in evolution, independently.
Similarly, an apparent reversion to the presumed ancestral condition in which
336 Species Phylogenies and Macroevolution

=
rAGA & AGG
.,..;;;...--
SIOP)-...
VERTEBRATA

ECHINODERMATA
rATA = lie)
rAAA = Asn)

ARTHROPODA
rAGA & AGG = Ser) rAGG t Ser)
rATA = Metl
.........
NEMATODA

(AAA = Asn)
to
"universal" ...___---1 PLATYHELMINTHES
code

CNIDARIA

Figure 8.12. Evolutionary scenario for alterations of the genetic code in metazoan
mtDNA (after Wolstenholme, 1992). Observed changes from the "universal genetic
code" are plotted on a presumed phylogeny for metazoan animals based mostly on
other evidence. Solid bars across branches indicate probable synapomorphies indica-
tive of various clades; open bars indicate probable homoplasious changes (conver-
gences or reversals).

ATA specifies isoleucine (rather than methionine) may have occurred in an

ancestor of the echinoderms.
These latter observations raise a cautionary note for all attempts to define
clades by any single genetic marker. No matter how secure a synapomorphy
might appear, the possibilities of evolutionary convergence or reversal seldom
can be eliminated entirely. Thus, confirmation from independent sources of data
should always be attempted before definitive phylogenetic conclusions are
drawn.

MTDNA SEQUENCES

A second approach to higher animal systematics through mtDNA involves

direct comparisons of more slowly-evolving nucleotide sequences or portions
thereof (such as transversional changes, or nonsynonymous substitutions in pro-
tein-coding regions). The identification of "universal" primers that can be used
to PeR-amplify particular segments ofmtDNA from numerous species (Box 3.3)
has facilitated greatly such sequencing studies. Prominent among the mtDNA
gene regions now sequenced routinely are those encoding the ribosomal RNA
 Species Phylogenies and Macroevolution 337

subunits, cytochrome oxidases, and cytochrome b. Several phylogenetic exam-

ples already have been presented (see the earlier sections on bats, crabs, whales,
coelacanths, and blackbirds), and more will follow later.

Chloroplast DNA and the Higher Systematics of Plants

The molecular appraisal of cpDNA recently has blossomed into a major in-
dustry within the field of plant phylogenetics. The chloroplast genome is well
suited for higher systematic studies because (a) it is a widely distributed and
relatively abundant component of plant total DNA, (b) much background infor-
mation is available (including complete sequences from at least three distantly
related species), which facilitates experimental and comparative work, (c) dis-
tinctive structural features of cladistic utility characterize the cpDNAs of some
taxa, and (d) the molecule generally exhibits a conservative rate of nucleotide
substitution (Clegg and Zurawski, 1992). Two distinct phylogenetic approaches
again have been employed (Olmstead et aI., 1990). The first involves monitoring
the taxonomic distributions of idiosyncratic, often fortuitously discovered struc-
tural characteristics of cpDNA, the rationale being that unusual or singularly-
arisen features are especially powerful for clade delineation. The second ap-
proach involves either RFLP analyses or direct nucleotide sequencing of
particular cpDNA genes or regions. These latter studies provide a much larger
number of potentially informative characters, but the data sets are generally
afflicted with a higher level of homoplasy.

EcCENTRIC cpDNA MARKERS

Relatively unusual or idiosyncratic features of cpDNA that have been em-

ployed as phylogenetic markers include inversions, losses of genes and introns,
and losses of a large inverted repeat region (Downie and Palmer, 1992). These
will be discussed in turn.
With a few notable exceptions [involving Pisum (Palmer et al., 1988b), Tri-
folium (Milligan et aI., 1989), and conifers (Strauss et al., 1988)], gene order
normally is a conservative feature of cpDNA in vascular plants. This is illus-
trated by the fact that the gene arrangement in tobacco (Nicotiana tabacum) is
similar to the presumed ancestral vascular plant gene order also found in most
other examined angiosperms, ferns, and the Ginkgo (Palmer et al., 1988a).
Among those cpDNA genomes that do differ in gene order, one or a few re-
sponsible inversions often can be deduced. For example, cpDNAs from bryo-
phytes (Marchantia), mosses (Physcomitrella), and lycopsids (Lycopodium, Se-
laginella, lsoetes) differ from that of most vascular plants by a 30-kb inversion
(Calie and Hughes, 1987; Ohyama et aI., 1986; Raubeson and Jansen, 1992a) ,
one of the few large structural alterations in cpDNA accepted over the 400-500
338 Species Phylogenies and Macroevolution

million years of evolution involved. This inversion appears to be a synapomor-

phy for vascular plants minus the lycopsids and identifies a basal split among
vascular plants.
One of the first and most comprehensive of phylogenetic studies of a cpDNA
rearrangement involved a 22-kb inversion found to be shared by 57 genera
representing all tribes of the Asteraceae (sunflowers), a large plant family with
more than 20,000 species and 1100 genera (Jansen and Palmer, 1987). The
absence of this inversion from the subtribe Barnadesiinae of the Mutisieae tribe,
and from all families allied to Asteraceae, suggested that Barnadesiinae repre-
sents the most basal lineage in the Asteraceae and that, contrary to earlier
opinion, Mutisieae is not monophyletic. These conclusions subsequently were
supported by congruent results obtained from phylogenetic analyses of cpDNA
restriction sites (Jansen and Palmer, 1988) and sequences (Kim et al., 1992; see
review in Jansen et aI., 1992).
Unlike the case for mtDNA in most higher animals, cpDNA genes carry
numerous introns [more than 20 have been identified at least tentatively in N.
tabacum (Shinozaki et aI., 1986)]. Systematic surveys have revealed that losses
of entire introns (in contrast to the more frequently observed mutations in intron
length) are relatively rare events that can be quite informative phylogenetically.
For example, the absence of an intron in the rpl2 gene marks all examined
members of the Caryophyllales (Downie et aI., 1991). However, caution in the
use of such markers alone is indicated also, because a loss of this intron appar-
ently has occurred independently in at least five other unrelated dicot lineages
(Downie et al., 1991). Occasional evolutionary losses of particular genes from
cpDNA (Perhaps to the nucleus) also have been employed as phylogenetic sig-
nals (Downie and Palmer, 1992).
Most land plant cpDNAs possess a 20-30-kb inverted repeat (IR) region, the
rare deletion of one copy of which has been employed to infer monophyly for six
tribes and one putatively allied genus Wisteria within the subfamily Papilion-
oideae (Lavin et aI., 1990). However, in a phylogenetic sense the character state
"IR loss" again may be informative locally but potentially misleading as a guide
to global phylogeny because IR losses have occurred independently in more than
one plant group (Doyle et al., 1992). In particular, conifers also possess only one
IR element (Lidholm et aI., 1988; Raubeson and Jansen, 1992b).
Generally speaking, convergent evolutionary gains of rare features are even less
likely than convergent losses (although both types of events can be troubled by
homoplasy), and hence the shared possessions of de novo genomic additions
should be of special significance in clade identification. The relationship of green
algae to land plants long has intrigued botanists. One green algal group from which
land plants were suspected to have arisen is the Charophyceae, a suggestion that
gained recent additional support from molecular observations on cpDNA. All
previously examined algae as well as eubacteria lack introns in their tRNA Ala and
 Species Phylogenies and Macroevolution 339

tRNA Ile genes, whereas all assayed land plants possess them. A recent discovery
of homologous introns in representatives of the Charophyceae (Coleochaete,
Nitella, and Spirogyra) was interpreted to indicate the evolutionary acquisition of
a genetic novelty, presumably some 400-500 mya, marking a clade that indeed
does link the land plants to the charophyceans (Manhart and Palmer, 1990).

cpDNA RESTRICTION SITES AND SEQUENCES

Because the cpDNA genome is large (relative to animal mtDNA, for example),
typical whole-genome studies based on Southern blotting procedures often include
a large number of six-cutter restriction sites (typically 200-1000). An example
of one such study, which deals with the magnificent woody giant-rosette plants
in the genus Lobelia, is summarized in Figure 8.13. This group has a nearly
pantropical distribution with spectacular evolutionary radiations in such places as
the mountains of eastern Africa and the Hawaiian Islands, but phylogeographic

L. holstll
M. lutea

L. fervens ] he,bace.us
L. cardinalls

]
100 L. po/yphyl/a
L. excelsa
100
Chile
L. tupa
L. brldgesll
100
L. nicotianifolla ] China
S. jayorum

71 L.

L.
hy~."u
L. bonlnensls

glorla-montls
]
Pacific
woody

46
L. glberroa
L.
p."."" ] East
Africa
L. strlcklandlae
L. organensls ] Brazil

Figure 8.13. Molecular phylogeny for the plant genus Lobelia and allies (S.
Sclerotheca; M. = Monopsis) (after Knox et al., 1993). The tree is derived from
132 informative cpDNA restriction-site mutations. Numbers indicate levels of
bootstrap support for putative clades. Superimposed on the phylogeny are struc-
tural rearrangements in cpDNA that also were monitored: deletions (open bars)
and inversions (closed bars).
340 Species Phylogenies and Macroevolution

relationships in the assemblage have remained controversial. Phylogenetic anal-

yses of 17 species using restriction-site mutations revealed that the woody lobelias
(a) are derived monophyletically from diploid herbaceous ancestors, (b) include
a hexaploid group in Chile that is a sister clade to a pan-tropical tetraploid clade,
(c) include the genus Sclerotheca, which now appears to be derived from a woody
Lobelia ancestor, and (d) contain a clade consisting of the giant lobelias of eastern
Africa plus a Brazilian species (Knox et al., 1993). Apart from these phylogenetic
insights, this study is important because it included also a phylogenetic analysis
of cpDNA structural rearrangements (deletions and inversions) that occur within
the group. These structural features could be overlaid on the restriction-site tree
topology without conflict (Fig. 8.13), indicating that the two sets of data yield
congruent (though not equally detailed) phylogenetic estimates.
In recent years, sequence analyses of cpDNA have become popUlar. A favorite
sequencing target is rbcL, a gene encoding the large subunit of ribulose-l,5-
biphosphate carboxylase. Reasons for this preference include the following
(D.E. Soltis et al., 1990): (a) much comparative information has accumulated [at
least 500 rbcL sequences are available at the time of this writing (Chase et al.,
1993)]; (b) rbcL is a large gene (>1400 bp) that provides numerous characters
for phylogenetic studies; and (c) the rate of rbcL evolution has proved especially
appropriate for addressing questions of plant phylogeny at the subfamilial level
or higher. One example of a tree generated from rbcL sequences (plus restriction-
site data) is presented in Figure 8.14. Interestingly, in this case, restriction-site
data from the entire cpDNA genome proved more useful than the rbcL se-
quences, presumably due to the sampling of more variation in the restriction-site
comparisons and the higher incidence of homoplasy in the sequence data (Kim
etal.,1992).
One advantage of sequence (or site) analyses over particular eccentric markers
(see above) in phylogenetic assessment is that general estimates of divergence
times (as well as branching patterns) can be attempted, using appeals to molecular
clock considerations. For example, from analyses of published sequences of
numerous chloroplast and nuclear genes in a variety of plant species, Wolfe et al.
(1989) estimated that the cycad-angiosperm split occurred about 340 my a and that
a monocot-dicot divergence occurred about 200 my a (with an uncertainty of about
40 my). Within the dicots, an evolutionary radiation of higher lineages was
estimated from rbcL sequences and the fossil record to have occurred during a
relatively short period about 85-90 my a (Olmstead et al., 1992). Within the
monocots, analysis of rbcL sequences from 104 species representing more than
50 taxonomic families produced a tree indicating a relatively rapid radiation of
major lineages within this group also, although the authors did not hazard a guess
as to the absolute age of this event (Duvall et al., 1993). In general, caution should,
of course, be exercised in interpreting conclusions about specific divergence times
from rbcL (or other) sequences because suspected rate differences of at least
fivefold have been reported for this gene across plant lineages (Gaut et al., 1992).
 Species Phylogenies and Macroevolution 341

Eupatorium
Chromolaena ] Eupatorleae

He/lanthus ] Heliantheae
Coreopsis ] Coreopsldeae
Tagetes ] Tageteae

87
Blennosperma
Senecio ] Senecloneae 3
ChrYSanthemum]
Anthemldeae
Achillea
24
Fel/c/a ] Astereae
Dlmorphotheca ] Calenduleae

]
Plptocarpha
Stokesla Vernonleae
Vernonia
Gerbera ] Mutlsleae

]
30
98 Echlnops
Cardueae 2
Carthamnus
76 Gazanla ] Arctoteae
Cacosmla ] Llabeae
10D Lactuca
Tragopogon ] Lactuceae

Barnadesla ] Barnadesleae ] 1

Figure 8.14. Molecular phylogeny for the angiosperm family Asteraceae (after
Kim et at., 1992). The tree is a consensus parsimony network based on more than
750 cpDNA characters (restriction sites and rbeL mutations). Numbers indicate
levels of bootstrap support for putative clades.

It has not gone unnoticed (Rieseberg and Brunsfeld, 1992) that much of the
current interest in the molecular systematics of higher plant taxa is based on the
same molecule (cpDNA) that has been used to document reticulate evolution due
to interspecific hybridization and introgression between closely related plant
species (Chapter 7). Could these or other phenomena that can lead to discrep-
ancies between cpDNA gene trees and organismal trees also cause gross errors
in higher phylogeny estimation? Clegg and Zurawski (1992) suggest that this is
unlikely: "It is reasonable to assume that the approximation to organismal his-
tory will improve as time increases, because the biases introduced by interspe-
cific hybridization or intraspecific polymorphism will diminish with an increase
in time scale." Nonetheless, the organismal phylogenies estimated by cpDNA
(or any other single-gene genealogy) will require corroboration from additional
lines of evidence.

Slowly Evolving Gene Sequences and Deep Phylogenetic Branches

Phylogenetic analyses of nucleotide sequences have revolutionized the study of

organismal relationships, particularly among higher taxa representing the deepest
342 Species Phylogenies and Macroevolution

branchings in the tree of life. Particularly noteworthy have been the large number
of studies conducted on ribosomal RNA genes (examples in Table 8.4). The main
function ofrRNA is protein synthesis, so it is not surprising that the genomes of
all organisms contain sequences that code for these essential molecules. Analyses
of rRN A genes have been facilitated by the fact that portions of the coding region
evolve very slowly [some nucleotide stretches are conserved across all species
examined, from microbes to higher plants and animals (Gerbi, 1985; Jorgensen
and Cluster, 1988)], whereas others evolve somewhat more rapidly and provide
phylogenetic markers at intermediate evolutionary depths. The occurrence of
ribosomal RNA genes and other evolutionarily conserved loci across widely
different organisms has opened the possibility for development of what Wheelis
et ai. (1992) refer to as a "global classification" for all of life.
Before turning to studies at such global biotic dimensions, two examples (one
each from plants and animals) will be presented to illustrate the utility of rRNA
gene sequence analyses at "meso" -evolutionary levels. Hamby and Zimmer
(1992) phylogenetically analyzed sequences collected from the nuclear 18S and
26S ribosomal RNA genes of 60 diverse plant taxa. Among the many conclu-
sions tentatively reached concerning the evolution of vascular seed plants were
the following (Fig. 8.15): (a) the "flowering" plants (angiosperms) are mono-
phyletic; (b) the "naked-seed" plants (gymnosperms) are not monophyletic, but
rather exhibit a paraphyletic relationship to the angiosperms; (c) within an-
giosperms, the monocots do not form a strictly monophyletic group, due prima-
rily to the inclusion of water lilies (Nelumbo and Ceratophyllum) along a mono-
cot branch; and (d) dicots, as traditionally viewed, exhibit a paraphyletic
relationship to the major monocot lineage(s). The latter conclusion, supported
further by analyses of rbcL sequences from the cpDNA genome (Chase et al.,
1993), is important because it suggests that the morphological traits character-
izing most monocots as a markedly distinctive group are probably shared-derived
(synapomorphic) conditions, rather than ancestral features of plants.
Another finding of the Hamby and Zimmer (1992) study was that in the
shortest parsimony trees, the order Gnetales [genera Welwitschia, Gnetum and
Ephedra (Fig. 8.15)] appeared to be the earliest diverging group of seed plants,
and that the other gymnosperms (conifers, cycads, and Ginkgo) constituted a
sister group to the angiosperms. However, this result is not in accord either with
other molecular information [from rbcL sequences (Chase et aI., 1993)], or with
cladistic analyses of morphological data (Crane, 1985), both of which place the
Gnetales as the sister group to the angiosperms. This result again emphasizes the
importance of basing any final phylogenetic conclusions on multiple genes (as
well as other sources of evidence).
Hedges et al. (1990) published a similar phylogenetic analysis of the riboso-
mal RNA gene sequences for the nuclear 18S and 28S molecules in more than 25
species of tetrapod vertebrates, with a particular emphasis on the amphibians
Table 8.4. A small sample of stimulating initial suggestions based on phylogenetic analyses of various rRNA gene sequences. a

Taxonomic Group RNA Subunit Reference Authors' Conclusions

Bacteria 16S Fox et al., 1980 Two deep clades in prokaryotes (archaebacteria and
eubacteria)
PI. Jtists Nuclear 5S Kumazaki et al., 1983 Green algae share common ancestor with vascular plants
Plants Nuclear 5S Hori et al., 1985 Cycas is a gymnosperm; land plants related to
charophyte algae
All living forms 16S Woese, 1987 Three clades of life (archaebacteria, eubacteria,
eukaryotes)
All living forms 5S and nuclear 5S Hori and Osawa, 1987 Archaebacteria and eukaryotes split off after eubacteria
Protozoa and fungi Nuclear 16S Edman et al., 1988 Pneumocystis carinii is a fungus
Animals Nuclear 18S Field et al., 1988 Cnidarians are separate from other animal lineages.
Eukaryotes Nuclear 18S Nairn and Fed, 1988 Angiosperms are monophyletic
Bacteria 16S Lake, 1988, 1989 New data analyses suggest archaebacteria are
paraphyletic
Bacteria 16S,23S Gouy and Li, 1989 Further statistical analyses support Woese (1987) above
Eukaryotes Nuclear 18S Sogin et al., 1989 Fungi, plants, and animals diverged relatively recently
Vertebrates Nuclear 28S Hillis and Dixon, 1989 Coelacanths allied to tetrapods; birds, mammals perhaps
linked
Fishes Nuclear 18S Stock and Whitt, 1992 Cyclostome fishes (lampreys and hagfishes)
monophyletic
Archaebacteria 16S Fuhrman et al., 1992 Novel archaebacterial lineage discovered in marine
plankton
Source: After Hamby and Zimmer (1992).
"These stated conclusions should be viewed as working hypotheses in need of further testing using data from other loci, rRNA gene data from additional
taxa, and further statistical evaluations.
344 Species Phylogenies and Macroevolution

Glycine
Pisum
Drimys
Liquidambar
Petroselinum
Trochodendron
Illicium
Hedycarya
Platanus
Liriodendron
Magnolia dicots
Asimina
Chloranthus
Calycanthus
A risto/ochia
Saruma
Ranunculus t/)
Duchesnea
Spinacia
Stellaria
...EG,)
Sagitta ria
Echinodorus
a.
t/)
Najas
Potamogeton
0
Co/ocasia 0)
Pistia I:
Zea m
Tripsacum
Saccharum
Sorghum monocots
Oryza
Hordeum
Avena
Triticum
Arundinaria
Sabal
Hosta
Ceratophyllum
Nelumbo
Piper
Peperomia
Saururus
Cabomba dicots
Nymphaea
Nuphar
Barclaya
t/)
Pinus
Juniperus
Cryptomeria
Cycas
...E
G,)
Encephalartoa
Zamia
a.
t/)
Zamia 0
Ginkgo I:
Welwitschia
Gnetum E
Ephedra >-
0)
Ephedra
Equisetum
Psilotum ] outgroups
Figure 8.15. Estimate of a maximum parsimony tree for seed plants based on nuclear
rRNA gene sequences (after Hamby and Zimmer, 1992). The original paper should be
consulted for additional details and for alternative tree topologies, many of which were
nearly as parsimonious as the one shown here.
 Species Phylogenies and Macroevolution 345

Typhlonectidae
Discogiossidae
Pelobatidae
Ichthyophiidae
en
53
Amphiumldae c
Caecillaidae
m
.0
Caecilialdae
J:
100 Plethodontidae C.
87 Sirenidae E
Ambystomatidae m
5 Sooglossidae C)
Leptodactylidae c
Microhylldae >
Bufonidae
Hylidae
Pipidae
, . . . - - - - - - - t u rt I e
r------ crocodilian

-
100
57 snake] squamate
lizard reptiles
en
64 CI)

64
Passerifor mes ]
birds
o
Galliformes c

~::an 1
E
m
mammals
mouse
rabbit
L -_ _ _ _ _ _ _ _ coelacanth

Figure 8.16. Consensus parsimony tree for amphibian

families and other tetrapod vertebrates, with the coela-
canth fish as outgroup, based on nuclear 18S rRNA gene
sequences (after Hedges et aI., 1990). Numbers indicate
levels of bootstrap support for putative clades.

(Fig. 8.16). Among the major conclusions reached include the following: (a) the
living amphibians represent a statistically significant monophyletic group; (b) so
too do the Amniota (vertebrates with an extra embryonic membrane-the rep-
tiles, birds, and mammals); and (c) most surprisingly, a sister-group relationship
might exist between the birds and mammals. This latter suggestion by Hedges
and colleagues contradicts conventional wisdom that the closest living relatives
of birds are crocodiles, but nonetheless can be accommodated with molecular
data from several other loci and thus will warrant further serious consideration
(e.g., Hillis and Dixon, 1989; see discussion in Hedges et al., 1990).
The greatest impact of ribosomal RNA analyses in evolutionary biology has
been in stimulating phylogenetic thought about the primary lineages of life. From
346 Species Phylogenies and Macroevolution

Greek antiquity to recent times, a common notion was that living organisms
could be divided into two kingdoms, animals and plants. In this century, another
primary division recognized widely was between the prokaryotes (microorgan-
isms lacking a membrane-bound nucleus) and eukaryotes (organisms consisting
of cells with true nuclei). In 1959, Whittaker proposed the existence of five
kingdoms (animals, plants, fungi, unicellular eukaryotes, and prokaryotes), and
this view gained widespread acceptance in the 1960s.
A breakthrough occurred in 1977, when Woese, Wolfe, and their colleagues
conducted analyses of 16S rRNA gene sequences and concluded that all living
systems should be divided in a different fashion-along what appeared to be
distinct phylogenetic lines of descent (Fox et al., 1977; Woese and Fox, 1977).
An updated version of this scenario (Woese et aI., 1990), based on analyses of
additional sequences from rRNA genes (and other loci), proposed that all forms
of life should be classified into one of three "domains" above the rank of
kingdom: Eucarya, including all eukaryotes (i.e., the basic eukaryotic cell
stripped of any contribution from its organelles); Bacteria (previously called
"eubacteria" or typical bacteria); and Archaea (formerly Archaebacteria), which
includes methanogens and thermophilic forms (Fig. 8.17). The molecular dif-
ferences separating extant representatives of these lineages appear to be "of a
more profound nature than the differences that separate typical kingdoms, such
as animals and plants" (Woese et aI., 1990). Although there have been some
alternative interpretations of the deep-branching structure of life as revealed by
molecular data [see Day (1991), Lake (1991), and references therein], there is no
doubt that major phylogenetic lineages, previously unrecognized, exist among
prokaryotic life forms.
One question of special interest concerns where the root should be placed on
the Eucarya-Bacteria-Archaea phylogeny. This issue has been addressed from a
cladistic standpoint using phylogenetic analyses of the sequences of primordially
duplicated genes: those encoding elongation factors and ATP-ase subunits (Gog-
arten et aI., 1989; Iwabe et aI., 1989). From observed distributions of shared-
derived features post-dating the gene duplications, both studies concluded that
the Archaea and Eucarya constitute an evolutionary clade to the exclusion of the
Bacteria.
Some taxonomists disagree that the prokaryotes should be divided into two
distinct groupings (Bacteria and Archaea), on the grounds that differences in
levels of biological organization between prokaryotes and eukaryotes remain so
profound that continued taxonomic recognition of these latter groups is desirable
(Mayr, 1990). Woese et al. (1991) retort that Mayr's scheme would represent a
return to an "artificial" and "flawed" classification based not on phylogeny.
This debate raises a more general point. Should classifications reflect solely the
phylogenetic branching order of taxa (as cladists suggest) or should perceived
grades of organismal differentiation somehow be incorporated as well (as many
 Species Phylogenies and Macroevolution 347

_ THERMOPHILIC

c::::::> AEROBIC ARCHAEA

c::::::!l CHLOROPHYLL-BASED Halococcus
PHOTOSYNTHESIS

Thermoproleus

Homo
Xenopus
Zea
Oxylricha
Saccharomyces
Diclyoslelium Chloroflexus
Trypanosoma Bacillus
Euglena

EUCARYA mitochondrill

BACTERIA
Figure 8.17. An updated version of one of the first reconstructions of the
deep structure in the tree of life [inferred from sequences of the small
subunit rRNA genes (G.J. Olsen and C.R. Woese, unpublished)]. Note the
positions of chloroplast and mitochondrial genes, embedded deep within
the Bacteria rather than the Eucarya where currently they are housed.
Codings indicate the distributions of three physiological traits in the net-
work (thermophilic adaptations, aerobic metabolism, and chlorophyll-
based photosynthesis), as indicated by their occurrence within at least
some members of the respective groups.

others would argue)? This is a subjective issue (though nonetheless important

operationally), such that an answer depends on one's orientation about the nature
of information that a formal classification should convey.
In any event, the original studies by Woese and colleagues provided a positive
stimulus for much of the subsequent molecular work on the deep phylogenetic
structure of life. One conclusion from analyses of many additional taxa is the
astounding phylogenetic diversity among eukaryotic protists (Knoll, 1992; So-
gin, 1991). Thus, extremely long evolutionary branches (inferred, for example,
from 16S-like ribosomal gene sequences) lead separately to the extant entamoe-
bas, euglenids, trichomonads, microsporidians, diplomonads, slime molds, and
several other protistan groups that therefore must have had long and independent
evolutionary histories (probably over a billion years).
Regardless of how the new molecular-based phylogenetic appraisals of life
eventually are translated into a formal classification scheme, the branching dia-
grams themselves remain of considerable interest for the phylogenetic mapping
348 Species Phylogenies and Macroevolution

of organismal traits (Figs. 8.17 and 8.18). For example, against the phylogenetic
backdrop proposed by the Woese group, aerobic metabolism occurs in widely
separated lineages of Bacteria and Archaea, suggesting a polyphyletic origin for
this metabolic trait (provided that anaerobic metabolism was the ancestral con-
dition, as most researchers now believe). On the other hand, the capacity for
chlorophyll-based photosynthesis appears only in the Bacteria (and their chlo-
roplast relatives), suggesting a single evolutionary origin for this capability (Fig.
8.17). Within the Bacteria, sulphur oxidation appears in at least two divergent
lineages, suggesting that interpretation of this potential phylogenetic marker as a
global synapomorph probably would be invalid (Fig. 8.18).
One of the most remarkable discoveries from the phylogenetic mapping of
characters on a molecular backdrop concerns the evolutionary origins of cpDNA
and mtDNA. Two theories previously were advanced to account for the existence
of separate and distinctive nuclear and cytoplasmic genomes within eukaryotic
cells. One theory stipulated that organellar genomes had an autogenous origin
within eukaryotes, as fragments from the nuclear genome became incorporated
into membrane-encased mitochondria or chloroplasts which subsequently as-

c::::=J SULPHUR OXIDATION

boldface ANAEROBIC
PHOTOSYNTHESIS
Escherichia coli

Rhodopseudomonas acidophlla
:::..-=--- Rhodopseudomonas palustris
Rhodopseudomonas marina

~~~~~~~~R~h~O~d~os~p~;r~il/~u~m~SB~/~eX~;g~e:ne:s::~_
Caulobacter crescentus
Zea mays mitochondrion

RhodospJrlllum fubrum
Rhodosp;r;llum molischianum
Rhodospirillum fulvum
Rhodospirillum sollnarum

Thiobacillus acidaphflus

Figure 8.18. Phylogenetic tree for the a-subdivision of the purple Bacteria inferred
from sequences of the small subunit rRNA genes (from C.R. Woese and G.J. Olsen,
unpublished). Codings indicate the distributions of two physiological adaptations
(sulphur oxidation and anaerobic photosynthesis) that had contributed to a more
traditional taxonomy for these organisms.
 Species Phylogenies and Macroevolution 349

sumed a quasi-independent existence (see Cavalier-Smith, 1975; Uzzell and

Spolsky, 1981). The second theory stipulated that organellar genomes had ex-
ogenous origins, stemming from bacterial-like ancestors that invaded (or were
engulfed by) proto-eukaryotic host cells bearing precursors of the nuclear ge-
nome (Margulis, 1981). The latter "endosymbiont theory" has received com-
pelling support from mUltiple lines of molecular evidence (Gray, 1992).
Much of the early molecular evidence in favor of the endosymbiont theory
came from c-type cytochromes (Schwartz and Dayhoff, 1978) and rRNA gene
sequences. In terms of molecular phylogenetic affinities, the rRNA gene se-
quences of both mitochondria and chloroplasts appear allied to bacterial se-
quences rather than to the nuclear sequences of the Eucarya (Fig. 8. 17). It is now
accepted that most Eucarya really are genetic chimeras containing a mixture of
truly distinctive evolutionary lineages.
Recent molecular attention has focused on closer details of the phylogenetic
origins for cpDNA and mtDNA. From analyses of rRNA gene sequences, chlo-
roplast sequences appear allied most closely to those of the photosynthetic cy-
anobacteria (Giovannovi et aI., 1988), and mitochondrial sequences may derive
from the a-subdivision of the purple bacteria (Proteobacteria) (Cedergren et aI.,
1988; Yang et al., 1985). Further proposals for the phylogenetic roots of mtDNA
and cpDNA have been made by many authors (e.g., Bremer and Bremer, 1989;
Gray et al., 1989; Howe et aI., 1992; Kishino et aI., 1990; Lockhart et aI., 1992;
W. Martin et aI., 1992; Van den Eynde et al., 1988; Van de Peer et al., 1990;
Villanueva et aI., 1985). A recent reexamination of chloroplast origins using the
inferred amino acid sequences from several protein-coding cpDNA loci (psbA,
rbcL, rbcS, tufA and atpB) not only supported the hypothesis of a bacterial origin
for chloroplasts but also suggested that the extant plastids in various algal and
plant lineages probably trace to a single endosymbiotic event that was followed
by at least one instance of a secondary transfer of an rbcLS operon from a purple
bacterium into a plastid lineage (Morden et aI., 1992).
The latter study by Morden et ai. (1992) is especially noteworthy because, as
has been argued repeatedly, final phylogenetic conclusions should be based on
multiple lines of genetic evidence. A general difficulty in evaluating the deep-
branching structure among life forms has been that until recently, relatively few
other genetic characters had been monitored at these deep evolutionary levels.
Thus, searches for other ancient molecular markers have been initiated. Among
the loci identified thus far that have appeared most promising for the study of
deep-branching topologies are those encoding ATP synthase subunits (Recipon
et aI., 1992), elongation factors EF-2/EF-G (Cammarano et al., 1992), and RNA
polymerases (Piihler et al., 1989). Phylogenetic analyses of these sequences have
further supported the alliance of cpDNA and mtDNA with Bacteria rather than
Eucarya (Recipon et al., 1992), as well as confirmed the general distinctiveness
of the Archaea as originally proposed by Woese and colleagues.
350 Species Phylogenies and Macroevolution

PROSPECTUS FOR A GLOBAL PHYLOGENY

In the foreseeable future, it should be possible to assemble molecular (and

other) data into a grand phylogenetic encyclopedia-a universal Tapestry linking
all life forms. This endeavor should include estimates of relationship at all
phylogenetic levels, from intraspecific kinships to the most ancient of evolution-
ary separations. The hierarchical structure inherent in organismal phylogeny
carries at least two ramifications for such an enterprise. First, life's hierarchical
arrangement will facilitate information storage and retrieval. Sections of the
global Tapestry could be stored and referenced as a nested series of phylogenies
at increasingly greater evolutionary depths (as in Fig. 8.19). Second, although a
complete Tapestry would necessitate the generation of millions of microphylog-
eny estimates (one for each species, clearly an impossible empirical task), the
pyramidal structure of organismal relationships means that fewer and fewer such
summaries will be required at increasingly higher taxonomic levels. Thus, it
should be feasible to achieve a consensus phylogenetic picture of the major
trunks and intermediate branches of life, plus occasional snapshots of microev-
olutionary relationships within selected species or genera of special interest.
A molecular phylogeny for fungi spanning all hierarchical levels (Fig. 8.19)
provides a small illustration of the kind of information that a global Tapestry
might include. This example also serves to illustrate at least two complications
that will arise. One complication is that different molecules and assay procedures
will have to be employed at different levels of the hierarchy, due to the varying
windows of resolution provided. In this case, mtDNA restriction fragments were
employed for the microevolutionary comparisons, and rRNA gene sequences for
the meso- and macro-evolutionary trees. No single molecular approach normally
will apply to all levels simultaneously. A second complication is that the sepa-
ration times among branches, as well as the topologies of each tree, will be
desirable. Purely cladistic appraisals aimed at resolving branching topology are
important, particularly for issues such as phylogenetic character mapping, but
they risk a diversion of attention from the equally important issue of divergence
times. As is often the case, the mesophylogenetic and macrophylogenetic sum-
maries for fungi shown in Figure 8. 19 were based on procedures designed
primarily to recover correct branching topology, but knowledge of absolute
divergence times would enrich these phylogenetic pictures greatly.
As this book testifies, many scattered pieces of the grand Tapestry puzzle
already have been gathered through molecular methods, and preliminary at-
tempts have been made to assemble these elements into coherent summaries for
particular taxonomic groups (e.g., Dutta, 1986; Fernholm et al., 1989; Sibley
and Ahlquist, 1990; P.S. Soltis et al., 1990). In a way, molecular phylogenetic
explorers are at a stage of biotic description analogous to that of the European
naturalists (including Charles Darwin) in the 1800s, during the early global
explorations of a newly discovered world-at that time too, many new bits of
 macrophylogeny meso phylogeny micro phylogeny

filamentous 1"'"-------- Saccharomyces 0130

Ascomycetes cerevislae
F. nlva/e
Ascomycete
yeasts Neurospora crasse 0131
Basidiomycetes F. solanl
0132
Chytrldlomycetes N. haematococca

kingdom Anlmalla F. solanl 0133

N. haematococca
kingdom Plantae
F. solanl 0135
diatoms and
Chrysophytes F. decemcellulBre
0134
Oomycote F. culmorum

red algae G. zaBe

A·6
F. gramlnearum
cellular slime molds
fuJlkurol ....-----0136
plasmodial slime molds
F. monlllforme
to Bacteria and Archaaa L-----0137
Fusarium oxysporum

major groups of fungi filamentous Ascomycetes strains of

and other forms of life ( Fusarium and related genera) Fusarium oxysporum

Figure 8.19. Empirical example of a tiered phylogenetic assessment (for fungi) based on molecular data. Shown are relationships among
strains of the wilt fungus Fusarium oxysporum and their position within the broader phylogenetic hierarchy of life (compiled from
diagrams and information in Bruns et al., 1991; Gaudet et aI., 1989; and Jacobson and Gordon, 1990). For additional details about
molecular relationships among the major fungal groups (Basidiomycetes, other Ascomycetes, and Chytridiomycetes), see Bowman et al.
(1992).
352 Species Phylogenies and Macroevolution

biological infonnation of relevance to systematics were coming in rapidly and an

important challenge was to catalog, assemble, and interpret the findings.

SPECIAL TOPICS IN MOLECULAR PHYLOGENETICS

Horizontal Gene Transfer

Genetic transmission is overwhelmingly "vertical"; i.e., from parents to off-
spring. Indeed, if this were not the case, phylogeny would have little meaning;
and furthennore, evolutionary trees built from independent characters would
seldom exhibit the coherent and consistent structures that have proved to char-
acterize most well-studied taxonomic groups. Nonetheless, several reports of
lateral transfer of genetic elements across seemingly inviolable taxonomic
boundaries have appeared in recent years (Table 8.5), and most molecular evo-
lutionists now accept that such "horizontal" movement occasionally does take
place. Note that horizontal exchange between taxa should not be confused with
hybridization-mediated introgression (Chapter 7), which is merely a special case
of vertical genetic transmission. [However, some semantic difficulties can arise,
as, for example, in the use of the word "conjugation" to describe the physical
joining between distantly related organisms, such as that between bacteria and
yeast which is known to result in the intertaxon transfer of DNA via conjugative
plasmids (Heinemann and Sprague, 1989).]
The evidence for horizontal transmission usually comes from a gross incon-
sistency between the phylogenies inferred from different sets of molecular mark-
ers, or between a molecular marker and a traditionally accepted phylogeny
(M.W. Smith et al., 1992). One of the first reported instances of a horizontal
genetic transfer involved a supposed movement of the gene encoding copper-zinc
superoxide dismutase (SOD), nonnally found predominantly in higher eukary-
otes, from the ponyfish Leiognathus splendens to its symbiotic bacterium Pho-
tobacterium leiognathi (Bannister and Parker, 1985; Martin and Fridovich,
1981). The unusual phylogenetic occurrence ofthis fonn of SOD in a bacterium,
and the observation that its amino acid composition resembled more closely the
ponyfish SOD than it did known SODs from other sources, led to the horizontal-
transfer scenario. However, further research over a broader phylogenetic scope
has called this conclusion into serious question (Leunissen and de long, 1986;
Steffens et al., 1983), due to two additional findings: the discovery of Cu-Zn
SOD in other prokaryotes, and the lack of significant statistical support for the
supposed phylogenetic grouping of the SOD of P. leiognathi with eukaryotic
sequences [indeed, the P. leiognathi SOD now appears allied more closely to
other prokaryotic SODs (Smith and Doolittle, 1992a)]. A similar controversy has
centered on a glutamine synthetase II gene (gin/I), which occurs in eukaryotes
and in a small subset of bacteria (genera Rhizobium, Agrobacterium, and allies)
Table 8.5. Reported instances of horizontal gene transfer between different species and unrelated taxa. The first two cases in the list have
been disputed seriously (see text).a

Taxonomic Groups Involved

and Direction of Movement
Gene Supposedly Transferred (Where Postulated) Reference

Prokaryote/Eukaryote Gene Transfers

Copper/zinc superoxide dismutase From fish to bacterium Martin and Fridovich, 1981
Glutamine synthetase II From plant to bacterium Carlson and Chelm, 1986
Glyceraldehyde-3 phosphate dehydrogenase From unspecified eukaryote to bacterium Doolittle et a\., 1990
Fibronectin type III From animal to bacterium Bork and Doolittle, 1992
Glucose-6-phosphate isomerase From plant to bacterium Smith and Doolittle, 1992b
Plasmid-mediated transfers From bacterium to plant Zambryski et a\., 1989
Plasmid-mediated transfers From bacterium to yeast Heinemann and Sprague, 1989
Eukaryotic Transfers of Transposable Elements
"mariner" From Drosophila to Zaprionus flies Maruyama and Hartl, 1991
"P" From D. willistoni complex to D. melanogaster Daniels et al., 1990
"hobo" Between Drosophila species Simmons, 1992
"jockey" Between Drosophila species Mizrokhi and Mazo, 1990
"hobo" and others Between animals and plants Calvi et al., 1991
"copia" -like Between numerous plant, animal, Flavell, 1992
and yeast hosts

aFor additional examples of lateral transfer, see Heinemann (1991), Kidwell (1992), Mazodier and Davies (1991), M.W. Smith et al. (1992), and Sprague
(1991).
354 Species Phylogenies and Macroevolution

that are symbiotic with higher plants. Sequence similarities between the glnII
loci in these bacteria and plants originally were interpreted to evidence a hori-
zontal transfer between kingdoms (Carlson and Chelm, 1986), but subsequent
analysis of a broader array of glutamine synthetases failed to support the con-
tention that glnII was of plant origin or that gene transfers across large taxonomic
gaps were required to explain the data (Shatters and Kahn, 1989).
Regardless of their final resolution, these case histories raise several important
points. First, thorough phylogenetic analyses are required before putative cases
of horizontal transfer can be confirmed (or refuted). An important step in such
analyses should involve statistical tests of the significance of the discordant tree
structures [for example, by bootstrapping procedures (Lawrence and Hartl,
1992)]. Second, it should be emphasized that a variety of evolutionary factors
other than horizontal transfer, in principle, can lead to apparent phylogenetic
discordancies among characters (Fig. 8.20). These include the shared retention
of ancestral states by the taxa in question, extreme molecular rate heterogeneities
across lineages, convergent evolution to a shared molecular condition, introgres-
sive hybridization, and a mistaken assumption of orthology when the loci in
question might truly be paralogous (Fig. 1.3). Third, the possibility of horizontal
transfer of particular genes adds yet another compelling rationale for the desir-
ability of including multiple lines of evidence in phylogenetic reconstructions.
Notwithstanding these considerable obstacles to the firm documentation of
horizontal genetic transfer, several reported instances (Table 8.5) do appear
convincing (or at least have not as yet been challenged seriously). Perhaps the most
compelling case for the occurrence of horizontal transfer in any eukaryotic system
involves the "P element" in fruit flies (Daniels et al., 1990). These transposable
genetic elements (see below) have a patchy phylogenetic occurrence confined
mostly to the genus Drosophila and related dipteran genera including Lucilia
blowflies (Perkins and Howells, 1992). A remarkable genetic discovery was that
P-element sequences in D. melanogaster are nearly identical to those in D.
willistoni, despite a suspected evolutionary separation of these host species of
more than 50 million years (Daniels et al., 1990). Furthermore, close relatives of
D. melanogaster appear to lack P elements entirely, whereas the elements are
widespread in species of the D. willistoni complex; and, there is strong circum-
stantial evidence for the recent spread of P elements within D. melanogaster
(Kidwell, 1992). The overall conclusion is that D. melanogaster probably ac-
quired P elements recently (within the last half-century!) via a horizontal genetic
transfer from the D. willistoni complex. Recent evidence suggests that a semi-
parasitic mite (Proctolaelaps regalis) may have been the mediating vector (Houck
et al., 1991).
Phylogeny of Retroviruses and Transposable Elements
Many of the suspected instances of horizontal genetic transfer involve trans-
posable elements (TEs) (Table 8.5), a class of DNA sequences that may be
 Species Phylogenies and Macroevolution 355

1
species

character
in qUrSIiOn

9=
y X A

-1
true horizontal
y B
x transfer
x C

x A
x
C
-1
mistaken species
1) x [9 phylogeny
y
y []]
x A
x
C retention of
2)
--Y x
y
x
B
C
ancestral state

x x A
~ extreme rate
3)
-1 x
y
x
B
C
heterogeneity

x A
X
C
-1
convergent
4) y B
x evolution
x C

Lf
y x A
introgressive
5)
-1 x
y
x
B
C
hybridization

a, x x A
C mistaken paralogous
6)
-1 a, x
a
x
B
C
comparison

Figure 8.20. Six competing hypotheses to account for an apparent

character-state discordance that otherwise might be attributable to
a horizontal gene-transfer event.

predisposed to such movement due to a known ability to shift from one chro-
mosomal position to another within cells. Among the mobile eukaryotic ele-
ments, some TEs transpose by reverse transcription of an RNA intermediate
("class I" retrotransposable elements), and others by a DNA to DNA transpo-
sition mechanism ("class II" elements) (Finnegan, 1989). Most TEs have char-
acteristic structures that include genes coding the enzymes involved in the trans-
position process, usually flanked by terminal repeat sequences of varying length.
In the retrotransposable elements (RTEs), one of these genes encodes reverse
transcriptase (RT), which catalyzes the transcription of RNA to DNA.
356 Species Phylogenies and Macroevolution

Particularly intriguing are the biological and structural similarities between

retrotransposable elements and retroviruses (RVs). Retroviruses are small, sin-
gle-stranded RNA viruses found mostly in mammals that resemble RTEs in
several features, including the production of a reverse transcriptase and the
presence of long terminal repeats (LTRs) flanking the coding region. However,
like other viruses and unlike RTEs, retroviruses can encase themselves in a
protective envelope that facilitates independent infectious transport across the
cells of the same or different organisms. These observations raise an interesting
evolutionary question (Doolittle et al., 1989; Finnegan, 1983). Might RTEs
represent "degenerate" retroviruses that secondarily lost much of this facility for
autonomous intercellular transport? Or alternatively, did retroviruses evolve
from ancestral RTEs by secondary acquisition of these capabilities?
In addition to its presence in both RTEs and RVs, a reverse transcriptase gene
also is found in several other genetic elements, including the hepadnaviruses of
animals and the caulimoviruses of plants. The RT gene also exhibits structural
similarities (suggestive of shared ancestry) to the RNA-directed RNA poly-
merases of some other viruses. Xiong and Eickbush (1990) have taken advantage
of these facts to estimate a molecular phylogeny for RT-containing genetic
elements, based on nucleotide sequences from the RT and RNA polymerase
genes. Results from this analysis for 82 sequenced retroelements are summarized
in Figure 8.21. Major conclusions included the following: (a) there appear to be
two fundamental branches in retroelement phylogeny, one leading to the non-
LTR retrotransposons, and the other leading to the LTR retrotransposons, ret-
roviruses, caulimoviruses, and hepadnaviruses; (b) most members within each of
these five named assemblages group together in terms of RT phylogeny; and (c)
due to the restricted position of retroviruses in the broader scheme of RT-
containing elements, it seems likely that retroviruses evolved from retrotrans-
posable elements, rather than the converse.
This example is presented not to suggest that the questions concerning
RTE-RV evolution are solved to everyone's satisfaction, but rather to illustrate
yet another arena (in this case involving some of the smallest and simplest forms
of life) in which molecular phylogenetic appraisals are playing a novel and
important role in evolutionary studies.

Moleculor Paleontology

Most molecular appraisals have been directed toward extant organisms, with
phylogenetic inferences representing extrapolations to the mutational changes
and cladistic events of the past. A longstanding dream of molecular evolutionists
has been to assess extinct biota more directly, through recovery of biological
macromolecules from fossil material.
In 1980, Prager and co-workers reported a phylogenetic signal retained in the
 Species Phylogenies and Macroevolution 357

[C=-~J
other RNA
viruses

retro-
transposons

LTR
retrotransposons
II (copia-Tyl group)
retroviruses elements

II
LTR retrotransposons
(gypsy-Ty3 group)

Figure 8.21. Phylogeny for retroelements based on RT sequences (after Xiong and
Eickbush, 1990). Various structural features of the RT-containing elements are su-
perimposed on the estimated phylogeny. Shaded boxes correspond to regions of the
coding sequence: solid shading, RT region; stippled, gag gene region; diagonal shad-
ing, integrase region; cross-hatched shading, envelope gene. Unshaded boxes repre-
sent LTRs. The ancestral structure (in parentheses) is hypothesized.

serum albumin proteins of a 40,OOO-year-old mammoth (Mammuthus primige-

nius) whose carcass had been preserved in the frozen soil of eastern Siberia. In
immunological tests, rabbits injected with ground mammoth muscle produced
antibodies that reacted strongly with albumins from extant Indian and African
elephants, weakly with sea cows (Sirenia, a taxonomic order thought to contain
living relatives of elephants), and still more weakly or not at all with other
mammalian albumins. Using similar assays, Lowenstein et al. (1981) showed that
albumins from the extinct Tasmanian wolf (Thylacinus cynocephalus) produced
phylogenetic ally informative levels of immunological reaction against the albu-
mins of other extant Australian marsupials. The preserved tissue in this study was
dried muscle that had adhered to museum specimens collected in the late 19th and
early 20th centuries_ Apart from a few such examples involving fortuitously
well-preserved or recent tissue remains, most other attempts to extract significant
genetic information from fossil proteins met with little success (Hare, 1980;
Wyckoff, 1972).
Early studies of "ancient DNA" (PiUibo, 1989) fared somewhat better. In the
358 Species Phylogenies and Macroevolution

first successful retrieval of phylogenetically informative DNA sequences from

museum material, Higuchi et al. (1984, 1987) recovered short mtDNA segments
from a salt-preserved study skin (140 years old) of the quagga (Equus quagga),
an extinct member of the horse family. Fragments of DNA isolated from dried
muscle and connective tissue were cloned into a X. vector, and sequences totaling
229 base pairs were obtained and compared against those of extant relatives. These
and subsequent molecular studies (George and Ryder, 1986; Piiiibo and Wilson,
1988) helped to solve a phylogenetic enigma regarding the quagga, which in terms
of general morphology exhibited an odd mixture of horselike and zebralike
features. Phylogenetic analyses of the molecular data indicated that the extinct
quagga was related closely to the Burchell zebra, Equus burchelli (Fig. 8.22).
In 1985, a new record for the evolutionary age of recovered DNA was estab-
lished with the isolation and biological cloning of nuclear DNA pieces from an
Egyptian mummy 2400 years old (Piiiibo, 1985). The record did not last long.
One year later, a report appeared on the extraction of DNA from human brain
tissue, 8000 years old, that had been buried in a swamp in central Florida (Doran
et al., 1986). Notwithstanding these and a few other success stories, traditional
isolation procedures seldom yielded ancient DNA sequences in a sufficient state
of preservation to be of practical utility for phylogenetic comparisons.
Much excitement, therefore, has attended recent developments in the appli-
cation ofPCR-based methods to the recovery of ancient DNA (Piiiibo et al., 1989).
The amplification of DNA suitable for sequencing from exceedingly small
amounts of fossil template, or from well-preserved museum materials, promises
to revolutionize molecular paleontology. Indeed, during the years 1989-1991
alone, following the introduction of peR, more publications appeared on fossil
DNA than had accumulated over all prior decades (Brown, 1992).

quagga

1
Burchell
Grevy zebras
mountain

wild ass
half ass

domestic J horses
Przewalski i

8 4 2 o
percent sequence divergence

Figure 8.22. Phylogenetic tree based on mtDNA sequences

from the extinct quagga and extant members of the genus Equus
(after Piiiibo et aI., 1989).
 Species Phylogenies and Macroevolution 359

Studies based on PCR approaches reportedly have extended the known tem-
poral range of DNA preservation by more than lOOO-fold. At the time of this
writing, the record for age of fossil DNA from a plant belongs to Golenberg et
ai. (1990), who extracted, PCR-amplified, and sequenced an 820-bp fragment of
the chloroplast rbcL gene from fossil Magnolia leaves dating to the Miocene
(some 18 million years bp). Phylogenetic comparisons against homologous DNA
sequences of modern magnolias and other plants indicated that the characterized
molecules really did reflect fossil material, rather than more recent laboratory or
field contamination.
Among animal fossils, even older records were established with the recovery
of DNA from insects preserved in amber (tree sap): a 30 million-year-old termite
[Mastotermes electrodominicus (DeSalle et al., 1992)]; a 25-40 million-year-old
stingless bee [Proplebeia dominicana (Cano et aI., 1992)]; and a 130 million-
year-old weevil [undescribed species of Coleoptera (Cano et aI., 1993)]. In the
termite study, comparisons of ancient DNA fragments from two ribosomal RNA
genes (16S from the mitochondrion, 18S from the nucleus) against homologous
sequences from living termites, cockroaches, and mantids helped to confirm a
morphological cladistic assessment of these same taxa and substantiated the
suspected monophyly of this extinct species with an extant termite (M. darwi-
niensis) currently abundant in Australia. Apart from the impressive time scales
over which dead DNA fragments were shown to persist, these studies also are of
special interest because of the detailed phylogenetic comparisons made possible
between molecules and morphology. Unlike most fossils, amber-preserved spec-
imens are displayed beautifully in three dimensions, enabling detailed appraisals
of "soft" morphological features such as mouthparts and genitalia.
Several other impressive examples of phylogenetic comparisons involving
PCR amplifications of fossil DNA have appeared. For example, P. S. Soltis et al.
(1992a) isolated rbcL sequences (= 1300 bp long) from a Miocene species of
bald cypress (Taxodium) and demonstrated that the fossil taxon is related more
closely to T. distichum than to the other extant species in Taxodiaceae and
Pinaceae. In an extension of the fossil-protein study cited above, Thomas et ai.
(1989) examined mtDNA sequences from the extinct Tasmanian wolf and dem-
onstrated that Thylacinus cynocephalus was related more closely to other Aus-
tralian marsupials than it was to a group of South American carnivorous mar-
supials. lanczewski et al. (1992) characterized mitochondrial and nuclear
sequences from 14,OOO-year-old bones of the saber-toothed cat (Smilodonfata-
lis) from tar pits in Los Angeles, and thereby uncovered the phylogenetic posi-
tion of this extinct species within the Felidae radiation.

SUMMARY
1. Phylogenetic hypotheses underlie virtually all conclusions in compara-
tive organismal evolution. To make these hypotheses more explicit and testable,
"phylogenetic character mapping" has become popular, whereby particular or-
360 Species Phylogenies and Macroevolution

ganismal features are matched with their associated species on a cladogram, with
the purpose of revealing the evolutionary histories of those traits. The indepen-
dent appraisals of phylogeny often are based on molecular markers.
2. Phylogenetic mapping against a molecular backdrop has been accom-
plished for numerous anatomical, physiological, and behavioral characteristics in
plants, animals, and microbes. Some organismal features have proved to have
arisen monophyletically, others polyphyletically. Concepts of gradients and
thresholds in the phylogeny of quantitative traits have been stimulated by the
exercise of phylogenetic character mapping.
3. Molecular markers are used widely in biogeographic assessment, for
example to test dispersalist versus vicariance explanations for the appearance of
related taxa in disjunct geographic regions, to distinguish between common
ancestry and convergence for organismal similarities between regional biotas,
and to reconstruct the biogeographic histories of island biotas.
4. In macrophylogeny assessment, especially noteworthy have been broad-
scale molecular studies based on DNA-DNA hybridization, nucleotide sequenc-
ing of slowly-evolving ribosomal RNA and other genes, and restriction-site and
sequence analyses of animal mtDNA and plant cpDNA. From the latter two
cytoplasmic systems, phylogenetic markers may involve sequence information
itself, or eccentric molecular features (such as gene order, presence versus ab-
sence of particular introns, or patterns of codon assignment) that appear to have
special significance for clade delineation.
5. Horizontal gene transfer, whereby genetic information is exchanged
among unrelated taxa, has been well-documented in several instances. However,
discordance between a gene tree and a species phylogeny alone is not sufficient
to document a horizontal transfer event because several alternative scenarios also
can produce the discordance.
6. Molecular studies have been extended to some of the simplest forms of
life, including the quasi-independent retroviruses and transposable elements
which have proved to be allied phylogenetically.
7. With advent of the PCR, molecular phylogenetic appraisals have been
extended to "ancient" DNA, in some cases extracted successfully from fossils
up to tens of millions of years old.
8. In the near future, prospects are great for developing a molecular-based
global or universal phylogeny for all of life.
 9

Conservation Genetics

Modern biology has produced a genuinely new way of

looking at the world . . . to the degree that we come to
understand other organisms, we will place a greater
value on them, and on ourselves.

E.O. Wilson, 1984

In the final analysis, concerns about the conservation of biodiversity represent

concerns about the conservation of genetic diversity. As we have seen, this
genetic diversity is arranged hierarchically, from the family units, extended
kinships, and geographic population structures within species, to a graded scale
of genetic differences among reproductively isolated taxa that have been sepa-
rated phylogenetically for various lengths of evolutionary time. As we have also
seen, the visible external phenotypes of organisms are not an infallible guide to
how this genetic diversity is partitioned. Ironically, even as we gain the molec-
ular tools to assess genetic heterogeneity in new and exciting ways, the marvel-
ous biodiversity that has carpeted our planet is being lost at a pace that is nearly
unprecedented in the history of life, due to direct and indirect effects of explosive
human population growth (Ehrlich and Ehrlich, 1991).
One goal of conservation biology is to preserve genetic diversity. Another goal
should be to preserve evolutionary processes. Hybridization, introgression, and
speciation are examples of natural and dynamic evolutionary processes that exert
great influence on how genetic diversity is organized, yet these forces are some-
times viewed with considerable naivety in conservation legislation (such as that
designed to protect "endangered species"). Not only must societies find ways to

361
362 Conservation Genetics

preserve existing genetic diversity, but they also must seek sustainable environ-
ments for life in which the evolutionary processes fostering biotic diversity are
maintained.
How might studies in molecular phylogenetics contribute to the assessment of
natural genetic diversity and evolutionary processes in ways that are serviceable
to the field of conservation biology? Answers to this question are the subject of
this concluding chapter.

ISSUES OF HETEROZYGOSITY

Most discussions of genetics in conservation biology have focused on how

best to preserve variability within rare or threatened populations, a common
assumption being that higher heterozygosity (H, one measure of the within-
population component of genetic variation) enhances the probability of a popu-
lation's survival over ecological or evolutionary time (Chapter 2). Traditional
approaches to heterozygosity assessment and management have been indirect.
Management for heterozygosity in captive populations (such as in zoos) normally
occurs in de facto fashion through controlled breeding programs designed to
avoid intense inbreeding, either by maintenance of closed populations above
some "minimum viable population size," or, where feasible, by the exchange of
breeding individuals among sites. For natural populations, the analogous con-
cerns have been to ensure adequate habitat such that local effective population
sizes remain above levels where inbreeding (and its associated fitness depression)
might become pronounced and/or to maintain habitat corridors for purposes such
as facilitating gene flow among local populations (Hobbs, 1992; Simberloff and
Cox, 1987; however, see Simberloff et al., 1992 for a critical appraisal of such
programs).
With the advent of molecular techniques, more direct estimates of heterozy-
gosity have become possible through assays of multiple marker loci. From these
new data bases, two major conservation-related issues regarding heterozygosity
have arisen: Is molecular variability reduced significantly in rare or threatened
populations? If so, is this reduction a cause for concern about that population's
future?

Molecular Heterozygosity in Rare and Threatened Species

In the mid-1800s, indiscriminate commercial harvests of the northern elephant

seal (Mirounga angustirostris) reduced this formerly abundant species to dan-
gerously low levels. Fewer than 30 individuals may have survived through the
1890s (on a single remote island west of Baja California), but following legis-
lative protection by the Mexican and U.S. governments, the species has re-
 Conservation Genetics 363

bounded and now numbers in the tens of thousands of individuals distributed

among several rookeries. Bonnell and Selander (1974) surveyed 24 allozyme loci
in 159 seals from five rookeries and observed absolutely no genetic variation, a
striking finding given the high heterozygosity estimates reported for most other
species assayed by similar protein-electrophoretic methods (Chapter 2). Recent
molecular analyses by Hoelzel et al. (1993) have confirmed and extended these
findings: At a total of 55 allozyme loci, no variability has as yet been detected
in the northern elephant seal, and at the normally variable control region of
mtDNA, only two haplotypes (sequence difference about 1%) were found. These
results cannot be attributed to abnormally low genetic variation intrinsic to all
seals because the southern elephant seal M. Leonina also included in the assays
displayed normal heterozygosity levels.
During the past decade, the isolated and endangered population of gray wolves
(Canis Lupus) on Isle Royale in Lake Superior declined from about 50 to fewer
than 12 remaining individuals. Molecular studies have revealed that approxi-
mately 50% of allozyme heterozygosity was lost in this island population, rel-
ative to mainland samples (Wayne et aI., 1991b). Furthermore, only a single
mtDNA genotype was present. In terms of multilocus nuclear DNA fingerprints,
the island wolves appeared to be about as similar genetically as are known full
siblings in a captive wolf colony, suggesting that the Isle Royale population is
inbred severely (Wayne et aI., 1991b).
Hillis et al. (1991) employed allozyme markers to estimate genetic variability
in the Florida tree snail (Liguus jasciatus), many of whose populations are
threatened or already extinct. Among the 34 loci monitored in 60 individuals,
only one was polymorphic, and mean heterozygosity overall was only H =
0.002. Perhaps a population bottleneck accompanied or followed the coloniza-
tion of Florida by this species from Cuba; or perhaps the snail's habit of partial
self-fertilization plays a role in heterozygosity loss (at least within local popu-
lations). Surprisingly, the lack of appreciable variation at the molecular (allo-
zymic) level in this snail contrasts diametrically with the exuberant morpholog-
ical variability exhibited, particularly with regard to the beautiful variants in shell
pattern that are known to be genetically based.
Genetic variation in remnant populations of the Sonoran topminnow (Poecil-
iopsis occidentalis) in Arizona, where the species is endangered, has been com-
pared to that in populations from Sonora, Mexico, where the fish is widespread
and abundant. At 25 allozyme loci, the geographically peripheral populations in
Arizona exhibited significantly less variation than did the Mexican populations
near the center of the species' distribution (Vrijenboek et al., 1985). The mo-
lecular analysis also revealed three major genetic groups within the species'
range. The authors suggest that these groups should be maintained as discrete
entities in nature, because the majority of overall genetic diversity in this species
is attributable to the intergroup differences. They also recommend that any
364 Conservation Genetics

restocking efforts in Arizona employ local populations, whose mixing could

increase within-popUlation heterozygosity without compromising the genetic dif-
ferences that characterize the broader geographic assemblages.
Another endangered species assayed extensively for molecular genetic varia-
tion is the cheetah (Acinonyxjubatus). The South African subspecies of this large
cat first was surveyed for genetic variation at 47 allozyme loci, all of which
proved monomorphic, and at 155 abundant soluble proteins revealed by two-
dimensional gel electrophoresis, where heterozygosity also proved low [H =
0.013 (O'Brien et al" 1983)]. Subsequent assays of additional allozyme markers
and of RFLP variation at the MHC (major histocompatibility complex) supported
the contention that this population is genetically depauperate (O'Brien et al.,
1985b; Yuhki and O'Brien, 1990), a result further confirmed by the failure of
these cats to acutely reject skin grafts from "unrelated" conspecifics. Again, the
low genetic variation observed in cheetahs cannot be attributed to some inherent
property characteristic of all cats because most other species of Felidae exhibit
normal to high levels of genic heterozygosity in each of the above assays. On the
basis of the genetic evidence, O'Brien et al' (1987) proposed that at least two
population bottlenecks occurred in cheetahs, one perhaps 10,000 years ago prior
to geographic isolation of two recognized subspecies (which are highly similar
genetically) and a second within the last century that may have led to the ex-
ceptional genetic impoverishment of the South African form. However, recent
assays of rapidly-evolving molecular systems (mtDNA and VNTR nuclear loci)
have uncovered moderate genetic variation in cheetahs, of a magnitude that
Menotti-Raymond and O'Brien (1993) calculate could be due to postbottleneck
mutational recovery over a time scale of about 6,000 to 20,000 years.
A similar scenario about bottleneck effects has emerged regarding the Asiatic
lion (Panthera leo persica), which now occurs as a remnant population in the Gir
Forest Sanctuary in western India. Allozyme surveys (46-50 loci) detected
absolutely no variation in a sample of 28 individuals from this subspecies,
whereas the Serengeti population of the African subspecies had much higher
genetic variability (Wildt et al" 1987). The relict group of lions in the Gir forest
descends from a population that was contracted to less than 20 animals in the first
quarter of this century. The obvious interpretation is that the population reduc-
tion profoundly impacted genomic variation.
Table 9. 1 lists additional examples of molecular studies on rare or endangered
populations in which exceptionally low levels of molecular heterozygosity have
been reported. In most of these instances, the authors provisionally attributed
results to effects of genetic drift attending historical bottlenecks in population
size. On the other hand, it also must be emphasized that many rare or endangered
species have proved not to be unusually depauperate genetically. Examples of
such species with more or less normal molecular heterozygosity levels include
the following: a federally protected spring-dwelling fish (Gambusia nobilis) en-
Table 9.1. Additional examples of extreme low genetic variability within rare or endangered species as documented by multilocus
protein-electrophoretic methods.

Species Observation Reference

Plants
Bensoniella oregona Complete absence of allozyme variation (24 loci) within or among P.S. Soltis et al., 1992b
(Saxifragaeceae) populations of this endemic herbaceous perennial in southwest
Oregon and northwest California
Pedicularis furbishiae Complete absence of allozyme variation (22 loci) within or among Waller et al., 1987
(Scrophulariaceae) populations of this endangered hemiparasitic lousewort in
northern Maine
Howellia aquaticus Complete absence of allozyme variation (18 loci) within or among Lesica et al., 1988
(Campanulaceae) populations of this rare and endangered aquatic plant in the
Pacific Northwest
Trifolium rejlexum Complete absence of allozyme variation (14 loci) in the only Hickey et al., 1991
(Fabaceae) known population of this rare native clover in Ohio [however,
allozyme assays (20 loci) of an endangered congener T.
stoloniferum did reveal low to moderate levels of genetic
variation]
Animals
Bison bison Only one allozyme locus (among 24 tested) was polymorphic in a McClenaghan et al., 1990
bison herd in South Dakota known to be descended from a small
founder group
Perameles gunnii Complete absence of allozyme variation (27 loci) within an Sherwin et al., 1991
endangered, isolated population of the eastern barred bandicoot
in Australia (however, a widespread and dense population of the
same species in Tasmania also lacked genetic variation at these
same loci)
Mustela nigripes Only one allozyme locus (among 46 tested) was polymorphic in the O'Brien et al., 1989
one known remaining population of the highly endangered
black-footed ferret
Strix occidentalis Complete absence of allozyme variation (23 loci) in six populations Barrowclough and Gutierrez, 1990
of the endangered spotted owl from Oregon and California
366 Conservation Genetics

demic to the Chihuahuan desert (A.F. Echelle et al., 1989); some populations of
a critically endangered flightless parrot (Strigops habroptilus) native to New
Zealand (Triggs et al., 1989); most populations of the endangered red-cockaded
woodpecker (Picoides borealis) in the southeastern United States (Stangel et al.,
1992); Przewalski's horse (Equus przewalskii) , which is extinct in the wild but
survived by 600 animals in zoos (Bowling and Ryder, 1987); and the endangered
manatee (Trichechus manatus) in Florida (McClenaghan and O'Shea, 1988).
In theory, the demographic details of population bottlenecks (such as their
size, duration, and periodicity) should exert important influence on the severity
of the expected reductions in neutral genetic variability. For example, the loss in
mean heterozygosity can be rather small if population size increases rapidly
following a single bottleneck of short duration (Nei et al., 1975). An empirical
example of a severe population reduction that for suspected demographic reasons
has not resulted in low heterozygosity involves the endangered one-horned rhi-
noceros (Rhinoceros unicornis). Prior to the 15th century, perhaps half a million
of these rhinos ranged across a broad area from northwestern Burma to northern
Pakistan. Land-clearing and human settlement then began to fragment and de-
stroy rhino habitat, and by 1962 fewer than 80 animals remained, all in what is
now the Royal Chitwan Park in Nepal. A surprising finding was that this herd
exhibits among the highest allozyme heterozygosities reported for any verte-
brate, near 10% (Dinerstein and McCracken, 1990). One possibility is that the
loss of rhino habitat across the Indian subcontinent compressed surviving pop-
ulations into the Chitwan area, thereby concentrating into a single locale genetic
variation, some of which formerly had been distributed among regions. Whether
or not this scenario is correct, these findings are significant because they again
demonstrate that not all endangered taxa are genetic paupers.

Does Reduced Molecular Heterozygosity Matter?

The examples cited earlier indicate that genic heterozygosity, indeed, is re-
duced in popUlations of many (but not all) rare or threatened species. Do these
findings carry any special significance for conservation efforts? Although it is
tempting to assume that a paucity of genetic variation jeopardizes a species'
future, the goal of firmly documenting a causal link between molecular heterozy-
gosity and population viability remains elusive (Chapter 2). In general, there are
several reasons for exercising caution in interpreting the low molecular heterozy-
gosities reported for rare species: (a) most of the reductions in genetic variation
presumably have been the outcomes rather than the causes of population bottle-
necks; (b) at least a few widespread and successful species also appear to have
low heterozygosities, as estimated by the same molecular methods (e.g., Fig.
2.2); (c) in some endangered species such as the northern elephant seal, low
genetic variation appears not to have seriously inhibited population recovery
 Conservation Genetics 367

from dangerously low levels (at least to this point in time); and (d) the fitness cost
of inbreeding (Box 9.1) is known to differ widely among species, with some taxa
highly susceptible but others relatively immune to fitness depression accompa-
nying inbreeding (Laikre and Ryman, 1991; Price and Waser, 1979; Ralls et aI.,
1988).
An additional concern about interpreting the evolutionary significance of mo-
lecular variation is that published estimates based on any single class of markers
(such as allozymes) may inadequately characterize genome-wide heterozygosity
(Hedrick et aI., 1986), including the variability that may underlie morphological
or physiological traits of potential adaptive significance (e.g., recall the discus-
sion of the Florida tree snails). Carson (1990) has gone further to suggest that
"genetic variance available to natural selection may actually increase following
a single severe bottleneck" and, thus, "character change in adaptation and
speciation may, in some instances, be promoted by founder events." This con-
clusion stemmed from observations and experiments with bottlenecked popula-
tions of fruit flies and house flies.
For many of these and related reasons, Lande (1988) has argued that demo-
graphic and behavioral considerations should be of greater immediate importance
than genetic (heterozygosity) concerns in the formulation of conservation plans
for endangered populations. Lande emphasized that individuals in many species
show decreased reproduction at low population densities for nongenetic reasons,
such as lack of social interactions necessary for breeding, difficulties of finding
a mate, or other density-dependent ecological factors collectively known as the
"Allee effect" (Andrewartha and Birch, 1954). Furthermore, when populations
are few in number and small in size, the possibility of species extinction through
"stochastic" demographic fluctuations (irrespective of heterozygosity) may be
of paramount immediate concern (Gilpin and Soule, 1986).
On the other hand, several authors have argued forcefully that heterozygosity
as measured by molecular markers is highly relevant to a population's health and
continued survival probability and must be monitored accordingly in enlightened
management programs (e.g., O'Brien and Evermann, 1988; Quattro and Vrijen-
hoek, 1989). In a few case studies, plausible arguments been been advanced for
a direct association between observed molecular variability and the viability of
an endangered taxon. For example, in the Sonoran topminnow, four measures of
fitness (survival, growth, early fecundity, and developmental stability) were
monitored experimentally among laboratory-reared progeny of fish representing
natural populations that differed widely in heterozygosity levels as measured by
allozymes (Quattro and Vrijenhoek, 1989). The authors found that all four fitness
traits were correlated positively with mean heterozygosities in the populations
from which they stemmed. For the Isle Royale population of gray wolves,
Wayne et aI. (1991b) speculated that an observed behavioral difficulty in the
pair-bonding of adults might be due to a recognition-triggered instinct for incest-
Box 9.1. Inbreeding Depression
Inbreeding depression is the decrease in growth, survival, or fertility often observed
following matings among relatives. The phenomenon is of special concern in conser-
vation biology because individuals in small populations are likely to be inbred. Ge-
netically, inbred populations have increased homozygosity (reduced heterozygosity)
due to increased probabilities that individuals carry alleles that are "identical by
descent" (stem from the same ancestral copy) in earlier generations of the pedigree.
This probability for individuall is the inbreeding coefficient, which for known ped-
igrees can be calculated as:
F[ = I (1/2); (1 + FA)'
where the summation is over all possible paths through all common ancestors, i is the
number of individuals in each path, and A is the common ancestor in each path [for
computational details, see Ballou (1983) and Boyce (1983)].
Two competing hypotheses for the genetic basis of inbreeding depression have
been debated for decades (see Charlesworth and Charlesworth, 1987). Under the
"dominance" scenario, lowered fitness under inbreeding results from homozygosity
at particular loci for deleterious recessive alleles that in outbred populations normally
are masked in expression by their more common dominant counterparts. According to
the "overdominance" or "heterozygous advantage" explanation, genome-wide het-
erozygosity levels per se are the critical influences on fitness. These two hypotheses
make different predictions about the relative tolerance of populations to inbreeding
(Lacy, 1992). If the expression of deleterious recessive alleles is the cause of inbreed-
ing depression, then selection will have removed most such alleles from populations
that have long histories of inbreeding, and those populations should be resistant to
further inbreeding impacts. In other words, populations that survive severe inbreeding
may be "purged" of deleterious recessive alleles. Under this scenario, mean het-
erozygosity (as estimated for example by molecular markers) should have little pre-
dictive value of a population's genetic "health." However, if inbreeding depression
occurs because of a selective advantage to genome-wide heterozygosity, then previ-
ously inbred (and homozygous) populations would have reduced fitness and would
fare no better under future inbreeding than would highly heterozygous populations.
In any event, different populations exhibit widely varying fitness costs associated
with inbreeding. For example, in a survey of captive populations of a variety of
mammalian species, relative reductions in survival in crosses between first-degree
relatives (such as full sibs) varied across more than two orders of magnitude (Ralls et
al., 1988, as summarized by Hedrick and Miller, 1992):
Species Cost of Inbreeding
Sumatran tiger (Panthera tigris sumatrae) 0.003
Bush dog (Speothos venaticus) 0.06
Short bare-tailed opossum (Monodelphis domestica) 0.10
Gaur (Bos gaurus) 0.12
Pygmy hippopotamus (Choeropsis liberiensis) 0.33
Greater galago (Galago c. crassicaudatus) 0.34
Dorcas gazelle (Gazella dorcas) 0.37
Elephant shrew (Elephantulus refuscens) 0.41
Golden lion tamarin (Leontopithecus r. rosalia) 0.42
Brown lemur (Lemur fulvus) 0.90
 Conservation Genetics 369

avoidance, because the molecular findings suggest that as a result of inbreeding

the extant wolves are related about as closely as siblings. For the isolated Gir
forest population of Indian lions, O'Brien and Evermann (1988) concluded that
the high levels of abnormal spermatozoa plus diminished testosterone concen-
trations observed in males (relative to lions of the African Serengeti) were
attributable to intense inbreeding, because similar damaging effects on sperm
development have been observed upon inbreeding of mice and livestock.
Perhaps the most intriguing case for a causal link between inbreeding, low
molecular heterozygosity, and diminished popUlation fitness involves the cheetah.
As mentioned above, molecular evidence for severely reduced heterozygosity in
the cheetah is multifaceted and includes RFLP assays of the major histocompat-
ibility complex (a DNA region encoding cell-surface antigens involved in the
immune response). Extreme monomorphism at the MHC is indicated further by
the remarkable acceptance of skin grafts among "unrelated" individuals. Re-
cently, an infectious disease (feline infectious peritonitis or FIP) caused by a
coronavirus swept through several captive cheetah colonies and caused 50-60%
mortality over a three-year period. The same virus in domestic cats (which have
normal levels of MHC variation as indicated by graft rejections and molecular
assays) has an average morbidity of only 1%. O'Brien and Evermann (1988)
speculate that a FIP virus might have acclimated initially to one cheetah, and then
spread rapidly to other individuals who were genetically uniform in their immu-
nological defenses. In general, enhanced susceptibility to infectious diseases or
parasitic agents probably constitutes one of the most serious of challenges faced
by a genetically depauperate population (O'Brien and Evermann, 1988).
Interestingly, emphasis on a special adaptive significance for immunorecog-
nition genes has led to the suggestion that captive breeding programs for endan-
gered species should be designed with the' 'specific goal of maintaining diversity
at MHC . . . loci," due to the possibility "that an individual heterozygous at all
or most MHC loci will be protected against a wider variety of pathogens than a
homozygous individual" (Hughes, 1991). Furthermore, according to Hughes
(1991), "at most loci loss of diversity should not be a cause for concern, because
the vast majority of genetic polymorphisms are selectively neutral." This pro-
vocative suggestion immediately was criticized on several grounds (Gilpin and
Wills, 1991; Miller and Hedrick, 1991; Vrijenhoek and Leberg, 1991), not the
least of which is the gross assumption that variability at genes other than the
MHC is adaptively irrelevant. Several other loci are known to contribute to
disease resistance itself, and polygenes underlying numerous other quantitative
traits of potential adaptive relevance should hardly be ignored so cavalierly
(Vrijenhoek and Leberg, 1991). Furthermore, selective breeding designed ex-
plicitly to maintain MHC diversity could have the counterproductive conse-
quence of accelerating inbreeding, with concomitant accelerated loss of diversity
elsewhere in the genome.
370 Conservation Genetics

At this point, we are left with the conclusion that de facto management for
genic heterozygosity through avoidance of unnecessary inbreeding and mainte-
nance of large Ne probably is an important element in conservation programs for
rare or threatened species. With sufficient effort, molecular markers can provide
a useful index to genome-wide levels of genetic variation, particularly when
multiple assays are employed and concordant results are discovered among the
different methods. Thus, molecular approaches can help to identify those natural
or captive populations that might be at special risk of extinction due to severe
genetic impoverishment from past population bottlenecks and/or inbreeding.
Nonetheless, implementation of heterozygosity guidelines for managed popula-
tions should not come at the expense of equally important behavioral or demo-
graphic considerations. Furthermore, when popUlation histories are known (as is
often the case in zoos), direct pedigree analyses rather than use of particular
molecular markers should in theory provide a more robust guide to the true
magnitude of heterozygosity loss through inbreeding, as well as to the breeding
priorities of particular individuals for purposes of maximizing genetic variation
within a population (Haig et aI., 1990; Hedrick and Miller, 1992). For example,
two breeding strategies that have been suggested for captive populations whose
pedigrees are known involve the design of crosses to (a) equalize the expected
genetic contributions from the original founder individuals (Lacy, 1989) or (b)
emphasize reproduction by individuals who are judged to be of special genetic
importance by virtue of unusual position in the pedigree (Geyer et aI., 1989).
In the early literature of conservation genetics, Franklin (1980) and Soule
(1980) suggested that a minimum effective population size of 50 would be
required to stem inbreeding depression, and Franklin (1980) added that an ef-
fective population size of 500 would prevent the long-term erosion of variability
by genetic drift. These specific management guidelines (Franklin and Soule,
1981), which became known as the "50/500 rule," have had great influence in
the field of practical conservation science (Simberloff, 1988). Nonetheless, as
should be clear from the above discussion, no single set of guidelines is likely to
be valid universally. Furthermore, as pointed out by Varvio et al. (1986), such
detailed instructions represent gross simplifications based on several untested
assumptions, such that "any single principle should not be imposed as a general
guideline for the management of small populations."

ISSUES OF PHYLOGENY

Debate over the relative importance of demographic versus genetic concerns

in endangered species management (see above) has had at least two undesirable
consequences: (a) it has tended to dichotomize genetics and demography,
thereby placing them in an antagonistic relationship (when in reality both classes
 Conservation Genetics 371

of concern are important, and indeed may sometimes be intimately interrelated)

and (b) it has left the unfair impression that the only task for molecular genetics
in conservation biology is in heterozygosity assessment (see Avise, 1989c).
However, molecular markers can play many additional roles, involving phylog-
eny estimation at any of the hierarchical levels that have formed the organiza-
tional format for this book. The following sections provide examples that involve
rare or endangered species.

Parentage and Kinship

Parentage assignments sometimes assume conservation or management rele-

vance. For example, Longmire et al. (1992) used DNA fingerprinting to assess
paternity within a small captive flock of the endangered whooping crane (Grus
americana) that had been maintained at the Patuxent Wildlife Center in Mary-
land since 1965. A substantial investment is required to produce young cranes in
captivity because of the species' long generation length and low reproductive
output. In an attempt to maximize production of fertile eggs, adult females often
had been inseminated with semen from several males. However, this procedure
also made paternity uncertain, with the undesirable consequence that breeding
plans based on maximizing Ne (i.e., avoidance of inbreeding) were compro-
mised. The molecular genetic study partially rectified this situation by providing
a posteriori knowledge of biological parentage within the flock. In turn, such
pedigree information may prove useful in the design of subsequent matings.
Studies of parentage and kinship through molecular genetic markers often can
provide useful background information about the biologies and natural histories
of threatened species. The rare blue duck (Hymenolaimus malacorhynchos) of
New Zealand inhabits isolated mountain streams, and recent DNA-fingerprinting
assays have provided new insight into the family-unit structure of this species.
From patterns of DNA band-sharing, significantly higher genetic relatedness was
documented within than between blue duck populations from different rivers,
likely due to a social system that includes limited dispersal from the natal site and
matings among relatives (Triggs et al., 1992).
In some cases, molecular genetic findings on extended kinship have clear and
immediate relevance for conservation strategies. For example, analyses of
mtDNA have shown that rookeries of the endangered green turtle are character-
ized by distinctive maternal lineages, indicating a strong propensity for natal
homing by the females of this highly migratory species (Chapter 6). Irrespective
of the level of interrookery gene flow mediated by males, this structure of
rookeries along matrilines indicates that green turtle colonies must be considered
independent of one another demographically [because any extirpated colony
would unlikely be reestablished naturally by females hatched elsewhere, at least
over the ecological timescales relevant to human interests (Fig. 9.1)]. This
372 Conservation Genetics

genetics-based deduction is consistent also with the observation that rookeries

exterminated by man over the past four centuries (including those on Grand
Cayman, Bermuda, and Alto Velo) have yet to be recolonized. Because the
continuing decline of many marine turtle colonies through overharvesting will
not be ameliorated significantly by recruitment from foreign rookeries, each
desired remaining colony will have to be protected individually (Bowen et al.,
1992).

Population Structure and Intraspecific Phylogeny

The extended kinship structures of blue ducks and green turtles are a reminder
that issues of kinship grade into those of geographic population structure and
intraspecific phylogeny. At these levels too, many applications of molecular
markers have been employed in a conservation context. For example, particu-

grounds

(a) rookeries
demographically
independent

I
(b) rookeries
demographically
non-independent

adult feeding grounds

Figure 9.1. Alternative scenarios for the genetics and demography of green turtle
rookeries (after Meylan et al., 1990). Coded arrows indicate possible migration
pathways of females between natal and feeding grounds. (a) In the diagram above
the heavy horizontal line, females are assumed to home to natal sites, in which
case rookeries would be independent both genetically (with respect to mtDNA)
and demographically. (b) In the diagram below the horizontal line, females com-
monly exchange among rookeries, yielding genetic and demographic non-inde-
pendence. Molecular data from mtDNA are consistent with the former scenario.
 Conservation Genetics 373

lady in agronomically important plant species much effort has been devoted to
the collection and long-term storage of "seed banks" from which future genetic
withdrawals may prove invaluable in development of needed strains (Frankel and
Hawkes, 1975). In organizing the deposits to such germplasm collections, ge-
netic diversity may be maximized through knowledge of how natural variation is
partitioned within and among plant populations, a task for which molecular
genetic markers are well suited (Schoen and Brown, 1991). In general, by
revealing how genetic variation is partitioned within any plant or animal species
(Chapter 6), molecular methods can help to characterize the intraspecific genetic
resources that conservation biology seeks to preserve.

GENETIC PARTITIONS WITHIN RARE AND THREATENED SPECIES

Numbers of the highly endangered black rhinoceros (Diceros bicornis) and

white rhinoceros (Ceratotherium simum) in Africa have been decimated by
poachers, who supply the lucrative markets that convert rhino horns to orna-
mental dagger handles and supposed medicines. Assuming that poaching may
somehow be brought under control, additional genetic and demographic prob-
lems in the few remaining populations also could threaten these species' con-
tinued survival. Should all remaining conspecific rhinos be considered members
of a single population for purposes of breeding and management? Or should the
recognized extant forms (including the geographic subspecies D.b. michaeli and
D.b. minor in the black rhino, and C.m. cotton; and C.m. simum in the white
rhino) be managed as separate populational entities, perhaps because of strong
genetic differentiation? The strategy of mixing and breeding conspecific rhinos
from separate geographic sources might enhance each species' chance of survival
by increasing effective population sizes, and thereby forestalling stochastic de-
mographic extinctions and inbreeding depression. On the other hand, attempted
amalgamations of well-differentiated genetic stocks could lead to outbreeding
depression, or cause an overall erosion of genetic variability most of which might
be distributed among rather than within geographic regions. Toward the goal of
empirically assessing genetic differentiation among rhino populations, prelimi-
nary studies of proteins and DNA have been conducted. Merenlender et al.
(1989) reported a small genetic distance (D = 0.(05) between the two white
rhino subspecies based on allozymes, and Ashley et al. (1990) reported only low
levels of mtDNA sequence divergence (p < 0.004) for three black rhino popu-
lations based on restriction-site comparisons. However, due to the preliminary
nature of the data, the lack of obvious genetic differentiation, and the fact that
more extensive genetic surveys are underway, the available results should not be
taken to provide an unambiguous guide to management strategy.
The piping plover (Charadrius melodus) of North America has been the sub-
ject of recent molecular analyses based on allozymes. Breeding birds have all but
374 Conservation Genetics

disappeared from the Great Lakes region in the past 50 years, but threatened or
endangered populations persist in the interior Great Plains and along the Atlantic
seaboard. Low variances in inter-populational allele frequencies at polymorphic
loci (mean FST == 0.02), and low overall genetic distances (D < 0.01) indicate
that geographically disjunct populations nonetheless have been in extensive and
recent genetic contact (Haig and Oring, 1988). This finding may not be too
surprising, given direct dispersal data suggesting low natal philopatry for mem-
bers of this species, as well as a considerable mixing of birds from different
breeding regions on the wintering grounds. Allozymic studies of the rare wood
stork (Mycteria americana) in Florida reached similar conclusions. This species
nests in spatially discrete colonies, but negligible differentiation among rookeries
(mean FST = 0.02; D < 0.001) indicated that the colonies were connected by
high gene flow and/or have separated only recently (Stangel et al., 1990).
Molecular studies on several other rare or endangered species have revealed
significant popUlation genetic structures apparently resulting from behavioral
and/or historical geographic influences. The striking matriarchal population
structure of endangered green turtle rookeries within ocean basins, attributable at
least in part to natal homing, already has been mentioned, and a similar though
less pronounced behavior-based genetic architecture also characterizes logger-
head turtles (Caretta caretta) in the North Atlantic Ocean and Mediterranean Sea
(Bowen et aI., 1993b). In the desert tortoise (Xerobates agassizi) of the Amer-
ican southwest, a dramatic difference in mtDNA haplotype composition between
populations east versus west of the Colorado River presumably reflects a histor-
ical biogeographic separation of populations, as well as the limited vagility of
members of this species (Lamb et al., 1989).
In a complex of endangered desert pupfishes (genus Cyprinodon) in and near
Death Valley, California, allozymic studies have revealed little polymorphism
within but considerable genetic divergence among most populations, which cur-
rently are confined to isolated springs and streams that are the remnants of inland
lakes and connected watercourses in former pluvial times (Turner, 1974). A
different pattern of geographic structure within one of the desert pupfish species
may be an exception that proves the rule. In C. macularius popUlations of the
Salton Sea area of southern California, polymorphism within remnant colonies
accounted for 70% of the total genetic variance, with differences among colonies
contributing only 30% [Echelle's et al. (1987) analysis of Turner's (1983) al-
lozyme data]. The hydrologic history of this region suggests an explanation: The
popUlations probably have been in repeated contact due to historical cycles of
flooding of the lake basin, perhaps most recently earlier in this century when
water broke out of the Colorado River irrigation system (Turner, 1983).
In general, fishes in the desert basins of North America are declining at an
alarming rate, with more than 20 taxa having gone extinct in the last few decades
and many more at risk of the same fate. Meffe and Vrijenhoek (1988) have
 Conservation Genetics 375

considered management recommendations that might stem from two different

types of population structure exhibited by the remaining species, and these
scenarios should apply to other biological settings as well. In the "Death Valley
model" (patterned after the desert pupfish), populations are small and isolated,
such that most of the overall genetic diversity is likely to be partitioned among
sites. In managing such populations, there is no need for concern about human-
mediated interruptions of gene flow because genetic contact has not existed
naturally since the ancestral watercourses desiccated. On the contrary, precau-
tions should be taken to avoid gene flow among populations that are isolated
naturally and may, therefore, be in the process of adaptive radiation. The main
concern should be the maintenance of high Ne within localities to alleviate both
the genetic and demographic dangers of small population size.
Under an alternative "stream hierarchy model" (modeled after the endangered
Poeciliopsis occidentalis and certain members of the Cyprinodon complex),
populations in dendritic water systems exhibit varying degrees of connectedness
and gene flow, such that a larger fraction of the overall genetic diversity occurs
within colonies. Meffe and Vrijenhoek (1988) suggest that management pro-
grams in such situations should be aimed at preserving the genetic integrity of
each species while at the same time maintaining its genetic variability. Thus,
movement of conspecific individuals among colonies within a river basin should
pose no special difficulties, because this probably occurs naturally. However,
precautions should be taken to avoid artificial movements between separated
drainages, particularly when this involves mixing of different species that might
compete or hybridize.
Molecular studies of popUlation genetic structure also have been conducted on
endangered plants. For example, the meadowfoam Limnanthes floccosa califor-
nica is a geographically restricted annual endemic to vernal pools in Butte
County, California. The taxon is of special agronomic interest as a source for a
sperm whale oil substitute. Among the total of 11 known populations, protein
electrophoretic analyses revealed that nearly all (> 95%) of the total genetic
diversity was distributed among (rather than within) populations, such that es-
timates of interpopulational genetic exchange were extremely low (Nm < 0.02;
Dole and Sun, 1992). Based on these findings, the authors recommended a
conservation plan that emphasizes preservation of as many populations as pos-
sible, even at the possible expense of fewer total individuals.

STOCK IDENTIFICATION

Particularly in the field of fisheries management, much discussion has centered

on questions of how best to recognize and characterize population "stocks." To
develop quotas and other management guidelines for a sustainable fishery, bi-
ologists must be able to identify and demographically characterize the exploited
376 Conservation Genetics

populations. The need for such population information has become more critical
each year, as growing numbers of commercial and sport fisheries approach
economic collapse through overharvesting. Genetic stock assessment [GSA (Ut-
ter, 1991)] represents a practical application of the principles and procedures of
population structure analysis (Chapter 6). A large literature devoted explicitly to
GSA in fishery biology has been reviewed elsewhere (e.g., Ovenden, 1990;
Ryman and Utter, 1987; Shaklee, 1983), so only selected examples and general
conclusions will be presented here. --
Mixed-Stock Fisheries Many commercial or sport fisheries entail mix-
tures of native and introduced (hatchery-produced or otherwise transplanted)
stocks. In several instances, genetic markers that distinguish the introduced from
native populations have been employed to monitor the fate of the introductions
and to assess whether hybridization and introgression subsequently have taken
place. Results of such genetic appraisals have varied. Among trout (Salmo trutta)
in the Conwy River of North Wales, the introduction of fry from anadromous
populations into landlocked populations resulted in considerable hybridization
between the two trout forms, as gauged by allozyme markers (Hauser et aI.,
1991). Thus, a stocking program that had been designed to bolster catches of
trout in landlocked bodies of water may have come at the risk of introgressive
loss of the unique genetic character of the native landlocked forms. On the other
hand, for this same species in Spain, hatchery supplementation appears to have
been a "failure": Allozymic analyses showed that genetically marked fry intro-
duced from hatcheries into indigenous populations failed to reach sexual maturity
and apparently did not contribute to the pool of catchable and reproductive fish
(Moran et aI., 1991). In the western United States, a widespread practice of
stocking and transplanting non-indigenous trout has resulted in extensive intro-
gressive hybridization with native forms, as judged by allozyme and mtDNA
evidence (see section on hybridization and introgression later in this chapter). On
the other hand, similar molecular studies of the Japanese ayu (Plecoglossus
altivelis) demonstrated that introduced stocks contributed little to reproduction in
a native river population of this fish species (Pastene et aI., 1991).
Several fisheries involve exploitation of mixed native stocks, an example
being the taking of anadromous salmon at sea. A long-sought goal in salmon
management has been to find diagnostic genetic or other markers that would
cleanly distinguish fish stemming from different rivers, such that the proportion-
ate contribution of various breeding populations to the mixed oceanic fishery
could be determined. With such knowledge in hand, harvesting strategies might
then be geared to the varying strengths (population sizes) of the respective
breeding stocks. Unfortunately, native salmon populations within a management
jurisdiction seldom have proved to exhibit fixed allelic differences between rivers
that would make such stock assignments unambiguous. Rather, observed genetic
differences normally involve shifts (often statistically significant) in frequencies
 Conservation Genetics 377

of alleles at polymorphic loci, such that overall relationships among the source
populations must be summarized merely in a quantitative phenetic sense (Fig.
9.2). Several statistical approaches have been developed for estimating the pro-
portionate contributions to a mixed fishery from breeding stocks that differ in
frequency distributions of alleles or other measured characters (e.g., Millar,
1987; Pella and Milner, 1987).
Inherited Versus Acquired Markers An important challenge, faced par-
ticularly in the field of fisheries management, has been to distinguish carefully
between the kinds of information provided by different classes of populational
markers (Ihssen et al., 1981). One useful start at organizing such thought is to
make a distinction between inherited (genetic) versus "acquired" or "environ-
mentally-induced" characters (Booke, 1981). The latter include a variety of
attributes commonly employed in stock discrimination; for example, parasite
loads, heavy metal concentrations, and perhaps many morphological character-
istics such as vertebral counts that may be developmentally or phenotypically
plastic (T.P. Quinn et aI., 1987). They also include the physical tags and radio
transmitters applied to individuals to monitor their movements. Acquired mark-
ers unquestionably serve an important role in population analysis because they
can reveal where individuals have spent various portions of their lives. Further-
more, acquired and genetic markers often should disclose concordant population
partitions. For example, Bermingham et al. (1991) used molecular markers to
assay physically tagged salmon caught in the West Greenland fishery-among 68
tagged fish examined, 67 were correctly assigned by mtDNA genotype to the true
continent of origin (North America versus Europe). Nonetheless, because phys-
ical tags or other acquired characteristics are not transmitted across generations,
they do not ineluctably delineate the reproductive units that also are relevant to
population management programs, nor for this reason can they reveal the prin-
cipal sources of phylogeographic diversity within a species.
Figure 9.3 illustrates four possible relationships between the apparent stock
structures registered by genetic versus environmentally induced markers. These
two classes of marker may reveal high and concordant structures, as for example
when populations have been geographically isolated (leading to genetic differ-
ences) and the disjunct habitats induce different acquired phenotypes. Such an
outcome might be anticipated for isolated or sedentary species (e.g., freshwater
fishes in different drainages). Or, both genetic and acquired markers might reveal
relative homogeneity over broad areas, as in many vagile marine species. How-
ever, discordant outcomes also are conceivable, as when significant population
structure is evidenced by genetic markers despite the absence of populational
differences in acquired characteristics. This outcome might arise in a natally-
homing anadromous species assayed at the freshwater adult life stage, provided
that the acquired characters were incorporated during the oceanic portion of the
life cycle. Conversely, significant geographic structure in acquired characters
378 Conservation Genetics

Hood , - - - - - - - - - Hood Canal H

Canal ~ L....._ _ _ _ _ _ _ Nooksack'

r-------- Dungeness
L....._ _ _ _ _ _ _ Gray Wolf
1.-_ _ _ _ _ _ _ _ Hood Canal W

r------ Puyallup
. - - - - - - Snohomish
, - - - - Stiliaguamish
L.-_ _ _ Skagit

, - - - - Fraser
River 1.-_ _ Harrison

I __...J L- - - - Vedder
• 1.-_ _ _ Coqulhalla
, - - - - - - Thompson
L.-_ _ _ _ Seton

1.-_ _ _ _ _ Bridge

, - - - - - - Indian
L.-_ _ _ _ _ Skwawka

South ....-" 1-------- Phillips

Coast ' - - - - - - - - Quinsam

\ r------ Adam
' - - - - - - - Kakweiken
L-_ _ _ _ _ Wakeman
L---------Keogh
, . . - - - - - - - Bablne
~:::.-L _____ Andesite

0.12 0.08 0.04 0.00

genetic distance
Figure 9.2. Genetic relationships among populations of pink
salmon (Oncorhynchus gorbuscha) from 26 streams in Wash-
ington and British Columbia (after Shaklee et aI., 1991). Shown
is a cluster phenogram based on allelic frequencies at more than
50 allozyme loci. Indicated by arrows are five major geographic
regions for which reasonably strong genetic clustering is evi-
dent. The Nooksack River (indicated by asterisk), however,
enters Puget Sound.
 Conservation Genetics 379
example breedi ng area non-breeding population structure registered by
area
genetic markers acquired markers

(a) freshwater
fishes
'?~ ~~
lakes lakes
high high

~ ~

(b) vagile
oceanic fishes low low

(c) anadromous
fishes (e.g., * high low*
salmon)

(d) catadromous ~*
streams
fishes (e.g., ~ low high*
eels)
~

Figure 9.3. Four possible categories of relationship between magnitudes of

apparent population structure as registered by the distributions of inherited
versus environmentally acquired markers (see text). For categories (c) and (d)
in the figure, it is assumed that the environmental markers were acquired
during the oceanic and freshwater life stages, respectively, as indicated by the
asterisks.

might be evidenced despite an absence of significant genetic differentiation. This

outcome might arise in a random-mating catadromous species sampled during
the freshwater portion of its life cycle, provided that the acquired characters were
incorporated during the freshwater phase. In other words, because acquired
phenotypic markers by definition are appropriated through contact with the en-
vironment, they may not reflect true population genetic partitions when the
appropriations took place at a locality or life stage other than where reproduction
occurs.
These distinctions between genetic and acquired markers could in some sit-
uations assume management and conservation relevance. For example, regarding
anadromous salmon originating in different rivers, diagnostic populational mark-
ers that happened to coincide with political (state or federal) boundaries could be
ideal for equitably apportioning oceanic catches to fishermen from the relevant
jurisdictions. For these purposes, any marker (genetic or otherwise) would do.
Thus, naturally-acquired stream-specific parasite loads, and/or dyes or tags ar-
tificially applied to smolts Uuvenile salmon in streams), would be perfectly
suitable for these management purposes. Yet, the "stocks" so identified could
have little or no evolutionary genetic significance if, for example, sufficient gene
380 Conservation Genetics

flow between stream populations occurs via "mistakes" in natal homing. Thus,
in principle, an anadromous species with dramatic spatial structure in acquired
characteristics nonetheless might remain panmictic throughout its range. Both
inherited and acquired markers can find gainful employment for various man-
agement objectives, but only genetic markers are of immediate relevance to
population genetic and evolutionary issues.
Shallow Versus Deep Stock Structures Another general point about pop-
ulation stocks relevant to conservation biology involves the distinction empha-
sized in Chapter 6 between shallow (contemporary) versus deep (long-term his-
torical) population genetic structures. Populations of many species may be isolated
strongly from one another at the present time (and at most points of time in the
past), but nonetheless remain tightly connected in a phylogenetic sense through
recent or pulsed episodes of gene flow. Both shallow and deep evolutionary
separations are relevant to management strategies. The shallow genetic separa-
tions are important because they indicate that populations not strongly connected
by gene flow at the present time are unlikely to recover from overexploitation by
significant natural recruitment from other populational sources. Deeper genetic
separations within a species are significant also, because they register the major
sources of evolutionary genetic diversity that ultimately may be of greatest
importance to long-term conservation efforts. These new phylogeographic per-
spectives on population genetic stocks are just beginning to make an appearance
in the literature of fisheries management (Avise, 1987; Dizon et al., 1992).

CONCLUSIONS ABOUT INTRASPECIFIC PHYLOGEOGRAPHY

Ehrlich (1992) recently lamented that time is running out for saving biological
diversity and that "The sort of intensive, species-focused research that I and my
colleagues have carried out . . . [on checkerspot butterflies] . . . appears to have
a very limited future in conservation biology. Instead, if a substantial portion of
remaining biodiversity is to be conserved, detailed studies of single species must
be replaced with "quick and dirty" methods of evaluating entire ecosystems,
designing reserves to protect them, and determining whether those reserves are
working." Willers (1992) goes further by cautioning that "To dwell endlessly on
the tasks of obtaining more and ever more data for the expressed purpose of
managing a biological reserve is to suggest that enough knowledge is just around
the corner. This is not so." In a sense, these authors are quite right. The severe
and broad problems faced by conservation biology cannot be solved solely by
detailed genetic and ecological studies of particular taxa or regions. Indeed, the
lack of political and social will to implement existing scientific understanding
(including the need for human population control) is certainly a far greater
impediment to the preservation of global biodiversity than is a lack of detailed
scientific information per se.
 Conservation Genetics 381

On the other hand, there are some important and general lessons to be gained
from detailed case histories of the sort described in this chapter. To emphasize
this point, consider again the genetic studies conducted over the past decade on
the phylogeography of the freshwater and maritime faunas of the southeastern
United States (Chapter 6). Several previously underappreciated perspectives of
general conservation relevance have flowed from these and related studies based
on molecular markers (Avise, 1992). Thus, it has become abundantly clear that
most species should not be viewed as undifferentiated monotypic entities, but
rather as consisting of a series of geographically varying populations with a
hierarchical and sometimes deep genetic structure. With recognition of this fact
come several general management guidelines for conservation of this phylogeo-
graphic diversity:
Limit Unnecessary Transplantations Although most biologists recog-
nize that introductions of exotic species can cause irreparable harm to regional
biodiversity by forcing extinctions of native species, they have been slower to
appreciate the problems that can stem from transplantation and mixing of genetic
stocks within species. In fact, many public and private management agencies
actively promote geographic transplantations from one area to another within a
species' range for purposes such as bolstering local population sizes, introducing
"desirable" genetic traits into a region, or increasing local heterozygosity. Un-
fortunately, several undesirable consequences also may arise from such geo-
graphic transplantations, including the possibility of disease or parasite spread,
the irretrievable loss of the rich historical genetic records of populations, and
inevitable erosion of the overall genetic diversity within a species (much of
which we now recognize to be generated and maintained through historical
geographic separations). Transplantations sometimes may be justified, as in the
reintroduction of a native species to its former range where it had been extirpated
by humans. However, a developing perspective is that the burden of proof in any
proposed transplantation program normally should rest on advocates of this
strategy rather than on those who would question the desirability of transplan-
tations on the grounds cited above.
Design Regional Reserves Molecular data on the fauna of the southeast-
ern United States indicate that particular areas are geographic centers for a
substantial fraction of regional, intraspecific biogenetic diversity. These biotic
provinces also have been recognized previously by a different type of biogeo-
graphic data-concentrations of species' distributional limits (see Avise et al.,
1987a). Such concordant lines of evidence for historical centers of biodiversity
already give ample support for special efforts to preserve the integrity of these
regional biotas. Such efforts might include the design of regional biological
preserves, as well as the implementation of strict guidelines to discourage un-
necessary transplantations between phylogeographic areas. Although such re-
gional perspectives on genetic diversity cannot hope to capture the idiosyncratic
382 Conservation Genetics

population structures and subdivisions of all species, they can provide useful
general guidelines for management strategies, particularly as natural environ-
ments come under increased pressure and decisions of conservation triage be-
come inevitable.

Speciation and Conservation Biology

Just as issues of close kinship grade into those of population structure and
intraspecific phylogeny, so too do the latter grade into issues regarding the
speciation process and associated taxonomic judgements. In the field of conser-
vation biology, these taxonomic issues often come into especially sharp focus in
discussions of "endangered species" (Box 9.2).

RECOGNITION OF ENDANGERED SPECIES

Many taxonomic assignments in use today (including the formal designations

of species and subspecies) were first proposed in the last century, often from
limited phenotypic information and preliminary assessments of geographic vari-
ation. How adequately do these traditional taxonomies summarize true bioge-
netic diversity? This question remains to be answered for most groups, through
continued systematic reappraisals to which molecular approaches can contribute
significantly. The problems and challenges are far more important than a mere
concern with nomenclature might at first imply. For better or for worse, nomen-
clatural assignments inevitably shape our most fundamental perceptions of how
the biological world is organized. Ineluctably, formal names summarize the
biotic units that are perceived and, therefore, discussed. In a conservation con-
text, these perceived entities provide the pool of candidates from which are
chosen the populations toward which special management efforts may be di-
rected. A "dusky seaside sparrow" by any other name is just as melanistic, but
without a taxonomic name this population would not likely have been recognized
as a biotic unit worthy of particular conservation attention.
In the case of the dusky seaside sparrow, this dark-plumaged population
endemic to Brevard County, Florida first was described in the late 1800s as a
species (Ammodramus nigrescens) distinct from other seaside sparrows (A. mar-
itimus) common along the Atlantic and Gulf coasts of North America. Although
the dusky later was demoted to formal subspecific status (A.m. nigrescens), the
nomenclatural legacy stemming from the original taxonomic description prompted
continued special focus on this federally "endangered species" when, during the
1960s, the Brevard County population declined severely due to deterioration of
its salt marsh habitat. In 1987, the last known dusky died in captivity, as last-ditch
efforts to save the population through captive breeding failed.
Following extinction of the dusky seaside sparrow, a retrospective molecular
study of nearly the entire seaside sparrow complex (which includes nine con-
 Conservation Genetics 383

Box 9.2. The United States Endangered Spedes Act of 1973

In 1966, passage of the Endangered Species Preservation Act (P.L. 89-669, Sections
1-3) initiated federal efforts in the United States to protect rare species from extinc-
tion. Three years later, attempts to remedy perceived deficiencies in this act (e.g., lack
of habitat protection and applicability only to native wildlife) resulted in the Endan-
gered Species Conservation Act of 1969 (P.L. 91-135, 83 Stat. 275). Again, short-
comings of the new act were recognized and, in 1973, Congress enacted a more
comprehensive "Endangered Species Act" (henceforth ESA) that today remains the
country's strongest conservation strategy for rare species.
The ESA was intended to "provide a means whereby the ecosystems upon which
endangered species and threatened species depend may be conserved, [and] to provide
a program for the conservation of such . . . species. . . ." (16 U .S.C. Section
1531[b)). To qualify for ESA protection, a species must appear on an official list,
which is prepared and updated under auspices of the U.S. Fish and Wildlife Service
(Interior Department) and the National Marine Fisheries Service (Commerce Depart-
ment). Regulations and official interpretations of the ESA are made by these agencies
as well, who receive legal advice from the Solicitor's Office and the Office of General
Council within the Departments of Interior and Commerce, respectively.
Listings under the ESA may be made for species, subspecies, or "distinct popula-
tion segments. " For example, grizzly bears in the lower 48 states are listed as threat-
ened, whereas the large Alaska population receives no protection under the ESA.
Criteria for listing are that the plant or animal in question occurs in numbers or habitats
sufficiently depleted to critically threaten survival. The current lists include over 600
species within U.S. boundaries and 500 species that occur elsewhere in the world.
Another 600 + species are acknowledged by federal agencies to be just as vulnerable
but have yet to be listed, and at least 3000 more remain on a waiting list pending
additional investigation (Gibbons, 1992).
The ESA has been criticized on numerous grounds (e.g., Geist, 1992; O'Brien and
Mayr, 1991; Rohlf, 1991), including (a) its focus on the preservation of particular
species rather than on ecosystem health or biodiversity, (b) its emphasis on a few
high-profile species such as birds and large mammals, (c) the lack of clear guidelines
as to what constitutes a distinct species or population, or precisely what constitutes
being threatened or endangered, and (d) the considerable timelag involved in the
formal listing process. On the other hand, some of these problems appear to be those
of implementation (including funding, political will, and incomplete scientific knowl-
edge) rather than the legislation itself (O'Connell, 1992). In the future, the species-
based approach of the ESA may well be replaced or supplemented with legislation
designed more explicitly to recognize and encompass the broader goals of ecosystem
maintenance and biodiversity protection. But at least as an interim measure, the ESA
has represented a revolutionary and enlightened piece of governmental legislation.
384 Conservation Genetics

ventionally recognized subspecies) produced a surprise (Avise and Nelson,

1989). In tenns of mtDNA sequence, the dusky proved essentially indistinguish-
able from other Atlantic coast genotypes, whereas all Atlantic coast genotypes
clearly were distinct phylogenetically from those observed along the Gulf of
Mexico coastline (Figs . 6.8 and 9.4). These results likely are attributable to an
ancient (Pleistocene) population separation, as evidenced further by a striking
similarity between the phylogeographic pattern of the seaside sparrow and those
of several other estuarine species complexes in the southeastern United States
(Chapter 6). Thus, the traditional taxonomy for the seaside sparrow complex
(upon which the endangered species status and the management efforts were
based) apparently had failed to capture the true phylogenetic partitions within the
group, in two respects: (a) by giving special emphasis to a presumed biotic
partition that was shallow or nonexistent and (b) by failing to recognize a deep
phylogeographic subdivision (between Atlantic and Gulf coast birds) that should
prove most important in any future conservation plans for remaining members of
the seaside sparrow assemblage.
Because of the special political and emotional sensitivities surrounding the
dusky seaside sparrow (or other well-publicized endangered species), an addi-
tional note should be provided here. The molecular findings in no way excuse or
justify any poor land-management practices [chronicled by Walters (1992)] that
may have led to the dusky's extinction, nor should publication of the molecular
results be interpreted as heartlessness over this population's loss. The extinction
of any natural population is regrettable, particularly in this age of rapid deteri-

>- 0.6
()
seaside sparrow
compa ri sons
c:
Q) betw een the Atla ntic
compar i sons
:::J and Gulf reg ions

-
C" 0.4 w ithin either th e
Q)
Atlantic or Gulf
'-
reg i ons

- /
Q)
> 0.2
(0
Q)
'- 0
0 0.2 0 .4 0.6 0.8 1.0 1.2 1.4
sequence divergence (%)
Figure 9.4. Frequency distribution of genetic distances among seaside sparrows. Shown
are estimates of mtDNA divergence between pairs of birds from locales along the Atlantic
and Gulf of Mexico coasts of the southeastern United States.
 Conservation Genetics 385

oration of natural habitats and biodiversity. However, the field of conservation

biology finds itself in a difficult situation, with hard choices to be made about
which taxa can be saved, and how best to allocate limited resources among
competing conservation needs. Especially in such circumstances, management
decisions should be based on the best available scientific information.
Another example of a misleading taxonomy for an "endangered species"
involved the pocket gopher Geomys colonus endemic to Camden County, Geor-
gia. This taxon was first described in 1898 on the basis of rather cursory de-
scriptions of pelage and cranial characteristics. The population remained essen-
tially unnoticed and unstudied until the 1960s, when gophers in Camden County
were "rediscovered." The population referable to G. colonus then consisted of
less than 100 individuals and was listed as an endangered species by the State of
Georgia. Subsequently, a molecular genetic survey was conducted using allo-
zyme assays, karyotypic examination, and RFLP analyses of mtDNA. None of
these genetic methods detected any consistent genetic distinction between "G.
colonus" and geographically adjacent populations of a common congener, G.
pine tis (Laerm et al., 1982). Results were not attributable to a lack of sensitivity
in the techniques employed because each molecular method revealed dramatic
genetic differences among a broader geographic array of G. pinetis populations
(particularly those in eastern versus western portions of the species' range; e.g.,
Fig. 6.6). The conclusion in this case was clear: "G. colonus" did not warrant
recognition as a distinct species. Either its description in 1898 was inappropriate,
or an original valid G. colonus species had gone extinct early in this century and
been replaced by recent G. pinetis immigrants into Camden County.
Of course, in principle as well as practice, no study can prove the null hy-
pothesis that genetic differences between putative taxa are absent. One example
of a "valid" species that appeared nearly indistinguishable in genetic composi-
tion from a close relative involved the endangered pallid sturgeon (Scaphirhyn-
chus albus). In allozyme assays of 37 monomorphic and polymorphic loci, S.
albus could not be separated from its more common congener, the shovelnose
sturgeon S. platorynchus (Phelps and Allendorf, 1983). Nevertheless, on the
basis of pronounced morphological differences between these taxa and a sym-
patric distribution, the pallid and shovelnose sturgeons appear to qualify as
"good" biological species [the possibility of a phenotypic polymorphism was
deemed unlikely (Phelps and Allendorf, 1983)]. The molecular results suggest
either some degree of introgressive hybridization between these sturgeons or that
a complete separation was accomplished only recently in evolution. In general,
molecular data should not be interpreted in isolation in making taxonorr.ic judg-
ments, but rather should be integrated with other available lines of evidence.
Molecular reappraisals of taxonomically suspect species may, of course, also
bolster the rationale for special conservation efforts. One case in point involves
the nearly extinct silvery minnow (Hybognathus amarus) endemic to the Rio
386 Conservation Genetics

Grande River in the southwestern United States. This species has had a troubled
taxonomic history, with some researchers viewing it as a distinct species and
others placing it in synonomy with H. nuchalis and H. placitus. However, based
on an allozyme survey of 22 loci, Cook et al. (1992) observed several fixed
allelic differences between these taxa, and overall levels of genetic distance (D
> 0.10) somewhat greater than those normally distinguishing conspecific pop-
ulations in other fish groups. The authors concluded that there is little justifica-
tion for considering H. amarus conspecific with other species with which it
previously had been placed in synonomy.
Another case in point involves the highly endangered Kemp's ridley turtle
(Lepidochelys kempi), which nests almost exclusively at a single locale in the
western Gulf of Mexico, and currently is the subject of the largest international
preservation effort for any marine turtle. Morphological similarities between L.
kempi and the related L. olivacea, and a geographic distribution of these sup-
posed sister taxa that makes little sense under modern conditions of climate and
geography (L. olivacea is distributed globally in warm waters), have raised
questions about the true evolutionary distinctiveness of L. kempi. Nonetheless, a
reappraisal of the complex based on mtDNA assays indicated that L. kempi is
more distinct from L. olivacea than are assayed Atlantic versus Pacific popula-
tions of the latter from one another (Bowen et aI., 1991). Furthermore, the
differentiation between the Kemp's and olive ridleys surpassed (slightly) levels
of genetic divergence observed among any conspecific populations of the glo-
bally distributed green or loggerhead turtles (Fig. 9.5).
Neglected taxonomies for endangered forms also can kill, as exemplified by
studies of the tuataras of New Zealand. These impressive lizards have been
treated by government and management authorities as belonging to a single
species, despite molecular (and morphological) evidence for three distinctive and
taxonomically described groups (Daugherty et aI., 1990). Official neglect ofthis
genetic diversity may unwittingly have consigned one form of tuatara ("Sphe-
nodon punctatus reischeki") to extinction, whereas another form ("S.
guntheri' ') appears to have survived to this point only by sheer good fortune. As
noted by Daugherty et al. (1990), good taxonomies "are not irrelevant abstrac-
tions, but the essential foundations of conservation practice."

MOLECULAR FORENSICS AND LAW ENFORCEMENT

A common problem in field enforcement of wildlife laws involves identifying

the species of origin of blood, carcasses, or meat when more obvious morpho-
logical characters such as feathers or fur are not available. Because of the
species-diagnostic power of several molecular methods (Chapter 7), assays of
proteins or DNA recovered from problematic tissues should find wide application
in wildlife forensics. For example, Cronin et al. (1991c) compiled a list of
 Conservation Genetics 387

maximum p maximum p
among global among global

loggerheads greens

~/
III

II
]
'C
Atlantic ca
CI)

...
J:
CI)
m
Pacific m
o

olive

J Kemp's ...

5 4 3 2 1 0
sequence divergence (p ,%)
Figure 9.5. Phylogeny for ridley and loggerhead marine turtles estimated from
mtDNA data (after Bowen et aI., 1991). Assayed olive ridleys from the Atlantic
and Pacific Oceans proved indistinguishable and, hence, are plotted here as a
single OTU. Also shown are the maximum levels of mtDNA genetic distance
observed among loggerhead turtles (Caretta caretta) and green turtles (Chelonia
mydas), respectively, from around the world.

diagnostic molecular characters (mtDNA digestion profiles in this case) for 22

species of large mammals that are the frequent objects of illegal poaching. These
molecular markers provide a potential means for law enforcement agencies to
identify meat or other tissues confiscated from illegal sources.
What follows are a few examples of law enforcement applications in molec-
ular wildlife forensics. In 1978, a Japanese trawler in U.S. coastal waters was
suspected of illegal harvest of a rockfish species Sebastes alutus. Tissues con-
fiscated by U. S. enforcement officers indeed proved upon protein-electrophoretic
examination to have come from S. alutus, thereby contradicting claims in the
trawler's log that no such specimens had been taken (Utter, 1991). A similar case
in Texas involved a suspected illegal sale of flathead catfish (Pylodictis olivaris)
and related species. Protein-electrophoretic analyses verified the species' identity
388 Conservation Genetics

of the frozen fish fillets and led to a fine levied against the seller (Harvey, 1990).
Findings in molecular forensics also can exonerate the falsely accused. In an-
other case in Texas, electrophoretic analyses of confiscated fillets revealed that
fishermen were innocent of the charge of illegal possession of red drum (Sci-
aenops ocellatus) and spotted sea trout (Cynoscion nebulosus) (Harvey, 1990).
Some forensic applications in law enforcement involve distinguishing conspe-
cific populations from different areas. For example, a commercial catch of king
crab (Paralithodes camtschatica) that was claimed to have been harvested in an
area of Alaska open to fishing proved upon protein-electrophoretic examination
to have come from a closed area northwest in the Bering Sea (Seeb et al., 1990).
A particularly interesting example of a molecular forensic case in a geographic
context involved the largemouth bass (Micropterus salmoides). Bass-fishing
tournaments in the southern United States are "big business," with first-place
cash awards often in excess of $50,000. In one recent tournament in Texas, the
apparent winner was suspected of having smuggled in a large bass from outside
the tournament site. Tissue samples of the prize-winning fish were examined
electrophoretically and shown to have come from the genetically distinctive
Florida subspecies of the largemouth bass (see Philipp et al., 1981), thereby
confirming that the trophy specimen had been imported illegally. Based on these
genetic findings, the fisherman was charged and subsequently found guilty of
fraud (Harvey, 1990).
In 1989, an organized and official approach to wildlife forensics became a
reality with the opening of a U.S. Fish and Wildlife Service laboratory in Ash-
ford, Oregon. The first of its kind in the world, this "Scotland Yard for animals"
conducts forensic investigations on wildlife products that have been confiscated
from professional poachers, overzealous sportsmen, illegal traders in wildlife
products, or even uninformed tourists merely wanting a souvenir from some
endangered species. The forensics facility is divided into three laboratory sec-
tions, Morphology, Criminalistics, and Serology, the latter devoted to the iden-
tification of samples by DNA and protein evidence. The task is daunting: Unlike
the 360 police crime labs in the United States which deal with a single species
(Homo sapiens), the Ashland facility must cope with the remainder of the bio-
logical world.
Hybridization and Introgression
In the context of rare and endangered species, both biological and legal issues
(Box 9.3) have arisen concerning instances of introgressive hybridization that
have been documented using molecular markers.
BIOLOGICAL ISSUES

One evolutionary threat to a rare species is genetic swamping through exten-

sive hybridization with related taxa. An empirical example involves the nearly
 Conservation Genetics 389

Box 9.3. The Hybrid PoBey of the Endangered Species Act

In a series of opinions issued by the Solicitor's office beginning in 1977, it was

concluded that natural or artificial hybrids occurring between endangered species,
subspecies, or populations should not receive protection under the Endangered Species
Act (ESA). The rationale appears to be that even if such hybrids were themselves to
breed, they would not produce purebreds of either parental form and, hence, would not
promote the purposes of the ESA. These decisions have become known as the "Hybrid
Policy. "
The Hybrid Policy has several serious ramifications. For example, it has prompted
formal petitions from various land-use constituencies to remove certain protected taxa
from the endangered species list, on the grounds that hybridization and introgression
have compromised the genetic integrity of the otherwise endangered forms. But as
noted by Grant and Grant (1992), (a) many if not most species of plants and animals
hybridize at least occasionally in nature, so that if hybridizing species fall outside the
limits of protection afforded by the ESA, few candidate taxa will ever qualify for
protection and (b) "if rarity increases the chances of interbreeding with a related
species, presumably because conspecific mates are scarce, then the species most in
need of protection, by virtue of their rarity, are the ones most likely to lose it under
current practice, by hybridizing."
O'Brien and Mayr (1991) have attacked the Hybrid Policy on additional grounds,
claiming that definitional and related operational difficulties "have led to confusion,
conflict, and, we believe, certain misinterpretations of the [Endangered Species] Act
by well-intentioned government officials." They conclude that whereas the Hybrid
Policy should discourage management programs based on hybridization between spe-
cies, it should not be applied to subspecies or populations because the latter retain the
potential to interbreed freely as part of ongoing natural processes. With regard to three
case histories involving hybridization of endangered taxa (described in the text),
O'Brien and Mayr (1991) conclude:
(a) the status of "endangered" should be retained for extant red wolves
because they are the only available descendants of a historic wolf sub-
species;
(b) there is no justification for eliminating protection of the gray wolf as a
species, because hybridization with the coyote is limited to a narrow zone
that developed recently;
(c) the Florida panther should receive continued protection because it clearly
qualifies as a subspecies.
390 Conservation Genetics

extinct mahogany tree Cercocarpus traskiae, an endemic to Santa Catalina Is-

land in Los Angeles County, California. Allozymic (and morphological) ap-
praisals of the seven remaining adults of this species revealed that some were, in
fact, the products of hybridization between C. traskiae and other more abundant
Cercocarpus species on the island (Rieseberg et al., 1989). However, several
seedlings were found that did exhibit "pure" C. traskiae genotypes. These
genetic discoveries led to two management suggestions intended to lower the
probability of further hybridization (Rieseberg et al., 1989): (a) eliminate indi-
viduals of other Cercocarpus species from near the remaining pure C. traskiae
specimens and (b) transplant seedlings and established cuttings from nonhybrid
individuals to more remote areas on Santa Catalina Island.
The cutthroat trout native to the western United States and Canada is a com-
plex of approximately 15 recognized subspecies, many of which are threatened
by human alteration of habitat and by artificial introductions of non-native trout
species. Eleven subspecies currently have protected legal status, and two already
may be extinct. Surveys of allozymes and mtDNA in scores of cutthroat popu-
lations have revealed a complex phylogeographic pattern, with some subspecies
virtually identical genetically and others as distinct as normal congeneric species
(Allendorf and Leary, 1988, and references therein). With regard to the current
discussion, these molecular markers also have documented extensive hybridiza-
tion between the native and introduced trout species, as well as between trans-
planted cutthroat forms. For example, introgression from rainbow trout was
observed in 7 of 39 assayed populations in Utah (Martin et al., 1985); in Mon-
tana, 40% of the 80 populations formerly thought to be pure "westslope" cut-
throats proved upon molecular examination to include products of hybridization
with either rainbow trout or the "Yellowstone" cutthroat form. In the Flathead
River drainage in Montana (considered one of the last remaining strongholds of
native westslope cutthroat trout), only 2 of 19 headwater lakes sampled con-
tained pure populations and detailed genetic analyses further revealed that the
hybridized headwater populations were "leaking" foreign genes into down-
stream areas (Allendorf and Leary, 1988). Such findings on the introgressed
structure of western cutthroats have led to two conservation-related concerns
(Allendorf and Leary, 1988). First, hybrids between genetically differentiated
trout forms often appear to exhibit reduced fitness due to developmental abnor-
malities. Second, extensive introgressive hybridization carries the danger of
genetic swamping and loss of locally adapted populations. As stated by Allen-
dorf and Leary (1988), "The eventual outcome of widespread introgression and
continued introduction of hatchery rainbow trout is the homogenization of west-
ern North American trout into a single taxon (Salrno ubiquiti?). Thus, we would
exchange all of the diversity within and between many separate lineages, pro-
duced by millions of years of evolution ... for a single new mongrel species."
Some endangered species currently recognized may themselves be the prod-
 Conservation Genetics 391

ucts of past hybridization and introgression. One example perhaps involves the
red wolf (Canis rufus), which fonnerly ranged throughout the southeastern
United States but declined precipitously in numbers after 1900 and became
extinct in the wild in about 1975. Molecular analyses of remaining captive
animals (as well as museum-preserved skins and blood samples from extinct wild
populations) revealed that extant red wolves likely contain some genetic material
derived through hybridization with coyotes (C. latrans), but are otherwise ex-
tremely similar to gray wolves (C. lupis) [Wayne, 1992; Wayne and Jenks, 1991;
but see also Ferrell et al. (1980), Nowak (1992), and Dowling et al. (1992) for
alternative interpretations of the data]. It is uncertain whether this hypothesized
introgression of coyote genes into red wolf populations would have occurred
naturally in ancient times and/or more recently following a range expansion of
coyote populations (facilitated by the human clearing of eastern forests). In any
event, on the basis of these genetic findings (as well as questions concerning the
distinctiveness of red wolves from gray wolves), much controversy has arisen
over taxonomy of the red wolf (Phillips and Henry, 1992) as well as the appro-
priateness of the intensive management program (Gittleman and Pimm, 1991),
which has included a call for reintroduction of this species into the wild.

LEGAL ISSUES

The "Hybrid Policy" of the Endangered Species Act [ESA-Box 9.3] has
been the focal point of several legal and ethical controversies surrounding the
topic of hybridization and introgression in conservation biology. The sentiment
of this policy, which denies fonnal protection for organisms of hybrid ancestry,
has served as a basis for calling into question several existing endangered species
designations and associated management programs. The red wolf situation de-
scribed above provides one example. So too does the gray wolf, which also
appears to have hybridized with the coyote. Across a small portion of the gray
wolf's range in the northern United States and southern Canada, the presence of
"coyote-type" mtDNA genotypes in populations that otherwise appear wolflike
has led to the conclusion that male wolves occasionally hybridized with female
coyotes in this area (Lehman et al., 1991) (Fig. 9.6). This genetics-based con-
clusion, when interpreted against the philosophical platfonn of the Hybrid Pol-
icy, in turn has prompted at least one petition to the Interior Department to have
the gray wolf removed from its status as an endangered species in the northern
United States. [This petition was, however, turned down by the U.S. Fish and
Wildlife Service (Fergus, 1991).]
Another example of a political controversy in conservation genetics involves
the Florida panther (Felis concolor coryi), a severely threatened relict population
of puma or mountain lion whose historic range included much of the southeastern
United States. The small remaining population occurs only in the Big Cypress
392 Conservation Genetics

]
Wl
W2 ~Q)
-"0
W3
W4
oCO
~-
W5 : (J
W6 *
W7
W8*
C24-W13 *
C23
C5
C2l
mtDNA Cll
Cl
lineages C18-W12*
C16
C13
C14-Wl0 * .! Q)
C15 0"0
C6
>0 co
0-
C2 (J (J
C4
C19

C7
C20
C12
C22

4 2 0
sequence divergence (0/0)
Figure 9.6. Phylogeny for mtDNA genotypes observed in
gray wolves and coyotes from North America (after Lehman
et aI., 1991). Note that whereas several genotypes observed
in wolves (W) group separately from those of coyotes (C),
others (indicated by asterisks) do not, these exceptions sup-
posedly being attributable to recent hybridizations between
wolf males and coyote females that led to introgression of
coyote mtDNA across the species boundary. This scenario
recently was bolstered by the discovery that mtDNA geno-
types in gray wolves from the Old World belong clearly to
the "wolf lineage" (Wayne et aI., 1992).
 Conservation Genetics 393

Swamp and the Everglades of extreme southern Florida. A molecular genetic

evaluation of this and other puma subspecies based on nuclear and mtDNA
markers indicated the existence of two distinct genetic stocks in the area
(O'Brien et al., 1990)--one confined to Big Cypress that appears to be a bona
fide descendant of the F.e. coryi lineage, and a second found largely in the
Everglades that appears to be descended from pumas that evolved in South or
Central America but probably were released (by man) into south Florida in this
century. Importantly, some individuals also appeared to be the products of hy-
bridization between these distinct genetic stocks. Thus, a strict interpretation of
the Hybrid Policy conceivably could result in removal of the Florida panther
from federal protection under the ESA.
Clearly, for several reasons including those mentioned in Box 9.3, the mere
documentation of hybridization involving an endangered population should not
alone be sufficient grounds for removal from the endangered or threatened spe-
cies lists. An additional consideration that may be especially relevant to the
Florida panther is the following. Most of the panthers in southern Florida appear
to be inbred severely, presumably as a result of the pronounced population
reduction. The evidence includes increased frequencies of several abnormal traits
that often are associated with known inbreeding, e.g., deformed tail vertebrae
(causing a 90° kink near the tip of the puma's tail), a "cowlick" whorl on the
animal's back, cryptorchism in males (a condition in which one testicle fails to
descend into the scrotum), and 90% defective sperm. Perhaps the infusion of
puma genes from South America into F.c. coryi, inadvertent though it was,
actually may increase the fitness and survival probabilities of the southern flor-
ida population (O'Brien et al., 1990).

Species Phylogenies and Macroevolution

Beyond the taxonomic level of species, phylogenetic considerations can also

be of relevance to conservation biology. Such interest may be partly academic.
For example, what is the ancestry and geographic origin of the endangered
Hawaiian goose (Nesochen sandvicensis)? Conventionally, this morphologically
distinctive species has been placed in a separate genus, with curiosity centering
on its phylogenetic and geographic roots. Recent comparisons based on mtDNA
restriction sites and sequences revealed that the Hawaiian goose is allied more
closely to the Canada goose (Branta canadensis) than it is to other extant can-
didate species-the black brant (Branta bernic/a) or the emperor goose (Chen
canagica) (Quinn et aI., 1991). From this molecular evidence on maternal lin-
eages, it was concluded that the Hawaiian goose's ancestors colonized the is-
lands from North America about 0.9 mya.
The black-footed ferret (Mustela nigripes) of the North American plains is a
highly endangered member of Mustelidae (weasel and skunk family). An abun-
394 Conservation Genetics

dant species with broad distribution in the last century, the black-footed ferret
was decimated primarily through human eradication of its principal prey base
and associate, the prairie dog (Cynonys sp.). In 1981, a few remaining specimens
of M. nigripes were discovered in Wyoming, and molecular analyses showed
that this small population was characterized by extremely low genetic variability
(Table 9.1), probably as a result of the severe population bottlenecks. The
black-footed ferret has more common relatives elsewhere, however, including
the presumed sister taxon M. eversmanni (steppe polecat) of Siberia. O'Brien et
al. (1989) employed allozyme assays to assess the phylogenetic position of M.
nigripes within the Mustelidae (Fig. 9.7). The molecular data confirmed that M.
eversmanni is the closest living relative of the black-footed ferret and suggested
that these sister taxa are only about as differentiated genetically (D == 0.08) as are
closely related congeners in other mammalian groups (Fig. 1.2). These two
species likely separated about 0.5-2.0 my a (O'Brien et al., 1989).
Most of the seven to eight species of marine turtles conventionally recognized
are listed as threatened or endangered in the official "Red Data Book" of the
IUCN (International Union for the Conservation of Nature and Natural Re-
sources). Several phylogenetic issues ranging from population-level distinctions
to deeper evolutionary alliances among species, genera, and families have

Eocene Oligocene "'Iocene Pliocene. -+- Pleis10cene

Mustela nigripes

Mustela eversmanni

Mustela putorius
Mustela vison

Mephitis mephitis

Spi/ogale putorius

Ictonyx striatus

Ursus americanus

0.8 0.6 0.4 0.2 0

genetic distance (Nei's D)
Figure 9.7. Phylogenetic position of the endangered black-footed ferret (Mustela nigripes)
within the Mustelidae, based on allozymic comparisons (after O'Brien et aI., 1989). Other
species assayed were the Siberian polecat (M. eversmanni), European polecat (M. puto-
rius), mink (M. vison), striped skunk (Mephitis mephitis), spotted skunk (Spilogale puto-
rius), African striped skunk (Ictonyx striatus), and as outgroup the American black bear
(Ursidae: Ursus americanus).
 Conservation Genetics 395

plagued the systematics of these reptilian mariners, with consequences some-

times extending to conservation priorities. Case histories involving the green and
ridley turtles already have been described (Figs. 9.1 and 9.5). Other troublesome
questions have included the following: Is the east Pacific black turtle (Chelonia
agassizO specifically distinct from the green turtle (C. mydas)? Is the spongiv-
orous hawksbill turtle (Eretmochelys imbricata) allied phylogenetically to the
herbivorous green turtle, or alternatively to the carnivorous loggerhead turtle
(Caretta caretta)? Is the Australian ftatback turtle (Natator depressa) allied
closely to greens (as its earlier placement within Chelonia suggested), or might
it be a relative of the loggerhead turtle? Are all marine turtles including the
distinctive leatherback (Dermochelys coriacea) of monophyletic origin? Not all
of these questions have as yet been answered definitively, but a molecular start
has been made using mtDNA restriction sites and nucleotide sequences from the
cytochrome b gene (Fig. 9.8). Based on these matrilineal markers, preliminary
answers to the above questions are as follows: The black turtle falls well within
the range of genetic differentiation exhibited among green turtle rookeries world-
wide; the hawksbill turtle is related more closely to the loggerhead complex than
to green turtles and, hence, probably evolved from a carnivorous rather than
herbivorous ancestor; the ftatback is highly distinct from both the loggerhead and
green complexes and is roughly equidistant to both; and, finally, both the leath-
erback and the freshwater snapping turtle (Chelydra serpentina) exhibit large
genetic distances from all other marine turtles, such that altogether an unresolved
phylogenetic trichotomy exists under the available molecular evidence (Bowen et
aI., 1992, 1993a).
Such phylogenetic appraisals of endangered (or other) assemblages are of
relevance to conservation biology in the general sense that they provide an
understanding of the evolutionary relationships among species and higher taxa.
Should the ramifications go farther and perhaps include phylogenetic position as
a guide to prioritizing taxa with regard to conservation value? For example, does
the sole living representative of an ancient and phylogenetically unique lineage
(e. g., the leatherback turtle) warrant greater conservation concern than another
species (e.g., the Kemp's ridley turtle) that belongs to a recent and more speciose
clade? Some authors have answered this humbling question in the affirmative, on
the grounds that species which are most distinctive in a phylogenetic sense make
a disproportionately large contribution to the world's extant evolutionary diver-
sity (Vane-Wright et al., 1991). On the other hand, each biological species is
unique, and currently independent from all others genetically. Furthermore,
every species alive today traces back through an unbroken chain of ancestry over
thousands of millions of years, irrespective of how many speciation events have
intervened along its phylogenetic journey. How can societies deign to play God
with any creatures of such evolutionary fortitude? The mere fact that such issues
of preservation priority must be raised represents a sad and shameful commen-
396 Conservation Genetics

) Lepidochelys olivacea

Lepldochelys kempi

) Caretta caretta

Eretmochelys imbricata

Natator depressus

Chelonia agassiz;

Chelonia mydas

1
Derm 0 chelys coriacea

Chelydra serpentina

20 15 10 5 o
sequence divergence (%)
Figure 9.8. Phylogeny for all recognized species of marine turtles
(plus the freshwater snapping turtle C. serpentina) estimated using
sequence data from the mtDNA cytochrome b gene (after Bowen
et aI., 1993a). For species represented by more than one sample,
the individuals came from different oceanic basins. Exact orders of
the nodes within the shaded boxes are uncertain, differing slightly
with alternative methods of data analysis.

tary on the degree to which the planet's natural heritage has succumbed to the
overwhelming impact of one species, humankind.

SUMMARY
1. Most discussions of genetics in conservation biology have centered on
the topic of heterozygosity or related measures of the within-population compo-
nent of genetic variation. Molecular heterozygosity indeed is exceptionally low
in many rare or endangered species, presumably because of genetic drift and
inbreeding accompanying severe population reductions.
 Conservation Genetics 397

2. Although it is tempting to manage endangered populations for enhance-

ment of genetic variation, in most cases causal links between heterozygosity (as
estimated by molecular markers) and fitness have proven difficult to establish
firmly. For these and other reasons, some authors have argued that behavioral
and demographic concerns should take priority over heterozygosity issues in
management programs for endangered species. In truth, both classes of concern
are important.
3. Molecular markers also can serve the field of conservation biology by
revealing phylogenetic relationships within and among rare or endangered spe-
cies. Such phylogenetic assessments can range from parentage evaluations in
captive breeding programs to the identification of the major sources of regional
phylogeographic diversity around which management guidelines and natural re-
serves might be established.
4. Some programs for endangered species have been directed toward taxa
whose evolutionary distinctiveness has been questioned by molecular genetic
reappraisals. In other cases, endangered "species" that were taxonomically
suspect have proved upon molecular reexamination to be highly distinct genet-
ically, thus adding to the rationale for special preservation efforts.
5. Particularly in the field of fisheries biology, molecular markers are em-
ployed widely to identify and characterize populational stocks under exploita-
tion. Contributions of genetic stocks to mixed fisheries can be quantified. Mo-
lecular approaches to stock assessment also have stimulated thought about the
nature of information provided by inherited versus acquired markers (such as
physical tags) and of the distinction between evolutionarily deep versus shallow
population genetic structures.
6. Molecular markers provide powerful forensic tools for government
agencies charged with enforcement of wildlife legislation.
7. Hybridization and genetic introgression documented by molecular mark-
ers have raised both biological and legal questions concerning the status of
several endangered species. Molecular appraisals of phylogenetic relationships
among reproductively isolated taxa also have contributed to management dis-
cussions.

CONCLUSION

I want to close this chapter, and the book, by reiterating a sentiment expressed
by E.O. Wilson in the quote that opened this chapter. Modern biology indeed has
produced a genuinely new way of looking at the world. The molecular perspec-
tives emphasized in this text do not supplant traditional approaches to the study
398 Conservation Genetics

of natural history and evolution, but rather they enrich our understanding of life.
Herein lies the greatest value of molecular methods in conservation biology or
elsewhere. To the degree that we come to understand and appreciate other or-
ganisms, we will increasingly cherish the earth's biological heritage, and our
own. "We cannot win this battle to save species and environments without
forging an emotional bond between ourselves and nature as well-for we will not
fight to save what we do not love ... We really must make room for nature in
our hearts" (Gould, 1991). Think back to even a few of the fascinating organ-
isms whose natural histories and evolutionary patterns have been elucidated
using molecular markers-the honey mushrooms and their giant clones on the
floors of northern forests; the hybridogenetic livebearing fishes in the arroyos of
northwestern Mexico and the substantial evolutionary ages that some of these
unisexual biotypes have achieved; the bluebirds in the pasturelands of the eastern
United States, and their unsuspected and sometimes devious means of achieving
parentage; the naked mole rats in the deserts of Africa, with their eusocial
behaviors and tight fabrics of kinship; and the female green turtles, who after
periods measured in decades return faithfully to natal sites across thousands of
kilometers of open ocean. If this book has accomplished nothing else, I hope that
it may have engendered an increased awareness, respect, and love, for the
marvelous biological diversity on this fragile planet.
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 Index to Taxonomic Genera

CHORDATA Macaca 10, 176, 177, 228

(mammals) Mammuthus 357
Acinonyx 364, 369 Megaptera 226
Ailuropoda 314-315 Microtus 186
Alouatta 177 Mirounga 362-363
Balaenoptera 281 Monodelphis 368
Bison 365 Mus 6, 205, 287-291
Bos 368 Mustela 365, 393-394
Canis 363, 391, 392 Neotoma 10
Ceratotherium 373 Odocoileus 290
Cercocebus 10 Orcinus 203
Choeropsis 368 Pan 9, 132, 329-330
Clethrionomys 290, 291 Panthera 200-202, 364, 368
Cynomys 200,394 Papio 10
Diceros 373 Peramelas 365
Dipodomys 10, 249 Peromyscus 10, 173,208,224,230-231,
Elephantulus 368 261, 265-266
Equus 358, 366 Phyllostomus 199
Felis 391, 393 Rhinoceros 366
Galago 368 Saimiri 187
Gazella 368 Smilodon 359
Geomys 10, 233-235, 385 Spalax 259
Globiocephala 202 Speothos 368
Gorilla 132, 329-330 Spermophilus 10, 184-185, 204
Heterocephalus 198-199 Spilogale 394
Homo 9,77, 132, 141-147, 186, Stenella 203
238,240-241,329-331,347, Symphalangus 329-330
388 Tadarida 187
Hylobates 329-330 Thomomys 10, 259
lctonyx 394 Thylacinus 357, 359
Lasiurus 10 Trichechus 366
Lemur 368 Urocyon 202
Leontopithecus 368 Ursus 315, 394

501
502 Index to Taxonomic Genera

CHORDATA (continued) Vermivora 10

(birds) Vireo 10
Actitis 185 Zonotrichia 10
Agelaius 6, 174, 175, 227 (reptiles)
Ammodramus 10,64,227,242-243, Amblyrhynchus 329
382, 384-385 Anolis 10, 324
Anas 10, 265-266, 333 Bipes 10
Anser 10 CareUa 374, 387, 395, 396
Aphelocoma 227 Chelonia 106, 122, 222, 224-226, 228,
Aythya 10 236,371-372,387,395,396
Branta 227, 393 Chelydra 395, 396
Calidris 227 Cnemidophorus 299-300, 303
Camphylorhynchus 176 Conolophus 329
Casuarius 56 Crotaphytus 10
Catharus 10 Dermochelys 395, 396
Charadrius 373 Dipsosaurus 246
Chen 168, 228-229, 393 Eretmochelys 395, 396
Contopus 275 Heteronotia 300
Dendroica 10, 323 Lacerta 10
Dromaius 56 Lepidochelys 386,387,395,396
Empidonax 275 Malaclemys 242
Ficedula 289-290 Natator 395, 396
Gallus 56 Sauromalus 246
Geospiza 10 Sceloporus 259
Grus371 Sphenodon 386
Hirundo 174, 175 Uma 10
Hymenolaimus 371 Xerobates 246, 374
Lanarius 275 (amphibians)
Melospiza 227 Ambystoma 157,302
Merops 174 Bufo 204
Molothrus 316 Ensatina 261,263
Mycteria 374 Gastrotheca 275
Nesochen 393 Hydromantes 10
Parus 10, 175 Hyla 10, 258, 284-286, 290, 291
Passerella 227 Litoria 10
Passerina 173, 174 Plethodon 10, 275, 298
Phylloscopus 173 Rana 10, 290, 301-302
Picoides 227, 366 Scaphiopus 10
Progne 175 Taricha 10
Prunella 170 Triturus 291
Rhea 56 Xenopus 347
Sialia 173, 175 (fishes)
Spizella 174 Acanthochromis 216
Strigops 366 Acipenser 232
Strix 365 Amia 244-246
Struthio 56 Anguilla 65,216-217
Teaniopygia 187 Anthias 192-193
Toxostoma 10 Auxis 318
Troglodytes 174, 188 Bathygobius 10
Tyrannus 174 Brevoortia 232
 Index to Taxonomic Genera 503

Campostoma 10 Sciaenops 388

Catostomus 53, 298 Scomber 318
Centropristis 232,242-243 Scomberomorus 318
Chanos 217 Sebastes 387
Cichlasoma 270 Sphyraena 318
Coregonus 10, 271 Stegastes 217
Coryphaena 318 Tetrapterus 318
Cottus 272 Thoburnia 10
Cynoscion 388 Thunnus 217,274,318
Cyprinodon 10, 281, 283, 374, 375 Trichiurus 318
Embiotoca 216 Xiphias 318
Etheostoma 10 (ascidians)
Euthynnus 318 Botryllus 221
Fundulus 27, 149, 238-239
Galaxias 265 PORIFERA (sponges)
Gambusia 205, 244, 246, 364 Niphates 154
Gasterochisma 318
Gempylus 318
CNIDARIA (corals, jellyfish, and allies)
Gila 298
Acropora 147, 149
Hesperoleucus 264
Actinia 148, 153
Hoplostethus 217
Epiactis 170
Hybognathus 385
Goniastrea 170
Hypentelium 10
Metridium 153
llyodon 270
Montastrea 274
lstiophorus 318
Montipora 154
Katsuwonus 217, 318
Oulactis 153
Latimeria 311-314, 343, 345
Pavona 153
Lavinia 264
Plexaura 154
Leiognathus 352
Pocillopora 153
Lepidocybium 318
Seriatopora 149
Lepomis 10, 223, 244-246, 261,
Tubastraea 148
262, 267-269, 281, 291-293
Luxilus 298
Malwira 318 NEMATODA (round worms)
Medialuna 216 Ostertagia 235
Menidia 10, 149, 300
Micropterus 388 MOLLUSCA (bivalves, snails, and allies)
Notropis 10, 267-269, 290 Biomphalaria 170
Oncorhynchus 270,378,390 Bulinus 170
Phoxinus 300 Cerion 283
Plecoglossus 376 Crassostrea 219-220, 242-243
Poecilia 299-300 Deroceras 171
Poeciliopsis 157, 159,299-301,303, Goniobasis 221
304, 363-364, 375 Helix 222
Pylodictis 387 Liguus 170,363
Rivulus 149 Littorina 216
Ruvettus 318 Mytilus 27, 62, 219
Salmo 226, 270, 376, 390 Rumina 211-213
Sarda 318 Siphonaria 218
Scaphirhynchus 385 Thiara 155
504 Index to Taxonomic Genera

ANNELIDA (segmented worms) Paralithodes 309-310,388

Capitella 274 Penaeus 274
Octolasion 155 Plagiodera 173
Phyllochaetopterus 215 Poecilimon 183
Spirorbis 215 Polybia 196
Proctolaelaps 354
ARTHROPODA (insects, crustaceans, and Proplebeia 359
allies) Rhagoletis 274
Aedes 276 Rhytidoponera 194, 196, 197
Agelaia 196 Sitobion 149
Alphaeus 218 Solenopsis 196
Anopheles 275-277 Tigriopus 27, 184, 218, 221
Apis 196, 197,229-230 Trachyphloeus 274
Aquarius 223 Zaprionus 253
Artemia 309-310
Bacillus 304 ECHINODERMATA (starfish, sea urchins,
Caledia 290 and allies)
Cerceris 196 Coscinasterias 153
Chthamalus 274 Cucumaria 275
Colias 27 Echinometra 218
Collops 224 Heliocidaris 216
Danaus 236-237 Psolus 275
Daphnia 155 Strongylocentrotus 217
Drosophila 6, 11, 12,24,27,77,103,
104, 134-137, 172, 173, 184,231, BRYOPHYTA (mosses and allies)
260-263, 265, 274, 290, 291, 328, Marchantia 337
353,354 Physcomitrella 337
Euhadenoecus 224 Plagiomnium 258
Formica 196
Gryllus 290 TRACHAEOPHYTA (vascular plants)
Hadenoecus 224 (horsetails)
Homarus 218 Equisetum 344
Jasus 217 (psilophytes)
Lasioglossum 197,316-317 Psilotum 344
Limnoporus 223 (clubmosses)
Limulus 218, 242-243 Isoetes 337
Lithodes 309-310 Lycopodium 337
Locusta 184 Selaginella 337
Lucilia 354 (ferns)
Magicicada 237 Asplenium 258
Malacosoma 199 (gymnosperms-cone-bearing seed plants)
Mastotermes 359 Cryptomeria 344
Microstigmus 196 Cycas 343, 344
Myrmecia 196 Encephalartos 344
Myzus 149 Ephedra 342, 344
Nothomyrmecia 196 Ginkgo 337, 342, 344
Oncopeltus 281 Gnetum 342, 344
Pagurus 309 Juniperus 344
Panulirus 275 Pinus 182, 203, 215, 344
Parachartergus 196 Taxodium 359
 Index to Taxonomic Genera 505

Welwitschia 342, 344 Glycine 258, 344

Zamia 344 Gossypium 290
(angiosperms-flowering plants) Hedycarya 344
Achillea 341 Helianthus 261, 290, 295-296, 298, 341
Aechmea 150 Heuchera 258,290
Aesculus 293 Hibiscus 185
Agrostis 205 Hordeum 344
Anthoxanthum 205 Hosta 344
Arabis 148 Howellia 365
Argyroxiphium 290 Illicium 344
Aristolochia 344 Ipomoea 185
Arundinaria 344 Iris 293-294, 297
Asimina 344 Lactuca 341
Avena 210-211, 213, 344 Lasthenia 297-298
Barclaya 344 Limnanthus 375
Barnadesia 341 Liquidambar 344
Bensoniella 365 Liriodendron 344
Betula 150 Lobelia 339
Blennospenna 341 Lophocereus 150
Brassica 290 Lupinus 180,221
Cabomba 344 Lycopersicon 264
Cacosmia 341 Magnolia 344, 359
Calycanthus 344 Monopsis 339
Carthamnus 341 Najas 344
Ceratophyllum 342, 344 Nelumbo 342, 344
Cercocarpus 390 Nicotiana 63, 337, 338
Chamaelirium 182 Nuphar 344
Chloranthus 344 Nymphaea 344
Chromolaena 341 Oryza 344
Chrysanthemum 341 Pedicularis 365
Clarkia 52, 180, 264 Peperomia 344
Colocasia 344 Persea 290
Coreopsis 341 Petroselinum 344
Cucurbita 182 Piper 344
Datisca 319 Piptocarpa 341
Dimorphotheca 341 Pistia 344
Drimys 344 Pisum 337, 344
Dubautia 290 Platanus 344
Duchesnea 344 Plectritis 211
Echinodorus 344 Populus 147, 152, 290
Echinops 341 Potamogeton 344
Erythronium 264 Quercus 290
Eucalyptus 294 Ranunculus 344
Eupatorium 341 Raphanus 179, 181, 185
Felicia 341 Rubus 148
Ficus 159 Sabal 344
Gaura 264 Saccharum 344
Gazania 341 Sagittaria 344
Gerbera 341 Saruma 344
Gilia 180 Saururus 344
506 Index to Taxonomic Genera

TRACHAEOPHYTA (continued) (algae)

Sclerotheca 339-340 Coleochaete 339
Senecio 258, 324, 341 Enteromorpha 148, 151
Solidago 150, 151 Nitella 339
Sorghum 344 Spirogyra 339
Spinacia 344 Symbiodinium 277, 278
Stellaria 344 (others)
Stephenomeria 259, 297 Dictyostelium 347
Stokesia 341 Euglena 347
Tagetas 341
Taraxacum 152 BACTERIA
Tellima 290 Acidiphillum 348
Tragopogon 258, 341 Agrobacterium 347,348, 352
Trifolium 337, 365 Anacystis 347
Tripsacum 344 Bacillus 166, 347
Triticum 205, 344 Bordatella 165
Trochodendron 344 Bradyrhizobium 104
Vernonia 341 Caulobacter 348
Vicia 77 ChloroJlexus 347
Wisteria 338 Escherichia 6,57,77, 104, 162,347,348
Zea 6, 290, 344, 347, 348 Haemophilus 165
Lactococcus 278
FUNGI (see also p. 350) Legionella 165
Armillaria 152 Neisseria 166
Crumenolopsis 148 Photobacterium 104, 352
Fusarium 351 Rhizobium 104, 352
Neurospora 351 Rhodocyclus 347
Pneumocystis 343 Rhodopseudomonas 348
Puccinia 161 Rhodospirillum 348
Saccharomyces 7, 347 Rochalimaea 348
Suillus 334 Shigella 165
Staphylococcus 12, 166
PROTISTS (single-celled organisms) (see also Thermotoga 347
pp. 338, 343) Thiobacillus 348
(protozoa)
Entamoeba 161 ARCHAEA
Giardia 161 Halococcus 347
Leischmania 161 Haloferax 347
Naegleria 148, 161 Methanobacterium 347
Oxytricha 347 Methanococcus 347
Plasmodium 161 Methanospirillum 347
Toxoplasma 160 Sulfolobus 347
Trichomonad 161 Thermococcus 347
Trypanosoma 160, 161, 347 Thermoproteus 347
 General Index

Acquired markers, of population structure 377, Chloroplast DNA (cpDNA)

379-380 structure, mode of transmission, and nature
Agamospenny 151-152 of data 63, 68-69
AIDS (acquired immune deficiency syndrome) in speciation analysis 259
249 in hybridization and introgression analysis
Albumin 46,47, 102, 104,281,282,325 290, 293-296, 298, 339-341
Alcohol dehydrogenase 12,27, 135, 136,231 in estimation of supraspecific phylogeny 42,
Allee effect 367 319,337-341,347, 349, 359
Allozymes (see Protein electrophoresis) Cladistics, Hennigian 34-39, 94, 121-122
Amylase, 135, 137 Cladogenesis 4, 35, 269 (see also Speciation)
Anagenesis 4, 35, 269 Cloning
Androgenesis 304 molecular 42, 59, 60, 70, 134, 358
Aneuploidy 52 organismal (see Asexual reproduction)
Apomixis (see Asexual reproduction) Coefficient of relatedness (r) (see also
Archaea 343, 346-349, 351 Parentage assessment, Kinship)
Asexual reproduction definition 191, 195
in higher animals and plants 147-155, 211 in eusocial species 194-199
in unisexual vertebrates 156-160, 299-305 in non-eusocial species 192-194, 199-203
in microorganisms 160-167 Concerted evolution 12, 77-78
Autogamy (see Self-fertilization) Creatine kinase 52
Cytochrome b 86,88,272,312,314,316,
Bacteria (Eubacteria) 6, 12, 57, 77, 104, 162, 318,337, 396
165-166, 278, 343, 346-349, 351-353 Cytoplasmic capture, via hybridization
Biogeography 38, 107,236,238,248, 289-290, 296
321-326, 328-330 (see also Cytoplasmic male sterility 295
Phylogeography)
Bottlenecks, in population size 24, 29, 78, Disequilibrium, gametic phase 75-76, 133,
128, 134,240,257,259,264-267, 135, 145, 162-165, 212 (see also Gene
364, 366, 367, 393, 394 trees)
Brood parasitism 169, 174, 187-188,315-316 Distance, genetic
concepts and definitions 94-%
Carnivory, in plants 319 as common yardsticks 9-11, 333
Ceiling principle, in human forensics 147 phylogenetic algorithms based on 111-120

507
508 General Index

Distance, genetic (continued) Forensics

associated with RIB evolution 261-264 human 8, 141-147, 186-187
between hybridizable species 281-282 wildlife 94, 386-388
and rate of speciation 267-269 Fossils, biological macromolecules from
Distance Wagner procedure 112, 115, 356-359
118-119
DNA-DNA hybridization Gender-biased dispersal 227-230, 293-295
techniques 53-57, 91 Gender identification 167
nature of data 93, 97-99 Gene conversion 12, 133
in estimation of supraspecific phylogeny 8, Gene duplications, as phylogenetic markers 8,
311,314,325-327,330-333 12-13, 52-53, 101, 346
DNA fingerprinting Gene expression patterns, as phylogenetic
techniques 59, 91 (see also Restriction markers 51, 52
fragment-length polymorphisms) Gene flow 94, 204-233 (see also Population
nature of data 78-82, 93, 97-99 structure)
in clonal recognition 41, 42, 149, 152, Generation effect, on evolutionary rates 103,
154 105, 106
in human forensics 142-147 Gene rearrangements, as phylogenetic markers
in gender identification 167 8, 12, 334-335, 337-340
in parentage assessment 8, 170-171, 173, Genetic code 14, 335-336
175-177, 183, 185-187 Genetic identity/non-identity 99, 101, 141-
in kinship assessment 198-204 167
in conservation biology 363, 364, 371 Genetic load 17, 18 (see also Inbreeding
DNA sequencing depression)
background 11, 23, 41, 42 Gene trees
techniques 82-84, 91 versus organismal phylogenies 13, 14,
nature of data 93, 97-99 126-137,241-242,247,265-266,
in population structure analysis 248-250 274, 352-355, 390-393
in speciation analysis 272, 276-278 for nuclear loci 134-137
in estimation of supraspecific phylogeny for organellar loci (see Mitochondrial DNA
308-316, 318-319, 326, 330, and Chloroplast DNA)
337-351, 354-356, 358-359, 395-396 Genets 147-150 (see also Asexual
reproduction)
Ecogeography 246-248 Genome size 7-8
Effective population size 28-33, 160, 232, Genome structure, classical and balance views
362,373 17-18
Endangered species 361-376, 382-386, Globins 52, 79, 102, 104
389-396 Glucose-6-phosphate dehydrogenase 27
Endangered Species Act 383 Glucosephosphate isomerase 27, 50, 52, 220
Endosymbiosis 4, 14, 347-349 Glutamate pyruvate transaminase 27
Endothermy, in fishes 316-318 Glutamine synthetase 352, 354
Esterase 12, 27, 220 a-Glycerophosphate dehydrogenase 27, 47
Eucarya 346-349 Gynogenesis 156-159, 299-304
Eusociality 194-199
Haldane's rule 289-291
F -statistics, of population structure Haplotype diversity 21
definitions 206 Hermaphroditism 149, 170-171, 178-180,
as a basis for estimating gene flow 207-208, 210-213
213-215,222-224,227,230-231 Heterozygosity
Fitch-Margoliash procedure 112-114, 120 definition 21
 General Index 509

background and concepts 17-31 invertebrate phyla 335-336

in rare and tbreatened species 362-371 king crabs and hermit crabs 309-310
Histocompatibility 149, 150, 153-154, 157, marine turtles 394-396
159,369 marsupial mammals 326
HIV (human immunodeficiency virus) megabats and microbats 307
249-250 pandas and bears 314-315
Horizontal gene transfer 40, 352-354 primates 329-331
Hybridization (organismal) 94, 126, 149,217, retroviruses, retrotransposons, and relatives
229, 260, 355, 361 354-357
frequencies and geographic settings 280-283 tetrapod vertebrates 342, 345
influence of mating behavior on 284-287, tunas, mackerais, and billfishes 316, 318
291-293 tunicates and other chordates 316-317, 319
differential introgression across zones of weasels, skunks, and allies 393-394
287-289 whales, dolphins, and ungulates 309-311
Haldane's rule 289-291 zebras and horses 358
differential gametic exchange 293-295 Major histocompatibility complex (MHC) 78,
leading to speciation 159, 297-299 129, 364, 369
involving rare or endangered species Malate dehydrogenase 51, 52
388-393 Maternity analysis (see Parentage assessment)
Hybridogenesis 156-159, 299-304 Mating systems 148, 179, 209-213, 259, 308
Hybrid policy, of the Endangered Species Act (see also Hybridization, Population
389 structure, Parentage assessment,
Self-fertilization)
Inbreeding 20, 25, 149, 204, 212, 362, Meiotic drive 34
369-371 (see also Mating systems) Microcomplement fixation (MCF) (see Protein
Inbreeding depression 179, 362, 367-368, immunological assays)
370,393 Microsatellite DNAs 82
Inclusive fitness 191, 198, 199 Minisatellite DNAs 78-81 (see also DNA
Introgression (see Hybridization) fingerprinting)
Introns 14,62,72, 101, 102,337-340 Mitochondrial DNA (mtDNA)
Isozymes (see Protein electrophoresis) background 21,31-33,41-42
novel concepts derived from 13-14
Kin recognition 203-204 PCR primer sequences from 86
Kin selection 191, 192,200 structure, mode of transmission, and nature
Kinship 94, 190-204, 371-372 of data 60-68, 88-89, 93, 98-104,
126, 133-134
Lactate dehydrogenase 27, 48, 50, 52, 193, endosymbiotic origin of 14, 347-349
238-239 in clonal recognition 155
Leucine aminopeptidase 27, 219, 220 in estimating clonal ages 157-159
in kinship assessment 204
Macroevolutionary trees, from molecular data in assessment of gene flow and
angiosperm and gymnosperm plants 319, phylogeography 208, 219-220,
337-344 224-249,350-351
avian taxa 8, 326-328, 331-333 in speciation analysis 265-267,271-272,
cnidarians 335 274-276
coelacanths, early fishes, tetrapods 311-312 in hybridization and introgression analysis
echinoderms 334 281-293, 298-304, 323
Eukarya, Archaea, Bacteria 319-320, in estimation of supraspecific phylogeny
346-349 309-316, 318, 326, 328, 330,
fungi 350-351 334-337, 347, 349, 358, 359
510 General Index

Mitochondrial DNA (mtDNA) (continued) Polyembryony 147

in conservation biology 363, 364, 371-374, Polygenic traits II, 320
384-386, 390-396 Polymerase chain reaction (PCR) (see also
Molecular clocks (see Rates of molecular DNA sequencing) 41, 42, 72, 73, 81,
evolution) 82, 134
Morphology, as a backdrop for molecular techniques 84-87, 91
comparisons 3, 6, 8, 9, 1I, 38-40, in amplifying fossil DNA 358-359
152, 154, 260, 270-272, 274, 275, Polyploidy 52, 53, 258, 300, 302-305
286-287,298,307-315 Population structure 146, 204-233, 363-364,
Mosaicism, genetic (see also Endosymbiosis) 372-380
14, 159-160 Protein electrophoresis
Muller's ratchet 155 background 10, 20-31, 41
techniques 47-49, 90
Natural selection, influence on molecular nature of data 49-53, 93, 98, 99, 121
markers 17, 18,23-28, 31,33-34, in clonal recognition 149-153, 155, 160-167
219-220,230-231,367 in mosaic recognition 159
Neighbor-joining procedure 1I2, 1I4-1I7 in mating system assessment 170
Nepotism 204 in parentage assessment 172-177, 179-188
Neutrality theory 26-34, 42 in kinship assessment 192-194, 196-200,
Nucleotide diversity 7, 21, 158 203,204
Numerical taxonomy (see Phenetics) in assessment of gene flow and population
structure 205, 206, 210-224, 226,
Orthology 13, 52, 77 228-231,238-239
in speciation analysis 258-265,267-271,
Paralogy 13, 77, 355 274-277
Parentage assessment 94, 99, 101 in hybridization and introgression analysis
maternity analysis 168-170, 174, 185-188 281-294, 298-304
paternity analysis 168 -185 in estimation of supraspecific phylogeny
in conservation biology 371 314-317,323-324,330
Parsimony 39, 94, 122 in conservation biology 363-367, 373-376,
Camin-Sokal 124 378, 385-388, 390, 394
Dollo 123-124 Protein immunological assays
Generalized 123 -124 techniques 44-46, 90
Wagner 123-124 nature of data 46,47,93,97-99
Parthenogenesis 147,149,154-159,171-172, in speciation analysis 275
299-304 in hybridization analysis 281
Paternal leakage, of mtDNA 62 in estimation of supraspecific phylogeny
Paternity analysis (see Parentage assessment) 314, 323-324, 328, 330, 357
Phenetics 34-35, 1I3, 124 Pseudogenes 53, 62, 101-102
Philopatry 222, 224-227, 236 Punctuated equilibrium 257, 267-269
Phosphoglucomutase 50, 192-193,220
6- Phosphogluconate dehydrogenase 27, 220 Quantitative traits (see Polygenic traits)
Phyletic gradualism 260, 267-269
Phylogenetic character mapping 12, 40, Ramets 147-150, 159
307-321,347-348 RAPDs (random amplified polymorphic
Phylogenetic trees, procedures for construction DNAs) 87, 152
109-126 Rates of molecular evolution 14, 31-33, 39,
Phylogeny, definition and scope 4-5 68, 69, 100-109, 355
Phylogeography 233-248, 321-326, 380-382 rbcL (ribulose-I ,5 biphosphate carboxylase)
Pollen competition 185 63, 319, 340-342, 349, 359
 General Index 511

Rectangular evolution (see Punctuated in parentage assessment 168 -169, 174, 175
equilibrium) in population structure analysis 219-220,
Relative rate test 107-108 222, 228-230
Reproductive isolating barriers (RIDS) in speciation analysis 278
254-255,261,263,279 Software packages, for phylogenetic
Restriction endonucleases, recognition reconstruction 125
sequences 57, 58 Southern blotting 42,59-60,69,71,73-75,
Restriction fragment-length polymorphisms 77,79,81,91 (see also Restriction
(RFLPs) (see also Mitochondrial DNA, fragment-length polymorphisms)
Chloroplast DNA, Ribosomal RNA Speciation
genes, DNA fingerprinting, Single-copy concepts and definitions 252-257, 278-280
nuclear DNA) sudden 258-259
background 41 genetic differentiation during 257-264
techniques for assay 57-61, 64-82, 91 sympatric 269-272
range of applications for 99 gradual versus rectangular 267-269
digestion profiles and site mapping 65 -67 , via population bottlenecks 264-267
71,72,74,76, 80, 125-126 by host- or habitat-switching 272-274
Reticulate evolution 4 (see also Hybridization, molecular diagnosis of 274-278
Horizontal gene transfer) by hybridization 297-299
Retrotransposable elements 355-356 and conservation biology 361,382-386
Retroviruses 249-250, 354-357 Species flocks, fishes 269-272
Reverse transcriptase 355-356 Sperm competition 182-185
Ribosomal RNA genes Sperm storage 172, 183-186
structure, and rates of evolution 75, 77-78, SSLPs (simple sequence length
101, 104 polymorphisms) (see Microsatellite
in paternity analysis 172 DNA sequences)
in species diagnostics 275-278 Stock assessment, in fishery biology 375-377,
in estimation of supraspecific phylogeny 379-380
109,308-309,311,314-316,319, Superoxide dismutase 192, 193,352
326,335,342-349,350-351,359
Rooting of trees 120, 125, 346 Transplantations, artificial 221-222, 281-282,
381,390
Self-fertilization 163,171,177-182,210-213, Transposable elements 11, 14, 353-357
215, 259, 264
Self-incompatibility loci 129, 178, 259
Sex-linked nuclear markers 134, 167, 172, Unisexual vertebrates 156-159, 299-305
227-228,241,288,291 UPGMA clustering procedure 111-113, 124
Single-copy nuclear DNA (scnDNA), RFLPs
from Vicariance, definition of 322 (see also
techniques for assay 69 -73, 91 Phylogeography)
nature of data 73-75, 93, 99 VNTR typing (see DNA fingerprinting)