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Population Genomics with R 1st Edition Emmanuel
Paradis Digital Instant Download
Author(s): Emmanuel Paradis
ISBN(s): 9781138608184, 1138608181
Edition: 1
File Details: PDF, 44.80 MB
Year: 2020
Language: english
Population Genomics
with R
Population Genomics
with R

by
Emmanuel Paradis
CRC Press
Taylor & Francis Group
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© 2020 by Taylor & Francis Group, LLC

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 To Laure and Sinta
for their constant support.
Contents

Preface xiii

Symbol Description xv

1 Introduction 1
1.1 Heredity, Genetics, and Genomics . . . . . . . . . . . . . . . 1
1.2 Principles of Population Genomics . . . . . . . . . . . . . . . 2
1.2.1 Units . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.2 Genome Structures . . . . . . . . . . . . . . . . . . . . 3
1.2.3 Mutations . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.2.4 Drift and Selection . . . . . . . . . . . . . . . . . . . . 11
1.3 R Packages and Conventions . . . . . . . . . . . . . . . . . . 12
1.4 Required Knowledge and Other Readings . . . . . . . . . . . 16

2 Data Acquisition 17
2.1 Samples and Sampling Designs . . . . . . . . . . . . . . . . . 17
2.1.1 How Much DNA in a Sample? . . . . . . . . . . . . . 17
2.1.2 Degraded Samples . . . . . . . . . . . . . . . . . . . . 18
2.1.3 Sampling Designs . . . . . . . . . . . . . . . . . . . . . 19
2.2 Low-Throughput Technologies . . . . . . . . . . . . . . . . . 22
2.2.1 Genotypes From Phenotypes . . . . . . . . . . . . . . 22
2.2.2 DNA Cleavage Methods . . . . . . . . . . . . . . . . . 23
2.2.3 Repeat Length Polymorphism . . . . . . . . . . . . . . 24
2.2.4 Sanger and Shotgun Sequencing . . . . . . . . . . . . 24
2.2.5 DNA Methylation and Bisulfite Sequencing . . . . . . 25
2.3 High-Throughput Technologies . . . . . . . . . . . . . . . . . 27
2.3.1 DNA Microarrays . . . . . . . . . . . . . . . . . . . . 27
2.3.2 High-Throughput Sequencing . . . . . . . . . . . . . . 27
2.3.3 Restriction Site Associated DNA . . . . . . . . . . . . 28
2.3.4 RNA Sequencing . . . . . . . . . . . . . . . . . . . . . 29
2.3.5 Exome Sequencing . . . . . . . . . . . . . . . . . . . . 29
2.3.6 Sequencing of Pooled Individuals . . . . . . . . . . . . 30
2.3.7 Designing a Study With HTS . . . . . . . . . . . . . . 30
2.3.8 The Future of DNA Sequencing . . . . . . . . . . . . . 30
2.4 File Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.4.1 Data Files . . . . . . . . . . . . . . . . . . . . . . . . . 31

vii
viii Contents

2.4.2 Archiving and Compression . . . . . . . . . . . . . . . 32

2.5 Bioinformatics and Genomics . . . . . . . . . . . . . . . . . . 35
2.5.1 Processing Sanger Sequencing Data With sangerseqR . 36
2.5.2 Read Mapping With Rsubread . . . . . . . . . . . . . . 36
2.5.3 Managing Read Alignments With Rsamtools . . . . . . 39
2.6 Simulation of High-Throughput Sequencing Data . . . . . . 41
2.7 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3 Genomic Data in R 47
3.1 What is an R Data Object? . . . . . . . . . . . . . . . . . . . 47
3.2 Data Classes for Genomic Data . . . . . . . . . . . . . . . . 49
3.2.1 The Class "loci" (pegas) . . . . . . . . . . . . . . . . 49
3.2.2 The Class "genind" (adegenet) . . . . . . . . . . . . . 50
3.2.3 The Classes "SNPbin" and "genlight" (adegenet) . . 51
3.2.4 The Class "SnpMatrix" (snpStats) . . . . . . . . . . . 52
3.2.5 The Class "DNAbin" (ape) . . . . . . . . . . . . . . . . 53
3.2.6 The Classes "XString" and "XStringSet" (Biostrings) 55
3.2.7 The Package SNPRelate . . . . . . . . . . . . . . . . . 56
3.3 Data Input and Output . . . . . . . . . . . . . . . . . . . . . 58
3.3.1 Reading Text Files . . . . . . . . . . . . . . . . . . . . 58
3.3.2 Reading Spreadsheet Files . . . . . . . . . . . . . . . . 59
3.3.3 Reading VCF Files . . . . . . . . . . . . . . . . . . . . 60
3.3.4 Reading PED and BED Files . . . . . . . . . . . . . . 65
3.3.5 Reading Sequence Files . . . . . . . . . . . . . . . . . 66
3.3.6 Reading Annotation Files . . . . . . . . . . . . . . . . 67
3.3.7 Writing Files . . . . . . . . . . . . . . . . . . . . . . . 67
3.4 Internet Databases . . . . . . . . . . . . . . . . . . . . . . . . 68
3.5 Managing Files and Projects . . . . . . . . . . . . . . . . . . 69
3.6 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

4 Data Manipulation 73
4.1 Basic Data Manipulation in R . . . . . . . . . . . . . . . . . 73
4.1.1 Subsetting, Replacement, and Deletion . . . . . . . . . 73
4.1.2 Commonly Used Functions . . . . . . . . . . . . . . . 74
4.1.3 Recycling and Coercion . . . . . . . . . . . . . . . . . 76
4.1.4 Logical Vectors . . . . . . . . . . . . . . . . . . . . . . 77
4.2 Memory Management . . . . . . . . . . . . . . . . . . . . . . 78
4.3 Conversions . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.4 Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.4.1 Mitochondrial Genomes of the Asiatic Golden Cat . . 82
4.4.2 Complete Genomes of the Fruit Fly . . . . . . . . . . 83
4.4.3 Human Genomes . . . . . . . . . . . . . . . . . . . . . 84
4.4.4 Influenza H1N1 Virus Sequences . . . . . . . . . . . . 85
4.4.5 Jaguar Microsatellites . . . . . . . . . . . . . . . . . . 87
4.4.6 Bacterial Whole Genome Sequences . . . . . . . . . . 87
Contents ix

4.4.7 Metabarcoding of Fish Communities . . . . . . . . . . 88

4.5 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

5 Data Exploration and Summaries 93

5.1 Genotype and Allele Frequencies . . . . . . . . . . . . . . . . 93
5.1.1 Allelic Richness . . . . . . . . . . . . . . . . . . . . . . 95
5.1.2 Missing Data . . . . . . . . . . . . . . . . . . . . . . . 96
5.2 Haplotype and Nucleotide Diversity . . . . . . . . . . . . . . 98
5.2.1 The Class "haplotype" . . . . . . . . . . . . . . . . . 98
5.2.2 Haplotype and Nucleotide Diversity From DNA Se-
quences . . . . . . . . . . . . . . . . . . . . . . . . . . 101
5.3 Genetic and Genomic Distances . . . . . . . . . . . . . . . . 103
5.3.1 Theoretical Background . . . . . . . . . . . . . . . . . 103
5.3.2 Hamming Distance . . . . . . . . . . . . . . . . . . . . 103
5.3.3 Distances From DNA Sequences . . . . . . . . . . . . 105
5.3.4 Distances From Allele Sharing . . . . . . . . . . . . . 105
5.3.5 Distances From Microsatellites . . . . . . . . . . . . . 106
5.4 Summary by Groups . . . . . . . . . . . . . . . . . . . . . . 107
5.5 Sliding Windows . . . . . . . . . . . . . . . . . . . . . . . . . 110
5.5.1 DNA Sequences . . . . . . . . . . . . . . . . . . . . . . 110
5.5.2 Summaries With Genomic Positions . . . . . . . . . . 112
5.5.3 Package SNPRelate . . . . . . . . . . . . . . . . . . . . 113
5.6 Multivariate Methods . . . . . . . . . . . . . . . . . . . . . . 114
5.6.1 Matrix Decomposition . . . . . . . . . . . . . . . . . . 115
5.6.1.1 Eigendecomposition . . . . . . . . . . . . . . 115
5.6.1.2 Singular Value Decomposition . . . . . . . . 117
5.6.1.3 Power Method and Random Matrices . . . . 118
5.6.2 Principal Component Analysis . . . . . . . . . . . . . 118
5.6.2.1 adegenet . . . . . . . . . . . . . . . . . . . . 119
5.6.2.2 SNPRelate . . . . . . . . . . . . . . . . . . . 121
5.6.2.3 flashpcaR . . . . . . . . . . . . . . . . . . . . 123
5.6.3 Multidimensional Scaling . . . . . . . . . . . . . . . . 124
5.7 Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
5.7.1 Mitochondrial Genomes of the Asiatic Golden Cat . . 125
5.7.2 Complete Genomes of the Fruit Fly . . . . . . . . . . 127
5.7.3 Human Genomes . . . . . . . . . . . . . . . . . . . . . 134
5.7.4 Influenza H1N1 Virus Sequences . . . . . . . . . . . . 138
5.7.5 Jaguar Microsatellites . . . . . . . . . . . . . . . . . . 142
5.7.6 Bacterial Whole Genome Sequences . . . . . . . . . . 149
5.7.7 Metabarcoding of Fish Communities . . . . . . . . . . 152
5.8 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
x Contents

6 Linkage Disequilibrium and Haplotype Structure 157

6.1 Why Linkage Disequilibrium is Important? . . . . . . . . . . 157
6.2 Linkage Disequilibrium: Two Loci . . . . . . . . . . . . . . . 159
6.2.1 Phased Genotypes . . . . . . . . . . . . . . . . . . . . 159
6.2.1.1 Theoretical Background . . . . . . . . . . . . 159
6.2.1.2 Implementation in pegas . . . . . . . . . . . 160
6.2.2 Unphased Genotypes . . . . . . . . . . . . . . . . . . . 162
6.3 More Than Two Loci . . . . . . . . . . . . . . . . . . . . . . 163
6.3.1 Haplotypes From Unphased Genotypes . . . . . . . . . 163
6.3.1.1 The Expectation–Maximization Algorithm . 164
6.3.1.2 Implementation in haplo.stats . . . . . . . . . 164
6.3.2 Locus-Specific Imputation . . . . . . . . . . . . . . . . 167
6.3.3 Maps of Linkage Disequilibrium . . . . . . . . . . . . 168
6.3.3.1 Phased Genotypes With pegas . . . . . . . . 168
6.3.3.2 SNPRelate . . . . . . . . . . . . . . . . . . . 170
6.3.3.3 snpStats . . . . . . . . . . . . . . . . . . . . . 171
6.4 Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
6.4.1 Complete Genomes of the Fruit Fly . . . . . . . . . . 172
6.4.2 Human Genomes . . . . . . . . . . . . . . . . . . . . . 176
6.4.3 Jaguar Microsatellites . . . . . . . . . . . . . . . . . . 177
6.5 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

7 Population Genetic Structure 185

7.1 Hardy–Weinberg Equilibrium . . . . . . . . . . . . . . . . . . 185
7.2 F -Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
7.2.1 Theoretical Background . . . . . . . . . . . . . . . . . 187
7.2.2 Implementations in pegas and in mmod . . . . . . . . 189
7.2.3 Implementations in snpStats and in SNPRelate . . . . 193
7.3 Trees and Networks . . . . . . . . . . . . . . . . . . . . . . . 196
7.3.1 Minimum Spanning Trees and Networks . . . . . . . . 197
7.3.2 Statistical Parsimony . . . . . . . . . . . . . . . . . . 199
7.3.3 Median Networks . . . . . . . . . . . . . . . . . . . . . 200
7.3.4 Phylogenetic Trees . . . . . . . . . . . . . . . . . . . . 201
7.4 Multivariate Methods . . . . . . . . . . . . . . . . . . . . . . 202
7.4.1 Principles of Discriminant Analysis . . . . . . . . . . . 202
7.4.2 Discriminant Analysis of Principal Components . . . . 203
7.4.3 Clustering . . . . . . . . . . . . . . . . . . . . . . . . . 207
7.4.4 Maximum Likelihood Methods . . . . . . . . . . . . . 207
7.4.5 Bayesian Clustering . . . . . . . . . . . . . . . . . . . 210
7.5 Admixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
7.5.1 Likelihood Method . . . . . . . . . . . . . . . . . . . . 214
7.5.2 Principal Component Analysis of Coancestry . . . . . 217
7.5.3 A Second Look at F -Statistics . . . . . . . . . . . . . 218
7.6 Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
7.6.1 Mitochondrial Genomes of the Asiatic Golden Cat . . 222
Contents xi

7.6.2 Complete Genomes of the Fruit Fly . . . . . . . . . . 225

7.6.3 Influenza H1N1 Virus Sequences . . . . . . . . . . . . 234
7.6.4 Jaguar Microsatellites . . . . . . . . . . . . . . . . . . 237
7.7 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

8 Geographical Structure 241

8.1 Geographical Data in R . . . . . . . . . . . . . . . . . . . . . 241
8.1.1 Packages and Classes . . . . . . . . . . . . . . . . . . 242
8.1.2 Calculating Geographical Distances . . . . . . . . . . 242
8.2 A Third Look at F -Statistics . . . . . . . . . . . . . . . . . . 243
8.2.1 Hierarchical Components of Genetic Diversity . . . . . 243
8.2.2 Analysis of Molecular Variance . . . . . . . . . . . . . 246
8.3 Moran I and Spatial Autocorrelation . . . . . . . . . . . . . 250
8.4 Spatial Principal Component Analysis . . . . . . . . . . . . . 251
8.5 Finding Boundaries Between Populations . . . . . . . . . . . 255
8.5.1 Spatial Ancestry (tess3r) . . . . . . . . . . . . . . . . . 255
8.5.2 Bayesian Methods (Geneland) . . . . . . . . . . . . . . 257
8.6 Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
8.6.1 Complete Genomes of the Fruit Fly . . . . . . . . . . 259
8.6.2 Human Genomes . . . . . . . . . . . . . . . . . . . . . 260
8.7 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

9 Past Demographic Events 265

9.1 The Coalescent . . . . . . . . . . . . . . . . . . . . . . . . . . 265
9.1.1 The Standard Coalescent . . . . . . . . . . . . . . . . 265
9.1.2 The Sequential Markovian Coalescent . . . . . . . . . 268
9.1.3 Simulation of Coalescent Data . . . . . . . . . . . . . 269
9.2 Estimation of Θ . . . . . . . . . . . . . . . . . . . . . . . . . 275
9.2.1 Heterozygosity . . . . . . . . . . . . . . . . . . . . . . 275
9.2.2 Number of Alleles . . . . . . . . . . . . . . . . . . . . 275
9.2.3 Segregating Sites . . . . . . . . . . . . . . . . . . . . . 276
9.2.4 Microsatellites . . . . . . . . . . . . . . . . . . . . . . 276
9.2.5 Trees . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
9.3 Coalescent-Based Inference . . . . . . . . . . . . . . . . . . . 278
9.3.1 Maximum Likelihood Methods . . . . . . . . . . . . . 278
9.3.2 Analysis of Markov Chain Monte Carlo Outputs . . . 280
9.3.3 Skyline Plots . . . . . . . . . . . . . . . . . . . . . . . 282
9.3.4 Bayesian Methods . . . . . . . . . . . . . . . . . . . . 282
9.4 Heterochronous Samples . . . . . . . . . . . . . . . . . . . . 284
9.5 Site Frequency Spectrum Methods . . . . . . . . . . . . . . . 286
9.5.1 The Stairway Method . . . . . . . . . . . . . . . . . . 288
9.5.2 CubSFS . . . . . . . . . . . . . . . . . . . . . . . . . . 289
9.5.3 Popsicle . . . . . . . . . . . . . . . . . . . . . . . . . . 289
9.6 Whole-Genome Methods (psmcr) . . . . . . . . . . . . . . . . 292
9.7 Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
xii Contents

9.7.1 Mitochondrial Genomes of the Asiatic Golden Cat . . 293

9.7.2 Complete Genomes of the Fruit Fly . . . . . . . . . . 298
9.7.3 Influenza H1N1 Virus Sequences . . . . . . . . . . . . 302
9.7.4 Bacterial Whole Genome Sequences . . . . . . . . . . 304
9.8 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

10 Natural Selection 309

10.1 Testing Neutrality . . . . . . . . . . . . . . . . . . . . . . . . 309
10.1.1 Simple Tests . . . . . . . . . . . . . . . . . . . . . . . 309
10.1.2 Selection in Protein-Coding Sequences . . . . . . . . . 310
10.2 Selection Scans . . . . . . . . . . . . . . . . . . . . . . . . . . 313
10.2.1 A Fourth Look at F -Statistics . . . . . . . . . . . . . . 313
10.2.2 Association Studies (LEA) . . . . . . . . . . . . . . . . 314
10.2.3 Principal Component Analysis (pcadapt) . . . . . . . . 314
10.2.4 Scans for Selection With Extended Haplotypes . . . . 315
10.2.5 F ST Outliers . . . . . . . . . . . . . . . . . . . . . . . 320
10.3 Time-Series of Allele Frequencies . . . . . . . . . . . . . . . . 324
10.4 Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
10.4.1 Mitochondrial Genomes of the Asiatic Golden Cat . . 326
10.4.2 Complete Genomes of the Fruit Fly . . . . . . . . . . 327
10.4.3 Influenza H1N1 Virus Sequences . . . . . . . . . . . . 335
10.5 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338

A Installing R Packages 341

B Compressing Large Sequence Files 345

C Sampling of Alleles in a Population 349

D Glossary 351

Bibliography 353

Index 373
Preface

For many years, population genetics was an immensely rich and pow-
erful theory with virtually no suitable facts on which to operate [. . . ]
Quite suddenly the situation has changed [. . . ] and facts in profusion
have been poured into the hopper of this theory machine. [. . . ] The
entire relationship between the theory and the facts needs to be recon-
sidered.
—Lewontin [159]

Sometimes, some researchers may say that they started to work on a topic
or issue incidentally before they spent a lot of their time and energy on it. This
could be said about my involvement in population genomics. My early training
in population genetics was very light, and my background in genomics (a very
new field when I was student) was even lighter. My long-time interest in evo-
lution and my investment in R started in late 1990s have eventually led me to
focus more and more of my scientific interest onto population genomics. Back-
ing on my experience with ape, I started the development of pegas which was
first released in May 2009. Developing an R package for evolutionary analysis
was not a new thing at this time and several colleagues had initiated similar
projects. The idea of this book emerged in part from discussions with some
of these colleagues. At this time high-throughput sequencing (HTS) was just
beginning its breakthrough and we were just starting to foresee the eventual
impacts of this technological revolution. The hackathon held at the National
Evolutionary Synthesis Center (NESCent) in Durham, USA, in March 2015
was for me a great opportunity to develop new tools to handle HTS data in
pegas. The idea of Population Genomics With R then grew progressively to
become a book project after discussions with John Kimmel started in Septem-
ber 2017.
There are three main driving ideas behind this book. The first one is to
consider all types of population genetic and genomic data, from the simplest
genetic data to the most large-scale genomic ‘big data’. The second idea is
to provide a single, common computing environment to address a wide range
of questions or tackle a wide range of analyses with population genetic and
genomic data. The third idea is to promote the use of free and open source
software. In the progress of writing, I found out that statisticians and develop-
ers in genomics use R more frequently than I thought. After having defended
the use of R for more than two decades, this is a fact that certainly provides
me some satisfaction.

xiii
xiv Preface

The basic materials of Population Genomics With R are the R packages

listed in Chaper 1. Clearly, this is not an exhaustive list of computing resources
for population genomics. I have tried as much as possible to consider packages
which are operational and integrate in the general framework of population
genomics outlined above. Theferore, I avoided to mention packages that are
clearly not maintained (e.g., orphaned packages on CRAN) or appeared to
not work correctly. During my research, I have certainly missed some packages
that should have been included in this book. As an example, DECIPHER, a
package distributed on BioConductor for managing very large databases of
sequence data, should have been cited in several chapters of this book. On
the other hand, I did not consider packages which are not distributed on a
server or which are too specialized: these include several R packages developed
to analyze human populations which are available only on request to their
authors—although it is not clear how this way to distribute software conflicts
with the free and open source software framework which I tried to follow.
Writing this book was a very progressive process and I benefited from
critical and very helpful comments on early drafts from Olivier François, San-
thosh Girirajan, Sarah Hendricks, and several anonymous reviewers. Hilmar
Lapp invited me to the hackathon held at NESCent in 2015 where I had one
of the most stimulating week of work and development: thanks to him and
to the colleagues and friends who were there too. Thibaut Jombart shared a
lot of great discussions on many occasions: thanks to him for organizing and
inviting me to another hackathon in London. I had the opportunity to give
several workshops on the packages I develop: these events are very rewarding
experiences and I want to thank particularly Frédéric Chiroleu, Soledad De
Esteban-Trivigno, Jérôme Goudet, and Nicolas Salamin for their invitations.
Many thanks to Agnès Mignot for her full support to develop my research
while she was head of the Institute of Evolutionary Sciences in Montpellier.
I am very grateful to John Kimmel for giving me the oppotunity to write
another book on using R to analyze evolution. Robin Lloyd Starkes enthusias-
tically worked out all the practical aspects handling my manuscript. Finally,
I am grateful to my wife Sinta and my daughter Laure for their permanent
support.

Bangsaen Emmanuel Paradis

March 2020
Preface xv

Symbol Description

E expectation. p number of variables; number of

H heterozygosity. columns in a table or in a matrix.
h haplotype diversity. pi proportion of allele i in the pop-
K number of populations, groups, ulation (i = 1, . . . , k).
or clusters. p̂i estimate of pi .
k number of alleles for a locus. p̃i predicted value of pi .
L likelihood. Pr probability.
N population size. µ mean; mutation rate.
Ne effective population size defined π nucleotide diversity.
as the number of alleles transmit- σ standard-deviation.
ted to the next generation. σ 2 variance.
n sample size (number of individu- Σ variance-covariance matrix.
als or of alleles); number of rows Θ the population genetic parameter
in a table or in a matrix. defined as Θ = 2Ne µ.
1
Introduction

1.1 Heredity, Genetics, and Genomics

One of the greatest achievements of biology during the twentieth century
was to discover the mechanisms of heredity. One can hardly imagine all the
theories formulated during many centuries before this discovery. Today, the
double helix of DNA structure is an icon of science, and DNA has now a wide
range of technological and commercial applications.
Heredity and its associated concepts are deeply rooted in the history of
mankind. The emergence of agriculture in different parts of the world between
10,000 and 5000 years ago clearly interacted with knowledge on the heredity of
some plants and animals. During thousands of years, breeders have observed
the consequences of heredity on the domesticated forms of these species. In the
nineteenth century, the scientific investigation of heredity took a significant
turn with the generalization of microscopic observations, the formulation of
the laws of heredity by Mendel, and Miescher’s discovery of “nuclein,” later
renamed nucleic acids. An often overlooked feature of the history of genetics
is that it took almost eight decades to demonstrate that DNA is the support
of heredity, and even the brillant experiments by Avery and his colleagues
were not convincing for some geneticists who thought that heredity was coded
by proteins [52]. Therefore, population genetics originated well before the
discovery of the physical support of heredity.

Historical Landmarks: Heredity, Genetics, and Genomics

1866: Mendel publishes his laws of heredity [184].
1869: Miescher discovers DNA [47].
1944: Avery et al. demonstrate that DNA is the support of heredity [10].
1953: Watson et al. discover the double helix structure of DNA [290].
1961: Crick et al. decipher the genetic code [44].
1973: Gilbert and Maxam publish the first DNA sequencing data [95].
1984: Discovery of microsatellites [295].
1996: First high-throughput sequencing technology [237].
2001: First human genome published [127].
2010: Completion of the first phase of the 1000 Genomes Project [270].

1
2 Introduction

During the twentieth century, the methods used by biologists to study

heredity and later DNA progressively increased in power (see Chap. 2). The
growth of high-throughput sequencing technologies has been a very significant
factor in the development of population genomics. Genomics has taken con-
siderable importance during the last decade as a scientific field and a subject
of considerable societal interest. This development has also impacted the field
of population genetics.
This book adopts the following definitions. Population genetics is the study
of the variation in genotypes among individuals across space and time, includ-
ing the forces behind this variation. Genomics is the study of the structure
and functions of genomes. Population genomics is similar to population ge-
netics but applied to a very large number of loci, usually across the whole
genome of a species. Thus, population genomics can be seen as a “scaled-up”
version of population genetics dealing with at least a large number of loci up
to the whole genome of the species of interest [20].

Historical Landmarks: Population Genetics

1930: Publication of Fisher’s Genetical Theory of Natural Selection [77].
1949: Publication of Wright’s paper on population genetic structure [303].
1955: Kimura’s paper on allele fixation under genetic drift [142].
1966: Empirical studies show the importance of molecular variation in
natural populations [107, 160].
1982: Kingman publishes three founding papers on the coalescent [147].
2005: Publication of the sequentially Markov coalescent facilitating the
analysis of genomic data with recombination [182].

1.2 Principles of Population Genomics

This section starts with some explanations on the units used in this book. The
biological meanings of some terms used here (bases, double-stranded, . . . ) are
explained in the following subsection.

1.2.1 Units
The basic unit of the genome is the base, the part of the nucleotide that
is variable: its symbol is ‘b.’ Genomes can be small or (very) big, thus it is
common to use prefixes borrowed from the International System of Units to
express the size of a genome or the length of a DNA sequence:
Principles of Population Genomics 3

one kilobase = 1 kb = 1,000 (103 ) bases

one megabase = 1 Mb = 1,000,000 (106 ) bases
one gigabase = 1 Gb = 1,000,000,000 (109 ) bases
one terabase = 1 Tb = 1,000,000,000,000 (1012 ) bases
one petabase = 1 Pb = 1,000,000,000,000,000 (1015 ) bases

Note that ‘base(s)’ is often used to actually mean ‘base pair(s)’ since DNA
is almost always double-stranded. Though this is inconsistent, ‘bp’ is usually
used as a symbol instead of ‘b’ when not prefixed, for instance: 1000 bp =
1 kb.
Modern genomics is tightly connected with computer science, so that we
often need to refer to quantity of information, memory usage, or file size. The
basic unit of information is the bit (or binary variable), and the practical unit
is the byte with symbol ‘B’ (one byte = eight bits). The most common units
of memory usage are:
one kilobyte = 1 kB = 103 bytes
one megabyte = 1 MB = 106 bytes
one gigabyte = 1 GB = 109 bytes
one terabyte = 1 TB = 1012 bytes
In this book, we will also use small units of mass because DNA is usually
present in very small quantities (see Chap. 2):
one microgram = 1 µg = 10−6 g
one nanogram = 1 ng = 10−9 g
one picogram = 1 pg = 10−12 g

1.2.2 Genome Structures

DNA is a polymer made of the repetition of nucleotides which are themselves
made of three molecules: phosphate, deoxyribose, and a base (Fig. 1.1). The
base of a nucleotide can be adenine (A), cytosine (C), guanine (G), or thymine
(T). The name “base” comes from the fact that these molecules are basic in
solution (i.e., they release hydroxide ions OH− , by contrast to acids which
release hydrogen ions H+ ). There are actually many bases in nature (e.g.,
caffeine, xanthine), but only those four are found in DNA. The sequence of
these bases in a DNA polymer stores the genetic information required to
carry out the basic functions of life, such as coding the sequences of proteins
or coding regulating sequences.
DNA is almost always double-stranded in a way that the bases of both
polymers (or strands) form specific pairs: A with T and G with C (Fig. 1.2).
The two strands are bound by weak forces sharing electrons between the bases
of a pair: two electrons for an A–T pair, three for a G–C pair.
There are a few exceptions to the rule of DNA as the support of genetic
information: in some viruses, ribonucleic acid (RNA) is the support of infor-
mation. RNA is similar to DNA but with two differences: uracyl (U) is used
Another Random Scribd Document
with Unrelated Content
part, but on absolute trust in the divine promises, so that the
redeemed man, while not ceasing to be a sinner, yet is freely and
fully forgiven, and from the new and joyous relationship to God in
Cl^ist, the new life of willing
 JUSTIFICATION BY FAITH 339 conformity to God's will
flows. It was a re-emphasis of a most important side of the Pauline
teaching. Yet it was not wholly Pauline. To Paul the Christian is
primarily a renewed moral being. To Luther he is first of all a
forgiven sinner ; but Luther, I j / y like Paul, made salvation in
essence a right personal relation* / 1 [ ship to God. The ground and
the pledge of tiiis right relation* ship is the mercy of God displayed
in the sufferings of Christ in man's behalf. Christ has borne our sins.
We, in turn, have imputed to us His righteousness. The German
mystics, especially Tauler, now helped Luther to the conclusion that
this transforming trust was not, as he had sup{>osed, a work in
which a man had a part, but wholly the gift of God. The work
preparatory to his lectures on Romans (1515-1516) but intensified
these convictions. He now declared that the common opinion that
God would infallibly infuse grace into those who did what was ia
their power was absurd and Pelagian. The basb of any work-
righteousness had been overthrown for Luther. While thus convinced
as to the nature and method of salvation, Luther's own peace of soul
was not yet secured. He needed the further conviction of certainty of
his own personal justification. That certainty he had, with Augustine,
denied. Yet as he labored on the latter part of his lectures on
Romans, and even more clearly in the closing months of 1516, his
confidence that the God-given' nature of faith involved personal
assurance became conviction. Thenceforth, in his own personal
experience the sum of the Gospel was the forgiveness of sins. It was
'^good news," filling the soul with peace, joy, and absolute trust in
God. It was absolute dependence on the divine promises, on God's
"word." Luther had not, thus far, consciously worked out a new
system of theology. He had had a deep, vital experience. It was an
experience, however, in no way to be squared with much of current
theories of salvation in which acts, penances, and satisfactions had a
prominent part. No theoretic considerations made Luther a reformer.
He was driven by the force of a profound inward experience to test
the beliefs and institutions which he saw about him. The profundity
and nobility of Luther's experience cannot be doubted. Yet its
applicability as a universal test may be questioned. To him faith was
a vital^ transforming power, a new and vivifying per 
 840 THE NINETY-FIVE THESES sonal relationship. Many
men, however, while sincerely desirous of serving God and their
generation, have no such sense of personal forgiveness, no such
soul-stirring depth of feeling, no such childlike trust. They desire,
with God's aid, to do the best they can. For them ''justification by
faith alone" is either well-nigh meaningless, or becomes an
intellectual assent to religious truth. To enter into the experience of
Luther or of Paul is by no means possible for all. By 1516 Luther did
not stand alone. In the University of Wittenberg his opposition to
Aristotelianism and Scholasticism and his Biblical theology foimd
much sympathy. His colleagues, Andreas Bodenstein of Karlstadt
(1480?-1541), who, unlike Luther, had represented the older
Scholasticism of Aquinas, and Nikolaus von Amsdorf (1483-1565),
now became his hearty supporters. In 1517 Luther had an
opportunity to apply his new conception of salvation to a crying
abuse. Pope Leo X had decided in favor of the claims of Albrecht of
Brandenburg to hold at the same time the archbishopric of Mainz,
the archbishopric of Magdeburg, and the admmistration of the
bishopric of Halberstadt, an argument moving thereto being a lai^
financial payment. To indenmify himself, Albrecht secured as his
share half the proceeds in his district of the indulgences that the
papacy had been issuing, since 1506, for building that new church of
St. Peter which is still one of the ornaments of Rome. A
commissioner for this collection was Johann Tetzel (14701519), a
Dominican monk of eloquence, who, intent on the largest possible
returns, painted the benefits of indulgences in the crassest terms.^
To Luther, convinced that only a right personal relation with God
would bring salvation, such teaching seemed destructive of real
religion. As Tetzel approached— he was not allowed to enter
electoral Saxony — Luther preached against the abuse of
indulgences and, on October 31, 1517, posted on the door of the
castle church, m Wittenberg, which served as the university bulletin
board, his ever memorable Ninety-five Theses.^ Viewed in
themselves, it may well be wondered why the ^ See extracts in
Kidd, Doeumenia lUuatralive of the Continental R^ormor Han, pp.
12-20. s Kidd, pp. 21-26 ; English tr., Wace and Buchheim, LiUher's
Primary Works, pp. 6-14.
 THE CONTROVERSY WIDENS 341 -Ninely-five Theses
proved the spark which kindled the explosion. They were intended
for academic debate. They do not deny the right of the Pope to
grant indulgences. They question the extension of indulgences to
purgatory, and make evident the abuses of current teaching —
abuses which they imply the Pope will repudiate when informed. Yet
though they are far from expressing the full roimd of Luther's
thought, certain principles are evident in them which, if developed,
would be revolutionary of the churchly practice of the day.
Repentance is not an act, but a life-long habit of mind. The true
treasury of the church is God's forgiving grace. The Christian seeks
rather than avoids divine discipline. ''Every Christian who feels true
compunction has of right plenary remission of pain and guilt, even
without letters of pardon." In the restless condition of Germany it
was an event of the utmost significance that a respected, if humble,
religious leader had spoken boldly against a great abuse, and the
Theses ran the length and breadth of the empire. Luther had not
anticipated the excitement. Tetzel answered at once,^ and stirred
Konrad Wimpina (?-1531) to make reply. A more formidable
opponent was the able and disputatious Johann Maier of Eck
(148&-1543), professor of theology in the University of Ingolstadt,
who answered with a tract circulated in manilscript and entitled
Obelisd. Luther was charged with heresy. He defended his position in
a sermon on "Indulgence and Grace" ;^ he replied to Eck. By the
beginning of 1518, complaints against Luther had been lodged in
Rome by Archbishop Albrecht of Mainz and the Dominicans. The
result was that the general of the Augustinians was ordered to end
the dispute and Luther was summoned before the general chapter of
the order met in Heidelberg, in April. There Luther argued against
free will and the control of Aristotle in theology and won new
adherents, of whom one of the most important was Martin Butzer
(Bucer). At about the same time Luther put forth a more elaborate
defense of his position on indulgences, the ReaoliUiones, Luther had
desired no quarrel with the papacy. He seems to have believed that
'the Pope might see the abuses of indulgences as he did, but the
course of events was leading to no choice save the sturdy
maintenance of his views or submission. 1 Kidd, pp. 30, 31. > Ibid.,
p. 29.
 0 y -- 342 LUTHER AND CAJETANUS In June, 1518, Pope
Leo X Issued a citation to Luther to appear in Rome, and
commissioned his censor of books, the Dominican Silvestro Mazzolini
of Prierio, to draw up an opinion on Luther's position. The siunmons
and the opinion reached Luther early in August. Prierio asserted that
''the Roman Church is representatively the college of cardinals, and
moreover is virtually the supreme pontiil," and that "He who says
that the Roman Church cannot do what it actually does r^[arding
indulgences is a heretic.^' ^ Luther's case would apparently have
speedily ended in his condemnation had he not had the powerful
protection of his prince, the Elector Frederick, "the Wise." In how far
Frederick sympathized with Luther's rdigious beliefs at any time is a
matter of controversy; but, at all events he was proud of his
Wittenberg professor, and averse to an almost certain condenmation
in Rome. His political skill effected a change of hearing from the
Roman court to the papal legate at the Reichstag in Augsbiu'g, the
learned commentator on Aquinas, Cardinal Thomas Vio (1469-1534),
known from his birthplace (Gaeta) as Cajetanus. Cajetanus was a
theologian of Eiu-opean repute and seems to have thought the
matter rather beneath his dignity. He ordered Luther to retract,
especially criticisms of the completeness of papal power of
indulgence. Luther refused,* and, on October 20, fled from
Augsburg, having appealed to the Pope "to be better informed."*
Not satisfied with this, Luther appealed from Wittenberg, in
November, 1518, to a future general council.* How little chance of a
favorable hearing he had in Rome is shown by the bull issued the
same month by Leo X defining indulgences in the sense which
Luther had criticised.^ Luther had no real hope of safety. If his
courage was great, his danger was no less so; but he was rescued
from immediate condemnation by the favorable turn of political
events. Meanwhile the summer of 1518 had seen the installation as
professor of Greek in Wittenberg of a young scholar, a native of
Bretten and grandnephew of Reuchlin, Philip Melanchthon (1497-
1560), who was to be singularly united with Luther in their after
work. Never was there a greater contrast. Melanchthon was timid
and retiring; but he was without a superior in scholarship, and under
the strong impress of Luther's per\Kidd, pp. 31, 32. « /Wd., pp. 33-
37. » /Wa., pp. 37-39. « /Wd., p. 40. » /Wd., p. 39.
 THE LEIPZIG DEBATE 343 sonality, he devoted his
remarkable abilities, almost from his arrival in Wittenberg, to the
furtherance of the Lutheran cause. The Emperor, Maximilian, was
now visibly nearing the end of his life, which was to come in
January, 1519, and the turmoil of a disputed election was
impending. Pope Leo X, as an Italian prince, looked with disfavor on
the candidacy of Charles of Spain, or Francis of France, as increasing
foreign influence in Italy, and sought the good-will of the Elector
Frederick, whom he would gladly have seen chosen. It was no time
to proceed against Frederick's favored professor. Leo, therefore, sent
his chamberlain, the Saxon Karl von Miltitz, as his nuncio, with a
golden rose, a present expressive of high papal favor, to the Elector.
Miltitz flattered himself that he could heal the ecclesiastical quarrel
and went far beyond his instructions. On his own motion he
disowned Tetzel, and held an interview with Luther, whom he
persuaded to agree to keep silent on the questions in dispute, to
submit the case, if possible, to learned German bishops, and to write
a humble letter to the Pope.^ Any real agreement was impossible.
Luther's Wittenberg colleague, Andreas Bodenstein of Karlstadt
(14807-1541), had argued in 1518, in opposition to Eck, that the
text of the Bible is to be preferred even to the authority of the whole
church. Eck demanded a public debate, to which Karbtadt agreed,
and Luther soon found himself drawn into the combat, proposing to
contend that the supremacy of the Roman Churdi is unsupported by
history or Scripture. In June and July, 1519, the great debate was
held in Leipzig. Karlstadt, who was an unready disputant, succeeded
but moderately in holding his own against the nimble-witted Eck.
Luther's earnestness acquitted itself much better; but Eck's skill
drove Luther to the admission that his positions were in some
respects those of Huss, and that in condemning Huss the revered
Council of Constance had erred. To Eck this seemed a forensic
triumph, and he believed victory to be his, declaring that one who
could deny the infallibility of a general council was a heathen and a
publican.' It was, indeed, a momentous declaration into which
Luther had been led. He had already rejected the final authority of
the Pope, he now admitted the fallibility of councils. Tliose steps
implied a break with the whole authoritative » Kidd, pp. 41-44. «
Ibid,, pp. 44-61.
 344 LUTHER'S GREAT TREATISES system of the Middle
Ages, and allowed final appeal only to the Scriptures, and to ^e
Scriptures, moreover, interpreted by the individual judgment. Eck felt
that the whole controversy might now be speedily ended by a papal
bull of condemnation, which he now set himself to secure and which
was issued on June 15, 1520.^ Luther was now, indeed, in the thick
of the battle. His own ideas were rapidly crystallizing. Humanistic
supporters, like I Ulrich von Hutten, were now rallying to him as one
who could I lead in a national conflict with Rome. Lather himself was
bei ginning to see his task as a national redemption of Germany
from a papacy which, rather than the individual Pope, he was
coming to regard as antichrist. His doctrine of salvation was bearing
larger fruitage. In his little tract. On Good Works, of May, 1520, after
defining "the noblest of all good works" to be "to believe in Christ,"
he affirmed the essential goodness of the normal trades and
occupations of life, and denounced those who " limit good works so
narrowly that they must consist in praying in church, fasting or
giving alms."* This vindication of the natiu*al hmnan life as the best
field for the service of God, rather than the unnatural limitations of
asceticism, was to be one of Luther's most important contributions
to Protestant thought, as well as one of his most significant
departures from ancient and mediaeval Christian conceptions.
Luther's great accomplishment of the year 1520 and his completion
of his title to leadership were the preparation of three epoch-making
works. The first of these treatises was published in August, entitled
To the Christian Nobility of the Oerman Nation.^ Written with
binning conviction, by a master of the German tongue, it soon ran
tiie breadth of the empire. It declared that three Roman walls were
overthrown by which the papacy had buttressed its power. The
pretended supmority of the spiritual to the temporal estate is
baseless, since all believers are priests. That truth of universal
priesthood casts down the second wall, that of exclusive papal right
to interpret the Scriptures; and the third wall, also, that a
reformatory council can be called by none but the Pope. "A true, free
council" for the reform of the church should be sum1 Kidd, pp. 74-
79. * Robinson, Readings, 2 : 6&-68. s Translated in full in Wace and
Buchheim's, Luther^a Primary Workt, pp. 17-«2.
 LUTHER'S GREAT TREATISES 345 moned by the temporal
authorities. Luther then proceeded to lay down a progranune for
reformatory action, his suggestions being practical rather than
theological. Papal misgovernment, appointments, and taxation are to
be curbed; burdensome offices abolished; German ecclesiastical
interests should be placed under a " Primate of Germany" ; clerical
marriage permitted ; the far too numerous holy days reduced in the
interest of industry and sobriety; beggary, including that of the
mendicant orders, forbidden; brothels closed; luxury curbed; and
theologibal education in the universities reformed. No wonder the
effect of Luther's work was profound. He had voiced what earnest
men had long been thinking. Two months later Luther put forth in
Latin his Babylonish CaptmJty of the Church,^ in which questions of
the highest theological import were handled and the teaching of the
Roman Church unsparingly attacked. The sole value of a sacrament,
Luther taught, is its witness to the divine promise. It seals or attests
the God-given pledge of union with Christ and forgiveness of sins. It
strengthens faith. Tried by the Scripture standard, there are only two
sacraments, baptism and the Lord's Supper, though penance has a
certain sacramental value as a return to baptism. Monastic vows,
pilgrimages, works of merit, are a man-made substitute for the
forgiveness of sins freely promised to faith in baptism. Luther
criticised the denial of the cup to the laity, doubted
transubstantiation, for which he would substitute a theory of
consubstantiation derived from d'Ailli, and especially rejected the
doctrine that the Supper is a sacrifice to God. The other Roman
sacraments, confirmation, matrimony, orders, and extreme unction,
have no sacramental standing in Scripture. It is one of the marvdis
of Luther's stormy career that he was able to compose and issue,
contemporaneously with these intensely polemic treatises, and while
the papal bull was being published in Germany, his third great
tractate of 1520, that On Christian Liberty^ In calm confidence he
presented the paradox of Christian experience: ''A Christian man is
the most free lord of aU, and subject to none; a Christian man is the
most dutiful servant of all, and subject to every one." He is free,
since justified by faith, no longer under the law of works * Luther*8
Primary Works, pp. 141-245. «/6ui.„ pp. 96-137.
 346 LUTHER BURNS THE PAPAL BULL and in new personal
relationship with Christ. He is a servant because bound by love to
bring his life into conformity to the will of God and to be helpful to
his neighbor. In this tract, in an elsewhere unmatched measure, the
power and the limitations of Lutheranism are evident. To Luther the
essence of the Gospel is the forgiveness of sins, wrought through a
faith, which, as with Paul, is nothing less than a vital, personal transf
ormmg relationship of the soul with Christ. It is unquestionr ably the
highest of Christian exi)eriences. Its limitation, as already pointed
out, is that this experience, if r^arded as the sole type of true
religion, is one beyond the practical attainment of many earnest
men. To this tract Luther prefaced a letter to Pope Leo X, which is a
most curious document, breathing good-will to the Pontiff personally,
but full of denunciation of the papal court and its daims for the
papacy, in which the Pope is represented as ''sitting like a lamb in
the midst of wolves." Though Luther's vision was to clarify hereafter
regarding many details, his theological system was thus practically
complete in its main outlines by 1520. Meanwhile Eck and Girolamo
Aleander (1480-1542) had come with the papal bull, as nimcios, to
Grennany. In Wittenberg its publication was refused, and its
reception in large parts of Germany was lukewarm or hostile, but
Aleander secured its publication in the Netherlands, and procured
the burning of Luther's books in Louvain, Li6ge, Antwerp, and
Cologne. On December 10, 1520, Luther answered by burning the
papal bull and the canon law, with the approving presence of
students and citizens of Wittenberg, and without opposition from the
civil authorities. It was evident that a considerable section of
Germany was in ecclesiastical rebellion, and the situation
demanaded the cognizance of the highest authorities of the empire.
On June 28, 1519, while the Leipzig disputation was in progress, the
imperial election had residted in the choice of Maximilian's grandson
Charles V (1500-1558). Heir of Spain, the Netherlands, the Austrian
territories of the house of Habsburg, master of a considerable
portion of Italy, and of newly discovered territories across the
Atlantic, his election as Holy Roman Emperor made him the head of
a territory vaster than that of any single ruler since Charlemagne. It
was an authority greatly limited, however, in Gerznany by the
territorial
 LUTHER AT WORMS 347 powers of the local princes. As yet
Charles was young and unknown, and both sides in the religious
struggles of the day had strong hope of his support. In reality he
was an earnest Roman Catholic, of the type of his grandmotheri
Isabella of Castile, sharing her reformatory views, desirous of
improvement in clerical morals, education, and administration, but
whoUy unsympathetic with any departure from the doctrinal or
hierarchical system of the Middle Ages. He had at last come to
Germany, and partly to regulate his government in that land, partly
to prepare for the war about to break out over the rival claims of
France and Spain in Italy, had called a Reichstag to meet in Worms
in November, 1520. Though there was much other business, all felt
the determination of Luther's case of high importance. The i>apal
nuncio, Aleander, pressed for a prompt condenmation, especially
after the final papal bull against Luther was issued on January 2,
1521. Since Luther was already condenmed by the Pope, the
Reichstag had no duty, Aleander urged, but to make that
condenmation effective. On the other hand, Luther had wide popular
support, and his ruler, the Elector Frederick the Wise, a master of
diplomatic intrigue, was, fortunately for Luther, of the opinion that
the condenmed monk had never had an adequate trial. Frederick,
and otiier nobles, believed that he should be heard before the
Reichstag previous to action by that body. Between the two counsels
the Emperor wavered, convinced that Luther was a damnable
heretic, but politician enough not to oppose German sentiment too
sharply, or to throw away the possible advantage of making the
heretic's fate a lever in bringing the Pope to the imperial side in the
struggle with France. The result was that Luther was smnmoned to
Worms un^er the protection of an imperial safe-conduct. His journey
thither from Wittenberg was well-nigh a popular ovation. On April
17, 1521, Luther appeared before the Emperor and Reichstag. A row
of his bool^ was pointed out to him and he was asked whether he
would recant them or not. Luther requested time for reflection. A
day was given him, and on the next afternoon he was once more
before the assembly. Here he acknowledged that, in the heat of
controversy, he had expressed himself too strongly against persons,
but the substance of what he had written he could not retract,
unless convinced of its wrongfulness by Scripture or adequate
argument. The
 348 LUTHER AT WORMS Emperor, who could hardly believe
that such temerity as to deny the infallibility of a general council was
possible, cut the discussion short. That Luther cried out, "I cannot
do otherwise. Here I stand. God help me, Amen," is not certain, but
seems not improbable. The words at least expressed the substance
of his unshaken determination. He had borne a great historic witness
to the truth of his convictions before the highest tribunal of his
nation. Of his dauntless courage he had given the completest proof.
The judgment of his hearers was divided, but if he alienated the
Emperor and the prelates by his strong and, as it seemed to them,
self-wiUed assertion, he made a favorable impression on many of
the German nobility and, fortunately, on the Elector Frederick. That
prince, though he thought Luther too bold, was confirmed in his
determination that no harm should come to the reformer. Yet the
result seemed a defeat for Luther. A month after Luther had started
on his homeward journey he was formally put under the ban of the
empire, though not till after many of the members of the Reichstag
had l^t. He was to be seized for punishment and his books
burned.^' This ban was never formally abrogated, and Luther
remained the rest of his life under imperial condemnation. Had
Germany been controlled by a strong central authority Luther's
career would soon have ended in martyrdom. Not even an imperial
edict, however, could be executed against the will of a vigorous
territorial ruler, and Frederick the Wise proved once more Luther's
salvation. Unwilling to come out openly as his defender, perhaps
somewhat afraid to do so, he had Luther seized by friendly hands, as
the reformer journeyed homeward from Worms, and carried secretly
to the Wartbitfg Castle, near Eisenach. For months Luther's hiding-
place was practically unknown; but that he lived and shared in the
fortunes of the struggle his ready pen made speedily apparent. His
attacks on the Roman practice grew more intense, but the most
lasting fruit of this period of enforced retirement was his translation
of the New Testament, begun in December, 1521, and published in
September of the following year. Luther was by no means the first to
translate the Scriptiu^s into German, but the earlier versions had
been made from the Vulgate, and were hard and awkward in
expression. Luther's work ^ Kidd, DocumenU, pp. 79-^.
 LUTHER TRANSLATES THE NEW TESTAMENT 349 was not
merely from the Greek, for which the labors of Erasmus gave the
basis, it was idiomatic and readable. It largely determined the form
of speech that should mark future German literature — that of the
Saxon chancery of the time — ^wrought and polished by a master
of popular expression. Few services greater than this translation
have ever been rendered to the development of the religious life of a
nation. Nor, with all his deference to the Word of God, was Luther
without his own canons of criticism. These were the relative
clearness with which his interpretation of the work of Christ and the
method of salvation by faith is taught. Judged by these standards,
he felt that Hebrews, James, Jvde, and Revelation were of inferior
worth. Even in Scripture itself there were differences in value. The
month which saw the beginning of Luther's work as a translator —
December, 1521 — ^witnessed the publication in Wittenberg of a
small volume by Melanchthon, the Loci Com-munes, meaning
Cardinal Points of Theology. With it the systematic presentation of
Lutheran theology may be said to have begun.^ It was to be
enlarged, developed, and modified in many later editions. SECTION
II. SEPARATIONS AND DIVISIONS Luther's sojourn in the Wartburg
left Wittenberg without his powerful leadership; but there were not
wanting many there to continue the ecclesiastical revolution. To his
earlier associates in the university, Karlstadt, Melanchthon, and
Nikolaus von Amsdorf (1483-1565), there had been added, in the
first half of the year 1521, Johann Bugenhagen (1485-1558) and
Justus Jonas (1493-1555). Of these, Karlstadt had unquestionably
greatest natural leadership, but was rash, impulsive, and radical.
Luther had as yet made no changes in public worship or in monastic
life. Yet it was inevitable that demand for such changes should
come. Luther's fiery fellow monk, Gabriel Zwilling (1487?-1558), by
October, 1521, was denouncing the mass and urging the
abandonment of clerical vows. He soon had a large following,
especially in the Augustinian monastery of Wittenberg, many of the
inmates of which now renounced their profession. With equal zeal
Zwilling was soon attacking images. At Christmas, 1521, Karlstadt
celebrated > £2xtracts in Kidd, Documenta, pp. QQ-M.
 350 RADICALISM. LUTHER'S CONSERVATISM the Lord's
Supperb» the castle church, without priestly garb, sacrificial
offeringffelevation of the host, and with the cup offered to the laity.
Auricular confession and fasts were abandoned. Karlstadt taught that
aU ministers should many, and, in January, 1522, took to himself a
wife. He was soon opposing the use of pictures, organs, and the
Gregorian chanting in public worship. Under his leadership the
Wittenberg dty government broke up the ancient rdigious fraternities
and confiscated their property, decreed that the services should be
in (xerman, condemned pictures in the churches, and forbad
beggary, ordering that really needy cases be aided from the city
treasury. The public commotion was augmented by the arrival, on
December 27, 1521, of three radical preachers from Zwickau, chief
of whom were Nikolaus Storch and Markus Thoma Stiibner. These
men claimed immediate divine inspiration, opposed infant baptism,
and prophesied the speedy end of the world. Melandithon was
somewhat shaken by them at first, though their influence in general
has been exaggerated. They undoubtedly added something to a
state of turmoil.^ These rapid changes, followed by a popular attack
on images, were highly displeasing to Elector Frederick the Wise,
and they drew forth the warning protests of German princes and the
imperial authorities. Though Luther was to further, within the next
three or four years, most of the changes whidi Karlstadt and Zwilling
had made, he now felt that his cause was in peril through a
dangerous radicalism. The city government appealed to Luther to
return. The Elector nominally fori>ad him, out of political
considerations, but on March 6, 1522, Luther was once more in
Wittenberg, which thenceforth was to be his home. Eight days of
preaching showed his power. The Gospel, he declared, consisted in
the knowledge of sin, in forgiveness through Christ, and in love to
one's neighbor. The alterations, which had raised the turmoil, had to
do with externals. They should be effected only in a spirit of
consideration of the weak. Luther was master of the situation.
Karlstadt lost all influence and had to leave the city. Many of the
changes were, for the moment, undone, and the
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