WHY

Molecular evolution matters?

Information, graphics and texts mostly after:

Dan Graur, Department of Zoology, Tel Aviv University, Israel
Itai Yanai, Molecular Genetics, Weizmann Institute of Science, Israel
Rose Hoberman, Carnegie Mellon University, USA

Molecular evolution

Two main subjects

The evolution of

molecular entities

e.g., genes, proteins, introns, chromosomal

arrangements
Molecular evolution Molecular evolution

Assumption:
The evolution of

organisms and
biological complexes Life is
e.g., species, higher taxa, coevolutionary
systems, ecological niches, and migratory
monophyletic
patterns, by using molecular data

Molecular evolution Molecular evolution

ancestor Any two ancestor
Some organisms
organisms (5 MYA) have very recent
share a
ancestors.
common
ancestor in
their past

descendant 1 descendant 2
Molecular evolution Molecular evolution
ancestor ancestor
(18 MYA) (120 MYA)
Some have less recent
ancestors…

Molecular evolution Molecular evolution

ancestor
ancestor But, any two organisms
share a common ancestor
(1,500 MYA) in their past

descendant 1 descendant 2
Molecular evolution Molecular evolution

K = number of
substitutions
T = divergence
time

The differences between 1 and 2 are the

result of changes on the lineage leading
to descendant 1 + those on the lineage
leading to descendant 2. K

Molecular evolution Molecular evolution

K = number of K = number of
substitutions substitutions
T = divergence T = divergence
time time

r = Nucleotide r = Nucleotide
substitution rate substitution rate
= número de = número de
substituições por substituições por
posição por ano posição por ano

K K
Molecular evolution Molecular evolution

Molecular evolution Molecular evolution

Homoplasias Homoplasias
Tempo Tempo
A A A A A A A A

A G C T A G C T

A A A A A A A A
 PAST PAST

G G

G C A C
One substitutions happened - one substitution is visible
Two substitutions happened - only one substitution is visible

PAST

A A One substitutions happened

one substitution is visible Two substitutions happened
no visible substitution

Two substitutions happened

Two substitutions happened - no visible substitution only one substitution is visible
 Molecular evolution
! Molecular Evolution !

Estimating Genetic Differences

If all nucleotides are equally
3.0 likely, the observed difference
Expected would plateau at 0.75
differences
Therefore, simply counting
2.0 differences underestimates
distances, because it

Models
Number of mutations

fails to count for multiple hits

1.0
Observed
differences

0
0 25 50 75

Million years

Molecular evolution
Impact of models: 3 sequences
AGC Sequences 1 and 2 differs at 1 out of 3 positions = 1/3
AAC Sequences 1 and 3 differs at 1 out of 3 positions = 1/3
ACC Sequences 2 and 3 differs at 1 out of 3 positions = 1/3
Models of
evolution
1 2 3
1 -
2 0.333 -
3 0.333 0.333 -
Page RDM, Holmes EC
(1998) Molecular Evolution:
a phylogenetic approach
Blackwell Science, Oxford.
http://artedi.ebc.uu.se/course/X3-2004/Phylogeny/Exercises/nj.html
 JC69 model (Jukes-Cantor, 1969) K80 model (Kimura, 1980) or
1
1
-
2 3 Kimura 2P
AGC 2 0.333 -
AGC Kimura's Two Parameter model (K2P) incorporates the
observation that the rate of transitions per site (a) may differ from
AAC 3 0.333 0.333 - AAC the rate of transversions (b), giving a total rate of substitiutions
per site of (a + 2b)(there are three possible substitutions: one
ACC ACC transition and two transversions).
Where P is the proportion of nucleotides that are different (the observed
differences above) in the two sequences and ln is the natural log The transition:transversion ratio a/b is often represented by the
function. To calculate the JC distances from the observed differences letter kappa (k).
above:
In the K2P model the number of nucleotide substitutions per site
is given by:

1 2 3
1 -
where:
2 0.441 - P the proportional differences between the two sequences due to
transitions
3 0.441 0.441 - Q are the proportional differences between the two sequences due to
transitions and transversions respectively.

http://www.bioinf.manchester.ac.uk/resources/phase/manual

K80 model (Kimura, 1980) or Note how the differences caused by the application of
different models give different distances
Kimura 2P Observed 1 2 3
AGC Sequences 1 and 2 differ one transition differences 1 -
AAC
2 0.333 -
AGC 3 0.333 0.333 -
Sequences 1 and 3 differ one transversion
ACC Sequences 2 and 3 differ one transversion
Jukes-Cantor 1 2 3
AAC model 1 -
ACC 2 0.441 -
1 2 3
3 0.441 0.441 -
1 -
2 0.549 - Kimura 2P 1 2 3

3 0.477 0.549 - 1 -
2 0.549 -
3 0.477 0.549 -
Molecular evolution Molecular evolution

Gene sequences accumulate

substitutions at a constant rate, therefore
we can use genes sequences to time
divergences.

This is referred to as a ‘Molecular Clock’

“the rate of molecular evolution is approximately constant

over time in all lineages”

Molecular evolution Molecular evolution

Molecular divergence is The idea of a molecular clock was initially

suggested by Zuckerkandl and Pauling in

ROUGHLY correlated 1962.

They noted that rates of amino acid

replacements in animal haemoglobin were
with divergence of time roughly proportional to real time, as judged
against the fossil record.
Molecular evolution Molecular evolution
Evidence for rate constancy in haemoglobin
1
Shark

Genetic Distances to human

0.75 Carp
Frog

0.5 Aligator
Chicken

0.25 Quoll
Cow (calibration point 80Myr ago)
The “constancy” of the molecular clock is particularly striking Baboon
0
when compared to the obvious variation in the rates of Species
morphological evolution (e.g. the existence of “living fossils”).
Molecular divergence time

from Zuckerkandl and Pauling (1965)

Molecular evolution Molecular evolution

Evidence for rate constancy in haemoglobin Evidence for rate constancy in haemoglobin
600 1 600
Shark Shark
500 500

Genetic Distances to human

Carp 0.75 Carp
Million Years

400

Million Years
Frog 400
Frog

Aligator 300 0.5 300

Aligator
Chicken Chicken
200 200
Quoll 0.25 Quoll
100
Cow (calibration point 80Myr ago) 100
Cow (calibration point 80Myr ago)
Baboon
0 Baboon
Species 0 0
Species
Fossil divergence time
Molecular divergence time
Fossil divergence time
Molecular evolution Molecular evolution

A Hipótese do
Relógio Molecular

Calibrations
• A quantidade de diferenças genéticas entre
sequências é função do tempo desde a
separação.
• A taxa de mutação é (suficientemente)
constante para estimar tempos de divergência

Molecular evolution
Isthmus of Panama
closure? closure

10MaMa
10 3 Ma
3 Ma
Molecular evolution
! Calibrating the molecular clock !

Phylogeny of Pacific (P) and

ATLANTIC
Caribbean (C) species pairs of Alpheus.
closure of Isthmus of Panama ≈ 3.1 MY

In 6 out of 7 cases, the closest

relative of a species was on the other
side of the Isthmus

PACIFIC

3.1% genetic distance

MUTATION RATE?? Knowlton, N., Weigt, L., Solorzano, L., Mills, D.,
& Bermingham, E. (1993). Science, 260 (5114),
1629.

Results Isthmus of Panama

West Atlantic
East Pacific
closure
Constraint

23 Ma 10MaMa
10 3 Ma

30 20 10 0 Ma
Time 3
Calibration Complexities

Molecular clock
Cannot date fossils perfectly not
Fossils usually not direct ancestors Universal
branched off tree before (after?) splitting
event.
Impossible to pinpoint the age of last
common ancestor of a group of living
species

Rate of molecular evolution can differ between

Mean Rate of nucleotide positions
genes
Nucleotide genomic regions
Substitution in genomes within species (nuclear vs organelle)
species
Mammalian Genomes over time

1% / 106 years
 Rate Heterogeneity
Rate of molecular evolution can differ between
nucleotide positions among lineages
genes
genomic regions
genomes within species (nuclear vs organelle) Cause Reason
species Repair e.g. RNA viruses have error-
over time mechanisms prone polymerases
If not considered, introduces bias into time
estimates Metabolic rate More free radicals
Generation time Copies DNA more frequently
Population size Effects mutation fixation rate

Evolution is a very slow process at

the molecular level
How different regions of
the genome may vary?
 Number of nucleotide substitutions per site
H

0
1
2
3
4
5
6
7
8
9
is
to
al na
ph H
H a-a 4
is c
H ton tin
is a
to
H na H3
is
to H2
na B
H
2A
H
G PT
as
N
eu In trin
Pa Me rop su
ra ta hy lin
th llo si
Fi yro thio n II
br id n
in h in
al H og orm II
ph em en o
a- og ga ne
G l
ly Me obi mm
co t n a
pr all alp
ot oth h
H ein ion a
em h in
o orm I
Se glob on
G rum in b e
r e
al owt alb ta
ph h u
a h m
al -La orm in
ph c o
a- tal ne
Fe bu
regions

to mi
Rates of

In pr n
o
te
rfe Pr tein
In ro ola
te n c
rfe a tin
In ron lpha
t

S
NS
R erfe alp 1
el
ax ron ha
in b 2
C eta
In pe 1
Protein-Coding

te p
Functional vs non-functional
Substitutions in

rfe R tide
ro el
n ax

Page & Holmes p240

ga in
m
m
Synonymous vs non-synonymous

Mean synonymous rate

Mean non-synonymous rate

substitutions per site per year

4.44 ± 1.36 × 10–9
0.84 ± 0.66 × 10–9
 Functional constraint
=
Degree of intolerance towards
The rate of synonymous mutations
substitution is much larger
than the non-synonymous
rate. The functional constraint defines the range of
alternative residues that are acceptable at a site
without affecting negatively the function or structure of
the gene or the gene product.

Apolipoproteins
Two different examples:
Transportadores de lípidos no sangue.
Domínios constituídos por resíduos
hidrofóbicos
Apolipoproteins

Histones 3
Alterações entre aminoácidos hidrofóbicos (valina – leucina)
permitidas em muitas posições.
 Apolipoproteins Função compatível com
Histones As histonas interagem directamente com
outras histonas ou com o DNA para a substituições entre
formação do nucleossoma.
aminoácidos hidrofóbicos

Histones Manutenção da
Manutenção da compactação e
compactação e
alcalinidade alcalinidade necessárias
necessárias = = poucas substituições.
poucas
substituições.
(H2A,H2B,
H3, H4) Histonas mutam 1000 vezes mais lentamente do
que as apolipoproteínas.
Histonas mutam 1000 vezes mais lentamente do que
as apolipoproteínas.

Functional regions evolve

slower than nonfunctional
regions.

Important proteins evolve

slower than unimportant
ones.
 Rates of
Substitutions in
Non-Coding
regions

Codificante Codificante

Não codificante

Perfil de semelhança de duas sequências de DNA.

Coding regions evolve slower
than noncoding regions.

Spalax ehrenberghi

FACT: S. ehrenberghi aA- WHEN:

crystallin lost its functional role
more than 25 MA ago
Water-soluble structural protein found in
the lens and the cornea of the eye
accounting for the transparency of the
structure (when the mole rat became
The main function of crystallins at least in
the lens of the eye is probably to
subterranean and presumably
increase the refractive index while not
obstructing light.
gradually lost use of its eyes)
FACT: FACT:
The aA-crystallin of S.
The aA-crystallin of S. ehrenberghi possess all the
ehrenberghi evolves 20 times prerequisites for normal function
faster than the aA-crystallins in and expression, including the
other rodents, such as rats, mice, proper signals for alternative
hamsters, gerbils and squirrel. splicing.

S. ehrenberghi aA-crystallin lost its We would expect a

functional role but it could
function... larger
equal
The functional role was lost a long lower
time ago: over 25 MA
mutational rate than the one from
The gene evolves 20 times faster pseudogenes?
than the aA-crystallins in other
rodents
 Notwithstanding…
Pseudogenes?
The aA-crystallin of S.
ehrenberghi evolves slower than
pseudogenes.

? Several explanations...

The genes are functional for the Explanation 1:

vision?
Are the genes functional?
Was the loss of vision more recent
(than 25 MY)? Maybe not all function is lost …

The gene has another function? (e.g.photoperiod perception)

Explanation 1: Explanation 2:

Contradicting evidence Slow evolving gene may be due to a more

recent (than 25 MY) loss of vision.
Photo-reception is lost.

The atrophied eye of Spalax does

NOT respond to light.

Explanation 2: Explanation 2:

Slow evolving gene may be due to a more Contradicting evidence:

recent (than 25 MY) loss of vision.
The aA-crystallin gene is intact as far as
Rate of mutation is affected by the rate of the essential molecular structures for its
mutation before loss of function and after expression are concerned.
nonfunctionalization. Therefore there is
an underestimation of the time of loss. The phylogenies indicate 25MY as the
probable timeframe for the mole vision
impairment.
Explanation 3: Facts:

The aA-crystallin-gene product 1.aA crystallin has been found in other tissues.
serves a function unrelated to that of
the eye (vision).

Facts: Facts:

1.aA crystallin has been found in other tissues. 1.aA crystallin has been found in other tissues.

2.aA crystallin functions as a chaperone that binds 2.aA crystallin functions as a chaperone that binds
denaturing proteins and prevents their aggregation. denaturing proteins and prevents their aggregation.

3.The regions within aA crystallin responsible for

chaperone activity are conserved in the mole rat,
therefore have a lower than expected substitution
rate.