Molecular evolution
Reminders:
• First writing assignment due in class on Wednesday
• Exam 2 next Monday, Nov 3
TA extra office hours this week:
Early (1960s) analyses of amino acid variation among different species
(Kimura; Zuckerkandl & Pauling)
e.g., Cytochrome c
Hemoglobin
human horse bird
fish
Kyra: Wed (10/29), 11 am - Noon
Nic: Fri (10/31), 9-10 am
Pu: Fri (10.31), 10-11 am
Cytochrome c
TA review session (Katie and Pu): 2:00 - 3:00 Friday, McMillan 149
Amount of aa differences between species ~correspond to the
length of time since species diverged from a common ancestor.
e.g., horses and humans, good fossil data on time of divergence
Suggested constant, steady rate of amino acid substitution
Molecular clock: constant, steady rate of change at the molecular level
(amino acids, DNA sequences).
e.g., for a hypothetical protein sequenced in several species
Humans and horses last
shared a common ancestor
~45 MY ago. Differ at 6 out of
100 aa sites
reptile-fish
Molecular clock: constant, steady rate of change at the molecular level
(amino acids, DNA sequences).
e.g., for a hypothetical protein sequenced in several species
Humans and horses last
shared a common ancestor
~45 MY ago. Differ at 6 out of
100 aa sites
Humans and fruitflies last
shared a common ancestor
~600 MY ago. Differ at 90 out
of 100 aa sites
reptile-fish
Linear relationship indicates
molecular clock
Humans and fruitflies last
shared a common ancestor
~600 MY ago. Differ at 90 out
of 100 aa sites
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Implications of molecular clock observations…
• Inconsistent with evolution by natural selection:
— Would expect far fewer changes (since the protein’s
function didn’t change — e.g., cytochrome c)
— Would expect changes to be episodic, not steady: associated with
periods of NS (e.g, environmental change, rapid speciation, etc.)
• Suggested that most mutations that arise and go to
fixation do so by genetic drift: selectively neutral
Neutral Theory of Evolution
Most mutations that arise and go to fixation are neutral with
respect to natural selection; fixation by genetic drift.
This doesn’t mean that natural selection isn’t acting on
these genes:
— We assume that most mutations that affect protein function are
deleterious and immediately selected against. Many mutations are
quickly eliminated by natural selection (not observed).
— The Neutral Theory focuses on mutations that arise and go to fixation
Revolutionary at the time, since it had been assumed that
natural selection was the primary mechanism of evolution
The proportion of a gene showing neutral evolution depends on
proportion of sites under functional constraint (where mutations
will likely be deleterious).
— Very few mutations are driven to fixation through natural selection
Molecular clock varies by protein: depends on functional constraint
e.g., functional constraint is lower for a gene that is mostly introns
e.g., functional constraint is lower for a gene where amino acid changes
are less likely to disrupt protein function
Variation in functional constraint within and among genes:
— pseudogenes (no expressed protein): no functional constraints
— noncoding regions (e.g., introns): very few functional constraints
— 3rd codon position (often synonymous): some functional constraints
—1st position (nonsynonymous): high functional constraints
Cyt c is under high
functional constraint:
a low proportion of
mutations evolve
neutrally
Molecular clock will vary for different
genes/proteins, gene regions
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Level of functional constraint varies within a gene
Degree of functional constraint on non-synonymous varies depending on
gene function:
Rates of nucleotide substitition between humans and mice/rats
Influenza A strains with different degrees of divergence
Gene
Lower proportion of
nonsynonymous sites
evolve neutrally: slower
molecular clock
Non Synonymous*
Synonymous*
Histone 3
0.00
6.38
Actin α
0.01
3.13
Thyrotropin
0.33
4.66
Immunoglobulin Ig VH
1.07
5.66
Interleukin I
1.42
4.60
Interferon γ
3.06
5.50
(*avg # substitutions per site per billion years)
For sites that are not under functional constraint…
 Evolution purely by genetic drift:
u = rate of mutation (per gamete per generation)
For a diploid population of size Ne, the number of new
mutations per generation = 2Neu
Rate of fixation at neutral sites equals the mutation rate (u)…
Implications:
(2Neu)(1/2Ne) = u
1. Does not depend on population size!
Large N: more mutants, but lower likelihood that any one is fixed
Recall: probability of fixation of an allele equals its frequency
For a newly arisen allele, this is 1/(2Ne)
(e.g., for N=5, 2N= 10, p=1/10 = 0.1)
So the number of mutations that arise per generation that
eventually get fixed is (2Neu)(1/[2Ne]) = u
Rate of fixation equals the mutation rate = u
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Rate of fixation at neutral sites equals the mutation rate (u)…
Implications:
(2Neu)(1/2Ne) = u
Rate of fixation at neutral sites equals the mutation rate (u)…
This would explain the molecular clock too, right?…
1. Does not depend on population size!
Large N: more mutants, but lower likelihood that any one is fixed
2. Often expect to find variation (polymorphism) at a site:
Recall: time to fixation by drift: 4Ne generations (don’t need to know derivation)
e.g., Ne = 1000
4000 generations to fixation of 1 new mutation…
If u = 1 X 10-9 mutations per gamete per generation,
And if we’re looking at a 1000 bp gene,
Then over a period of 4000 generations we expect 8 new mutations:
Higher
proportion
of neutrally
evolving
sites.
Lower proportion
of neutrally
evolving sites.
(1 X 10-9)(2000 gametes)(4000 generations)(1000 bp) = 8 mutations
2Ne
 Variation!
(=No fixation)
Tomoko Ohta saves the day: she proposes Nearly Neutral Model
Wait! We have a problem…
Rate of fixation equals the mutation rate = u
u = rate of mutation per gamete per generation
NOT per absolute time
But the molecular clock seems to follow absolute time…
Even though
species vary widely
in generation time
Why aren’t more
mutations
accumulating in
lineages with short
generation time??
If we allow for the possibility that most mutations are slightly
deleterious instead of strictly neutral, then the probability of
drifting to fixation will depend on population size:
Small N = drift overrides weak selection, so most mutations
are evolving as if neutral: ‘effectively neutral’
Large N = drift is weak, so most mutations not neutral and are
selected against
Mutations are effectively neutral (i.e., s=0) for s <
1
2Ne
So why does this save the day?…
Absolute time
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Because species with short generation times tend to have larger
populations:
Detecting natural selection on DNA sequences
Using (nearly) neutral evolution as the null hypothesis: look
for deviations from neutral expectations
In other words, look for patterns of evolution that don’t fit
evolution by genetic drift alone.
Short generation time  many mutations per year
 but fewer mutations effectively neutral
s<
1
2Ne
Long generation time  few mutations per year
 but many mutations are effectively neutral
So the difference in generation time is balanced out by N
Method 1. dN/dS ratios
Nature Vol. 335, 8 September 1988
dN = rate of non-synonymous substitutions per site
(measured as # nonsynonymous polymorphisms)
dS = rate of synonymous substitutions per site
(measured as # synonymous polymorphisms)
MHC: major histocompatibility complex
ARS receptor: antigen recognition site
Antigen recognition
site (57 codons)
dN/dS < 1  aa replacements largely deleterious
(e.g., normal functioning gene)
dN/dS = 1  aa replacements are neutral
(e.g., pseudogene, no functional constraint)
dN/dS > 1  aa replacements are advantageous
and are favored by selection
dN
13.3 per 100 sites
ds
3.5 per 100 sites
dN/dS: 3.8
Selection favors amino
acid changes in ARS
(new alleles favored)
MHC protein
Remaining codons
in Exons 2 and 3
1.6
per 100 sites
2.5 per 100 sites
dN/dS: 0.64
Typical for nonsynonymous vs
synonymous sites (functional constraint)
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Nature Genetics 25, 410 - 413 (2000)
Adaptive evolution of the tumour suppressor BRCA1 in humans and chimpanzees
Gavin A. Huttley, Simon Easteal, Melissa C. Southey, Andrea Tesoriero, Graham G. Giles, Margaret R.E. McCredie,
John L. Hopper & Deon J. Venter
One problem with using dN/dS ratios to infer selection: it’s
extremely conservative if you average across the entire gene
e.g., 363 genes examined in mice/rats: only one with dN/dS > 1
Most useful for comparing different domains within a protein:
e.g., abalone lysin protein domains
Recall: sperm
competition to
penetrate egg
rapid evolution.
Suggests selection
dN/dS ratios
Exposed protein
regions are
evolving rapidly:
Black: dN/dS >3.0
Gray: dN/dS <1.0
White: dN/dS ~1.0
dN/dS >3.0
MK test…
Detecting natural selection on DNA sequences…
Adh (alcohol dehydrogenase)
gene in Drosophila species
Method 2. McDonald-Kreitman (MK) test:
Neutral theory: # polymorphic sites within species should be
directly proportional to number of # differences fixed
between species
Might expect
selection on Adh
for alcohol
tolerance in
species whose
larvae live in
fermenting fruit
Within species: look at polymorphic sites (# nonsynon., # synon.)
Between species: look at fixed differences (# nonsynon., #synon.)
MK test reveals selection where dN/dS alone does not:
Within-species
polymorphism
Differences fixed
between species
Nonsynonymous:
2
8
Synonymous:
10
40
These ratios should be equal
under neutral evolution
An excess of
non-synonymous
fixed differences
would indicate
selection driving
amino acid
change.
Within-species
polymorphism
Differences fixed
between species
Nonsynonymous:
2
7
Synonymous:
42
17
By itself, dN/dS just
looks like normal
functional constraint
0.048
(=dN/dS)
Excess nonsynonymous fixed
differences
0.412
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Detecting natural selection on DNA sequences…
Method 3: test for excess of old or new mutations…
With no selection (genetic drift
acting alone), we expect
alleles (=haplotypes) to
continuously arise and to go
extinct
Haplotype tree with no selection:
Haplotype tree with positive selection:
Most alleles will be recent
descendents of favored
allele
Expect mixture of closely related
alleles (=recently diverged from
an ancestral allele), and those
that are more distantly related
(=older common ancestor allele)
* = selectively favored mutation
With directional selection for
an advantageous mutation
(= Positive Selection), we
expect fewer older alleles
than with neutrality
time
Method 3: test for whether there is an excess of old or new
polymorphisms compared to neutral expectations.
Alleles closely related, no
long branches: shallow
haplotype tree
(blue= extinct alleles)
(blue= extinct alleles)
Method 3: test for excess of old or new mutations…
With selection to maintain two
or more allele classes
(=balancing selection), find
maintenance of allele lineages
that would otherwise go extinct
Haplotype tree with balancing selection:
Some alleles will be very
distantly related: long
branches
e.g., heterozygote advantage
negative freq-dep. selection
diversifying selection
(blue= extinct alleles)
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