17-02-2020

MOLECULAR EVIDENCES OF
EVOLUTION
Dr. Simran Luthra

EVOLUTIONARY BIOLOGY
CORE COURSE XIV THEORY (CREDITS 4)

Unit 1: 7

Life’s Beginnings: Chemogeny, RNA world, Biogeny, Origin of photosynthesis, Evolution of eukaryotes
Unit 2: 4

Historical review of evolutionary concept: Lamarckism, Darwinism, Neo-Darwinism
Unit 3: 10

Evidences of Evolution: Fossil record (types of fossils, transitional forms, geological time scale, evolution of horse, Molecular
(universality of genetic code and protein synthesising machinery, three domains of life, neutral theory of molecular evolution,
molecular clock ,example of globin gene family, rRNA/cyt c
Unit 4: 8

Sources of variations: Heritable variations and their role in evolution
Unit 5: 13

Population genetics: Hardy-Weinberg Law (statement and derivation of equation, application of law to human Population);Evolutionary forces
upsetting H-W equilibrium.

Natural selection (concept of fitness, selection coefficient, derivation of one unit of selection for a dominant allele, genetic load, mechanism of
working, types of selection, density-dependent selection, heterozygous superiority, kin selection, adaptive resemblances, sexual selection.
Genetic Drift (mechanism, founder’s effect, bottleneck phenomenon; Role of Migration and Mutation in changing allele frequencies
Unit 6: 7

Product of evolution: Micro evolutionary changes (inter-population variations, clines, races, Species concept, Isolating mechanisms, modes of
speciation—allopatric, sympatric, Adaptive radiation / macroevolution (exemplified by Galapagos finches
Unit 7: 2

Extinctions, Back ground and mass extinctions (causes and effects), detailed example of K-T extinction
Unit 8: 6

Origin and evolution of man, Unique hominin characteristics contrasted with primate characteristics, primate phylogeny from Dryopithecus
leading to Homo sapiens, molecular analysis of human origin
Unit 9: 2

Phylogenetic trees, Multiple sequence alignment, construction of phylogenetic trees, interpretation of trees
SUGGESTED BOOKS
1. Ridley, M. Evolution. III Edition.
2. Hall, B.K. and Hallgrimsson, B. Evolution. IV Edition.
3. Campbell, N.A. and Reece J.B. Biology. IX Edition
4. Douglas, J. Futuyma. Evolutionary Biology.
5. Pevsner, J. Bioinformatics and functional genomics. II Edition.

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Molecular evolution: changes in DNA (RNA) of chromosomes which take place over
the history of a species and distinguish the species from its ancestors.

Studied through evolutionary or molecular systematics or phylogenetics.

• Almost every gene in the vertebrate

genome exists in multiple copies
• Gene duplication allows for new functions
to arise without having to start from scratch
• Studies suggest the early in vertebrate
evolution the entire genome was duplicated
at least twice

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MULTIGENE FAMILIES

Groups of similar genes that have arisen by duplication from a common

ancestral gene and that generally retain similar functions.

Related genes may be organized in several clusters at different locations.

These are known as gene families e.g. Globin genes.

Gene clusters and gene families vary in importance in different taxonomic
groups; they seem to be much rarer, for example, in insects than in mammals.

Multigene families are defined as groups of genes with sequence homology
and related overlapping functions, whereas superfamilies are defined as a
group of proteins or genes of common origin with nonoverlapping functions.

If a group of proteins or genes contains a domain of common origin, it is a
superfamily; a protein or a gene may belong to two or more superfamilies.

EVOLUTION OF GENE CLUSTERS

Many genes occur as multigene families (e.g., actin, tubulin, globins, Hox)

Inference is that they evolved from a common ancestor

Families can be
 Clustered-nearby on chromosomes (α-globins, HoxA)
 Dispersed-on various chromosomes (actin, tubulin)
 Both-related clusters on different chromosomes (α,β-globins, Hox
A,B,C,D)

Members of clusters may show stage or tissue-specific expression
- Implies means for co-regulation as well as individual regulation

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Hox genes are more similar in closely related

species and less similar in more distantly related
species. By comparing sequence similarity,
scientists can determine when in evolutionary
history certain duplication events happened, and
where some Hox genes were lost along the way.

Two (or more) identical genes present on the same chromosome are described
as nonallelic copies.

Nonfunctional genes are defined as such by their inability to code for proteins; the
reasons for inactivity vary, and the deficiencies may be in transcription or translation (or
both). They are called pseudogenes and given the symbol Ψ.

Processed pseudogene is an inactive gene copy that lacks introns, contrasted with the
interrupted structure of the active gene. Such genes presumably originate by reverse
transcription of mRNA and insertion of a duplex copy into the genome.

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THE FATE OF DUPLICATED GENES

1. All the copies may retain the original function but may differ in spatial and temporal
expression.
2. Duplicated gene may become functionless-pseudogene.
3. Diverge functionally-neofunctionalization or subfunctionalization.

An alternative fate for gene duplications is for both copies to remain functional, while diverging
in their sequence and pattern of expression and taking on different roles.

This process of “duplication and divergence” almost certainly explains the presence of large
families of genes with related functions in biologically complex organisms, and it is thought to
play a critical role in the evolution of increased biological complexity.

EVOLUTION & MULTIGENE FAMILIES

Gene families with several members show a high level of nucleotide sequence
conservation, eg 16S rRNA, ribosomal genes.

But may have different sequence and functions.

Necessary prerequisites for the evolution of new structures and functions.

May involve large or small regions of the genome

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EVOLUTION OF GLOBIN SUPERFAMILY

The globins are the best studied
Fig. family in terms of sequence
conservation, partly because
21.16 they were one of the first
families for which multiple
members were sequenced, and
partly because some of the
earliest protein structures
solved were globins.

The unmistakable homologies in amino acid sequence and structure among the present-
day globins indicate that they all must derive from a common ancestral gene

ORGANIZATION OF GLOBIN GENES

Related clusters on
different chromosomes
Fig.
21.16

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Developmental variation in gene expression

Gene expression controlled by location

MOLECULAR CLOCK

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• Molecules can estimate the date of common ancestors for which no fossils are known (filling in gaps
in the fossil record)….. invertebrates, bacteria, and viruses
• Molecules can estimate divergence dates when there is no obvious morphological change
(particularly important for microorganisms).
• It also allows us to estimate the timing of events that are too recent to be resolved by fossil
evidence, such as divergences among conspecific populations.

Linus Pauling Emile Zuckerkandl Motoo Kimura

In 1962, Emile Zuckerkandl and Linus Pauling noticed that the rate of amino
acid substitution in hemoglobin is constant over time

In 1965, after several protein sequence (cytochrome c, hemoglobin and
fibrinopeptides) seemed to show this pattern, they proposed the molecular
clock hypothesis

In 1968, neutral theory gives a theoretical backing to this concept

Zuckerkandl & Pauling (1962) noted that the number of amino acid
differences between animal haemoglobins was proportional to species
divergence time, as defined by the fossil record. Thus sequences seem to
provide an accurate evolutionary molecular clock.

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A Constant Rate of Sequence Divergence Is a Molecular Clock

Rates of amino acid substitution in three proteins: fibrinopeptides, hemoglobin, and cytochrome c. The number of amino acid differences (per
100 residues, and corrected for multiple changes at the same residue) is plotted for comparisons between various mammals, birds and reptiles,
mammals and reptiles, reptiles and fish, carp and lamprey, and vertebrates and insects, all lineages of organisms for which fossil data provided
estimates of the time since divergence. Note that while some comparisons of the three proteins are from the same pair of organisms and time
of divergence, the three proteins are evolving at very different rates (i.e., fibrinopeptide the fastest, and cytochrome c the slowest). In addition,
the rough linearity of the rate of accumulation of molecular divergence with time illustrates the molecular clock concept.

The more EARLY in the past an ancestral stock diverged into present day
species, MORE CHANGES accumulate in the amino acids sequences of the
proteins in two contemporary species.

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WHAT IS A MOLECULAR CLOCK?

The molecular clock is based on the molecular clock hypothesis(MCH)

Requires information from:
 Fossil constraints
 Rates of molecular change

The molecular data :
 nucleotide sequences for DNA or
 amino acid sequences for proteins.

Provides information for:
 to deduce the time in geologic history when two species or other taxa diverged.
 to estimate the time of occurrence of events called speciation or radiation. It is
sometimes called a gene clock or evolutionary clock.

Measured by comparing present day species derived from common
ancestors.

Requires estimating change quantitatively.

Requires accurate dating of common ancestors.

MOLECULAR CLOCK

Highly conserved proteins- cyctochrome c, haemoglobin- provide best
molecular clocks for determining
 Rate of evolution
 Trace evolutionary relationships between different groups.

The more early in the past an ancestral stock diverged into present day
species, more changes accumulate in the amino acids sequences of the
proteins in two contemporary species.

Number of amino acid modifications in the line of descent can be used as a
measure of time of divergence of two species from a common ancestor.

E.g. cytochrome c from horses and other mammals differs in 5.1 amino acids.
Since horses diverged from other mammals about 90 million years ago, it
means on an average one amino acid substitution has occurred every 17.6
million years (90/5.1= 17.6).

When molecular changes accumulate at a constant rate the phenomenon is

called as molecular clock.

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Each protein has a distinct rate of evolution depending upon how important its function is.

The less functional constraint on a molecule, the faster it evolves in terms of mutant substitution
than those molecules subject to stronger constraint.

For example, histones bind DNA in chromosomes and regulate DNA activity. Thus, a histones structure
is strictly defined because its ability to bind DNA depends upon its particular structure and shape. The
103 amino acids in this protein are identical for nearly all plants and animals.

Fibrinopeptides can perform their role in blood clotting with almost any amino acid change. The 20
amino acids in this protein differ by 86% between a horse and a human. Fibrinopeptides exhibit a very
fast rate of change because they are subject to less functional constraint.

Therefore, when doing a study, it is essential to select a molecule that is appropriate to the time span
of interest.

Different rates even within a molecule
 16S rRNA-conserved and variable regions

 Proteins-conserved domains, variable loops.

Different rates between proteins and RNAs

ORFs: codon position 3 changes faster than position 1 and 2

Introns change faster than exons.

DNA: transitions more frequent than transversions mutations.

ASSUMPTIONS OF MOLECULAR CLOCK

Mutations at molecular level are incorporated at fixed or regular rates over time.

Fixation of molecular mutations does not occur on their adaptive or selective value but
on a fixed rate.

Rate of fixation of mutation in some gene remains the same throughout any line of
descent.

The lines of descent leading from a common ancestor to all its descendants have similar
rates of fixed mutation.

Molecular clock hypothesis postulates that for any macromolecule (a protein or DNA
sequence), the rate of evolution (measured as the mean number of amino acids or
nucleotide sequence changes per site per year) is approximately constant over time in
all evolutionary lineages.

For example, the gene that codes for the protein alpha-globin experiences base
changes at a rate of 0.56 changes per base pair per billion years.

Constant rates of evolution as a constant clock makes it possible to estimate
divergence times, and to date specific events (such as migration, transmission, etc).

The number of nucleotide substitutions in related genes is estimated to be
proportional to the time since they last shared a common ancestor.

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Nucleotide sequence of a coding region into potential replacement sites and silent
sites:
• At replacement sites, a mutation alters the amino acid that is coded. The effect of the
mutation (deleterious, neutral, or advantageous) depends on the result of the amino
acid replacement.
• At silent sites, mutation only substitutes one synonym codon for another, so there is no
change in the protein.

From the slope: average rate of ~0.096% per million

years (or a UEP of 10.4).

BASIS FOR THE EVOLUTIONARY CLOCK

Sequence divergences form the basis for the molecular clock:
• The sequences of homologous genes in different species vary at:

REPLACEMENT SITES SILENT SITES

(non synonymous changes) (synonymous changes)
• Mutation causes amino acid substitutions • Mutation does not affect the protein sequence
• The effect of the mutation (deleterious, • Mutation only substitutes one synonym codon
neutral, or advantageous) depends on the for another, so there is no change in the
result of the amino acid replacement. protein.
• Account for 75% of a coding sequence • Account for 25% of a coding sequence
• The mutations in replacement sites should • Silent mutations are neutral with regard to the
correspond with the amino acid protein they could affect gene expression via
divergence. the sequence change in RNA.

• Mutation accumulates slowly. • Mutation accumulates 10 times faster.

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• The more distant the taxonomic relationship between

the listed organisms, the more evolutionary time
elapsed from their common ancestor, and the greater
the number of synonymous ("silent") mutations that
do not cause amino acid substitutions.

• Silent mutations occur at random at a constant rate.

• A highly critical selective process is the primary agent

restricting or permitting particular amino acid
replacements.

The rate of divergence can be measured as the percent difference per million
years, or as its reciprocal, the unit evolutionary period (UEP), the time in
millions of years that it takes for 1% divergence to develop.

An average divergence of 10% in the replacement sites of either the a- or β-globin genes of
mammals that have been separated since the mammalian radiation occurred ~85 million years
ago. This corresponds to a replacement divergence rate of 0.12% per million years.

Average replacement divergence between corresponding mammalian and chicken globin genes is
23%. Relative to a separation -270 million years ago, this gives a rate of 0.09% per million years.

Compare the α- with the β-globin genes: >500 million years ago. They have an average
replacement divergence of 50%, which gives a rate of 0.1% per million years.

From the slope: average rate of ~0.096% per million years (or a UEP of 10.4).

The difference between the human β and δ genes is 3.7% for replacement sites. At a UEP of 10.4,
these genes must have diverged 10.4 X 3.7 = 40 million years ago—about the time of the separation
of the lines leading to New World monkeys, Old World monkeys, great apes, and man. All of these
higher primates have both β and 8 genes, which suggests that the gene divergence commenced just
before this point in evolution.

γ and ε genes is 10%, which corresponds to a time of separation ~100 million years ago.

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EVIDENCE FOR PHYLOGENETIC RELATIONSHIPS AMONG PRIMATES, BASED ON THE

GLOBIN PSEUDOGENE

Macaca (old world monkey) and Ateles (New world

Monkey) are outgroups wrt Hominoidea super family that
include remaining forms.

• 76 changes between Homo and its common ancestor with Pan

• 14 changes between that common ancestor and the Gorilla branch
• 70 changes between that common ancestor and the Pongo branch
• Since these hominoids diverged from the lineage leading to Old World monkeys (Cercopithecidae, represented by the
rhesus monkey, Macaca), there have been 76 + 14 + 70 + 150 = 310 base pair changes in the lineage leading to Homo.

Calculate the number of differences (e.g., in base pairs) that have accrued among pairs of
species since their common ancestor.
The average rate of base pair substitution in any lineage can be estimated if we have an
estimate of the absolute time of divergence.
• For example, the oldest fossils of cercopithecoid monkeys are dated at 25 million years
(my) ago, providing a minimal estimate of time since divergence between the rhesus
monkey and the hominoids.
• The number of substitutions per base pair per million years for the rhesus monkey lineage
is 457/10,000 base pairs sequenced/25 My = 1.83 X 10-3 per My, or 1.83 x 10-9 per year.
From this common ancestor to Homo, the average rate has been 310/10,000/25 = 1.24 x
10-3 per My.
• The average rate at which substitutions have occurred in each Iineage is therefore (457 +
310)/2 = 383.5/10,000/25 My, or 1.534 X 10-3 per My.

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suppose the proportion of base pairs that differ between the globin pseudogene sequences of
two primate species is 0.0256.

D= 2rt

 where D is the proportion of base pairs that differ between the two sequences,
 r is the rate of divergence per base pair per My,
 t is the time (in My) since the species' common ancestor,
 the factor 2 represents the two diverging lineages.

If D = 0.0256 and

r = 0.001534, as estimated from the earlier data,

then t= D/2r· = 8.3,

8.3 My is our best estimate of when the two species diverged from their common ancestor.

PROBLEMS WITH MOLECULAR CLOCK:

I. Estimates of evolutionary divergences older than fossil records have a higher
rate of uncertainty.
II. species, genes, parts of genes evolve at different rates(natural selection) for
example,

1. Male-driven evolution: more cell division 2. The mtDNA of warm blooded animals
in the male germ line than the female, seems to evolve faster than that of cold-
leading to faster Y chromosome evolution blooded ones
than the X chromosome (in mammals).

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MOLECULAR CLOCK CALIBRATIONS

Fossil- The earliest known fossil assigned to a lineage provides a minimum age constraint on
the divergence event at the base of its clade. Charles Langley and Walter Fitch (1974) were
among the first to use data from fossils to test the molecular clock hypothesis.

Geologic event: Geological calibrations are assigned to internal nodes based on the
assumption that phylogenetic divergence was caused by a geogrophic barrier.

Sampling Date: Data sets containing sequences isolated at different times, i.e.,
heterochronous data, are calibrated by assigning known sample ages to terminal nodes in the
phylogeny.

Substitution rate: In the absence of external calibrations, a known substitution rate may be
applied to sequence data to convert genetic distance into time. This rate can be estimated by
direct observation of genetic change, provided that the temporal range over which
sequences are sampled is large relative to the rate of mutation.

Secondary calibrations: Secondary calibrations are node ages derived from previous
analyses, applied to an independent data set without reference to the original calibration
used to generate them .

To estimate the evolutionary rate of the mitochondrial gene encoding cytochrome b in

birds, Weir and Schluter chose 74 different calibrations to estimate that cytochrome b
genes in birds evolve at an average rate of approximately 2% per 1 million years,
meaning that any two bird species are diverging from each other at a rate of 2% per 1
million years. This has long been regarded as a standard quantity in genetic studies of
birds and is known as the "2% rule."

For example, the 2% rule has been used to test the hypothesis that many modern
songbird species originated during pronounced glacial cycles over the past 250,000
years.

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ORGANIZATION OF GLOBIN GENES

• Ancestral globin gene (present in primitive animals) was duplicated ~500 mya.
• Mutations accumulated in both genes to differentiate them - α and β present in all higher vertebrates
Fig.
• Further gene duplications produced alternative forms in mammals and in primates
21.16

1. THE PRIMITIVE HEMOGLOBIN

The most primitive oxygen-carrying molecule in animals is a globin polypeptide chain of about 150 amino acids,
which is found in many marine worms, insects, and primitive fish. The hemoglobin molecule in higher
vertebrates, however, is composed of two kinds of globin chains.

It appears that about 500 MYA, during the evolution of higher fish, a series of gene duplications and mutations
occurred. These events established two slightly different globin genes, coding for the α- and β-globin chains in
the genome of each individual. In modern higher vertebrates each hemoglobin molecule is a complex of two α
chains and two β chains.

The four oxygen-binding sites in the α2β2 molecule interact, allowing a cooperative allosteric change in the
molecule as it binds and releases oxygen, which enables hemoglobin to take up and to release oxygen more
efficiently than the single-chain version.

2. THE β-CHAIN EVOLVES

Still later, during the evolution of mammals, the β-chain gene apparently underwent duplication and mutation to
give rise to a second β-like chain that is synthesized specifically in the fetus.

The resulting hemoglobin molecule has a higher affinity for oxygen than adult hemoglobin and thus helps in the
transfer of oxygen from the mother to the fetus.

The gene for the new β-like chain subsequently mutated and duplicated again to produce two new genes, ε and
γ, the ε chain being produced earlier in development (to form α2ε2) than the fetal γ chain, which forms α2γ2.

A duplication of the adult β-chain gene occurred still later, during primate evolution, to give rise to a δ-globin
gene and thus to a minor form of hemoglobin (α2δ2) found only in adult primates

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GLOBIN GENE FAMILY

1. Gene number tends to increase with

evolutionary complexity (fish to humans)
2. Clusters evolve by duplication and
divergence

Total number and types of β-globin genes, and the numbers and
structures of pseudogenes are different.

All of these changes must have occurred since the mammalian
radiation, ~85 million years ago (the last point in evolution common
to all the mammals).

Gene duplication, rearrangement, and variation is as important a
factor in evolution as the slow accumulation of point mutations in
individual genes.

A gene cluster can expand or contract by unequal crossing-over, when
recombination occurs between nonallelic genes .

When a recombination event occurs between the mispaired gene
copies, it generates nonreciprocal recombinant chromosomes, one of
which has a duplication of the gene and the other a deletion.

A similar general organization is found in other vertebrate globin

gene clusters, but details of the types, numbers, and order of
genes all vary.

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From the organization of globin genes in a variety of species,

we should be able to trace the evolution of present globin
gene clusters from a single ancestral globin gene.

The leghemoglobin gene of plants, which is related to the

globin genes, may represent the ancestral form.

The furthest back that we can trace a globin gene in modern

form is provided by the sequence of the single chain of
mammalian myoglobin, which diverged from the globin line
of descent ~800 million years ago. The myoglobin gene has
the same organization as globin genes, so we may take the
three-exon structure to represent their common ancestor.

Some "primitive fish" have only a single type of globin

chain, so they must have diverged from the line of evolution
before the ancestral globin gene was duplicated to give rise
to the α and β variants. This appears to have occurred ~500
million years ago, during the evolution of the bony fish.

The next stage of evolution is represented by the state of

the globin genes in the frog X. laevis, which has two globin
clusters. However, each cluster contains both α and β
genes, of both larval and adult types. The cluster must
therefore have evolved by duplication of a linked α-β pair,
followed by divergence between the individual copies.
Later the entire cluster was duplicated.

The amphibians separated from the mammalian/avian line

~350 million years ago, so the separation of the α- and β-
globin genes must have resulted from a transposition in the
mammalian/avian forerunner after this time. This probably
occurred in the period of early vertebrate evolution.

Since there are separate clusters for α and β globins in both

birds and mammals, the α and β genes must have been
physically separated before the mammals and birds
diverged from their common ancestor, an event that
occurred probably ~270 million years ago.

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• Cytochrome c is an enzyme whose function depends most critically on a few amino acid residues
at the active site, which bind to its heme cofactor.
• Consequently, these active site residues rarely vary, even though amino acids around them
change.
• Of 104 residues, only Cys-17, His-18 and Met-80 are totally invariant.
• In other places variation is low; large, nonpolar, amino acid residues always fill positions 35 and
36.
• Cytochrome c molecules may vary by as many as 88% of their residues, they retain the same 3-D
conformation

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• 16S rRNA is present in all cells

• It has exactly the same function in all cells
• It is conserved enough in sequence & structure to be readily & accurately aligned.
• It contains both rapidly & slowly evolving regions - the fast regions are useful for determining
closely related species, whereas the slow regions are useful for determining distant relationships
• Horizontal transfer of rRNA genes is absent or rare.
• There is a large database (>100,000) of aligned sequences available

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Evolution is a two step process

1. Mutation (random)
2. Selection (non-random)

Detrimental mutation Negative selection Mutation not seen

Beneficial mutation Positive selection Mutation seen

Detrimental mutation Negative selection Mutation not seen

Neutral mutation No selection Mutation may be seen genetic drift

Beneficial mutation Positive selection Mutation seen

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NEUTRAL THEORY

In 1968, Motoo Kimura, propose the neutral theory of molecular evolution
(not organism level….pertains to aa substitutions in DNA)

The molecular clock hypothesis was based on empirical observations, but it
soon received theoretical backing when biologist Motoo Kimura developed the
neutral theory of molecular evolution in 1968.

Kimura suggested that a large fraction of new mutations do not have an effect
on evolutionary fitness, so natural selection would neither favor nor disfavor
them.

Eventually, each of these neutral mutations would either spread throughout a
population and become fixed in all of its members, or they would be lost
entirely in a stochastic process called genetic drift.

Neutral theory applies to molecules and not to the phenotypes of organism

1. Polymorphism within a species, and evolutionary change between species, can be

explained by two processes: natural selection and drift.

Kimura observed that the total rate of nucleotide substitution in a mammalian genome far
exceeds the upper limit of adaptive evolution suggested by Haldane which led him to propose
that most genetic changes have been fixed by random drift rather than positive Darwinian
selection.

He further states that the large amounts of fruit fly and human genetic polymorphism
discovered by using protein gel electrophoresis is consistent with the hypothesis that natural
polymorphisms are largely neutral.

King and Jukes also suggested that most evolutionary changes in DNA sequence are neutral.

2. Molecular clock

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The neutral theory of molecular evolution suggests that most of the genetic variation in
populations is the result of mutation and genetic drift and not selection.

According to this theory, if a population carries several different alleles of a particular

gene, odds are that each of those alleles is equally good at performing its job-in other
words, that variation is neutral: whether you carry allele A or allele B does not affect your
fitness.

FATE OF MUTATIONS
The evolution of living organisms is the consequence of two processes:
• genetic variability generated by mutations, which continuously arise within populations.
• changes in the frequency of alleles within populations over time.
A. Natural selection: partly determines the fate of those mutations that affect the fitness of their carrier
1. New alleles that confer a higher fitness tend to increase in frequency over time until they reach fixation, thus
replacing the ancestral allele in the population. This evolutionary process is called positive or directional
selection.
2. Conversely, new mutations that decrease the carrier's fitness tend to disappear from populations through a
process known as negative or purifying selection.
3. Finally, it may happen that a mutation is advantageous only in heterozygotes but not in homozygotes. Such
alleles tend to be maintained at an intermediate frequency in populations by way of the process known as
balancing selection.

B. Genetic Drift: consider a theoretical population in which all individuals, or genotypes, have exactly the same
fitness. In this situation, natural selection does not operate, because all genotypes have the same chance to
contribute to the next generation. Given that populations do not grow infinitely and that each individual produces
many gametes, it follows that only a fraction of the gametes that are produced will succeed in developing into adults.

Thus, in each generation, allelic frequencies may change simply as a consequence of this random process of
gamete sampling. This process is called genetic drift.

The difference between genetic drift and natural selection is that changes in allele frequency caused by genetic
drift are random, rather than directional.

Ultimately, genetic drift leads to the fixation of some alleles and the loss of others.

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Detrimental mutation Negative selection Mutation not seen

Neutral mutation No selection Mutation may be seen genetic drift

Beneficial mutation Positive selection Mutation seen

A DNA position at which all alleles are selectively equivalent, and where the rate of mutation per
generation is µ.

In a haploid population of size N, Nµ mutations occur at this site at each generation.

Given that there is no selection, all genotypes have the same probability to reach fixation. Under a
neutral model, the probability that an allele or mutation fixes is simply its relative frequency in the
population. For a new mutation in a haploid population, this relative frequency is 1/N; thus, the
probability that a new mutation reaches fixation is simply 1/N (the same reasoning also holds for
diploid species).

The rate of substitution per generation (K) is obtained simply by multiplying the number of
mutations that occur at each generation by their probability of fixation. Thus, for neutrally evolving
sites, the equation becomes the following:
K = Nµ × 1/N = µ

Of course, because of natural selection, advantageous mutations have a higher probability of
fixation than neutral mutations, and deleterious mutations have a lower probability of fixation.
1. It therefore follows that sequences subject to positive selection evolve faster than neutral sites (K > µ),
2. whereas sequences subject to negative selection evolve more slowly (K < u).

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• Kimura suggested that a large fraction of new mutations do not have an effect on evolutionary fitness,
so natural selection would neither favor nor disfavor them. Eventually, each of these neutral mutations
would either spread throughout a population and become fixed in all of its members, or they would be
lost entirely in a stochastic process called genetic drift.
• Kimura then showed that the rate at which neutral mutations become fixed in a population (known as
the substitution rate) is equivalent to the rate of appearance of new mutations in each member of the
population (the mutation rate). Provided that the mutation rate is consistent across species, the
substitution rate would remain constant throughout the tree of life.

• At the molecular level most evolutionary changes and most of the variation within and between
species is not caused by natural selection but by random drift of mutant alleles that are neutral.
• A neutral mutation is one that does not affect an organism's ability to survive and reproduce.
• The neutral theory allows for the possibility that most mutations are deleterious, but holds that
because these are rapidly purged by natural selection, they do not make significant contributions to
variation within and between species at the molecular level. Mutations that are not deleterious are
assumed to be mostly neutral rather than beneficial.
• The theory applies only for evolution at the molecular level, and phenotypic evolution is controlled by
natural selection, as postulated by Charles Darwin.

The neutral theory of molecular evolution holds that although a small minority
of mutations in DNA or protein sequences are advantageous and are fixed by
natural selection, and although many mutations are disadvantageous and are
eliminated by natural selection, the great majority of those mutations that are
fixed are effectively neutral with respect to fitness and are fixed by genetic drift.

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TWO EXTREME VIEWS- SELECTIONIST AND NEUTRALIST

The difference between the two ideas can be understood in terms of the frequency
distribution for the selection coefficients of mutations, or genetic variants.

If the selection coefficient is positive- natural selection favours variant ;

If it is negative, it is elimnated ;

If it is zero, the gene frequencies drift.

SELECTIONISTs NEUTRALISTs PAN-NEUTRALISM

• Exactly neutral mutations •
are rare and there are
enough favorable mutations •
to account for all molecular
evolution.
• Uses natural selection to
explain why •
disadvantageous mutations
are lost & advantageous are
fixed.
Kimura's original neutral • The theory of pan-
theory. neutralism, according to
Neutralists believe there which all mutations are
are more neutral, and selectively neutral.
hardly any selectively • Almost certainly false.
favored mutations. • Cannot explain why
Uses selection to explain different genes & different
why disadvantageous parts of genes evolve at
mutations are lost and drift different rates.
to explain how new
mutations are fixed.

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Neutral theory says-

 That majority of molecular evolution is driven by neutral drift but that does
not mean that majority of mutations are neutral.
 that evolution at the level of DNA and proteins is dominated by random
processes. Natural selection is still needed to explain adaptation.

An important fraction of molecular evolution is almost certainly driven by
selection i.e.,the fraction of molecular evolution that occurs during the evolution
of adaptations.

Selectionists and neutralists agree that selection drives adaptive evolution. The
disagreement is over what fraction of molecular evolution is adaptive.

Kimura’s neutral theory holds that effectively neutral mutations that rise to
fixation by drift vastly outnumber beneficial mutations that rise to fixation by
natural selection. Genetic drift, not natural selection, is thus the mechanism
responsible for most molecular evolution.

SUPPORT FOR NEUTRAL THEORY:

1. Pseudogenes Establish a Canonical Rate of Neutral Evolution…. Pseudogenes are functionless
stretches of DNA that result from gene duplications. Because they do not encode proteins,
mutations in pseudogenes should be neutral with respect to fitness. When such mutations
achieve fixation in populations, it should happen solely as a result of drift.

2. Silent Sites Change Faster than Replacement Sites in Most Coding Loci…Both kinds of
substitution accumulated in a linear, clocklike fashion, but the rate of evolution for silent
changes is much higher than the rate of evolution for replacement changes. Because the rate of
neutral substitution equals the rate of neutral mutation, neutral theory can explain the
molecular clock phenomenon if the neutral mutation rate is constant per year.

3. Variation among Loci: Evidence for Functional Constraints…. Kimura and Ohta shows that the
functionally important and constrained histone H4 evolves much more slowly than the
functionally relatively unimportant and unconstrained fibrinopeptides, consistent with the
prediction of the neutral theory.

4. Natural populations are highly polymorphic

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PROBLEMS WITH NEUTRAL THEORY:

1. The stronger influence of generation times on the rate of synonymous evolution than the
rate of non-synonymous evolution.
2. The molecular clock, which is not constant enough.
3. Levels of heterozygosity, which are too constant between species and too low in species with
large population sizes.
4. Observed levels of genetic variation and of evolutionary rates, which are not related in the
predicted way.

Synonymous evolution Non synonymous evolution

• Synonymous changes are nucleotide
changes that do not alter the amino acid.
• Synonymous substitutions occur faster in
species with shorter generation times.
• Synonymous evolution fits the neutral
theory.
• Nucleotide changes that do alter the
amino acid are called non-synonymous.
• Generation time may influence the rate
of non-synonymous evolution in some
genes, or some lineages, but not others.
• Non-synonymous evolution either does
not fit the neutral theory, or does not fit
it so well as synonymous evolution

RATE OF DIVERGENCE

The rate of divergence can be measured as the percent difference per million years, or
as its reciprocal, the unit evolutionary period (UEP), the time in millions of years that it
takes for 1% divergence to develop.

percent difference
Rate of divergence
time of separation

For example, the average replacement divergence between corresponding mammalian

and chicken globin genes is 23%. Relative to a separation of approximately 270 mya,
this gives a rate of 0.09% per million years.
rate= 23% / 270 =0.09%

Similarly, α&βhave been diverging since the individual gene types separated >500
million years ago. They have an average replacement divergence of approximately 50% ,
which gives a rate of 0.1 % per million years.

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1. The neutral theory of molecular evolution suggests that molecular evolution is mainly due to
neutral drift. Alternatively, molecular evolution may be mainly driven by natural selection.
2. Four main observations were originally interpreted in favor of the neutral theory: molecular
evolution has a rapid rate, its rate has a clock-like constancy, it is more rapid in functionally less
constrained parts of molecules, and natural populations are highly polymorphic.
3. Kimura argued that the high rate of evolution, and the high degree of variability of proteins,
would, if caused by natural selection, impose a high genetic load. Neutral drift, however, can drive
high rates of evolution, and maintain high levels of variability, without imposing a genetic load.
4. The constant rate of molecular evolution gives rise to a 'molecular clock'.
5. Neutral drift should drive evolution at a stochastically constant rate; Kimura pointed to the
contrast between uneven rates of morphological evolution and the constant rate of molecular
evolution and argued that natural selection would not drive molecular evolution at a constant
rate.
6. The molecular clock for proteins ticks over according to absolute time rather than generational
time. But for silent changes in DNA, lineages with shorter generation times probably evolve faster.
Neutral drift should cause the molecular clock to run according to generational, not absolute,
time.
7. Selection can operate without producing impossible genetic loads, and Kimura's original case for
the neutral theory is no longer convincing.

• The neutral theory explains the higher evolutionary rate of functionally less
constrained regions of proteins by the greater chance that a mutation there will be
neutral.
• Selectionists explain the higher evolutionary rate of functionally less constrained
regions of proteins by the greater chance that a mutation there will be a small,
rather than a large, change.
• Pseudogenes and silent changes in third codon positions may be relatively
functionally unconstrained. These parts of the DNA evolve faster than do the first
two positions in codons, and meaningful third base changes. Neutralists attribute
this high rate of evolution to enhanced neutral drift.
• For amino acids encoded by more than one codon, there are consistent biases in the
frequencies of the codons. Changes between the silent codons are therefore not
completely unconstrained.
• The neutral theory predicts a positive relation between the degree of variability of a
molecule and its rate of evolution.

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There are two ways to determine:

Calibration is done using fossil record. Fossil record tells the

time of divergence of two species

Assume that there is 5% differences between two species

This means they have each diverged 2.5%

since their common ancestor
5%

If a fossil or other evidence will let us
calibrate this clock we can convert %
difference to years

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Rate of mutation = change

time

Change = no of nucleotide substitution

total no of nucleotides
Nucleotide substitution

Million of years ago (mya)

Nucleotide substitution versus time since divergence, showing constancy of rate

of molecular evolution

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Drawback

To calibrate a clock, a good fossil record is required because

there are many problems with the fossils like they do not give
complete information ,limited no of characters available for
dentification of a species ,etc.

RELATIVE RATE TEST

In 1973, Sarich and Wilson proposed this test

Test shows that a molecule evolved at same rate in the two lineages connecting
the two modern species with their common ancestor.

This test does not require to know the absolute date of common ancestor. - no
need of fossils

To compare the rate of molecular substitution in lineage a and b, a third species

c, will be used as a reference or an outgroup

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• The relative rate test for constancy of the rate of molecular divergence. Sequences
are obtained for living species A and B
• and for outgroup species E. Y and X represent ancestral species. Lowercase italic
letters represent the number of character differences (e.g., nucleotide changes)
along each branch. The genetic distance between A and E is DAE = a + c + d. That
between Band E is DBE = b + c
• + d. If the rate of nucleotide substitution
• is constant, then a = b, so DAE = DBE. If rate constancy holds throughout the tree,
the distance between any pair of species that have species X as a common ancestor
will equal that between any other such pair of species.

RELATIVE RATE TEST

The time that has elapsed from any common ancestor(lie., any branch point on
a phylogenetic tree) to each of the living species derived from that ancestor is
exactly the same.

Therefore, if lineages have diverged at a constant rate, the number of
changes (sometimes called the GENETIC DISTAnce) along all paths of the
phylogenetic tree from one descendant species to another through their
common ancestor should be about the same.

In the hominoid example, the number of differences between the rhesus
monkey and the various hominoids ranges from 806 (to orangutan) to 767
(human). These numbers are so close that they indicate a fairly constant rate
of divergence, although the human lineage appears to have slowed down
somewhat.

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Drawbacks
• For closely related species test applies but not for distantly related
species. Distantly related taxa often have rather different evolutionary
rates
 For example, the rate of sequence evolution in rodents is two to three
times greater than in primates.
 Can only show that a molecule evolved at a same rate in two lineage
but this Does not prove that molecules always have a constant rate

IMPORTANCE OF MOLECULAR CLOCK

Use to estimate the time of divergence where the fossil record is inadequate or
absent. For eg, fungi don’t make fossils well

Different genes evolve at different rates, which gives us flexibility to date
events throughout the history of life
 evolution of important genes – slow For e.g., histones

 less important gene – fast For e.g., fibrinopeptides

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Histones
1 amino acid change
Pea Cow
Function –bind to DNA
Amino acid – 103
Rate of evolution – slow

Fibrinopeptides
86% amino acid change
Horse Human
Function – role in blood clotting
Amino acid – 20 Rate of evolution – fast

A. GENETIC VARIATION/HERITABLE VARIATION

1. Gene/chromosome duplication/abberations
1. Euploidy

2. Aneuploidy

3. Deletions

4. Duplications

5. Inversions

6. Translocations

7. Gene duplication and divergence

2. Mutation

1. Point mutation

1. Transition

2. Transversion

2. Frame shift mutation

1. Insertion

2. deletions

3. Changes in gene regulation

4. Transposons

5. Horizontal gene transfer

6. Endosymbiotic origin of organelles and their genes.

B. ENVIRONMENTAL VARIATION

C. GENOTYPE-BY-ENVIRONMENT INTERACTION

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