Tracking adaptive evolutionary events in genomic sequences
David A Liberles* and Marta L Wayne'
Addresses: *Department of Biochemistry and Biophysics and Stockholm Bioinformatics Center, Stockholm University, 10691 Stockholm,
Sweden. Department of Zoology, University of Florida, Gainesville, FL 32611, USA.
Correspondence: David A Liberles. E-mail: firstname.lastname@example.org
Published: 29 May 2002
Genome Biology 2002, 3 (6):reviews 1018.1-1018.4
The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2002/3/6/reviews/1018
BioMed Central Ltd (Print ISSN 1465-6906; Online ISSN 1465-6914)
As more gene and genomic sequences from an increasing assortment of species become available, new
pictures of evolution are emerging. Improved methods can pinpoint where positive and negative
selection act in individual codons in specific genes on specific branches of phylogenetic trees. Positive
selection appears to be important in the interaction between genotype, protein structure, function,
and organismal phenotype.
Today's genomes have been shaped both by random evolu-
tionary processes and by selection. Positive selective pressure,
in which particular changes give an organism an advantage
over other organisms, can quickly alter the sequences found
at selected sites within a genome. At the same time, a slower
dynamic process called genetic drift, in which mutations
spread through a whole population (are 'fixed') by chance
rather than because of selection, dictates the changes at
other sites. The combination of positive selection and genetic
drift, plus the purifying selection that keeps advantageous
sequences from being changed, can be analyzed to assess
the effects of selection on genomes and to detect where
specific nucleotide (or codon) positions have been under
different pressures at different points in evolutionary
history. There are many mechanisms by which function
can evolve adaptively in genomes, including changes in
gene expression, splice-site usage, gene duplication, and
many other processes, but we will focus in this article on
There are several methods that can be used to evaluate
selective pressures on protein-coding genes. The most
straightforward approach is to determine the ratio of the rate
of substitutions that change the amino acid (nonsynonymous
substitutions, K a) to those that do not (synonymous substi-
tutions, Ks); the fraction Ka/Ks is also known as dN/dS or co
and provides a quantitative measure of selection . This
approach has been expanded to include consideration of
how different the chemical structure of a substituted amino
acid is to the original one , by comparing more sophisticated
measures of protein and DNA distance (point accepted
mutations and neutral evolutionary distance) , and by
examining sub-regions of a gene that either are close
together along the primary sequence, are close in the three-
dimensional structure, or are picked out as variable or
invariable positions from a multiple sequence alignment
[4,5]. More sophisticated tests, like the Tajima D statistic
and McDonald-Kreitman test, consider Ka/Ks but also take
advantage of single-nucleotide polymorphism data [6,7].
For example, in the McDonald-Kreitman test, the difference
between the ratio of nonsynonymous to synonymous
divergence and the ratio of nonsynonymous to synonymous
polymorphism is used to measure the fraction of sites under
positive selective pressure.
A long debate has raged in the molecular evolution literature
over what fraction of mutations are fixed through drift and
what fraction are fixed through positive selection. In the
196os, Kimura  proposed that molecular change is
selectively neutral, a stance known as 'neutralism'. More
recent studies show clear examples of positive selection of
molecular sequences, leading some researchers to disagree
with Kimura and revert to the previously prevalent view,
selectionism. Using the Ka/Ks approach, an early study 
systematically compared orthologous genes in rat and mouse
and identified only one, the interleukin-3 gene, that was
2 Genome Biology Vol 3 No 6 Liberles and Wayne
clearly under positive selective pressure. A later, similar
study found only two chordate genes under positive selective
pressure prostatic steroid binding protein and snake
neurotoxin . Subsequently, an innovative approach was
introduced that combined phylogeny with Ka/Ks analysis,
allowing the identification of specific branches of phylogenetic
trees that have been under positive selective pressure [10,11].
Systematic analysis using this approach has identified 643
branches of gene family trees in chordates and 228
branches in land plants (embryophytes) that appear to be
under positive selective pressure (when Ka/Ks was calculated
averaging over the whole gene length) . Further, the
functions of the genes we found were not random, but
appeared to be linked to organismal functions for which
selective pressures are thought to play a major role, including
the immune and reproductive systems.
Such genome-wide rate-testing approaches, along with
individually accumulated examples  and a systematic
analysis of divergence after gene duplication in completed
genomes , have begun to point to the importance of
positive selective pressures in shaping the protein-coding
content of genomes. Three recent studies [14-16] have
taken advantage of single nucleotide polymorphism data in
primates and Drosophila and have provided additional
evidence for the importance of positive selection in coding-
sequence evolution. All three conclude that there is evidence
for far more adaptation than had previously been suspected
from various statistical measures.
Smith and Eyre-Walker  started by collecting a sample of
genes for which there were data on sequence polymorphisms
between Drosophila simulans and D. yakuba orthologs.
They excluded some genes that were thought to be under
selection a priori and others because they contained no
polymorphisms and thus were uninformative; this left
35 genes. They then worked from the assumption that all
polymorphic amino-acid variation within a species is
selectively neutral, whereas amino-acid differences
between species (substitutions) could be neutral or
strongly advantageous. A certain number of substitutions
would be expected from the neutral polymorphism rate;
the authors reasoned that if there were more substitutions
than this, the excess must indicate adaptive evolution.
From the original sample of 35 genes, they estimated using
this method that 24% of amino-acid substitutions between
the species were adaptive; this was not statistically signifi-
cant, however. Smith and Eyre-Walker  then excluded
the genes that were contributing most of the variance and
that were viewed as outliers, as they had five or fewer syn-
onymous polymorphic sites. This left a set of 30 genes and
an estimate of adaptive substitutions of 43%. Finally, they
also excluded three genes that individually showed evidence
of adaptive evolution, leaving a set of 27 genes and a sta-
tistically significant estimated adaptive substitution rate
Fay, Wyckoff, and Wu  took the comparison of ratios of
nonsynonymous to synonymous changes (Ka/Ks) within
and between species to a new level of sophistication. They
built on an initial result that the ratio is twice as large for
divergence as for polymorphism, and tested a sophisticated
hypothesis: that the difference between polymorphism and
divergence can be attributed to only a few genes, rather
than being a whole-genome phenomenon. This distinction
is important because genome-wide phenomena could have
multiple alternative explanations, such as changes in selective
constraint, changes in population size or a number of
other possibilities in addition to selection. In contrast, any
phenomenon that varies between the genes in a genome is
more likely to be due to selection. The authors  considered
substitutions between D. melanogaster and its sibling
species, D. simulans. They began with an initial set of
45 genes for which there were data on both polymorphism
and divergence. No genes were excluded for a priori reasons,
as the point of this test was to detect variation in patterns of
selection among genes, precisely the sort of variation that was
excluded by Smith and Eyre-Walker . Fay et al. 
concluded that there are two classes of gene: neutral genes
that are evolving predominantly by drift, and rapidly evolving
genes. The latter comprised approximately 25% of the genes
surveyed and included genes involved in the Drosophila
immune system and reproduction, among others (similar to
the results of a previous study ).
Fay et al.  are less whole-heartedly selectionist in their
conclusions than Smith and Eyre-Walker , in part
because they find evidence that some substitutions that
Smith and Eyre-Walker consider adaptive to actually be
mildly deleterious. In a different study, Fay et al.  found
evidence for mildly deleterious segregating variation in the
human genome as well. Their evidence is that a significant
excess of rare amino-acid polymorphism was found in auto-
somal but not X-linked genes, suggesting that these rare
polymorphisms are mildly deleterious and are being eliminated
more efficiently from the X chromosome because they are
Methods have recently been developed to detect shifts over
time in selective pressures on particular amino acids (sites)
within a protein sequence [17-20]. One study on the mito-
chondrial cytochrome b, ATP synthase A chain, NADH
dehydrogenase subunit 3, and cytochrome oxidase subunit 2
gene families went so far as to indicate that such shifts in
selective pressure are the rule rather than the exception, also
in line with a selectionist model of evolution . Whether
over a short or a long evolutionary time, such adaptive
evolution must be ultimately governed by selective pressures
acting on three-dimensional protein structures. Different sites
within a protein will be subject to different selective pressures
and modeled with a different substitution matrix. This concept
has led to the development of context-dependent substitu-
tion matrices  and subsequently site-class-dependent
substitution matrices  to explicitly model structure
through evolution, and evolution from structure, respectively.
Interestingly, the evolutionary site-dependent substitution
matrices resulting from these studies have no clear correlation
with biophysical properties of the amino acids. The ultimate
general connection between secondary structure, specific
mutations between amino acids with different chemical
functionalities, and changes in binding properties and
enzymatic activities remains an active area of research.
Approaches linking sequence evolution, structural evolution,
and organismal selective pressures have become increasingly
common. For example, Naylor and Gerstein  examined
patterns of variation in the globin genes in different mam-
malian families using both sequence alignments and secondary
and tertiary structural considerations. They found that
regions of the myoglobin sequence differed in their variability
between whales and primates, possibly reflecting the differ-
ences in selective pressure for oxygen binding between the
muscles of the aquatic whales and terrestrial primates. A
preliminary systematic analysis has identified 749
sequences for which automated homology model construction
has produced protein structural context data for mutations
occurring on branches with high Ka/Ks ratios; these structures
now need an automated method to generate and test ecological
hypotheses to explain specific mutations in specific proteins
(, D.A.L. and A. Elofsson, unpublished observations).
Systematic, genome-wide analyses combining sequence,
structural, and organismal evolution will undoubtedly
reveal much of interest about both the mechanisms and
results of selection as we progress further into the genome
Scientific theories can go in cycles, even with the steady
progression of data. Before Kimura presented his controversial
mathematical analysis on the limited newly available protein
sequence data from that time , selectionist arguments
dominated molecular evolution. As genome sequencing data
accumulate, the tide seems to be shifting back to a selectionist
view. The reason the neutral theory had such a tremendous
impact on evolutionary biology is not just that it explained
much of the data well, but that it provided a testable null
hypothesis . Selection at specific times on specific
positions within specific genes in each species may account
for much of the functionally significant change in proteins
that correlates with species diversification, particularly in
genes subject to evolutionary 'arms races' in the fight for
survival and reproduction. Molecular evolution shows a
backdrop of negatively selected and slowly drifting sites
combined with positive selected sites evolving rapidly to
produce new protein-binding specificities, binding kinetics,
and enzymatic catalysis. A core of residues in each protein
might be conserved to retain fold structure and stability,
while other residues may be freer to vary (as seen, for
example, in leptin  and the globins ). As mutations
are fixed during divergence, different core residues may have
to be selected in the new genetic background (sequence
landscape) to retain the desired fold and stability. Ultimately,
this results in distantly related proteins with similar folds
performing chemically similar yet biologically divergent
The 45 genes analyzed by Fay et al.  are a tiny fraction
of the estimated 13,601 proteins in D. melanogaster. We
must wait for truly genome-wide estimates of molecular
polymorphism and divergence if we are to estimate rates
and patterns of adaptive substitutions with confidence. The
datasets that could scarcely be imagined ten years ago will
soon be at our fingertips. As coding-sequence analyses are
carried out on more and more genes, we will see whether the
current support for selection is merely another pendulum
swing in the selectionist/neutralist debate or a true change
in our understanding of evolution. Moreover, future
genome-sequencing efforts will enable us to define better the
molecular evolutionary processes shaping the genomes
themselves, ultimately yielding a better understanding of
functions in genomes at all levels of resolution.
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