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Darwinian evolution in the light of genomics


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Comparative genomics and systems biology offer unprecedented opportunities for testing central tenets of evolutionary biology formulated by Darwin in the Origin of Species in 1859 and expanded in the Modern Synthesis 100 years later. Evolutionary-genomic studies show that natural selection is only one of the forces that shape genome evolution and is not quantitatively dominant, whereas non-adaptive processes are much more prominent than previously suspected. Major contributions of horizontal gene transfer and diverse selfish genetic elements to genome evolution undermine the Tree of Life concept. An adequate depiction of evolution requires the more complex concept of a network or ‘forest’ of life. There is no consistent tendency of evolution towards increased genomic complexity, and when complexity increases, this appears to be a non-adaptive consequence of evolution under weak purifying selection rather than an adaptation. Several universals of genome evolution were discovered including the invariant distributions of evolutionary rates among orthologous genes from diverse genomes and of paralogous gene family sizes, and the negative correlation between gene expression level and sequence evolution rate. Simple, non-adaptive models of evolution explain some of these universals, suggesting that a new synthesis of evolutionary biology might become feasible in a not so remote future.
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Published online 12 February 2009 Nucleic Acids Research, 2009, Vol. 37, No. 4 1011–1034
Darwinian evolution in the light of genomics
Eugene V. Koonin*
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health,
Bethesda, MD, USA
Received January 9, 2009; Revised January 30, 2009; Accepted February 4, 2009
Comparative genomics and systems biology
offer unprecedented opportunities for testing cen-
tral tenets of evolutionary biology formulated by
Darwin in the Origin of Species in 1859 and
expanded in the Modern Synthesis 100 years later.
Evolutionary-genomic studies show that natural
selection is only one of the forces that shape
genome evolution and is not quantitatively domi-
nant, whereas non-adaptive processes are much
more prominent than previously suspected.
Major contributions of horizontal gene transfer and
diverse selfish genetic elements to genome evolu-
tion undermine the Tree of Life concept. An ade-
quate depiction of evolution requires the more
complex concept of a network or ‘forest’ of life.
There is no consistent tendency of evolution
towards increased genomic complexity, and when
complexity increases, this appears to be a non-
adaptive consequence of evolution under weak pur-
ifying selection rather than an adaptation. Several
universals of genome evolution were discovered
including the invariant distributions of evolutionary
rates among orthologous genes from diverse gen-
omes and of paralogous gene family sizes, and the
negative correlation between gene expression level
and sequence evolution rate. Simple, non-adaptive
models of evolution explain some of these univer-
sals, suggesting that a new synthesis of evolution-
ary biology might become feasible in a not so
remote future.
Charles Darwin’s book On the Origin of Species that
appeared in London in 1859 (1) was the first plausible,
detailed account of biological evolution ever published,
along with the simultaneous and independent brief out-
lines by Darwin and Alfred Russell Wallace published
the previous year (2–3). Of course, Darwin did not dis-
cover evolution and did not even offer the first coherent
description of evolution—arguably, that honor belongs to
Jean-Baptiste Lamarck whose magnum opus Philosophie
Zoologique (4) was, uncannily, published in the year of
Darwin’s birth. However, Lamarck’s picture of evolution
was based on an innate drive of evolving organisms
toward perfection, an idea that cannot be acceptable
to a rationalist mind. Besides, Lamarck did not proclaim
the universal character of evolution: he postulated multi-
ple acts of creation, apparently, one for each species.
Darwin was the first to present a rational, mechanistic,
and arguably, magnificent picture of the origin of the
entire diversity of life forms ‘from so simple a beginning’,
probably, from a single common ancestor (1). Darwin’s
vision of the evolution of life was sufficiently complete and
powerful to win over or, at least, deeply affect the minds of
most biologists (and scientists in general, and the educated
public at large), so that all research in biology during the
last 150 years developed within the framework set by the
Origin (even when in opposition to Darwin’s ideas).
Darwin’s vision lacked the essential foundation in
genetics because mechanisms of heredity were unknown
in his day (Mendel’s work went unnoticed, whereas
Darwin’s own ideas in this area were less than productive).
The genetic basis of evolution was established after the
rediscovery of Mendel’s laws, with the development of
population genetics in the first third of the 20th century,
primarily, through the pioneering work of Fisher, Wright
and Haldane (5–7). The new, advanced understanding
of evolution, informed by theoretical and experimental
work in genetics, was consolidated in the Modern
Synthesis of evolutionary biology, usually, associated
with the names of Dobzhansky, Julius Huxley, Mayr and
Simpson (8–11). Apparently, the Modern Synthesis (neo-
Darwinism) adopted its mature form during the 1959
centennial celebration for the Origin in Chicago (12–14).
Now, 50 years after the consolidation of the Modern
Synthesis, evolutionary biology undoubtedly faces a
new major challenge and, at the same time, the prospect
of a new conceptual breakthrough (15). If the Modern
Synthesis can be succinctly described as Darwinism
in the Light of Genetics (often referred to as
*To whom correspondence should be addressed. Tel: 301 496 2477 (Ext 294); Fax: 30 480 9241; Email:
ß 2009 The Author(s)
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neo-Darwinism), then, the new stage is Evolutionary
Biology in the Light of Genomics. In this article, I attempt
to outline the changes to the basic tenets of evolutionary
biology brought about by comparative and functional
genomics and argue that, in many respects, the genomic
stage could be a more radical departure from the
neo-Darwinism than the latter was from classic
Darwinism. Of course, to do so, it is necessary first to
recapitulate the principal concepts of evolution proposed
by Darwin and amended by the architects of the Modern
Synthesis. In the rest of the article, I return to each of
these points.
(i) Undirected, random variation is the main process
that provides the material for evolution. Darwin
was the first to allow chance as a major factor
into the history of life, and arguably, that was one
of his greatest insights.
(ii) Evolution proceeds by fixation of the rare beneficial
variations and elimination of deleterious variations:
this is the process of natural selection that, along
with random variation, is the principal driving force
of evolution according to Darwin and the Modern
Synthesis. Natural selection which is, obviously,
akin to and inspired by the ‘invisible hand’ (of the
market) that ruled economy according to Adam
Smith, was the first mechanism of evolution ever
proposed that was simple, plausible, and did
not require any mysterious innate trends. As such,
this was Darwin’s second key insight. The founders
of population genetics, in particular, Sewall Wright,
emphasized that chance could play a substantial
role in the fixation of changes during evolution
not only in their emergence, via the phenomenon
of genetic drift that entails random fixation of neu-
tral or even deleterious changes. Population-genetic
theory indicates that drift is particularly important
in small populations that go through bottlenecks
(6,16). However, the Modern Synthesis, in its ‘har-
dened’ form (13), effectively, rejected drift as an
important evolutionary force, and adhered to a
purely adaptationist model of evolution (17). This
model inevitably leads to the concept of ‘progress’,
gradual improvement of ‘organs’ during evolution,
an idea that Darwin endorsed as a general trend,
despite his clear understanding that organisms are
less than perfectly adapted, as strikingly exemplified
by rudimentary organs, and despite his abhorrence
of any semblance of an innate strive for perfection
of the Lamarckian ilk.
(iii) The beneficial changes that are fixed by natural
selection are ‘infinitesimally’ small, so that evolution
proceeds via the gradual accumulation of these tiny
modifications. Darwin insisted on strict gradualism
as an essential staple of his theory: ‘Natural
selection can act only by the preservation and accu-
mulation of infinitesimally small inherited modifi-
cations, each profitable to the preserved being ...If
it could be demonstrated that any complex organ
existed, which could not possibly have been formed
by numerous, successive, slight modifications, my
theory would absolutely break down.’ [(1), chapter
6]. Even some contemporaries of Darwin believed
that was an unnecessary stricture on the theory. In
particular, the early objections of Thomas Huxley
are well known: even before the publication of the
Origin Huxley wrote to Darwin ‘‘You have loaded
yourself with an unnecessary difficulty in adopting
Natura non facit saltum so unreservedly’ (18).
(iv) An aspect of the classic evolutionary biology that
is related but not identical to the principled gradu-
alism is uniformitarianism (absorbed by Darwin
from Lyell’s geology), that is, the belief that the
evolutionary processes remained, essentially, the
same throughout the history of life.
(v) Evolution of life can be presented as a ‘great tree’,
as epitomized by the single, famous illustration of
the Origin [(1), Chapter 4].
(vi) A corollary of the single tree of life (TOL) concept
that, however, deserves the status of a separate prin-
ciple: all extant diversity of life forms evolved from
a single common ancestor [or very few ancestral
forms, under Darwin’s cautious formula (1), chapter
14] that much later was dubbed the Last Universal
Common (Cellular) Ancestor (LUCA) (19).
Obviously, evolutionary biologists did not stay idle during
the 40-year span that separated the consolidation of the
Modern Synthesis and the coming of age of evolutionary
genomics; below I briefly summarize what appear to be the
key advances (undoubtedly, this brief account is incom-
plete and might be considered somewhat subjective).
Molecular evolution and phylogeny
The traditional phylogeny that fleshed out Darwin’s con-
cept of the TOL was based on comparisons of diagnostic
features of organisms’ morphology, such as, for instance,
skeleton structure in animals and flower architecture in
plants (20). The idea that the actual molecular substrate
of evolution that undergoes the changes acted upon by
natural selection (the genes, simply put) could be com-
pared for the purpose of phylogeny reconstructions
did not enter the minds of evolutionary biologists for
the obvious reason that (next to) nothing was known on
the chemical nature of that substrate and the way it
encoded the phenotype of an organism. Moreover, the
adaptationist paradigm of evolutionary biology seemed
to imply that genes, whatever their molecular nature,
would not be well conserved between distant organisms,
given the major phenotypic differences between them, as
emphasized in particular by Mayr, one of the chief archi-
tects of the Modern Synthesis (21).
The idea that DNA base sequence could be employed
for evolutionary reconstruction seems to have been first
expressed in print by Crick, appropriately, in the same
seminal article where he formulated the adaptor hypothe-
sis (22). The actual principles and the first implementation
of molecular evolutionary analysis were given a few years
Nucleic Acids Research, 2009, Vol. 37, No. 4
later by Zuckerkandl and Pauling who directly falsified
Mayr’s conjecture by showing that the amino-acid
sequences of several proteins available at the time, such
as cytochrome c and globins, were highly conserved even
between distantly related animals (23,24). Zuckerkandl
and Pauling also proposed the concept of molecular
clock, a relatively constant rate of evolution of the
sequence that they predicted to be characteristic of each
protein in the absence of functional change. In the next
few years, primarily, through the efforts of Dayhoff and
coworkers, it has been demonstrated that protein sequence
conservation extended to the most diverse life forms, from
bacteria to mammals (25–27).
The early phase of molecular evolution research culmi-
nated in the work of Woese and coworkers who revealed
the conservation of the sequences of certain molecules,
above all, ribosomal RNA in all cellular life forms, and
their suitability for phylogenetic analysis (28). The crown-
ing achievement in this line of study was the entirely unex-
pected discovery of the third domain of life—archaea—
that includes organisms previously lumped with bacteria
but shown to be highly distinct by the phylogenetic ana-
lysis of rRNA (29,30). As a result of these studies, a grow-
ing tendency developed to equate the phylogenetic tree
of rRNA, with its three-domain structure (31), with the
‘TOL’ envisaged by Darwin and first explicated by
Haeckel (28,32,33). However, even in the pre-genomic
era, it became clear that not all trees of protein-coding
genes have the same topology as the rRNA tree; the
causes of the discrepancies remained murky but there
thought to involve horizontal gene transfer (HGT) (34).
The neutral theory and purifying selection
Arguably, the most important conceptual breakthrough in
evolutionary biology after the Modern Synthesis was the
neutral theory of molecular evolution that is usually asso-
ciated with the name of Kimura (35,36) although a similar
theory was simultaneously and independently developed
by Jukes and King (37). Originally, the neutral theory was
derived as a development of Wright’s population-genetic
ideas on the importance of genetic drift in evolution.
According to the neutral theory, a substantial majority
of the mutations that are fixed in the course of evolution
are selectively neutral so that fixation occurs via random
drift. A corollary of this theory is that gene sequences
evolve in an approximately clock-like manner (in support
of the original molecular clock hypothesis of Zuckerkandl
and Pauling) whereas episodic beneficial mutations subject
to natural selection are sufficiently rare to be safely dis-
regarded for a quantitative description of the evolutionary
process. Of course, the neutral theory should not be taken
to mean that selection is unimportant for evolution. What
the theory actually maintains is that the dominant
mode of selection is not the Darwinian positive selection
of adaptive mutations, but stabilizing, or purifying selec-
tion that eliminates deleterious mutations while allowing
fixation of neutral mutations by drift (17).
Subsequent studies refined the theory and made it more
realistic in that, to be fixed, a mutation needs not to be
literally neutral but only needs to exert a deleterious
effect that is small enough to escape efficient elimination
by purifying selection—the modern ‘nearly neutral’ theory
(38). Which mutations are ‘seen’ by purifying selection as
deleterious critically depends on the effective populations’
size: in small populations, drift can fix even mutations
with a significant deleterious effect (16). The main empir-
ical test of the (nearly) neutral theory comes from mea-
surements of the constancy of the evolutionary rates in
gene families. Although it was repeatedly observed that
molecular clock is significantly over-dispersed (39,40),
such tests strongly suggest that the fraction of neutral
mutations among the fixed ones is, indeed, substantial
(36). The (nearly) neutral theory is a major departure
from the Modern Synthesis selectionist paradigm as it
explicitly posits that the majority of mutations fixed
during evolution are not affected by Darwinian (positive)
selection (Darwin seems to have presaged the neutralist
paradigm by remarking that selectively neutral characters
would serve best for classification purposes (1); however,
he did not elaborate on this idea, and it has not become
part of the Modern Synthesis).
Importantly, in the later elaborations of the neutral
theory, Kimura and others realized that mutations that
were (nearly) neutral at the time of fixation were not indif-
ferent to evolution. On the contrary, such mutations
comprised the pool of variation that can be tapped into
by natural selection under changed conditions, a phenom-
enon that could be potentially important for macroevolu-
tion (17,41).
Selfish genes, junk DNA and mobile elements
Although this was rarely stated explicitly, classic genetics
certainly implies that (nearly) all parts of the genome
(all nucleotides in more modern, molecular terms) have
a specific function. However, this implicit understanding
came into doubt in the 1960–70s owing to accumulating
data on the lack of a direct correspondence between geno-
mic and phenotypic complexity of organisms. It was
shown that organisms of about the same phenotypic com-
plexity often had genomes that differed in size and com-
plexity by orders of magnitude (the so-called c-value
paradox) (42,43). This paradox was conceptually resolved
by two related, fundamental ideas, those of selfish genes
and junk DNA. The selfish gene concept was first devel-
oped by Dawkins in his eponymous classic book (44).
Dawkins realized, in a striking departure from the organ-
ism-centric paradigm of the Modern Synthesis, that natu-
ral selection could act not only at the level of the organism
as a whole but also at the level of an individual gene.
Under a somewhat provocative formulation of this view,
the genome and the organism are, simply, vehicles for the
propagation of genes. This concept was further advanced
by Doolittle and Sapienza (45), and by Orgel and Crick
(46), who proposed that much if not the most of the geno-
mic DNA (at least, in complex organisms) consisted of
various classes of repeats that originate from the replica-
tion of selfish elements (ultimate parasites, according
to Orgel and Crick). In other words, from the organism’s
standpoint, much of its genomic DNA should be consid-
ered junk. This view of the genome dramatically differs
Nucleic Acids Research, 2009, Vol. 37, No. 4 1013
from the picture implied by the selectionist paradigm
under which most if not all nucleotides in the genome
would be affected by (purifying or positive) selection
acting at the level of the organism.
A conceptually related major development was the dis-
covery, first in plants by McClintock in the 1940s (47), and
subsequently, in animals (48), of ‘jumping genes’, later
known as mobile elements, that is, genetic elements that
were prone to frequently changing their position in the
genome. The demonstration of the ubiquity of mobile ele-
ments suggested the picture of highly dynamic genomes,
ever changing genomes even before the advent of modern
genomics (49,50).
Evolution by gene and genome duplication
The central tenet of Darwin, the gradualist insistence on
infinitesimal changes as the only material of evolution,
was challenged by the concept of evolution by gene dupli-
cation that was developed by Ohno in his classic 1970
book (51). The idea that duplication of parts of chromo-
somes might contribute to evolution goes back to some of
the founders of modern genetics, in particular, Fisher (52),
but Ohno was the first to propose that gene duplication
was central to the evolution of genomes and organisms,
and to support this proposition by a qualitative theory.
Starting from the evidence of a whole-genome duplication
early in the evolution of chordates, Ohno hypothesized
that gene duplication could be an important, if not the
principal, path to the evolution of new biological func-
tions, because after a duplication, one of the gene copies
would be free of constraints imposed by purifying selec-
tion, and would have the potential to evolve a new func-
tion (a phenomenon later named neofunctionalization).
Clearly, the emergence of a new gene as a result of a
duplication, let alone duplication of a genomic region
including multiple genes or whole genome duplication,
are far from being ‘infinitesimal’ changes, and if such
larger events are indeed important for evolution, the gra-
dualist paradigm comes into jeopardy.
Spandrels, exaptation, tinkering and the deficiency of the
Panglossian paradigm of evolution
A spirited, sweeping critique of the adaptationist program
of evolutionary biology was mounted by Gould and
Lewontin in the famous ‘Spandrels of San Marco’ paper
(53). Gould and Lewontin sarcastically described the
adaptationist worldview as the Panglossian paradigm,
after the notorious character in Voltaire’s Candide who
insisted that ‘everything was to the better in this best of
all worlds’ (even major disasters). Gould and Lewontin
emphasized that, rather than hastily concoct ‘just so sto-
ries’ of plausible adaptations, evolutionary biologists
should seek explanations of the observed features of bio-
logical organization under a pluralist approach that takes
into account not only selection but also intrinsic con-
straints, random drift and other factors. The spandrel
metaphor holds that many functionally important ele-
ments of biological organization did not evolve as specific
devices to perform their current functions but rather are
products of non-adaptive architectural constraints—much
like spandrels that inevitably appear at arches of cathe-
drals and other buildings, and can be employed for vari-
ous functions such as housing key elements of the imagery
adorning the cathedral. The process of utilization of span-
drels for biological functions was given the special name
exaptation and was propounded by Gould as an impor-
tant route of evolution (54).
In an even earlier, conceptually related development,
Jacob promoted the metaphor of evolution as tinkering
(55). Jacob’s argument, based, primarily, of the results
of comparative analysis of developmental mechanisms,
that evolution did not act as an engineer or designer but
rather as a tinkerer that is heavily dependent on previous
contingencies for solving outstanding problems and whose
actions, therefore, are unpredictable and unexplainable
without detailed knowledge of preceding evolution.
Evolution in the world of microbes and viruses
Perhaps, the development in biology that had the most
profound effect on the changes in our understanding of
evolution was the extension of evolutionary research into
the realm of bacteria (and archaea) and viruses. Darwin’s
account of evolution and all the developments in evolu-
tionary biology in the subsequent few decades dealt exclu-
sively with animals and plants, with unicellular eukaryotes
(Protista) and bacteria (Monera) nominally placed near
the root of the TOL by Haeckel and his successors (56).
Although by 1950s, genetic analysis of bacteriophages
and bacteria was well advanced, making it obvious that
these life forms had evolving genomes (57), the Modern
Synthesis made no notice of these developments. That
bacteria (let alone viruses) would evolve under the same
principles and by the same mechanisms as animals and
plants, is by no means obvious given all their striking bio-
logical differences from multicellular organisms, and spe-
cifically, because they lack regular sexual reproduction
and reproductive isolation that is crucial for speciation
in animals and plants.
Effectively, prokaryotes became ‘visible’ to evolutionary
biologists in 1977, with the groundbreaking work of
Woese and colleagues on rRNA phylogeny that led to
the identification of archaea and major groups of bacteria
(28,29,58). Shortly afterward, the field of comparative and
evolutionary genomics was born as multiple, complete
genome sequences of diverse small viruses became avail-
able. Despite the fast sequence evolution that is character-
istic of viruses, this early comparative-genomic research
was successful in the delineation of sets of genes that are
conserved in large groups of viruses (59–62). Moreover, a
general principle became apparent: whereas some genes
were conserved across an astonishing variety of viruses,
genome architectures, virion structures, and biological fea-
tures of viruses showed much greater plasticity, so that
gene exchange, even between highly dissimilar viruses,
emerged as a major factor of evolution (62).
The hypothesis that certain organelles of eukaryotic cells,
in particular, the plant chloroplasts, evolved from bacteria
is not that much younger than the Origin: it was proposed
Nucleic Acids Research, 2009, Vol. 37, No. 4
by several researchers in the late 19th century on the basis
of microscopic study of plant cells that revealed con-
spicuous structural similarity between chloroplasts and
cyanobacteria (then known as blue-green alga) and
was presented in a coherent form by Mereschkowsky
in the beginning of the 20th century (63). For the first
two-thirds of the 20th century, this hypothesis of endo-
symbiosis remained a fringe speculation. However, this
perception changed shortly after the appearance of the
seminal 1967 publication of Sagan (Margulis) who sum-
marized the then available data on the similarity between
certain organelles and bacteria, in particular, the striking
discovery of organellar genomes, and came to the conclu-
sion that not only chloroplasts but also the mitochondria
evolved from endosymbiotic bacteria (64). Subsequent
work, in particular, phylogenetic analysis of both genes
contained in the mitochondrial genome and genes encod-
ing proteins that function in the mitochondria and appar-
ently were transferred form the mitochondrial to the
nuclear genome turned the endosymbiosis hypothesis
into a well-established fact (65). Moreover, these phyloge-
netic studies convincingly demonstrated the origin of
mitochondria from a particular group of bacteria, the
a-proteobacteria (66,67). The major evolutionary role
assigned to effectively unique events like endosymbiosis
is, of course, incompatible with both gradualism and
The treasure trove of genomic, metagenomic and
post-genomic data
The fundamental principles of molecular evolution
were established, and many specific observations of
major importance and impact on the fundamentals of
neo-Darwinism were made in the pre-genomic era, the
rRNA-based phylogeny being the premier case in point.
However, the advent of full-fledged genome sequencing
qualitatively changed the entire enterprise of evolutionary
biology. The importance of massive amounts of sequences
for comparison is obvious because this material allows
researchers to investigate mechanisms and specific events
of evolution with the necessary statistical rigor and
to reveal even subtle evolutionary trends. In addition, it
is worth emphasizing that collections of diverse complete
genomes are enormously useful beyond the sheer amount
of sequence data. Indeed, only by comparing complete
genomes, it is possible to clearly disambiguate ortholo-
gous (common descent from a single ancestral gene) and
paralogous (gene duplication) relationship between genes;
to convincingly demonstrate the absence of a particular
gene in a genome, and to pinpoint gene loss events; to
perform a complete comparison of genome organizations
and reconstruct genome rearrangement events (68–71).
Furthermore, for the maximum benefit of evolutionary
biology, it is crucial to sample the genome space both
deeply (that is, obtain genome sequences of multiple, clo-
sely related representatives of the same taxon) and broadly
(obtain representative sequences for as many diverse taxa
as possible). Genomes separated by different evolutionary
distances are most suitable for different tasks, e.g., to
reveal the range of the conservation of a particular gene
or to attempt reconstruction of major evolutionary events,
distantly related genomes have to be compared, whereas
for the quantitative characterization of the selection
process affecting genomes, sets of closely related genomes
are indispensable (72–75). The collection of completely
sequenced genomes that is available on Darwin’s 200th
anniversary consists of thousands of viral genomes, close
to 1000 genomes of bacteria and archaea, and close to 100
eukaryotic genomes (76,77). Although, certainly, not all
major taxa are adequately represented, this rapidly grow-
ing collection increasingly satisfies the demands of both
microevolutionary and macroevolutionary research.
Complementary to the advances of traditional genomics
is the more recent accumulation of extensive metagenomic
data. Although metagenomics typically does not yield
complete genomes, it provides invaluable information on
the diversity of life in various environments (78,79).
Beyond genomics and metagenomics, one of the hall-
marks of the first decade of the new millennium is the
progress of research in functional genomics and systems
biology. These fields now yield high quality, genome-wide
data on gene expression, genetic and protein–protein
interactions, protein localization within cells, and more,
opening new dimensions of evolutionary analysis,
what is sometimes called Evolutionary Systems Biology
(80–82). This new field of research has the potential to
yield insights into the genome-wide connections between
sequence evolution and other variables, such as the rate of
expression, and to illuminate the selective and neutral
components of the evolution of these aspects of genome
Below I attempt to briefly synthesize the main insights
of evolutionary genomics, with an emphasis on the ways
in which these new findings affect the central tenets of
evolutionary biology, in particular, with regard to the rel-
ative contributions of selective and neutral, random
The evolutionary conservation of gene sequences and
structures versus the fluidity of gene composition and
genome architecture
A fundamental observation supported by the entire body
of evidence amassed by evolutionary genomics is that the
sequences and structures of genes encoding proteins and
structural RNAs are, generally, highly conserved through
vast evolutionary spans. With the present collection of
sequenced genomes, orthologs in distant taxa are found
for the substantial majority of proteins encoded in each
genome (83). For instance, recent genome sequencing of
primitive animals, sea anemone and Trichoplax, revealed
extensive conservation of the gene repertoire compared to
mammals or birds, with the implication that the charac-
teristic life span of an animal gene includes (at least) hun-
dreds millions of years (84–86). The results of extensive
comparative analysis of plant, fungal and prokaryotic
genomes are fully compatible with this conclusion
(87,83). Moreover, deep evolutionary reconstructions
Nucleic Acids Research, 2009, Vol. 37, No. 4 1015
suggest that ancestors of hundreds of extant genes were
already present in LUCA (88–92). Conservative recon-
structions of the gene sets of the common ancestors of
the two domains of prokaryotes, bacteria and archaea,
seem to indicate that these ancestral forms that, probably,
existed over 3 billion years ago, were comparable in
genetic complexity, at least, to the simpler of modern
free-living prokaryotes (88,93). From an evolutionary
biology perspective, it appears that the sequences of
many genes encoding core cellular functions, especially,
translation, transcription, replication and central meta-
bolic pathways, are subject to strong purifying selection
that remained in place for extended time intervals, on
many occasions, throughout the 3.5 billion year history
of cellular life.
Remarkably, it is not only the sequence and structure of
the encoded proteins but also features of gene architecture
that are not necessarily directly relevant to the gene func-
tion that are highly conserved across lengthy periods of
life history. In particular, the positions of a large fraction
of introns are conserved even between the most distant
intron-rich genomes of eukaryotes (25–30% conservation
in orthologs from plants and chordates) (94–96), and the
great majority of intron positions are shared by mammals
and basal animals, such as Trichoplax and the sea anem-
one (84,86).
The striking conservation of gene sequences and struc-
tures contrasts the fluidity of the gene composition of
genomes of all forms of life that is revealed by compara-
tive genomics and evolutionary reconstruction. The
(nearly) universal genes make up but a tiny fraction
of the entire gene universe: altogether, this central core
of cellular life consists of, at most, 70 genes, that is, no
more than 10% of the genes in even the smallest of the
genomes of cellular life forms, but typically, closer to 1%
of the genes or less (90,97,98). Although in each individual
genome, the majority of the genes belong to a moderately
conserved genetic ‘shell’ that is shared with distantly
related organisms, within the entire gene universe, the
core and shell genes (or more precisely, sets of orthologous
genes) are a small minority (83). Given this distinctive
structure of the gene universe, evolutionary reconstruc-
tions inevitably yield a dynamic picture of genome evolu-
tion, with numerous genes lost and many others gained via
HGT (mostly, in prokaryotes), and gene duplication (see
Even to a greater extent than the gene composition of
the genomes, the genome architecture, that is, arrange-
ment of genes in a genome shows evolutionary instability
compared to gene sequences (99). With the exception of
the organization of small groups of functionally linked
genes in operons that are, in some cases, shared by dis-
tantly related bacteria and archaea, in part, probably,
owing to extensive HGT (see below), there is, generally,
relatively little conservation of gene order even among
closely related organisms (100,101). In particular, in pro-
karyotes, the long range conservation of gene order com-
pletely disappears even in some groups of closely related
genomes which retain an almost one-to-one correspon-
dence of orthologous genes and over 99% mean sequence
identity between orthologous proteins (75). Thus, in
prokaryotes, the organization of genes beyond the level
of operons is, mostly, determined by extensive random
shuffling, in particular, via inversions centered at the
origin of replication (75,102,103). Eukaryotes show a
somewhat greater conservation of long range genomic
synteny but, even in this case, there are few shared ele-
ments of genome architecture between, for instance, dif-
ferent animal phyla, and none at all between different
kingdoms (99).
The variability of the genome architectures presents an
interesting dilemma to evolutionary biologists: do organ-
isms possess unique genome architectures that are specif-
ically adapted to satisfy unique functional demands of the
respective organisms, or is evolution of genome architec-
ture a mostly neutral process? Although local clustering
of functionally related genes and other patterns suggestive
of functionally relevant gene coexpression were repeatedly
observed, these trends are relatively weak and by no
means ubiquitous (104,105). Thus, the dominant factor
in the evolution of genome architecture appears to be
random, non-adaptive rearrangement rather than purify-
ing or positive selection.
Horizontal gene transfer, the network of evolution and
the Forest replacing of the TOL
Even long before the genomic era, microbiologists realized
that bacteria had the capacity to exchange genetic infor-
mation via HGT, in some cases, producing outcomes of
major importance, such as antibiotic resistance (106).
Multiple molecular mechanisms of HGT have been eluci-
dated including plasmid exchange, transduction (HGT
mediated by bacteriophages) and transformation (107).
These discoveries notwithstanding, HGT was generally
viewed as a minor phenomenon that is important only
under special circumstances and, in any case, was not con-
sidered to jeopardize the concept of the TOL that could be
reconstructed by phylogenetic analysis of rRNA and other
conserved genes. This fundamental belief was challenged
by early results of genome comparisons of bacteria and
archaea which indicated that, at least, in some prokaryotic
genomes, a major fraction of genes were acquired via
demonstrable HGT. The pathogenicity islands and similar
simbiosis islands that comprise over 30% of the genome in
many pathogenic and symbiotic bacteria are the prime
case in point (108–110). Moreover, comparative analysis
of the genomes of hyperthermophilic bacteria and archaea
suggested that even interdomain HGT can be extensive
given shared habitats (111,112).
It can be difficult to demonstrate HGT unambiguously,
and in particular, to differentiate from extensive gene loss,
so the extent of horizontal genetic mobility between pro-
karyotes is still debated (113–115). Nevertheless, as the
genomic database grows, extensive comparative-genomic
and phylogenetic analyses increasingly lead to the conclu-
sion that HGT is virtually ubiquitous in the prokaryotic
world in the sense that there are very few if any ortholo-
gous gene sets whose history is free of HGT (116,117).
The rate of HGT substantially differs for different genes
depending on the gene functions, in part, according to the
so called complexity hypothesis which posits that barriers
Nucleic Acids Research, 2009, Vol. 37, No. 4
might exist for HGT of genes encoding subunits of protein
complexes because dosage imbalance and mixing of
heterologous subunits resulting from such events could
be deleterious (118,119). However, phylogenetic analyses
indicate that even such genes, for instance, those for ribo-
somal proteins and RNA polymerase subunits, are not
immune to HGT (120–122).
The high prevalence of HGT in prokaryotes might, in
part, explain the persistence of the organization of many
operons across broad ranges of organisms, under the self-
ish operon hypothesis (123,124). Although the operons
might be initially selected for the beneficial coexpression
and coregulation of functionally linked genes, it is likely
that they are maintained and disseminated in the prokary-
otic world owing to the increased likelihood of fixa-
tion of an operon following HGT, compared, e.g. to a
non-operonic pair of genes. This scenario presents a nota-
ble case of a combination of selective (coregulation) and
neutral (HGT) forces contributing to the evolution of a
major aspect of genome organization (76,104).
Eukaryotes are different from prokaryotes with respect
to the role played by HGT in genome evolution. In multi-
cellular eukaryotes, where germline cells are distinct from
the soma, HGT appears to be rare (125) although not
impossible (126). Under certain special circumstances,
such as persistence of endosymbiotic bacteria in animals,
transfer of large segments of bacterial genomes to the
genome of the host are indeed common (127,128).
Unicellular eukaryotes do seem to acquire bacterial
genes and exchange genes between themselves on rela-
tively frequent occasions (129–131). Far more crucial,
however, is the major contribution of the genomes of
endosymbionts to the gene complements of all eukaryotes.
The discovery of mitochondria-like organelles and genes
of apparent mitochondrial origin in all thoroughly char-
acterized unicellular eukaryotes, essentially, ascertain that
the last common ancestor of the extant eukaryotes already
possessed the mitochondrial endosymbiont (132,133). In
terms of their apparent phylogenetic affinities, eukaryotic
genes that possess readily identifiable prokaryotic ortho-
logs are sharply split into genes of likely archaeal origin
(primarily, but not exclusively, components of informa-
tion processing systems) and those of likely bacterial
origin (mostly, metabolic enzyme and components of var-
ious cellular structures) (134,135). It is often assumed on
general grounds that the majority of ancestral ‘bacterial’
genes in eukaryotes are of mitochondrial origin but this is
hard to demonstrate directly because in phylogenetic ana-
lysis, these genes cluster with diverse groups of bacteria
(134). These findings are difficult to interpret because the
gene composition of the endosymbiont and its host are not
known, and conceivably, either or both might have already
amassed numerous genes from diverse sources (136). An
even bigger point of uncertainty is the actual scenario of
the origin of eukaryotes [a detailed discussion of this major
subject is outside the scope of this article, see recent reviews
and discussions (133,137–140)]. In a nutshell, the compet-
ing and hotly debated hypotheses are as follows:
(i) The symbiogenetic scenario according to which the
a-proteobacterial ancestor of mitochondria invaded
an archaeal host, and this event triggeredeukaryo-
genesis including the formation of the signature
structural features of the eukaryotic cell such as
the endomembrane system, the cytoskeleton and
the nucleus (138,141).
(ii) The archezoan scenario under which the host of
the mitochondrial endosymbiont was a primitive
eukaryote that already possessed all the principal
features of the eukaryotic cell that evolved without
any relation to endosymbiosis but facilitated the
latter through the phagocytic capability of the
protoeukaryote (137,142).
Regardless of the exact role played by endosymbiosis
in eukaryogenesis, there is no reasonable doubt that the
gene complement of eukaryotes is a chimera comprised of
functionally distinct genes of archaeal and bacteria des-
cents (134,143). Moreover, endosymbiosis apparently
made substantial contributions to the gene complements
of some of the individual major groups of eukaryotes.
Thus, strong evidence was presented of massive HGT of
thousands of genes from a cyanobacterial endosymbiont
(the chloroplast) to the host (plant) genomes (144).
Similarly, genes of apparent algal origin were detected in
chromalveolates that engulfed a red alga in an act of sec-
ondary endosymbiosis (145).
The observations of extensive, ubiquitous and occurring
via multiple routes HGT outlined above lead to a funda-
mental generalization: the genomes of all life forms are
collections of genes with diverse evolutionary histories.
The corollary of this generalization is that the TOL con-
cept must be substantially revised or abandoned because a
single tree topology or even congruent topologies of trees
for several highly conserved genes cannot possibly repre-
sent the history of all or even the majority of the genes
(146–149). Thus, an adequate representation of life’s
history is a network of genetic exchanges rather than a
single tree, and accordingly, the ‘strong’ TOL hypothesis,
namely, the existence of a ‘species tree’ for the entire his-
tory of cellular life, is falsified by the results of compara-
tive genomics.
Certainly, this conclusion is not to be taken as an indi-
cation that the concept of evolutionary tree introduced by
Darwin (1) should be abandoned altogether. First, trees
have the potential to accurately represent the evolution of
individual gene families. Secondly, there exist, beyond
doubt, expansive parts of life’s history for which congru-
ent trees can be obtained for large sets of orthologous
genes, and accordingly, the consensus topology of these
trees qualifies as a species tree. Evolution of major groups
of eukaryotes, such as animals or plants, is the most obvi-
ous case in point but tree-like evolution seems to apply
also to many groups of prokaryotes at relatively shallow
phylogenetic depths. The question remains open whether
evolution of life in its entirety is best depicted as:
(i) a consensus tree of highly conserved genes that
represents a ‘central trend’ in evolution, with HGT
events, including massive ones associated with
endosymbiosis, comprising horizontal connections
between the tree branches [Figure 1A; (150)], or
Nucleic Acids Research, 2009, Vol. 37, No. 4 1017
(ii) a complex network where phases of tree-like evolu-
tion (with horizontal connections) are interspersed
with ‘Big Bang’ phases of rampant horizontal
exchange of genetic information that cannot be
represented as trees in principle [Figure 1B; (151)].
Metagenomics, the expanding world of selfish replicons
and replicon fusion
Metagenomics is a major new direction of genomic
research that pursues (typically, partial, at this stage)
sequencing of the genomes of all life forms that thrive in
a certain habitat. Although a young field, metagenomics
can already claim major advances in characterizing the
bacterial diversity of a variety of habitats, in particular,
those in the oceans (152–154). The direction that I would
like to emphasize as being of particular conceptual impor-
tance for evolutionary biology is metagenomics of viruses
(155). The striking conclusion of several viral metage-
nomic studies is that, at least, in some, particularly,
marine habitats, viruses (bacteriophages) are the most
abundant biological entities, with the number of viral par-
ticles exceeding by an order of magnitude the number
of cells (156,157). Although viral genomes are small com-
pared to genomes of cellular life forms, these metagenomic
results indicate that viral genomes comprise a major part
of the genetic universe that is, at least, comparable in size
with the part taken by genomes of cellular organisms.
Moreover, given that, in viruses with large genomes, a
substantial fraction of genes do not have detectable homo-
logs in current sequence databases (158–160), it seems
most likely that viruses encompass most of the genetic
diversity on this planet. These findings reverberate with
the high prevalence of various classes of mobile elements
within the genomes of many cellular organisms. Indeed,
in mammalian genomes, sequences derived from
mobile elements, primarily, retrotransposons (SINEs and
LINEs) appear to constitute, at least, 40% of the genomic
DNA (161).
Viruses and various other selfish replicons (defined as
genetic elements that do not encode a complete translation
system), such as diverse plasmids and transposons, com-
prise an interconnected genetic pool that is variously
known as the mobilome, the virosphere or the virus
world (76,162–164). The identity of the virus world is
manifested in the existence of a set of ‘hallmark genes’
that encode proteins with key roles in the reproduction
of selfish elements (including viral capsid proteins) and
are present in extremely diverse elements that propagate
in a broad variety of hosts, but not in cellular life forms.
The existence of the distinct pool of hallmark genes that
includes, among others, RNA-dependent RNA and DNA
polymerase, replication enzymes that, probably, antedate
large DNA genomes, strongly suggests that the virus
Bacteria Eukaryota ArchaeaBacteria Eukaryota Archaea
Figure 1. Two views of life history to replace the Tree of Life. (A) The ‘TOL as a central trend’ model. The history of life is represented as a tree,
with connecting lines between branches depicting HGT and shaded trapezoids depicting phases of compressed cladogenesis (276). The origin of
eukaryotes is depicted according to the archezoan hypothesis whereby the host of the mitochondrial endosymbiont was a proto-eukaryotes (arche-
zoan). A cellular Last Universal Common Ancestor (LUCA) is envisaged. (B) The ‘Big Bang’ model. The history of life is represented as a succession
of tree-like phases accompanied by HGT and non-tree-like, Big Bang phases. Connecting lines between tree branches depict HGT and colored
trapezoids depict Big Bang phases (151). The origin of eukaryotes is depicted according to the symbiogenesis model whereby the host of the
mitochondrial endosymbiont was an archaeon. A pre-cellular Last Universal Common Ancestral State (LUCAS) is envisaged. Ar, archaeon (host
of the mitochondrion in b), AZ, archezoan (host of the mitochondrion in a), BB, Big Bang, C, chloroplast, CC, compressed cladogenesis, M,
1018 Nucleic Acids Research, 2009, Vol. 37, No. 4
world coexists with cellular life forms throughout their
history, and possibly, even originates from a primordial,
pre-cellular pool of genetic elements (164).
Although distinct, the virus world constantly interacts
with the genomic pool of cellular life forms, as illustrated
by constant movement of genes between transducing bac-
teriophages, plasmids and bacterial chromosomes (83), or
by the capture of cellular genes (protooncogenes) by
animal retroviruses (165). Recent observations of bacter-
iophage-mediated gene transfer between distantly related
bacteria, even without the phage propagation in the recip-
ient organism, suggest that the gene flow mediated by self-
ish replicons could be more extensive than so far suspected
(166). Importantly, parts of mobile elements are fre-
quently recruited (exapted) by host genes as regulatory
elements (167,168) and, in some cases, parts of protein-
coding sequences (169). Individual cases of exaptation of
complete genes from mobile elements are also known as
strikingly exemplified by the evolution of the hedgehog
gene, a key regulator of animal development, from an
intein (170,171).
All prokaryotic genomes, without exception, contain
traces of integration of multiple plasmids and phages.
Even more revealingly, the archaeal genomes typically
carry multiple versions of an operon that encodes key
components of the plasmid partitioning machinery, and
often possess more than one origin of replication (172).
Thus, fusion of distinct replicons appears to routinely
occur in prokaryotes, and over the course of evolution,
such fusion might have been a major factor in shaping
the observed architecture of prokaryotic chromosomes
In summary, comparative genomics and metagenomics
reveal a vast, dynamic, interconnected world of selfish
replicons that interacts with genomes of cellular life
forms and, over long spans of evolution, makes major
contributions to the composition of chromosomes. In pro-
karyotes, the interaction between bacterial and archaeal
chromosomes and selfish replicons is so intensive, and the
distinction between chromosomes and megaplasmids is
blurred to such an extent that chromosomes are, probably,
best viewed as ‘islands’ of relative stability in the turbulent
‘sea’ of mobile elements (83). In eukaryotes, especially,
in multicellular forms that evolved the separation between
the germline and soma, the distinction between chromo-
somes and selfish replicons is sharper. Nevertheless, intra-
genomic mobility of selfish transposable elements is
extensive, and intergenomic mobility, at least, within a
species, is actually facilitated by sex, with bursts of trans-
posable element propagation likely marking evolutionary
transitions (16). The central role of mobile elements in
genome evolution further undermines the TOL concept,
although phylogenetic trees of individual hallmark genes
can be highly informative for the reconstruction of the
evolution of the selfish elements themselves (174,175).
The nature of the Last Universal Common Ancestor
and early evolutionary transitions
Comparative genomics vindicates Darwin’s conjecture on
the origin of all extant life forms from a single common
ancestor. Indeed, evolutionary reconstructions suggest
that hundreds of conserved genes, most likely, trace
back to LUCA (88,89–91). More specifically, these recon-
structions indicate that LUCA already possessed a com-
plete system of translation that was not dramatically
different from (at least) the simpler versions of the
modern translation machinery (that is, consisted of,
roughly, 100 RNA and protein molecules) as well as the
core transcription system and several central metabolic
pathways, such as those for purine and pyrimidine nucleo-
tide biosynthesis (90). However, the sets of genes assigned
to LUCA in these reconstructions lack certain essential
components of the modern cellular machinery. In particu-
lar, the core components of the DNA replication machin-
ery are non-homologous (or, at least, non-orthologous) in
bacteria, on the one hand, and archaea and eukaryotes, on
the other hand (176). In another sharp divide, the mem-
brane lipids have distinct structures, and the membrane
biogenesis enzymes are accordingly non-homologous
(non-orthologous) (177).
These major gaps in the reconstructed gene set of
LUCA support the idea that different cellular systems
‘crystallized’ asynchronously and are suggestive of
‘phase transitions’ in the early phases of cellular evolution
(151,178). One class of hypotheses holds that LUCA was
radically different from modern cells, possibly, not a cell at
all, but rather a pool of genetic elements that employed
diverse replication and expression strategies, and might
have populated inorganic compartments like those seen
at hydrothermal vents (179,180). Under these scenarios,
the modern-type DNA replications systems and mem-
branes evolved at least twice independently in two
domains of life (assuming a symbiogenetic origin for
eukaryotes). In this case, the very concept of a distinct
LUCA becomes ambiguous, and it might be more appro-
priate to speak of LUCAS, the Last Universal Common
Ancestral State (181). The alternative class of scenarios
postulate that LUCA was a modern-type cell with either
the archaeal or the bacterial varieties of the DNA replica-
tion systems and membranes, or even mixed systems
(177,182). This class of scenarios implies that there were
switches from one type to the other in the evolution of
each of these key cellular systems or differential loss of the
respective genes.
Regardless of which scenario is preferred, the lack of
conservation of central cellular systems among the
domains of life indicates that the early stages of cell evo-
lution involved radical changes which are hardly compat-
ible with uniformitarianism.
Genome-wide quantification of selection and junk DNA:
distinct evolutionary regimes for different genomes
There are major differences in the genome layouts
between different lines of life evolution. Prokaryotes
and, especially, viruses have ‘wall-to-wall’ genomes that
consist, mainly, of genes encoding proteins and structural
RNAs, with non-coding regions comprising, with a few
exceptions, no more than 10–15% of the genomic DNA.
The genomes of unicellular eukaryotes have lower charac-
teristic gene densities but, on the whole, do not depart
Nucleic Acids Research, 2009, Vol. 37, No. 4 1019
too far from the prokaryotic principles, with most of the
DNA dedicated to protein-coding, despite the distinct,
exon–intron gene architecture. The genomes of multicel-
lular eukaryotes are drastically different in that only a
minority (a small minority in vertebrates) of the genomic
DNA is comprised of sequences encoding proteins or
structural RNAs. Generally, across the entire range of
life forms, there is a notable negative exponential depen-
dence between the density of protein-coding genes and
genome size although significant deviations from this
overall dependence are seen as well, particularly, in pro-
karyotes (Figure 2).
This dramatic difference in genome organization
between the genomes of (most) unicellular and multicellu-
lar organisms demands an explanation, and the simplest,
plausible one is given by the population-genetic theory
according to which the intensity of purifying selection
affecting a population is proportional to the effective pop-
ulation size. Fixation of non-coding sequences, such as
introns or mobile elements is, at best, neutral but, more
likely, at least, slightly deleterious, even if only because
of the extra burden on the replication machinery.
Therefore, extensive accumulation of such sequences is
possible only in relatively small populations in which the
intensity of purifying selection falls below the ‘complex-
ification threshold’. More specifically, theory predicts that
all mutations with selection coefficient (s) less than 10
would accumulate as neutral in genomes of multicellular
eukaryotes, and many cases of insertion of non-coding
sequences indeed are associated with such low s values
Considering the genome-scale study of evolution, the
next series of important questions has to do with the dis-
tribution of selection coefficients across genomes: how
much of the non-coding DNA is actually junk, what is
the pressure of purifying selection in different genes, and
how common positive (Darwinian) selection actually is?
Although measurement of selection for individual genes,
let alone individual sites, especially, in non-coding regions
is technically challenging (185,186), several genome-wide
analyses have been reported. A comprehensive analysis of
the human protein set that combined data on pathogenic
mutations, non-synonymous SNPs, and divergence in
human-chimpanzee orthologs led to the estimate that
only 12% of the amino-acid residues are associated
with s < 10
, whereas about half of the sites have s
values between 10
and 10
(187). Thus, the majority
of the protein sequences seem to be subject to substantial
Figure 2. Dependence between genome size and gene density for large viruses and diverse cellular life forms. The plot is semi-logarithmic. Points
corresponding to selected organisms are marked: Af, Archaeoglobus fulgidus (archaeon), Cp, Cryptosporidium parvum (unicellular eukaryote, alveo-
late), Hs, Homo sapiens, Os, Oryza sativa (rice), Mg, Mycoplasma genitalium (obligate parasitic bacterium), Mv, mimivirus, Tv, Trichomonas vaginalis
(unicellular eukaryote, excavate).
1020 Nucleic Acids Research, 2009, Vol. 37, No. 4
purifying selection. A complementary study on the evolu-
tion regimes of multiple groups of closely related bacteria
and archaea also revealed typically strong purifying selec-
tion, with the genome wide means of the dN/dS ratios
(the ratio of non-synonymous to synonymous nucleotide
substitution rates that is the traditional measure of selec-
tion in protein-coding sequences) between 0.02 and 0.2
(dN/dS <<1 is the signature of purifying selection) (75).
A genome-wide search for positive selection (measured
as the gene-specific dN/dS ratio) in protein-coding genes
from six mammalian species revealed 400 genes (2.5%)
that seem to have experienced positive selection in at least
one branch of the phylogenetic tree of the analyzed spe-
cies; the values for most of the individual branches were
very small (188). These estimates, although conservative,
show that, at least, in mammals, positive selection affect-
ing entire gene sequence is quite rare although many genes
that are, generally, subject to purifying selection are likely
to include positively selected sites. Comprehensive analy-
ses of amino-acid coding sites in 12 Drosophila genomes
yielded very different results, suggesting that a substantial
fraction and, perhaps, the majority of amino-acid replace-
ments are driven by positive selection although the bene-
ficial effects of most of these replacements seem to be quite
small (189,190). Notably, the distribution of positively
selected sites is strongly non-random among functional
categories of genes, with genes involved in immunity and
other defense functions, reproduction, and sensory per-
ception being particularly amenable to positive selection;
this distribution seemed to be stable among widely differ-
ent animals including mammals, flies, and nematodes
A burning question in genome-wide evolutionary stu-
dies, especially, for mammals with their huge genomes,
what fraction of the non-coding DNA is ‘real’ junk, and
how much is subject to yet unknown functional con-
straints. The possibility that, despite the lack of detectable
evolutionary conservation, a large fraction if not most of
the human DNA is, in fact, functionally important and
hence maintained by selection is often discussed, espe-
cially, in the light of the demonstrations that a very
large fraction of the genome is transcribed (192–194).
The discovery of the so-called ultraconserved sequences
that appear to be subject to an exceptionally strong pur-
ifying selection (195,196) is compatible with this idea.
Furthermore, a considerable fraction of the ‘junk’ DNA
could be involved in functional roles that entail only lim-
ited sequence conservation but nevertheless are important,
in particular, for chromatin structure maintenances and
remodeling such as scaffold/matrix attachment regions
(SARs/MARs) (197,198). Nevertheless, a recent genome-
wide analysis of the distribution of insertion and deletions
(in comparisons of human, mouse and dog genomes) sug-
gests that only 3% of the human euchromatin DNA
is under selective constraints (199). Given that protein-
coding sequences comprise only 1.2% of the euchroma-
tin, these results indicate that the majority of functionally
important DNA sequences in mammals do not code for
proteins, but also vindicate the early conjectures that most
of the human genome is non-functional that is, after all,
junk (45,46). Of course, it should be kept in mind that any
definition of junk is conditional in that yesterday’s gar-
bage tomorrow can be recruited for a functional role.
In contrast, interspecies comparisons of non-coding geno-
mic regions in Drosophila indicate that the majority (70%
or more of the nucleotides) of these sequences evolve
under selective constraints, and a significant fraction (up
to 20%) seems to be affected by positive selection (200–
202). Certainly, these studies are based on different sim-
plifying assumptions (that cannot be here discussed in
detail), so the conclusion on major differences in selective
regimes between different lineages should be assessed with
caution and is subject to further validation. However,
the very fact that organisms with comparable sizes of
the gene sets and levels of organizational complexity,
such as insects, on the one hand, and mammals, on the
other hand, differ so dramatically in terms of gene density
and the amount of the apparent genomic ‘junk’ (Figure 2)
suggests that their genomes evolve under different selective
The study of the interplay between neutral processes,
purifying selection, and positive selection is still in its
early stages. The collection of sets of closely related gen-
omes from diverse taxa that is essential for this analysis is
currently small, although rapidly growing, and the meth-
ods for discriminating different modes of evolution are
still under active development. Nevertheless, even the
already available results make it abundantly clear that
the contributions of each of these factors are highly vari-
able among organisms, depending on the effective popu-
lation size, the characteristic rates of mutation and
recombination, and probably, other factors that are not
yet elucidated.
Gene and genome duplication: the principal route of
genomic innovation
Analysis of the numerous sequenced genomes vindicated
Ohno’s vision of gene duplication as a major evolutionary
mechanism (51), perhaps, even to a greater extent than the
originator of the concept could anticipate. The majority of
the genes in most genomes of cellular life forms (except for
the smallest genomes of obligate parasites) possess para-
logs indicative of duplication at some point during evolu-
tion (16,69), and many genes belong to large families of
paralogs which form a characteristic power-law distribu-
tion of the number of members [(203,204); see discussion
below]. With regard to the contribution of duplication to
the origin of new genes, it is important to note that there is
little compelling evidence of de novo emergence of genes
from non-coding sequences; although genes can expand by
recruiting small adjacent segments of non-coding sequence
[for instance, from an intron (205), birth of a complete
novel gene via this route seems to be an exceptional
event (206)]. Hence it is tempting to generalize that gene
duplication is not just an important but indeed the dom-
inant route that leads to the origin of new genes, with the
important addition that duplication is often followed by
accelerated sequence evolution as well as rearrangement of
a gene, an evolutionary mode that obliterates detectable
connections to the original source.
Nucleic Acids Research, 2009, Vol. 37, No. 4 1021
Ohno’s idea on the elimination or relaxation of selection
following a gene duplication, allowing accelerated evolu-
tion that has the potential to produce functional novelty,
also was supported by comparative-genomic data, albeit
with a significant twist. It was argued theoretically and
then demonstrated by empirical measurement of the selec-
tion pressure on recently duplicated gene sequences that
relaxation of purifying selection was more likely to be
symmetrical, to affect both duplicates more or less equally
(207,208). Thus, the more common path of evolution
of duplicated genes might not be neofunctionalization
postulated by Ohno but rather subfunctionalization
whereby new paralogs retain distinct subsets of the origi-
nal functions of the ancestral gene whereas the rest of the
functions differentially deteriorate (209,210). More sophis-
ticated analyses seem to suggest that both regimes of evo-
lution could realize at different stages of the history of
paralogous genes, with fast subfunctionalization immedi-
ately after duplication succeeded by subsequent, slower
neofunctionalization (211–213).
Gene duplications occurs throughout the evolution of
any lineage but the rate of duplication is not uniform on
large evolutionary scales, so that organizational transi-
tions in evolution seem to be accompanied by bursts of
gene duplication, conceivably, enabled by weak purifying
selection during population bottlenecks (see below).
Perhaps, the most illustrative case in point is the emer-
gence of eukaryotes that was accompanied by a wave
of massive duplication, yielding the characteristic many-
to-one co-orthologous relationship between eukaryotic
genes and their prokaryotic ancestors (214). Similarly, dif-
ferential duplication of Hox gene clusters and other devel-
opmental regulators is thought to have played a pivotal
role in the differentiation of animal phyla (215,216).
Arguably, the most dramatic cases of ‘saltatory’ gene
duplication involve whole-genome duplication (WGD)
events (217). Following the original hypothesis of Ohno,
genome analysis revealed traces of independent WGD
events retained in the size distribution of paralogous
gene families and/or genomic positions of paralogous
regions, despite the extensive loss of genes after WGD,
in yeasts (218,219), chordates (220–223) and plants
(224,225). Mechanistically, the high prevalence of WGD
in eukaryotes might not be particularly surprising because
it results from a well known, widespread genetic phenom-
enon, polyploidization. However, evolutionary conse-
quences of WGD appear to be momentous as these
events create the possibility of rapid sub/neofunctionaliza-
tion simultaneously in the entire gene complement of an
organism (226). In particular, WGD is thought to have
played a central role in the primary radiation of chordates
(220). It is difficult to rule out the possibility that more
ancient WGD events are no longer readily detectable
owing to numerous gene losses that obscure the WGD
signal; in particular, the burst of duplications that fol-
lowed eukaryogenesis but antedates the last common
ancestor of extant eukaryotes might have been brought
about by the first WGD in eukaryotic evolution (214).
Considering the wide occurrence of WGD in multiple
eukaryotic lineages, it is notable that so far no such
events were detected by analysis of the numerous available
prokaryotic genomes although transient polyploidy was
repeatedly observed (227,228). Conceivably, the absence
of detectable WGD in prokaryotes is due to the efficient
purifying selection that acts in large prokaryotic popula-
tion (see below) and leads to rapid elimination of duplicate
genes that would obliterate traces of WGD should such an
event occur.
At the level of general concepts of evolutionary biology
with which I am primarily concerned here, genomic stu-
dies on gene duplication lead to, at least, two substantial
generalizations. First, the demonstration of the primary
evolutionary significance of duplications including dupli-
cations of large genome regions and whole genomes is a
virtual death knell for Darwinian gradualism: even a
single gene duplication hardly qualifies as an infinitesi-
mally small variation whereas WGD qualifies as a bona
fide saltatory event. Secondly, the primacy of gene dupli-
cation with the subsequent (sometimes, rapid) diversifica-
tion of the paralogs as the route of novel gene origin
reinforces the metaphor of evolution as a tinkerer: evolu-
tion clearly tends to generate new functional devices by
tinkering with the old ones after making a backup copy
rather than create novelty from scratch.
Emergence and evolution of genomic complexity:
the non-selective paradigm and the fallacy of
evolutionary progress
Undoubtedly, multicellular eukaryotes, such as animals
and plants, are characterized by a far greater organiza-
tional complexity than unicellular life forms, and in the
spirit of the Modern Synthesis, this complexity is generally
seen as a result of numerous adaptive changes driven by
natural selection, and, being so regarded, can be viewed as
a manifestation of ‘progress’ in evolution. The correspon-
dence between the organizational complexity and genomic
complexity is an open issue, in part, because genomic com-
plexity is not easy to define. A simple and plausible defi-
nition can be the number of nucleotides that carry
functionally relevant information, that is, are affected by
selection (229,230). Under this definition, genomes of mul-
ticellular eukaryotes, of course, are much more complex
than genomes of unicellular forms, and this higher geno-
mic complexity translates into functional complexity
as well.
A striking case in point is alternative splicing that is
a crucial functional device in complex organisms like
mammals where it creates several-fold more proteins
than there are protein-coding genes (231–233) (thus,
the fact that humans have 20 000 genes compared to
10 000 genes in the bacterium Myxococcus xanthus
should not be translated into the claim that ‘the human
proteome is twice as complex as that of a bacterium’: the
real difference is greater owing to alternative splicing).
Alternative splicing is made possible by weak splice signals
that are processed or skipped by the spliceosome with com-
parable frequencies (234). In a sense, functionally impor-
tant alternative splicing events are encoded in these
splice junctures and, to some extent, also in additional
intronic sequences. However, did alternative splicing
evolve as a functional adaptation? In all likelihood, no.
Nucleic Acids Research, 2009, Vol. 37, No. 4
Indeed, it was shown that intron-rich genomes typically
possess weak splice signals whereas intron-poor genomes
(mostly, those of unicellular eukaryotes) have tight
splice junctions, presumably, ensuring high fidelity of
splicing (235). Recent detailed studies demonstrated
low splicing fidelity in intron-rich organisms, so that
numerous misspliced variants are produced and are,
mostly, destroyed by the nonsense-mediated decay
(NMD) system (236). Evolutionary reconstructions
strongly suggest that ancient eukaryotes including the
last common ancestor of extant forms possessed high
intron densities comparable to those in the most intron-
rich modern genomes, such as vertebrates (237–239) and,
by inference, had weak splice signals yielding numerous
alternative transcripts (235). The conservation of the
NMD machinery in all eukaryotes (240) is fully compati-
ble with this hypothesis. Thus, it appears that alternative
splicing emerged as a ‘genomic defect’ of which the respec-
tive organisms could not get rid, presumably, because of
weak purifying selection, and evolved a special mechanism
to cope with, namely, NMD. Gradually, they also evolved
ways to utilize this spandrel for multiple functions.
The above account of the origin of alternative splicing
could epitomize the non-adaptationist population-genetic
theory of evolution of genomic complexity that was
recently expounded by Lynch (16,183,184). As already
alluded to in the preceding section, the central tenet of
the theory is that genetic changes leading to an increase
of complexity, such as gene duplications or intron inser-
tions are slightly deleterious, and therefore can be fixed at
an appreciable rate only when purifying selection in a pop-
ulation is weak. Therefore, given that the strength of pur-
ifying selection is proportional to the effective population
size, substantial increase in the genomic complexity is pos-
sible only during population bottlenecks. Under this con-
cept, genomic complexity is not, originally, adaptive but
is brought about by neutral evolutionary processes when
purifying selection is ineffective. In other words, complex-
ification begins as a ‘genomic syndrome’ although com-
plex features (spandrels) subsequently are co-opted for
various functions and become subject to selection. By con-
trast, in highly successful, large populations, like those of
many prokaryotes, purifying selection is so intense that no
increase in genomic complexity is feasible, and indeed,
genome contraction is more likely.
Of course, there are exceptions to these principles, such
as bacterial genomes with more than 12 000 genes (241),
viral genomes with extensive proliferation of duplicated
genes (158), and genomes of unicellular eukaryotes [e.g.
Chlamydomonas (242) or Trichomonas (243)] that, by most
criteria, are as complex as the genomes of multicellular
animals or plants. Furthermore, some prokaryotic gen-
omes [e.g. the crenarchaeon Sulfolobus solfataricus (244)]
and genomes of unicellular eukaryotes [e.g. Trichomonas
vaginalis (243)] are among those with the highest content
of transposable elements. Apparently, the outcome of
genome evolution depends on the balance between the
pressure of purifying selection, itself dependent on
the population size and mutation rate, the intensity of
recombination processes, the activity of selfish elements,
and adaptation to specific habitats (99). An attractive
hypothesis is that, at least, in prokaryotes, the upper
bound for the number of genes in a genome, a good
proxy for genomic complexity, is determined by the ‘reg-
ulatory (bureaucratic) overhead’ (83,245,246). The exis-
tence of such an overhead is implied by the notable
observation that different functional classes of genes
scale differently with respect to the total number of
genes in a genome, and in particular, regulatory genes
(such as transcription repressors and activators) show a
(nearly) quadratic scaling (83,245,247,248). Conceivably,
at some ratio of the number of regulators to the number of
regulated genes, perhaps, close to 1:1, the burden of reg-
ulators becomes unsustainable. Thus, evolution of genome
complexity, undoubtedly, depends on a complex combina-
tion of stochastic (neutral) and adaptive processes. It
appears, however, that at present, the most consistent,
simple null hypothesis of genomic evolution is that
genome expansion, a pre-requisite for complexification,
is not a result of adaptation but rather a consequence of
weak purifying selection.
The next big question that begs to be asked with regard
to complexity, both organizational and genomic, is: was
there a consistent trend towards increasing complexity
during the 3.5 billion years of life evolution on earth?
The most likely answer is, no. Even very conservative
reconstructions of ancestral genomes of archaea and bac-
teria indicate that these genomes were comparable in size
and complexity to those of relatively simple modern forms
(88,89,91,93). Furthermore, reconstructions for some indi-
vidual groups, and not only parasites, point to gene loss
and genome shrinking as the prevailing mode of evolution
(249). Considering that numerous prokaryotic groups
undoubtedly have gone extinct in the course of life history,
there is every reason to believe that, even prior to the
radiation of all major lineages known today, the distribu-
tion of genome sizes and the mean complexity in prokary-
otes was (nearly) the same as it is now. Of course, it is
conceivable that the most complex forms known evolved
relatively late in evolution but, should that be the case, it
could be accounted for by purely stochastic processes,
given that life, in the pre-LUCAS stages of its evolution,
must have started ‘from so simple a beginning’ (1250).
In the same vein, the discovery of large and complex
genomes in stem animals (that is, animals with radial sym-
metry, such as Cnidaria, that branched off the trunk of
metazoan evolution prior to the origin of the Bilateria)
(84–86) suggests that there was little if any increase
in genomic complexity during the evolution of the meta-
zoa (although organizational complexity did increase);
instead, recurrent gene loss in different lineages was the
most prevalent evolutionary process.
Certainly, episodes of major increase in complexity
are known, such as the origin of eukaryotes, and the
origin of multicellular forms, to mention obvious exam-
ples. However, these seem not to be parts of a consistent,
gradualist trend, but rather singular, more or less cata-
strophic events triggered by rare, chance occurrences
such as the domestication of the endosymbiont in the
case of the origin of eukaryotes.
On the whole, the theoretical and empirical studies
on the evolution of genomic complexity suggest that
Nucleic Acids Research, 2009, Vol. 37, No. 4 1023
there is no trend for complexification in the history of life
and that, when complexity does substantially increase, this
occurs not as an adaptation but as a consequence of weak
purifying selection, in itself, paradoxical as this might
sound, a telltale sign of evolutionary failure. It appears
that these findings are sufficient to put to rest the notion
of evolutionary ‘progress’, a suggestion that was made
previously on more general grounds.
Functional genomics, systems biology and the determinants
of gene evolution rate
Just like the final decade of the 20th century was the age of
genomics when the quantity of genome sequences was
transformed into a new quality, allowing novel general-
ization, such as the ‘uprooting’ the TOL, the first decade
of the new century became the age of functional genomics
and systems biology. These disciplines yielded increasingly
reliable data of a new kind that start to fill the previously
glaring gap between the genome and the phenotype of an
organism (hereinafter denoted phenomic variables). The
phenomic variables include genome-wide profiles of gene
expression levels, comprehensive maps of protein–protein
and genetic interactions, information on the effects of gene
knockout (gene dispensability, typically, defined as essen-
tiality of a gene for growth on rich media), and more
(81,82). The first comparative analyses that became possi-
ble when sufficient information on gene expression became
available for multiple organisms revealed an interplay
between neutral and selective processes. Although the
levels of expression between orthologous genes in human
and mouse show significant conservation (compared to
random gene pairs), the divergence in expression is more
pronounced than that between protein sequences of the
orthologs (251,252). Thus, although, in general terms,
evolution of gene expression is similar to sequence evolu-
tion in that purifying selection is the principal constraining
force (253), the genuinely neutral, unconstrained com-
ponent is likely to contribute more to the evolution of
Joint analysis of the novel class of phenomic variables
characterized by systems biology and the measures of gene
evolution such as sequence evolution rate and propensity
for gene loss revealed a rather unexpected structure of
correlations [(81,254,255); Figure 3A]. Despite the intu-
itive link between the rate of evolution and gene dispen-
sability [‘important’ genes would be expected to evolve
slower than less important ones (256)], only a weak
link (at best) between these characteristics was detected
(257–259). The link between evolution rate and functional
importance of a gene deserves further investigation
because comprehensive analysis reveals a measurable phe-
notypic effect of knockout of virtually each yeast gene
under some conditions (260). However, regardless of the
outcome of such studies, clearly, this link is subtle, even
if it turns out to be robust. In contrast, the strongest
correlation in all comparisons between evolutionary and
phenomic variables was seen between gene expression
level and sequence evolution rate or propensity for gene
loss: highly expressed genes, indeed, tend to evolve sub-
stantially slower than lowly expressed genes (254,261).
This finding is buttressed by the observations of a positive
correlation between sequence divergence and the diver-
gence of expression profiles among human and mouse
orthologous genes (252) and the comparatively low
rates of expression profile divergence in highly expressed
genes (262).
The overall structure of the correlations between evolu-
tionary and phenomic variables is succinctly captured in
the concept of a gene’s ‘status’ in a genome (255). High-
status genes evolve slow, are rarely lost during evolution
and are, typically, highly expressed, with numerous pro-
tein–protein and genetic interactions, and many paralogs
(Figure 3B). It should be noted, however, that despite this
appearance of order in the correlation structure, all corre-
lations are relatively weak, and do not seem to signifi-
cantly increase with the improvement of the data quality
(254,255). These observations point to the multiplicity of
the determinants of the course of a gene’s evolution and
suggest that truly random, stochastic noise could be an
important factor.
The emergence of the link between sequence evolution
rate as the most prominent connection between evolution-
ary and phenomic variable led to a new concept of the
principal determinants of protein evolution. In the pre-
genomic era, it was generally assumed that the sequence
evolution rate should be a function of, firstly, the intrinsic
structural-functional constraints that affect the given
(slow) Evolutionary variables (fast)
(low) Phenomic variables (high)
Figure 3. Evolutionary genomics and systems biology. (A) Evolution-
ary and phenomic variables. The phenomic variables are viewed as
mutually dependent and affecting evolutionary variables (left). Positive
correlations are shown by red arrows and negative correlations are
shown by blue arrows. (B) The concept of gene status. The red
points schematically denote data scatter.
1024 Nucleic Acids Research, 2009, Vol. 37, No. 4
protein and, secondly, the importance of the biological
role of the protein in the organism (256). With the
advent of the systems biology data, it was realized that
phenomic variables, in particular, gene expression could
be equally or even more important than the traditionally
considered factors (263,264). This realization led to the
Mistranslation-Induced Misfolding (MIM) hypothesis
according to which expression level or, more precisely,
the rate of translational events is indeed the dominant
determinant of the sequence evolution rate. The cause of
the covariation between the sequence evolution rate and
expression level is thought to be selection for robustness to
protein misfolding that is increasingly important for
highly expressed proteins owing to the toxic effects of
misfolded proteins (265,266). The MIM hypothesis could
additionally explain the rather puzzling but consistent and
strong positive correlation between the rates of evolution
in synonymous and non-synonymous positions (dN
and dS, respectively) of protein-coding sequences (267).
Indeed, this correlation is likely to be a consequence of
the slow evolution in both classes of sites in highly
expressed genes which, in the case of synonymous sites,
is likely to be caused by selection for codons that minimize
mistranslation (268,269). Detailed computer simulations
of protein evolution suggest that the toxic effect of protein
misfolding, indeed, could suffice to explain the observed
covariation of expression level and sequence evolution
rate (269). An analysis of the evolution of multidomain
proteins revealed substantial homogenization of the
domain-specific evolutionary rates compared to the same
pair of domains in separate proteins, conceivably, attrib-
utable to the equalized translation rates, but significant
differences between domain-specific evolution rates per-
sisted even in multidomain proteins (270). Hence the gen-
eralized MIM hypothesis according to which the rate of
protein evolution, primarily, depends on two factors:
(i) Intrinsic misfolding robustness that depends on the
characteristic stability and designability of the given
protein (domain).
(ii) Translation rate that can be viewed as an amplifier
of the fitness cost of misfolding and, accordingly,
of the selection for the robustness to amino-acid
Evolutionary systems biology revealed a new layer of
connections between the evolution and functioning of the
genome. It is becoming clear that processes that link the
genome and the phenotype of an organism, in particular,
gene expression exert a substantial feedback on gene evo-
lution. The rate of evolution of protein-coding genes
might depend more on constraints related to the preven-
tion of deleterious effects of misfolding than on con-
straints associated with the specific protein function.
Universals of genome evolution
Comparative genomics and systems biology yield enor-
mous amounts of data, and this wealth of information
begs for a search for patterns and regularities. Indeed,
several such regularities that are widespread and could
even be universal for the entire course of life evolution
were discovered. In the preceding section, I discussed
one of such apparent universals, the negative correlation
between gene sequence evolution rate and expression level
that seems to hold in all organisms for which the data are
available and leads to a reappraisal of the factors that
affect gene evolution (269).
Other potentially important regularities come in the
form of conserved distributions of evolutionary and func-
tional variables. Strikingly, the distributions of the
sequence evolution rates of orthologous genes between
closely related genomes were found to be highly similar
in distant taxa (271); when standardized, these distribu-
tions are virtually indistinguishable in bacteria, archaea
and eukaryotes and are best approximated by a
log-normal distribution (Figure 4A). Considering the dra-
matic differences in the genomic complexity and architec-
ture (see above) as well as the biology of these organisms,
the near identity of the rate distributions is surprising and
demands an explanation in terms of universal factors that
affect genome evolution. Robustness to protein misfolding
discussed above seems to be a good candidate for such a
universal factor although quantitative models explaining
the rate distribution remain to be developed.
As discussed above, gene duplication shapes all gen-
omes, and the distribution of family size in all sequenced
genomes follows a power-law-like distribution, with the
only appreciable difference being the exponent (203,204),
so this distribution comes across as a universal of genomic
evolution. This distribution is closely fit by a simple birth-
and-death model of gene evolution with balanced birth
and death rates and without direct involvement of any
form of selection [(204,272); Figure 4B].
The differential scaling of functional classes of genes
with genome size that is mentioned above suggests the
existence of an entire set of fundamental constants of evo-
lution. The ratios of the duplication rates to gene elimina-
tion rates that determine the exponents of the power laws
for each class of genes appear to be the same for all tested
lineages of prokaryotes and invariant with respect to
time, so the functional classes of genes appear to possess
universal ‘evolutionary potentials’ (245,273).
The apparent universality of these and other central
characteristics of genome evolution suggests that relatively
simple, non-selective models might be sufficient to form
the framework of a general evolutionary theory with
respect to which purifying selection would provide bound-
ary conditions (constraints) whereas positive, Darwinian
selection (adaptation) would manifest itself as a quantita-
tively modest, even if functionally crucial modulator of the
evolutionary process.
Two centuries after Darwin’s birth, 150 years after the
publication of his ‘Origin of Species’, and 50 years after
the consolidation of the Modern Synthesis, comparative
analysis of hundreds of genomes from many diverse taxa
offers unprecedented opportunities for testing the conjec-
tures of (neo)Darwinism and deciphering the mechanisms
of evolution. Comparative genomics revealed a striking
Nucleic Acids Research, 2009, Vol. 37, No. 4 1025
1 10 100 1000
1 10 100 1000
1 10 100 1000 10000
1 10 100 1000 10000
relative evolution rate (log S.D. scale)
Figure 4. Universals of evolution. (A) Distributions of evolutionary rates between orthologs in pairs of closely related genomes of bacteria, archaea
and eukaryotes. The evolutionary distances between aligned nucleotide sequences of orthologous genes were calculated using the Jukes–Cantor
correction and standardized so that the mean of each distribution equaled to 0, and the standard deviation equaled to 1. The plot is semi-logarithmic.
Metma—Methanococcus maripaludis C5 versus M. maripaludis C7 (Euryarchaeota); Bursp—Burkholderia cenocepacia MC0-3 versus B. vietnamiensis
G4 (Proteobacteria); Salsp—Salinispora arenicola CNS-205 versus S. tropica CNB-440 (Actinobacteria). All sequences were from the NCBI RefSeq
database. The probability density curves were obtained by Gaussian-kernel smoothing of the individual data points. (B) Fit of empirical paralogous
gene family size distributions to the balanced birth-and-death model. The results are shown for yeast Saccharomyces cerevisiae (Sc, left) and humans
(Hs, right). Upper panels, binned distributions of paralogous family sizes; middle panels, paralogous family size distributions in double logarithmic
coordinates; bottom panels, cumulative distribution function of paralogous family sizes. The lines show the predictions the balanced birth-and-death
model. The figure is from (204).
1026 Nucleic Acids Research, 2009, Vol. 37, No. 4
diversity of evolutionary processes that was unimaginable
in the pre-genomic era. In addition to point mutations
that can be equated with Darwin’s ‘infinitesimal changes’,
genome evolution involves major contributions from gene
and whole genome duplications, large deletions including
loss of genes or groups of genes, horizontal transfer
of genes and entire genomic regions, various types of
genome rearrangements, and interaction between genomes
of cellular life forms and diverse selfish genetic elements.
The emerging landscape of genome evolution includes the
classic, Darwinian natural selection as an important com-
ponent but is by far more pluralistic and complex than
entailed by Darwin’s straightforward vision that was soli-
dified in the Modern Synthesis (16,184). The majority of
the sequences in all genomes evolve under the pressure of
purifying selection or, in organisms with the largest gen-
omes, neutrally, with only a small fraction of mutations
actually being beneficial and fixed by natural selection
as envisioned by Darwin. Furthermore, the relative con-
tributions of different evolutionarily forces greatly vary
between organismal lineages, primarily, owing to differ-
ences in population structure.
Evolutionary genomics effectively demolished the
straightforward concept of the TOL by revealing the
dynamic, reticulated character of evolution where HGT,
genome fusion, and interaction between genomes of cellu-
lar life forms and diverse selfish genetic elements take the
central stage. In this dynamic worldview, each genome is
a palimpsest, a diverse collection of genes with different
evolutionary fates and widely varying likelihoods of being
lost, transferred, or duplicated. So the TOL becomes a
network, or perhaps, most appropriately, the Forest of
Life that consists of trees, bushes, thickets of lianas, and
of course, numerous dead trunks and branches. Whether
the TOL can be salvaged as central trend in the evolution
of multiple conserved genes or this concept should be
squarely abandoned for the Forest of Life image remains
an open question (274).
Table 1 outlines the status of the central tenets of clas-
sical evolutionary biology in the age of evolutionary geno-
mics and systems biology. All the classical concepts have
undergone transformation, turning into much more com-
plex, pluralistic characterizations of the evolutionary pro-
cess (15). Depicting the change in the widest strokes
possible, Darwin’s paramount insight on the interplay
between chance and order (introduced by natural selec-
tion) survived, even if in a new, much more complex
and nuanced form, with specific contributions of different
types of random processes and distinct types of selection
revealed. By contrast, the insistence on adaptation being
the primary mode of evolution that is apparent in the
Origin, but especially in the Modern Synthesis, became
deeply suspicious if not outright obsolete, making room
for a new worldview that gives much more prominence to
non-adaptive processes (184).
Beyond the astonishing, unexpected diversity of genome
organization and modes of evolution revealed by compar-
ative genomics, is there any chance to discover underlying
general principles? Or, is the only such principle the cen-
tral role of chance and contingency in evolution, elegantly
captured by Jacob (55) in his ‘evolution as tinkering’ for-
mula? In a somewhat tongue-in-cheek manner, one is
inclined to ask: is a Postmodern Synthesis conceivable
and, perhaps, even in sight?
Table 1. The status of the central propositions of Darwinism-Modern Synthesis in the light of evolutionary genomics
Proposition Current status
The material for evolution is provided,
primarily, by random, heritable
True. The repertoire of relevant random changes greatly expanded to include duplication of genes,
genome regions, and entire genomes; loss of genes and, generally, genetic material; HGT including
massive gene flux in cases of endosymbiosis; invasion of mobile selfish elements and recruitment
of sequences from them; and more
Fixation of (rare) beneficial changes by
natural selection is the main driving
force of evolution that, generally, pro-
duces increasingly complex adaptive
features of organisms; hence progress
as a general trend in evolution
False. Natural (positive) selection is an important factor of evolution but is only one of several
fundamental forces and is not quantitatively dominant; neutral processes combined with purifying
selection dominate evolution. Genomic complexity, probably evolved as a ‘genomic syndrome’ cause
by weak purifying selection in small population and not as an adaptation. There is no consistent
trend towards increasing complexity in evolution, and the notion of evolutionary progress is
The variations fixed by natural selection
are ‘infinitesimally small’. Evolution
adheres to gradualism
False. Even single gene duplications and HGT of single genes are by no means ‘infinitesimally small’ let
alone deletion or acquisition of larger regions, genome rearrangements, whole-genome duplication,
and most dramatically, endosymbiosis. Gradualism is not the principal regime of evolution
Uniformitarianism: evolutionary
processes remained, largely, the same
throughout the evolution of life
Largely, true. However, the earliest stages of evolution (pre-LUCA), probably, involved distinct
processes not involved in subsequent, ‘normal’ evolution. Major transition in evolution like the origin
of eukaryotes could be brought about by (effectively) unique events such as endosymbiosis
The entire evolution of life can be
depicted as a single ‘big tree’
False. The discovery of the fundamental contributions of HGT and mobile genetic elements to genome
evolution invalidate the TOL concept in its original sense. However, trees remain essential templates
to represent evolution of individual genes and many phases of evolution in groups of relatively close
organisms. The possibility of salvaging the TOL as a central trend of evolution remains
All extant cellular life forms descend
from very few, and probably, one
ancestral form (LUCA)
True. Comparative genomics leaves no doubt of the common ancestry of cellular life. However, it
also yields indications that LUCA(S) might have been very different from modern cells
The six fundamental tenets of (neo)Darwinism examined here are the same as listed in the ‘Introduction’ section. Here, I lump together the
propositions made by Darwin in the Origins and those of the Modern Synthesis. The distinction between these are instructive but belong in a
much more complete historical account; a deep, even if, possibly idiosyncratic discussion of these differences is given by Gould (13).
Nucleic Acids Research, 2009, Vol. 37, No. 4 1027
Several recent developments in evolutionary genomics
can be candidates for the roles of high-level generaliza-
tions underlying the diversity of evolutionary processes.
Perhaps, the most far-reaching of these is the popula-
tion-genetic concept of genome evolution developed by
Lynch (16). According to this concept, the principal fea-
tures of genomes are shaped not by adaptation but by
stochastic evolutionary processes that critically depend
on the intensity of purifying selection in the which, in
turn, is determined by the effective population size and
mutation rate of the respective organisms. In particular,
the complexity of the genomes of multicellular eukaryotes
is interpreted as evolving, primarily, not as an adaptation
ensuring organizational and functional complexity but as
a ‘genomic syndrome’ caused by inefficient purifying selec-
tion in small populations. Some of the sequence elements
accumulated via neutral processes are then recruited for
biological functions that collectively, indeed, provide for
the evolution of structurally and functionally complex
organisms. Conversely, the compact genomes of prokary-
otes and some unicellular eukaryotes might not be
shaped by selection for ‘genome streamlining’ but rather
by effective amelioration of even slightly deleterious
sequences in large populations (83). The non-adaptive
view of the evolution of genomic complexity by no
means implies that no complex features ever evolve as
direct adaptations or that genome streamlining can
never be a major driving force of genome evolution.
However, I believe that the evidence amassed by evolu-
tionary genomics is sufficient to necessitate the change of
the central null hypothesis of genome evolution from
adaptationist to neutral, with the burden of proof shifted
to the adepts of pervasive adaptation (230).
The concept of the substantially non-adaptive character
of genome evolution indeed seems to affect our basic
understanding of the meaning of conservation of genomic
features. As a case in point, the rather enigmatic conser-
vation of the positions of a large fraction of intron posi-
tions throughout the evolution of eukaryotes might not be
a consequence of strong purifying selection that would
cause elimination of variants in which the respective
introns were lost (the default interpretation implied by
the very notion of purifying selection and fully compatible
with the neutral theory). On the contrary, the conserva-
tion of introns and other genomic features without obvi-
ous functions could be the consequence of weak purifying
selection in small populations of complex organisms that
is insufficient to efficiently remove these elements. This is
not meant to claim that many genomic characters (such as
individual genes, amino-acid residues or nucleotides) are
not conserved during evolution owing to their functional
importance but to suggest that even this ‘sacred’, central
tenet of evolutionary biology—‘what is conserved is
functionally relevant’—is not an absolute, and the non-
adaptive alternative is to be taken seriously. Together
with the realization that genome contraction is at least
as common in evolution as genome expansion, and the
increase of genomic complexity is not a central evolution-
ary trend, the concept of non-adaptive genome evolution
implies that the idea of evolutionary progress can be safely
put to rest.
It is sometimes argued that recent developments
in genomics and systems biology produce a maze of con-
nections between different type of data that is intractable
in any explicit form, thus eliminating any hope for the
discovery of simple, ‘law-like’ regularities and reducing
much of the research in these areas to the development
of predictive algorithms (275). However, it is exactly this
type of simple and apparently universal regularities that
emerge from the joint analysis of comparative-genomic
and systems biology data. The distribution of evolution-
ary rates across sets of orthologous genes, the distribution
of the sizes of paralogous gene families, the negative cor-
relation between the expression level and the sequence
evolution rate of a gene, and other relationships between
key evolutionary and phenomic variables seem to be gen-
uine universals of evolution. The simplicity of these uni-
versal regularities suggests that they are shaped by equally
simple, fundamental evolutionary processes, rather than
by selection for specific functions. In some cases, explicit
models of such processes have already been developed and
shown to fit the data. These models either do not include
selection at all or give selection a new interpretation.
A good case in point is the generalized mistranslation-
induced misfolding hypothesis that explains the covaria-
tion of gene expression and sequence evolution rate by
selection for robustness to misfolding that comes across
as a major determinant of protein evolution. The unex-
pected corollary of this model is that the primary driving
force of purifying selection might not be the maintenance
of a biological function but rather prevention of non-
specific deleterious effects of a misfolded protein.
Collectively, the developments in evolutionary genomics
and systems biology outlined here seem to suggest
that, although at present only isolated elements of a
new, ‘postmodern’ synthesis of evolutionary biology are
starting to be formulated, such a synthesis is indeed fea-
sible. Moreover, it is likely to assume definitive shape long
before Darwin’s 250th anniversary.
I thank Valerian Dolja, Allan Drummond, David
Lipman, Michael Lynch, Tania Senkevich, Claus Wilke
and Yuri Wolf for many helpful discussions, Tania
Senkevich for critical reading of the manuscript, and
Yuri Wolf for help with the preparation of the figures.
DHHS (NIH, National Library of Medicine) intramural
funds. Funding for open access charge: DHHS (NIH,
National Library of Medicine) intramural funds.
Conflict of interest statement. None declared.
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