666 E vo l u ti o n o f G e n e F a m i l i e s
effects, over relatively long times. This viewpoint has
been challenged, by champions of punctuated equilib-
rium with rapid speciation and by proposals that more
drastic genetic events might be responsible for quite
dramatic changes in morphology. It is clear that very
small effects, as demonstrated by selection experi-
ments with animals such as Drosophila, can account
for the large morphological changes observed in fossil
series (Stebbins and Ayala, 1981). Furthermore, spe-
ciation that appears to be rapid on the geological time
scale may actually require tens of thousands of years, a
period perfectly consistent with small, slow genetic
events. On the other hand, studies of developmental
genetics have revealed genes, such as homeotic genes,
that govern major morphological changes, and the
growing marriage of developmental biology with
evolution may reveal ways that rapid evolutionary
change might result from changes in these regulatory
genes.
Microbial Evolution
There are clearly instances of lateral transfer, where
viruses, for example, carry genes from one species to
another or insert themselves in the middle of a gene
making substantial changes. That may further cloud
the picture at times, and it clearly plays a far greater
role in complexities of microbial evolution. The
current sequencing of large numbers of microbial
genomes is facilitating rapid growth in our under-
standing of that process, presenting very different
pictures of the early stages of cellular evolution and
the development of the three kingdoms than those
based primarily on ribosomal RNA data. The pos-
sibility has been raised that viruses may even provide
windows into some of the ancient organisms that dis-
appeared in the bottleneck of the `last common ances-
tor' of the three kingdoms; only a fraction of the genes
of the large viruses look like anything seen to date
in cellular organisms, and a significant number of
similarities have been seen between genes of bacterio-
phages and eukaryotic viruses. There is much evi-
dence, at least, that most families of viruses are very
ancient in origin and have coevolved with their vari-
ous hosts.
Further Reading
Dobzhansky TS (1941) Genetics and the Origin of Species. New
York: Columbia University Press.
Huxley JS (1942) Evolution: The Modern Synthesis. New York:
Harper & Sons.
Mayr E (1942) Systematics and the Origin of Species. New York:
Columbia University Press.
Stebbins GL (1950) Variation and Evolution in Plants. New York:
Columbia University Press.
References
Guttman BS (1999) Biology. Dubuque, IA: WCB/McGraw-Hill.
Stebbins GL and Ayala FJ (1981) Is a new evolutionary synthesis
necessary? Science 213: 967±971.
See also: Darwin, Charles; Dobzhansky,
Theodosius; Evolutionary Rate; Speciation;
Wallace, Alfred Russel
Evolution of Gene Families
L Silver
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.0433
Genomic Complexity Increases by Gene
Duplication and Selection for New
Function
Mice, humans, the lowly intestinal bacterium Escheri-
chia coli, and all other forms of life evolved from the
same common ancestor that was alive on this planet a
few billion years ago. We know this is the case from
the universal use of the same molecule ± DNA ± for
the storage of genetic information, and from the
nearly universal genetic code. But E. coli has a genome
size of 4.2 megabases (Mb), while the mammalian
genome is nearly 1000-fold larger at ~3000 Mb. If one
assumes that our common ancestor had a genome size
that was no larger than that of the modern-day E. coli,
the obvious question one can ask is where did all of
our extra DNA come from?
The answer is that our genome grew in size and
evolved through a repeated process of duplication and
divergence. Duplication events can occur essentially at
random throughout the genome and the size of the
duplication unit can vary from as little as a few nucleo-
tides to large subchromosomal sections that are tens,
or even hundreds, of megabases in length. When the
duplicated segment contains one or more genes, either
the original or duplicated copy of each is set free to
accumulate mutations without harm to the organism
since the other good copy with an original function
will still be present.
Duplicated regions, like all other genetic novelties,
must originate in the genome of a single individual and
their initial survival in at least some animals in each
subsequent generation of a population is, most often, a
simple matter of chance. This is because the addition
of one extra copy of most genes ± to the two already
present in a diploid genome ± is usually tolerated
without significant harm to the individual animal. In
the terminology of population genetics, most dupli-
cated units are essentially netural (in terms of genetic

selection) and thus, they are subject to genetic drift,
inherited by some offspring but not others derived
from parents that carry the duplication unit. By
chance, most neutral genetic elements will succumb
to extinction within a matter of generations. But
even when a duplicated region survives for a sig-
nificant period of time, random mutations in what
were once-functional genes will almost always lead
to nonfunctionality. At this point, the gene becomes a
pseudogene. Pseudogenes will be subject to continu-
ous genetic drift with the accumulation of new muta-
tions at a pace that is so predictable (~0.5% divergence
per million years) as to be likened to a `molecular
clock.' Eventually, nearly all pseudogene sequences
will tend to drift past a boundary where it is no longer
possible to identify the functional genes from which
they derived. Continued drift will act to turn a once-
functional sequence into a sequence of essentially ran-
dom DNA.
Miraculously, every so often, the accumulation of a
set of random mutations in a spare copy of a gene can
lead to the emergence of a new functional unit ± or
gene ± that provides benefit and, as a consequence,
selective advantage to the organism in which it resides.
Usually, the new gene has a function that is related to
the original gene function. However, it is often the
case that the new gene will have a novel expression
pattern ± spatially, temporally, or both ± which must
result from alterations in cis-regulatory sequences that
occur along with codon changes. A new function can
emerge directly from a previously functional gene or
even from a pseudogene. In the latter case, a gene can
go through a period of nonfunctionality during which
there may be multiple alterations before the gene
comes back to life. Molecular events of this class can
play a role in `punctuated evolution' where, according
to the fossil or phylogenetic record, an organism or
evolutionary line appears to have taken a `quantum
leap' forward to a new phenotypic state.
Duplication by Transposition
With duplication acting as such an important force in
evolution, it is critical to understand the mechanisms
by which it occurs. These fall into two broad cat-
egories: (1) transposition is responsible for the disper-
sion of related sequences; (2) unequal crossing-over is
responsible for the generation of gene clusters. Trans-
position refers to a process in which one region of the
genome relocates to a new chromosomal location.
Transposition can occur either through the direct
movement of original sequences from one site to
another or through an RNA intermediate that leaves
the original site intact. When the genomic region itself
(rather than its proxy) has moved, the `duplication' of
E vo l u t i o n o f G en e F a m i l i es 667
genetic material actually occurs in a subsequent gen-
eration after the transposed region has segregated into
the same genome as the originally positioned region
from a nondeleted homolog. In theory, there is no
upper limit to the size of a genomic region that can
be duplicated in this way.
A much more common mode of transposition
occurs by means of an intermediate RNA transcript
that is reverse-transcribed into DNA and then
inserted randomly into the genome. This process is
referred to as retrotransposition. The size of the retro-
transposition unit ± called a retroposon ± cannot be
larger than the size of the intermediate RNA tran-
script. Retrotransposition has been exploited by vari-
ous families of selfish genetic elements, some of
which have been copied into 100 000 or more locations
dispersed throughout the genome with a self-encoded
reverse transcriptase. But, examples of functional,
intronless retroposons ± such as Pgk2 and Pdha2 ±
have also been identified. In such cases, functionality
is absolutely dependent upon novel regulatory ele-
ments either present at the site of insertion or created
by subsequent mutations in these sequences.
Duplication by Unequal Crossing-Over
The second broad class of duplication events result
from unequal crossing-over. Normal crossing-over,
or recombination, can occur between equivalent
sequences on homologous chromatids present in a
synaptonemal complex that forms during the pachy-
tene stage of meiosis in both male and female mam-
mals. Unequal crossing-over ± also referred to as
illegitimate recombination ± refers to crossover events
that occur between nonequivalent sequences. Unequal
crossing-over can be initiated by the presence of re-
lated sequences ± such as highly repeated retroposon-
dispersed selfish elements ± located nearby in the
genome. Although the event is unequal, in this case,
it is still mediated by the homology that exists at the
two nonequivalent sites.
So-called nonhomologous unequal crossovers can
also occur, although they are much rarer than homo-
logous events. They are ``so-called'' because even these
events may be dependent on at least a short stretch of
sequence homology at the two sites at which the event
is initiated. The initial duplication event that produces
a two-gene cluster may be either homologous or non-
homologous, but once two units of related sequence
are present in tandem, further rounds of homologous
unequal crossing-over can be easily initiated between
nonequivalent members of the pair as illustrated in the
Figure 1. Thus, it is easy to see how clusters can
expand to contain three, four, and many more copies
of an original DNA sequence.
668 E vo l u ti o n o f G e n e F a m i l i e s

Initial duplication of single copy region Further expansion from a two repeat cluster
A B' B C
A B C genetic
exchange B′ B C
sites region of crossover
A' B' pairing site

A B' B/B' B C
reciprocal products
divergence
deleted chromosome
A C'
A B1 B2 B3 C
A' B' B C
three-member gene family cluster
duplicated segments

Figure 1 Unequal crossing-over generates gene families. The left side illustrates an unequal crossing-over event and
the two products that are generated. One product is deleted and the other is duplicated for the same region. In this
example, the duplicated region contains a second complete copy of a single gene (B). The right side illustrates a
second round of unequal crossing-over that can occur in a genome that is homozygous for the original duplicated
chromosome. In this case, the crossover event has occurred between the two copies of the original gene. Only the
duplicated product generated by this event is shown. Over time, the three copies of the B gene can diverge into three
distinct functional units of a gene family cluster.

In all cases, unequal crossing-over between homo-
logs results in two reciprocal chromosomal products:
one will have a duplication of the region located be-
tween the two sites and the other will have a deletion
that covers the same exact region (Figure 1). It is im-
portant to remember that, unlike retrotransposition,
unequal crossing-over operates on genomic regions
without regard to functional boundaries. The size of
the duplicated region can vary from a few base pairs to
tens or even hundreds of kilobases and it can contain
no genes, a portion of a gene, a few genes, or many.
Genetic Exchange between Related
DNA Elements
There are many examples in the genome where genetic
information appears to flow from one DNA element
to other related ± but nonallelic ± elements located
nearby or even on different chromosomes. In some
special cases, the flow of information is so extreme as
to allow all members of a gene family to coevolve with
near-identity as in the case of ribosomal RNA genes. In
at least one case ± that of the class I genes of the major
histocompatibility complex (MHC or H2) ± informa-
tion flow is unidirectionally selected, going from a
series of 25 to 38 nonfunctional pseudogenes into two
or three functional genes. In this case, intergenic infor-
mation transfer serves to increase dramatically the
level of polymorphism that is present at the small
number of functional gene members of this family.
Information flow between related DNA sequences
occurs as a result of an alternative outcome from the
same exact process that is responsible for unequal
crossing-over. This alternative outcome is known as
intergenic gene conversion. Gene conversion was ori-
ginally defined in yeast through the observation of
altered ratios of segregation from individual loci that
were followed in tetrad analyses. These observations
were fully explained within the context of the Holli-
day model of DNA recombination which states that
homologous DNA duplexes first exchange single
strands that hybridize to their complements and
migrate for hundreds or thousands of bases. Resolu-
tion of this `Holliday intermediate' can lead with
equal frequency to crossing-over between flanking
markers or back to the status quo without crossing-
over. In the latter case, a short single strand stretch
from the invading molecule will be left behind within
the DNA that was invaded. If an invading strand
carries nucleotides that differ at any site from the
strand that was replaced, these will lead to the produc-
tion of heteroduplexes with base pair mismatches.
Mismatches can be repaired (in either direction) by
specialized `repair enzymes' or they can remain as-is
to produce non-identical daughter DNAs through the
next round of replication.
By extrapolation, it is easy to see how the Holliday
model can be applied to the case of an unequal cross-
over intermediate which can be resolved in one of two
directions with equal probability. With one resolution,
unequal crossing-over will result; with the alternative
resolution, gene conversion can be initiated between
nonallelic sequences. Remarkably, information trans-
fer ± presumably by means of gene conversion ± can

also occur across related DNA sequences that are even
distributed to different chromosomes. There have
been numerous modifications of the Holliday model
± including those proposed by Meselson and Rading ±
that allow a better fit to the actual data, and there is
still lack of consensus on the some of the details
involved. However, the central feature of the Holliday
model ± single-strand invasion, branch migration, and
duplex resolution ± is still considered to provide the
molecular basis for gene conversion.
See also: Gene Conversion; Holliday's Model;
Major Histocompatibility Complex (MHC);
Molecular Clock; Unequal Crossing Over
Evolutionarily Stable
Strategies
E Pianka
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.0431
When natural selection acts on several different alter-
native behaviors, the most optimal should be favored.
If costs and benefits of alternatives depend on choices
made by other individuals, optimal solutions are not
always as obvious as they are in simpler situations. An
evolutionarily stable strategy, or ESS, is a mathemat-
ical definition for an optimal choice of strategy under
such conditions.
Interactions between two individuals can be de-
picted as a mathematical game between two players.
A branch of mathematics, called game theory, seeks to
find the best strategy to play in any given carefully
defined game. The central problem of game theory is
to find the best strategy to take in a game that depends
on what other players are expected to do.
Originally used in studies of economics and human
conflicts of interest, game theoretical thinking was
first used in biology by Hamilton (1967) to study evo-
lution of sex ratios. Later, game theory was explicitly
applied to behavioral biology by Maynard Smith (1972)
and Maynard Smith and Price (1973). Maynard Smith
coined the term ESS for a refinement of the Nash
equilibrium used by economists to define a solution
to a game.
The notion of a Nash equilibrium makes some tacit
assumptions about rational foresight on the part of the
player. An ESS must meet a stricter set of require-
ments than Nash equilibria; the mathematical differ-
ence boils down to whether a tie between strategies
leads to a new strategy being considered better. An
E vo l u t i o n a r i l y S ta b l e S t r a t e g i e s 669
ESS attempts to define conditions under which blind
evolution will return to the strategy in question, rather
than requiring rational foresight to dissuade the ex-
ploration of alternatives.
An ESS is a strategy that cannot be beaten by any
other strategy. An individual adopting it outperforms
any individual adopting any alternative tactic. No
other strategy can outperform an ESS. Individuals
adopting an ESS tactic have a higher reproductive
success than individuals adopting other tactics. Such
an unbeatable tactic can go to fixation (100%) in a
population and such a population cannot be invaded
by any other tactic. Inevitably, an ESS ends up
encountering itself more often than it confronts any
other strategy, and it must therefore perform better
against itself than any other strategy can perform
against it.
Game theory involves conflicts of interest in which
the value of a given action by a decision maker
depends both on its own choices as well as on those
of others. A `payoff' matrix of values of outcomes is
postulated based on the respective behaviors of two or
more contestants under all possible situations. Payoffs
are frequency dependent. Decision rules that repre-
sent an evolutionarily stable solution to such an
evolutionary game constitute an ESS (Axelrod and
Hamilton, 1981).
As an example, consider a well-known game theor-
etical model called the `prisoner's dilemma.' In this
hypothetical situation, two partners in crime have
been arrested. The police interrogate each person
alone. Each party could cooperate with the other and
steadfastly refuse to squeal on their friend. If both
cooperate and remain silent, the authorities cannot
establish guilt and both get off scott free (loyalty
pays off). Alternatively, each could betray their part-
ner and confess. Now consider respective rewards and
punishments received by each partner for making each
decision. If only one party confesses while the other
remains quiet, this betrayal is rewarded by giving the
confessor a light sentence for providing `state's evi-
dence' and testifying as to the guilt of their loyal silent
partner, who is then found guilty and receives a much
longer prison term (he gets the `sucker's payoff').
However, if both partners tell, the authorities put
both on trial and both receive moderate, but not
long, sentences of imprisonment. In a `zero sum'
game, all losses add up to equal all gains. Not so in
this game, where each partner can gain considerably
without as much loss to the other (indeed, by working
together, both could escape conviction altogether).
But they are not allowed to work together and neither
knows what the other will do.
Here then, is the classic `prisoner's dilemma': each
prisoner must decide what to do without knowing