2

THE IMPORTANCE OF EXPERIMENTAL

STUDIES IN EVOLUTIONARY BIOLOGY

Douglas J. Futuyma and Albert F. Bennett

METHODS OF STUDYING EVOLUTION

Experimental Evolution

Studies on Natural Populations

Comparative Methods

TWO EXAMPLES OF THE CONTRIBUTION OF EXPERIMENTAL

STUDIES TO UNDERSTANDING EVOLUTION

Adaptive Gain and Correlated Loss

Studying the Adaptive Role of Genetic Drift

Fitting the Experiment to the Question

CONCLUSION

Experimental Evolution: Concepts, Methods, and Applications of Selection Experiments, edited by Theodore
Garland, Jr., and Michael R. Rose. Copyright © by the Regents of the University of California. All rights of
reproduction in any form reserved.

15
METHODS OF STUDYING EVOLUTION

Experimental studies complement the several other major approaches to analyzing

evolutionary processes. Each approach has both advantages and limitations. We first
describe the advantages of experimental evolution, and then we contrast it with other
approaches.

EXPERIMENTAL EVOLUTION

The essence of experimental evolution is conceptually quite simple. For many genera-
tions, a series of replicated populations is exposed to a novel environment, while a paral-
lel series of populations is maintained within the ancestral environment, thereby serving
as experimental controls. The environmental novelty may involve alteration of any as-
pects of the abiotic, biotic, or demographic condition of the ancestral population. Usually
only a single environmental variable is altered to keep the experiment as simple as possi-
ble. It is, however, feasible to change multiple environmental factors simultaneously. The
novel experimental environment provides new selective conditions and hence promotes
evolution. New genetic variants (produced through recombination, mutation, or other
processes) may be differentially advantaged or disabled in the altered conditions, produc-
ing differential reproduction and increase of the favored genotypes.
Several different methods of environmental alteration are used in experimental
evolutionary studies, including artificial truncation, culling, and laboratory natural selec-
tion (Rose et al. 1990; Garland 2003; Rose and Garland this volume). The distinctions
among and advantages and limitations of each of these methods are discussed more
fully elsewhere in this volume (especially Huey and Rosenzweig). They are all similar,
however, in creating conditions favoring evolutionary alteration of the condition of the
ancestral population. After a sufficient number of generations, the novel experimental
populations may be compared directly with the controls (or in some cases directly to
their own ancestors), and any variety of a priori hypotheses concerning evolution may
thereby be tested. The longer the antecedents of the ancestral population have been
maintained in the ancestral (control) condition, the more likely any differences observed
within the novel experimental populations will be specific to the novel environmental
alteration (see also Travisano this volume).
What is particularly advantageous about this approach to evolutionary studies? Its
special strengths lie in the essence of any experiment: replication and control. By repli-
cating the number of populations exposed to the novel environment, an investigator can,
in effect, repeat the opportunity for evolutionary change and determine if the outcome
has consistency. In the metaphor of S. J. Gould (1989), the tape of life and evolution can
be replayed as often as desired to determine the similarity of its resulting products.
Evolutionary lineages of natural populations are a series of unique events, and it is never
possible to determine if things could have turned out differently or what factors were
responsible for the observed differentiation. In experimental evolution, however, the

16 • I N T R O D U C T I O N T O E X P E R I M E N TA L E V O L U T I O N
diversity of responses can be examined directly. Through replicated measurements
among experimental populations, this diversity can be analyzed statistically. Further, the
novel experimental populations can as a group be statistically compared with the group
of control populations (or, in some situations, with the ancestral population itself), with
the number of degrees of freedom determined by the number of independent replicates
of each. The statistical significance of any differences between the experimental and con-
trol groups can be evaluated to test evolutionary hypotheses.
Experimental evolution has certain additional attractive features. Unlike studies of
historical evolution in the natural world, there is never any uncertainty about ancestral
condition: the experimenters know the condition and composition of the ancestral pop-
ulation because they chose and observed it. Also in contrast to historical evolution, ex-
perimental evolution can define and control the degree of environmental change, limit-
ing it to a single factor or any desired combination of factors. So many environmental
aspects change simultaneously in the natural world that it is difficult to know to which
aspect of the environment the population is adapting. Experimental evolution can isolate
and analyze the adaptive response to specific environmental factors.
Experimental studies can also produce entirely new types of organisms for biological
study, literally building a better mouse (Bennett 2003; Garland 2003; Swallow et al. this
volume). By its nature, experimental evolution produces populations with traits that
enhance function and overall fitness in the new selective environment. The resulting
“improved” organisms are then subsequently available for functional and genetic analy-
ses. The different experimental replicate populations may contain a diversity of adaptive
solutions to their common environmental challenge, sometimes solutions not previ-
ously observed or expected (Bennett 2003; Garland 2003; Swallow et al. this volume).
Given that biological systems are complex and their evolution is unpredictable, the
emergent variability among selected populations can be especially informative about the
possible range of adaptive mechanisms.
As with any methodology, experimental evolution has its limitations and drawbacks
(see also Huey and Rosenzweig this volume; Oakley this volume). This approach,
because of its emphases on control and replication, is more suited to laboratory rather
than to natural situations. Although experimental evolutionary studies have been under-
taken successfully in the field on natural populations of organisms (see, e.g., Reznick
and Endler 1982; Reznick et al. 1990; Irschick and Reznick this volume), the large
majority have been laboratory based. Consequently, they lack the multidimensionality of
natural environments and sacrifice the ecological realism provided by studies carried out
in nature. Further, because of their requirements for large population size and rapid
reproduction, they have to be restricted to certain kinds of organisms that can be easily
maintained and bred in quantity in the laboratory. Although the range of such organ-
isms is phylogenetically broad, from viruses to mice, it is nonetheless limited, and many
interesting taxa are largely excluded from consideration as experimental evolutionary
subjects. Experimental evolution is not meant to reproduce, mimic, or predict evolution

T H E I M P O R TA N C E O F E X P E R I M E N TA L S T U D I E S • 17
in nature precisely. Its principal utility is its ability to test evolutionary theory and
hypotheses, and in that it is unmatched by alternative approaches (Bennett and Lenski
1999).
Experimental evolution can be contrasted with studies of natural populations of indi-
vidual species and with comparisons among species.

STUDIES ON NATURAL POPULATIONS

Most studies of natural populations draw inferences either from “snapshot” descriptions
of patterns of variation or from temporal data on actively evolving populations. The great
majority of studies of natural populations document patterns of variation, such as allele
and genotype frequencies, or statistical data on phenotypic characters, within and
among populations of a species. In some cases, the genetic component of phenotypic
variation is described from samples brought into the laboratory or garden for controlled
breeding. It is often attempted to infer the processes or history responsible for the ob-
served patterns by comparing data with expectations from theory. For example, if a defi-
ciency of heterozygotes and an excess of homozygotes is observed at many loci, it is rea-
sonable to infer that the population is subject to inbreeding, and such patterns are
indeed often seen in plants that practice some self-fertilization. A deficiency of genetic
variation in a population, relative to other populations of the species, may signal recent
reduction in population size or recent colonization; more detailed histories of popula-
tion size and movement can be inferred by applying coalescent analysis and phylogeo-
graphic study of “gene trees.”
There are several ways, moreover, in which inferences can be made about the effects
of natural selection in natural populations on characters of interest (summarized in
Endler 1986). For example, features may have evolved very recently, in response to iden-
tifiable selective factors, in populations introduced into new environments, or in
response to anthropogenic environmental changes. The study of geographic variation
within species, and its correlation with environmental variables, has been one of the
most widely used and successful approaches to demonstrating adaptation (reviewed in
Garland and Adolph 1991). Several methods of selection analysis have been used to
describe the mode and strength of selection on variable loci or phenotypic traits. In
almost all studies of natural populations, the analysis of factors that impinge on natural
variation is used to make inferences about the causal history by which a feature has
evolved. For instance, is the coloration (or size or behavior or thermal tolerance, etc.) of
this organism a consequence of genetic drift or of natural selection? If selection, then
what have been the selective factors?
As successful as these approaches have been, they have certain limitations that exper-
imental evolutionary studies can often overcome (see also Irschick and Reznick this
volume). The most important are concerns about adequacy of replication and control

18 • I N T R O D U C T I O N T O E X P E R I M E N TA L E V O L U T I O N
over confounding variables. Recent or ongoing evolution is often described for a single
population or ecological event (e.g., spread of an introduced species from a single colo-
nization), or for multiple populations that may not represent independent evolutionary
changes if they are connected by gene flow. A similar problem of lack of independence
may likewise be a concern in some studies of geographic variation. In contrast, experi-
mental evolution enables replication of populations and therefore greater statistical con-
fidence. Moreover, population replication may provide insights into the role of random
events, such as the impact of different mutations in different lines.
In studying natural populations, investigators must be concerned that correlated
variables might be responsible for genetic changes, rather than the variable they have
identified. The confounding variables may be external to the population of organisms—
environmental variables that are correlated with the one of interest—or internal, such as
selection on genes physically linked to those that affect the character studied, or an
unknown demographic history in which genetic drift has loomed large. Correlated vari-
ables are especially important if only a single population or event is studied, for there is
then no possibility of separating the postulated independent variable from those with
which it might be confounded (see also Garland and Adolph 1994). In contrast, the
effects of historical contingency can be minimized, or at least identified, in replicated
experimental populations. By manipulating population size, it may be possible to
identify the relative effects of selection and genetic drift, a problem that is exceedingly
difficult in natural populations. By the very nature of an experiment, environmental vari-
ables can be controlled or randomized, so that the effects of individual variables of inter-
est can be isolated and intentionally altered, and assessed with reference to control pop-
ulations. Consequently, the inferences that may be drawn about the factors responsible
for evolution in experimental populations can be far more direct, and often can be
asserted with greater confidence, than we can usually draw from natural populations.

COMPARATIVE METHODS

Character similarity among taxa has long been taken as evidence of common evolution-
ary history, and the divergence of characters in otherwise similar groups, as evidence of
adaptation. Studies of this type are generally termed “the comparative method.” It is only
within the past several decades, however, that it has been recognized that the comparative
method must take into account the statistical nonindependence of taxa because of their
common evolutionary history (Felsenstein 1985). A variety of different types of analytical
methodologies (e.g., independent contrasts, generalized least-squares models, Monte
Carlo computer simulations) has been developed for this purpose (see Garland et al. 2005
for a recent summary of modern comparative methods). These methods differ in their
analytical details, sometimes in ways that affect the inferences that can be drawn. In some
approaches, characters of interest (discrete or quantitative) are “mapped” onto a preexisting,

T H E I M P O R TA N C E O F E X P E R I M E N TA L S T U D I E S • 19
independently derived phylogeny. With certain assumptions, ancestral nodal conditions
can be estimated, and instances of evolutionary character reversal can be identified. The
total pattern of historical diversification can then be examined for concordance of charac-
ter change with historical or environmental or biotic changes within the taxa. In other ap-
proaches, statistical models (e.g., multiple regression) are applied in a way that acknowl-
edges the likely nonindependence of residuals and can model that nonindependence in a
variety of ways (reviewed in Lavin et al. in press).
The modern comparative method is our best tool for trying to understand the histor-
ical evolution of characters in the natural world, and it is widely accepted and employed.
It has, however, some shortcomings (Garland and Adolph 1994; Garland et al. 2005).
First, it is explicitly correlational and cannot directly demonstrate causality. Second, it
depends crucially on the assumption of parsimony in character diversification, and we
do not know if evolution is in fact parsimonious. Research on reverse evolution (see
Estes and Teotonio this volume) suggests that this assumption will in fact lead to system-
atic error when evolution reverses direction. Finally, many of these methods are depen-
dent on the assumption of a particular phylogeny of the group of interest (but see, e.g.,
Huelsenbeck and Rannala 2003). This phylogeny is only a hypothesis about ancestral re-
lationships that cannot be directly observed, and it is susceptible to extensive revisions in
the light of new information. Such phylogenetic revisions may completely change previ-
ous conclusions about the evolution of characters.

TWO EXAMPLES OF THE CONTRIBUTION OF EXPERIMENTAL

STUDIES TO UNDERSTANDING EVOLUTION

Here, to illustrate the power of the experimental approach, we discuss two areas of
evolutionary thought and theory to which experimental studies have made substantial
contributions: the presence and generality of trade-offs and the role of genetic drift
during adaptation.

ADAPTIVE GAIN AND CORRELATED LOSS

A long tradition in evolutionary thought claims that adaptive gain can be achieved only at
a price. That is, as fitness increases in one respect (reproduction, environment, age, etc.),
it decreases in other respects. These decrements are termed trade-offs (see also Frankino
et al. this volume; Gibbs and Gefen this volume), which can be more precisely defined as
a decline in nonselected fitness characters that accompanies adaptation to new selective
conditions. Darwin (1859) referred to trade-offs as “compensation” and said that “in order
to spend on one side, nature is forced to economise on the other side.” The concept of a
necessary reallocation of resources resulting from adaptation is still current; for example,
“improvements cannot occur indefinitely, because eventually organisms come up against
limitations. . . . At that point, improvements in one trait may be achievable only at the

20 • I N T R O D U C T I O N T O E X P E R I M E N TA L E V O L U T I O N
expense of others—there is a trade-off between the traits” (Sibly 2002; see also Futuyma
and Moreno 1988; Stearns 1992; Futuyma 1998). The underlying basis for some trade-offs
is relatively straightforward as the differential allocation of some limited resource, such as
energy, space, or time. If a kilocalorie of energy is expended in one activity, then it is not
available to support another activity; this is the sense in which Darwin discussed trade-
offs. However, trade-offs may take other forms, including functional constraints (e.g.,
multiple effects of changes in circulating hormone levels, aerobic vs. anaerobic muscle
structure) (see also Swallow et al. this volume).
Trade-offs are the presumptive basis for niche shifts (rather than niche expansion)
along an environmental range, such as temperature, salinity, or acidity. If a population is
adapting to one extreme of its realized niche, then the niche is expected to shift toward
that extreme, with a consequent loss of function and fitness at the other end of the niche.
Consequently, the niche is expected to shift along that environmental continuum. Adap-
tation to cold environments would therefore be expected to be accompanied by a decline
in ability to persist in hot environments. Levins’s (1968) principle of allocation provided
the theoretical basis for such niche shifts, and many mathematical models concerning
adaptive shifts during environmental change incorporate this principle (e.g., Lynch and
Gabriel 1987; Pease et al. 1989; but see Gilchrist 1995).
Although the concept that constructive change may also entail loss has an intuitive
appeal, operationally investigating trade-offs can be difficult. For a multidimensional
niche, the existence of a trade-off cannot be conclusively disproved: if decrements in all
functions examined cannot be demonstrated, then it can always be asserted that the
decline occurred in some function that is yet to be investigated. What can be investigated
is whether performance has declined in any portion of the range of the specific environ-
mental variable under selection. For instance, during specialization, does performance
decline at both niche extremes during adaptation to a constant intermediate environ-
ment? Selection experiments are a particularly powerful tool for investigations of such
trade-offs because of their unique advantages of experimental replication and control of
the selective environment. Fitness increments at the selective environmental variable
can be measured and compared with correlated changes in fitness in other portions of
the same environmental axis.
An example of an investigation of trade-offs using a selection experiment is provided
by research on evolutionary adaptation in the bacterium Escherichia coli. A bacterial
clone was first adapted to 37°C and 7.2 pH (Lenski et al. 1991) and then used as the
ancestor for replicated experimental groups at a variety of different culture temperatures
(Bennett et al. 1992; Mongold et al. 1996) and acidities (Hughes et al. 2007a, 2007b).
The niche of the common ancestor, defined as the range over which it could grow and
maintain itself in serial dilution culture of 100-fold per day, extended from 19.5°C to
42.2°C and from 5.4 to 8.0 pH. Experimental groups of six replicated populations were
cultured in different environments (including the niche extremes of 20°C, 41.5°C,
pH 5.4, and pH 8.0) for 2,000 generations. The fitness of each of these experimental

T H E I M P O R TA N C E O F E X P E R I M E N TA L S T U D I E S • 21
groups relative to their common ancestor was measured directly by competition experi-
ments in their new selective environments. The fitness increments provide a quantitative
measure of their adaptation. At 20°C, fitness increased 8.7 percent; at 41.5°C, 33.5 per-
cent; at pH 5.4, 20.0 percent; and at pH 8.0, 7.6 percent (Bennett and Lenski 1996;
Hughes et al. 2007a).
Was this adaptation to one extreme of the niche accompanied by a loss of fitness at
the other end of the niche? To determine whether trade-offs occurred, relative fitness of
each group was measured at the other niche extreme (40°C for the 20°C group; 20°C for
the 41.5°C group; pH 8.0 for the 5.4 pH group; and pH 5.4 for the 8.0 pH group). In con-
trast to the uniform pattern of fitness increments associated with the adaptive response,
the trade-off response varied widely in both presence and magnitude. For the 20°C
adapted group, fitness at 40°C declined significantly by 17 percent (p ⫽ 0.02) (Mongold
et al. 1996), but the 41.5°C group did not experience any fitness loss at 20°C (Bennett
and Lenski 1993). The acid-adapted (5.4 pH) group had a significant fitness decline of
23 percent (p ⬍ 0.001) at pH 8.0, while the base-adapted (8.0 pH) group measured at
5.4 pH experienced a significant decline in fitness of three of its six lines, an increment
in one, and no significant change in the other two, resulting in no significant group
trade-off effect (Hughes et al. 2007a).
Another, more extensive examination of thermal trade-offs (Bennett and Lenski
2007) was done on four groups of six lines each that were secondarily adapted to 20°C
for 2,000 generations after previously adapting to diverse warmer thermal environ-
ments (32°, 37°, and 41.5°C, and an environment alternating between 32° and 41.5°C)
(Mongold et al. 1996). Fitness relative to the progenitor of each line was measured at
40°C. Because of the multiple levels of replication possible within this system, it was
possible to determine whether the trade-off effect was significant across all experimen-
tal populations and whether it occurred within each experimental population. Fitness at
40°C declined significantly in fifteen of the twenty-four lines; it did not change signifi-
cantly in eight of the lines, and it actually increased significantly in one. For the entire
group of twenty-four lines, mean fitness at 40°C declined 9.4 percent (p ⬍ 0.001), so
there was a significant pattern of trade-off, even though it did not occur in 38 percent of
the lines examined.
What can we learn from these results about adaptation and trade-off? It is apparent that
trade-offs sometimes do occur as a correlated response to adaptation, as they did in these
experiments during evolution in cold and acidic environments. In some situations, how-
ever, such as in adaptation to a hot environment, no discernible trade-offs were detected.
In other situations (e.g., adaptation to alkalinity), there was such extreme variation among
populations that the overall trade-off effect, while readily apparent in some lines, was not
statistically significant overall. Even when an overall trade-off effect is significant, it may
not be general. That is, even when evolution generally operates to produce trade-offs, it
may not do so universally, and a large number of populations may demonstrate adaptation

22 • I N T R O D U C T I O N T O E X P E R I M E N TA L E V O L U T I O N
without correlated loss of function. These experimental results engender a more stochas-
tic and nuanced view of adaptive evolution, in which generalities can be specifically tested
and exceptions documented (see also Huey and Rosenzweig this volume).

STUDYING THE ADAPTIVE ROLE OF GENETIC DRIFT

One of Ernst Mayr’s most influential ideas was his proposal (1954) of founder-effect spe-
ciation, which he developed further in 1963 and christened “peripatric speciation” in
1982. Mayr suggested that evolution proceeds slowly in large, stable populations because
of strong fitness interactions (epistasis) among loci; specifically, the prevalent alleles will
be those that confer high fitness on the many different genetic backgrounds that they
encounter in a large, genetically variable population. However, an isolated population
founded by a few colonists will be much less genetically variable (the “founder effect,” an
instance of genetic drift), so selection may instead favor alleles that are good “soloists,”
those that increase fitness in concert with fewer segregating alleles at interacting loci.
Consequently, a founder event (or bottleneck in population size) may initiate new paths
of adaptive evolution, and these genetic changes, initiated by drift but directed by selec-
tion thereafter, might incidentally result in pre- or postzygotic reproductive isolation—
speciation. Some years later, Hampton Carson (1968, 1975; also Carson and Templeton
1984) proposed a rather similar idea, the “founder-flush-crash” scenario, in which expo-
nential population growth followed by severe population reduction results in major
genetic reorganization: the relaxation of selection during population growth enables
many recombinant genotypes to proliferate, of which only a few, perhaps very different
from previously prevalent genotypes, survive the population crash. The population may
thereby be set on a new trajectory of molding by natural and sexual selection that may
result in speciation.
Many subsequent authors discussed these ideas in the context of Wright’s adaptive
landscape. The effect of a bottleneck on a population’s genetic composition could be
viewed as drift to a different position on the landscape; if this point were on the slope of
a different adaptive peak than that of the parent population, selection would lead the
population to a different equilibrium constitution. Mathematical theoreticians used this
framework to analyze models that they thought represented Mayr’s or Carson’s verbal
model. Depending on their assumptions, they concluded that speciation is quite likely
(e.g., Gavrilets and Hastings 1996) or not at all likely (e.g., Barton and Charlesworth
1984; Charlesworth and Rouhani 1988). The differences in these authors’ conclusions
depend partly on assumptions about the nature of epistasis and the number of loci or
characters involved, which have been very difficult to examine empirically.
Experiments, therefore, may be the best way to judge the likelihood that stochastic
genetic changes owing to bottlenecks may initiate adaptive divergence and/or repro-
ductive isolation from the large “parent” population. Such experiments typically have

T H E I M P O R TA N C E O F E X P E R I M E N TA L S T U D I E S • 23
involved initiating replicate experimental populations with different numbers of
founders, and scoring characters of interest after the passage of a number of genera-
tions at the same, large population size. (That is, the treatments differ in founder num-
bers, not equilibrium population numbers.) In one of the first and demonstrably less
memorable such experiments, Futuyma (1970) exposed replicate populations of
Drosophila melanogaster, initiated with two, ten, or thirty pairs of flies from the same
base population, to competition with Drosophila simulans, and after ten generations
scored them for population-level indicators of competitive ability (e.g., numbers or bio-
mass produced from a standard number of parents placed with the competing species).
Compared with control populations that were not subjected to interspecific competi-
tion, a few experimental lines displayed differences in competitive ability, but no effect
of founder number was discerned.
Starting in the 1970s, many investigators tested for the evolution of incipient repro-
ductive isolation in bottleneck experiments; most imposed multiple bottlenecks, more
or less mimicking Carson’s scenario (see reviews in Rice and Hostert 1993, Coyne and
Orr 2004, and Rundle 2003; see also Fry this volume). For example, Agustí Galiana,
Andrés Moya, and Francisco Ayala (1993; also Moya et al. 1995) set up multiple popula-
tions of Drosophila pseudoobscura, derived from flies collected in either Utah or Mexico,
at several bottleneck sizes (n ⫽ one, three, five, or nine pairs), and subjected them to
multiple flush/crash cycles: in each cycle, populations grew exponentially for an average
of six generations and then were reduced to n pairs. The “ancestral” stocks were main-
tained at large size throughout. In cycles 4, 5, and 7, various experimental populations
were tested for sexual isolation, either from one another or from their ancestral stock, by
placing twelve pairs of virgin flies from each of two populations in an arena and scoring
the numbers of matings. (Flies from the two populations were made distinguishable by
clipping a small piece out of the wing, a procedure that did not alter the results.) Galiana
et al. found, overall, that twenty-three of the forty-five experimental populations, viewed
individually, displayed positive assortative mating (i.e., incipient reproductive isolation)
when tested against other populations, and ten remained statistically significant when
subjected to the Bonferroni correction for multiple tests. Significantly more populations
in the small-bottleneck (n ⫽ one or three) treatments showed assortative mating than in
the large-bottleneck (n ⫽ five, seven, or nine) treatments. Only positive assortative mat-
ing (i.e., mating within the same population) was observed; no population mated prefer-
entially with members of different populations. (Such an outcome might have been ex-
pected by chance; indeed, positive and negative assortative mating were equally frequent
in a rather similar experiment with D. melanogaster, performed by Howard Rundle
[2003].) However, most individual pairs of populations did not evince assortative mating,
and the authors concluded that their results “do not support the claim that the founder-
flush-crash model identifies conditions very likely to result in speciation events.”
Perhaps the support or lack of support for the hypothesis lies in the eye of the beholder,
for Templeton (1996) argued that this study does support Carson’s scenario, but later

24 • I N T R O D U C T I O N T O E X P E R I M E N TA L E V O L U T I O N
experiments in Drosophila have led most students of speciation toward a similar negative
conclusion (Coyne and Orr 2004).
As theoreticians turned their attention to the effects of population bottlenecks,
another interesting hypothesis bearing on adaptation and speciation surfaced. It has
long been known that if genetic variation in a trait is based on additively acting alleles,
the additive genetic variance (VA), which measures the correlation between parents and
offspring and is consequently the kind of genetic variation that underlies the response to
selection, is diminished within a population that is reduced in size. However, Charles
Goodnight (1988; also Whitlock et al. 1993; Cheverud and Routman 1996) showed that
if the phenotype is based substantially on interactions between alleles at different loci
(i.e., on epistasis), the epistatic variance, VI (the component of the population’s genetic
variance that is caused by such interaction), is commonly transformed into additive ge-
netic variance if the allele frequencies are altered by genetic drift during a bottleneck.
Consequently, directional selection on a characteristic may be more effective in a bottle-
necked population than in a stable, large population! If such characteristics, or the
genes underlying them, affect reproductive isolation, steps toward speciation might be
enhanced in founder populations for a quite different reason than either Mayr or Carson
had envisioned.
Whether traits have the epistatic variance postulated by these models can be deter-
mined only by empirical study. Increases in VA in bottlenecked populations have been
found in some, but not all, traits that have been studied. For example, Michael Wade
et al. (1996) imposed artificial selection for pupal weight in fifteen populations of the
flour beetle Tribolium castaneum after zero, two, or five generations of single-pair
sister-brother matings. After eight generations of selection, they estimated heritability
of both pupal weight and total fitness (as the number of adult offspring per individual)
by offspring/parent regression. They found that the heritability of pupal weight
declined in accord with the additive model, but the heritability of fitness in the bottle-
necked lines equaled or exceeded that of outbred control populations. Similarly, Edwin
Bryant, Lisa Meffert, and their colleagues have found that for several morphological
measurements on houseflies (Musca domestica), VA increases in populations that have
passed through one or more bottlenecks (e.g., Bryant and Meffert 1993). An important
question is whether such increases are caused by conversion of VI to VA (which could
enhance responses to natural selection) or simply by an increase, due to inbreeding, of
homozygotes for deleterious recessive alleles, which would not contribute to adapta-
tion. In a painstaking analysis of the components of genetic variance, Bryant and
Meffert (1996) found that two characteristics that showed increased VA after bottle-
necks have a high level of epistatic variance in the outbred base population, whereas
this is not the case for two other characteristics, for which the bottleneck effect was not
observed.
Experimental studies, in which genetic variance may be altered by genetic drift and
natural selection, have revived the possibility that founder effects might initiate new

T H E I M P O R TA N C E O F E X P E R I M E N TA L S T U D I E S • 25
evolutionary trajectories. The next question is whether or not such changes might also
initiate evolution of reproductive isolation. Meffert and collaborators explored this ques-
tion in a series of experiments with houseflies. In one study (Meffert and Bryant 1991),
male courtship behavior was analyzed in fifteen bottlenecked lines. Although only one
pair displayed significant assortative mating, many of the lines diverged in the frequency
with which various courtship components were displayed. From a statistical point of
view, these results do not encourage confidence that founder effects initiate reproductive
isolation, but they are nonetheless biologically intriguing. One could entertain the anal-
ogy that a statistically trivial fraction of mutations enhance fitness, but mutation is
nonetheless a foundation of adaptive evolution.

FITTING THE EXPERIMENT TO THE QUESTION

These two examples illustrate the necessity of tailoring the design of an experimental
evolutionary study to the question posed. The experiments with bacteria reviewed here
could address questions about adaptation based on newly arisen mutations, because of
the large populations and numbers of generations. Mutation-accumulation experiments
have been carried out with Drosophila in order to estimate the rate at which variation
arises, but most experimental evolutionary studies of Drosophila, such as the bottleneck
experiments we have described, have characterized the effects of standing genetic varia-
tion in the ancestral population. A fly experiment modeled on the E. coli experiments we
have described would call for a very long-term commitment, if the same number of gen-
erations were used. On the other hand, the rate of phenotypic response of an outbred
population undergoing laboratory evolution can be two orders of magnitude faster per
generation than that achievable with microbes (see Rose et al. 2004 for examples).
Conversely, the ideas tested in the bottleneck experiments described here pertain pri-
marily to sexually reproducing populations, and it is generally supposed that, if founder
effects are to have important consequences for subsequent evolutionary changes, the
consequences should be evident within a few generations. If one postulated, instead,
that drift-induced changes in a founder population’s genetic constitution determined the
selective advantage or disadvantage of subsequent new mutations, then bottleneck
experiments would require extension or redesign. Before launching into the great commit-
ment that an experimental evolutionary study typically requires, the investigator would
do well to consider the question, and the background theory, very carefully.

CONCLUSION

Of all methods used in evolutionary studies, experimental evolution mostly closely

approximates the “strong inference” approach of studying science (Platt 1964). It satis-
fies all of the elements of the classical scientific method and provides, therefore, the
most unarguable and convincing empirical analysis of evolutionary processes. It is not

26 • I N T R O D U C T I O N T O E X P E R I M E N TA L E V O L U T I O N
suited to answering all questions about evolution, particularly evolution in the natural
world. But because of its powerful use of population replication and control, it can
provide rigorous testing of evolutionary hypotheses and theories that formerly were
matters only of assumption or speculation. It will become an approach of increasing
utility and importance in future synthetic studies of evolutionary science.

SUMMARY

The goals of experimental evolutionary research are to enhance our understanding of

the bases and dynamics of evolution, and also to elucidate limitations on evolutionary
processes. Experiments can be designed to estimate the effects of such factors as muta-
tion, genetic drift, gene flow, and selection on genetic variation and on heritable pheno-
typic traits. Experiments can also cast light on variation in responses to selection and on
the factors that may bias or constrain the ability of populations to respond to selection.
Many evolutionary experiments probe the process of adaptation to specific environmen-
tal challenges. An important aspect of such studies is that they can provide insight into
the genetic, molecular, cellular, and developmental bases and components of adaptive
change—that is, into the mechanistic bases of adaptation.

ACKNOWLEDGMENTS

A.F.B.’s research was supported by the NASA Astrobiology Institute (NASA Grant
632731, Center for Genomic and Evolutionary Studies on Microbial Life at Low Temper-
atures) and currently by National Science Foundation grant IOS-0748903.

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