, 737 (2009);
et al.Dolph Schluter,
Evidence for Ecological Speciation and Its
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Evidence for Ecological Speciation
and Its Alternative
Natural selection commonly drives the origin of species, as Darwin initially claimed. Mechanisms of
speciation by selection fall into two broad categories: ecological and mutation-order. Under
ecological speciation, divergence is driven by divergent natural selection between environments,
whereas under mutation-order speciation, divergence occurs when different mutations arise and
are fixed in separate populations adapting to similar selection pressures. Tests of parallel evolution
of reproductive isolation, trait-based assortative mating, and reproductive isolation by active
selection have demonstrated that ecological speciation is a common means by which new species
arise. Evidence for mutation-order speciation by natural selection is more limited and has been
best documented by instances of reproductive isolation resulting from intragenomic conflict.
However, we still have not identified all aspects of selection, and identifying the underlying genes
for reproductive isolation remains challenging.
(2–5). Not all species appear to evolve by
selection, but the evidence suggests that most of
them do. The effort leading up to this conclusion
involved many experimental and conceptual ad-
vances, including a revision of the notion of
speciation itself, 80 years after publication of On
the Origin of the Species, to a definition based on
reproductive isolation instead of morphological
differences (6, 7).
The main question today is how does selec-
of natural selection, what genes are affected, and
how do changes at these genes yield the habitat,
behavioral, mechanical, chemical, physiological,
and other incompatibilities that are the reproduc-
tive barriers between new species? As a start, the
many ways by which new species might arise by
selection can be grouped into two broad catego-
but at last we can agree with Darwin that the
origin of species, “that mystery of mysteries”
ries: ecological speciation and mutation-order
speciation. Ecological speciation refers to the
evolution of reproductive isolation between pop-
ulations or subsets of a single population by ad-
aptation to different environments or ecological
niches (2, 8, 9). Natural selection is divergent,
acting in contrasting directions between environ-
ments, which drives the fixation of different
alleles, each advantageous in one environment
but not in the other. Following G. S. Mani and
B. C. Clarke (10), I define mutation-order specia-
tion as the evolution of reproductive isolation by
the chance occurrence and fixation of different
alleles between populations adapting to similar
because populations fix distinct mutations that
would nevertheless be advantageous in both of
their environments. The relative importance of
these two categories of mechanism for the origin
of species in nature is unknown.
standing the general features of speciation by se-
lection.I do notdifferentiate speciation bysexual
divergence of mate preferences, by either eco-
logical or mutation-order mechanisms, in most
theories of the process (8, 11). I leave out dis-
instead identify the likelihood of ecological and
mutation-order speciation when there is gene
flow. I ignore reinforcement, a special type of
natural selection thought to favor stronger pre-
mating reproductive isolation once postzygotic
isolation has evolved. I also ignore speciation by
in the early stages.
Speciation and Adaptation from
Darwin to Dobzhansky
Appreciation of the connection between adapta-
tion and speciation began with Darwin when a
morphological concept of species largely pre-
that “I look at the term species, as one arbitrarily
given for the sake of convenience to a set of
individuals closely resembling each other...” and
“The amount of difference is one very important
criterion in settling whether two forms should be
ranked as species or varieties” (1). Under this
sufficiently many differences between popula-
tions to warrant their classification as separate
taxonomic species. Darwin understood the im-
(1), but the study of speciation after the pub-
lication of this work focused mainly on the evo-
lution of species differences, particularly of
morphological traits but also of behavioral and
other phenotypic traits.
Under this Darwinian perspective, linking
speciation with adaptation was relatively straight-
ic differences between species were caused by
natural selection. For example, at the American
Association for the Advancement of Science 1939
speciation symposium [the last major symposium
on speciation before the biological species concept
(7)], an extensive comparative and biogeographic
study showcased instances in which derived mor-
phological and life history forms of fishes had
arisen over and over again from the same ancestral
type (12). The repeated, parallel origin of non-
parasitic lamprey in streams from the same migra-
tory, parasitic ancestor showed that “Again and
Biodiversity Research Centre and Zoology Department,
University of British Columbia, Vancouver, BC V6T 1Z4,
Canada. E-mail: email@example.com
VOL 3236 FEBRUARY 2009
on April 6, 2010
parasitic forms...correlated with life in small
streams, where a suitable food supply in the way
of large fish is scarce or seasonal” (12). When cor-
related with environmental factors, such repetition
is unlikely to result from chance; environmental
selection pressures must therefore be the cause of
speciation. “As a result of our recent studies on
fishes...weight is constantly being added to the
theory that speciation is...under the rigid control of
the environment” (12). However, this case is only
referring to the origin of morphological species.
The turning point for speciation studies came
with the modern concept of speciation “Species
separation is defined as a stage of the evolu-
tionary process at which physiological isolat-
ing mechanisms become developed” (6) (here,
“physiological” is interpreted to mean evolved
reproductive isolation between populations, as
distinct from geographical barriers to interbreed-
ing). Subsequently, species were defined as
“groups of interbreeding natural populations that
are reproductively isolated from other such
groups” (7). From this point on, the study of
speciation was the study of the evolution of
reproductive isolation (3). Progress up to then in
understanding the link between morphological
speciation and adaptation was largely forgotten,
its contributions uncertain under the new concept.
The biological species concept must surely
between speciation and natural selection. T.
lying differencesbetween populations inordinary
phenotypic traits were unlikely to be the basis of
but at the time this viewpoint, and the generally
greater difficulty of studying reproductive isola-
tion than morphology, must have discouraged
many from pursuing the connection. Virtually no
research effort followed that tested the role of
adaptation in speciation.
Models of Speciation by Selection
again receiving attention. The two most general
hypotheses involving selection are ecological and
mutation-order speciation. Ecological speciation
is defined as the evolution of reproductive iso-
lation between populations by divergent natural
selection arising from differences between eco-
logical environments (2, 8, 9, 14). It predicts that
reproductive isolation should evolve between
populations adapting to contrasting environments
but not between populations adapting to similar
environments.The basicidea hasbeenaroundfor
a while (7), although it was tested only recently.
resources, climate, habitat, and interspecies inter-
actions such as disease, competition, and behav-
ioral interference. Ecological speciation can lead
to the evolution of any type of reproductive
isolation, including premating isolation, hybrid
sterility, and intrinsic hybrid inviability as well as
extrinsic, ecologically based pre- and postzygotic
isolation. Speciation by sexual selection is
ecological speciation if ecologically based diver-
gent selection drives divergence of mating
preferences, for example by sensory drive (15).
In accordance with (10), mutation-order spe-
isolation by the fixation of different advanta-
geous mutations in separate populations expe-
riencing similar selection pressures. Whereas
different alleles are favored between populations
under ecological speciation, the same alleles
would be favored in different populations under
way because,by chance,thepop-
ulations do not acquire the same
chastic but the process is distinct
from genetic drift. It can occur in
both small and large (though not
infinite) populations. Selection
can be ecologically based under
mutation-order speciation, but
ecology does not favor diver-
gence as such. It can lead to the
evolution of any type of repro-
ductive isolation, with the excep-
Speciation resulting from in-
tragenomic conflict such as mei-
otic drive or cytoplasmic male
sterility (Fig. 1B) is likely to be
mutation-order speciation be-
tions causing drive and those
countering it are unlikely to be
the same in separate populations.
Speciation by sexual selection is
mutation-order speciation if di-
vergence of mate preferences or
gamete recognition occurs by the
fixation of alternative advanta-
geous mutations in different pop-
ulations, as by sexual conflict
(16). Divergence in song and
other learned components of be-
havior under purely social selec-
tion, not molded by selection for
efficient signal transmission (5),
is the cultural equivalent of the
al scenarios are elaborated in (5).
Both models of speciation,
ecological and mutation-order,
are theoretically plausible, and
only data can determine their rel-
ative importance in nature. The
key is to figure out by which
first evolved (3). Once the earliest genetic differ-
ences have accumulated between populations by
either process, subsequent mutations might be
favored in one population and not the other
because of epistatic interactions with genetic
background (10). Hence, epistasis, including that
producing Dobzhansky-Muller incompatibilities
in hybrids between species (3), can result from
either ecological or mutation-order speciation.
Speciation can be rapid under both speciation
models, because alleles are driven to fixation by
natural selection in both cases. However, under
the mutation-order process, the same alleles, if
present, would be favored in every population, at
least in the early stages of divergence. For this
reason, mutation-order speciation is difficult when
Fig. 1. (A) Example of ecological speciation. Repeatedly and
independently, the mosquito fish, Gambusia hubbsi, inhabiting
blue holes in the Bahamas has evolved a larger caudal region and
smaller head in the presence of predators (top) than in their
absence (bottom) (29). In laboratory trials, the probability of two
individuals mating was higher when they were from different
populations having the same predation environment (and similar
body shape) than when they were from opposite predation
environments. [Photo credit: Brian Langerhans (29)]. (B) Example
of reproductive isolation evolving under the mutation-order
mechanism. Male-fertile (left) and male-sterile (right) flowers of
F2 hybrids between an Oregon population of monkey flowers (M.
guttatus) having a cytoplasmic male sterility element and nuclear
restorer and a closely related species (M. nasutus) having neither
(46, 47). Both flowers shown have M. guttatus cytoplasm. The
flower on the left also has the nuclear restorer, whereas the one on
the right, with undeveloped anthers, lacks the restorer. [Photo
credit: Andrea Case (47)]
6 FEBRUARY 2009VOL 323
on April 6, 2010
there is gene flow, because gene flow increases the
possibility that favorable mutations occurring in
one population will spread to other populations,
preventing divergence (17, 18). Any process
resulting in low levels of gene flow, including
selection, facilitates subsequent divergence by the
mutation-order process (19). In contrast, ecological
speciation can proceed with or without gene flow,
although it is easiest when gene flow is absent.
Experiments with laboratory populations of
Drosophila and yeast demonstrate the plausibility
of ecological speciation. In those instances when
measurable pre- and postmating reproductive
isolation evolved, it was greater between lines
subjected to different environments than between
tained under homogeneous conditions for many
sistent with the mutation-order process (22), but
effects on reproductive isolation have not been
Two approaches investigate the mechanisms of
speciation by natural selection in nature. The
bottom-up approach involves (i) genetic mapping
of reproductive isolation between closely related
species, (ii) testing whether discovered genes
exhibit a genomic signature of positive selection,
and (iii) identifying the phenotype and source of
fitness effects of alternative alleles at selected loci.
The approach has been hugely successful in
identifying major genes implicated in hybrid
inviability (Hmr, Lhr, Nup96), sterility (Odsh,
JYAlpha), and sexual isolation (ds2) between
Drosophila species. Most of these genes show
natural selection’s role (3), provided that fixation
occurred before complete reproductive isolation
rather than afterward. The top-down approach
involves identifying (i) the phenotypic traits un-
der divergent selection, (ii) those traits associated
with reproductive isolation, and (iii) the genes
underlying traits and reproductive isolation. Step
(iii) has been challenging under both approaches
to reproductive isolation.
Evidence for ecological speciation has accumu-
lated from top-down studies of adaptation and
reproductive isolation [reviewed in (2, 8, 9)]. We
in part, evolved by divergent natural selection
ductive isolation are often strong and straight-
forward. It follows that much of the genetic basis
of reproductive isolation should involve ordinary
genes that underlie differences in phenotypic
traits. But we still know little about the genetics
of ecological speciation.
One line of evidence comes from tests of
parallel speciation, whereby greater reproductive
isolation repeatedly evolves between indepen-
dentpopulationsadapting tocontrasting environ-
ments than between independent populations
adapting to similar environments (20, 23). A
major challenge in applying the test to natural
ecotype has originated just once and has spread to
multiple locales. This is difficult because gene
flow of neutral markers between closely related
but nearby populations can result in the false
appearance of multiple independent origins of
these populations when evaluated by phylogenies
several examples of parallel speciation, including
the sympatric benthic-limnetic species pairs of
threespine stickleback in young lakes of British
Columbia (25, 26), the repeated origin of diver-
gent marine and stream populations of threespine
ecotypes of Timema walking stick insects living
(24), and mosquito fish inhabiting blue holes with
and without fish predators in the Bahamas (29)
and females are more likely to mate if they are of
the same ecotype, regardless of relatedness as
indicated by phylogenetic affinity.
Ecological speciation is also
supported by examples of premat-
ing reproductive isolation in which
encounter mates on the basis of
phenotypic traits that are under
ecologically based divergent selec-
tion. Examples include assortative
mating by host choice in insects,
body size and coloration in fish,
beak size in birds, pollinator pref-
erences for specific phenotypic
ments [see examples in (8, 30, 31)].
Ecologically based divergent
selection has also been directly
ness of each ecotype in the envi-
ronment of the other [immigrant
inviability; reviewed in (31, 32)]
the parental environments [extrin-
sic postzygotic isolation (33)]. For
example, each of the coastal peren-
nial and inland annual races of the
monkey flower (Mimulus guttatus)
along the west coast of North
America has low fitness when
transplanted to the habitat of the
other (31). This is an example of
active selection on phenotypic dif-
ferences, and it also constitutes di-
rect reproductive isolation because
it is an evolved barrier to gene flow between
parental populations. Multiple traits are probably
involved, including flowering time and tolerance
of salt and drought. This type of reproductive
isolation is context-dependent and is weakened in
intermediate environments. On the other hand,
differences between populations, which may
strengthen the barrier to gene flow (20).
It is unclear how much reproductive isolation
typically evolves by ecologically based divergent
from estimates of the combined contribution of
mating, as compared with other forms of re-
productive isolation (Fig. 2 and table S1). These
estimates are incomplete because individual
studies may lack data on components of repro-
independent, and the strength of barriers between
species may not be symmetric (34). Nevertheless,
compilation of the data shows that the amount of
reproductive isolation attributable to active selec-
strong, on average, as the amount from compo-
nents of reproductive isolation lacking identifiable
causes (Fig. 2). The unidentified component of
speciation, if built by selection and not genetic
Cumulative reproductive isolation
Fig. 2. Estimates of the magnitude of reproductive isolation
resulting from divergent selection components (top), compared
with other components lacking identifiable causes (bottom).
Divergent selection components include those attributable to
active selection on traits (immigrant inviability and extrinsic
preference, floral isolation, and breeding time). The unattributed
components include intrinsic hybrid inviability, sexual selection
against hybrids, pollen competition, and reduced hybrid fecundity.
Data were taken from (32, 31) (table S1). A negative value
indicates that hybrids had higher fitness than the parental species
of –2.66 was left out of the bottom panel.
Number of studies
Components – divergent selection
Components – unknown cause
0 0.20.4 0.60.81-0.2 -0.4-0.6
VOL 323 6 FEBRUARY 2009
on April 6, 2010
drift, could be the result of either ecological or
These examples indicate a growing knowledge
of the mechanisms of selection and its conse-
most glaring deficiency is our knowledge of the
impact of selection on genes. Optimistically, pro-
quantitative trait loci (QTLs) and genes or regu-
latory control regions that affect individual pheno-
typic traits on which components of reproductive
isolation depend. Examples include the yup QTL,
which affects flower color differences between the
(35). Swapping alleles of this QTL between the
in pollinator preference and, hence, indirectly af-
fected premating isolation. Survival and salt toler-
ance of second-generation hybrids between the
sunflowers Helianthus annuus and H. petiolaris
ly to a QTL identified as the salt tolerance gene
Another form of investigation involves the
analysis of genome scans of ecologically different
populations and species. These scans compare
many marker loci spaced throughout the genome
(37). Markers that show excessive differentiation
between populations (outliers) may indicate selec-
tion on nearby genes. The method is particularly
informative when applied to populations with
ongoing hybridization, because outlier markers
may identify points in the genome that resist the
homogenizing influence of gene flow, perhaps
indicating genomic regions under divergent selec-
tion. However, sets of genes that diverged under a
mutation-order process can produce the same
pattern (17, 18), which makes analysis of such
studies more difficult. Clues to whether ecologi-
cally based divergent selection is involved are
gained if outliers at the same genomic locations
turn up repeatedly in scans between populations
that inhabit contrasting environments (38) and by
identifying phenotypic traits under divergent
(36, 39, 40). As genomic resources increase for
transplantingotherwise relatively homogenous ex-
into the environments of the parent species (35).
Mounting evidence for divergent selection in
speciation does not diminish the potential role of
mutation-order divergence. It may be that the
mutation-order process is more difficult to detect,
or perhaps we have not looked hard enough at
We still do not know much about the selective
factors causing mutation-order speciation.
from instances in which reproductive isolation
apparently evolved as a by-product of conflict
resolution between genetic elements within indi-
viduals (intragenomic conflict), such as cyto-
plasmic male sterility in hermaphroditic plants
(Fig. 1B), and genetic elements conferring
meiotic drive. Under both mechanisms, a muta-
tion arises that can distort representation in
gametes and spreads in a selfish manner, even
though such an element reduces overall fitness of
the organism that bears it. This, in turn, places
selection on mutations in other genes that counter
the selfish element’s effects and restore more
equal genetic representation in gametes. Distorter
and restorer mutationsare unlikely tobe thesame
in different populations regardless of environ-
ment; thus the process leads to divergence. The
mismatch between the distorter in one population
and the restorer in the other can result in hybrid
sterility or inviability and, thus, reproductive
isolation (3, 41). Numerous examples of selfish
elements, such as those observed in cytoplasmic
male sterility of plants, support these hypotheses
generated by meiotic drive has been identified in
Drosophila [reviewed in (3, 41)]. Sexual conflict
is also expected to lead to mutation-order spe-
ciation, but there are few compelling examples
(3). The contribution by these mechanisms to
speciation is still uncertain, however. The alleles
responsible for meiotic drive and cytoplasmic
fixation because selection on such elements is
frequency-dependent (43) and because restorer
alleles arise and weaken selection on the distorter
elements (44). Second, if divergent populations
come into secondary contact, the alleles within
each population causing cytoplasmic male steril-
ity or meiotic drive (and the corresponding
restorer alleles) will spread between the popula-
reproductive isolation (43). Hence, for these
of hybrids must be reduced to very low levels, or
other incompatibilities must arise that interact
with these genes to prevent their spread after
Our understanding of the role of natural selection
time. If he were here to witness, he would most
likely be staggered by the discoveries of genes
and molecular evolution and astonished at the
could generate reproductive isolation (45). Most-
evidence for the role of natural selection on
phenotypic traits in the origin of species. This is
really what On the Origin of Species was all
about.Between1859and the present,thegeneral
acceptance of the biological species concept
altered the focus of speciation studies. Yet, the
discovery that reproductive isolation can be
brought about by ecological adaptation in ordi-
nary phenotypic traits bridges Darwin’s science
of speciation and our own.
The most obvious shortcoming of our current
understanding of speciation is that the threads
connecting genes and selection are still few. We
have many cases of ecological selection generat-
the genetic changes that allow it. We have strong
signatures of positive selection at genes for repro-
ductive isolation without enough knowledge of
hardly have time to complain. So many new
that the filling of major gaps is imminent. By the
time we reach the bicentennial of the greatest
book ever written, I expect that we will have that
much more to celebrate.
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48. We thank M. Arnegard, R. Barrett, A. Case, H. Hoekstra,
M. Noor, P. Nosil, S. Otto, T. Price, L. Rieseberg,
S. Rogers, A. Schluter, S. Via, M. Whitlock, J. Willis, and
a reviewer for assistance and comments. This work
was supported by grants from the Natural Sciences and
Engineering Research Council of Canada and the Canada
Foundation for Innovation.
Supporting Online Material
Tables S1 to S3
The Bacterial Species Challenge:
Making Sense of Genetic and
Christophe Fraser,1* Eric J. Alm,2,3,4Martin F. Polz,2Brian G. Spratt,1William P. Hanage1
The Bacteria and Archaea are the most genetically diverse superkingdoms of life, and techniques
for exploring that diversity are only just becoming widespread. Taxonomists classify these
organisms into species in much the same way as they classify eukaryotes, but differences in their
biology—including horizontal gene transfer between distantly related taxa and variable rates of
homologous recombination—mean that we still do not understand what a bacterial species is. This
is not merely a semantic question; evolutionary theory should be able to explain why species
exist at all levels of the tree of life, and we need to be able to define species for practical
applications in industry, agriculture, and medicine. Recent studies have emphasized the need to
combine genetic diversity and distinct ecology in an attempt to define species in a coherent and
convincing fashion. The resulting data may help to discriminate among the many theories of
prokaryotic species that have been produced to date.
reveals about our ignorance of how evolutionary
forces form, shape, and extinguish bacterial ge-
netic lineages, of the mechanisms of differen-
tiation between subpopulations sharing common
descent, and of the process of adaptation to new
niches and changing environments. Animal spe-
cies are defined by their morphological and be-
havioral traits and by their ability or inability to
interbreed, but such categories cannot easily be
applied to the Bacteria or Archaea (or indeed to
many eukaryoticmicrobes). Instead,taxonomists
pose. Naturally, biochemical characters have been
selected for the convenience of taxonomists; they
he species debate in microbiology is not
only about a human desire to catalog bac-
terial diversity in a consistent manner, but
reflect only a tiny subset of those characters that
allow bacteria to use different resources in the
the true diversity in this superkingdom of life.
More recently, molecular methods [particularly
DNA-DNA hybridization and ribosomal RNA
but these methods have serious limitations and
cannot reliably assign a large collection of similar
strains to species (e.g., rRNA sequences are too
conserved to resolve similar species). rRNA se-
ordinary variety of microbial life, much of it
distinguished and circumscribed by rRNA se-
quences have revealed further diversity through
multilocus sequence analysis (MLSA) (2) and
to be explained by theory. Thus, practical dif-
ficulties, lack of theory, and observations of vast
have all fueled the controversy of how one de-
fines a bacterial species (3–8).
Darwin commented that “all true classification
is genealogical” [(9), p. 404]. Taxonomists have
thus used sequence relatedness to define cutoff
values that place two bacterial isolates into the
same or different species. The overall genetic
relatedness of isolates may be measured by the
extent of DNA hybridization between them, and
those that show 70% or more DNA hybrid-
ization are defined as the same species (2, 10).
Such cutoffs imply that sequences that cluster
together with a certain amount of similarity
must be from the same species, and moreover
that this cutoff value is applicable to all groups
of bacteria or archaea. Recent MLSA studies,
which use the concatenated sequences of mul-
tiple housekeeping genes to discern clustering
patterns among populations of closely related
taxa, suggest that species defined by taxono-
mists in many cases correspond to well-resolved
sequence clusters. However, these studies also
show that there is no universal cutoff or descrip-
tor of clusters that characterizes a species. Fur-
thermore, inspection of the clusters does not
always clearly reveal which level in the hierarchy
is more fundamental than any other (Fig. 1) (7).
As an example, Fig. 1A shows the relation-
ships among multiple isolates of three closely
related streptococcal species. Streptococcus
pneumoniae is a major human pathogen, S. mitis
is a commensal bacteria with a history of taxo-
these data (12). There are striking differences in
the amount of sequence diversity observed within
homologous housekeeping genes in these named
species, ranging from 1.2% for S. pneumoniae to
S. mitis. The distance between two randomly se-
lected S. mitis genotypes is similar to the average
distance between S. pneumoniae and S. pseudo-
pneumoniae genotypes (5.1%) (2). This implies
for differentiating species would tend to either
rejoin S. pneumoniae and S. pseudopneumoniae,
a species of its own. This is clearly unsatisfactory.
Habitats and Ecological Differentiation
A clear natural criterion to identify clusters of
evolutionary importance, which we might want
to call species, is to find ecological features that
distinguish them from close relatives. Among
pathogens, the ability to cause a distinctive dis-
ease has historically been used to define species,
1Department of Infectious Disease Epidemiology, Imperial
College London, London W2 1PG, UK.2Department of Civil
and Environmental Engineering, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA.3Department of
Biological Engineering, Massachusetts Institute of Technol-
ogy, Cambridge, MA 02139, USA.4Broad Institute of MIT
and Harvard University, Cambridge, MA 02139, USA.
*To whom correspondence should be addressed. E-mail:
VOL 3236 FEBRUARY 2009
on April 6, 2010