A test of ecologically dependent postmating isolation between sympatric sticklebacks.

Abstract

Ecological speciation occurs when reproductive isolation evolves ultimately as a result of divergent natural selection between populations inhabiting different environments or exploiting alternative resources. I tested a prediction of the ecological model concerning the fitness of hybrids between two young, sympatric species of threespine sticklebacks (Benthics and Limnetics). The two species are ecologically and morphologically divergent: the Benthic is adapted to feeding on invertebrates in the littoral zone of the lake whereas the Limnetic is adapted to feeding on zooplankton in the open water. The growth rate of two types of hybrids, the Benthic backcross and the Limnetic backcross, as well as both parent species, was evaluated in enclosures in both parental habitats in the lake. The use of backcrosses is ideal because a comparison of their growth rates in the two habitats estimates an ecologically dependent component of their fitness while controlling for any intrinsic genetic incompatibilities that may exist between the Benthic and Limnetic genomes. The backcross results revealed a striking pattern of ecological dependence: in the littoral zone, Benthic backcrosses grew at approximately twice the rate of Limnetic backcrosses, while in the open water, Limnetic backcrosses grew at approximately twice the rate of Benthic backcrosses. Such a reversal of relative fitness of the two cross-types in the two environments provides strong evidence that divergent natural selection has played a central role in the evolution of postmating isolation between Benthics and Limnetics. Although the rank order of growth rates of all cross-types in the littoral zone was Benthic > Benthic backcross > Limnetic backcross > Limnetic, neither backcross differed significantly from the parent from which it was mainly derived. Implications of this result are discussed in terms of ecological speciation and possible introgressive hybridization between the species. Results in the open water were less clear and were not fully consistent with the ecological model of speciation, mainly as a result of the low growth rate of Limnetics. However, analysis of the diet of the fish in the open water suggests that these enclosures may not have been fully successful at replicating the food regimes characteristic of this habitat.

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? 2002 The Society for the Study of Evolution. All rights reserved.
Evolution, 56(2), 2002, pp. 322–329
A TEST OF ECOLOGICALLY DEPENDENT POSTMATING ISOLATION BETWEEN
SYMPATRIC STICKLEBACKS
HOWARD D. RUNDLE1
Department of Zoology and Centre for Biodiversity Research, University of British Columbia, Vancouver,
British Columbia V6T 1Z4, Canada
E-mail: rundle@zoology.ubc.ca
Abstract.
selection between populations inhabiting different environments or exploiting alternative resources. I tested a prediction
of the ecological model concerning the fitness of hybrids between two young, sympatric species of threespine stick-
lebacks (Benthics and Limnetics). The two species are ecologically and morphologically divergent: the Benthic is
adapted to feeding on invertebrates in the littoral zone of the lake whereas the Limnetic is adapted to feeding on
zooplankton in the open water. The growth rate of two types of hybrids, the Benthic backcross and the Limnetic
backcross, as well as both parent species, was evaluated in enclosures in both parental habitats in the lake. The use
of backcrosses is ideal because a comparison of their growth rates in the two habitats estimates an ecologically
dependent component of their fitness while controlling for any intrinsic genetic incompatibilities that may exist between
the Benthic and Limnetic genomes. The backcross results revealed a striking pattern of ecological dependence: in the
littoral zone, Benthic backcrosses grew at approximately twice the rate of Limnetic backcrosses, while in the open
water, Limnetic backcrosses grew at approximately twice the rate of Benthic backcrosses. Such a reversal of relative
fitness of the two cross-types in the two environments provides strong evidence that divergent natural selection has
played a central role in the evolution of postmating isolation between Benthics and Limnetics. Although the rank
order of growth rates of all cross-types in the littoral zone was Benthic ? Benthic backcross ? Limnetic backcross
? Limnetic, neither backcross differed significantly from the parent from which it was mainly derived. Implications
of this result are discussed in terms of ecological speciation and possible introgressive hybridization between the
species. Results in the open water were less clear and were not fully consistent with the ecological model of speciation,
mainly as a result of the low growth rate of Limnetics. However, analysis of the diet of the fish in the open water
suggests that these enclosures may not have been fully successful at replicating the food regimes characteristic of
this habitat.
Ecological speciation occurs when reproductive isolation evolves ultimately as a result of divergent natural
Key words.
isolation, sticklebacks.
Backcross, ecological dependence, ecological speciation, hybrid fitness, postmating isolation,reproductive
Received January 31, 2001. Accepted October 31, 2001.
Although natural selection has long been thought to play
a central role in the formation of new species, examples from
nature in support of this hypothesis are scarce (Coyne 1992;
Schluter 1996a; Futuyma 1998). The study of speciation has
instead focused on other issues including the role of founder
events in the evolution of reproductive isolation and the geo-
graphic context (i.e., sympatric vs. allopatric) of speciation.
However, renewed interest in mechanisms of speciation has
revived questions concerning selection’s role in the evolution
of reproductive isolation.
‘‘Ecological speciation’’ is a classic scenario for speciation
in which selection plays a central role. Ecological speciation
occurs when reproductive isolation evolves ultimately as a
result of divergent natural selection between populations in-
habiting distinct environments or exploiting alternative re-
sources (Mayr 1942; Muller 1942; Dobzhansky 1951; Endler
1977; Rice and Hostert 1993; Schluter 1996a, 1998, 2000).
Reproductive isolation (pre- and/or postmating) builds as
populations climb separate adaptive peaks under the influence
of natural selection. While laboratory experiments using Dro-
sophila have demonstrated the feasibility of this model (re-
viewed in Rice and Hostert 1993), there are limited data
concerning its importance in nature (but see MacNair and
Christie 1983; Craig et al. 1997; Feder 1998, Nagel and
1Present address: Department of Biological Sciences, Simon Fra-
ser University, Burnaby, British Columbia V5A 1S6, Canada.
Schluter 1998; Filchak et al. 1999; Hatfield and Schluter
1999; Rundle et al. 2000).
Here I focus on the evolution of postmating isolation by
divergent selection. Two types of postmating isolation are
recognized (Rice and Hostert 1993; Coyne and Orr 1998;
Rundle and Whitlock 2001). The first is ecologically depen-
dent isolation (equivalent to Rice and Hostert’s [1993] ‘‘en-
vironment-dependent’’ postzygotic isolation) and occurs
when hybrids, because they are intermediate in phenotype,
are less efficient at exploiting the dominant parental envi-
ronment (or resource) and an intermediate environment (or
resource) is lacking. In effect, these hybrids fall between
niches and thus suffer reduced fitness. The second type of
postmating isolation occurs when hybrids suffer reduced vi-
ability and/or fertility because of intrinsic genetic incom-
patibilities between the parental genomes independent of
their ecological context (equivalent to Rice and Hostert’s
[1993] ‘‘unconditional’’ isolation). Although intrinsic ge-
netic incompatibilities are an expected outcome of all mech-
anisms of speciation, ecologically dependent isolation is a
unique expectation of ecological speciation (Fig. 1). I test
this prediction using a recently diverged sympatric species-
pair of threespine stickleback.
My study builds on previous studies of the stickleback
species-pair in Paxton Lake, British Columbia, Canada
(McPhail 1984, 1992, 1994; Schluter and McPhail 1992). The
species are one of several pairs that evolved following the
invasion of fresh water by the marine threespine stickleback

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ECOLOGY OF BACKCROSS FITNESS
FIG. 1.
hybrids (open circles) relative to both parent species (closed circles)
as a result of two alternative mechanisms. Reproductive isolation
can be a function of either or both of these factors. (A) Postmating
isolation results only from intrinsic genetic incompatibilities be-
tween parent populations independent of their ecological context.
The relative fitness of hybrids may vary and depends on the extent
of heterosis, genetic incompatibilities, and the breakup of favorable
gene combinations interacting epistatically in the parents. (B) In-
trinsic genetic incompatibilities are absent and reproductive iso-
lation is solely the product of ecological mechanisms. Shown is the
simplest case in which morphology is inherited in a purely additive
fashion. Population P1is native to environment A and P2to B.
Relative fitness increases as genotypes approach the species native
to each habitat. Lines connect the same backcrosses in the two
environments, demonstrating the reversal in relative fitness ex-
pected under the ecological model. Nonlinear patterns may result
when dominance and epistatic effects contribute to hybrid fitness.
Hypothetical examples of a reduction in the fitness of
TABLE 1. Mean (? SE) initial body mass and seven other morphological traits for the actual fish used in the enclosure experiment. Standard
length, body depth, gape width, and gill raker length were ln-transformed prior to analysis. Body depth, gape width, and gill raker length were
size corrected by regression on ln(standard length).
Trait Benthic
Benthic
backcross
Limnetic
backcrossLimnetic
Mass (g)
Standard length
Body depth
Gape width
Gill rake number1
Gill raker length2
Pelvic spine length
Plate number3
1.08 (0.03)
3.85 (0.01)
0.032 (0.007)
0.12 (0.01)
12.75 (0.14)
?0.16 (0.02)
0.24 (0.14)
0.42 (0.16)
1.12 (0.02)
3.86 (0.01)
0.028 (0.008)
0.05 (0.02)
13.70 (0.23)
?0.06 (0.02)
1.54 (0.36)
2.70 (0.25)
0.99 (0.03)
3.83 (0.01)
?0.013 (0.011)
?0.08 (0.02)
16.23 (0.21)
0.06 (0.02)
3.77 (0.15)
5.18 (0.18)
0.95 (0.02)
3.80 (0.01)
?0.050 (0.012)
?0.10 (0.02)
16.86 (0.20)
0.19 (0.02)
4.18 (0.14)
6.59 (0.11)
1Total number on first gill arch.
2Length of longest raker on first gill arch.
3Includes any plate, regardless of size, measured on the right side of the individual.
(Gasterosteus aculeatus) after the retreat of the Pleistocene
glaciers 10,000–12,000 years ago (McPhail 1994). One spe-
cies of every sympatric pair, referred to as the Benthic, is
larger and more robust, has a wider gape and fewer, shorter
gill rakers, and feeds on invertebrates in the littoral zone of
the lake. The other species, referred to as the Limnetic, is
smaller and more fusiform, feeding primarily on zooplankton
taken in the open water of the lake. It has a narrow gape and
long and more numerous gill rakers (protuberances on the
gill arches that sieve food particles or direct the movement
of water through the buccal cavity [Sanderson et al. 1991]).
These phenotypic differences have a polygenic basis and per-
sist over multiple generations in a common laboratory en-
vironment (McPhail 1984, 1992; Hatfield 1997).
Premating isolation between the sympatric species is
strong and previous studies indicate that it has evolved ul-
timately as a result of ecological mechanisms (Nagel and
Schluter 1998; Rundle et al. 2000). Despite this premating
isolation, F1hybrids are found occasionally in the wild (?
1% adults; McPhail 1992) and can be readily made in the
laboratory using artificial crossing techniques. When raised
in the laboratory, these hybrids are intermediate in mor-
phology between the two parent species and are fully viable
and fertile, showing no evidence of intrinsic genetic break-
down (McPhail 1984, 1992; Hatfield and Schluter 1999).
However, individuals from a population established from F1
hybrids ten generations earlier, which are also intermediate
in morphology, have a foraging and growth disadvantage
relative to each parent species in the habitat of that parent
(Schluter 1993, 1995). Similarly, a transplant experiment in
the wild revealed that F1hybrids between Benthics and Lim-
netics from Paxton Lake, despite showing no growth reduc-
tion in the laboratory, grew at 73% the rate of Benthics in
the littoral zone and at 76% the rate of Limnetics in the open
water (Hatfield and Schluter 1999).
The present study tests a further prediction of the ecolog-
ical model, one that concerns the fitness of the backcrosses.
If postmating isolation is ecologically dependent, hybrid fit-
ness should depend on their phenotypic resemblance to the
parent species. Fitness should be higher in hybrids that more
closely resemble the native parent species in the habitat of
that parent. The Benthic backcross and Limnetic backcross
hybrids each resemble more closely the parent species from
which it was mainly derived (Table 1; Hatfield 1997). Thus,
the ecological model makes a clear prediction concerning
their fitnesses in the wild: the Benthic backcross should out-
perform the Limnetic backcross in the littoral zone (the hab-
itat preferred by the Benthic parent species), whereas the

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HOWARD D. RUNDLE
TABLE 2. Crossing design and number of families used in the recip-
rocal transplant. First letter of a cross-type indicates the species of its
mother and the second indicates the species of its father. Numbers
indicate the number of separate families. Wild and lab refer to whether
individuals of that cross-type were wild-caught or laboratory-reared
fish.
Female
Male
BB (wild)BL (lab)LB (lab)LL (wild)
BB (wild)
BL (lab)
LB (lab)
LL (wild)
8
2
2
2
2
2
2
2
2
8
converse should be true in the open water zone (e.g., see the
lines connecting the two backcrosses in Fig. 1B).
The use of backcrosses has an additional advantage in that
it allows us to discriminate between two possible hypotheses
to explain the earlier F1transplant results of Hatfield and
Schluter (1999). The first is that the reduced fitness of F1
hybrids in the wild is the result of ecologically dependent
isolation arising from their intermediate phenotype. Alter-
natively, reduced F1hybrid fitness is the result of intrinsic
genetic incompatibilities between the Benthic and Limnetic
genomes, but is expressed only in the stressful natural en-
vironment and not in the benign conditions of the laboratory
(where predation and disease are absent and food is abun-
dant). Indeed, this possibility is suggested by two past ex-
periments. In the first, an increased difference in fitness be-
tween control and mutation accumulation lines of Drosophila
was found when fitness was assayed under more harsh con-
ditions (Kondrashov and Houle 1994). In the second, new
mutations in diploid yeast strains that had no detectable fit-
ness consequences in a benign environment were shown to
have significant deleterious effects under a more harsh en-
vironment (Szafraniec et al. 2001; see Hoffmann and Merila ¨
[1999] for a review of the expression of genetic variation in
favorable and unfavorable environments). Thus, under this
second hypothesis, the expression of reduced F1hybrid fit-
ness is context dependent (benign vs. harsh conditions), but
fitness does not conform to the ‘‘ecological model’’ in which
hybrids suffer because they fall between the two niches of
the parent species. Backcrosses, but not F1hybrids alone,
can be used to distinguish these alternatives.
Backcrosses provide a measure of ecologically dependent
isolation while controlling for intrinsic genetic isolation that
may be present (Rundle and Whitlock 2001). This is because
backcrosses are phenotypically different yet are similarly af-
fected by genetic incompatibilities. Thus, a comparison of
the fitness of the backcrosses in the habitat of each parent
species allows the contribution of these incompatibilities to
be removed. This expectation derives from an analysis of a
quantitative genetic model of the phenotype of various in-
dividuals under the influence of outcrossing. The model, orig-
inally developed by Lynch (1991), was expanded to include
two environments and the resulting genetic effects that act
in an environment-dependent manner. Details are presented
in Rundle and Whitlock (2001). Analysis of this model re-
veals that a comparison of the fitness of both backcrosses in
both environments estimates the additive effect of genes that
act in an environment-dependent manner, independent of the
contribution of any intrinsic genetic incompatibilities. This
method is conservative as the comparison does not estimate
all genetic effects that may contribute to ecologically de-
pendent isolation (e.g., estimation of dominance effects that
act in an environmentally dependent manner requires addi-
tional crosses including the F1). The current study is the first
to use this method to estimate the extent of ecologically
dependent isolation while controlling for any intrinsic genetic
incompatibilities.
In addition to the prediction concerning the relative fitness
of the backcrosses, ecological speciation also requires that
hybrid fitness be reduced related to the native parent species
in each habitat (i.e., selection must be divergent). Thus, I
included both parent species in both habitats as a reference.
Including the parent species also allows a test of a more
general prediction of ecological speciation. In the absence of
any intrinsic genetic isolation, ecological speciation predicts
the rank order of the fitnesses of all cross-types. Basically,
the closer in phenotype a cross-type is to the parent species
native to that habitat, the higher its fitness. Thus, the rank
order of fitness in the littoral zone of the lake is predicted
to be: Benthic ? Benthic backcross ? Limnetic backcross
? Limnetic. This order should be reversed in the open water.
MATERIALS AND METHODS
Experimental Crosses
All crosses were made in the laboratory in the summer of
1999. Eight families each of both parent species (Benthic and
Limnetic) and both backcrosses (F1crossed with a Benthic
or with a Limnetic) were made and raised separately using
the fully balanced design shown in Table 2. All Benthic and
Limnetic individuals used to make these crosses were wild-
caught fish recently trapped from Paxton Lake on Texada
Island, British Columbia. F1hybrids used in the crosses were
laboratory-reared fish made the previous summer (1998) us-
ing wild-caught individuals from Paxton lake.
To make crosses, gentle pressure was applied to the ab-
domen of a gravid females causing her eggs to be released
into a petri dish filled with water. Fertilization was achieved
by macerating the testes dissected from a single male in the
same petri dish. Each clutch was then hatched separately in
an aerated, mesh-bottomed plastic cup suspended in one half
of a divided 100 L aquarium. Hatchlings were fed daily a
diet of live Artemia nauplii. As they grew, their diets were
supplemented with frozen bloodworms (chironomid sp.) and
frozen adult brine shrimp (Artemia sp.). Approximately two
to three months after hatching, 30 individuals from each
clutch were transferred into separate, undivided 100 L aquaria
to equalize densities among families.
Juveniles were overwintered in the laboratory at a variable
temperature (laboratory open to outside ambient temperature,
8–16?C) and a constant light regime (10L:14D). Fish were
fed daily a mixed diet of live Artemia nauplii, frozen blood-
worms and adult brine shrimp. These conditions were main-
tained until the fish were used in the transplant experiment
to reduce the likelihood of individuals entering reproductive
condition. At approximately nine months of age, family sizes
were reduced to 20 individuals each, with individuals selected

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ECOLOGY OF BACKCROSS FITNESS
nonrandomly within each family to minimize body size dif-
ferences among families and among cross-types. In addition,
the first dorsal spine of every fish was clipped to permit
identification at the end of the transplant experiment. From
the remaining 20 fish in each family, 12 were haphazardly
selected for use in the transplant experiment.
Reciprocal Transplant
The design of the transplant experiment was similar to
earlier experiments (Schluter 1995; Hatfield and Schluter
1999). The fitness surrogate was growth rate, measured over
a three-week period, of individual fish held separately in
enclosures in either parental habitat (littoral zone or open
water) in the lake. In May 2000, a total of 96 enclosures were
placed in Paxton Lake: 48 in the littoral zone and 48 in the
open water of the lake. The study was conducted at this time
because open water enclosures have the fewest benthic or-
ganisms colonizing them and previous studies have shown
that the diet of fish inside the enclosures is similar to wild
caught fish outside the enclosures at this time (Schluter 1995;
Hatfield and Schluter 1999).
Littoral zone enclosures were 1 m ? 1 m square, made of
6–10 mm knotless nylon mesh, with open bottoms and tops
and a metal frame attached around the bottom margin. They
were secured by sinking the metal frame into the sediment
of the lake bottom with the top edge of each enclosure held
out of the water on wooden stakes placed in each of the four
corners. The enclosures were located haphazardly around the
margin of the lake at a depth of approximately 1 m and were
emptied of wild fish prior to the experiment. Open water
enclosures were cylinders, made of the same nylon mesh, 1
m in diameter and 5 m deep, with a closed bottom and an
open top encircled by a metal hoop. Eight enclosures were
suspended from each of six rafts anchored in the deepest part
of the lake.
Prior to introduction, each fish was individually weighed
to within 0.01 g using an Ohaus CT 200 portable balance
(Ohaus Corporation, Florham, NJ). On May 1, 2000, a single
individual was placed in each enclosure. Twelve individuals
(from eight families) of each of the four cross-types were
used in each habitat. All fish were approximately 11 months
old when introduced to the enclosures and were large enough
to prevent their escape but small enough to allow for sig-
nificant further growth. Past studies have shown that calanoid
copepods, a large component of the diet of wild Limnetics,
tend to abandon open water enclosures (Schluter 1995). For
this reason, the contents of a single plankton tow, taken in
the open water of the lake at a depth of 0–2 m over an
approximately 100 m distance, was proportioned among the
open water enclosures daily.
After three weeks, all fish were removed from their en-
closures, weighed, anaesthetized, and then placed in 10%
Formalin. Growth rate (mg day?1) of the fish from each en-
closure was used as the independent observation. Fish were
recovered from 45 of the littoral zone enclosures (two Lim-
netics and one Limnetic backcross were not retrieved) and
from all 48 of the open water enclosures. Although none of
the fish were in reproductive condition at the start of the
experiment, a few were by the end. The weight of both males
and females may change when they enter reproductive con-
dition, so to avoid confounding effects, two gravid back-
crosses (one Benthic and one Limnetic) were deleted from
the littoral zone analysis, leaving 43 replicates. Conclusions
are similar when they are included.
Stomach contents were examined from all fish in both hab-
itats. Variation in diet was measured as percent organism
number with whole prey items identified and grouped into
broad categories (e.g., insect larvae, ostracods, chydorids,
pelagic copepods) indicative of the habitat in which they are
primarily found (e.g., littoral vs. open water).
RESULTS
Individual growth rates varied widely, within and between
cross-types, in both the littoral zone and open water enclo-
sures (Fig. 2A). In the littoral zone, Benthics grew fastest on
average (17.2 mg day?1), followed very closely by Benthic
backcrosses (16.7 mg day?1), with Limnetic backcrosses
growing more slowly (7.2 mg day?1), but still outperforming
Limnetics (3.7 mg day?1; Fig. 2B). In the open water, Benth-
ics and Benthic backcrosses grew slowly and at similar rates
(5.2 and 4.2 mg day?1, respectively). Limnetic backcrosses
performed better (10.1 mg day?1), whereas Limnetics per-
formed relatively poorly, growing at the same rate as Benthics
(5.2 mg day?1; Fig. 2B).
In accord with the prediction of ecological speciation, the
fitnesses of the two backcrosses were ecologically dependent
and reversed in the two habitats. In the littoral zone Benthic
backcrosses grew at more than twice the rate of Limnetic
backcrosses, whereas in the open water Limnetic backcrosses
outperformed Benthic backcrosses by a similar margin (Fig.
2B). This difference was confirmed by a significant inter-
action between habitat (littoral vs. open water) and backcross
type (Benthic vs. Limnetic) in a two-way ANOVA on growth
rates (F1,41? 17.61, P ? 0.0001). The main effect of habitat
was also significant, indicating that overall the growth rate
of backcrosses was higher in the littoral zone than in the open
water (F1,41? 6.72, P ? 0.013). The other main effect of
backcross was not significant, indicating that the difference
in growth rates of the backcrosses was similar in the two
habitats (F1,41? 0.91, P ? 0.346). These conclusions are
not altered if the data are ln-transformed (with the addition
of a constant to make every growth rate positive), with the
exception that the habitat term becomes marginally nonsig-
nificant (P ? 0.057).
Regarding the second prediction of ecological speciation
concerning the rank order of fitnesses of the cross-types in
each habitat, results were mixed. In the littoral zone, cross-
type (i.e., Benthic, Benthic backcross, Limnetic backcross,
Limnetic) had a significant effect on growth rate (F3,39?
9.18, P ? 0.0001) and, although the rank order of the cross-
types was as expected, not all differences were significant.
A Tukey posthoc comparison of means (Zar 1996) indicated
that the growth rates of Benthics and Benthic backcrosses
were significantly higher than that of Limnetic backcrosses
and Limnetics, but no other comparisons were significant
(Fig. 2B). Thus, Benthic backcross fitness was not signifi-
cantly lower than that of Benthics, the type native to the
littoral zone. In addition, growth rates of Benthic backcross

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HOWARD D. RUNDLE
FIG. 2.
in the littoral zone and open water of Paxton Lake. Individuals belong to one of four cross-types: Benthic, B; Benthic backcross, BB;
Limnetic backcross, BL; and Limnetic, L. (B) Mean growth rate (? SE) of each cross-type. Lines connect the same backcrosses in the
two habitats. Letters (a,b and c,d) indicate significantly different means using Tukey’s post hoc comparison of means (Zar 1996).
Comparisons are performed among the four cross-types separately for each habitat.
(A) Growth rates of hybrid (open circles) and parent species (closed circles) individuals when held for three weeks in enclosures
individuals were highly variable, with some individuals out-
performing the average Benthic (the type native to the littoral
zone; Fig. 2A).
In the open water, growth rate was also dependent on cross-
type (F3,44? 3.69, P ? 0.018) but results were less clear
and the rank order of cross-types was not fully as expected
under the ecological model of speciation (Fig. 2B). As ex-
pected, Benthics and Benthic backcrosses grew slowly; these
were the only cross-types in which some individuals lost
weight. Also as expected, Limnetic backcrosses performed
better. However, contrary to expectation Limnetics, the type
native to this habitat, performed relatively poorly and grew
at the same rate as Benthics. A Tukey posthoc comparison
of means (Zar 1996) indicated that the only significant dif-
ference in growth rate was between Benthic backcrosses and
Limnetic backcrosses.
Analysis of the diet of the fish indicated that the littoral
enclosures were successful at replicating the food regimes of
this habitat. The gut content of fish in these enclosures con-
sisted primarily of benthic invertebrates such as insect larvae
(41%, 36%, 38%, 33%; mean percent in diet for Benthics,
Benthic backcrosses, Limnetic backcrosses, and Limnetics,
respectively) and ostracods and chydorids (56%, 52%, 38%,
57%). For neither of these prey types do the proportions in
the diet differ significantly among cross-types when tested
using a one-way ANOVA (proportions were arcsine trans-
formed prior to testing; insect larvae: F3,37? 0.11, P ? 0.95;
ostracods and chydorids: F3,37? 0.78, P ? 0.51).
In the open water enclosures, diets were similar to that of
wild Limnetics in the lake, consisting mainly of zooplankton
such as pelagic copepods (51%, 37%, 77%, 62%). Differ-
ences in these proportions among cross-types approachedsig-
nificance however (F3,37? 2.44, P ? 0.08), suggesting that
different cross-types may have been exploiting different food
resources. These differences in diet among cross-types mirror
their differences in growth rate, with Benthic backcrosses
consuming the lowest fraction of zooplankton and also grow-
ing slowest, while Limnetic backcrosses consumed the high-
est proportion of zooplankton and also grew fastest (Fig. 2B).
Ostracods were also found in the diets of the open water fish
(29%, 40%, 15%, 26%), although the proportion in the diet
does not differ significantly among cross-types (F3,37? 1.56,
P ? 0.21). Ostracods are a prey item generally associated
with the littoral zone of the lake and not normally found in
significant numbers in the diet of wild Limnetics (Schluter
1993). Their presence also suggests that the environment
within these enclosures was not fully representative of the
open water environment of the lake.
DISCUSSION
Under the hypothesis of ecological speciation, reproduc-
tive isolation arises ultimately as a consequence of divergent
natural selection between environments or niches (Schluter
1996a, 2000). Here I have focused on the evolution of eco-
logically dependent postmating isolation, a unique prediction