MCPEEK, MARK A., AND GARY A. WELLBORN. Genetic variation

Genetic variation and reproductive isolation among phenotypically
amphipod populations
divergent
Mark A. McPeek
Department of Biological
Sciences, Dartmouth College, Hanover, New Hampshire 03755
Gary A. Wellborn1
Department of Biology, Yale University,
New Haven, Connecticut 06520
Abstract
We examined the degree of reproductive isolation and patterns of genetic structure and diversification within and
among seven populations of the freshwater amphipod Hyalella azteca. Hyalella occurs acrossecologically dissimilar
habitats in southeasternMichigan and exhibits substantial, and apparently adaptive, genetically based phenotypic
variation among populations. Habitats with predatory centrarchid fish contain a small-bodied Hyalella ecotype,
whereas habitats lacking these predators contain a large-bodied ecotype. Hierarchical F statistics showed that allele
frequency variation at six electrophoretic loci is greater among populations within an ecotype than between ecotypes,
a result also reflected in a cluster analysis based on genetic distances among the seven populations. Despite the
lack of clear genetic differentiation between ecotypes, gene flow between ecotypes appearedrestricted or absent.
Physically adjacent large- and small-ecotype populations in the same drainage differed appreciably in allele frequencies. Within-population genetic structuring also differed between ecotypes.Whereasgenotype frequencies never
differed from Hardy-Weinberg expectations in large-ecotype populations, all small-ecotype populations exhibited a
significant deficiency of heterozygous genotypes at one or more loci. Interbreeding trials demonstrated that individuals from different populations of the same ecotype readily interbreed, but no interbreeding was observed in
crossesinvolving individuals of dissimilar ecotypes.Interbreeding results, considered together with between-ecotype
differences in allele and genotype structure, suggestthat Hyalella ecotypes in southeastMichigan form at least two
separate species. Furthermore, the lack of distinct allelic differences and the relative levels of allele frequency
differentiation within and between ecotypes suggestrecent local speciation in this taxon.
Lentic freshwater habitats are arrayed along a gradient
from small, fishless ponds and marshes to large lakes containing well-developed fish communities. This freshwater
habitat gradient has provided an important vehicle for exploring the ecology and evolution of freshwater taxa, particularly ways in which ecological processes shape community
structure in freshwater ecosystems (Brooks and Dodson
1965; Zaret 1980; Wellborn et al. 1996). Numerous comparative studies have provided insight into the ecological
mechanisms apparently underlying evolutionary diversification in taxa that exist across the habitat gradient (reviewed
in Wellborn et al. 1996). Few studies in this system, however, have focused on explicit evolutionary mechanisms of
diversification, such as genetic mechanisms of speciation or
intraspecific divergence, genetic structuring of populations
within and across regions of the habitat gradient, gene flow,
and degree and mechanisms of reproductive isolation among
divergent populations. Given the extensive understanding of
ecological processes affecting individuals and populations
1To whom correspondenceshould be addressed.Present address:
Department of Zoology and Biological Station, University of Oklahoma, Norman, Oklahoma 73019.
Acknowledgments
We thank S. J. Tonsor and S. Kalisz for allowing us to use the
“gel lab” at the Kellogg Biological Station to complete part of this
work. We also thank J. Brown, T. Kane, M. Pace,and an anonymous
reviewer for their helpful comments on the manuscript. This work
was supported by the National Science Foundation (grant DEB9307033).
across this gradient, exploration of evolutionary mechanisms
can shed light on how ecological processes may foster diversification in taxon lineages distributed over this habitat
gradient.
Here we explore some of these evolutionary issues for a
phenotypically variable and taxonomically problematic amphipod, Hyalella azteca (Amphipoda: Hyalellidae), that occurs in many permanent freshwater habitats throughout
much of North America. Hyalella exhibits genetically based
divergence among populations in body size (Strong 1972;
Wellborn 1994a), behavior (Strong 1973; Wellborn 1995b),
life history (Strong 1972; France 1992; Wellborn 1994a),
and morphological characteristics (Bousfield 1958; Cole and
Watkins 1977; Stevenson and Peden 1973) that, in at least
some cases (Cole and Watkins 1977; Stevenson and Peden
1973), is associated with speciation. Ecological studies of
life history diversification suggest that this divergence is
adaptive (Strong 1972; Wellborn 1994a). H. azteca is a problematic taxon because the level of genetically based divergence among populations and apparent lack of hybridization
between ecotypes (Wellborn 1995a) suggests H. azteca is a
species complex. The lack of qualitative morphological differences (Strong 1972, Wellborn 1995a), however, suggests
recent diversification within the taxon.
At least two Hyalella ecotypes occur in southeast Michigan. These ecotypes differ in body size, life history, and
mating behavior (Wellborn 1994a, 1995a, b). Fishless habitats contain a large-bodied ecotype, and habitats with fish
communities dominated by centrarchid fishes contain a
1162
Amphipod evolution
small-bodied ecotype (Wellborn 1995a). The size disparity
between ecotypes is substantial, with little overlap in adult
body size distributions (Wellborn 1995a). Compared with
the small-bodied ecotype, individuals of the large-bodied
ecotype mature at a larger size and exhibit a lower sizespecific reproductive effort (Wellborn 1994a). Hyalella in
the two habitat types experience qualitatively different regimes of size-biased predation (Wellborn 1994a), and the
observed evolutionary divergence in body size and life history is consistent with adaptive divergence in response to
these disparate mortality schedules (Law 1979; Michod
1979; Taylor and Gabriel 1992; Wellborn 1994a).
For Hyalella and other taxa that exist across ecologically
disparate habitat types, spatial heterogeneity in the selective
environment may drive interpopulation
trait divergence
(Reznick and Endler 1982; Reznick et al. 1990; Wellborn
1994a), which, in turn, can generate important genetic effects. Most obviously, adaptive divergence among populations may restrict gene flow among the populations, allowing
secondary genetic differentiation and perhaps fostering speciation. The reduction in gene flow may result, e.g., when
dispersing individuals experience low survival in new habitats because they possess traits that are inappropriate in the
colonized habitat (McPeek and Holt 1992). Additionally, reproductive success of dispersing individuals may be diminished because of mating incompatibilities associated with the
trait divergence (Schluter and Nagel 1995). Changes in within-population
genetic structure might also result from
changes in breeding structure associated with adaptive divergence. For example, changes in assortative mating (Wellborn 1995b) or local spatial mating structure may affect levels of inbreeding.
Here we address four issues concerning the genetic diversity and structure of Hyalella populations and ecotypes.
First, we describe the among-population genetic structure of
several Hyalella populations, including populations of both
ecotypes. Second, we examine the congruence of genotype
frequencies with Hardy-Weinberg expectations to determine
whether processes shaping intrapopulation genetic structure
differ among the populations. Third, we examine the pattern
of genetic diversification for ecotypes and populations using
a measure of genetic distance. Fourth, we use interbreeding
experiments to determine the extent of interfertility and potential gene flow among populations within and between ecotypes.
1163
and Kaiser Road Marsh appear to be isolated but lie within
the Huron River watershed. Winnewanna drains into the
Portage River system.
Methods
Allozyme analysis- Hyalella individuals were collected at
multiple locations within each habitat by sweeping littoral
vegetation with a dip net. Individuals for allozyme studies
were placed in microcentrifuge tubes and stored at -80°C.
We used cellulose acetate electrophoresis to examine the genetic structure of the seven Hyalella populations. We characterized the genotypes of individuals at six polymorphic
loci, The loci were mannose-phosphate isomerase (MPI, EC
5.3.1.8), triose-phosphate isomerase (TPI, EC 5.3.1.l), phosphoglucomutase (PGM, EC 2.7.5.l), phosphoglucose isomerase (PGI, EC 5.3.1.9), arginine phosphokinase (APK, EC
2.7.3.3), and malate dehydrogenase (MDH, EC 1.1.1.37). A
Tris-glycine (pH 8.5) continuous buffer system was used for
all enzymes except MDH, which was run in a Tris-maleate
(pH 7.8) buffer. Individuals were ground in one drop of 0.1
M Tris-HCl pH 8.0 and plated onto cellulose acetate plates
(Helena Laboratories). Gels were run for 20 min at room
temperature (22-24°C) and stained according to protocols
provided in Hebert and Beaton (1989) and Richardson et al.
(1986).
We used two kinds of analyses to examine patterns of
genetic differentiation and structure. First, we calculated
three-level hierarchical F statistics (Weir 1990), an analysis
that uses genotype frequencies to quantify population structure at hierarchical levels. Population structure at each hierarchical level arises from nonrandom mating and is quantified by the degree of departure from the expected
heterozygosity for a panmictic population. The highest level
in the hierarchy is ecotype, the next level is populations
within ecotypes, and finally individuals within populations.
We generate variance estimates for each hierarchical F statistic by jackknifing across loci (Weir 1990). Second, we
used cluster analysis to examine the relative distance relationships among populations in allele frequency space. Our
clustering algorithm used the method of unweighted pair
groups using arithmetic averages (UPGMA), and was based
on Nei’s (1978) genetic distances among the seven populations. To examine within-population
structure for each population, we applied x2- tests to determine whether genotype
frequencies at each locus occur in Hardy-Weinberg proportions (Weir 1990). Because many genotype classes had small
expected frequencies, we pooled genotypes into all homozygotes and all heterozygotes and performed tests for these
two classes.
Study populations -We collected Hyalella from seven
populations in Livingston and Washtenaw Counties, Michigan, in July and August 1994, including four lakes (Duck,
Mill, Winnewanna, and Sullivan) that support populations of
the small ecotype and have diverse fish faunas and three
fishless habitats (Kaiser Road Marsh, Duck Marsh, and
George Pond) that support populations of the large ecotype
(Wellborn 1995a). All populations are within 22 km of each
other in this hydrologically complex region. Duck Lake, Mill
Lake, and Duck Marsh have outflows draining eventually
into the Huron River system. Sullivan Lake, George Pond,
Interfertility
trials- We conducted interfertility trials to
examine the potential for gene flow among populations and
between ecotypes. One to a few days before the female molt,
males grasp females in a precopulatory mate-guarding behavior (Borowsky 1984). Pairs remain attached until the female’s molt, at which time any first instar offspring from a
previous mating are released and the new clutch of eggs is
fertilized by the guarding male as eggs pass from the female
genital pore into a ventral brood chamber (i.e., marsupium).
Fertilization is external, and females have no mechanism for
sperm storage. Pairs separate after fertilization.
1164
McPeek and Wellborn
Table 1. Allele frequencies of the seven Hyalella azteca populations. All population/locus combinations are based on a sample
size of 48 individuals, except PGM in Duck Marsh and MDH in
Duck Lake, which are based on a sample size of 36 individuals.
Populations exhibited little variation at APK and MDH, so those
data were omitted here.
Frequency
SulliGene Al- Kaiser Duck George Duck van
locus lele Marsh Marsh Pond Lake Lake
MPI
A
0.104 0.000 0.000 0.000 0.000
B
0.240 0.063 0.115 0.281 0.302
C
0.479 0.250 0.323 0.406 0.427
D
0.177 0.667 0.396 0.313 0.271
E
0.000 0.021 0.167 0.000 0.000
TPI
A
0.000 0.000 0.000 0.000 0.000
B
0.229 0.208 0.104 0.469 0.729
C
0.760 0.729 0.896 0.521 0.260
D
0.010 0.063 0.000 0.010 0.010
PGM A
0.010 0.181 0.063 0.083 0.021
B
0.156 0.528 0.823 0.656 0.531
C
0.750 0.292 0.083 0.188 0.229
D
0.083 0.000 0.031 0.073 0.219
E
0.000 0.000 0.000 0.000 0.000
PGI
A
0.000 0.000 0.000 0.010 0.000
B
0.313 0.281 0.375 0.469 0.677
C
0.469 0.521 0.375 0.104 0.302
D
0.063 0.063 0.177 0.406 0.021
E
0.156 0.125 0.073 0.010 0.000
F
0.000 0.010 0.000 0.000 0.000
I
0.20
NEI’S GENETIC
I
I
0.15
0.10
DISTANCE
I
0.05
I
0.00
KAISERMARSH
DUCK MARSH
(L)
(L)
GEORGE
POND(L)
SULLIVAN
(S)
WinneMill wanna
Lake Lake
0.000
0.156
0.656
0.188
0.000
0.010
0.448
0.510
0.031
0.031
0.656
0.135
0.177
0.000
0.010
0.417
0.104
0.448
0.021
0.000
0.021
0.135
0.802
0.042
0.000
0.000
0.958
0.031
0.010
0.135
0.510
0.281
0.063
0.010
0.094
0.875
0.021
0.000
0.010
0.000
Mature males and females carrying fertilized eggs in their
marsupia were collected from two small-ecotype (Sullivan
Lake and Duck Lake) and two large-ecotype (George Pond
and Duck Marsh) populations in July 1994. Sexes were separated from one another and held in several 1 liter jars for
5-6 d. The jars, which contained sediment and Potomogeton,
were capped with fine-mesh screen and submerged 40 cm
deep on a platform in a reservoir at the E.S. George Reserve’s experimental pond facility. The 5- to 6-d holding
period was sufficient for females to release their developing
broods (all females were examined under a dissecting microscope to ensure that they carried no eggs at the beginning
of the experiment).
After this isolation period, individuals were placed in experimental beakers in all possible intra- and inter-population
combinations (10 total combinations). Two individuals of
each sex were placed a 150-ml beaker that had been prepared
with 25 ml of pond sediment and a 30-cm-long stem of
Potomogeton with roots. Beakers were sealed with fine-mesh
screens and submerged on the reservoir platform. Head
length, a metric highly correlated with Hyalella body length
and mass (Edwards and Cowell 1992), of females and males,
respectively, used in the interbreeding experiment was 0.49
mm ± 0.049 (mean ± SD) and 0.44 ± 0.035 for Sullivan
Lake, 0.54 ± 0.051 and 0.49 ± 0.049 for Duck Lake, 0.72
± 0.031 and 0.80 ± 0.035 for George Pond, and 0.76 ±
0.058 and 0.79 ± 0.056 for Duck Marsh. The beakers were
recovered after 13 d. Each beaker was examined for the presence of offspring, and females were examined for the pres-
WINNEWANNA
0.20
I
0.15
I
0.10
0.65
(S)
0.00
Fig. 1. Phenogram of genetic similarity among Hyalella populations in southeast Michigan. The phenogram was constructed by
UPGMA clustering of a genetic distance matrix generatedfrom allele frequency variation at six loci. Letters in parenthesesindicate
ecotype: L, large-bodied ecotype; S, small-bodied ecotype.
ence developing embryos, which would indicate that the egg
had been fertilized and initial development had occurred.
Either finding was considered indicative of a successful mating.
Results
Allozyme analysis and population structure- Allele
frequencies in the seven populations at the four most informative electrophoretic loci are presented in Table 1. We detected 3-6 alleles per locus. Four loci, MPI, TPI, PGM, and
PGI, exhibited at least moderate levels of allelic polymorphism. The remaining two loci, APK and MDH, had one
allele at a frequency L 0.90 in every population.
The hierarchical analysis of population structure indicated
significant population subdivision at each hierarchical level,
but most differentiation among populations occurred within
the two ecotypes rather than between them. The lowest hierarchical level was individuals within populations, and substantial subdivision was observed at this level. The value of
the F statistic at this level is 0.483 (95% confidence interval
= 0.45 1-0.513), indicating a high degree of genetic structure
within populations due to inbreeding. The overall measure
of differentiation
among populations within ecotypes is
0.224 (0.199-0.249), and the measure of differentiation between ecotypes is 0.112 (0.084-0.138). Thus, differentiation
among populations within the two ecotypes is about twice
as great as the added level of differentiation between ecotypes.
The cluster diagram (Fig. 1) derived from Nei’s genetic
distance estimates (Table 2) also suggests that the large- and
small-ecotype populations do not form two genetically distinct groups. The small-ecotype Sullivan Lake and Winnewanna populations form one primary cluster, and the three
large-ecotype
populations
and the remaining
two
small-ecotype populations form the other primary cluster.
1165
Amphipod evolution
Table 2. Nei’s genetic distances among the seven Hyalella populations included in the allozyme study of genetic differentiation.
* L, large-ecotype population; S, small-ecotype population.
loci (MPI, TPI, PGM, and PGI; x2 test, P < 0.005 for each
comparison).
Populations of the two ecotypes clearly differed in their
correspondence with Hardy-Weinberg expectations (Table
3). No locus in any population of the large-ecotype displayed
genotype frequencies that were inconsistent with HardyWeinberg expectations (Table 3). In contrast, 12 of the 16
possible locus-population combinations for small-ecotype
populations had genotype frequencies that were inconsistent
with Hardy-Weinberg expectations at the MPI, TPI, PGM,
and PGI loci (Table 3). All these loci were deficient in heterozygotes, suggesting that inbreeding is substantial in the
small-ecotype populations. APK and MDH have very little
power to test Hardy-Weinberg expectations because each has
one allele near fixation in every population.
Although the large-ecotype populations do not form a distinct group, they do all lie within one of the two primary
clusters. Geographic arrangement of habitats may play some
role in patterns of genetic distance. With the exception of
Sullivan Lake, the two primary clusters define populations
within different watersheds, with Winnewanna in the Portage
River system and all other habitats in the Huron River system. Because Sullivan Lake appears to be isolated, it is possible that this habitat was colonized from the Portage River
drainage, explaining the genetic similarity of Hyalella in
Sullivan Lake and Winnewanna Lakes.
One notable result was that Duck Lake (small ecotype)
and Duck Marsh (large ecotype) populations were genetically dissimilar despite a physical arrangement of habitats
that makes movement of individuals between habitats very
likely. These water bodies are only 15 m apart and are contiguous along a 125-m earthen dam that separates the two.
Moreover, Duck Lake drains directly and continuously into
Duck Marsh during ice-free months. Thus, ample opportunity exists for exchange of individuals between these habitats due to the drainage flow, as well as transport by waterfowl and human activities. The genetic distance between
these populations (0.098), however, suggests that they are
genetically distinct. Also, these populations differ significantly in allele frequency at four of the six electrophoretic
Interfertility trials- Some mortality occurred during the
13 d of the interfertility experiment. Survival was higher for
individuals from the large-ecotype populations (86.0% of
both males and females survived) than for those from the
small-ecotype populations (47.0% of males and 60.5% of
females survived) over the course of the experiment. The
following analyses are therefore based only on replicates in
which at least one individual of each sex was alive at the
end of the experiment.
Interbreeding was observed in 11 of the 13 replicates
(84.6%) with males and females from the same population
(Table 4). Interbreeding was observed in 9 of the 14 replicates (64.3%) in which males and females from different
populations but the same ecotype were placed together. In
contrast, no offspring were observed in any of the 19 replicates (0.0%) in which males and females of different ecotype were placed together. A contingency table analysis of
these results indicated that the chances of obtaining offspring
in replicates when the same versus different ecotypes are
placed together differ significantly ( x2 = 25.76, df = 1, P
< 0.001). These results suggest that interbreeding between
ecotypes either does not occur or occurs at sufficiently low
levels to remain undetected with the sample sizes we used.
Because interbreeding at low levels can be genetically important, we used the binomial probability distribution to de-
Genetic distance*
SulliMill
van
Lake Lake
Lake*
(S)
(S)
Duck Lake (S)
0.059 0.016
Sullivan Lake (S)
0.077
Mill Lake (S)
Winnewanna (S)
Duck Marsh (L)
George Pond (L)
Winnewanna
Lake
(S)
0.142
0.058
0.130
Duck
Marsh
(L)
0.098
0.155
0.136
0.337
George
Pond
(L)
0.069
0.161
0.087
0.334
0.044
Kaiser
Marsh
(L)
0.145
0.166
0.156
0.290
0.100
0.148
Table 3. Inbreeding coefficients (Hartl and Clarke 1989) for each locus. x2 tests were used to determine whether genotype frequencies
differed from Hardy-Weinberg expectations. The test was performed on the genotype frequencies in two classes: homozygotes and heterozygotes (df = 1). Asterisks indicate a significant deviation from Hardy-Weinberg expectations. Lines indicate loci that are at or near
fixation for one allele (<5 expected heterozygotes) and therefore uninformative for determination of inbreeding coefficients and our test
for fit to Hardy-Weinberg expectations.
Population?
Kaiser Road Marsh (L)
Duck Marsh (L)
George Pond (L)
Duck Lake (S)
Sullivan Lake (S)
Mill Lake (S)
Winnewanna Lake (S)
MPI
0.130
0.105
-0.134
0.810***
0.774***
0.877***
0.443*
*P < 0.05, **p < 0.01, ***p < 0.001.
-1L, large-ecotype population; S, small-ecotype population.
TPI
0.041
0.060
-0.116
0.918***
0.896***
0.845***
0.224
PGM
0.053
-0.012
- 0.005
0.202
0.493***
0.303*
0.053
PGI
-0.019
-0.034
0.145
0.394**
0.629***
0.580*** 0.259
APK
-
MDH
0.023
-
1166
McPeek and Wellborn
Table 4. Population crossesmade, the number of replicates that
had at least one individual of each sex at the end, and the number
of replicates in which offspring were produced.
Replicates
with
reproduction
Replicates* observed (%)
Population cross
Crosses within a population
Sullivan Lake X Sullivan Lake
3(4)
Duck Lake X Duck Lake
2(4)
George Pond X George Pond
4(4)
Duck Marsh X Duck Marsh
4(4)
Crosses between populations within the same ecotype?
Sullivan Lake X Duck Lake
6(8)
Duck Marsh X George Pond
8(8)
Crosses between different ecotypest
Sullivan Lake X Duck Marsh
4(8)
Sullivan Lake X George Pond
3(8)
Duck Marsh X Duck Lake
7(8)
Duck Lake X George Pond
5(8)
66
50
100
100
33
87
0
0
0
0
* Number in parentheses is the original number of replicates. The first number is the number of replicates at the end of the experiment that had at
least one individual of each sex alive.
-t In these crosses, half of the replicates were initiated with males of the first
population listed and females of the second; the other half were initiated
with females from the first population listed and males from the second.
termine our confidence level in the interbreeding result by
asking at what background levels of interbreeding is the
probability of obtaining our result <0.05. This analysis indicated that the interbreeding probability per experimental
unit was unlikely to be >0.15.
Discussion
This study sheds light on possible mechanisms by which
Hyalella amphipods have diversified across the freshwater
habitat gradient and may aid in understanding the more general role of ecological processes in shaping diversification.
Recent investigations of several taxonomic groups undergoing diversification suggest that adaptation in the context
of a habitat template may play a central role in the process
of diversification (Schluter 1996). These investigations include studies of cave amphipods (Culver et al. 1995), Darwin’s finches (Grant 1986), sticklebacks (Schluter 1996),
sockeye salmon (Foote et al. 1989; Wood and Foote 1990),
and whitefish (Bernatchez et al. 1996). Study of such groups
can be especially revealing of ecological mechanisms in evolution (e.g., Schluter and McPhail 1992, 1993) because processes fostering incipient divergence are less likely to be
obscured by extensive postdivergence dispersal or accrual of
secondary changes (Brooks and McLennan 1991; Avise
1994). We first discuss our results, then explore their implications for diversification in Hyalella.
Genetic structure and diversification
among populations- Allele
frequencies at the six loci examined do not
clearly distinguish populations of the two ecotypes. Hierarchical F statistics indicated greater genetic variation among
populations within ecotypes than between ecotypes. This
pattern is also evident in the UPGMA cluster analysis, in
which the deepest division separates two small-ecotype populations from all other populations. Additionally, almost all
allelic variation is due to quantitative differences in frequencies rather than qualitative differences in allele composition
(Table 1). Although phylogenetic analysis is needed, these
genetic patterns suggest recent ecotypic divergence in these
populations.
Genetic structure within populations- Ecotypes evince a
dramatic difference in their fit to Hardy-Weinberg expectations for genotype frequencies (Table 3). Whereas large-ecotype populations did not deviate significantly from HardyWeinberg expectations, most small-ecotype populations displayed a clear and consistent deficiency in heterozygotes.
Heterozygote deficiency is quantified by inbreeding coefficients (Table 3). The degree of heterozygote deficiency in
Duck, Mill, and Sullivan Lakes is substantial. For example,
the average ratio of observed-to-expected heterozygote frequencies for MPI, TPI, PGM, and PGI loci in these populations is 0.36; thus, we observed only about one-third of
the number of heterozygotes expected with random mating.
The exception among the small-ecotype populations was
Winnewanna, which deviated from Hardy-Weinberg only at
the MPI locus.
The degree and consistency of the departure of genotype
frequencies from Hardy-Weinberg expectations in Duck,
Mill, and Sullivan Lakes suggest a common causative factor.
The most plausible explanation for this departure is inbreeding, which can cause extreme departure from Hardy-Weinberg expectations (Falconer 1981; Hartl and Clark 1989).
Although further study is required to resolve this somewhat
perplexing result, we suggest that one possible cause is that
low movement rates of small-ecotype individuals (Wellborn
1993) may result in the development of highly localized subpopulations, or demes. Hyalella primarily inhabit submerged
macrophytes in these habitats. Because predatory fish in the
small-ecotype habitats use visual cues to detect prey (Healey
1984), dispersal from plant to plant may be avoided because
of its high mortality risk. If dispersal rates are very low,
demes may develop from a few founder individuals at the
spatial scale of individual plants or plant stems. Such demic
structure may not develop in the fishless habitats because
prey motion is generally less costly in fishless habitats
(McPeek 1990; Wellborn et al. 1996).
Reproductive isolation between ecotypes- Available evidence indicates that gene flow between Hyalella ecotypes in
southeast Michigan is restricted, possibly to the degree of
complete reproductive isolation. First, no successful reproduction was observed in the between-ecotype interbreeding
trials, but successful reproduction was observed in 74% of
within-ecotype trials. Second, Duck Lake (small ecotype)
and Duck Marsh (large ecotype) populations were genetically dissimilar, despite the physical proximity of the habitats that makes movement of individuals between the habitats very likely. Third, reproductive isolation probably
explains the persistence of both ecotypes in Winnewanna
Reservoir (Wellborn 1995a). Individuals of the two ecotypes
coexist in sympatry at roughly equal densities in Winne-
Amphipod evolution
wanna Reservoir, yet there is no apparent hybridization, as
evidenced by the absence of morphologically intermediate
individuals (Wellborn 1995a). Because even low levels of
gene flow between ecotypes should lead to their genetic
amalgamation under such conditions, reproductive isolation
between the ecotypes is likely to play a strong role in their
persistence as distinct ecotypes.
Although the mechanism of reduced reproductive success
between ecotypes has not yet been examined, two factors, a
size bias in female mating preferences and the difference in
body size between ecotypes, may play important roles. In
the large ecotype, male reproductive success is strongly dependent on body size, with large males obtaining the great
majority of copulations (Wellborn 1995b). This size bias in
male reproductive success is apparently due to female choice
of larger males (Wellborn 1995b). Thus, a small-ecotype
male dispersing into a large-ecotype habitat may be effectively prevented from mating in that habitat simply because
of his small body size. Similarly, a large-ecotype female dispersing into a small-ecotype habitat may avoid mating with
smaller males. Although male reproductive success is also
size-dependent for the small ecotype, larger males do not
have greater mating success than males of intermediate size.
Thus, a large-ecotype male dispersing into a small-ecotype
habitat, while possibly being able to mate, will not have a
special mating advantage conferred by its large size. In addition to behavioral differences in the size dependency of
male mating success, the large disparity in body size between ecotypes might preclude interbreeding because of mechanical incompatibilities
in fertilization. Males grasp females in a stereotypical fashion and must position their
genital pore close to the posterior edge of the female’s ventral brood pouch at the time of egg release (Wilder 1940).
Because this positioning of the genital pore requires that
males bend their bodies around females, body size differences between ecotypes may hinder proper positioning and
thus fertilization.
Are ecotypes separate species?- Four lines of evidence
suggest that large and small Hyalella ecotypes in southeast
Michigan are separate species. First, as discussed above, this
study and field patterns imply a high degree of reproductive
isolation between populations of dissimilar ecotype, but not
between populations of the same ecotype. Second, Duck
Lake and Duck Marsh populations are genetically distinct
despite their close spatial proximity and the substantial opportunity for interbreeding. Similarly, both ecotypes coexist
sympatrically in Winnewanna Reservoir (Wellborn 1995a)
yet remain morphologically distinct, indicating little if any
gene flow between ecotypes in this habitat. Third, withinpopulation genetic structure differed between ecotypes but
was similar within ecotypes. Fourth, ecotype differences in
body size and important life history traits are nonplastic and
genetically based (Strong 1972; Wellborn 1994a). The presence of genetically based phenotypic differences, differences
in allelic frequencies, and absence of gene flow between ecotypes indicate speciation under current species concepts
(Cracraft 1989; Templeton 1989).
Although we think the weight of current evidence suggests that the large and small Hyalella ecotypes in southeast
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Michigan are separate species, the lack of consistent genetic
differences between ecotypes points to ambiguity on the issue of speciation. Although the average genetic distance between populations of different ecotypes is consistent with
the hypothesis of separate species (Thorpe 1982; Nei 1987),
the range of distance values is high and overlaps with withinecotype genetic distances. This lack of distinct genetic differentiation between apparently different species probably
reflects a relatively recent divergence time for the ecotypes
and suggests the possibility of repeated parallel evolution of
the ecotypes. A recent divergence is also suggested by the
absence of distinct morphological differences between ecotypes (Wellborn 1995a).
study helps to elucidate
Evolutionary patterns - This
some evolutionary patterns in Hyalella, at both geographic
and local spatial scales. For example, an important largescale pattern of divergence in these amphipods is that Hyalella ecotypes similar to the large and small ecotypes found
in southeastern Michigan also occur in Oregon (Strong
1972). In Oregon, coastal lakes with bluegill and other fish
have small-bodied Hyalella populations, whereas mountain
lakes, which contain only salmonid fish, contain large-bodied Hyalella (Strong 1972). One explanation for the parallel
pattern in both geographic regions is that divergence in Hyalella into two primary ecotypes occurred only once, with
their current distributional pattern due to postdivergence dispersal to appropriate habitat types. The absence of distinct
allelic differences and the inconsistent pattern of allele frequency differentiation between ecotypes in Michigan, however, makes this scenario doubtful. That genetic differentiation within the small ecotype is greater than differentiation
between ecotypes in southeast Michigan suggests that the
divergence occurred more locally. Thus, a more plausible
scenario is recent divergence in Michigan, with the Oregon
ecotypes issuing from a similar but independent divergence
event. When considered at the local spatial scale of southeast
Michigan, our results suggest that ecotypic divergence may
have occurred at least once within this group of habitats. As
described earlier, the patterns of genetic differentiation do
not clearly distinguish populations of the two ecotypes in
this area.
Whether considered at local or geographic spatial scales,
our results suggest that Hyalella may have diversified
through the mechanism of parallel speciation (Schluter and
Nagel 1995). Parallel speciation involves repeated independent evolution of the same reproductive isolating mechanism
in separate, but closely related, lineages. The process of parallel speciation involves the active or passive dispersal of
individuals from an ancestral source population into ecologically similar habitats where descendent populations are established. Each descendent population evolves the same reproductive isolating mechanism, thus becoming isolated
from the ancestral population but not necessarily reproductively isolated from other descendent populations (species).
The same reproductive isolating mechanism evolves because
descendent populations occupy ecologically similar habitats
and thus evolve similarly by natural selection. In Hyalella,
if body size differences between ecotypes are responsible for
reproductive isolation of large and small ecotypes, then evo-
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McPeek and Wellborn
lution of one ecotype from the other will produce reproductive isolation between them. For Hyalella, it is unclear
whether one ecotype is always the ancestral “source” population, but allowing either ecotype to give rise to the other
does not alter the fundamental processes that characterize
parallel speciation.
The possibility that the pattern of association between Hyalella ecotype and habitat type (fish-containing versus fishless) may have arisen through multiple independent evolutionary divergences at local or geographic scales is
strengthened by the observations that size and life history
changes appear to be (1) due to relatively simple shifts in
quantitative traits and (2) driven by similar selective regimes
that may cause rapid divergence within a lineage and trait
convergence between lineages. The major differences between populations in both body size and life history appear
to derive from a single, fundamental difference between the
ecotypes in the way they allocate resources to growth versus
reproduction across their ontogeny (Wellborn 1994b). When
compared with the large-bodied ecotype, the small-bodied
ecotype initiates allocation of resources to reproduction at a
relatively small body size (and thus has a smaller terminal
body size) and invests relatively heavily in reproduction
(Wellborn 1994a,b). Furthermore, divergence appears to be
an adaptive response to habitat differences in size-biased
predation and the resultant schedules of size-specific mortality (Wellborn 1994a). Disparate predation regimes can
drive rapid divergence in these life history traits (Reznick et
al. 1990, 1997). Studies involving other aquatic species have
suggested that parallel or convergent evolution, rather than
common ancestry, is responsible for morphological similarity among isolated populations. For example, populations of
the amphipod Gammarus minus in the southeastern United
States inhabit both surface waters and subterranean pools.
Cave populations have reduced eyes and large antennae relative to surface water populations (Culver 1987; Kane et al.
1992), and morphological similarity among populations occupying similar habitats has apparently arisen through multiple episodes of parallel evolution (Jones et al. 1992; Kane
et al. 1992; Sarbu et al. 1993; Culver et al. 1995).
Conclusions
The gradient of freshwater habitats from small ponds to
large lakes may play an important role in diversification of
freshwater taxa (McPeek et al. 1996, Wellborn et al. 1996).
Many freshwater animal genera contain species that are distributed among ecologically disparate regions of the gradient
(Wellborn et al. 1996), and such spatial heterogeneity in ecological regime can drive trait divergence among spatially
segregated populations (Reznick and Endler 1982; Reznick
et al. 1990; Neill 1992; Wellborn 1994a; Wellborn et al.
1996). In such an ecological setting, parallel speciation or
related processes may be important mechanisms of diversification. Future study of explicit evolutionary mechanisms
of diversification across the habitat gradient, including studies of population genetics, phylogenetics, and selection,
should contribute importantly to our understanding of the
role of ecological processes in shaping lineage diversification.
References
Amphipod
evolution
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