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Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066The Linnean Society of London, 2005*** 2005
842
161175
Original Article
HYALELLA
ECOMORPHS
G. A. WELLBORN
Biological Journal of the Linnean Society, 2005, 84, 161–175. With 4 figures
Life history and allozyme diversification in regional
ecomorphs of the Hyalella azteca (Crustacea: Amphipoda)
species complex
GARY A. WELLBORN1,2,*, RICKEY COTHRAN1 and SUZANNE BARTHOLF2
1
Department of Zoology and 2Biological Station, University of Oklahoma, Norman OK 73019, USA
Received 29 September 2003; accepted for publication 10 June 2004
In North America, several species in the freshwater amphipod genus Hyalella occur as one of two alternative phenotypic types, or ‘ecomorphs’, each possessing life history traits that allow success in alternative habitats that differ
in predation regime. This study documents life history diversification, reproductive isolation and allozyme differentiation of Hyalella ecomorphs in Oklahoma, and compares these results to previously reported patterns of phenotypic
and systematic diversification in Michigan. As in Michigan, two ecomorphs are common in Oklahoma, with an early
maturing, small sized ecomorph found in habitats containing Lepomis sunfish, which prey on Hyalella, and a late
reproducing, large sized ecomorph found in fishless habitats and in very shallow margins of large reservoirs. Allozyme analysis and laboratory interbreeding trials demonstrated that ecomorphs in Oklahoma are reproductively isolated species. Phenotypically, these species are very similar to species of the same ecomorph in Michigan. Large
ecomorph species in the two regions differ substantially in allozyme allele composition in a pattern consistent with
reproductive isolation, yet these species did not differ in a comparison of phenotype. The small ecomorph in Oklahoma is similar in phenotype to two of three small ecomorph species in Michigan. Overall, this study supports the
hypothesis that Hyalella diversification in North America is characterized by the evolution of similar phenotypic
solutions to comparable ecological challenges. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175.
ADDITIONAL KEYWORDS: adaptation – endemic species – evolution – Lepomis – predation.
INTRODUCTION
Convergent and parallel evolution are hallmarks of
ecologically mediated species diversification (Losos
et al., 1998; Ruber, Verheyen & Meyer, 1999; Bossuyt
& Milinkovitch, 2000; Schluter, 2000). Because ecologically similar habitats are often replicated across
landscapes (Wellborn, Skelly & Werner, 1996), the evolution of similar phenotypes in spatially recurring ecological settings should be common in adaptive
diversification (Schluter, 2000). Convergent or parallel
diversification may occur when habitats are similar in
biotic processes, such as competition (Grant, 1986;
Schluter, 1996) and predation (Reznick, Bryga &
Endler, 1990; Wellborn et al., 1996), or abiotic qualiET AL
.
*Corresponding author. E-mail: [email protected]
ties of the environment, such as light (Endler, 1987;
Culver, Kane & Fong, 1995). Current challenges to our
understanding of adaptive diversification include
identifying and understanding the major ecological
processes that drive adaptive diversification, determining which traits are targets of selection, and ascertaining the extent to which this adaptive process
influences the evolution of reproductive isolation and
speciation in a lineage.
This study examines life history diversification
within the Hyalella azteca species complex
(Amphipoda: Hyalellidae) to determine whether similar ecological settings promote similar expressions of
life history traits. Hyalella amphipods are common
grazers in freshwater habitats of the New World
(Bousfield, 1996), and recent studies have documented extensive diversification within the H. azteca
species complex in North America (Wellborn, 1994a,
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
161
162
G. A. WELLBORN ET AL.
1995a; Thomas, Blinn & Keim, 1997; Hogg et al., 1998;
McPeek & Wellborn, 1998; Witt & Hebert, 2000; Wellborn, 2002; Witt, Blinn & Hebert, 2003; Wellborn &
Cothran, 2004). Most notably, Witt & Hebert (2000)
used allozyme and mitochondrial DNA analyses to
document the occurrence of at least seven species distributed across recently deglaciated regions of North
America, with major clades separated by pronounced
genetic divergence. These species, and those examined
in this study, are currently undescribed.
Studies in south-eastern Michigan demonstrate
that species diversification has involved a marked pattern of habitat-specific morphological and life history
differentiation that is consistent with adaptive diversification (Wellborn, 1994a, 1995a). In fishless habitats, where mortality decreases with body size,
Hyalella mature at a large size, and obtain a large
adult size that provides a size refuge from predation
(Wellborn, 1994a) and enhances competitive ability
(Wellborn, 2002). In habitats with centrarchid fish
such as bluegill (Lepomis macrochirus) and related
species, mortality increases with body size due to
size-selective fish predation (Wellborn, 1994a), and
Hyalella mature at a small size and maintain a small
adult body size. These differences between ‘ecomorphs’
are consistent with adaptation to disparate mortality
schedules (Law, 1979; Edley & Law, 1988; Taylor &
Gabriel, 1992). A comparable pattern of Hyalella ecomorph diversification occurs in Oregon (Strong, 1972).
Here, we document a similar occurrence of Hyalella
ecomorph diversity within Oklahoma in the southcentral United States. We asked the following ques-
tions: (1) are large and small ecomorphs in Oklahoma
reproductively isolated species; (2) is life history and
morphological diversification of ecomorphs in Oklahoma quantitatively similar to that documented in
Michigan; and (3) what are the genetic relationships
among ecomorphs in Oklahoma and Michigan? Our
approach is to examine patterns of phenotypic diversification, genetic differentiation and reproductive isolation in large and small ecomorphs in Oklahoma, and
compare these results to those for Michigan ecomorphs. Additionally we examine an apparently endemic
species with traits that do not resemble either the
large or small ecomorphs.
MATERIAL AND METHODS
COLLECTION
SITES AND SPECIES
Hyalella collection sites in Oklahoma are described
in Table 1, and characteristics of Michigan sites are
described elsewhere (Wellborn, 1995a; Wellborn &
Cothran, 2004). Amphipods were collected during the
summer from shallow regions of the habitats. In Oklahoma, large and small ecomorphs are not known to
occur in the same habitats. Small ecomorph populations in Oklahoma are found in ponds and streams
that contain centrarchid fish, including one or more
species of Lepomis sunfish. As in Michigan (Wellborn,
1995a), large ecomorph populations in Oklahoma are
common in permanent fishless habitats where predatory invertebrates, such as dragonfly larvae, are the
dominant predators. The large ecomorph in Okla-
Table 1. Collection sites in Oklahoma, USA. Watershed nomenclature follows that of the United States Environmental
Protection Agency
Habitat (watershed)
Latitude, longitude
Ecomorph
Habitat ecology
Parameters examined
Antelope Pond (Middle Washita)
Blue River (Blue)
Briar Creek (Lake Texoma)
Choctaw Creek (Lower North
Canadian)
Washington Pond (Lower
Canadian)
Cowan Creek (Lake Texoma)
Thunderbird Lake (Little)
34∞30¢ N, 96∞56¢ W
34∞22¢ N, 96∞35¢ W
33∞60¢ N, 96∞49¢ W
35∞30¢ N, 97∞15¢ W
Small
Small
Small
Small
Small pond, Lepomis
Stream pools, Lepomis
Stream pools, Lepomis
Stream pools, Lepomis
Phenotype
Allozyme, Phenotype
Allozyme, Phenotype
Allozyme
34∞60¢ N, 97∞31¢ W
Small
Small pond, Lepomis
Allozyme, Phenotype
33∞54¢ N, 96∞51¢ W
35∞14¢ N, 97∞15¢ W
Large
Large
Allozyme, Phenotype
Allozyme, Phenotype
35∞30¢ N, 97∞18¢ W
Large
33∞50¢ N, 96∞48¢ W
34∞27¢ N, 96∞37¢ W
Large
Unique
Fishless seep and stream
Reservoir, Lepomis,
shallow benthosa
Reservoir, Lepomis,
shallow benthosa
Fishless stream and pools
Fishless spring
Ten Acre Lake (Lower North
Canadian)
UOBS Creek (Lake Texoma)
Cummings Spring (Blue)
Allozyme
Allozyme, Phenotype
Allozyme, Phenotype
a
Hyalella in Thunderbird and Ten Acre Lakes are restricted to the benthos at the extreme shallow edge of these habitats,
at water depths of approximately 1–2 cm.
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
HYALELLA ECOMORPHS
homa is also found in some large reservoirs that contain predatory fish, including Lepomis, but are
restricted to the benthos at the extreme edge of the
reservoir in water depths less than approximately 1–
2 cm (G. A. Wellborn, pers. observ.), where they
appear to be protected from predatory fish. The two
ecomorphs in Oklahoma are easily distinguished by
the presence of a dorsal mucronation on pleon segment 2 of the large ecomorph that is lacking in the
small ecomorph.
In addition to large and small ecomorphs, we also
examine an apparently unique Hyalella population
from a permanent freshwater spring, Cummings
Spring, with individuals that do not resemble the
large or small ecomorph. Physical characteristics of
the spring, along with Hyalella density estimates
and other observations are reported in Milstead &
Threlkeld (1986). Although small in body size, amphipods in this population have the dorsal mucronation
that is lacking in the small ecomorph, and differs
from both ecomorphs in life history (see Results). We
include the Cummings Spring population in this study
to capture the known scope of Hyalella diversification
in Oklahoma.
ALLOZYME
ANALYSIS
We used allozymes to assay nuclear genetic variation
among Hyalella ecomorphs in Oklahoma and Michigan, with the goal of assessing both evidence for reproductive isolation among the groups and broad
patterns in relationships among them. Although we
employ phylogenetic tools to evaluate patterns of
genetic association, we do not intend this analysis to
generate a rigorous phylogenetic hypothesis because
our allozyme study was designed primarily to assess
evidence for reproductive isolation among groups.
Nonetheless, this analytical approach can provide a
useful assessment of some broad patterns of genetic
relationship, such as determining the extent to which
regional ecomorphs form monophyletic groups.
The analysis included individuals from 11 habitats, with four populations each of the large and
small ecomorphs in Oklahoma, two populations of
the large ecomorph in Michigan, and individuals
from the unique population in Cummings Spring,
Oklahoma (Table 1). In order to understand relationships among these groups and Michigan small ecomorph species, our analyses incorporate allozyme
data from a study of three small ecomorph species
that occur sympatrically in Michigan lakes (Wellborn
& Cothran, 2004).
We evaluated allozyme variation using cellulose
acetate electrophoresis with protocols described in
Hebert & Beaton (1993). We scored allozyme variation
at seven polymorphic loci: aldehyde oxidase (Ao) (EC
163
1.2.3.1), glyceraldehyde-3-phosphate dehydrogenase
(G3pdh) (EC 1.2.1.12), glucose-6-phosphate isomerase
(Gpi) (EC 5.3.1.9), lactate dehydrogenase ( Ldh1,
Ldh2) (EC 1.1.1.27), mannose-6-phosphate isomerase
(Mpi) (EC 5.3.1.8) and phosphoglucomutase ( Pgm)
(EC 5.4.2.2). A Tris-citrate (pH = 8.0) continuous
buffer system was used for all enzyme systems. To
facilitate alignment of alleles, each 12-lane cellulose
acetate plate contained individuals from two habitats
loaded in alternate lanes, allowing individuals of each
regional ecomorph to be run alongside every other
regional ecomorph on several plates. Conformity of
genotype frequencies to Hardy–Weinberg equilibrium
was tested for each locus in each population using
Genetic Data Analysis (Lewis & Zaykin, 2001).
We used inspection of allele frequency data to assess
evidence for reproductive isolation among groups. In
particular, we evaluated the degree to which alleles
are common in one group, but are not detected in
another group, indicating an absence of gene flow
between the groups (Avise & Ball, 1990; Mallet, 1995).
This criterion is conservative, especially for geographically overlapping groups, in that it focuses on qualitative differences in allele composition (Mallet, 1995;
Sites & Crandall, 1997).
To assess relationships among regional ecomorphs,
we evaluated allozyme data using both continuous
maximum likelihood and parsimony approaches.
Wiens (2000) found that continuous maximum likelihood was among the most accurate methods for recovering a known topology with allozyme data, and that
parsimony approaches tended to be less robust, but
were effective in some cases. Continuous maximum
likelihood (Felsenstein, 1981) uses gene frequency
data and a Brownian motion model of evolution to formulate a phylogenetic hypothesis. We used the
CONTML program in PHYLIP (Felsenstein, 2002) to
derive the maximum likelihood tree. Bootstrap confidence in tree topology was assessed with PHYLIP. We
first generated 1000 bootstrapped data sets using
SEQBOOT, then determined the maximum likelihood
tree for each bootstrapped sample using CONTML,
and constructed the consensus of these trees with
CONSENSE. Maximum likelihood analysis includes
all alleles detected, regardless of frequency. Our parsimony analysis used loci as characters and each
unique allele combination as character states (Mabee
& Humphries, 1993), a method based on the presence
or absence of alleles at a locus. For our analysis, an
allele was included as present in a population if it
occurred at a frequency of 0.1. This restriction was
necessary to maintain the number of character states
at a computationally feasible level, but additionally
had the effect of reducing the influence of rare alleles.
Step matrices for character state transitions used a
weight of one for each allele gained or lost at a locus.
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
164
G. A. WELLBORN ET AL.
We used PAUP* 4.0 (Swofford, 1998) to conduct the
parsimony analysis, and evaluated support for clades
within the tree using bootstrap analysis. All trees
were unrooted.
LIFE
HISTORIES
To assess life history diversification of Hyalella in
Oklahoma we measured traits of females and males
from four small ecomorph populations, three large ecomorph populations, and the unique population from
Cummings Spring (Table 1). Only mature individuals
are used in the analysis. Traits examined were (1)
head length, an index of body size that is correlated
with total body length and mass (Edwards & Cowell,
1992; Pickard & Benke, 1996), (2) size at maturity,
estimated as the head length of the smallest gravid
female in the population sample, (3) clutch size of
gravid females measured as the number of embryos
carried in the ventral brood pouch, (4) mean embryo
size and (5) (in males) width of propodus of posterior
gnathopods, a strongly sexually dimorphic appendage
enlarged in males (Wellborn, 2000). Measurements to
the nearest 0.022 mm were obtained using an ocular
micrometer fitted on a dissecting microscope. We
determined mean embryo size for each female by measuring the long axis of a sample of at least three
embryos from the clutch, then applying the formula
for a prolate spheroid to calculate embryo volume. The
dimension for the short axis used in the calculation
was determined by measuring long and short axes on
samples of 15–29 females from each ecomorph, then
using regression to estimate short axis length from the
long axis length. Data for all traits were ln-transformed prior to statistical analysis.
We used life history data to address three primary
issues. Firstly, we evaluate whether the large and
small ecomorphs in Oklahoma differ in life history
phenotypes. Secondly, we examine how each of the
large and small ecomorphs in Oklahoma differs from
the corresponding ecomorph in Michigan. Thirdly, we
consider how the Cummings Spring population differs
from the large and small ecomorphs in Oklahoma. We
used populations as primary units of analysis, and the
sexes were considered separately. Generally, phenotypes were compared using multivariate analysis of
variance (MANOVA), with dependent variables for
females being maturation size, and population means
of head length, clutch size and embryo size. For males,
dependent variables were population means of head
length and gnathopod width. Comparisons of Oklahoma ecomorphs with Michigan ecomorphs used data
for the Michigan large ecomorph populations from
Wellborn (1995a) and for the three Michigan small
ecomorph species from Wellborn & Cothran (2004).
When the MANOVA was significant, indicating a dif-
ference in overall life history phenotype, we explored
the nature of this difference by evaluating traits in
separate univariate analyses. For the univariate analyses, maturation size, head length and embryo size
were analysed with one-way ANOVA, and clutch size
and gnathopod width were analysed with nested analysis of covariance using population as a nested factor
within ecomorphs, and head length as a covariate.
Head length was included as a covariate because
clutch size and gnathopod width are correlated with
body size within populations (Wellborn, 1995a).
Because life history characteristics of the three small
ecomorph species in Michigan have been assessed in
only a single habitat (Wellborn & Cothran, 2004) we
could not use MANOVA to compare characteristics of
the small ecomorph species in Oklahoma to those in
Michigan. Instead, we examine each trait separately
and simply ask whether traits of each small ecomorph
species in Michigan falls outside the 95% (and 99%)
confidence interval (calculated from population
means) of the Oklahoma small species. Given that life
history features of the recently discovered Michigan
small species have been assessed in only one habitat,
these comparisons do not incorporate among-population variation in traits of these species, and thus
results should be regarded as preliminary. We also
examined patterns of sexual size dimorphism for each
Oklahoma ecomorph using a paired t-test based on
the population mean size of each sex.
The unique Cummings Spring population was evaluated separately from the other populations in Oklahoma. We assessed its life history by comparing its
mean life history traits to the 95% (and 99%) confidence intervals of the large and small ecomorphs
from Oklahoma. Sexual size dimorphism was
assessed by analysis of variance.
INTERBREEDING
We employed laboratory mating trials to evaluate the
level of reproductive isolation between the large and
small ecomorphs in Oklahoma. Trials were initiated
by placing one male and one female in a 150 mL beaker containing a sand substrate and filled with lake
water. Beakers were housed in a greenhouse and
were allowed to accumulate a film of periphyton
before the trials. Before they were added to beakers,
females were separated from males until they
released their current clutch of offspring; thus any
developing embryos observed during trials must have
been fertilized by males used in the mating trials
(females have no mechanism for sperm storage).
Crosses used all possible combinations of males and
females from two small species populations (Antelope
Spring Pond, Briar Creek) and two large species
populations (UOBS Spring Creek and Thunderbird
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
HYALELLA ECOMORPHS
Lake). After initiating a trial, the beaker was examined once each day for precopulatory pairing and for
the presence of eggs in the marsupium. Four days
after eggs were first observed in the marsupium they
were examined daily under a microscope to check for
signs of development. Fertilized eggs have conspicuous patterning indicative of development, while
unfertilized eggs remain homogeneously opaque and
eventually disintegrate. Trials were ended when eggs
showed distinct signs of either development or disintegration. Trials in which the male or female died
were discarded.
Oklahoma large
Ten Acre
Cowan
UOBS
ALLOZYME
ANALYSIS
Michigan small
species C
Choctaw
Washington
Sullivan C
Duck Lake C
Michigan small
species A
Deep A
Duck A
100
Sullivan A
61
Blue
58
Duck Lake B
61
Michigan small
species B
Ten Acre
Oklahoma UOBS
Cowan
large
Thunderbird
77
Sullivan B
62
Duck
Marsh George
Michigan large
Duck Marsh
60
Cummings
Spring
91
97
90
55
Duck
Lake C
52
Michigan Sullivan C
small species C
Deep B
Michigan small
Sullivan B species B
Duck Lake B
74
Duck A
Sullivan A
Deep A
82
Michigan
small species A
Blue
Briar
Choctaw
Oklahoma small
Analysis of allozyme variation revealed substantial
levels of genetic divergence among groups (Appendix),
and the pattern of this allelic differentiation provides
substantial support for the species status of most
regional ecomorphs. Both the maximum likelihood
(Fig. 1) and parsimony (Fig. 2) analyses were similar
in placing populations of each regional ecomorph and
known species together in clades, and in consistently
placing some clades together. Within populations
there was much allelic diversity in some cases, and
deviation from Hardy–Weinberg equilibrium was
observed for at least one locus in each population
(Appendix). In all cases these deviations were due to
an excess of homozygotes, suggesting that some
microspatial population structuring may be common
in these species. Below, we use both inspection of
allelic composition and phylogenetic analyses to eval-
Oklahoma Briar
small
George Michigan large
Thunderbird
Washington
RESULTS
165
Deep B
Cummings Spring
Figure 1. Maximum likelihood tree as determined by
CONTML. Numbers are bootstrap values based on 1000
bootstrap replicates. Nodes without values are below 50%.
Figure 2. Majority rule parsimony tree from branch and
bound search that produced 35 most parsimonious trees of
length 66. Numbers are bootstrap values based on 1000
bootstrap replicates. Nodes without values are below 50%
and are represented as polytomies.
uate genetic patterns for regional ecomorphs and the
Cummings Spring population.
Large and small ecomorphs in Oklahoma differ substantially in allozyme composition, with a pattern of
genotypic differentiation indicative of two reproductively isolated species. At each locus, there is at least
one case in which an allele is common in one ecomorph
but is not detected in the other ecomorph (indicated in
Appendix). This difference in allelic composition is
echoed in results of the phylogenetic analyses in which
the large and small ecomorphs form monophyletic
clades (Figs 1, 2).
The Cummings Spring population appears reproductively isolated from both the large and small Oklahoma ecomorphs. There are numerous cases, broadly
represented across loci, in which alleles occurring at
high frequency in one or both of the ecomorphs is not
detected in the Cummings Spring population (indicated in Appendix). Both phylogenetic analyses suggest that the Cummings Spring population may be
most closely related to the Oklahoma large ecomorph,
but the long branch lengths separating them in the
maximum likelihood analysis suggests substantial
genetic divergence.
The genetic analysis suggests that the large ecomorphs in Oklahoma and Michigan are separate species with substantial genetic divergence. That the
ecomorphs from the two regions are separate species
is supported by the substantial difference in allelic
composition of the two regional ecomorphs. At three of
the seven loci they share no alleles in common, and at
each remaining locus there is at least one case of an
allele common in one group that is not detected in the
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
166
G. A. WELLBORN ET AL.
other (indicated in Appendix). Both phylogenetic analyses support placement of the Michigan large ecomorph in a clade with the small-bodied Michigan
species B. Oklahoma large ecomorph populations
form a distinct clade in both phylogenetic analyses,
with bootstrap support in the parsimony analysis, but
its placement relative to other groups is not well
resolved.
In both phylogenetic analyses, the Oklahoma small
ecomorph is closely related to Michigan species C, and
is distinct from other regional ecomorphs (Figs 1, 2).
Although the Oklahoma small ecomorph and Michigan species C are genetically similar, some distinct
differences in allele composition exist, with cases of
alleles at high frequency in one group, but absent in
the other group occurring at Mpi allele E, and Ao alleles A and B (Appendix; allele frequencies for Michigan
small ecomorph species are reported in Wellborn &
Cothran, 2004). Allele composition of the Oklahoma
small ecomorph differs broadly from all other species,
including the Michigan small ecomorph species A and
B (compare Appendix with Wellborn & Cothran,
2004), suggesting that it is not conspecific with these
other species.
LIFE
HISTORIES
Life history and morphology data for the large and
small species in Oklahoma and for the Cummings
Spring population are summarized in Table 2. The
Oklahoma large and small ecomorphs differed in overall life history phenotype for females (MANOVA;
d.f. = 4, 2; Wilks’ Lambda = 0.011; F = 45.47;
P = 0.022) and in traits examined for males
(MANOVA; d.f. = 4, 2; Wilks’ Lambda = 0.017;
F = 117.59; P < 0.001). Considering individual traits
for Oklahoma large and small ecomorphs, the large
ecomorph had larger adult body size for both females
(Fig. 3; ANOVA; d.f. = 1, 5; F = 238.72; P < 0.001) and
males (ANOVA; d.f. = 1, 5; F = 281.91; P < 0.001). The
large ecomorph also had a larger maturation size
(Fig. 4; ANOVA; d.f. = 1, 5; F = 234.45; P < 0.001). The
large and small ecomorphs did not differ in embryo
size (Fig. 4; ANOVA, d.f. = 1, 5; F = 4.94; P = 0.077) or
size-specific clutch size (Fig. 4; nested ANCOVA,
d.f. = 1, 5; F = 0.50; P = 0.48). Males of the small species had larger size-specific gnathopod size than the
large species (Fig. 4; nested ANCOVA, d.f. = 1, 5;
F = 16.85; P < 0.001).
Table 2. Phenotypic traits of Oklahoma populations of Hyalella. Generally, data are population means ± SD of untransformed measurements. Parenthetical data are size-specific data presented as least square means ± SE from ANCOVA (see
text). Maturation size is the size of the smallest gravid female in the sample. Only mature individuals were included in
the study
Females
Males
Population
N
Head length
(mm)
Maturation size
(mm head
length)
Small ecomorph
Antelope Pond
22
0.49 ± 0.032
0.44
Blue River
50
0.48 ± 0.039
0.42
Briar Creek
61
0.49 ± 0.053
0.42
Washington Pond
38
0.51 ± 0.064
0.44
Large ecomorph
Cowan Creek
36
0.71 ± 0.083
0.58*
29
0.68 ± 0.066
0.60
35
0.69 ± 0.095
0.58
46
0.47 ± 0.051
0.40
Thunderbird
Lake
UOBS Creek
Unique species
Cummings
Spring
a
Clutch size
5.2 ± 1.56
(7.1 ± 0.65)
5.3 ± 1.62
(7.4 ± 0.43)
6.2 ± 3.02
(8.5 ± 0.38)
5.2 ± 2.27
(6.3 ± 0.51)
19.2 ± 5.35
(10.5 ± 1.04)
14.7 ± 4.78
(9.4 ± 0.70)
10.3 ± 4.93
(5.0 ± 0.71)
2.2 ± 1.06
(4.8 ± 0.45)
Embryo size
(mm3 ¥ 100)
N
Head length
(mm)
Gnathopod
width (mm)
1.5 ± 0.31
19
0.48 ± 0.030
1.4 ± 0.46
30
0.45 ± 0.033
1.7 ± 0.52
21
0.46 ± 0.046
1.6 ± 0.38
11
0.48 ± 0.042
0.43 ± 0.046
(0.53 ± 0.015)
0.31 ± 0.054
(0.45 ± 0.013)
0.37 ± 0.048
(0.51 ± 0.015)
0.35 ± 0.044
(0.47 ± 0.019)
1.6 ± 0.63
31
0.78 ± 0.144
1.8 ± 0.60
10
0.72 ± 0.080
2.1 ± 0.59
34
0.76 ± 0.093
2.2 ± 0.76
24
0.54 ± 0.056
0.54 ± 0.169
(0.36 ± 0.014)
0.60 ± 0.074
(0.48 ± 0.019)
0.59 ± 0.122
(0.44 ± 0.013)
0.40 ± 0.051
(0.46 ± 0.012)
Maturation size of Cowan Creek population is that determined in Cothran (2002).
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
0.7
Female
0.6
0.5
0.4
OKS
OKL
CS
MIL
MI A
MIB
MIC
Species
Figure 3. Head length, and index of body size, of adult
females and males. OKS, OKL, and MIL are means (± SD)
calculated across multiple populations, others are means
of a single population (see text). OKS, Oklahoma
small ecomorph; OKL, Oklahoma large ecomorph;
MIL, Michigan large ecomorph; CS, Cummings Spring population. MI A, MI B and MI C are Michigan small ecomorph species A, B and C, respectively.
Embryo size
(mm3 x 100)
Clutch size
(adjusted LSM)
Male
Gnathopod size
(mm adjusted LSM)
Body size (mm head length)
0.8
Maturation size
(mm head length)
HYALELLA ECOMORPHS
167
0. 6
0. 5
0. 4
10
8
6
2. 0
1. 5
0. 5
0. 4
OKS
OKL
CS
MIL
MI A
MI B
MI C
Species
Large ecomorph populations in Oklahoma did not
differ from large ecomorph populations in Michigan in
overall life history phenotype for females (MANOVA,
d.f. = 4, 2; Wilks’ Lambda = 0.095; F = 4.76; P = 0.18)
or in traits examined in males (MANOVA, d.f. = 4, 2;
Wilks’ Lambda = 0.409; F = 4.88; P = 0.17). Individual
trait comparison of the Oklahoma small ecomorph
with the three small ecomorph species in Michigan
(Table 3, Figs 3, 4) suggests that the Oklahoma small
ecomorph is very similar to Michigan’s species A and
C, but generally differs from species B. Specifically, the
Oklahoma small ecomorph species did not differ from
species A in any trait, and differed from species C only
in having a slightly smaller maturation size. The
Oklahoma small ecomorph species differed from species B in having a smaller body size for both males and
females, and in having a smaller maturation size and
larger embryos.
Large and small ecomorphs in Oklahoma differ in
the direction of sexual size dimorphism (Fig. 3). In the
large ecomorph species, males are larger than females
(paired t-test, d.f. = 2, t = 6.89, P = 0.020), but in the
small ecomorph species, males are smaller than
females (paired t-test, d.f. = 3, t = 3.23, P = 0.048).
Hyalella in Cummings Spring exhibit a life history
phenotype distinct from those of the large and small
ecomorphs in Oklahoma (Table 3). Individuals of the
Cummings Spring population are generally small in
size, with both sexes smaller than the Oklahoma large
Figure 4. Mean trait values for adults of regional ecomorphs and species. OKS, OKL and MIL are means (± SD)
calculated across multiple populations, others are means
of a single population (see text). Notations for regional
ecomorphs and species are the same as in Fig. 3.
ecomorph. Cummings Spring males are larger than
the Oklahoma small ecomorph, but females do not differ in size from the small ecomorph. Within the Cummings Spring population, males are larger than
females (ANOVA; d.f. = 1, 68; F = 23.82; P < 0.001).
Maturation size was smaller in Cummings Spring
individuals than either of the Oklahoma ecomorphs
(Table 3). Furthermore, females from Cummings
Spring had unusually small clutches of large embryos.
Gravid females averaged only 2.4 (± 1.16 SD) embryos
per clutch, significantly less than that of either the
large or small Oklahoma ecomorphs, even after
adjustment for differences in body size. Embryo size,
however, was significantly larger than that of the
small ecomorph and did not differ from the large ecomorph.
INTERBREEDING
Interbreeding trials between large and small
Hyalella species in Oklahoma demonstrated a sub-
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
168
G. A. WELLBORN ET AL.
Table 3. Comparison of single population with species confidence interval for each trait. OKS, Oklahoma small ecomorph;
OKL, Oklahoma large ecomorph; CS, Cummings Spring population. MI A, MI B, and MI C are Michigan small ecomorph
species A, B and C, respectively
Trait
Comparison
Female body
size
Maturation
size
Clutch size
Embryo size
Male body size
Male gnathopod
size
OKS vs. CS
OKL vs. CS
OKS vs. MI A
OKS vs. MI B
OKS vs. MI C
NS
OKL > CS**
NS
OKS < MI B**
NS
OKS > CS*
OKL > CS**
NS
OKS < MI B**
OKS < MI C*
OKS > CS**
OKL > CS**
NS
NS
NS
OKS < CS**
NS
NS
OKS > MI B**
NS
OKS < CS**
OKL > CS**
NS
OKS < MI B**
NS
NS
NS
NS
NS
NS
*P < 0.05, **P < 0.01, NS, not significant.
Table 4. Results of interbreeding trials for large and small ecomorphs in Oklahoma. N, number of experimental pairings
Cross
N
Embryo development observed (%)
Precopulatory pairing observed (%)
Small ¥ small, same population
Small ¥ small, different populations
Large ¥ large, same population
Large ¥ large, different populations
Small ¥ large
46
39
42
47
90
91.3
92.3
73.8
89.4
1.1
47.8
61.5
50.0
40.4
3.3
stantial level of reproductive isolation between
the species (Table 4). Crosses between conspecifics
resulted in successful fertilization and early embryo
development in a majority of trials, regardless of
whether crosses involved individuals from the same
or different population. In contrast, only one of 90
heterospecific crosses produced developing embryos.
Similarly, precopulatory pairing was often observed
in trials involving conspecific crosses, but pairs were
observed in only three of the 90 heterospecific crosses
(Table 4).
DISCUSSION
This study lends support to the hypothesis that life
history evolution in North American Hyalella amphipods has been shaped by adaptation to ecological
interactions that vary discontinuously across habitats
(Wellborn, 1994a, 1995a, 2002; Witt et al., 2003). Evidence presented here points to evolution of similar
size and life history phenotypes for species within two
ecomorphs in Oklahoma and Michigan, and these similarities in phenotype are associated with ecological
similarity of the habitats occupied by each ecomorph,
suggesting similar adaptive responses to similar ecological challenges.
DIVERGENCE AND REPRODUCTIVE ISOLATION
OKLAHOMA ECOMORPHS
IN
The large and small ecomorphs in Oklahoma are
distinct species, rather than phenotypic variants of a
single species. The qualitatively different allele composition of the ecomorphs (Appendix) is consistent with
reproductively isolated species, especially given that
populations of the two ecomorphs are geographically
interspersed in Oklahoma and in some cases their
habitats are permanently hydrologically connected
(Table 1, G. A. Wellborn & R. D. Cothran, pers. observ.).
Laboratory interbreeding trials also support the
conclusion that ecomorphs in Oklahoma are distinct
species, and additionally suggest that reproductive
isolation is prezygotic. In these experimental crosses,
only one of the 90 trials involving individuals of different ecomorphs resulted in embryo development.
Because trials were ended when embryo development
was observed, it is not known whether this cross would
have resulted in viable, fertile offspring. Nonetheless,
the very low frequency of successful fertilization in
these laboratory trials, together with the genetic evidence of reproductive isolation in natural habitats,
indicates the absence of any relevant gene flow
between large and small ecomorphs. Furthermore,
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
HYALELLA ECOMORPHS
these trials point to prezygotic reproductive isolation
of ecomorphs. Precopulatory pairing was observed in
approximately 50% of trials involving the same ecomorph, but in only 3% of trials involving different ecomorphs. Mechanisms of species and sex recognition are
poorly understood in amphipods (Jormalainen, 1998),
but chemical cues, such as contact pheromones, may be
widespread (Borowsky, 1991). Because of their potentially critical role in the evolution of reproductive isolation, assessing the factors that mediate mate
recognition in Hyalella may be essential for understanding diversification in this group.
Maturation and body size differences between large
and small ecomorphs in Oklahoma are substantial.
Compared to the small ecomorph, large ecomorph
females mature at approximately 35% greater head
length, or approximately 230% difference in dry mass
(based on head length–mass relationships from Pickard & Benke, 1996) and obtained a 40% greater mean
adult head length, or 260% greater dry mass. For
males, differences in body size were even more pronounced, owing to ecomorph differences in sexual size
dimorphism (Fig. 3). Overall, the difference in adult
size produced essentially non-overlapping adult size
ranges, with only 5% of small ecomorph individuals
falling within the adult size range of the large ecomorph. Although size ranges of amphipods in these
field samples are subject to influences of size-selective
predation, substantial size differences persist in
experimental populations that are protected from predation and reared over generations (Wellborn, 2002).
Differences in maturation size, rather than differences in juvenile growth rate, probably explain ecomorph differences in adult size within Hyalella. A
study of size-specific growth in Michigan ecomorphs
found that ecomorphs grew at similar rates while
juveniles, and that growth rates of both ecomorphs
slowed at maturity. Because the small ecomorph
matured earlier than the large ecomorph, however,
the large ecomorph continued rapid pre-reproductive
growth for a longer duration, resulting in substantial
differences in adult size (Wellborn, 1994b). This pattern is consistent with the broader observation that
maturation size and maximum adult body size are
generally correlated in animal taxa (Shine & Charnov,
1992; Winemiller & Rose, 1992). The Oklahoma ecomorphs did not, however, differ significantly in embryo
size or size-specific clutch size, suggesting that change
in maturation size, together with associated differences in adult body size represents the primary functional difference between these ecomorphs.
ECOMORPHS
IN
MICHIGAN
AND
OKLAHOMA
The genetic analysis strongly suggests that large ecomorphs in Oklahoma and Michigan are separate spe-
169
cies, and further raises the possibility that they have
independently converged on their very similar life history phenotypes. Although it is not possible in this
study to entirely eliminate the possibility that ecomorphs in the two regions are a single species with genetic
differences between them arising from extreme geographical variation in gene frequencies, this explanation is improbable given the pattern of genetic
differentiation among known species. To consider this
issue, we used the allozyme data to calculate Nei’s
(1978) unbiased genetic distance (Genetic Data Analysis; Lewis & Zaykin, 2001) between populations. The
range of pairwise genetic distances between large ecomorph populations in Oklahoma and Michigan is
2.33–2.80, a much greater genetic divergence than the
range of 1.33–1.83 observed between populations of
the large and small ecotype species in Oklahoma, and
the range of 1.07–1.37 observed between both ecomorphs in Oklahoma and the Cummings Spring species.
Thus the substantial genetic divergence between large
ecomorphs in Michigan and Oklahoma is consistent
with reproductively isolated species. In the light of the
extensive genetic differentiation of these species, the
close similarity of their life history phenotypes is
remarkable. The large ecomorph species in Oklahoma
and Michigan do not differ in the multivariate analysis of traits for either males or females. We are also
unaware of any distinct morphological traits that differ between these species.
The small ecomorph in Oklahoma is commensurate
in size and life history with small ecomorph species in
Michigan, and these species occupy ecologically similar habitats. The Oklahoma small ecomorph is very
similar in body size and life history to the small ecomorph species A and C in Michigan, differing only in
that Oklahoma small ecotype females mature at a
slightly smaller size than Michigan species C
(Table 4). The Oklahoma small species is distinct from
Michigan species B, being generally smaller in size
and having larger embryos, differences consistent
with the pattern of differentiation observed among the
three small ecomorph Michigan species (Wellborn &
Cothran, 2004). It is clear from inspection of gene frequencies and phylogenetic analyses that the Oklahoma small ecomorph is genetically very similar to
Michigan species C, but it is unclear whether these are
the same or distinct species. The Oklahoma small species differs morphologically and genetically from the
Michigan species C, suggesting that they may be different species. The Oklahoma small species lacks a
spine or mucronation on pleon segment 2 that is typically found in species within the H. azteca species
complex, including the Michigan species C. Although
similar in allozyme allele composition, at two loci
there are cases in which an allele common in one
regional ecomorph is not detected in the other (Mpi
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
170
G. A. WELLBORN ET AL.
allele E and Ao alleles A and B, Appendix). These
morphological and genetic differences may reflect
intraspecific geographical divergence, but might also
arise through reproductive isolation. Resolution of
this issue requires further studies focusing on interbreeding potential and biogeographical genetic analysis (Mallet, 1995).
LIFE
HISTORY PHENOTYPE AND ECOLOGICAL SUCCESS
Ecomorph differences in size and life history probably
contribute substantially to differential success of ecomorphs among ecologically disparate habitat types,
and to their complementary distributions across these
habitats. Our current understanding of ecomorph ecology suggests that ecomorph phenotypes are adaptive
solutions to disparate regimes of predation and competition (Wellborn, 1994a, 1995a, 2002) that vary discretely across the landscape (Wellborn et al., 1996). In
Oklahoma, as in Michigan and Oregon the small ecomorph is found in habitats that contain sunfish in the
genus Lepomis (Table 1; Strong, 1972; Wellborn,
1995a), predators that impose selection for small body
size. These fish locate prey visually and are strongly
size-selective, consuming larger individuals within
populations of Hyalella (Wellborn, 1994a) and other
invertebrates (Werner et al., 1983). In Michigan,
larger Hyalella individuals within small ecomorph
species had about five times greater per capita risk of
being consumed by a fish than did smaller individuals
(Wellborn, 1994a). This fish predation is intense, regulating Hyalella population density (Crowder & Cooper, 1982; Mittelbach, 1988; Wellborn & Robinson,
1991). Thus, the early maturity and small body size of
the small ecomorph species is adaptive under this
form of size-biased predation (Law, 1979; Taylor &
Gabriel, 1992), and selection experiments with microcrustaceans have confirmed the evolution of early
maturity and small size when mortality is greater for
larger individuals (Edley & Law, 1988).
Large ecomorph Hyalella occur in somewhat more
ecologically varied habitats than do small ecomorph
species, but in each region large ecomorph populations
appear similar in that they occur in habitats where
they experience no, or less intense, fish predation.
Large ecomorph populations are found in fishless habitats in Oklahoma and Michigan. In Oregon, large ecomorph Hyalella are found in Cascade Mountain lakes
where they are at risk of predation from trout only in
the early spring (Strong, 1972) before the seasonal
onset of reproduction, a time when Hyalella in these
habitats tend to be relatively uniform in size (G. Wellborn, unpubl. data). In Oklahoma, large ecomorph
Hyalella have also invaded extremely shallow marginal areas of some large reservoirs. This microhabitat, in benthos at water depths of 1–2 cm, may be a
refuge from fish predation due to mechanical impairment of foraging in such shallow water. In the absence
of strong size-selective fish predation, larger size is
likely to be beneficial for both avoiding predatory
invertebrates and enhanced success in intraspecific
interactions. Large Hyalella individuals are less susceptible to predation by many predatory invertebrates
(Wellborn, 1994a) that can be especially common in
fishless habitats (McPeek, 1990a). Accordingly, in a
field study of Michigan species, large ecomorph
Hyalella in a fishless habitat experienced decreasing
mortality as they grew in size from juveniles to adults
(Wellborn, 1994a). Large size may also provide a competitive advantage to individuals of the large ecomorph. Large ecomorph Hyalella strongly outcompete
small ecomorph Hyalella (Wellborn, 2002), perhaps
due in part to an increase in resource consumption
rate with body size (Wellborn, 1994b). Finally, male
mating success increases monotonically with body size
in the large ecomorph species in Oklahoma and Michigan, but not in the small ecomorph species (Wellborn,
1995b; Wellborn & Bartholf, in press). Thus in the
absence of significant mortality costs for large size,
ecological and behavioural processes favour the evolution of large body size, suggesting that large ecomorph
phenotype is adaptive in these habitats.
EVOLUTION
OF ECOMORPHS
This study suggests a prominent role for natural selection in shaping life history diversification of Hyalella
ecomorphs. Although detailed understanding of the
evolutionary history of diversification must await a
robust phylogenetic hypothesis, results of this and
other studies (Wellborn, 1994a, 1995a; Witt et al.,
2003; Wellborn & Cothran, 2004) point to adaptive
diversification as the explanation for both the dichotomous diversification into distinct ecotypes and
within-ecotype phenotypic similarity of species. Diversification of a lineage into two or more discrete
‘ecotypes’ may be common in freshwater taxa.
Although freshwater habitats vary along continuous
gradients of hydroperiod and size, transitions in composition of aquatic communities are often abrupt
(Wellborn et al., 1996; Stoks & McPeek, 2003). The
transition from fishless habitats to habitats containing predatory fish is characterized by substantial species turnover because the phenotypic characteristics
that make many species successful in fishless habitats
also make them highly vulnerable to predatory fish
(McPeek, 1990a, b; Wellborn, 1994a; Wellborn et al.,
1996; Stoks & McPeek, 2003). Larval damselflies in
the genus Enallagma, for example, exhibit complete
species turnover between fishless and fish-containing
habitats in Michigan because behaviours that are
advantageous in one habitat type are detrimental in
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
HYALELLA ECOMORPHS
the alternative habitat (McPeek, 1990a, b). Because
we often understand mechanisms underlying species
success in alternative habitats, such systems can be
especially illuminating of the ways that ecological
interactions contribute to distribution and abundance
of species, and to lineage diversification (Wellborn
et al., 1996; Brown, McPeek & May, 2000; McPeek &
Brown, 2000; Richardson, 2001).
Although species diversification of North America
Hyalella is characterized by substantial genetic differentiation (this study, Witt & Hebert, 2000; Witt et al.,
2003; Wellborn & Cothran, 2004), phenotypic diversification among species tends to be limited to two basic
ecomorphs, with each ecomorph comprised of more
than one species. Species not conforming to one of the
two typical ecomorph phenotypes have been described,
but these occur in isolated spring and hypogean habitats (Stevenson & Peden, 1973; Cole & Watkins, 1977;
Baldinger, Shepard & Theroff, 2000). Our current
understanding of more widely distributed species is
that they conform to either the large or small ecotypes
(Fig. 1; Strong, 1972; Wellborn, 1995a; Wellborn &
Cothran, 2004), and each ecotype is associated with
specific ecological conditions of the environment.
Thus, similar ecomorph phenotypes appear to represent similar phenotypic solutions to similar ecological
challenges. Although it is currently unclear whether
species similarity within ecotypes is due primarily to
convergent or parallel evolution, strong ecological
interactions, combined with the discontinuous nature
of aquatic habitats, appears to limit the potential
scope of phenotypic diversification even across substantial levels of molecular evolution.
CUMMINGS SPRING
The population of Hyalella amphipods in Cummings
Spring is distinct in both allozyme allele composition
and life history, and we suggest that this population is
a previously unrecognized species of Hyalella, and
may be endemic to this single spring habitat. The
prevalence of distinct allele differences between this
population and the widely distributed ecomorphs
clearly suggests the complete absence of gene flow
between the Cummings Spring population and populations of both the large and small ecomorphs in Oklahoma. Alternative explanations for the genetic
pattern, such as local selection on alleles and extensive drift due to low dispersal opportunity are unlikely
for a few reasons. Firstly, in addition to genotypic differences, the Cummings Spring population also differs
from the large and small ecomorphs in phenotype.
Although it is similar in size to the Oklahoma small
ecomorph, the Cummings Spring population has a
dorsal mucronation on pleon segment 2 that is lacking
in the small ecomorph in Oklahoma. Cummings
171
Spring also differs from the small ecomorph in clutch
and embryo size. Compared to the large ecomorph in
Oklahoma, the Cummings Spring population is much
smaller in body size and differs in other traits as well
(Table 3). Secondly, low opportunity for gene flow also
seems an unlikely explanation for the distinct genetic
pattern of the Cummings Spring population because
Cummings Spring drains into the Blue River only
about 0.1 km from the spring source, and Hyalella
from the Blue River are genetically and phenotypically typical of the Oklahoma small ecomorph.
Although current evidence suggests that Cummings
Spring Hyalella represent new species within the
H. azteca species complex, further studies are
required to determine the extent of its distribution.
Cummings Spring is located in a karst region, where
freshwater springs are common, and thus other populations of the species in Cummings Spring may exist
in regionally in other spring habitats.
The distinct life history of the Cummings Spring
population may be an adaptive response to its spring
environment. One pronounced life history feature of
this population is its comparatively large embryo size
(Table 3, Fig. 4). Offspring investment theory predicts
that females should produce larger eggs when the offspring’s survival advantage arising from large size
outweighs the concomitant decrease in maternal
fecundity (Smith & Fretwell, 1974; Lloyd, 1987). Thus,
larger egg size is favourable in an environmental setting in which smaller body size entails a comparatively high mortality cost for juveniles, as might occur
under resource stress or size-biased predation. Predators are uncommon in this habitat (Milstead &
Threlkeld, 1986; G. A. Wellborn, pers. observ.). The
spring is fishless, but does contain a relatively low
population density of the amphipod Gammarus lacustrus that might prey on juvenile Hyalella. Resource
stress, however, may be common in this habitat, which
has a rapid renewal time [approximately 00.09 hours
(Milstead & Threlkeld, 1986)] and thus limited opportunity for nutrient accrual and primary production.
Hyalella occur at high density in the spring, approximately 20 000–40 000 m-2 (Milstead & Threlkeld,
1986), perhaps in part due to low predation rates. This
high population density coupled with low habitat productivity may cause low survival for smaller juveniles
and drive the evolution of large egg size. Low productivity environments are often characterized by species
or populations with comparatively large egg size (Culver, 1982; Lessios, 1990), and the production of larger
eggs as a phenotypically plastic response to low
resource levels is interpreted as an adaptive life history shift (Gliwicz & Guisande, 1992; Urabe & Sterner,
2001). The small maturation size and generally small
body size of the Cummings Spring population may
also be an evolutionary response to resource stress if
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
172
G. A. WELLBORN ET AL.
the benefits of initiating reproduction at a relatively
small size outweigh the cost of lower future fecundity
due to decreased adult size (Kozlowski & Wiegert,
1987; Stearns, 1992). Optimal physiological allocation
models suggest that lower somatic growth rates,
which may result from chronic nutrient stress, favour
smaller maturation size (Kozlowski & Wiegert, 1987).
Ultimately, assessment of life history evolution in the
Cummings Spring species will require detailed demographic and ecological analysis of the population.
ACKNOWLEDGEMENTS
We thank S. Carson for allowing us to work at Cummings Spring and E. Kessler for access to Washington
Pond. We thank J. Witt and an anonymous reviewer
for providing very helpful comments on the manuscript. This research was supported by the National
Science Foundation (DEB 98–15059).
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APPENDIX
Allele frequencies for Gpi, Pgm, Mpi, G3pdh, Ao, Ldh1 and Ldh2 for Hyalella populations (Table 1). Alleles at each locus
are designated by letters, arranged alphabetically from slowest to fastest migrating allele. Comparable allele frequencies
for Michigan small ecotype species A, B and C are reported in Wellborn & Cothran (2004). Sample size is given in
parenthesis. Significant deviations from Hardy–Weinberg equilibrium were observed for the loci shown in the following
populations: Blue River (Pgm, G3pdh, Ao, Ldh2); Briar Creek (Pgm, Mpi, Ao); Choctaw Creek (Pgm, Mpi); Cowan Creek
(Ao); Cummings Spring (Pgm); Duck Marsh (Gpi, Pgm); George Pond (Pgm, Mpi, Ao, Ldh2); Thunderbird Lake (Ao); Ten
Acre Lake (G3pdh, Ao); UOBS Creek (Mpi, Ao); Washington Pond (Ao).
Locus
Allele
Blue
River
(47)
Gpi
A
B
C
Da
Ea,b,c
Fa,b,c
G
A
Ba
Cc
Db,c
Ea
F
G
H
A
Ba,b
Cc
Dc
Eb
Fa,c
G
Aa,b,c
B
Ca,b,c
D
E
Ab
B
Cc
Da
Ea
Fa
Gb,c
H
Ia
0
0
0.098
0
0
0.728
0.174
0
0
0.202
0.298
0.351
0.149
0
0
0
0.283
0
0.250
0.457
0.011
0
0
0.087
0
0.902
0.011
0.489
0
0.278
0
0.167
0
0
0
0.067
Pgm
Mpi
G3pdh
Ao
Briar
Creek Choctaw Washington
(53)
(46)
(42)
Cowan
(60)
Thunderbird
(42)
Ten
Acre
(62)
UOBS
(41)
Cummings
(36)
Duck
Marsh
(41)
George
(59)
0
0
0.047
0
0
0.792
0.160
0
0
0.178
0.289
0.456
0.078
0
0
0
0.227
0
0.205
0.568
0
0
0
0
1.000
0
0
0.550
0
0.380
0
0.070
0
0
0
0
0
0
0.241
0
0.750
0
0.009
0
0
0
0
0.862
0.138
0
0
0
0
0
0.792
0
0.208
0
1.000
0
0
0
0
0
0
0
0
0.316
0
0.105
0
0.579
0
0
0.159
0
0.817
0
0.024
0
0
0
0
0.850
0.150
0
0
0
0
0
0.619
0
0.357
0.024
1.000
0
0
0
0
0
0
0
0
0.300
0
0.038
0
0.663
0
0.066
0.107
0
0.762
0
0.0656
0
0
0.018
0
0.891
0.064
0.009
0.018
0
0
0
0.669
0
0.331
0
0.984
0.016
0
0
0
0
0
0.020
0
0.402
0
0.392
0
0.186
0
0
0.200
0
0.775
0
0.025
0
0
0.075
0
0.750
0.175
0
0
0
0
0
0.613
0
0.388
0
1.000
0
0
0
0
0
0
0.013
0
0.224
0
0.303
0
0.461
0
0
0.972
0
0
0
0.028
0
0
0
0
0.229
0.771
0
0
0
0.056
0.889
0
0.056
0
0
0
1.000
0
0
0
0.057
0
0
0
0.943
0
0
0
0
0.013
0.075
0.050
0.463
0
0.400
0
0
0.487
0.513
0
0
0
0
0
0.038
0.613
0
0.350
0
0
0
0
0
1.000
0
0
0
0
0
0.402
0
0.549
0
0.049
0
0
0.059
0.059
0.510
0
0.373
0
0.065
0.380
0.556
0
0
0
0
0
0.175
0.395
0
0.430
0
0
0
0
0
0.992
0.008
0
0
0
0
0.345
0
0.526
0
0.129
0
0
0
0.278
0
0
0.556
0.167
0
0
0.185
0.424
0.380
0.011
0
0
0
0.079
0
0.605
0.250
0.066
0
0
0
1.000
0
0
0.670
0
0.205
0
0.125
0
0
0
0
0
0
0.015
0
0
0.727
0.258
0
0
0.292
0.319
0.25
0.139
0
0
0
0.143
0
0.429
0.298
0.131
0
0
0
1.000
0
0
0.390
0
0.439
0
0.085
0
0
0
0.085
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175
HYALELLA ECOMORPHS
175
APPENDIX Continued
Locus
Allele
Blue
River
(47)
Ldh1
A
B
Ca,b,c
Da,b
E
A
B
C
D
Ea
F
Gb,c
H
0
0
0
0.989
0.011
0
0
0
0.065
0.054
0.261
0.609
0.011
Ldh2
Briar
Creek Choctaw Washington
(53)
(46)
(42)
Cowan
(60)
Thunderbird
(42)
Ten
Acre
(62)
UOBS
(41)
Cummings
(36)
Duck
Marsh
(41)
George
(59)
0
0.009
0
0.906
0.085
0
0
0
0.088
0
0.216
0.686
0.010
0
0
1.000
0
0
0
0
0
0.088
0
0.912
0
0
0
0
1.000
0
0
0
0
0
0.171
0
0.829
0
0
0
0
1.000
0
0
0
0
0
0.356
0
0.644
0
0
0
0
1.000
0
0
0
0
0
0.225
0
0.775
0
0
0
0
0
1.000
0
0
0
0
0.986
0
0.014
0
0
0
0
0
1.000
0
0.038
0
0.154
0.218
0.564
0.026
0
0
0
0
0
1.000
0
0.009
0
0.167
0.325
0.500
0
0
0
0.200
0
0
0.8
0
0
0
0
0
0
0.307
0.693
0
0
0
0
1.000
0
0
0
0
0.012
0
0.226
0.762
0
a
Allele common in populations of the large ecomorph in either Oklahoma or Michigan, but not detected in the other. bAllele common
in populations of one ecomorph in Oklahoma, but not detected in other ecomorph. cAllele common in either the large or small
ecomorph in Oklahoma but not detected in the Cummings Spring population, or common in the Cummings Spring population but
not detected in at least one Oklahoma ecomorph.
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 161–175