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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). 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The recent evolutionary origin of the phenotypically novel amphipod Hyalella montezuma offers an ecological explanation for morphological stasis in a closely allied species complex. Molecular Ecology 12: 405–413. Witt JDS, Hebert PDN. 2000. Cryptic species diversity and evolution in the amphipod genus Hyalella within central glaciated North America: a molecular phylogenetic approach. Canadian Journal of Fisheries and Aquatic Sciences 57: 687– 698. 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