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Geographic variation in nuclear genes of the eastern oyster, Crassostrea virginica Gmelin.

ABSTRACT The eastern oyster, Crassostrea virginica Gmelin, is a common inhabitant of estuarine and coastal waters from maritime Canada through the Gulf of Mexico. Because mitochondrial DNA haplotypes exhibit a distinct genetic break between Atlantic and Gulf oysters at Cape Canaveral, Florida, the degree of divergence between Atlantic and Gulf oysters in nuclear genes is less well known. We examined patterns of variation in four nuclear loci using restriction fragment analysis of amplified DNA (PCR-RFLP) in oysters (n = 317) from 16 locations spanning the geographic range of C. virginica. Marked differentiation was observed between Atlantic and Gulf populations, with smaller differences detected between North Atlantic and South Atlantic populations. Intermediate populations were observed in both eastern and northwest Florida. Regional population structure was also evident in the Gulf Coast, with Texas oysters highly divergent from all other populations.

KEY WORDS: Crassostrea virginica, oyster, genetics, nuclear DNA, RFLP

INTRODUCTION

The eastern oyster, Crassostrea virginica Gmelin, supports an important shellfish industry along the coasts of the Atlantic Ocean and the Gulf of Mexico. Its native range extends from the Gulf of St. Lawrence, Canada to the Gulf of Mexico. Reports of C. virginica from the Caribbean and South America (e.g., Nirchio et al. 2000) must be considered provisional, because the morphologically similar C. gasar has been observed in Brazil (Lapegue et al. 2002) and Venezuela (Gaffney, unpubl.).

Given the economic value of C. virginica, much interest has arisen in improving oyster stocks decimated during the last century by overfishing and disease (Galtsoff 1964, Ford & Haskin 1982, Rothschild et al. 1994). However, to allow effective enhancement of native oyster stocks, a better understanding of their basic population structure is essential. In particular, a more accurate picture of how intraspecific genetic diversity is distributed geographically will provide biologic guidance for regulating the transport of oysters, and for the selection of broodstock for hatchery-based restoration programs. Delineation and preservation of genetic variation is a central element in conservation biology programs, where the main goal is the survival and continued evolution of a species (Avise 1994, Driscoll 1998). Molecular genetic data, along with ecologic and morphologic data, can help inform efforts to preserve genetic and evolutionary diversity in a threatened or declining species (Mace et al. 1996).

Like most benthic marine invertebrates, C. virginica has a planktonic larval stage, which is in principle capable of widespread dispersal. If this occurs, then neutral (nonselected) genes should exhibit homogeneity across the geographic range of the species. However, evidence of geographic heterogeneity has been reported in both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) in C. virginica (Reeb & Avise 1990, Karl & Arise 1992, Hare & Arise 1996). Mitochondrial DNA haplotypes form 2 major assemblages, Atlantic (Gulf of St. Lawrence to Cape Canaveral) and Gulf Coast (including the Atlantic coast of Florida south of Cape Canaveral).

A similar genetic break has been observed in the distributions of other organisms such as the horseshoe crab, toadfish, and diamondback terrapin (Saunders et al. 1986, Avise et al. 1987). The geographical concordance in mtDNA divergence across species suggests a common set of historical factors initiating population genetic divergence and similar restrictions to gene flow (Reeb & Avise 1990). Considering the amount of mitochondrial DNA sequence divergence, Reeb and Avise (1990) estimated that the two oyster populations separated about 1.2 million years ago.

In addition, regional genetic discontinuities have been observed, particularly in peripheral populations. For example, oysters inhabiting Laguna Madre, Texas showed substantial genetic divergence from adjacent populations at 6 of 15 allozyme loci (King et al. 1994). Similarly, a comparison of samples from Florida and Nova Scotia showed fixation for alternative alleles at several allozyme loci (Buroker et al. 1979). This differentiation may be attributed to random genetic drift and/or natural selection in populations isolated by hydrogeographic barriers. For example, the distinct genetic composition of the Laguna Madre population has been attributed to prevailing currents that prevent transport of larvae in or out of the Laguna Madre (King et al. 1994). Genetic divergence may also be facilitated by natural selection. Laguna Madre oysters exhibit enhanced tolerance to hypersalinity, a factor which may serve both as a barrier to gene flow and as an agent of natural selection.

However, unlike mtDNA, studies of intraspecific differentiation in nuclear genes have yielded mixed results. Early allozyme surveys suggested little geographic differentiation over the entire species range, with the exception of peripheral populations (Buroker 1983, Gaffney 1996), although a reanalysis of Buroker's original data revealed a genetic break between Gulf and Atlantic populations (Cunningham & Collins 1994). In this case, the breakpoint occurred in northwest Florida, in contrast to that of mtDNA, which occurred on the Atlantic coast of Florida.

In the first study of nDNA polymorphisms in this species, Karl and Avise (1992) reported significant RFLP allelic frequency differences between Atlantic and Gulf populations at four anonymous nuclear loci. In contrast, McDonald et al. (1996) found no differences between Gulf and Atlantic oysters at six anonymous loci. In a DNA sequence analysis of alleles at three nuclear loci, Hare and Avise (1998) noted that the multiple haplotypes observed could not be aligned phylogenetically into distinct Atlantic and Gulf lineages, and argued that although nuclear loci might often display incomplete lineage sorting, differences in haplotype frequencies are consistent with current barriers to gene flow. They concluded that "Because so few intraspecific studies that attempt comparisons among multiple gene genealogies are as yet available, we do not know whether the hetereogeneity of phylogeographic patterns across loci in oysters will prove to be a common or rare outcome."

In this study, we examined patterns of geographic variation in four nonanonymous (Type I) nuclear loci, using targeted restriction endonuclease fragment length (RFLP) analysis. Primers were designed from 2 types of DNA sequence data: (1) complementary DNA (cDNA) and (2) putative protein-coding genomic sequence data. Our primary objective is to determine whether these loci exhibited a distinct Atlantic-Gulf break and at the same time to elucidate smaller-scale regional patterns of variation. As the number of independent nuclear loci sampled increases, it can be expected that a more robust and coherent phylogeographic picture of oyster population structure will emerge.

MATERIALS AND METHODS

Sample Collection and DNA Extraction

A total of 317 oysters from 16 sites between Canada and the Yucatan Peninsula, Mexico were used in this study (Fig. 1, Table 1). Tissues were preserved in 95% ethanol and stored at 4[degrees]C until the time of DNA extraction. DNA was extracted from adductor muscle or gill tissue using a Qiagen DNeasy extraction protocol for animal tissues. This method was chosen for its ease of use and high DNA yield (Robledo et al. 2000).

[FIGURE 1 OMITTED]

PCR Amplification of DNA

Five nuclear amplicons were screened for polymorphisms by targeted RFLP analysis, using restriction enzymes chosen on the basis of available sequence data. A standard PCR protocol included an initial denaturation step of 2 rain at 94[degrees]C followed by 35 cycles of 45 sec denaturation at 94[degrees]C, 45 sec annealing at either 55[degrees]C or 61[degrees]C, and 1:30 min extension at 72[degrees]C, with a final extension of 5 min at 72[degrees]C. Reaction volumes of 25 [micro]L contained 1 [micro]L of DNA extract, 1.5 mM Mg[Cl.sub.2], 200[micro]M each dNTP, 2.5 units of Taq polymerase (Sigma Jumpstart), and 0.2 [micro]M of each primer.

Primers O-40/41 amplified a 292 bp fragment derived from a random genomic clone containing putative coding sequence for a serotonin receptor (Fig. 2). The region amplified by these primers will henceforth be called Locus A, for simplicity. A second DNA fragment (Locus B), approximately 600 bp in size, was amplified using primers 0-42/43, derived from an annexin cDNA sequence (GenBank accession number BG624416). Because the amplicon is larger than the predicted 284 bp, it presumably contains one or more introns (Fig. 3).

[FIGURES 2-3 OMITTED]

Primer sets O-50/51 and 0-52/53 were designed to amplify overlapping fragments of a putative GABA-associated receptor protein (Fig. 4). Both primer pairs yielded products considerably larger than the expected sizes, consistent with the presence of one or more introns. Because each amplicon was subjected to RFLP analysis, it was necessary to verify that the additional (intron) sequence was not present in the region of overlap, or the same polymorphisms might inadvertently be scored in each amplicon. Amplification of the region of overlap using primers O-52/51 (Fig. 4) yielded a small fragment of the predicted size (173 bp), indicating that the introns were located outside of the region of overlap, and thus different introns were contained in the two amplicons. Because both were derived from the same cDNA sequence, they were combined into a single locus (Locus C) for purposes of haplotype analysis.

[FIGURE 4 OMITTED]

Primers O-91 and O-92 amplified a 350 bp fragment representing coding sequence from a putative acetylcholine receptor gene (Locus D, Fig. 5).

[FIGURE 5 OMITTED]

RFLP Analysis

Enzymes for RFLP analysis were chosen on the basis of available sequence data; if the complete sequence was not known (i.e., when introns were present in the amplified DNA), amplicons were digested with a selection of 4- and 5-base cutters. For each amplicon, a panel of 24 oysters representing the geographic range was screened initially; enzymes revealing readily scored polymorphisms were used to screen the entire Collection of oysters.

For each reaction, 4.5 [micro]L deionized water, 1.2 [micro]L 10x buffer, 5 units restriction enzyme, and 6 [micro]L PCR product were incubated for 3 h to overnight in a water bath at temperatures appropriate for each enzyme. All digests were examined on 2% agarose gels with the exception of Sac I and Nla III digests of Locus D, which were run on precast 5% ar 10% TBE polyacrylamide gels (Bio-Rad Criterion). All gels were stained with ethidium bromide for visualization of DNA bands. Restriction enzyme profiles were examined using a UV transilluminator (GibcoBRL, TFX-20M). Photographs of each gel were taken using a Kodak DC290 digital camera. Genotypes were inferred from the pattern of DNA bands resulting from enzyme digestion (Fig. 6). Only clearly interpretable polymorphisms (those in which the sum of the band sizes equaled the size of the uncut product) were scored.

[FIGURE 6 OMITTED]

Statistical Analysis of RFLP Data

Restriction digest patterns for each enzyme were combined into a multilocus genotype for each oyster. The computer program PHASE (Stephens et al. 2001) was used to infer haplotype phase status for individuals scored as heterozygotes at two or more loci. For each PHASE genotype assignment, confidence values are provided for each variable site. Individuals having one or more RFLP sites with confidence values below 60% were dropped from the data set. The inclusion of certain variable sites caused many individuals to have confidence values at one or more RFLP sites below 60%; these restriction sites were excluded from the analysis. PHASE genotype assignments were converted into NEXUS format for use in the GDA v. 1.1 (Lewis & Zaykin 2001). GDA was used to estimate single- and multi-locus statistics, including pairwise gametic phase disequilibrium, and Wright's F-statistics.

Alleles with FST values above an arbitrary cutoff level of 0.1 were examined for geographic patterns of variation in allele frequencies. In cases where fewer than two alleles met the 0.1 cutoff value, a value of 0.05 was used instead.

Exact tests of Hardy-Weinberg equilibrium were calculated using the program HWE, based on the Markov chain algorithm of Guo and Thompson (1992). A critical table-wide value of [alpha]' = 0.0031 (0.05 divided by 96 tests) was used as the initial significance cutoff for multiple testing using a sequential Bonferroni procedure (Holm 1979).

Exact row by column (R x C) tests (StatXact 4.0.1, Cytel, Inc.) were used to determine homogeneity of allelic frequencies among different geographic sites. R x C tests were also used to test homogeneity of allelic frequencies in geographically proximate collecting sites (e.g., within the Chesapeake Bay) as a condition for pooling subsamples.

A genetic distance matrix (Nei 1978) was calculated and used to construct a neighbor-joining phylogenetic tree using GDA and viewed using TreeView (Win32 version 1.6.6). Multidimensional scaling (MDS) of the genetic distance matrix using SYSTAT 9.0 (Wilkinson 1990) provided a 2-dimensional clustering of populations for the analysis of regional variation.

RESULTS

Locus A

Enzyme digestion of PCR products with enzymes Dpn II, Msp I, and Tsp509 I revealed a total of four cut sites consistent with the known sequence (Fig. 2). Phase analysis of the RFLP data resulted in seven different alleles (haplotypes). Of these seven alleles, three had significant [F.sub.ST] values (Fig. 2). Allele 1 was the most common, with frequencies greater than 0.6 at all sites except Texas, where its frequency dropped to <0.09 and the frequency of allele 6 rose to 0.35. Allele 7 was observed only at the Indian River, Florida site, with a frequency of 0.33.

The Tsp509 I restriction site polymorphism was eliminated from the PHASE analysis because its inclusion yielded genotype assignment confidence values below 0.6. Examination of data for restriction site 138 at each geographic area revealed heterozygote excesses at most sites, suggesting the possibility that some homozygotes were incorrectly scored as heterozygotes due to incomplete digestion of the PCR product. Homozygotes lacking the Tsp509 I cut site could be accurately scored, however, and these showed no geographic heterogeneity (R x C Monte Carlo test, P = 0.177), suggesting that significant information was not lost by elimination of this polymorphism.

Locus B

Enzyme digestion of PCR products with enzymes Dpn II and Hpa II resulted in a total of two cut sites. Phase analysis of restriction site genotypes yielded four different haplotypes (see Table 3). Of these, three met the 0.05 [F.sub.ST] cutoff (Fig. 3). Allele 1 was the most common, with frequencies fluctuating between 0.47 and 0.93. Allele 2 had frequencies between 0.04 and 0.40, with noticeable peaks at Cedar Key, Florida and Texas. Allele 4 ranged in frequency from 0.0 to 0.28, with highest frequencies in the mid-Atlantic, and was not observed in the Gulf coast.

Locus C

Enzyme digestion of PCR products with enzymes Ase I, Dra I, and Csp6 I resulted in a total of nine cut sites. Phase analysis resulted in 21 different alleles (Table 4). Four of the 21 alleles (1, 3, 10, and 17) met a 0.1 [F.sub.ST] cutoff (Fig. 4). Allele 1 exhibited a clinal pattern, with highest frequencies in the northern sites. Allele 3 was observed only in Florida, Louisiana, and Mexico. Allele 10 was most common in the southern Atlantic sites; allele 17 was found only in two Gulf coast sites. Six alleles were found only in the Texas population.

Locus D

Enzyme digestion of PCR products with enzymes Hind III, Sac I, and Nla III resulted in a total of six cut sites. Due to poor electrophoretic resolution of restriction digests, Sac I cut sites 75 and 308 were discarded. Nla III site 130 was discarded because its inclusion resulted in genotype assignment confidence values below 0.6.

PHASE analysis of the remaining three cut sites resulted in five alleles (haplotypes), of which three fell above the 0.05 [F.sub.ST] cutoff (Fig. 5). Allele 1 was the most common allele, present at all geographic sites, with frequencies between 0.5 and 0.98. The remaining two alleles, 4 and 5, were found only in the Texas sample.

Population Statistics

Genotypic frequencies at all four loci were generally in agreement with Hardy-Weinberg expectations (Tables 2, 3, 4, 5). Overall, genetic differentiation was substantial, with [F.sub.ST] values ranging from 0.061-0.166; 95% bootstrap confidence intervals for [F.sub.ST] averaged across loci were 0.071-0.144. No significant pairwise linkage disequilibrium was detected.

An UPGMA phylogenetic tree constructed using Nei's genetic distance (1978) shows distinct population structure within both Atlantic and Gulf (Fig. 7). Consistent with previous allozyme studies, the most divergent population sampled was Texas. Regional structure was also apparent in the MDS plot (Fig. 8), which suggests cluster of populations corresponding to North Atlantic, South Atlantic, and Gulf of Mexico.

[FIGURES 7-8 OMITTED]

DISCUSSION

Previous studies using anonymous nuclear DNA markers have obtained conflicting evidence on the extent of an Atlantic-Gulf genetic break in C. virginica. In this study, we examined patterns of variation in four Type I loci, derived from cDNA sequence data or from putative coding sequence in genomic DNA.

Genetic differentiation over subpopulations was moderate to high, with [F.sub.ST] values ranging from 0.061-0.166. These are considerably higher than average [F.sub.ST] values reported for allozymes in C. virginica, estimated to be 0.029 in the Atlantic and 0.034 in the Gulf of Mexico. On a global scale, marked genetic differentiation was observed between Atlantic and Gulf sites. Modest differentiation was also observed between North and South Atlantic.

The location of the Gulf-Atlantic transition zone is not well defined for the loci examined here. In mtDNA and anonymous nDNA, steep gene frequency clines occur near Cape Canaveral, (Reeb & Avise 1990, Hare & Avise 1996), whereas for allozymes the shift occurs in northwest Florida (Buroker 1983, Cunningham & Collins 1994). Our data (Fig. 7) show distinct genotypic profiles for central eastern Florida (Indian River, the transition zone between Atlantic and Gulf Coast genotypic assemblages) and northwest Florida (Cedar Key).

Although multidimensional scaling of the multilocus genetic distance data revealed an Atlantic-Gulf split, patterns shown by individual loci were less clear-cut. At Locus A, allelic frequencies were similar for all populations except TX and IR. Locus B showed a Gulf-Atlantic split, with allele 4 absent from all Gulf populations (CK, AP, LA, TX, MX). Locus C showed a clinal decrease in the frequency of allele 1 from northern Atlantic to Gulf populations, whereas allele 3 was found only in the Gulf (AP, LA, MX) and allele 10 was absent from Gulf populations except CK. Locus D showed similar allelic frequencies at all sites except TX.

To estimate the extent of differentiation between Gulf and Atlantic oysters, we pooled populations into Gulf and Atlantic groups. (Texas was excluded from the Gulf group in view of its obvious genetic separation from other Gulf sites. Likewise, Indian River was excluded from the Atlantic group due to its transition zone status.) Within the two groups, overall genetic differentiation ([F.sub.ST]) was moderate (0.050 for Gulf Coast populations, 0.051 for Atlantic populations). For the Gulf-Atlantic comparison, an [F.sub.ST] value of 0.083 was observed, comparable to [F.sub.ST] values of -0.006-0.116 reported by McDonald et al (1996) but lower than those reported by Karl and Avise (1992), which ranged from 0.074-0.603.

To examine the distribution of variation over several levels of geographic differentiation, a hierarchical analysis of molecular variance (AMOVA) was carried out using Arlequin (Schneider et al. 1997). Among-groups variation (Gulf vs. Atlantic populations) accounted for 7.5% of the total variation. Variation among populations within groups represented 4.5% of total variation, whereas within-population variation accounted for 88.0% of the variance. P values for each of these variance components were found to be highly significant (P < 0.000005 within populations, P < 0.000005 among populations within groups, and P = 0.0039 for among-group [Gulf vs. Atlantic] variation).

Oysters collected from Texas were consistently different from all other populations. King et al. (1994) proposed the presence of a major transition zone between Corpus Christi Bay and Upper Laguna Madre. Likewise, Buroker (1983) reported a genetically distinct population at Brownsville, Texas, with a dramatic break observed in several allozyme loci between Corpus Christi Bay and Upper Laguna Madre. The oysters used in the current study were collected just east of Corpus Christi Bay at Port Aransas and may represent an admixture of two genetically distinct populations, leading to heterozygote deficiencies (Wahlund Effect) as noted for Locus A.

The PCR-RFLP method used in this study was technically straightforward but imperfect on several counts. For some enzymes, incomplete digestion of amplicons made it difficult to separate homozygotes for the cut allele from heterozygotes. For some loci, intronic size polymorphisms make interpretation of digests difficult; several candidate loci were excluded for this reason. Finally, presence/absence of a recognition site is a crude distinction that may lump several sequence variants into a single category. For example, sequence analysis shows that three of the six nucleotides comprising the Sac I site in Locus D (Fig. 5) are variable, creating several distinct alleles that are indistinguishable by Sac I digestion (Varney & Gaffney, unpubl.). Because the quantity of C. virginica sequence data continues to expand, genotypic analysis of single-nucleotide polymorphisms (SNPs) may represent the best method for obtaining robust and easily analyzed data from a large number of independent loci (Morin et al. 2004).

The data presented here are consistent with the phylogeographic patterns of nuclear loci discussed by Hare and Avise (1998), which suggest a history of vicariant separation of Gulf and Atlantic populations and contemporary barriers to gene flow, evidenced by a clear break in mtDNA but incomplete lineage sorting at nuclear loci. Because Gulf and Atlantic populations generally fall into distinct clusters, patterns of allelic frequency variation at individual loci are highly variable. Within the Atlantic assemblage, population structure (North vs. South Atlantic) is evident (Fig. 7), although not as clearly demarcated as the Atlantic-Gulf split. Sequence analysis of mtDNA has also shown distinct North and South Atlantic haplotypes with clinal distributions on the Atlantic coast (Wakefield & Gaffney 1996, Milbury & Gaffney, unpubl.). Finally, we note that although this study uses a small number of nuclear markers, it is consistent with the general conclusion of previous studies--modest differentiation between Atlantic and Gulf Coast populations, and a highly divergent Texas population--whereas also pointing to the genetic distinctness of the Cedar Key population, which has also been observed in mitochondrial DNA (Milbury & Gaffney, unpubl.). Future efforts to more accurately delineate regional population structure will be most effective if multiple independent loci (nuclear and mitochondrial) and adequate sample sizes are used.
TABLE 1.
Crassostrea virginica collections.

 Locality N

 1 Ellerslie, Prince Edward Island (EL) 25
 2 Bras d'Or, Prince Edward Island (BD) 20
 3 Piscataqua River, Maine (ME) 18
 4 Mullica River, New Jersey (NJ) 20
 5 Lewes, Delaware (DE) 24
 6 Upper Chesapeake Bay, Maryland (MD) 19
 7 Wachapreague, Virginia (VA) 20
 8 Cape Fear, North Carolina (NC) 18
 9 Georgetown, South Carolina (SC) 24
10 Fernandina Beach, Florida (FB) 24
11 Indian River, Florida (IR) 24
12 Cedar Key, Florida (CK) 20
13 Apalachicola Bay, Florida (AP) 10
14 Grand Isle, Louisiana (LA) 10
15 Port Aransas, Texas (TX) 19
16 Tabasco, Mexico (MX) 22

TABLE 2.
Allelic frequencies at locus A. N = number of individuals.

Site N Allele Frequency

 1 2 3 4 5
 1 25 0.920 -- -- 0.020 0.060
 2 20 0.600 0.050 0.125 0.025 0.175
 3 16 0.719 0.031 -- -- 0.250
 4 20 0.725 0.100 0.050 0.025 0.100
 5 24 0.708 0.063 -- 0.042 0.188
 6 19 0.947 0.026 0.026 -- --
 7 20 0.975 0.025 -- -- --
 8 18 0.861 0.139 -- -- --
 9 24 0.979 0.021 -- -- --
10 24 0.958 0.021 -- 0.021 --
11 24 0.750 0.083 -- -- --
12 20 0.825 0.050 -- 0.100 0.025
13 10 0.850 0.100 0.050 -- --
14 10 1.000 -- -- -- --
15 17 0.088 0.088 0.147 0.206 0.118
16 22 0.818 0.023 -- -- 0.159

Site Allele Frequency P value [F.sub Total [F. Total [F.
 HWE .IS] sub.IS] sub.ST]

 6 7 -0.068 0.166
 1 -- -- 1.000 -0.057
 2 0.025 -- 0.700 -0.333
 3 -- -- 0.459 -0.339
 4 -- -- 1.000 -0.194
 5 -- -- 0.175 -0.072
 6 -- -- 1.000 -0.014
 7 -- -- 1.000 0.000
 8 -- -- 0.273 0.329
 9 -- -- 1.000 0.000
10 -- -- 0.633 -0.221
11 -- 0.167 1.000 -0.011
12 -- -- 0.316 0.208
13 -- -- 0.158 0.294
14 -- -- 1.000 --
15 0.353 -- 0.002 * 0.202
16 -- -- 1.000 -0.171

* Significant at tablewide [alpha]' = 0.0031.

TABLE 3.
Allelic frequencies at locus B. N = number of individuals.

Site N Allele Frequency

 1 2 3 4
 1 18 0.750 0.139 0.056 0.056
 2 20 0.750 0.150 0.100 --
 3 18 0.639 0.222 0.056 0.083
 4 20 0.800 0.100 0.025 0.075
 5 24 0.854 0.042 0.042 0.063
 6 19 0.789 0.158 0.053 --
 7 20 0.550 0.150 0.100 0.200
 8 18 0.861 -- -- 0.139
 9 24 0.563 0.125 0.104 0.208
10 23 0.674 -- 0.043 0.283
11 15 0.600 0.033 0.100 0.267
12 20 0.725 0.275 -- --
13 10 1.000 -- -- --
14 8 0.875 0.125 -- --
15 17 0.471 0.412 0.118 --
16 22 0.932 0.068 -- --

Site P value [F.sub Total [F. Total [F.
 HWE .IS] sub.IS] sub.ST]

 -0.074 -0.077
 1 0.736 0.085
 2 1.000 -0.086
 3 0.556 0.089
 4 1.000 -0.139
 5 1.000 -0.088
 6 1.000 -0.180
 7 0.723 0.066
 8 0.273 0.329
 9 0.063 0.003
10 0.789 -0.283
11 0.660 -0.102
12 0.256 -0.357
13 1.000
14 1.000 -0.077
15 0.048 -0.257
16 1.000 -0.050

TABLE 4.
Allelic frequencies at locus C. N = number of individuals.

Site N Allele Frequency

 1 2 3 4 5
 1 24 0.625 0.063 -- -- 0.042
 2 20 0.575 0.100 -- -- 0.050
 3 17 0.618 0.059 -- -- --
 4 19 0.632 0.105 -- -- 0.026
 5 23 0.696 0.043 -- -- --
 6 18 0.556 0.139 -- -- --
 7 19 1.553 0.184 -- -- --
 8 16 0.531 0.156 -- 0.031 --
 9 24 0.333 0.208 -- 0.063 0.021
10 22 0.250 0.136 -- -- 0.091
11 14 0.107 0.179 -- -- 0.036
12 20 0.150 0.250 -- -- 0.200
13 10 0.050 0.450 0.250 0.100 0.150
14 7 0.071 0.500 0.143 0.071 --
15 17 0.029 0.059 -- -- --
16 21 0.071 0.429 0.048 -- 0.095

Site Allele Frequency

 6 7 8 9 10
 1 0.140 0.104 0.021 -- 0.042
 2 0.150 0.050 0.075 -- --
 3 0.088 0.029 0.029 -- 0.176
 4 0.105 0.053 -- -- 0.026
 5 0.022 0.109 -- -- 0.087
 6 0.139 -- 0.028 0.056 --
 7 -- 0.026 -- 0.079 0.105
 8 0.156 0.031 0.031 -- 0.031
 9 0.021 0.104 0.042 -- 0.063
10 0.068 -- -- 0.023 0.341
11 0.179 0.036 -- 0.107 0.286
12 0.050 -- -- 0.150 0.175
13 -- -- -- -- --
14 0.071 0.071 -- -- --
15 0.324 -- -- -- --
16 0.048 0.119 -- 0.071 --

Site Allele Frequency

 11 12 13 14 15
 1 -- -- -- -- --
 2 -- -- -- -- --
 3 -- -- -- -- --
 4 -- 0.026 -- 0.026 --
 5 -- -- 0.043 -- --
 6 0.028 0.028 -- -- --
 7 0.053 -- -- -- --
 8 -- -- -- 0.031 --
 9 0.146 -- -- -- --
10 0.068 0.023 -- -- --
11 -- 0.071 -- -- --
12 0.025 - -- -- --
13 -- -- -- -- --
14 -- -- -- 0.071 --
15 -- -- -- -- 0.088
16 -- -- 0.024 0.048 --

Site Allele Frequency

 16 17 18 19 20
 1 -- -- -- -- --
 2 -- -- -- -- --
 3 -- -- -- -- --
 4 -- -- -- -- --
 5 -- -- -- -- --
 6 -- -- -- -- 0.028
 7 -- -- -- -- --
 8 -- -- -- -- --
 9 -- -- -- -- --
10 -- -- -- -- --
11 -- -- -- -- --
12 -- -- -- -- --
13 -- -- -- -- --
14 -- -- -- -- --
15 0.059 0.265 0.118 0.029 --
16 -- 0.048 -- -- --

Site Allele p [F.sub Total [F. Total [F.
 Frequency value .IS] sub.IS] sub.ST]

 21 HWE 0.027 0.119
 1 -- 0.077 0.159
 2 -- 0.277 -0.092
 3 -- 0.965 0.006
 4 -- 0.899 -0.075
 5 -- 0.009 0.401
 6 -- 0.683 -0.103
 7 -- 0.770 0.040
 8 -- 0.541 -0.003
 9 -- 0.082 0.139
10 -- 0.108 -0.003
11 -- 0.219 -0.067
12 -- 0.354 -0.197
13 -- 1.000 -0.237
14 -- 0.532 0.062
15 0.029 0.134 0.214
16 -- 0.031 0.098

TABLE 5
Allelic frequencies at locus D. N = number of individuals.

Site N Allele Frequency

 1 2 3 4
 1 25 0.780 0.220 -- --
 2 20 0.975 0.025 -- --
 3 16 0.875 0.125 -- --
 4 20 0.725 0.275 -- --
 5 24 0.833 0.146 0.021 --
 6 19 0.868 0.132 -- --
 7 18 0.694 0.306 -- --
 8 18 0.778 0.222 -- --
 9 24 0.979 0.021 -- --
10 24 0.917 0.083 -- --
11 24 0.729 0.271 -- --
12 20 0.975 0.025 -- --
13 10 0.900 0.100 -- --
14 10 0.900 0.100 -- --
15 16 0.500 0.219 0.031 0.125
16 22 0.750 0.250 -- --

Site Allele P value [F.sub. Total [F. Total [F.
 Frequency HWE IS] sub.IS] sub.ST]

 5 0.202 0.061
 1 -- 0.300 -0.263
 2 -- 1.000 0.000
 3 -- 0.190 0.455
 4 -- 0.256 -0.357
 5 -- 0.001 * 0.717
 6 -- 0.259 0.333
 7 -- 0.015 0.625
 8 -- 0.159 0.382
 9 -- 1.000 0.000
10 -- 0.135 -0.353
11 -- 0.126 0.471
12 -- 1.000 0.000
13 -- 0.053 1.000
14 -- 1.000 -0.059
15 0.125 0.257 0.099
16 -- 0.006 0.650

* Significant at tablewide [alpha]' = 0.0031.


ACKNOWLEDGMENTS

The authors thank Keith Bayha, Bill Fisher, Geoff Flimlin, Nancy Hadley, Sharon McGladdery, and Don Meritt for providing samples. This research was supported by Delaware Sea Grant NA96RG0029, a Lerner-Gray Grant for Marine Research, and a University of Delaware Presidential Fellowship to CAH. This work is part of the M.S. thesis of the first author.

LITERATURE CITED

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CINDI A. HOOVER AND PATRICK M. GAFFNEY

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Author:Gaffney, Patrick M.
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Date:Jan 1, 2005
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