Population structure in two marine invertebrate species (Panopea abrupta and Strongylocentrotus franciscanus) targeted for aquaculture and enhancement in British Columbia.

ABSTRACT As the number of native species targeted for aquaculture and enhancement in British Columbia (BC) rises, fisheries managers must develop policies and species-specific management plans that facilitate industry expansion while protecting the integrity and fitness of wild stocks. Whereas genetic introgression of domesticated stocks with wild stocks cannot be completely eliminated, some control can be gained by limiting the collection and culture of domesticated stocks to geographic units defined by the genetic structure of wild stocks. We describe the genetic structure of two species that are targeted for intensive enhancement and aquaculture in BC: Geoduck clams, Panopea abrupta and red sea urchins, Strongylocentrotus franciscanus. Based on a survey of eight polymorphic microsatellite loci, P. abrupta contained significant geographic structure within BC, with the distribution of genetic variation consistent with stepping stone gene flow under an isolation-by-distance model. A survey of seven polymorphic microsatellite loci covering a similar geographic range revealed genetic homogeneity of S. franciscanus in BC. Based on the different levels of structure displayed in the two species, we propose three geographically-based management units for P. abrupta and two for S. franciscanus in BC.

KEY WORDS: geoduck, clam, red sea urchin, Panopea abrupta, Strongylocentrotus franciscanus, population structure, enhancement, genetic, microsatellite

INTRODUCTION

In British Columbia (BC), many native (mussels) and nonnative (oysters, manila clams, Japanese scallops, Atlantic salmon) marine species are under intensive aquaculture. Additional species that are important capture fisheries are also becoming prospects for aquaculture and artificial enhancement (e.g., geoduck, urchins, blackcod, halibut and rockfish species). Different genetic issues accompany the widespread hatchery production of native and nonnative species. In the culture of nonnative species, competition and hybridization with native species are potential impacts, whereas in the culture of native species, reduction of the genetic fitness of wild populations through introgression with aquaculture strains is a primary concern (Utter & Epifanio 2002). There are three ways in which hatchery-reared animals may differ genetically from their wild counterparts. They may have a geographically distant origin and may be adapted to different natural conditions. Such organisms released from the hatchery may be relatively unfit in their new environment and hybridization with wild conspecifics may lead to outbreeding depression, a reduction in fitness of hybrids in the wild (Allendorf & Ryman 1987). When local organisms are used for hatchery propopagation, the reduced number of parents successfully contributing to hatchery offspring can lead to founder effects, genetic drift and future inbreeding (Tave 1993). Even when large numbers of broodstock are collected locally, domestication, or adaptation to hatchery culture, may reduce the fitness of hatchery-produced organisms and their hybrids in the wild (Lynch & O'Hely 2001, Ford 2002).

Few species have been subjected to the detailed studies required to detect and quantify fitness differences between wild, cultured and hybrid organisms in the wild environment. For Atlantic salmon, one of the most intensively cultured and studied species worldwide, domesticated fish and their first-generation hybrids with wild fish have lower fitness than wild fish in natural environments (McGinnity et al. 1997, Fleming et al. 2000, McGinnity et al. 2003). For invertebrate species, hatchery-produced organisms often contain reduced genetic variability compared with their wild conspecifics (Hedgecock et al. 1992, Boudry et al. 2002), but few studies have investigated the fitness of cultured individuals in different environments (see Tremblay et al. 2001, Jorstad et al. 2005, Sekino et al. 2005).

Because most cultured marine invertebrate species have pelagic larval dispersal stages, complete isolation of wild and hatchery-reared conspecifics could only be achieved by limiting culture to expensive on-land facilities, rearing of nonreproductive animals (e.g., triploids) or complete harvesting of cultured animals before they become reproductively mature. However, introgression of geographically distinct cultured stocks can be avoided by managing broodstock collection and outplanting within regions identified on the basis of wild population structure. Loss of variation and domestication in hatchery strains can be reduced by maintaining large broodstock numbers of wild origin organisms for hatchery production. Alternately, in the case where hatchery broodstocks are genetically distinct from wild stocks, either having undergone extensive genetic selection, bottlenecks or genetic marking, prior knowledge of the genetic characteristics of the wild populations and local hatchery broodstocks may enable monitoring in the vicinity of aquaculture sites and beyond to detect genetic interactions.

Most marine invertebrate species currently cultured or enhanced are characterized by high levels of fecundity and planktotrophic larvae with high dispersal capabilities, both features that are associated with genetic panmixia (Bohonak 1999). However, although panmixia is often observed in marine invertebrate species with these life history attributes, many species do not display panmixia over all spatial scales (Hellberg et al. 2002). The three additional types of structure most often observed include chaotic genetic patchiness, isolation by distance (IBD) and abrupt phylogenetic breaks associated with geographic barriers (reviewed in Hellberg et al. 2002). Chaotic genetic patchiness, defined by non geographically based genetic variance over space and/or time, has been observed over small to medium geographic scales (0-500 km) in a large range of marine invertebrate species (Johnson & Black 1982, Benzie & Williams 1997, Michinina & Rebordinos 1997, Moberg & Burton 2000, Hamre & Burton 2000). An IBD model of structure results from stepping stone gene flow (Slatkin 1993), and has been observed over macrogeographic (100s to 1,000s of km) scales in some marine invertebrate species (Shaklee & Bentzen 1998, Hellberg et al. 2002, Palumbi 2002). Alternately, abrupt genetic boundaries have been observed most often in species occupying bays and estuaries, or those crossing strong phylogeographic boundaries limited by ocean current circulation (Reeb & Avise 1990, Hellberg et al. 2002).

Geoduck clams, Panopea abrupta (hereafter Pab) and red sea urchins, Strongylocentrotus franciscanus (hereafter Sfr) are both long lived (up to 100-150 y) mainly dioecious species that form dense aggregations in the wild (Bureau et al. 2002, Ebert & Southon 2003). Larval duration (approximately 6 wk) is similar in both species, and repeat broadcast spawning events synchronized among individuals clustered over small spatial scales occur from spring through summer (Ebert et al. 1994, Goodwin & Bradbury 2001, Flowers et al. 2002). Because recruitment is sporadic, natural replenishment of over-fished sites is slow, with an estimated 30-50 y natural recovery time to preharvest levels for geoduck clams (Goodwin & Bradbury 2001). Enhancement with hatchery-reared juveniles to facilitate rebuilding has been in effect for intertidal geoduck clams in Washington State since 1982 and for subtidal geoduck clams in BC (limited to the Strait of Georgia) since 1993 (http://www-sci.pac.dfo-mpo.gc.ca/geoduck/ intro_e.htm). Recent experimental work on outplanting methods on red sea urchins has also been undertaken (Alan Campbell, pers. comm.).

Little is known about the genetic structure of Pab and Sfr in BC, although some genetic information exists on these species from other geographic locations, and genetic data is available on species closely related to Sfr. In this study we examine the genetic structure of geoduck clams and red sea urchins in BC with highly variable microsatellite loci (Kaukinen et al. 2004, Miller et al. 2004) and use the information to provide genetic guidelines for culture activities.

METHODS

Pab siphon tissue was collected from 16 sites ranging from Washington through northern BC and spanning over 1100 km of coastline (Fig. 1). Collections took place over a 3-y period, from 2000-2003. Ethanol preserved siphon tissue was extracted using the chelex method from Small et al. (1998). Variation at 2 dinucleotide (Pab 5, Pab 132) and 6 tetranucleotide (Pab 6, Pab 117, Pab 101e, Pab 105e, Pab 106e, Pab 112e) microsatellite loci isolated from Pab was surveyed using the primers and protocols outlined in Kaukinen et al. 2004 (note Pab 5 and 6 are from Vadopalas & Bentzen 2000).

[FIGURE 1 OMITTED]

Sfr were collected over 3 years from 13 sites spanning over 1,000 km of coastline ranging from southern BC to Southern Alaska (Fig. 1 and Table 1). For Sfr, gonad tissue was collected destructively and frozen at -80[degrees]C until use. Genomic DNA was extracted using DNeasy kits (Qiagen, Valencia, CA). Variation at six polymorphic dinucleotide (Sfr 06, Sfr 13, Sfr 22, Sfr 34, Sfr 64 and Sfr 90) loci and one trinucleotide (Sfr 55) repeat microsatellite locus isolated from Sfr was surveyed using the primers and protocols outlined in Miller et al. (2004).

Allelic and genotypic frequency data were analyzed using the Genetic Data Analysis (GDA) (Lewis & Zaykin 2001), GENEPOP version 3.1d (Raymond & Rousset 1995) and FSTAT version 2.9.3.2 (Goudet 2001). Conformance to Hardy Weinberg equilibrium (HWE) distributions of genotypic frequencies at each locus in each sample was tested using GENEPOP. Levels of differentiation among populations using [F.sub.ST] values (Weir & Cockerham 1984) were computed over all samples and on a pairwise basis between samples using FSTAT. For loci that were not in HWE, [F.sub.ST] distances were calculated by randomized genotypes rather than alleles (Goudet 2001). The significance of the multilocus [F.sub.ST] value over all samples was determined by jackknifing over loci. Allelic richness was calculated by standardizing allelic diversity to a sample size of 50 individuals in FSTAT. Homogeneity of allele richness and heterozygosity among sites was tested using a chi-square ([chi square]) test. An IBD model of structure was tested by regression of pairwise [F.sub.ST] values with geographic distance (shortest waterway distances between sites), calculated in FSTAT following Mantel (1967). The stability of the IBD model structure was tested using the web-based IBD program from http://www.bio.sdsu.edu/pub/ andy/BohonakCV.html#Computer%Programs, with all values set to default (Bohonak 2002).

Genetic relationships among samples of Sfr and of Pab were determined using a dendrogram based on the neighbor-joining algorithm, constructed using pairwise differences (Nei 1978). The effective population size ([N.sub.E]), which is an estimate of the number of breeding adults in a population, for Sfr and Pab was calculated from expected heterozygosity (HE) values over all loci using the relationship [N.sub.E] - (1/[[1-[H.sub.E]].sup.2] -1)/8[micro], where [micro] is the mutation rate for the microsatellite loci (Lehmann et al. 1998). The mammalian mutation rate of [micro] = [10.sup.-4] was used as a conservative estimate, recognizing that [N.sub.E] values could be 100 times greater if [micro] approaches [10.sup.-6], as observed in Drosophila (Shug et al. 1997).

Hierarchical analysis of allele frequency variation was conducted in Arlequin with a nested analysis of molecular variance (AMOVA; random effects model; Weir 1996). The significance of allele frequency differences attributable to geographic regions identified in the dendrograms based on [F.sub.ST] distances was tested in a hierarchical model with sample sites nested within regions.

RESULTS

Genetic Variation Within Samples

Pab microsatellite loci were all highly polymorphic. Allelic richness ranged from 16-38 alleles per 50 individuals, and expected heterozygosities ranged from 0.91-0.98 among loci (Table 2). Four of the eight Pab loci contained excess homozygotes resulting in significant deviations from HWE over all sites, whereas Pab 112e contained an excess at 11/16 sites (overall P < 0.001 for each of these loci). Deviations from HWE were minimal in the remaining three loci (P > 0.1 over all sites). Estimates of the inbreeding coefficient, [F.sub.IS], over all sites ranged from 0.03 for Pab 6 to 0.30 for Pab 117 (Table 2).

Sfr microsatellite loci were slightly less polymorphic than Pab loci, with a range in allelic richness of 9-35 (mean 20) and a range in expected heterozygosity of 0.63-0.96. Fewer deviations from HWE were observed in Sfr than for Pab, with only two of the seven loci containing significant excesses of homozygotes in over 50% of the Sfr sites. As a result, the mean [F.sub.IS] over all loci was lower in Sfr (0.11 [+ or -] 0.03) than in Pab (0.15 [+ or -] 0.04).

Using the mammalian microsatellite mutation rate ([10.sup.-4]) and [H.sub.E] values estimated from the loci in this study, locus-specific estimates of effective population sizes ([N.sub.E]) for Pab ranged from 1.4-13.8 million (mean of 5.8 million), and for Sfr ranged from 0.08-9.7 million (mean of 2.1 million).

Genetic Variation Among Samples

In Pab, levels of heterozygosity were homogeneously distributed among sites (P > 0.05), whereas spatial heterogeneity in allelic richness was observed (P < 0.01), with a greater number of alleles observed in southern sites (Table 1). Alternately, no significant heterogeneity in allelic richness or heterozygosity was observed among sites for Sfr (P > 0.05). Similarly, less variation in [F.sub.IS] was observed among sites than among loci for both species ([F.sub.IS] among sites: 0.12-0.13 Pab, 0.08-0.13 Sfr; [F.sub.IS] among loci 0.05-0.30 Pab, 0.03-0.23 Sfr). Despite the high degree of polymorphism displayed at all loci of both species, few private alleles (i.e., alleles observed in only one site) were observed. Out of 355 alleles for Pab observed over all loci, only 6 were private. For Sfr, 24 of the 265 alleles were private. Private alleles were not found in either species at frequencies above 0.03, nor were they found disproportionately in specific sites or regions.

For both species, [F.sub.ST] calculated over all loci and all populations was significantly greater than zero (P < 0.01); however, only the Pab [F.sub.ST] was significant after jackknifing (P < 0.0004). The global [F.sub.ST] estimate for Pab (0.004 [+ or -] 0.001) was twice as high as that observed for Sfr (0.002 [+ or -] 0.001). [F.sub.ST] values for single Pab loci ranged from 0.002-0.005, and were significantly greater than 0 for all eight loci (P < 0.05) (Table 2). Alternately, for Sfr, [F.sub.ST] values for single loci ranged from -0.001-3.006, and four of the seven loci (Sfr 34, 55, 64 and 90) were significantly greater than 0. When calculated on a pairwise basis, after bonferroni correction, over 85% of population comparisons were significant for Pab, versus only 10% for Sfr (Table 3).

The majority of the genetic variation in Pab was found within sites (99.5%), with only 0.4% partitioned among sites and 0.08% among regions. Sites represented a significant portion of the variation in the AMOVA (F-ratio = 1.88, df 12,16482, P = 0.03), but permutations of models based on different regional configurations did not reveal any significant effects of region (P > 0.2 in all models). However, in the neighbor-joining dendrogram, geoduck samples generally clustered into four geographically-based units (Fig. 2A), and the mean [F.sub.ST] values calculated among the four depicted regions (range of 0.003-0.005) were generally twice as high as those observed within regions (range of 0.002-0.003). The geographic cluster containing the three Queen Charlotte Island (QCI) samples, Gowgaia Bay, Hippa Island, and Tasu Sound, was the most distinctive. Although the two Washington samples, Point Heyer and Hood Canal, clustered together, they were fairly distinct from one another (significantly different at 3/8 loci), and were not highly differentiated from two of the East Coast of Vancouver Island (ECVI) sites, Marina Island and North Savory. However, they were well differentiated from all other sites on the BC coast. The remainder of the ECVI samples clustered together. The West Coast of Vancouver Island (WCVI) sites and single North Coast of Vancouver Island (NCVI) site formed the fourth cluster. In all, the distribution of genetic variation in Pab was consistent with an IBD model of structure ([R.sup.2] = 25.5, P < 0.0001; FSTAT), but it was clear from the low [R.sup.2] value that all sources of variation were not accounted for by this model (Fig. 3A). An IBD model of structure was also supported using the IBD program of Bohonak (2002), but the [R.sup.2] and P values were generally lower. A higher degree of significance was obtained when log values of genetic distance were used, and when the two Washington sites were removed (P < 0.002 versus P < 0.023). Alternately, IBD structure was not supported when the three QCI sites were removed (P > 0.35).

[FIGURES 2-3 OMITTED]

Despite the fact that a lower percentage of population pairs were significantly different in Sfr versus Pab, hierarchical gene diversity analysis revealed a slightly greater amount of variation partitioned among sites than observed in Pab (1.24% vs. 0.48%). This difference is likely because of the higher number of private alleles observed in Sfr. Hence, sample site accounted for a significant portion of the variation in Sfr (F-ratio = 3.14, df 11,2415, P = 0.0003). However, no strong geographic clustering of Sfr samples was observed in the neighbor joining dendrogram (Fig. 2B), nor was regional structure supported in hierarchical gene diversity analysis (North/South regions, F-ratio = 1.44, df1,11, P = 0.26). Given that the highest pairwise [F.sub.ST] value (0.007; P = 0.01, not significant after bonferroni correction) was observed between the two north coast populations, South West Price Island and Stryker Island, that are only 40 km apart, it is not surprising that an IBD model of structure was also not supported ([R.sup.2] = 1.07, P = 0.36; FSTAT; Fig. 3B).

DISCUSSION

Current and potential culture operations for geoduck clams and red sea urchins in BC generally aim to enhance harvestable populations (Alan Campbell, pers. comm.). The degree of genetic risk to wild populations associated with these activities is largely influenced by: (1) the degree of assimilation of cultured releases into natural populations; (2) genetic differences between the natural and supplementation populations and (3) the degree of substructure of the natural population (Utter & Epifanio 2002). Our spatial analysis of microsatellite variation in Pab and Sfr provide the first view of substructure in natural populations of these species in BC, although additional sampling over time will be required to verify conclusions about stable population structure. In addition, data from this study provide a basis for the monitoring and evaluation activities recommended by Utter & Epifanio (2002).

For Sfr in BC, we found no evidence of a stepping stone pattern of connectedness (IBD model). Although the lack of geographically-based structure and "randomly" distributed private alleles could fit a model of chaotic genetic patchiness, more data focusing on genetic variation among settling cohorts would be required to provide strong support for this model. In general, our data do not refute the null hypothesis of genetic panmixia of Sfr within BC and SE Alaska.

Previous genetic studies of Sfr have focused on the southern portion of their range (Edmands et al. 1996, Debenham et al. 2000, Moberg & Burton 2000). Our results are consistent with these studies and with genetic studies of other sea urchin species. Generally, panmixia is not rejected at macrogeographic scales (Palumbi & Wilson 1990, Palumbi & Kessing 1991, Debenham et al. 2000, Addison & Hart 2004), but chaotic genetic patchiness is often observed at microgeographic scales (1-200 km) (Edmands et al. 1996, Flowers et al. 2002). None of the studies to date have supported an IBD model of structure. However, genetic structure caused by phylogeographic boundaries was observed in three species (Mladenov et al. 1997, Addison & Hart 2004).

Patterns of geographical genetic differentiation may reflect events in the distant past (thousands to millions of years ago) as much or more than ongoing patterns of dispersal (Brown et al. 2001, Hellberg et al. 2002). Hence, whereas the lack of genetic differentiation of Sfr within BC is consistent with significant gene flow over evolutionary time (thousands of years), recruitment data suggest that over ecological time (tens to hundreds of years), sites may not be as connected by larval dispersal as they appear (Ebert et al. 1994). Recruitment of Sfr is sporadic, with large pulses of recruitment occurring only every 3-5 y (ibid), and variation in the distribution of size classes among sites indicates high variability in recruitment over small spatial scales (0.5-8 km) (Sloan et al. 1987). These observations suggest that over ecological time scales, exchange among sites may be more limited than genetic analyses suggest.

Pab inhabits coastal and estuarine sites from California to Alaska and may occur in small numbers in southern Japan (Coan et al. 2000). Pab and can be found in the low intertidal zone down to depths of 100 m (Goodwin & Pease 1989). We observed a higher level of spatial genetic structuring in Pab among the BC sites than reported among sites within Puget Sound and between Puget Sound and an Alaskan site by Vadopalas et al. (2004). Although different microsatellite loci were examined in the two studies, both included Pab 5 and Pab 6. The [F.sub.ST] values for Pab 5 and Pab 6 were higher in BC (0.005 and 0.003, respectively) than in Puget Sound (0.001 and 0.0001, respectively), indicating greater genetic variability among BC than Puget Sound sites. Whereas the overall geographic range of the two studies was similar, the IBD structure resolved in our study was not supported in the Vadopalas et al. (2004) study. This discrepancy is likely because of differences in the sampling distribution of the two studies: most of the samples in Vadopalas' study were collected within 200 km of one another within the Puget Sound basin and only a single distant Alaskan sample was compared, whereas the samples in our study were more evenly distributed across a span of over 1,100 km, with an average pairwise distance among sites of 445 km. By combining the data from both studies, we can conclude that Pab exhibits panmixia at small spatial scales of 50-300 km, and stepping stone gene flow at intermediate scales of 500-1,000 km.

The only Pab sites that did not cluster regionally were Marina and Savory Islands, which are located adjacent to one another in the Strait of Georgia (ECVI) but clustered with Puget Sound instead of other ECVI sites. Both of these sites were exposed to heavy commercial fishing in the past, and the Marina Island population was so depleted that in 1990 it was closed to fishing and became a research site (Campbell et al. 2004). In the mid 1990s Marina and Savory Islands underwent intensive reseeding efforts, with records indicating that broodstock were collected locally (ibid). However, seed from Puget Sound hatcheries was used in reseeding some west coast Vancouver Island sites at a similar time. Thus, the apparent genetic similarity of the Marina and Savory Island samples with Puget Sound samples observed in this study may indicate the introductions records for these two sites are not complete.

Genetic differentiation between rockfish species inhabiting sites in southern Puget Sound and Strait of Georgia/Strait of Juan de Fuca (collectively called the Georgia Basin) (Stout et al. 2001, Buonaccorsi et al. 2002) indicates that the shallow sill at Admiralty Inlet and complicated bathymetry within Puget Sound lead to larval retention within the Sound. For most species, insufficient sampling has been conducted to define the precise location of the biogeographic boundary (Stout et al. 2001, Vadopalas 2003, Vadopalas et al. 2004). Although little differentiation was observed among most pairs of sites in Vadopalas et al. (2004), the sample from Juan de Fuca Strait was distinct from Puget Sound sites. Our study did not contain a sample from the Juan de Fuca Strait, but sites within Puget Sound were significantly differentiated from 6/8 ECVI sites and all sites located in other BC regions. Further, 16/21 comparisons between ECVI and WCVI were significant. Given the vastly different ecological environments between ECVI and WCVI (one wave swept, one sheltered), genetic exchange between these waters may be further limited because of adaptive constraints.

In northern BC, the marine biota of the QCI tend to be distinctive from southern coastal areas (Arndt & Smith 1998, Kyle & Boulding 2000, Withler et al. 2003) due either to the existence of historical refugia or to isolation of the QCI from the mainland by eddies in Hecate Strait (Briggs 1974, Allen & Smith 1988, Hetherington & Reid 2003, Di Lorenzo et al. 2005). The distinction of the QCI region was also relatively well supported for Pab in our study, and we hypothesize that oceanographic factors (i.e., disjunction between the California Current and the Alaska Gyre located just south of QCI) have limited recent migration of Pab between QCI and more southerly sites. Whereas we cannot discount completely the possibility that historic recolonization was also a factor in the generation of a genetically distinct QCI population, the lack of private alleles and statistical support for QCI as a distinct region in hierchical diversity analysis suggests that the differences observed are not deep enough to have been derived through historical isolation. This is an important point because statistical support for the IBD structure in Pab was dependent on the inclusion of the QCI sites, and if historical events have influenced their relationships, then true stepping-stone gene flow is not necessarily occurring. However, future analyses using data from a broader geographic range of sites and markers could be used to test this hypothesis.

For Pab, we suggest the adoption of at least 3 management areas within BC (QCI, Georgia Strait, and west coast Vancouver Island) and one for Puget Sound. This regional structure corresponds well with five general shellfish transfer zones proposed for BC by the Introductions and Transfer Committee (Dorothee Kieser, Fisheries and Oceans Canada). These zones are Haida Gwaii (our QCI), Georgia Strait, West Coast Vancouver Island, Queen Charlotte Strait (represented by a single sample in our study, E. Daphne Point) and the north/central coast (not sampled in our study). Additional Pab sampling from the Queen Charlotte Strait and north/central coasts of BC is required to examine population structure in these regions.

For Sfr, the genetic data provide no evidence of strong sub-structuring within BC waters but do not preclude the presence of geographically-based adaptive differences in growth and survival in the wild. We recommend that at least two management areas be identified within BC associated with the bifurcation of the Sub-arctic Current into the California and Alaska currents off the west coast of Vancouver Island, and the likely influence of these current systems on recruitment and productivity of marine organisms. Sir culture in northern BC should be conducted with organisms originating within the Haida Gwaii and north/central coast shellfish transfer zones, whereas those in southern BC should be based on organisms originating from waters contiguous with Vancouver Island (i.e., Queen Charlotte Strait, Georgia Strait and west coast Vancouver Island zones).

ACKNOWLEDGMENTS

The authors thank Alan Campbell and two anonymous reviewers for constructive advice relating to the manuscript. Urchin sample collections were coordinated by the Pacific Underwater Harvesters Association (PUHA), Alan Campbell and Dominique Bureau from the Fisheries and Oceans, Canada and Kyle Hebert from the Alaska Department of Fish and Game. Geoduck sample collections were coordinated by the Underwater Harvesters Association (UHA), Fan Seafoods, Island Scallops Ltd. and the Washington Dept. of Fish and Wildlife. This research was funded by the Department of Fisheries and Oceans, Canada through a Canadian Biotechnology Strategy Grant and industry partners, PUHA and UHA.

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KRISTINA M. MILLER, * K. JANINE SUPERNAULT, SHAORONG LI AND RUTH E, WITHLER

Fisheries and Oceans Canada Pacific Biological Station, 3190 Hammond Bay Rd. Nanaimo, BC Canada V9T 6N7

* Corresponding author. E-mail: [e-mail]

TABLE 1.

Distribution of collection sites for P. abrupta and S. franciscanus in
BC, Washington (WA), and Alaska (AK), with site-specific data on
sample size, allelic richness ([A.sub.R]), and expected heterozygosity
(He).

                      Province                      Sample
 Collection Site      or State        Region         Size     [A.sub.R]

Pab
  Point Heyer            WA       S. Puget Sound     98         28.0
  Hazel Point            WA       Hood Canal        134         27.0
  Marina Island          BC       ECVI              122         28.6
  Savory Island          BC       ECVI               95         28.7
  Cortes Island          BC       ECVI               92         27.7
  Porpoise Bay           BC       ECVI               98         27.5
  Round Island           BC       ECVI               99         28.1
  Boatswain Bank         BC       ECVI               97         26.3
  Thormanby Island       BC       ECVI               97         28.2
  East Daphne Pt         BC       N-ECVI             98         26.3
  Nootka Sound           BC       WCVI              103         26.7
  Coomes Banks           BC       WCVI              102         27.8
  Maggie River           BC       WCVI               96         26.4
  Hippa Island           BC       QCI               137         27.5
  Tasu Sound             BC       QCI               103         27.4
  Gowgaia Bay            BC       QCI               107         26.8
  Mean                                              105.3       27.4
Sfr
  Shute Reef             BC       SCVI               84         19.8
  Denman                 BC       ECVI               68         20.3
  Campbell River         BC       ECVI               78         19.2
  Johnstone Strait       BC       N-ECVI             92         20.0
  Broken Group           BC       WCVI               58         19.7
  Tofino                 BC       WCVI               79         19.4
  Stryker                BC       CC                 67         19.2
  SW Price               BC       CC                 63         19.7
  Louscoone              BC       QCI                87         20.4
  Windy Bay              BC       QCI                76         21.2
  Kelp Bay               AK       SEAK               81         21.0
  Blank                  AK       SEAK               77         20.3
  Meares Pass            AK       SEAK               72         19.4
  Mean                                               75.5       20.0

 Collection Site        He

Pab
  Point Heyer         0.967
  Hazel Point         0.970
  Marina Island       0.970
  Savory Island       0.967
  Cortes Island       0.961
  Porpoise Bay        0.962
  Round Island        0.970
  Boatswain Bank      0.958
  Thormanby Island    0.971
  East Daphne Pt      0.962
  Nootka Sound        0.967
  Coomes Banks        0.969
  Maggie River        0.966
  Hippa Island        0.970
  Tasu Sound          0.969
  Gowgaia Bay         0.963
  Mean                0.966
Sfr
  Shute Reef          0.933
  Denman              0.943
  Campbell River      0.937
  Johnstone Strait    0.942
  Broken Group        0.919
  Tofino              0.943
  Stryker             0.946
  SW Price            0.944
  Louscoone           0.938
  Windy Bay           0.948
  Kelp Bay            0.947
  Blank               0.941
  Meares Pass         0.944
  Mean                0.941

Abbreviations include: Washington (WA), BC (BC), Alaska (AK), East
coast of Vancouver Isalnd (ECVI), north ECVI (N-ECVI), west coast of
Vancouver Island (WCVI(, Queen Charlotte Islands (QCI), central coast
(CC), and south east Alaska (SEAK). Allel frequencies for all samples
surveyed in this study are available at:
http://www.pac.dfo-mpo.gc.ca/sci/aqua/bgsid_e.htm.

TABLE 2.

Statistics on S. franciscanus and P. abrupta microsatellite loci,
including allelic richness ([A.sub.R]), expected (He), and observed
(Ho) heterozygosity, inbreeding coefficient ([F.sub.IS]), number of
populations that deviate significantly (P < 0.05) from HWE after
bonferroni correction, Coancestry coefficient ([F.sub.ST]), and the
associated P-value for [F.sub.ST]. HWE was not assumed in the
calculation of the Coancestry coefficient ([F.sub.ST]) when 20% or
more of the populations significantly deviated from HWE (highlighted
with an asterisk).

     Locus          N      [A.sub.R]     He      Ho     [F.sub.IS]

P. abrupta
  Pab 5 (1)        1118       16        0.91    0.71       0.22
  Pab 6 (1)        1113       22        0.93    0.90       0.03
  Pab 117          1019       35        0.97    0.68       0.30
  Pab 132          1019       38        0.94    0.70       0.26
  Pab 101e         1113       19        0.93    0.88       0.05
  Pab 105e         1113       38        0.98    0.72       0.22
  Pab 106e         1018       18        0.93    0.91       0.06
  Pab 112e         1113       34        0.96    0.88       0.09
  Mean                        27        0.94    0.80       0.15
S. franciscanus
  Sfr 06           1016       12        0.87    0.85       0.03
  Sfr 13           1026       16        0.91    0.81       0.11
  Sfr 22           1102       25        0.94    0.82       0.12
  Sfr 34           1074       28        0.81    0.69       0.15
  Sfr 55            930       35        0.96    0.74       0.23
  Sfr 64           1039        9        0.83    0.76       0.09
  Sfr 90            895       14        0.63    0.66       0.04
  Mean                        20        0.85    0.76       0.11

                    # Pop
     Locus         P < 0.05    [F.sub.ST HW]    GP-Value

P. abrupta
  Pab 5 (1)           16         0.0040 *         0.002
  Pab 6 (1)            0         0.0032           0.002
  Pab 117             16         0.0020 *         0.002
  Pab 132             15         0.0040 *         0.002
  Pab 101e             3         0.0024           0.002
  Pab 105e            16         0.0020 *         0.002
  Pab 106e             5         0.0041 *         0.002
  Pab 112e            11         0.0050 *         0.002
  Mean                           0.0036
S. franciscanus
  Sfr 06               0         0.0000           0.468
  Sfr 13               6         0.0000 *         0.667
  Sfr 22               6         0.0010 *         0.135
  Sfr 34               9         0.0010 *         0.007
  Sfr 55              13         0.0030 *         0.002
  Sfr 64               2         0.0023           0.005
  Sfr 90               0         0.0051           0.002
  Mean                           0.0020

(1) Locus primers from Vadopalas & Bentzen (2000).

TABLE 3.

Pairwise [F.sub.ST] distances and their significance for A. Pab and B.
Sfr. Values above the diagonal indicate significance of each pairwise
[F.sub.ST] at P < 0.05, after bonferroni correction, with an asterisk
(*) indicating significant and "ns" nonsignificant tests. [F.sub.ST]
values are shown below the diagonal. Site names are abbreviated from
Table 1, and abbreviated region names are indicated above the site
names. Note that the regions within Washington (WA) are not shown for
the two WA sites.

                          WA         WA         ECVI        ECVI
A.                     Pt Heyer     Hazel      Marina      Savory

WA        Pt Heyer                    *          *            *
WA        Hazel         0.0037                   *            *
ECVI      Marina        0.0029     0.0024                    ns
ECVI      Savory        0.0040     0.0026      0.0013
ECVI      Cortes        0.0045     0.0043      0.0034      0.0017
ECVI      Porpoise      0.0059     0.0047      0.0043      0.0037
ECVI      Round         0.0046     0.0023      0.0032      0.0034
ECVI      Botswain      0.0064     0.0045      0.0043      0.0035
ECVI      Thormanby     0.0059     0.0054      0.0030      0.0023
N-ECVI    E. Daphne     0.0068     0.0044      0.0048      0.0053
WCVI      Nootka        0.0036     0.0034      0.0026      0.0033
WCVI      Coomes        0.0045     0.0034      0.0028      0.0023
WCVI      Maggie        0.0035     0.0025      0.0024      0.0043
QCI       Hippa         0.0056     0.0031      0.0039      0.0055
QCI       Tasu          0.0040     0.0041      0.0037      0.0038
QCI       Gowgaia       0.0049     0.0031      0.0032      0.0044

            ECVI         ECVI       ECVI        ECVI        ECVI
A.         Cortes      Porpoise     Round     Botswain     Thorman

WA            *           *           *          *            *
WA            *           *           *          *            *
ECVI          *           *           *          *            *
ECVI          *           *           *          *            *
ECVI                      *           *          *           ns
ECVI       0.0023                     *          *           ns
ECVI       0.0018       0.0018                   *            *
ECVI       0.0024       0.0039     0.0035                     *
ECVI       0.0011       0.0031     0.0026      0.0027
N-ECVI     0.0033       0.0049     0.0044      0.0034      0.0047
WCVI       0.0032       0.0027     0.0021      0.0023      0.0031
WCVI       0.0021       0.0030     0.0022      0.0018      0.0024
WCVI       0.0030       0.0028     0.0025      0.0039      0.0045
QCI        0.0046       0.0056     0.0039      0.0041      0.0059
QCI        0.0031       0.0059     0.0038      0.0042      0.0050
QCI        0.0030       0.0043     0.0030      0.0044      0.0046

           N-ECVI        WCVI       WCVI        WCVI         QCI
A.         Daphne       Nootka     Coomes      Maggie       Hippa

WA            *           *           *          *            *
WA            *           *           *          *            *
ECVI          *           *           *          *            *
ECVI          *           *           *          *            *
ECVI          *           ns          *          *            *
ECVI          *           ns          *          *            *
ECVI          *           ns          *          *            *
ECVI          *           ns          *          *            *
ECVI          *           ns         ns          *            *
N-ECVI                                *          *            *
WCVI       0.0032                    ns          *            *
WCVI       0.0021       0.0015                   *            *
WCVI       0.0019       0.0018     0.0022
QCI        0.0042       0.0028     0.0045      0.0041
QCI        0.0032       0.0029     0.0027      0.0054      0.0022
QCI        0.0034       0.0032     0.0027      0.0046      0.0011

             QCI         QCI
A.          Tasu       Gowgaia

WA            *           *
WA            *           *
ECVI          *           *
ECVI          *           *
ECVI          *           *
ECVI          *           *
ECVI          *           *
ECVI          *           *
ECVI          *           *
N-ECVI        *           *
WCVI          *           *
WCVI          *           *
WCVI          *           *
QCI           *           ns
QCI                       ns
QCI        0.0022

                         SCVI       ECVI        ECVI       N-ECVI
B.                      Shute      Denman     Campbell    Johnstone

SCVI      Shute                      ns          ns          ns
ECVI      Denman        0.0003                   ns          ns
ECVI      Campbell     -0.0007      0.0012                    ns
N-ECVI    Johnstone    -0.0008      0.0006    -0.0008
WCVI      Broken        0.0000     -0.0014     0.0017      0.0026
WCVI      Tofino        0.0028     -0.0006     0.0008      0.0014
CC        Stryker       0.0044      0.0000     0.0042      0.0036
CC        SW Price      0.0049      0.0021     0.0044      0.0059
QCI       Louscoone     0.0008      0.0001     0.0014     -0.0006
QCI       Windy         0.0045      0.0009     0.0042      0.0030
SEAK      Kelp          0.0005     -0.0002     0.0004     -0.0002
SEAK      Blank         0.0027     -0.0010     0.0012      0.0009
SEAK      Meares        0.0018      0.0022     0.0018      0.0009

            WCVI         WCVI        CC          CC          QCI
B.         Broken       Tofino     Stryker    SW Price    Louscoone

SCVI         ns           ns         ns          ns          ns
ECVI         ns           ns         ns          ns          ns
ECVI          *           ns         ns          ns          ns
N-ECVI        *           ns          *          *           ns
WCVI                      ns         ns          ns          ns
WCVI      -0.0002                    ns          ns          ns
CC         0.0030       0.0057                   ns          ns
CC         0.0010      -0.0004     0.0071                    ns
QCI        0.0014       0.0013     0.0045      0.0019
QCI        0.0007       0.0001     0.0052     -0.0010      0.0005
SEAK       0.0015       0.0015     0.0012      0.0055      0.0014
SEAK       0.0017       0.0004     0.0038      0.0024     -0.0002
SEAK       0.0032       0.0002     0.0050      0.0033     -0.0001

             QCI         SEAK       SEAK        SEAK
B.          Windy        Kelp       Blank      Meares

SCVI         ns           ns         ns          ns
ECVI         ns           ns         ns          ns
ECVI         ns           ns         ns          ns
N-ECVI       ns           ns         ns          ns
WCVI         ns           ns         ns
WCVI         ns           ns         ns          ns
CC            *           ns          *          ns
CC           ns           ns         ns
QCI          ns           ns         ns          ns
QCI                       ns         ns
SEAK       0.0033                    ns          ns
SEAK       0.0028       0.0013                   ns
SEAK       0.0042       0.0010     0.0017