The genetic stock structure of the American lobster (Homarus americanus) in Long Island Sound and the Hudson Canyon.

ABSTRACT The genetic population structure of the American lobster (Homarus americanus) was examined in populations collected in Long Island Sound (LIS) and the Hudson Canyon of the Northeastern United States with recently developed microsatellite DNA loci probes. Pereiopods, a thoracic appendage used for movement, feeding and defense, were collected from egg-bearing female lobsters from three sites within LIS--an eastern, central and western site--and from sites within the Hudson Canyon. Genomic DNA was isolated from each pereiopod and examined for nine microsatellite loci. Microsatellite allele frequencies, corrected for the presence of null alleles, were used to determine genetic differences between sampled groups. In agreement with earlier studies that used mitochondrial DNA and allozyme markers, there was little genetic differentiation between eastern and central LIS sites and the Hudson Canyon site. However, the genetic differences between western LIS populations and other sampled populations were 10 times higher. These were greater differences than could be attributed to geographical separation. These differences may have arisen as a result of the massive lobster die-off that occurred in 1998/1999 in western LIS.

KEY WORDS: Homarus americanus, American lobster, genetic, populations

INTRODUCTION

An understanding of the genetic population structure of commercially important fisheries is critical for the conservation and management of exploited fish and crustacean species (Thorpe et al. 2000). The American lobster (Homarus americanus H. Milne Edwards, 1837) is found at intertidal depths to 720 m, but most frequently at 4-50 m, along the continental shelf throughout much of western North Atlantic from southern Labrador to offshore North Carolina (Herrick 1909). Major coastal concentrations of lobster are in the Gulf of Maine and the coastal waters of New Brunswick and Nova Scotia, Canada (Cooper & Uzmann 1980). Major offshore concentrations are along the outer edge of the Continental Shelf and upper slope between the eastern part of Georges Bank and the Delaware Bay (Schroeder 1959). Small numbers inhabit the outer edge of the Nova Scotia shelf (Cooper & Uzmann 1980).

American lobster is a commercially important species and effective management of exploited species requires identification of biologically relevant management units that reflect the degree of reproductive isolation (Carvalho & Hauser 1994). Previous work that characterized American lobster populations with allozyme markers and randomly amplified polymorphic DNA (RAPD) (Tracey et al. 1975, Harding et al. 1997), suggested that little population structure existed. The lack of noted population structure may have been influenced by the limited resolution of these approaches. Recently, high-resolution microsatellite loci have been characterized for the American lobster (Jones et al. 2003). These loci have been shown to have relatively high levels of heterozygosity (compared with allozyme markers and RAPD but not as high as for some marine fish) and have been shown to be suitable for the characterization of lobster populations.

Previous work has suggested that the enormous potential for larval dispersion, the wide-ranging movement of adult lobsters (from tagging experiments) and the anthropogenic influence of humans in the placement of adult lobsters have acted to muddy the genetic waters. There are several factors that suggest that lobsters may indeed have a heterogeneous genetic distribution through areas of the eastern United States and specifically within Long Island Sound (LIS). A lobster die-off reduced the 1999 fall landings of lobsters in western LIS by <99% (CT DEP 2003). The die-off corresponded with several years of above-normal water temperatures, application of pesticides for West Nile virus-carrying mosquito control and Paramoeba sp. infections (CT DEP 2003). Certain factors suggest that there may have been a genetic component for survivorship.

In this study, experiments are carried out to determine the genetic population structure of lobsters within LIS and from the Hudson Canyon area, an area long believed to supply lobsters to LIS. The results of these studies are discussed in the context of lobster management.

METHODS

Collections

Egg-bearing female lobster pereiopods (a thoracic appendage used for feeding, walking, and defense) were collected from four sites in the spring, and two sites in the summer in 2001 within LIS and from a site in the spring in the Hudson Canyon by scientists from the Connecticut DEP and the Millstone Environmental Laboratory (Table 1 & Fig. 1). Pereiopods were collected from eggbearing females because natal homing increases the likelihood of detecting genetic differences between populations. The sites within LIS are approximately 30-50 km apart, whereas the Hudson Canyon collection site is over 50 km away from the other collection sites. Lobsters were collected from baited traps, and the pereiopods removed and placed in 70% ethanol and then transferred to the laboratory. Up to 150 pereiopods were collected from each site, one from each lobster (Table 1). The spring and summer lobster collections were treated as separate groups even if they were collected from the same sites, because it has been suggested that spawning lobster populations (spring) are different from summer and fall populations (P. Howell, CT DEP, pers. comm.).

[FIGURE 1 OMITTED]

Genomic DNA Isolation

In the laboratory, a segment was cut from each pereiopod, and the inner soft tissue removed and processed for the isolation of genomic DNA as described (Crivello et al. 2004). The genomic DNA was quantified with PicoGreen (Molecular Probes Inc., Eugene, Oregon) and each sample was adjusted to 2 ng/[micro]L genomic DNA.

Microsatellite Loci

Lobster-specific microsatellite locus and flanking PCR primer sequences described in Jones et al. (2003) were used to characterize microsatellite alleles at 9 loci (Table 2). To analyze each microsatellite loci, 10 ng of genomic DNA was mixed with a stock solution containing 0.5 [micro]M forward & reverse primer (with the forward primer tagged with a [D.sub.2], [D.sub.3] or [D.sub.4] fluorescent tag, Beckman Coulter, Palo Alto, California), 0.2 mM dNTPs, 10 mM Tris, 50 mM KCl, 2.5 mM Mg[Cl.sub.2] and 0.5 units of a thermostable DNA polymerase in a final 10 [micro]L volume. Each sample was heated to 94[degrees]C for 30 s, to the annealing temperature for 30 s and then 72[degrees]C for 45 s for 35 cycles.

The reaction products were diluted with 30 [micro]L of water and then 10 [micro]L of a [D.sub.2], [D.sub.3] and [D.sub.4] reaction were combined and precipitated. The precipitated products were washed with 70% ethanol and redissolved in formamide that contained 60-400 base pair size markers labeled with [D.sub.1] (Beckman Coulter, Palo Alto, California). The samples were then analyzed in a Beckman Seq2000 capillary electrophoresis system. Microsatellite alleles were identified by size with a resolution of 0.25 bp by comparison with size standards.

Statistical Analysis of Genetic Differences

Observed heterozygosity, mean number of alleles, and conformity to Hardy-Weinberg Equilibrium (HWE) were analyzed for all loci with GenePop version 3.2 (Raymond & Rousset 1995). For loci with more than four alleles in a sampled population, a Markov chain method was used to estimate the exact P value. Each microsatellite loci was tested for the presence of null alleles by the method of Brookfield (1996) using the freely available MicroChecker software (http://www.microchecker.hull.ac.uk). Null alleles are one or more alleles that fail to amplify during PCR, or incorrect scoring of alleles because of stuttering, or if large alleles do not amplify as efficiently as small alleles-allele dropout. The allele frequencies of loci showing evidence of null alleles were corrected statistically prior to analysis of conformity to HWE and for genetic differences between populations (van Oosterhout et al. 2004).

Determination of population genetic structure was made by pair-wise statistical examination of the differences in allele frequencies in each population. The statistic [F.sub.ST] reflects the proportion of the observed genetic variation that can be explained by partitioning between populations (Wright 1969). Several approaches have been designed for the analysis of microsatellite differences between populations; [R.sub.ST] is an [F.sub.ST] analogue based on allele size, and [delta][[mu].sup.2] a [D.sub.S] analogue based on allele size (Slatkin 1995, Goldstein & Pollock 1997, Ronfort et al. 1998). [F.sub.ST] and [R.sub.ST] are computed using an ANOVA approach following, respectively, Cockerham & Weir (1986), Ronfort et al. (1998) and Roussett (1996). To obtain estimates of RST using multilocus data, the appropriate variances for each locus was calculated and then averaged over all loci before calculating [R.sub.ST] (Slatkin 1995). However, in datasets with widely different variances, loci with low variance contribute little to [R.sub.ST] even if they show high levels of differentiation. The loci are made compatible by globally standardizing the dataset so alleles are expressed in terms of standard deviations from the global mean rather than the allele repeat number. All population-based statistics were computed for each locus and a multilocus weighted average was also calculated (Hardy & Vekemans 1999). A jackknife procedure over loci (Sokal & Rohlf 1995) provides approximate errors for multilocus estimates. Spatial genetic structure estimates, population differentiation and inbreeding coefficients are tested by resampling procedures whereby spatial locations, individuals or genes are permuted. Permuting locations is equivalent to a Mantel test between matrices of pairwise genetic statistics.

RESULTS

A total of 507 lobster pereiopods were collected from an equal number of egg-bearing female lobsters from three sites within LIS and a site within the Hudson Canyon (Table 1 and Fig. 1). The pereiopods were collected in spring and summer. The spring and summer pereiopod collections from eastern and western LIS were treated as separate groups for all statistical analyses.

Lobster genomic DNA analyzed for nine microsatellite loci revealed a high level of heterozygosity among all collection sites and seasons (average [H.sub.obs] = 0.7144) (Table 3). There were no significant differences in heterozygosity among the sampled populations. All microsatellite loci produced multiple products (15 alleles on average per locus). The number of alleles produced per locus was greater than that reported by Jones et al. (2003) from a smaller number of samples. Analysis for the presence of null alleles revealed that 4 out of the 9 microsatellite loci showed evidence of null alleles (Table 4). Loci Ham 6, 9, 15 and 48 showed evidence of null alleles. The allele frequencies for those loci were corrected as described (van Oosterhout et al. 2004). The data were corrected by estimating of the null allele frequency and adjusting the allele and genotype frequencies accordingly. The adjusted allele frequencies were subsequently re-examined for HardyWeinberg deviations (van Oosterhout et al. 2004) (Table 3). Using the corrected values, several of the alleles were now shown to conform to Hardy-Weinberg equilibrium.

The overall corrected allele frequencies are given in Figure 2. The allele frequencies are similar to those reported by Jones et al. (2003) for American lobsters collected in Canada and for the European lobster (Homarus gammarus Linnaeus, 1758).

[FIGURE 2 OMITTED]

The corrected allele frequencies were used to determine conformity to HWE (Table 3). Two-thirds of the loci--on a population basis--showed lack of conformity to HWE. The lack of conformity to HWE was related to the geographic area from which the lobsters were collected. The lobsters collected from eastern and central LIS and the Hudson Canyon area showed lack of conformity to HWE but lobsters collected in western LIS showed conformance to HWE but only in spring and with only a sample of 11 animals.

Population-level differences in allele frequencies were examined by pair-wise statistical approaches (Table 5). The spring and summer eastern and western LIS populations showed little genetic differentiation among themselves, suggesting that there is little difference between lobster breeding populations and populations later in the year. The eastern (spring and summer) and the central LIS populations showed little genetic differentiation from the Hudson Canyon population. In contrast, even through the central LIS population is approximately the same geographical distance from the western and Stratford Shoal LIS populations as it is from the eastern LIS population, it showed 10 times the level of genetic differentiation. The eastern LIS and Hudson Canyon populations also showed high levels of genetic differentiation from the western LIS and Stratford Shoal populations. The Stratford Shoals and western LIS populations showed high levels of genetic differentiation between themselves even though they are geographically closer together than the central and eastern LIS populations.

DISCUSSION

Evidence has suggested that lobster populations in LIS may be genetically differentiated because of anthropogenic selective pressures (CT DEP 2003). Western LIS was the site of large commercial lobster catches and the area also receives high levels of anthropogenic impacts from the surrounding land, raising the possibility for development of pollution resistance in resident lobsters. There is ample evidence in the literature for the rapid development of insecticide resistance in insects (Baker & Argobast 1995, ffrench-Constant et al. 2000). Trapping studies (DNC 2002) have shown that lobsters released in eastern LIS (>150,000) were not collected in western LIS, but were found predominantly (>98%) in eastern LIS. Finally, a shell disease prevalent in eastern LIS is much less prevalent in lobsters collected from western LIS.

Anthropogenic pressures may select for lobster populations more resistant to pollutants but with reduced heterozygosity. The possible reduced lobster population heterozygosity may make them more susceptible to unusual stresses, such as the application of pesticides or elevation in water temperature, and could have led to their massive die-off in 1999.

Previous examination of lobster population structure in coastal American waters has not revealed the presence of extensive genetic differentiation. Previous work relied on mitochondrial DNA and allozyme markers with less resolution than the highly polymorphic microsatellite loci used in this study (Tracey et al. 1975, Harding et al. 1997). The recent development of highly polymorphic microsatellite loci for H. americanus allows for the examination of lobster population structure on a finer geographic range (Jones et al. 2003).

This is the first report of the examination of H. americanus genetic population structure through the use of highly polymorphic and heterozygotic microsatellite loci. In contrast to the work reported by Jones et al. (2003), the microsatellite loci examined in these experiments produced more alleles. This was likely caused by an increase in samples number (over 500 vs. 50 in the Jones work) that any changes in diversity.

Four out of the nine-microsatellite loci showed evidence of null alleles in samples collected from LIS and Hudson Canyon. This is in contrast to the low levels of null alleles reported by Jones et al. (2003) in the examination of H. americanus and H. gammarus collected in Canada and European waters. Null alleles--or nonamplified alleles can cause deviations from Hardy-Weinberg equilibrium and may bias both spatial and temporal population genetic analyses (Pemberton et al. 1995, Jones et al. 1998, Holm et al. 2001). The cause of high levels of null alleles in these lobsters is unclear. After correction of the allele frequencies for null alleles by estimation of the null frequency and adjustment of allele frequencies (van Oosterhout et al. 2004), there was a decrease in the alleles not at the Hardy-Weinberg equilibrium. The remaining nonconformity was found specifically in lobsters collected from central and eastern LIS and the Hudson Canyon area. The fact that some loci were in HWE and others were not is often interpreted as evidence for random mating and panmixia. In such cases, deviation from HWE is assumed to be a locus-specific phenomenon, possibly a scoring error or null allele.

The analysis of genetic population differences with corrected allele frequencies showed that the eastern and central LIS lobster populations show slight evidence of genetic differentiation, suggesting ample gene flow between populations. The eastern and central LIS lobster populations also showed greater--but not significant--genetic differentiation with Hudson Canyon lobsters, suggesting a geographical component. Genetic subpopulations have been identified in the European lobster H. gammarus that reflect the levels of geographic isolation (Ulrich et al. 2001).

Examination of western LIS and Stratford Shoal lobster populations revealed a much greater level of genetic differentiation--by a factor of 10--from the eastern and central LIS and Hudson Canyon populations. This genetic differentiation is six times greater than what would be expected on the basis of geographic distance using the genetic differences between eastern and central Long Island Sound as a guide. Examination of microsatellite heterozygosity difference did not indicate that the western LIS populations were less heterozygous than the Hudson Canyon or other LIS populations.

The higher levels of genetic differentiation may be caused by processes, such as, development of pollution resistance, commercial fishing pressure or unique ecologic conditions, naturally occurring in western LIS but not eastern LIS (Howell et al. 2003). The differences may also be a result of the massive die-off in 1999, and the remaining lobsters provided the founder population for the subsequent generations. These experiments cannot differentiate between these two possibilities.

Additional experiments are required to determine if these genetic differences are temporally stable, and, if so, the factors responsible for maintaining these genetic differences. The restoration of lobster populations in western LIS will require a better understanding of the genetic population structure and the linkage between egg-bearing female lobsters and lobster larvae recruitment to establish which female populations are responsible for larvae populations.

ACKNOWLEDGMENTS

The authors thank Penny Howell and the scientists at the Connecticut DEP for the collection of egg-bearing female lobster pereiopods. This work was funded by the Connecticut DEP and the National Marine Fisheries Service.

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JOSEPH F. CRIVELLO, (1) * DONALD F. LANDERS JR. (2) AND MILAN KESER (2)

(1) Department of Physiology and Neurobiology, University of Connecticut, 3107 Horse Barn Hill Road, Storrs, Connecticut 06269; (2) Millstone Power Station, Resource Services Inc., Environmental Laboratory, Waterford, Connecticut 06385

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

TABLE 1.
Sites for the collection of lobster pereiopod.

Site                          Lat/Long (a)               Season (b)

Hudson Canyon      39[degrees] 31' to 39[degrees] 45'    Spring
Eastern LIS        41[degrees] 17' to 41[degrees] 20'    Spring
                   72[degrees] 09' to 72[degrees] 10'    Summer
Central LIS        41[degrees] 02' to 41[degrees] 11'    Spring
Stratford Shoals   41[degrees] 04' to 41[degrees] 07'    Spring
Western LIS        40[degrees] 59' to 41[degrees] 14'    Spring
                   72[degrees] 59' to 73[degrees] 50'    Summer
Total

                   Number of Collected
Site               Lobster Pereiopods         Label

Hudson Canyon              137           Hudson
Eastern LIS                105           Eastern-1
                            28           Eastern-2
Central LIS                135           Central
Stratford Shoals             9           Stratford Shoals
Western LIS                 11           Western-1
                            82           Western-2
Total                      507

(a) Latitude and longitude values are given for an approximate
rectangular area in which collections occurred.

(b) Lobser pereiopods collected before July 1st were included in the
spring group. those collected after July 1st were included in the
summer group.

TABLE 2.
Primers and PCR conditions, microsatellite sizes and number of alleles.

Loci     Primer Sequences (5' [right arrow] 3')

Ham-6    D2-CATGCAGGTATACACAGACACACTC
         ACTGTGTTGACTTAATCTGGAGAAA
Ham-9    D3-CTGGCTCCATGCATACCC
         CAAGCGGCTACATAACTTTTCGTG
Ham-10   D4-CTATCTACAAGGTCATATGTTCAGTT
         CACAACACACCTTTTATACGATT
Ham-15   D2-CTGCGCCATTAGAGGACA
         GTTGCCATCAGGGTGTTC
Ham-21   D3-TTACTCACTCAACGGCACT
         GACTTGCGGTGTGAAAA
Ham-22   D4-GAGGCAAACATACAAATAGACACA
         GTTTGTCCCTTATTTTCTGGT
Ham-30   D2-CCTTTTATATTCTATCTATCTATCTCTG
         GTTTAACCGGACCAGAC
Ham-48   D3-TTCTGAAAGTTTGACGGGTTA
         ACACGTACACACAGGGATTG
Ham-53   D4-GGCATCCCATAGTGAAGG
         ATTTGCGTTTTTGTTTCATTT

         Product
         Length
         (base      Annealing    Allele
Loci     pairs)    T[degrees]C   Number

Ham-6    102-160       60          19

Ham-9    140-206       58           9

Ham-10   120-188       50          18

Ham-15   68-148        52          17

Ham-21   68-136        53          16

Ham-22   68-144        55           9

Ham-30   68-144        55          13

Ham-48   76-148        54          15

Ham-53   106-160       58          19

[D.sub.2], [D.sub.3] & [D.sub.4] refer to fluorescent tags on the
forward primer.

TABLE 3.
Summary statistics for nine microsatellite loci surveyed in lobsters
at indicated locations.

                               Ham-6             Ham-9
Hudson
N = 137
[Het.sub.Obs]                      0.8759            0.5547
[Het.sub.Est]                      0.9218            0.7507
HWE P value [+ or -] SE            0.0010            0.0000
                          [+ or -] 0.0004   [+ or -] 0.0000
Corrected *                        0.0033            0.0053
                          [+ or -] 0.0001   [+ or -] 0.0006
Eastern-1
N = 105
[Het.sub.Obs]                      0.9047            0.4762
[Het.sub.Est]                      0.9228            0.7300
HWE P value [+ or -] SE            0.4387            0.0000
                          [+ or -] 0.0090   [+ or -] 0.0000
Corrected *                        0.4567            0.0421
                          [+ or -] 0.0094   [+ or -] 0.0011
Eastern-2
N = 28
[Het.sub.Obs]                      0.8571            0.5714
[Het.sub.Est]                      0.8982            0.7836
HWE P value [+ or -] SE            0.3391            0.0375
                          [+ or -] 0.0055   [+ or -] 0.0018
Corrected *                        0.4256            0.0512
                          [+ or -] 0.0061   [+ or -] 0.0017
Central
N = 135
[Het.sub.Obs]                      0.8222            0.5037
[Het.sub.Est]                      0.9286            0.7959
HWE P value [+ or -] SE            0.0003            0.0000
                          [+ or -] 0.0001   [+ or -] 0.0000
Corrected *                        0.0512            0.0331
                          [+ or -] 0.0004   [+ or -] 0.001
Stratford
N = 9
[Het.sub.Obs]                      0.8888            0.4444
[Het.sub.Est]                      0.9542            0.5622
HWE P value [+ or -] SE            0.3741            0.0592
                          [+ or -] 0.0061   [+ or -] 0.0008
Corrected *                        0.4321            0.0672
                          [+ or -] 0.0054   [+ or -] 0.0012
Western-1
N = 11
[Het.sub.Obs]                      0.9090            0.8182
[Het.sub.Est]                      0.9263            0.7727
HWE P value [+ or -] SE            0.5786            0.8320
                          [+ or -] 0.0043   [+ or -] 0.0019
Corrected *                        0.6432            0.8453
                          [+ or -] 0.0067   [+ or -] 0.0007
Western-2
N = 82
[Het.sub.Obs]                      0.9012            0.5062
[Het.sub.Est]                      0.9065            0.6728
HWE P value [+ or -] SE            0.0181            0.0465
                          [+ or -] 0.0020   [+ or -] 0.0017
Corrected *                        0.0561            0.0673
                          [+ or -] 0.0067   [+ or -] 0.0066

                              Ham-10            Ham-15
Hudson
N = 137
[Het.sub.Obs]                      0.5766            0.8102
[Het.sub.Est]                      0.8507            0.8936
HWE P value [+ or -] SE            0.0000            0.0328
                          [+ or -] 0.0000   [+ or -] 0.0035
Corrected *                     --                   0.0543
                                            [+ or -] 0.0031
Eastern-1
N = 105
[Het.sub.Obs]                      0.7810            0.6667
[Het.sub.Est]                      0.8696            0.8082
HWE P value [+ or -] SE            0.0000            0.0001
                          [+ or -] 0.0000   [+ or -] 0.0001
Corrected *                     --                   0.0231
                                            [+ or -] 0.0033
Eastern-2
N = 28
[Het.sub.Obs]                      0.6786            0.8214
[Het.sub.Est]                      0.8039            0.8807
HWE P value [+ or -] SE            0.0000            0.1699
                          [+ or -] 0.0000   [+ or -] 0.0053
Corrected *                     --                   0.2345
                                            [+ or -] 0.0064
Central
N = 135
[Het.sub.Obs]                      0.7407            0.8593
[Het.sub.Est]                      0.8907            0.9018
HWE P value [+ or -] SE            0.0000            0.0585
                          [+ or -] 0.0000   [+ or -] 0.0048
Corrected *                     --                   0.0617
                                            [+ or -] 0.0004
Stratford
N = 9
[Het.sub.Obs]                      0.6667            0.6667
[Het.sub.Est]                      0.5756            0.9211
HWE P value [+ or -] SE            0.8552            0.0201
                          [+ or -] 0.0009   [+ or -] 0.0008
Corrected *                     --                   0.0456
                                            [+ or -] 0.0078
Western-1
N = 11
[Het.sub.Obs]                      0.7273            0.8182
[Het.sub.Est]                      0.8364            0.8636
HWE P value [+ or -] SE            0.1513            0.2263
                          [+ or -] 0.0012   [+ or -] 0.0027
Corrected *                     --                   0.2235
                                            [+ or -] 0.0034
Western-2
N = 82
[Het.sub.Obs]                      0.6543            0.8025
[Het.sub.Est]                      0.7988            0.8790
HWE P value [+ or -] SE            0.0000            0.0318
                          [+ or -] 0.0000   [+ or -] 0.0025
Corrected *                     --                   0.0578
                                            [+ or -] 0.0011

                              Ham-21            Ham-22
Hudson
N = 137
[Het.sub.Obs]                      0.7299            0.5912
[Het.sub.Est]                      0.8458            0.8363
HWE P value [+ or -] SE            0.0000            0.0000
                          [+ or -] 0.0000   [+ or -] 0.0000
Corrected *                     --                --

Eastern-1
N = 105
[Het.sub.Obs]                      0.7524            0.7333
[Het.sub.Est]                      0.8007            0.8294
HWE P value [+ or -] SE            0.0011            0.0001
                          [+ or -] 0.0005   [+ or -] 0.0001
Corrected *                     --                --

Eastern-2
N = 28
[Het.sub.Obs]                      0.8214            0.6429
[Het.sub.Est]                      0.8700            0.7921
HWE P value [+ or -] SE            0.0719            0.0026
                          [+ or -] 0.0026   [+ or -] 0.0002
Corrected *                     --                --

Central
N = 135
[Het.sub.Obs]                      0.7259            0.7556
[Het.sub.Est]                      0.8016            0.8381
HWE P value [+ or -] SE            0.0003            0.0000
                          [+ or -] 0.0003   [+ or -] 0.0002
Corrected *                     --                --

Stratford
N = 9
[Het.sub.Obs]                      0.4444            0.6667
[Het.sub.Est]                      0.8367            0.7256
HWE P value [+ or -] SE            0.0010            0.3481
                          [+ or -] 0.0001   [+ or -] 0.0013
Corrected *                     --                --

Western-1
N = 11
[Het.sub.Obs]                      0.8182            0.8182
[Het.sub.Est]                      0.9273            0.7818
HWE P value [+ or -] SE            0.1645            0.8222
                          [+ or -] 0.0032   [+ or -] 0.0019
Corrected *                     --                --

Western-2
N = 82
[Het.sub.Obs]                      0.5556            0.7284
[Het.sub.Est]                      0.7593            0.8111
HWE P value [+ or -] SE            0.0000            0.0002
                          [+ or -] 0.0000   [+ or -] 0.0000
Corrected *                     --                --

                              Ham-30            Ham-48
Hudson
N = 137
[Het.sub.Obs]                      0.5255            0.4380
[Het.sub.Est]                      0.7588            0.8042
HWE P value [+ or -] SE            0.0000            0.0000
                          [+ or -] 0.0000   [+ or -] 0.0000
Corrected *                     --                   0.0059
                                            [+ or -] 0.0001
Eastern-1
N = 105
[Het.sub.Obs]                      0.5619            0.5905
[Het.sub.Est]                      0.7502            0.8209
HWE P value [+ or -] SE            0.0003            0.0000
                          [+ or -] 0.0002   [+ or -] 0.0000
Corrected *                     --                   0.0378
                                            [+ or -] 0.0051
Eastern-2
N = 28
[Het.sub.Obs]                      0.4286            0.5714
[Het.sub.Est]                      0.7036            0.8193
HWE P value [+ or -] SE            0.0000            0.0460
                          [+ or -] 0.0000   [+ or -] 0.0027
Corrected *                     --                   0.0567
                                            [+ or -] 0.0039
Central
N = 135
[Het.sub.Obs]                      0.6074            0.6148
[Het.sub.Est]                      0.8068            0.8324
HWE P value [+ or -] SE            0.0000            0.0001
                          [+ or -] 0.0000   [+ or -] 0.0001
Corrected *                     --                   0.0345
                                            [+ or -] 0.0007
Stratford
N = 9
[Het.sub.Obs]                      0.7778            0.6667
[Het.sub.Est]                      0.7911            0.9089
HWE P value [+ or -] SE            0.6488            0.0144
                          [+ or -] 0.0020   [+ or -] 0.006
Corrected *                     --                   0.0345
                                            [+ or -] 0.0089
Western-1
N = 11
[Het.sub.Obs]                      0.4545            0.9091
[Het.sub.Est]                      0.6000            0.8818
HWE P value [+ or -] SE            0.0594            0.5329
                          [+ or -] 0.0010   [+ or -] 0.0026
Corrected *                     --                   0.5676
                                            [+ or -] 0.0034
Western-2
N = 82
[Het.sub.Obs]                      0.3580            0.8395
[Het.sub.Est]                      0.5642            0.8531
HWE P value [+ or -] SE            0.0000            0.0038
                          [+ or -] 0.0000   [+ or -] 0.0006
Corrected *                     --                   0.0356
                                            [+ or -] 0.0009

                              Ham-53
Hudson
N = 137
[Het.sub.Obs]                      0.8540
[Het.sub.Est]                      0.9202
HWE P value [+ or -] SE            0.0006
                          [+ or -] 0.0003
Corrected *                     --

Eastern-1
N = 105
[Het.sub.Obs]                      0.8857
[Het.sub.Est]                      0.9371
HWE P value [+ or -] SE            0.0751
                          [+ or -] 0.0044
Corrected *                     --

Eastern-2
N = 28
[Het.sub.Obs]                      0.7500
[Het.sub.Est]                      0.9214
HWE P value [+ or -] SE            0.0092
                          [+ or -] 0.0005
Corrected *                     --

Central
N = 135
[Het.sub.Obs]                      0.8222
[Het.sub.Est]                      0.9124
HWE P value [+ or -] SE            0.0000
                          [+ or -] 0.0000
Corrected *                     --

Stratford
N = 9
[Het.sub.Obs]                      0.6667
[Het.sub.Est]                      0.6144
HWE P value [+ or -] SE            0.0068
                          [+ or -] 0.0006
Corrected *                     --

Western-1
N = 11
[Het.sub.Obs]                      0.7273
[Het.sub.Est]                      0.8727
HWE P value [+ or -] SE            0.0733
                          [+ or -] 0.0020
Corrected *                     --

Western-2
N = 82
[Het.sub.Obs]                      0.7531
[Het.sub.Est]                      0.9049
HWE P value [+ or -] SE            0.0005
                          [+ or -] 0.0002
Corrected *                     --

Hardy-Weinberg equilibrium assumes the "null hypothesis" that the
observed genotype frequencies are not significantly different from
those predicted for a population in equilibrium. A P value less than
0.05 indicate that they are significantly different and the loci are
not at HWE. Loci not at HWE equilibrium are in italics.

* The Hardy-Weinberg equilibrium was recalculated for several of the
loci after correction for null alleles as described by Van Oosterhout
et al. (2004). Only those loci shown to have null alleles (Table 4)
were reanalyzed for compliance to Hardy-Weinberg equilibrium.

TABLE 4.
Presence of null alleles in microsatellite loci.

             Presence
             Null of
Loci         Alleles    Brookfield-1   Corrected *

Ham-6          +        0.0693           0.0514
Ham-9          +        0.1495           0.0725
Ham-10         -        0.0469             --
Ham-15         +        0.1104           0.0511
Ham-21         -        0.0252             --
Ham-22         -        0.0315             --
Ham-30         -        0.0411             --
Ham-48         +        0.1144           0.0612
Ham-53         -        0.0461             --

A value greater than 0.05 indicates the presence of null alleles.
The Brookfield-1 algorithm ignores all null alleles as degraded DNA,
human error, or other reasons for nonamplification other than the
presence of a  true null allele homozygote.

* Statistically corrected values as described by Van Oosterhout
et al (2004).

TABLE 5.
Genetic differences between sampled populations from LIS and the
Hudson Canyon.

                                              [delta]
Location           Vs.         [F.sub.ST]   [[mu].sup.2]   [R.sub.ST]

Hudson      Stratford Shoals     0.0275        0.1085       0.0321
  Canyon    Western-1            0.2000        0.1058       0.0236
Eastern-1   Central              0.0033        0.0141       0.0045
            Hudson Canyon        0.0033        0.0168       0.0049
            Stratford Shoals     0.0391        0.1618       0.0441
            Western-1            0.0192        0.1009       0.0233
Eastern-2   Eastern-1            0.0029        0.0021       0.0034
            Central              0.0033        0.0141       0.0043
            Hudson Canyon        0.0045        0.0283       0.0055
            Stratford Shoals     0.0227        0.0696       0.0328
            Western-1            0.0289        0.0786       0.0277
            Western-2            0.0277        0.0654       0.0331
Central     Hudson Canyon        0.0051        0.0248       0.0051
            Stratford Shoals     0.0406        0.1745       0.0452
            Western-1            0.0215        0.1377       0.0313
Stratford
  Shoals    Western-1            0.0366        0.1166       0.0412
Western-2   Eastern-1            0.0145        0.0366       0.0241
            Central              0.0111        0.0451       0.0216
            Hudson Canyon        0.0205        0.0731       0.0289
            Stratford Shoals     0.0106        0.0393       0.0156
            Western-1            0.0169        0.0690       0.0278