Journal of Shellfish Research, Vol. 30, No. 3, 583–608, 2011.

NATURAL AND ANTHROPOGENIC FORCES SHAPE THE POPULATION GENETICS AND RECENT EVOLUTIONARY HISTORY OF EASTERN UNITED STATES BAY ( IRRADIANS )

THERESA M. BERT,* WILLIAM S. ARNOLD,† ANNE L. MCMILLEN-JACKSON, AMI E. WILBUR‡ AND CHARLES CRAWFORD Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 Eighth Avenue Southeast, St. Petersburg, Florida 33701

ABSTRACT Bay scallops ( Lamarck) are ecologically important in U.S. Atlantic waters off northeastern states and in the Florida Gulf of Mexico, and have been intensely harvested from both of those regions for decades. However, a detailed study comparing their basic population genetic structures using more than a single type of genetic marker has not been conducted. Through such a study, key phylogeographic, taxonomic, and fisheries issues can be addressed. We used variation in allozyme loci and mitochondrial DNA restriction fragment length polymorphisms to evaluate and compare the population genetic structures of bay scallops from those two regions, to propose a new interpretation for the composition of the North Carolina bay population, to resolve the taxonomic quandary of Argopecten irradians taylorae, and to evaluate the apparent and potential genetic effects of the common fishery practice of hatchery-based stock enhancement on the genetic diversity and relatedness of Atlantic bay scallop populations. Atlantic (North Carolina through New York) bay scallop populations are genetically more distant from each other than are Florida Gulf bay scallop populations, except those in Florida Bay. Each Atlantic population has a different phylogeographic history, is quasi-independent, and should be treated as a genetically unique entity. The North Carolina bay scallop population is composed of Argopecten irradians irradians individuals, but also has genetic input from Argopecten irradians concentricus. Bay scallops occurring in Florida Bay constitute a population of A. i. concentricus that has diverged from other Florida Gulf populations because it has undergone repeated contractions and expansions of varying magnitude and is nearly isolated from other bay scallop populations. For the common practice of hatchery-based stock enhancement in the Atlantic, broodstock bay scallops should be taken from the same genetic population, and all stock enhancement efforts should include comprehensive genetic monitoring programs. In some cases, improving the abundance and density of bay scallop aggregations through habitat improvement may be preferable to stock enhancement for bay scallop restoration, but in other cases genetically conscientious stock supplementation or restoration may be the only alternative.

KEY WORDS: aquaculture, Argopecten irradians, Atlantic, bay scallop, evolution, fishery, Florida, Gulf of Mexico, population genetics, stock enhancement,

INTRODUCTION in the extreme southeastern United States and eastern Gulf, and Argopecten irradians amplicostatus occurs in the western Gulf The charismatic, environmentally, and economically impor- (Waller 1969) (Fig. 1A). Since the first publication of the sub- tant bay scallop Argopecten irradians (Lamarck) inhabits shallow- species’ ranges, and despite numerous taxonomic studies that water seagrass flats and algal beds in the eastern United States. In evaluated variation in morphometrics, meristics, and multiple the western North Atlantic Ocean (henceforth, Atlantic), bay types of genes, the locations of sympatry between A. i. irradians scallops range in the United States from Massachusetts (Clarke and A. i. concentricus in the Atlantic, the taxonomic identity of 1965) through North Carolina (NC) and, below a 10° latitudinal A. irradians in NC, and the existence of a fourth subspecies gap extending from South Carolina to east–central Florida (Argopecten irradians taylorae (Petuch 1987)) in and around FB (Heffernan et al. 1988), occur again in southeastern Florida from have remained ambiguous. The confusion is understandable, West Palm Beach to Biscayne Bay (Marelli et al. 1997a) (Fig. 1). given the considerably different results that can emerge among In the Gulf of Mexico (henceforth, Gulf), bay scallops range these character sets, even when all data are taken from the same from Florida Bay (FB) northward through Florida Gulf waters individuals (e.g., Wilbur 1995, Wilbur & Gaffney 1997). and westward to the Chandeleur Islands, LA (Waller 1969). Population genetics analyses have been woven into these Westward of a gap along the northern Gulf, they occur again principally taxonomic studies, and also into other ecological from northeastern Texas southward through Mexico (Waller and biological studies on bay scallops, but a thorough compar- 1969, Wakida-Kusunoki 2009) to Colombia (Abbott 1974). ison of the population genetic structure of bay scallops in the Within its range, A. irradians has been divided into several Atlantic and Gulf basins using multiple genetic markers has not subspecies. Argopecten irradians irradians occurs in the north- yet been conducted. Understanding the population genetic eastern United States, Argopecten irradians concentricus occurs structure of bay scallops is important for conservation and restoration of populations as well as for fisheries management *Corresponding author. E-mail: [email protected] because destructive natural and anthropogenic phenomena, †Current address: National Marine Fisheries Service, 203 13th Avenue South, St. Petersburg, FL 33701 coupled with intense commercial and recreational fisheries, ‡Current address: Department of Biology and Marine Biology and the have greatly reduced the number of dense aggregations and Center for Marine Science, University of North Carolina-Wilmington, overall population sizes of bay scallops in both the Atlantic and 5600 Marvin K. Moss Lane, Wilmington, NC 28409 Gulf during the past few decades (Peterson & Summerson 1992, DOI: 10.2983/035.030.0302 Tettelbach & Wenczel 1993, Arnold et al. 1998).

583 584 BERT ET AL.

pleted wild populations or suitable unpopulated areas with aquacultured young individuals or placing aquacultured mature individuals in cages in the ocean and allowing them to spawn) (Tettelbach & Wenczel 1993, Peterson et al. 1996, Arnold et al. 2005, Tettelbach 2009, Tettelbach et al. 2010). The potential genetic impacts of both the depletions and the restorations are numerous, varied, and potentially threatening to the species’ ability to survive over evolutionary time (Bert et al. 2007). Here, we address these three conundrums. We evaluate and compare the population genetic structures of bay scallops from Atlantic and Florida Gulf waters using allozyme loci and a segment of the mitochondrial DNA (mtDNA) molecule. Col- lectively considering the information provided by analysis of both nuclear and mitochondrial genes often provides a more complete picture of population genetic structure and the mechanisms driving that structure (Edmands et al. 1996, Rigaa et al. 1997, Shaklee & Bentzen 1998, Busack & Lawson 2008). Together, the allozyme data and mtDNA data enabled us to provide a fresh interpretation of the taxonomic composition of bay scallops in the Atlantic area of sympatry between A. i. irradians and A. i. concentricus, and to probe the phylogeographic history of the genetic differentiation between the two subspecies. We then draw upon both data sets to resolve the taxonomic ambiguity of A. i. taylorae and deepen our understanding of its population history. Last, we discuss the potential influence of the common fishery management practice of hatchery-based stock enhancement on Atlantic bay scallop population genetic struc- ture and speculate about its impacts on Atlantic bay scallop populations. Our results are particularly relevant because they are based on genetic data collected prior to stock enhancement efforts for Florida Gulf bay scallops, and much stock enhance- ment has continued in Atlantic bay scallop populations since our samples were collected. Future studies using the same loci and collecting sites could reveal the effects of the numerous, sub- sequent stock enhancement programs on the population genetic structures of both Florida Gulf and Atlantic bay scallops.

METHODS AND MATERIALS

Field and Laboratory

From 1995 through 1998, we obtained collections of bay scallops from 15 locations in eastern U.S. nearshore waters (Table 1, Fig. 1B). Whole wild bay scallops were collected from Atlantic locations during summers and were shipped alive to us by colleagues. Scuba divers collected adult bay scallops from 12 locations in the Florida Gulf during annual surveys. All bay Figure 1. Bay scallop, Argopecten irradians, ranges and sampling scallops were dissected, and samples of adductor muscle, gills, locations. (A) Subspecies distributions. Asterisks after abbreviations and digestive gland were excised, wrapped, immediately frozen denote collecting locations. FL, Florida; GA, Georgia; LA, Louisiana; in liquid nitrogen, and stored at –80°C. MA, Massachusetts; MD, Maryland; ME, Maine; NJ, New Jersey: NY, New York; NC, North Carolina; SC, South Carolina; TX, Texas; VA, For allozyme electrophoresis, small pieces of the three tissue Virginia. (B) Florida collecting locations ($ subpopulations; Bert et al. in types were combined and homogenized in 0.1 M Tris–EDTA, prep.) grouped into the 4 populations used in this report. pH 7.0; the supernatant was used as the source. Hori- zontal starch gel electrophoresis was conducted using standard protocols (Selander et al. 1971). Eighteen loci were resolved using Historically, A. irradians was generally abundant but patch- 4 buffer systems and were visualized using appropriate staining ily distributed throughout its ranges. Its high fishery value has procedures (Table 2). All gels were scored and checked by at least resulted in considerable attention toward increasing the num- two researchers. Alleles were identified by their mobility relative bers of individuals and geographic expanses of existing aggre- to that of the most common allele, which was designated as gations and regenerating lost aggregations, principally through 100; the numerical code was translated into an alpha code for some form of hatchery-based stock enhancement (seeding de- statistical analysis (e.g., 100 ¼ A). POPULATION GENETICS OF EASTERN UNITED STATES BAY SCALLOPS 585

TABLE 1. Each PCR (10 mL) was digested with a battery of Numbers of individuals and collecting locations for a study 9 : Alu I, Ban II, Bgl II, BsiHKA I, HinFI,Rsa I, ScrF a of population genetic structure and genetic relationships I, Tsp 509 I, and Taq I (5 U enzyme, 20-mL total volume) of eastern U.S. bay scallops. according to the manufacturer’s specifications (New England BioLabs, Beverly, MA). The digests were incubated for 3 h and stopped with 5 mL of loading dye (20% Ficol 400, 0.1 M Na Collecting Years Total EDTA, pH 8), 1% sodium dodecyl sulfate, 0.25% bromophenol Group ERERblue). Entire digests were loaded onto 20-cm, 2%, low-melting Atlantic Ocean subpopulation/ point agarose gels and electrophoresed for 3–4 h at 90–100 V. collection Fragment patterns were visualized by ethidium bromide stain- North Carolina (NC) ing and were photographed under UV light. Fragment sizes Bogue Sound/Core Sound 1997 1997 55 24 were determined from migration distances relative to known area (NC97) standards. Bogue Sound (NCB98) 1998 1998 25 22 Core Sound (NCC98) 1998 1998 20 27 New Jersey, Little Egg Harbor 1998 1998 20 15 Statistical Analysis (NJ98) Many statistical analyses of Atlantic samples were per- New York, Peconic Bay, eastern 1997 1997 65 20 Long Island Sound (NY97) formed at both the collection level and the subpopulation level. Florida Gulf of Mexico population/component subpopulations The three NC collections were combined to form that sub- Panhandle (PN) population; the single New Jersey (NJ) and New York (NY) Saint Andrew Bay (SA) 1995–1998 1998 129 25 collections also served as those subpopulations. Sample desig- Crooked Island Sound (CI) 1995–1996 — 64 — nations for collection-level analyses include both the location Saint Joseph Bay (SJ) 1995–1998 1995–1998 154 69 and year of collection (e.g., NC97); designations for subpopu- Core (CO) lation-level analyses include only the location abbreviation Steinhatchee (ST) 1995–1998 1995–1998 174 69 (e.g., NC). For brevity, we refer to all subpopulations and Cedar Key (CK) 1997 — 53 — populations by their location names. Sample sizes differed Homosassa Bay (HO) 1995–1998 1995–1998 180 87 between the two genetic techniques used, and they are given in Hernando County (HE) 1997–1998 — 41 — Anclote Estuary (AN) 1996–1998 1998 113 41 Table 1. Southwest Florida (SF) Allozyme Locus Analysis Tampa Bay (TB) 1997 1997 57 23 Sarasota Bay (SS) 1998 1998 35 24 Our Florida Gulf allozyme and mtDNA data were drawn Pine Island Sound (PI) 1995–1998 1995–1998 149 66 from a complementary study (Bert et al. in prep.). In that study, Florida Bay (FB) 1998 1998 35 21 the multiple annual collections were combined to form sub- Total 1,335 534 population samples from each of 12 locations. Based on com- Subpopulation and population abbreviations in parentheses. E, samples mon genetic characteristics, the subpopulations were grouped used for allozyme electrophoresis; R, samples used for restriction fragment into 4 populations which, except for FB, were composed of bay length polymorphism analysis of mitochondrial DNA; —, no sample. scallops from several locations (Fig. 1B). Here, we synthesized Collection locations shown in Figure 1. the allele frequency data and related statistics, including genetic distances, at the population level. Unless otherwise noted, we obtained population-level values by averaging the subpopula- For the mtDNA analysis, purified mtDNA extracts (see tion values presented in Bert et al. (in prep.), which compen- Blake and Graves (1995) for methods of extraction) were sated for the sometimes highly unequal sample sizes among amplified using primers Patinopecten yessoensis primers 40-F subpopulations. For the mtDNA data, we merged haplotypes and 40-R (Boulding et al. 1993) generating a 610-base pair (bp) from all locations used in Bert et al. (in prep) into population- fragment that was manually sequenced for 28 individuals (19 level haplotype frequencies. from Florida, 5 from NC, and 4 from Massachusetts). An internal We used BIOSYS-1 (Swofford & Selander 1981) to calculate Argopecten–specific primer was generated and used to acquire allele and genotype frequencies. For each Atlantic collection, an additional ;490-bp mtDNA sequence by genome walking we compared observed genotype frequencies of each locus with (vectorette PCR protocols; Sigma-Genosys, Woodlands, TX). Hardy-Weinberg (H-W) expected genotype frequencies using The ;1-kb fragment was sequenced, and new primers were Fisher’s exact test and determined heterozygote deficiency designed (AI99F: ATT CCC CCT CAA CAA ART CA and or excess using the D statistic (Selander 1970). To describe de- AI912R: ACA AAC TGC CCG TCG CTC TC), and amplifica- viations from H-W genotype frequency expectations for each tion yielded an 833-bp fragment, which we used during the RFLP Florida Gulf population, we provide the proportion of the analysis. Analysis by a BLAST search indicates the fragment to appropriate Florida Gulf subpopulations that did not conform include portions of the 12s ribosomal subunit, transfer RNAs for to H-W expectations in Bert et al. (in prep). We tested for sig- glutamine and valine, and the NADH 1 coding regions. The PCR nificant differences in proportion of loci deviating from H-W amplification conditions to yield this fragment were as follows: 40 equilibrium among Atlantic subpopulations and Florida Gulf cycles, each of 30 sec at 94°C, 30 sec at 53°C,and1minat72°C. populations separately and between those 2 groups using the Reactions were performed in 100-mL volumes of 13 PCR buffer R3C G-test followed by the simultaneous test procedure (STP) containing 1.5 mM MgCl2,0.2mM each primer, 800 mM dNTP, for frequencies (BIOMstat, version 3; http://www.ExeterSoftware. and 2.5 U Taq polymerase. com; Sokal & Rohlf (1995)). We used GENEPOP (version 3.2a; 586 BERT ET AL.

TABLE 2. Proteins used to investigate population genetic structure of Argopecten irradians.

No. of Loci Reference Abbreviation Protein Enzyme No. (EC) Resolved Buffer System for Stain AAP Alanyl aminopeptidase 3.4.1.- 2 PC A AAT Aspartate aminotransferase 2.6.1.1 2 PC E dPEP Nonspecific aminopeptidase 3.4.1.- 1 TCI G EST Esterase 3.1.-.- 1 TCII E GP General proteins NA 1 LiOH F GPI Glucose-6-phosphate dehydrogenase 5.3.1.9 1 LiOH G HDH Hexanol dehydrogenase 1.1.1.71 1 TCI C IDH Isocitrate dehydrogenase 1.1.1.42 1 TCII F LAP a-amino acyl peptide 3.4.1.- 1 LiOH D MDH Malate dehydrogenase 1.1.1.37 2 TCII G MPI Mannose 6-phosphate 5.3.1.8 1 TCII A PGD 6-phosphogluconate dehydrogenase 1.1.1.44 1 PC G PGM Phosphoglucomutase 5.4.2.2 1 PC G OPDH D-octopine dehydrogenase 1.5.1.11 1 TCI B SOD Superoxide dismutase 1.15.1.1 1 TCII D

Buffer systems are as follows. LiOH: electrode, 0.03 M lithium hydroxide, 0.19 M boric acid, pH 8.1; gel, 0.05 M Tris, 0.008 M citric acid, pH 8.4, PC: electrode, 0.214 M potassium phosphate, 0.027 M citric acid, pH 6.7, gel, 0.0061 M potassium phosphate, 0.0012 M citric acid, pH 7.0; TCI: electrode, 0.30 M borate, pH 8.2; gel, 0.076 M Tris, 0.005 M citric acid, pH 8.7, TCII: electrode, 0.687 M Tris, 0.157 M citric acid, pH 8.0; gel, 0.023 M Tris, 0.005 M citric acid, pH 8.0. References: A, Bricelj and Krause (1992); B, Dando et al. (1981); C, Dillon and Davis (1980); D, Koehn et al. (1988); E, Murphy et al. (1990); F, Selander et al. (1971); G, Shaw and Prasad (1970). NA, not applicable.

Raymond & Rousset (1995)) to calculate the following genetic the Ryan-Einot-Gabriel-Welsch multiple range test. If needed, variability statistics: average (direct-count) heterozygosity per we transformed QST values using log n + 1. This approach capi- locus (Ho), percentage of polymorphic loci at the P95 and the P99 talizes on the idea that the nonrandom distribution of small levels (frequency of rarest allele was 0.05 or 0.01, respectively), genetic differences is more convincing than a single tablewide FST and mean number of alleles per locus (na). or GST value calculated for an entire data set (Palumbi 2003), and We searched for patterns of significance in allele frequency also allows for exploration of the connectivity of collections and differences at each polymorphic locus (P95 level) among North subpopulations separated by varying geographic distances while Carolina collections, Atlantic subpopulations, and Florida Gulf avoiding the unreliability of single pairwise FST values (Hellberg populations independently and among Atlantic subpopulations 2006). Relational patterns can be detected in high-dispersal and Florida Gulf populations together using R3CG-tests to species (e.g., those with pelagic larvae) using this approach establish the significance followed by pairwise R3CG-tests to (Palumbi 2003). For bay scallops, establishing connections locate the sources of the differences. To eliminate cells with 0 fre- within and between ocean basins can, by inference, reveal un- quencies, we combined rare alleles so that no cell had a frequency derlying successful larval dispersal patterns. of 0. Then, using the exact probability test, we tested allele fre- We also used the pairwise Nei’s D values calculated for quencies collectively over all loci for homogeneity between all Atlantic (presented herein) and Florida subpopulations (from pairwise combinations of Atlantic subpopulations and Florida Bert et al. in prep) to generate unweighted pair group method populations. In each set of analyses, we compensated for multiple of analysis (UPGMA) and neighbor-joining (N-J) phenograms testing of the null hypothesis of homogenous allele frequencies by (UPGMA Tree software,(Jin & Ferguson 1990)). To estimate adjusting the significant probability values accordingly using the statistical confidence in the phenograms, we calculated standard sequential Bonferroni method (Rice 1989). errors for the nodes in the UPGMA phenogram (Nei et al. (1985), We examined geographic relationships by first calculating as implemented in UPGMA Tree) and bootstrap probability pairwise Nei’s genetic distances (Nei’s D; Nei (1972)) and pairwise values for the nodes in the N-J phenogram (1,000 replicates QST (Wright’s FST analogue; Weir & Cockerham (1984)) values DISPAN; Ota (1993)). over all loci, following Slatkin (1993; in GENEPOP), for all mtDNA Analysis pairwise combinations of Atlantic collections and of Atlantic subpopulations and Florida populations. Values for Atlantic/ We calculated haplotype frequencies using REAP (version 4, Florida Gulf comparisons were obtained by averaging Atlantic McElroy et al. (1992)). We used both Monte Carlo simulation collection/Florida subpopulation values. We analyzed spatial (in REAP) and the R3C G-test followed by the STP to search variation of the pairwise Nei’s D and QST genetic distance values for significant differences in haplotype frequencies among by calculating the means of various combinations of pairwise Atlantic collections and subpopulations. For the R3CG-tests, values and testing those means for significant differences using haplotypes other than the common haplotype were combined. the Kruskal-Wallis test or Wilcoxon’s 2-sample test followed by For each Atlantic collection and subpopulation, we calculated POPULATION GENETICS OF EASTERN UNITED STATES BAY SCALLOPS 587 haplotype diversity (h) and nucleotide diversity (p) (Nei 1987), and other accessory statistics, including Tajima’s (1989) D and and calculated pairwise p for subpopulations (REAP). For Fu’s (1997) FS, to generate an interpretation of the full MSN Florida Gulf population-level h and p,weaveragedthe different from that produced solely by the NCPA. collection-level values reported in Bert et al. (in prep.); for population-level pairwise p values, we averaged the subpopu- RESULTS lation-level values presented in that report. The h and p values of Atlantic collections and Florida Gulf populations were tested Allozyme Electrophoresis Analysis for significant outliers within each group using Dixon’s test; the Genetic Diversity means of the Atlantic and Florida Gulf values for those statistics (omitting FB) were compared using Wilcoxon’s 2-sample test We consider genetic diversity to be composed of 2 types of (Sokal & Rohlf 1995). genetic variability: the number of alleles present (genetic varia- We investigated genetic relationships between pairs of Atlantic tion) and the frequencies of those alleles (genetic composition). subpopulations, Florida populations, and Atlantic–Florida com- Genetic diversity measures for the 18 loci are presented in Table binations by testing the significance of Cockerham’s (1969, 1973) 3. Although no loci differed significantly in allele frequencies pairwise genetic distances (FST; exact test, Arlequin, version 2.0; among Atlantic subpopulations, NJ had unusual allele frequen- Schneider et al. (2000)) and of pairwise nucleotide divergences cies at several loci, notably LAP. (d; Nei (1987)) (REAP). We used the sequential Bonferroni test Patterns of rare-allele (frequency, <0.1) occurrence revealed (Rice 1989) to correct for multiple tests of the null hypothesis that relationships and provided information for inferring the recent no significant differences existed. history of the subpopulations. At 7 of the 10 highly polymorphic To search for genetic population structure, we analyzed loci, a total of 8 rare alleles were shared only by NY and NC. isolation by distance by regressing the geographic distances of In contrast, only 1 rare allele was shared by NJ and NC, and no all pairwise combinations of Atlantic subpopulations and the rare alleles were shared by NJ and NY. Sixteen NC individuals subpopulations composing the Florida Gulf populations (Bert (16% of the sample) and 3 NY individuals (5% of the sample) et al. in prep.) against the pairwise FST values (Slatkin 1993). possessed rare alleles otherwise found only in Florida Gulf We estimated geographic distances by following the major populations (NC: AAT-1*C, D; EST*B, F; LAP*B, G; MDH- contours of the coastline using the ruler tool in Google Earth. 1*C; MPI*E; OPDH*E; NY: AAT-2*B, MDH-2*C, SOD*B). We tested groupings of pairwise FST values for significant Four NC individuals carried 2 alleles; 2 of the individuals were differences within and between Atlantic subpopulations and homozygotes, 1 was a heterozygote for 2 EST alleles, and 1 was Florida Gulf populations, as we did for the allozyme pairwise heterozygous for a rare allele at 2 loci. The probabilities that any genetic distance measures; and, using the pairwise FST values, of these individuals would exist in our NC sample ranged from we generated an N-J tree (PHYLIP version 3.5c; distributed by 0.0004–0.0009. Two of the NY individuals were also homozy- J. Felsenstein, Department of Genetics, University of Washington, gotes; their probabilities of existence in that sample were each Seattle, WA). We also conducted AMOVA analyses (Arlequin) also 0.0004. In contrast, NJ was depleted of rare alleles at multiple using all pairwise FST values; and, exclusively, Atlantic values and loci (AAT-1, AAT-2, DPEP, GPI, OPDH, LAP) and had no Florida Gulf values. private rare alleles in common with Florida Gulf populations. To explore mtDNA haplotype variation in depth, we first Genotype frequencies at the DPEP, EST, and MPI loci did used the pairwise d values to produce a minimum spanning not conform to H-W expectations in nearly all samples, par- network (MSN; Arlequin), and visualized the haplotype relation- ticularly in the subpopulations that composed the Florida Gulf ships using the VGJ graph-drawing tool in http://www.eng. populations (Table 3). All significant deviations from H-W auburn.edu/department/csse/research/research_groups/research/ expectations but one (NJ, at GPI) were the result of heterozy- graph_drawing/vgj.html. Using the TCS program (Clement et al. gote deficits. The incidence of nonconformity did not differ 2000), we checked for parsimony probabilities indicative of the significantly among either Atlantic or Florida Gulf subpopula- number of mutational steps between haplotypes that we can tions, or between subpopulations from the 2 ocean basins. NY’s accept in the MSN to generate the step 1 groupings for a nested relatively high proportion (39%) of deviating loci was the result clade phylogeographic analysis (NCPA; Templeton (1998)). To of the high incidence of rare-allele homozygotes (at 10 of 13 perform the TCS, we changed our 0/1-coded haplotype data polymorphic loci) that had low probabilities of existing (one- to an A/T code (D. Posada, Universidad de Vigo, Spain; pers. half to one-hundredth of their actual frequencies; data not comm.). We used the GeoDis program (Posada et al. 2000) to test shown); this also was the major contributor to the relatively low for significant geographic associations among haplotypes and dcHo value and high na value for NY (Table 3). The relatively applied the NCPA to the clades with significant associations. We low dcHo value and na value for NJ were attributable to higher also performed sequence mismatch analyses (Slatkin & Hudson frequencies of common-allele homozygotes at multiple loci. 1991, Rogers & Harpending 1992, Templeton 1998) (Arlequin; Most measures of genetic variability were related to sample 1,000 bootstrap replicates) on, separately, all samples, Atlantic size in the NC collections, but measures of genetic variability samples, and Gulf samples, as well as on each level-3 NCPA and H-W conformation were inversely related to heterozygos- clade. The NCPA provides information useful for inferring past ity. NC97 had the highest values for both levels of polymor- phylogeographic events, and the mismatch analysis performed phism, na, and number of loci deviating from H-W expectations, on clades provides insight into the contribution of lineages but the lowest value for dcHo, whereas NCB98 and NCC98 had through time to current phylogeographic patterns. To assist with lower values for the 4 former measures but higher values for interpretations further, we produced separate MSNs for Atlantic heterozygosity. This relationship may be associated with the and Florida Gulf haplotypes and used those, together with origins of the bay scallops that formed the aggregations we informationgainedfromtheNCPAandmismatchanalyses sampled rather than the consequence of the absence of rare 588 BERT ET AL.

TABLE 3. Allozyme locus allelic frequencies and measures of genetic variability for bay scallops.

Atlantic Ocean Collection/y or Subpopulation Florida Gulf of Mexico Population NC NC NC NC NJ NY Allozyme

Diversity 97 98B 98C SP 98 97 PN CO SF FB Locus/alleles (Number of individuals) AAP-1 0/3 2/5 0/3 A 0.53 ND ND 0.53 0.63 0.49 0.48 0.41 0.45 0.33 B — ND ND — — — 0.01 — 0.04 — C 0.04 ND ND 0.04 0.02 0.05 0.03 0.02 0.04 — D 0.10 ND ND 0.10 0.13 0.15 0.29 0.28 0.24 0.32 E 0.12 ND ND 0.12 0.10 0.19 0.10 0.18 0.15 0.30 F 0.21 ND ND 0.21 0.12 0.12 0.10 0.11 0.10 0.05 (N) 55 55 20 65 318 544 221 30 AAP-2 * * * 3/3 4/5 1/3 A 0.78 ND ND 0.78 0.81 0.88 0.91 0.79 0.80 0.62 B 0.10 ND ND 0.10 0.03 0.08 0.01 0.04 0.10 — C 0.12 ND ND 0.12 0.16 0.04 0.06 0.16 0.12 0.21 D — ND ND — — — 0.01 0.03 0.03 0.17 E — ND ND — — — — 0.01 — — (N) 24 24 19 25 235 425 161 29 AAT-1 0/3 1/5 1/3 A 0.90 1.00 0.92 0.94 1.00 0.98 0.99 0.99 0.98 1.00 B 0.06 — — 0.02 — 0.02 <0.01 0.01 0.01 — C 0.01 — 0.06 0.02 — — 0.01 <0.01 0.01 — D 0.03 — 0.02 0.02 — — — — <0.01 — (N) 36 23 25 84 18 48 303 508 219 33 AAT-2 * 0/3 0/5 1/3 A 0.99 1.00 1.00 0.99 1.00 0.97 0.98 >0.99 0.98 0.97 B — — — — — 0.02 0.02 <0.01 0.01 — C 0.01 — — 0.01 — 0.01 <0.01 <0.01 0.01 0.03 (N) 45 25 25 95 19 63 335 526 234 35 DPEP * * * * 3/3 4/5 3/3 * A 0.65 0.74 0.70 0.69 0.47 0.78 0.59 0.63 0.58 0.46 B — — — — — — 0.01 0.01 — 0.01 C 0.04 — 0.08 0.04 0.06 0.09 0.07 0.04 0.02 0.17 D 0.29 0.22 0.20 0.24 0.47 0.10 0.24 0.24 0.28 0.27 E 0.02 0.04 0.02 0.03 — 0.03 0.09 0.08 0.12 0.09 F — — — — — — 0.01 — — — (N) 50 25 25 100 17 60 303 456 233 5 EST * * 3/3 4/5 2/3 A 0.62 0.63 0.50 0.59 0.65 0.58 0.51 0.57 0.51 0.54 B 0.03 — — 0.02 — — 0.06 0.04 0.03 0.03 C 0.26 0.24 0.31 0.27 0.17 0.39 0.37 0.31 0.37 0.31 D 0.06 0.13 0.17 0.10 0.10 0.02 0.05 0.05 0.07 0.07 E 0.01 — 0.02 0.01 0.08 0.01 0.01 0.05 0.04 0.04 F 0.02 — — 0.01 — — 0.01 0.01 — 0.01 (N) 46 23 24 93 20 58 336 494 221 35 GP 0/3 0/5 1/3 A 1.00 1.00 1.00 1.00 1.00 1.00 >0.99 >0.99 >0.99 0.96 B——————<0.01 <0.01 — — C————————<0.01 0.02 D————————<0.01 0.02 (N) 45 22 25 92 8 59 322 514 216 31 GPI 0/3 0/5 0/3 A 0.44 0.36 0.34 0.40 0.48 0.35 0.59 0.61 0.57 0.70 B — 0.02 — 0.01 — — — — — — C 0.22 0.26 0.26 0.24 0.12 0.08 0.04 0.05 0.05 0.07 D 0.30 0.34 0.34 0.32 0.40 0.52 0.37 0.33 0.36 0.23 E 0.03 0.02 0.04 0.03 — 0.04 — 0.01 0.01 — F 0.01 — 0.02 <0.01 — 0.01 0.01 0.01 0.01 — G — — — — — — — — 0.01 —

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TABLE 3. continued

Atlantic Ocean Collection/y or Subpopulation Florida Gulf of Mexico Population NC NC NC NC NJ NY Allozyme

Diversity 97 98B 98C SP 98 97 PN CO SF FB (N) 55 25 25 105 20 65 247 561 241 35 HDH 1/3 2/5 1/3 A 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.99 0.99 0.98 B — — — — — — 0.01 0.01 0.01 — C ——— — —— <0.01 <0.01 <0.01 — D ——— — —— <0.01 <0.01 — 0.02 (N) 44 19 21 84 16 47 287 308 197 34 IDH 0/3 0/5 0/3 A 1.00 1.00 1.00 1.00 ND 1.00 >0.99 >0.99 0.99 1.00 B ——— —ND— <0.01 <0.01 <0.01 — C ——— —ND— <0.01 <0.01 0.01 — (N) 46 22 24 92 53 293 428 183 22 LAP * * * 2/3 2/5 3/3 A 0.31 0.08 0.24 0.24 0.05 0.36 0.44 0.47 0.40 0.19 B — — 0.02 0.01 — — 0.01 — 0.01 — C 0.36 0.26 0.22 0.30 0.75 0.41 0.28 0.19 0.20 0.08 D 0.32 0.28 0.20 0.28 0.15 0.16 0.19 0.29 0.21 0.48 E 0.01 0.12 0.24 0.09 0.05 0.05 0.08 0.04 0.11 0.25 F — 0.24 0.08 0.07 — 0.02 0.01 — 0.10 — G — 0.02 — 0.01 — — — 0.01 — — (N) 53 25 25 103 10 65 332 394 222 24 MDH-1 0/3 0/5 0/3 A 1.00 0.98 1.00 0.99 1.00 1.00 >0.99 0.99 0.99 0.99 B ——— — —— <0.01 — — — C — 0.02 — 0.01 — — <0.01 <0.01 0.01 D — — — — — — — 0.01 <0.01 — (N) 55 25 25 105 20 65 349 558 240 35 — MDH-2 0/3 0/5 0/3 0.01 A 1.00 1.00 1.00 1.00 1.00 0.99 >0.99 >0.99 0.99 1.00 B ——— — —— <0.01 <0.01 — — C — — — — — 0.01 — <0.01 0.01 — D ——— — —— <0.01 — — — (N) 51 25 25 101 20 63 340 542 231 30 MPI * * * 3/3 5/5 3/3 A 0.87 0.98 0.98 0.92 0.90 0.95 0.85 0.87 0.85 0.84 B 0.01 — — 0.01 0.05 — 0.04 0.02 0.02 0.03 C 0.09 0.02 — 0.05 0.02 0.01 0.06 0.04 0.06 0.01 D 0.01 — 0.02 0.01 0.03 0.04 0.06 0.06 0.05 0.06 E 0.02 — — 0.01 — — 0.02 0.02 0.03 0.06 F — — — — — — — — 0.01 — (N) 54 25 25 104 20 64 326 550 227 35 PGD 1/3 1/5 0/3 * A 0.99 0.94 1.00 0.98 0.95 0.98 0.97 0.95 0.96 0.92 B 0.01 0.06 — 0.02 0.05 0.02 0.01 0.03 0.03 0.03 C — — — — — — 0.01 0.01 0.01 — D — — — — — — 0.01 0.01 <0.01 0.05 (N) 44 25 25 94 20 55 246 523 224 30 PGM 3/3 3/5 1/3 A 0.20 0.34 0.30 0.25 0.10 0.04 0.47 0.43 0.48 0.42 B 0.21 0.21 0.26 0.22 0.30 0.40 0.01 — 0.01 0.01 C 0.07 0.06 0.06 0.07 0.15 0.08 0.03 0.04 0.05 — D 0.09 0.04 0.06 0.07 — 0.14 0.25 0.20 0.21 0.13 E 0.18 0.08 0.04 0.12 0.10 0.12 0.15 0.17 0.17 0.17 F 0.22 0.23 0.28 0.24 0.23 0.16 0.05 0.09 0.03 0.17 G 0.02 0.04 — 0.02 0.10 0.06 0.02 0.05 0.04 0.06 H 0.01 — — 0.01 0.02 — 0.02 0.02 0.01 0.04

continued on next page 590 BERT ET AL.

TABLE 3. continued

Atlantic Ocean Collection/y or Subpopulation Florida Gulf of Mexico Population NC NC NC NC NJ NY Allozyme

Diversity 97 98B 98C SP 98 97 PN CO SF FB (N) 55 24 25 104 20 55 341 543 231 35 OPDH * 1/3 0/5 0/3 A 0.82 0.90 0.94 0.88 0.90 0.91 0.82 0.80 0.73 0.74 B——————<0.01 <0.01 <0.01 0.04 C 0.14 0.06 0.06 0.08 0.08 0.05 0.10 0.13 0.22 0.10 D 0.02 — — 0.01 — 0.02 0.02 0.02 0.01 — E — 0.02 — 0.01 — — <0.01 0.01 0.01 — F — 0.02 — 0.01 0.02 0.01 0.05 0.02 0.02 0.06 G 0.02 — — 0.01 — 0.01 <0.01 0.01 0.01 0.06 H — — — — — — 0.01 0.01 <0.01 — I———————<0.01 — — (N) 55 25 25 105 20 65 349 550 241 35 SOD * 0/3 0/5 0/3 A 1.00 1.00 1.00 1.00 1.00 0.97 1.00 1.00 >0.99 1.00 B — — — — — 0.02 — — <0.01 — C — — — — — 0.01 — — — — (N) 55 24 25 104 20 64 348 551 241 35 Dev. H-W expectations No. 4 0 1 4 2 6 20/54 28/90 17/54 2 % 33 0 13 31 20 43 37 31 31 14 Genetic variability

dcHo 0.20 0.23 0.27 0.23 0.18 0.18 0.19 0.21 0.21 0.25 P95 56 44 50 56 56 44 50 50 50 56 P99 67 56 56 61 56 72 63 61 74 78 na 3.2 2.5 2.6 3.6 2.6 3.2 3.5 3.4 3.5 3.2

Allozyme abbreviations are defined in Table 2. Collections, subpopulations (SP), and populations are defined in Table 1 and shown in Figure 1. Fractions above allelic frequencies for Florida Gulf populations are the proportions of the population’s component subpopulations (Fig. 1) deviating from Hardy-Weinberg (H-W) genotype equilibrium expectations. Dev. H-W expectations, number of loci and percent of all polymorphic loci deviating significantly from H-W expectations; ND, no data; * , individual locus deviated significantly (P # 0.05) from H-W expectations after correction for multiple tests at that locus; dc Ho, direct count heterozygosity; P95, P99, percentage of loci polymorphic when frequency of most common allele is, respectively, #0.95 or #0.99; na, average number of alleles per locus; —, frequencies of 0.00. alleles in the 1998 collections resulting from their smaller sample many pairwise R3CG-tests were significant in pairs with PN as sizes. At the highly polymorphic GPI and LAP loci, rare alleles a pair member compared with all other pairs (Table 4A). are replete in the 1998 collections. Although Atlantic subpopulations and Florida Gulf pop- All measures of heterozygosity and polymorphism in Florida ulations shared most or all alleles at all loci, 6 loci differed sig- Gulf populations, including FB, were comparable with Atlantic nificantly in allele frequencies between bay scallops from the values. Values for FB were also usually the highest among the 2 ocean basins (AAP-1, GPI, LAP, PGM: P < 0.0001; OPDH: Florida populations, despite the absence of rare alleles at nearly P < 0.001; AAP-2: P < 0.005). Nearly twice as many NY/Florida every locus and a comparatively very small sample size (Table 3). Gulf pairs and NC/Florida Gulf pairs differed significantly in FB differed from other Florida Gulf populations in that allele allele frequencies at individual loci than did NJ/Florida Gulf pairs frequencies at highly polymorphic loci (AAP-1, AAP-2, DPEP, (Table 4B), principally because NJ was lacking in rare alleles. PGM, OPDH) were more equivalently distributed and, at loci All Atlantic subpopulation pairs differed significantly from with low levels of polymorphism (AAT-2, GP, HDH), the rare each other in overall genetic composition (Table 4C), as did all alleles were present at higher frequencies. Florida pairs except the Core (CO)–Southwest Florida (SF) com- parison, which was marginally nonsignificant. NY was the most Population Genetic Relationships differentiated among the Atlantic subpopulations. Of the Florida The only significant difference in single-locus allele frequen- populations, the pivotal core was more closely related to SF than cies among Atlantic collections or subpopulations was at LAP; to PN, and the uniqueness of FB was reconfirmed. All compar- NC97 allele frequencies differed significantly from those of both isons between Atlantic subpopulations and Florida Gulf pop- NC 1998 collections (P < 0.001 for both). Allele frequencies in ulations were highly significant. Florida Gulf populations differed significantly at 5 loci (AAP-2, Pairwise Nei’s D and QST values for Atlantic collections were LAP: P < 0.0001; DPEP, OPDH: P < 0.005, AAP-1: P < 0.01). very low for NC collection pairs (Table 5A). Indeed, although The difference among Florida Gulf populations was principally QST values are usually higher than Nei’s D values, all pairwise the result of uniqueness of Panhandle (PN). More than twice as QST values for NC collection pairs were 0. In our grouping POPULATION GENETICS OF EASTERN UNITED STATES BAY SCALLOPS 591

TABLE 4. Significance values for pairwise tests for genetic homogeneity of bay scallop allozyme allelic frequencies at polymorphic loci. (A) Paired Florida Gulf of Mexico populations.

Locus Pop. 1 Pop. 2 AAP-1 AAP-2 DPEP LAP OPDH PN CO P < 0.01 P < 0.0001 NS P < 0.0001 P ; 0.05 PN SF NS P < 0.0001 P < 0.005 P < 0.001 P < 0.001 PN FB P < 0.01 P < 0.0001 NS P < 0.0001 NS CO SF NS NS P ; 0.05 P < 0.0001 P < 0.05 CO FB NS NS P < 0.025 P < 0.0001 NS SF FB P < 0.05 NS P < 0.005 P < 0.01 NS

(B) Atlantic subpopulations paired with Florida populations. Locus Atlantic Florida Subpop. Gulf Pop. AAP-1 AAP-2 GPI LAP PGM OPDH NC PN P < 0.005 NS P < 0.0001 P < 0.005 P < 0.0001 NS NC CO P < 0.005 NS P < 0.0001 P < 0.0001 P < 0.0001 NS NC SF P < 0.0001 NS P < 0.0001 P < 0.025 P < 0.0001 P < 0.01 NC FB P < 0.001 NS P ~ 0.005 P < 0.05 NS NS NJ PN NS NS NS P < 0.05 P < 0.005 NS NJ CO NS NS NS P < 0.005 P < 0.01 NS NJ SF P < 0.001 NS NS P < 0.01 P < 0.001 NS NJ FB P < 0.05 NS NS P < 0.005 P < 0.05 NS NY PN P < 0.05 NS P < 0.005 NS P < 0.0001 P ; 0.05 NY CO NS NS P < 0.001 P < 0.001 P < 0.001 P < 0.05 NY SF P << 0.0001 NS P ~ 0.005 P < 0.001 P < 0.0001 P < 0.001 NY FB P < 0.05 P < 0.05 P ~ 0.005 P < 0.0001 P < 0.0001 P ¼ 0.05

(C) All pairwise combinations of Atlantic subpopulations and Florida Gulf populations, all loci. Atlantic Subpop. Florida Pop. Group NC NJ NY Panhandle Core Southern Atlantic subpop. NC — NJ 0.037 — NY *** *** — Florida pop. PN *** *** *** — CO *** *** *** 0.002 — SF *** *** *** *** 0.057 — FB *** *** *** *** *** ***

*** Cell significance of P # 0.001 and tablewide significance of P # 0.05, after correction for multiple tests of the null hypothesis. Subpopulations (Subpop.) and populations (Pop.) defined in Table 1, shown in Figure 1; loci defined in Table 2. Bold print: (A) and (B), significant after sequential Bonferroni adjustment for multiple tests of the null hypothesis of no cell significance; (C) cell significance only. NS, not significant.

analyses of pairwise Nei’s D and QST, the mean genetic distance distant from the Florida populations was NJ; NJ/Florida Gulf between NC collections was significantly smaller than the means genetic distances were generally 20–33% greater than were NY/ of those collections paired with NJ97 or NY98 (Table 6A). Florida Gulf genetic distances (Table 5B), and the means of some Genetic distances between collection pairs from different NJ/Florida Gulf pairs were significantly higher than the means of Atlantic locations varied among the collections (Table 5A). The some NY/Florida Gulf pairs (Table 6B; D1, QST1). QST values for NJ98/NCC98 and NJ98/NY97 were notably higher The means with Atlantic subpopulations and FB as pair than the values for all other collection pairs and were as high as members were among the most differentiated of all pairwise the values between NC and FB (Table 5B). combinations (Tables 5B and 6B; D1, QST1), in part because FB Other than pairs with FB, pairwise genetic distances for is enriched in rare alleles specific to Florida subpopulations Atlantic bay scallop subpopulations were, significantly, 2–9 (i.e., AAP-2*D, DPEP*B, GP*C,D, HDH*D, OPDH*B; Table times higher than the means for the very low and statistically 3). Interestingly, the genetic distances of FB paired with other homogeneous Florida Gulf bay scallop populations (Tables 5B Florida Gulf populations was equivalent to the genetic dis- and 6B; D1, QST1). The Atlantic subpopulation most genetically tances of NC paired with those populations (Table 5B), and the 592 BERT ET AL.

TABLE 5. Matrices of bay scallop allozyme–allele pairwise genetic distances (3103). (A) Atlantic collections.

Collection NC97 NCB98 NCC98 NJ98 NY97 NC97 — 0 0 22 24

NCB98 5 — 0 24 26 NCC98 1 3 — 42 18 NJ98 10 17 16 — 48 NY97 9 17 6 18 —

QST, above bars; Nei’s D, below bars. (B) Atlantic subpopulations (Subpop.) and Florida populations (Pop.). Atlantic subpop. Florida pop. Group NC NJ NY Panhandle Core Southern FB Atlantic subpop. NC—292440± 436± 938± 848 NJ 10 — 48 81 ± 886± 13 79 ± 17 91 NY 9 18 — 60 ± 364± 969± 12 101 Florida pop. PN 15 ± 332± 222± 1— 9± 49± 544± 1 CO 14 ± 436± 624± 44± 2—8± 526± 7 SF 16 ± 335± 727± 44± 22± 1—30± 4 FB 22 42 42 17 ± 211± 214± 2—

QST, above bars; Nei’s D, below bars. Groups shown in Figure 1. See text for method of calculating values presented as mean ± SD. mean genetic distances of FB or NC paired with other Florida Gulf izing those haplotypes are in Table 7, and their frequencies are populations were approximately 25–50% smaller than the mean in Table 8, as are h and p values. Except for NY, which had distances between other Atlantic/Florida Gulf pairs (Table 6B; D1, codominant haplotypes (H2, H3), the common haplotype in QST1). However, FB and NC were not closely related; their genetic Atlantic collections was H2. Haplotype 11 clearly predomi- distances from each other were equivalent to those of either one nated in Florida Gulf populations and, except for one other, paired with other Florida Gulf populations (Table 5B). was the only haplotype in the FB sample. Haplotype frequen- Grouping the genetically similar PN, CO, and SF populations cies did not differ significantly among groups within either into a single peninsular Florida (FLA) population provided ocean basin. additional insight into intra- and interbasin relationships (Table Although they were elevated, the h value for NY97 and the p 6B; D2, QST2). The very low mean genetic distances between the value for NJ98 did not differ significantly from the corresponding population pairs forming FLA emphasizes their genetic similar- values for other Atlantic collections, but both h and p for FB were ity compared with the far more distant Atlantic subpopulations. significantly lower (P < 0.05 and 0.01, respectively) than the mean The high mean genetic distances between NJ and FLA are the values for the collections forming other Florida Gulf populations. result of the unique absence of rare Florida Gulf alleles in NJ. Discounting FB, the mean p value for Atlantic subpopulations In both allozyme cluster phenograms (Fig. 2, A, B), A. i. was significantly lower than the mean for Florida Gulf populations irradians and A. i. concentricus were clearly separated, NC grouped (P < 0.01) and, compared with the average molluscan p value (0.85 with A. i. irradians and FB with A. i. concentricus, and some or all (Bazin et al. 2006)), Atlantic subpopulation values were much CO subpopulations grouped together. The Florida Gulf popula- lower and Florida Gulf population values much higher. Pairwise p tions advocated by Bert et al. (in prep) were fully maintained in the values for all Atlantic pairs were equivalent, as were those for all UPGMA phenogram; the N-J phenogram may more reflect Florida Gulf pairs except those with FB as a member (Table 9). source-sink genetic relationships in that metapopulation. FB has The near fixation of the common Florida Gulf haplotype in FB unique positions in both phenograms—the most basal of the decreased all within-Gulf, and increased all Atlantic–Gulf, pair- Florida subpopulations in the UPGMA phenogram and the most wise p values that included that sample. The mean p values for highly derived in the N-J phenogram. Those placements further populations paired with FB were significantly lower than the emphasize the genetic distance of FB from the rest of the Florida means of pairs with other Gulf collections (P ¼ 0.0003). Gulf. The FST values for Atlantic subpopulation pairs were significantly higher than those for Florida Gulf population mtDNA RFLP Analysis pairs (P < 0.0001); and the NY/NC pair differed significantly at the tablewide level (Table 9). Within the Florida Gulf, the Genetic Diversity genetic distance between FB and other populations was 2–3 We found 53 haplotypes (H) in the 534 bay scallops that we times greater than the distance between the other populations. examined. The estimated restriction fragment sizes character- Genetic distances between all pairs of Atlantic subpopulations TABLE 6. Significant statistical groupings of average pairwise genetic distances (X 103) between bay scallop subpopulations and populations.

Category Data set: pairs used, n values Statistic Means and statistically similar groupings Probability level A. Between Atlantic subpopulations Allozyme loci; all pairs: n ¼ 3 Nei’s D NC/NC NC/NY NC/NJ P < 0.05 31114 (NJ/NY ¼ 18) P OPULATION QST NC/NC NC/NY NC/NJ P < 0.001 02329 (NJ/NY ¼ 48) B. Between Atlantic and Florida Gulf bay scallops G Allozyme loci; Atlantic Nei’s D1* CO/CO PN/PN PN/CO CO/SF PN /SF SF/SF P # 0.0001 subpopulations and Florida 12 4 4 4 6 OF ENETICS populations, all pairwise combinations: CO/CO, n ¼ FB/CO NC-NY/NC-NY FB/SF NC/CO NC/PN NC/SF FB/PN 10; PN/CO, CO /FS, n ¼ 15; 11 12 14 14 15 16 17 NY/PN NY/CO NY/SF NJ/PN NJ/SF NC-NY/FB NJ/CO

PN /FS, n ¼ 9; CO/FB, NC/CO, E

NJ/CO, NY/CO, n ¼ 5; all 22 24 27 32 35 35 36 ASTERN others, n ¼ 3

QST1 * CO/CO PN/PN SF/CO PN/CO PN/SF SF/SF P # 0.0001 U

2 4 8 9 10 11 NITED

FB/CO FB/SF NC-NY/NC-NY NC/CO NC/SF NC/PN FB/PN S

26 30 34 35 38 40 44 TATES NY/PN NY/CO NY/SF NJ/PN NJ/SF NC-NY/ FB NJ/CO 59 65 69 79 79 81 86 B AY S

Nei’s D2 FLA/FLA FB/FLA NC-NY/NC-NY NC/FLA NY/FLA NJ/FLA NC-NY/FB P < 0.0001 CALLOPS 3121315243535

QST2 FLA/FLA FB/FLA NC/ FLA NC-NY/ NC-NY NY/FLA NC-NY/FB NJ/FLA P < 0.0001 7323845648182

mtDNA; Atlantic subpopulations FST FLA/FLA FLA/FB NC-NY/NC-NY FLA/NC-NY FB/NC-NY P < 0.0001 and Florida populations; FLA/ 12 8 5363 FLA, n ¼ 28; FLA /FB, n ¼ 8, FLA /NC-NY, n ¼ 24; NC-NY/ NC-NY, FB/NC-NY, n ¼ 3

3 Nei’s D, QST , and FST (all defined in Methods) mean values310 . Atlantic subpopulation means are averages of appropriate pairwise distance values in Table 5A. Florida Gulf means were obtained 593 from Bert et al. (in prep.). Except for Nei’s D* and QST * , where only the single lowest and 3 highest groupings are underlined, all statistically homogeneous groupings are underlined. Subpopulations and populations are defined in Table 1 and are shown in Figure 1. 594 BERT ET AL.

Figure 2. Cluster phenograms depicting genetic distances among bay scallop subpopulations (abbreviations defined in Table 1, shown in Figure 1). (A) UPGMA phenogram based on allozyme loci. x-axis: Nei’s D values (3103); numbers at nodes: SEs for Nei’s D values. (B) N-J phenogram based on allozyme loci. Numbers at nodes: bootstrap confidence values greater than 50%. (C) N-J phenogram based on mtDNA RFLPs. Scale: FST $ 0.1. POPULATION GENETICS OF EASTERN UNITED STATES BAY SCALLOPS 595

TABLE 7. the magnitude of the difference between Atlantic and Florida Bay scallop mitochondrial DNA nucleotide base-pair Gulf mtDNA RFLPs, as well as the greater genetic distance fragment sizes for the 12s/ND1 fragment. between Atlantic subpopulations compared with the distance between Florida Gulf populations. For the AMOVA analysis involving all pairwise F values, Enzyme Identifier Pattern ST we partitioned the data into Atlantic and Gulf groups. That ScrF I A 488 145 200 grouping explained 58% (P < 0.0001) of the variation in B 488 345 haplotype diversity. Variation among individuals within pop- C 488 145 100 100 ulations explained nearly all remaining differentiation (41%; P < D 310 178 200 145 0.0001). Variation among populations within groups was signif- E 406 200 145 82 icant (P ¼ 0.03), but explained only 1% of the population genetic Tsp509 I A 413 264 156 B 264 241 172 156 structure. The data were not partitioned for the within-ocean C 314 264 156 99 basin AMOVA analyses. Among Atlantic subpopulations, a D 413 200 156 64 small but significant amount (5%; P ¼ 0.01) of the genetic E 833 structuring was explained by variation among the subpopula- Alu I A 682 72 51 28 tions. Among Florida Gulf populations, virtually all variation B 625 72 57 51 28 was explained by differences among individuals within popula- C 392 290 72 51 28 tions; the populations were not genetically structured (P ¼ 0.45). D 682 123 28 The TCS program revealed that level 1 clades (C) that in- E 710 72 51 cluded up to 2 mutations between haplotypes could be gener- F 504 178 72 51 28 ated. Only 1 haplotype (H27) exceeded this limit. The MSN was Rsa I A 437 228 112 56 B 665 112 56 composed of 3 level 3 clades (C3-1, C3-2, C3-3; Fig. 3A), each C 437 228 168 containing 2 level 2 clades. Within C3-1, C2-1 included princi- D 437 176 112 56 52 pally Atlantic haplotypes, whereas C2-2 contained only Florida E 273 228 164 112 56 Gulf haplotypes except for H15, which also occurred in North HinF I A 544 281 8 Carolina (2 individuals). Both level 2 clades in C3-2 were a B 544 194 87 8 mixture of Gulf and Atlantic haplotypes from widely scattered C 833 locations. Within C3-3, C2-5 had, by far, the most Gulf D 474 281 70 8 haplotypes; nearly all were 1 or 2 steps removed from the very E 454 194 90 87 8 common H11, which was also found in 3 NC individuals. C2-6, BsiHKA I A 621 212 a small clade with only NC haplotypes, was derived from H13, B 833 C 621 192 20 a member of C2-5. Thus, C3-3 was essentially a Florida Gulf Bgl II A 418 415 clade with a few haplotypes found also or exclusively in North B 833 Carolina. C 415 298 120 Clades at all levels were significant in the NCPA (Fig. 3A, Ban II A 481 323 29 Table 10). Independently significant level 1 clades were exclu- B 833 sive to C3-1. The inferred demographic event for both C2-1 and C 510 323 its constituent C1-2 was contiguous range expansion. Within Taq I A 598 235 C2-1, the significance of H2, the most common Atlantic haplo- B 833 type within C1-2, and of C1-1, which contained principally C 598 225 10 Atlantic haplotypes (Fig. 3A, Table 8), indicated that C2-1 D 375 235 223 originated in the Atlantic. C2-4, the only significant component Underlined estimates were inferred from discrepancies between the of C3-2, and for which contiguous range expansion was also uncut PCR product and the sum of the visualized fragments. inferred, was composed of haplotypes from widely ranging locations (Fig. 3A, Table 8). Because no haplotypes in that clade paired with Florida Gulf populations were comparatively quite were shared between the Gulf and Atlantic, it seems more likely large. Nucleotide divergences between Atlantic subpopulations that some other process, perhaps dispersal within regions and were small (NC/NJ ¼ 0.0001, NC/NY ¼ 0.0020, NJ/NY ¼ fragmentation between regions, shaped C2-4. C2-5 was signifi- 0.0007), but both pairs with NY were highly significant, table- cant, due principally to the significance of nested clades 1–10 and wide (P < 0.001; data not shown). All pairwise d values for 1–14 (Table 10). The phylogeographic processes inferred for C2-5 Florida Gulf populations were 0, except those with FB (range, were restricted gene flow and dispersal, some of which was long 0.0009–0.0016). All d values for Atlantic/Florida Gulf pairs distance. Long-distance dispersal to NC is evidenced by C1-14, were highly significant (range, 0.0092–0.0215). Pairs with FB as but limited gene flow is not consistent with the previous analyses a member were highest because FB lacked Atlantic haplotypes. of Florida Gulf mtDNA population genetic structure, which supported panmixia for all populations, perhaps other than Population Genetic Relationships FB.Alternatively,theC2-5‘‘starburst’’ haplotype array may Isolation by distance explained a significant component of have been generated by another phylogeographic process: the pairwise genetic distances between Atlantic subpopulations demographic expansion (Slatkin & Hudson 1991, Fu 1997). and Gulf populations (y ¼ 0.0008x – 0.11; r ¼ 0.84, P < 0.001), Demographic expansion may also be the process operating in and both the grouping analysis of mean pairwise genetic dis- C2-2, a clade deemed to be inconclusive in the NCPA and with tances (Table 6, FST) and the NJ dendrogram (Fig. 2C) illustrate a less diverse but similar structure to C2-5. 596 BERT ET AL.

TABLE 8. RFLP haplotype frequencies and diversity measures for the bay scallop mtDNA 12s/ND1 segment.

Atlantic Ocean Collection/y or Subpopulation Florida Gulf of Mexico Population

NC1 NC2 NC NJ NY Hap.No. Designation 98 98 SP 98 97 PN CO SF FB n 22 27 49 20 15 94 198 113 21 (14–25) (14–31) (12–25) h 0.49 0.58 0.54 0.56 0.76 0.52 0.50 0.54 0.09 (0.36–0.58) (0.36–0.58) (0.36–0.63) p 0.58 0.68 0.62 0.92 0.54 1.21 1.17 1.23 0.28 (0.90–1.39) (0.85–1.34) (0.90–1.42) 1 AAAEAAAAA — — — 1 1 1 1 — — 2 BABABAAAA 17 16 33 13 4 2 5 4 — 3 BAAABAAAA 1 1 2 — 6 — 1 1 — 4 BADABAAAA — — — — 1 — — — — 5 BAAACAAAA — — — — 2 — — — — 6 BABABBAAA — — — — 1 — — 1 — 7 AAAECAAAA — — — 3 — — — — — 8 AAAEBAAAA — — — 1 — — — — — 9 BABACAAAA — 2 2 1 — — — — — 10 BAAEAAAAA — — — 1 — — — — — 11 AAAAAAAAA 1 2 3 — — 67 139 76 20 12 BABABAABA 1 — 1 — — — — — — 13 AAAAAAABA 1 1 2 — — — 5 1 — 14 AAAEAAABA — 1 1 — — — — — — 15 BABAAAAAA 1 1 2 — — 3 3 4 — 16 BABEAAAAA — 1 1 — — — — — — 17 AABABAABA — 2 2 — — — — — — 18 AAFAAAAAA — — — — — 1 2 1 1 19 AAAAABAAA — — — — — 4 4 6 — 20 AAAAAAAAC — — — — — — 1 2 — 21 AAAABAAAA — — — — — 2 1 5 — 22 AADAAAAAB — — — — — — 1 1 — 23 AADADAAAA — — — — — — — 1 — 24 AAABAAAAC — — — — — — — 1 — 25 AAAAAAACA — — — — — — — 1 — 26 BABBAAAAD — — — — — — — 1 — 27 ABDAEAAAA — — — — — — — 1 — 28 AADAAAAAA — — — — — 2 1 1 — 29 AAAACAAAA — — — — — 1 1 1 — 30 AAAAAABAA — — — — — — — 1 — 31 AAABAAAAA — — — — — 1 3 1 — 32 AAAADAAAA — — — — — — — 1 — 33 AACAAAAAA — — — — — 2 3 1 — 34 BAFABAAAA — — — — — — 1 — — 35 BAAAAAAAA — — — — — — 1 — — 36 AABAAAAAA — — — — — 1 1 — — 37 AAAAAAAAB — — — — — 3 8 — — 38 AAEAAAAAA — — — — — — 2 — — 39 AAAAABAAC — — — — — 1 1 — — 40 AACAABAAA — — — — — — 1 — — 41 ABAAAAAAA — — — — — 1 1 — — 42 BABAAACAA — — — — — — 1 — — 43 AEAAAAAAB — — — — — — 1 — — 44 AAFBAAAAA — — — — — — 1 — — 45 BABAACAAA — — — — — — 1 — — 46 AADAABAAA — — — — — — 1 — — 47 DADAAAAAA — — — — — — 1 — — 48 ACAAAAAAA — — — — — 1 2 — — 49 ADAAAAAAA — — — — — — 1 — — 50 BBBAAAAAA — — — — — — 1 — — 51 BABAABAAA — — — — — — 1 — — 52 BABABAAAC — — — — — — 1 — — 53 CAAAAAAAA — — — — — 1 — — —

Numbers in parentheses are the ranges of values for collections composing Florida populations (Bert et al. in prep.). Composite haplotypes correspond to restriction endonuclease fragment patterns in Table 7. Collection, subpopulation (SP), and population abbreviations are defined in Table 1; locations are shown in Figure 1. Hap. no., haplotype number; n, number of individuals; h, haplotype diversity (SD range, 0.05–0.1); p, nucleotide diversity (3102); h, p in bold print are values significantly higher or lower than others. POPULATION GENETICS OF EASTERN UNITED STATES BAY SCALLOPS 597

TABLE 9. population demographic and spatial contractions and expan- Bay scallop pairwise nucleotide diversities (p) and genetic sions and/or selective sweeps through a longer time period in the 3 past (Rogers et al. 1996), whereas Florida Gulf bay scallops distances (FST) (both 310 ) based on RFLP analyses of mtDNA 12S/ND1 segment. have the steep, unimodal mismatch distribution indicative of a dramatic population expansion in the recent past (Rogers & Harpending 1992). Moreover, the y-axis scales for the Atlantic Atlantic Subpopulation Florida Population and C3-2 demographic and spatial expansion profiles are Group NC NJ NY PN CO SF FB much lower compared with the Florida Gulf and C3-3 profiles, Atlantic subpopulation showing that expansions in the Atlantic have been less NC — 8 8 22 22 22 26 dramatic than the Florida Gulf expansion (Schneider & NJ 6 — 8 23 23 22 27 Excoffier 1999). Fourth, the high modal number of nucleotide NY 13 5 — 20 20 19 24 differences of clade C3-2 shows that it is ancient (Fauvelot et al. Florida population 2003) and has undergone a previous population expansion and PN 576 507 553 —12121decline (Fig. 4B); that clade now contains haplotypes that CO 579 518 547 3 — 12 1 persisted through that demographic event. Florida Gulf C3-3 SF 571 457 496 43—1 has the sharply declining mismatch profile (Fig. 4B) and FB 635 578 666 20 11 23 — numerous, closely related haplotypes associated with a recent p, above bars; FST, below bars. expansion (Slatkin & Hudson 1991, Rogers & Harpending Abbreviations are defined in Table 1; locations are shown in Figure 1. 1992). Fifth, positive Tajima’s D values (Table 11) indicate that Bold print represents FST values significant after correction for multiple C3-2 and its component C2-4 may have undergone an overall tests. reduction in population size ((Harpending et al. 1993, Schneider & Excoffier 1999), whereas all Florida Gulf clades have highly Overall, the NCPA supports the idea of recent, contiguous negative (all P < 0.001) Tajima’s D values, indicative of sub- range expansion in Atlantic bay scallops and both limited stantial demographic expansion following a bottleneck or se- dispersal (with population expansion) in the Gulf, and occa- lective sweep (Rand 1996). Sixth, and last—and unique to the sional long-distance dispersal from Florida Gulf populations Atlantic—the declining hierarchical M values of Atlantic into NC. The significant higher level clades 3-1 and 3-3 illustrate groups (infinite for level 2 clades, intermediate for level 3 clades, that long-distance dispersal and colonization were the past small for Atlantic; Table 11) indicate that short-term gene flow processes that shaped present-day bay scallop phylogeographic is much higher than long-term gene flow as geographic expanse structure. At the highest clade level (4-1) and most distant past increases. (Templeton 1998), allopatric fragmentation between the Gulf Despite their many differences, Atlantic and Gulf bay scallops and Atlantic clades generated the genetically differentiated have one feature in common. All values for both q0 (demographic subspecies existing today. expansion) and q (spatial expansion), and for their confidence Multiple lines of evidence from MSNs generated separately intervals, were very small. The reductions in effective population for Atlantic and Florida Gulf haplotypes (Fig. 3B, C) and from size experienced by both groups have been severe, near-extinction the studywide, regional, and level 3 clade mismatch frequency events; aggregations have recolonized or regenerated from very profiles (Fig. 4A, B) and accompanying and accessory mis- few founders (Grant 2005). match statistics (Table 11) indicate that Atlantic subpopula- Three features of the NCPA analyses and mismatch statistics tions are older and demographically more static than Florida generated values that were difficult to reconcile with the pre- Gulf populations, which are younger and collectively have dominant Atlantic and Florida Gulf patterns. First, the exis- experienced recent large population expansions, as follows. tence of the older, principally Atlantic, C2-1 together with the First, the Atlantic haplotype MSN (Fig. 3B) bears the intricacy starburst Florida Gulf C2-2 in C3-1 generated infinite Q1 and M and geographic expanse of an ancient lineage in which haplo- values, and the appearance of recent demographic and spatial types have gone extinct, and multiple stepwise mutations within expansions in the C3-1 mismatch distribution (Fig. 4B). Separate distinct lineages have generated sublineages with haplotypes mismatch frequency distributions for those clades (not shown) related to each other by sequential mutations (Bargelloni et al. were strikingly different. Second, the much greater number of 2005, Grant 2005, Teske et al. 2005). In sharp contrast, the Florida Gulf haplotypes compared with the number of Atlantic highly abundant H11 and numerous closely related, rare hap- haplotypes biased some of the Gulf and Atlantic group mismatch lotypes in the Florida Gulf lineage (Fig. 3C) are characteristic statistics such that they resembled those of the Florida Gulf of a recent lineage marked by a rapid demographic expansion alone. Third, because Florida Gulf subpopulations individually (Bowen & Grant 1997, Grant & Bowen 1998, Avise 2000; Teske expand, contract, and go extinct (Bert et al. in prep), the et al. 2005). Second, the mean numbers of haplotype differ- demographic expansion model C2-2, C2-5, and C3-3 Q and ences and the T values of Atlantic groups are generally greater HR (for C2-5 and C3-3) statistics did not show the demo- than those of Florida Gulf groups (Table 11), and the Atlantic graphic expansion demonstrated by the Florida Gulf and C3-3 values are generally hierarchical (clade level 2 < clade level 3 < mismatch distributions (Fig. 4). Atlantic). Haplotype differences accumulate through time; The separate MSNs for Atlantic and Florida Gulf haplotypes, higher numbers of differences characterize older populations, considered together with the results of the NCPA and mismatch and T values are related to the mean numbers of haplotype analyses, the knowledge that older haplotypes tend to have differences between groups (Bargelloni et al. 2005). Third, the broader distributions and to be present in higher frequencies, multimodal mismatch distributions (Fig. 4A) for the Atlantic and the knowledge that older lineages tend to be complex groups imply that Atlantic bay scallops have undergone several (Castelloe & Templeton 1994, Excoffier and Smouse 1994, 598 BERT ET AL.

Figure 3. Minimum spanning networks of Atlantic (light shading) and Florida Gulf (dark shading) bay scallop mtDNA haplotypes listed in Table 8. Circles are generally sized to represent haplotype frequencies, and are partitioned according to proportions of Atlantic and Gulf bay scallops within each haplotype. Squares represent missing haplotypes. (A) All level 2 and higher clades and significant or noteworthy level 1 clades as determined by NCPA (Table 10). Inferred demographic events of significant clades are presented within the clade boundaries. NC, North Carolina. (B) Atlantic bay scallop haplotypes. (C) Florida Gulf bay scallop haplotypes. (D) Hypothesized ancestral Atlantic/Florida Gulf lineage (highlighted). For certain haplotypes, numbers of individuals and Atlantic subpopulations where they occurred are given below haplotypes numbers.

Pruett et al. 2005), prompted us to formulate an alternative basins. These differences reflect the diverse array of demographic interpretation for the bay scallop MSN. We considered the and ecological events that have affected these subpopulations lineage that includes all shared Gulf/Atlantic haplotypes, all and populations, as well as the numerous, substantial anthropo- intermediate haplotypes linking those haplotypes in Figures genic activities that, over time, impinged principally on Atlantic 3A–C, and all branches in which Atlantic and Florida Gulf subpopulations prior to the time of our sampling. The individ- haplotypes are interspersed as representing the ancestral lineage uality of these influences on each Atlantic subpopulation war- (Fig. 3D). This interpretation clearly illustrates the proliferation rants close examination. We present our deductions about NY of Florida Gulf haplotypes belonging to or derived from core and NJ in this section, and about NC in Taxonomic Puzzles. haplotypes with widespread distributions. It also suggests that NC haplotypes H12, H14, and H17 were derived from the ancestral New York Florida Gulf H11 lineage. NY bay scallops comprise a relatively sequestered subpop- DISCUSSION ulation located in an area with a low probability of gene flow from more southerly subpopulations. Thus, not surprisingly, Population Genetic Structure our NY sample emerged as the most genetically distant among Atlantic subpopulations in the pairwise tests of overall allozyme Atlantic and Florida Gulf bay scallops have very different allele frequencies and of mtDNA nucleotide divergences. NY population genetic structures, both within and between ocean also had low allozyme heterozygosity and P95 values coupled POPULATION GENETICS OF EASTERN UNITED STATES BAY SCALLOPS 599

TABLE 10. Significant bay scallop clades as determined by nested clade phylogeographic analysis performed on mtDNA restriction fragment length patterns.

Chain of Inference Sig. Clade Significant Components P Value [inference direction] Inferred Demographic Event 1–2 1–2 – 11b – 12 – No

Hap DC [#] 0.03; DN [#] 0.04 Contiguous range expansion I-T DN [#] 0.03 1–3 1–2 – 11–17 – No

Hap 15 DN [$] 0.02 Inconclusive 2–1 1–2 – 11ac – 12 – No 2–1 0.01 Contiguous range expansion

1–1 (Haps. 3, 4, 34) DC [$] 0.04, DN [$] 0.06 I-T DC [#] 0.05 2–4 1–2 – 11a – 12 – No

1–7 (Haps. 7, 29) DC [$] 0.02; DN [$] 0.02 Contiguous range expansion 2–5 1–2a – 3c – 5–6 – 7 - Yes

1–10 (Haps. 11, 30, 31, 32, 36, 41, DC [#] 0.04, DN [#] 0.04 Restricted gene flow and dispersal 48, 53, 21, 25, 37, 38) with some long-distance dispersal

1–14 (Haps. 13, 14) DC [$] <0.00; DN [$] <0.00 3–1 1–2a – 3a – 5–6 – 13–14 - Yes 3–1 <0.00 Long-distance colonization

2–1 DC [#] <0.00; DN [#] <0.00 2–2 DC [#] <0.00; DN [#] <0.00 I-T DC [#] 0.01 I-T DN [$] <0.00 3–3 1–2a, B – 3abc – 5–6 – 13–14 – Yes 3–3 <0.00 Long-distance colonization

2–5 DC [#] <0.00; DN [#] <0.00 2–6 (Haps. 12, 17) DC [#] <0.00; DN [$] <0.00 I-T DC [$] 0.05 I-T DN [#] <0.00 4–1 1–2b – 3b – 5–15–16 – Yes 4–1 <0.00 Allopatric fragmentation

3–1 DC [$] <0.00; DN [$] <0.00 3–2 DC [$] <0.01; DN [$] <0.00 3–3 DC [#] <0.00; DN [#] <0.00 I-T DC [$] 0.05 I-T DN [$] <0.00

Haplotypes [Hap(s.)] and their distributions defined in Table 8, significant (Sig.) clades are shown in Figure 3A. with high frequencies of common alleles at nearly all loci, high Multiple factors could have contributed to the high inci- P99 values resulting from a high incidence of rare-allele homo- dence of rare homozygotes, limited genetic diversity, predom- zygotes, a high mean number of alleles over all loci, a high fre- inance of NY haplotypes in a single small clade (C2–1), and quency of loci deviating from H-W equilibrium, the lowest mean codominance of mtDNA haplotypes H2 and H3 seen in the pairwise difference between haplotypes, and high mtDNA NY bay scallops. First, all NY bay scallop aggregations inhabit haplotype diversity resulting from codominant haplotypes, territory formerly covered by the Laurentide glacier up to about one of which is otherwise rare in Atlantic subpopulations. 20,000 y ago (Balco & Schaefer 2006). Remnants reflecting the Together, these features indicate that the Peconic Bay aggrega- genetic diversity of the colonizing (presumably) larvae, which tion from which we obtained our NY98 collection experienced likely constituted a relatively small subset of the total Atlantic a recent genetic bottleneck or colonization from a few founders larval pool, could still remain. Post-Pleistocene founding pop- (Grant 2005), and an infusion of new genotypes from genetically ulations often have limited or altered genetic diversity compared differentiated individuals within the recent past. Two NY in- with comparatively older, established populations (e.g., Dillon & dividuals were homozygous for rare Florida Gulf alleles, and NY Manzi 1992). Second, many NY aggregations, including that in- shared some rare alleles with NC, but shared none with NJ. A habiting Peconic Bay, have been particularly besieged by some- commonality of rare alleles indicates gene flow (Slatkin 1985, times multiyear blooms of the ‘‘brown tide’’ alga Aureococcus Saavedra et al. 1993), suggesting that our hypothesized infusion anophagefferens and, to a lesser degree, by outbreaks of shell- of bay scallops into the Peconic Bay aggregation came directly or boring parasites, and starfish and crab predators, all of which indirectly from NC or the Florida Gulf, and that gene flow can cause repeated reductions or local extirpations (Tettelbach between NY and NJ bay scallops is very rare, at least within the & Wenczel 1993). Repeated demographic flushes and crashes time frame of our study. can change population genetic diversity significantly (e.g., Haag 600 BERT ET AL.

Figure 4. (A, B) Observed (solid lines) and expected (dashed lines) frequencies (F) of nucleotide differences (mismatches; i) between regional (A) and level 3 clade (B; Fig. 3A) bay scallop mtDNA haplotype pairs. Because mutations accumulate over time, increasing values of i signify increasing distances into the past. Associated mismatch statistics are reported in Table 11. *Significant deviation from the sudden expansion model. et al. 2005). Third, because the time of recovery to high density bay scallops using allozyme loci indicates that the hatchery levels can be protracted in bay scallops (Peterson & Summerson scallops were significantly genetically differentiated from the 1992, Tettelbach 2009), the Peconic Bay aggregation has been in- native aggregation. A bay scallop sample (CR) obtained from termittently supplemented since 1985—until 2004 (S. Tettelbach, a hatchery and used for mtDNA RFLP analysis by Blake and Long Island University; pers. comm.)—with bay scallops prin- Graves (1995) also had codominant haplotypes, suggesting cipally from an aquaculture-based aggregation in Maine and that the codominance of H2 and H3 we observed resulted from from the wild aggregation in Massachusetts (Tettelbach & supplementation with hatchery bay scallops. Wenczel 1993). At least occasionally, these enhancements were New Jersey successful; seeded scallops contributed an estimated one quarter of the Peconic Bay scallop set in 1989 (Krause 1992). Supple- At multiple loci, our NJ sample was depleted in rare allozyme menting with bay scallops from either of these sources could have alleles otherwise present in our NC and/or NY samples; and NJ affected the genetic diversity of NY bay scallops. Unless gener- was underrepresented in Florida Gulf alleles compared with NC ally large numbers of broodstock are used and care is taken to and NY. NJ had a high incidence of homozygous genotypes for maintain the genetic diversity of the recipient population, sup- common Atlantic alleles (data not shown), and uniquely different plementation with aquacultured broods can significantly alter the allele frequencies at some loci. The comparatively low sample size genetic diversity of the admixed (hatchery component + wild of NJ could have contributed to the low number of alleles, but component) population, particularly if aquaculture-based sup- other measures of heterozygosity and polymorphism were not plementation is successful and repeated (Bert et al. 2007). Al- well correlated with overall Atlantic collection sample sizes, and though the Massachusetts aggregation has been stable for decades NJ’s high mtDNA nucleotide diversity suggests that the sample (S. Tettelbach, pers. comm.), it is at the northern limit of the bay sizes adequately represented that subpopulation. Together, NJ’s scallop range (Waller 1969), and it, too, is post-Pleistocene in age– genetic attributes signal a genetic bottleneck in the NJ aggrega- both reasons that may contribute to limited or altered genetic tion in the recent past, and no subsequent input of Florida Gulf diversity compared with aggregations farther south (e.g., Hellberg genes. et al. 2001). NJ bay scallops were harvested intensely from the 1950s to At least some of the unusual features of our NY sample likely late 1960s (Campanella et al. 2007), which depleted the sub- are not sampling artifacts or temporary conditions. The fact that population. Since then, sufficient bay scallops to support a Krause (1992) could distinguish hatchery bay scallops from wild fishery have been present only infrequently (Bologna et al. 2001, TABLE 11. Mismatch and related statistics relative to the expansion model for bay scallop mtDNA haplotype groupings shown in Figure 3A.

Group or Clade (geographic region) Model or Indicator 3–1* Statistic Gulf 2–2 (Gulf) 2–5* (Gulf) 3–3* (Gulf) Atl 2–1* (Atl) 2–3 (Atl) 2–4* (Atl) 3–2 (Atl) Gulf & Atl (Gulf & Atl) P Mean no. 0.9 0.7 0.6 0.6 1.9 0.4 1.3 1.4 2.5 1.5 0.8 OPULATION hap. difs. Variance 1.4 0.7 0.7 0.9 3.8 0.4 0.8 1.0 2.3 2.2 0.7 Demographic expansion G

SSD 0.32 0.00 0.00 0.00 0.02 0.00 0.01 0.00 0.00 0.49 0.00 OF ENETICS P value 0.00 0.92 0.90 0.93 0.53 0.47 0.56 0.62 0.94 0.00 0.91 Q0 0.0 (0.0–0.0) 0.0 (0.0–0.3) 0.6 (0.0–0.0)† 0.2 (0.0–0.3) 0.0 (0.0–1.0) 0.0 (0.0–0.0) 0.0 (0.0–0.0) 0.0 (0.0–0.4) 0.0 (0.0–1.1) 0.0 (0.0–0.1) 0.0 (0.0–0.1) Q1 4289 (4296–4296)† 42.2 (1.3–N) 1.5 (4.9–N)‡ 0.7 (0.0–N) 2.1 (0.5–N) 0.5 (0.0–N) N (4.1–N) N (11.1–N) 29.5 (8.4–N) N (N-N) N (24.6–N) T(¼ 2 t 3 m) 0.0 (0.0–0.0) 0.7 (0.2–1.7) 0.1 (0.0–1.4) 1.3 (0.0–3.3) 5.1 (0.6–91.1) 3.0 (0.4–3.5) 1.5 (0.3–3.0) 1.6 (0.5–2.8) 2.8 (1.2–3.8) 0.0 (0.0–0.2) 0.8 (0.5–1.2)

Spatial expansion E SSD 0.00 0.00 0.00 0.00 <0.01 0.00 0.02 <0.01 <0.01 0.01 0.00 ASTERN P 0.90 0.95 0.85 0.96 0.85 0.55 0.52 0.68 0.89 0.77 0.61 Q (Nem 3 m) 0.4 (0.0–1.0) 0.1 (0.0–0.2) 0.1 (0.0–0.1) 0.1 (0.0–0.1) 0.7 (0.0–1.3) 0.0 (0.0–0.3) 0.0 (0.0–1.0) 0.0 (0.0–1.0) 0.0 (0.0–1.7) 0.2 (0.0–1.0) 0.0 (0.0–0.5) T 1.3 (0.2–3.7) 0.6 (0.3–1.7) 0.8 (0.7–2.3) 0.8 (0.1–2.2) 2.8 (0.8–6.9) 0.4 (0.2–0.7) 1.5 (0.3–2.8) 1.6 (0.6–2.9) 2.7 (1.2–3.9) 2.2 (0.5–4.4) 0.8 (0.3–1.1) U Migration 1.1 (0.1–N) N (1.3–N) 2.1 (0.2–N) 1.9 (0.3–N) 1.2 (0.3–N) N (0.9–N) N (1.8–N) N (2.8–N) 37.5 (12.0–N) 2.1 (0.4–N) N (3.2–N) NITED (M ¼ 2Nm)

0.09 0.08 0.15 0.14 0.07 0.20 0.11 0.09 0.03 0.07 0.07 S

HR TATES Tajima’s D Value –2.10 –2.02 –2.14 –2.13 –0.10 –1.36 –0.73 1.32 1.53 –1.72 –1.70 B

P 0.001 0.008 0.001 0.000 0.50 0.07 0.29 0.91 0.93 0.008 0.020 AY Fu’s FS S

Value –28.5 –3.7 –33.8 –34 3 1038 –7.7 –3.6 –2.2 0.0 –2.6 –28.1 –8.6 CALLOPS P 0.000 0.000 0.000 0.000 0.002 0.15 0.015 0.47 0.08 0.000 0.000

* Clade significant in nested clade phylogenetic analysis. † Bounds reflect low mean value of 0.01 compared with reported estimated value. ‡ Mean value exceeded reported estimated value; bounds reflect mean value. Clade 2–6 was not included because it is a 2-step clade restricted to North Carolina. As recommended by Schneider and Excoffier (1999), confidence intervals (in parentheses) for mismatch statistics are for alpha ¼ 0.10 because the mismatch test is very conservative. Mismatch statistic values greater than 99,000 are reported as N. Gulf, Florida Gulf of Mexico bay scallops; Atl, Atlantic Ocean bay scallops; both are defined in Table 1 and shown in Figure 1. no. hap. difs., number of haplotype differences; SSD, sum of squared deviations; P, significance level; q,2Nef m, the estimated expansion parameter; q0 and q1, respectively, estimated preexpansion and postexpansion population size; T, 2 t3m, time in generations since 2 populations last exchanged migrants (Rogers & Harpending 1992), which is a relative estimate of time since expansion event; M, 2Nef m, the scaled migration rate (Nm, effective number of migrants); HR, Harpending’s raggedness index (Harpending 1994). Tajima’s D and Fu’s FS are described in Methods. 601 602 BERT ET AL.

Campanella et al. 2007). NJ bay scallops may be a small, self- The presence of the ancestral lineage in Atlantic bay scallops seeding population (Bologna et al. 2001), or the Little Egg Harbor and the cool-water ancestry of Argopecten (Waller 1969) sup- colony may have originated principally from a small group of port the concept that the genus evolved in cool waters. The immigrants swept in as larvae or transplanted at some life stage predominance of H11 could have resulted from a selective from another location. The location of the aggregation—close to sweep of an alternative mtDNA haplotype better adapted to the mouth of an ocean-to-bay inlet—supports the former in- function in the warmer Gulf waters. ATP production by terpretation. Pairwise allozyme relationships, water current pat- mtDNA is temperature sensitive in marine molluscs because, terns, coastal geography, and the inferred time of settlement (fall) in high water temperatures, becomes limited, mitochon- (Bologna et al. 2001) suggest that NC was a source of that NJ dria become hypoxic, and anaerobiosis affects ATP production aggregation. In contrast to the summer spawning peak of NY, (Sokolova & Po¨ rtner 2003). Oxygen is important for functioning NC bay scallops have a fall spawning peak (Bologna et al. 2001). of both the 12S RNA gene, which assists in assembling amino Differences in the inferred origins of NJ bay scallop aggregations acids to build proteins used in oxidative phosphorylation, and (Bologna et al. 2001, Campanella et al. 2007, this study) may be the ND-1 gene, which provides instructions for making a com- the result of actual disparities in source subpopulations over time. ponent of an enzyme complex essential in the oxidative phos- Although we view NC as a likely source of immigrants into NJ, phorylation process (Po¨ rtner et al. 1999, Abele et al. 2002). larvae may also occasionally be swept in from NY or other Positive selection drives mtDNA evolution in (Bazin areas that harbor aggregations temporarily (e.g., Maryland et al. 2006), and a selected haplotype tends to become the major waters, where small aggregations have recently been seen in the haplotype (Mousset et al. 2004). A contribution of mtDNA H11 seaward areas of bays; S. Tettelbach, pers. comm.). Regardless to greater efficiency of ATP production in warm water could of the source, the overall paucity of genetic variation in the NJ have facilitated that proliferation of that haplotype in Florida subpopulation indicates that it was parented by few individ- Gulf bay scallops. uals and is isolated. The abundance of H11, generation and persistence of low- frequency mutations derived from that haplotype, and far Florida Gulf greater mtDNA FST values (nearly an order of magnitude) The allozyme and mtDNA RFLP analyses yielded radically compared with the allozyme QST values signal a selective sweep different images of the genetic relationships among Florida and recovery from a population crash sufficiently in the past to Gulf populations. The allozyme analysis confirmed the hierar- allow 1-step mutations to flourish (Saavedra & Pen˜a 2005). The chical structure of the Florida Gulf metapopulation first pro- H15 haplotype cluster (Fig. 3A) may also be such a selective posed by Arnold et al. (1998) and confirmed by Bert et al. (in sweep/mutational proliferation in an earlier stage of progress. prep). Here, at the population level, significant differences in Collectively, the MSN structure and mismatch statistics reflect allele frequencies at multiple loci, overall differences in allele all of the processes affecting Florida Gulf bay scallop popula- frequencies, and the structure of the UPGMA phenogram tion genetic structure—severe, sporadic population declines or confirmed the validity of the 4 populations and supported the local extinctions and rapid recovery to variable, sometimes high, closer relationship of CO and SF than of other population pairs. numbers of individuals; selective sweeps, possibly for metabolic The occurrence of many significant allele frequency differences processes in which specific mtDNA haplotypes are beneficial; among conspecific populations can be the result, in part, of large generation of numerous, closely related haplotypes resulting sample sizes (Gold et al. 2001); but in this case, differences in from relaxed selection during population explosions (Rand recruitment sources and patterns and in population dynamics 1996, Lessios et al. 2001, Bargelloni et al. 2005); and a core– (Bert et al. in prep) are also contributing factors. PN is principally periphery metapopulation structure perpetually in nonequi- differentiated from the CO-SF population complex because of its librium (Slatkin 1977). apparently independent recruitment in some years (Bert et al. in prep). The relationship of FB to this metapopulation structure is Atlantic–Florida Gulf Comparison even more peripheral (see A. i. taylorae, discussed later). Bay scallop mtDNA diversity, homogeneous throughout the Although allozymes differentiate Atlantic subpopulations Florida Gulf except for FB, reflects phylogenetic history rather and Florida Gulf populations only by allele frequencies, mtDNA than current population genetic structure. Collectively, the differentiation is quite distinct. Although Atlantic and Florida NCPA, MSN, and mismatch analyses support a large, regional, Gulf bay scallops could be distinguished by allozymes, we found demographic expansion that occurred recently in the past. This no fixed allozyme allele frequency differences between the 2 sudden expansion and the NCPA conclusion of limited gene flow groups, measures of genetic variability were similar, and both seem to be reflecting permanent attributes of the Florida Gulf groups exhibited a north-to-south trend in increasing heterozy- bay scallop core–periphery metapopulation structure (Bert et al. gosity. In contrast, mtDNA differentiation between the groups in prep.). The implications of continued exponential growth are appeared as isolation by distance, widely separated lineages in the indistinguishable from those of a sudden burst of population cluster and AMOVA analyses, and highly differentiated haplo- growth (Rogers et al. 1996). The stable, CO population is likely type frequencies, lineage structures, and demographic histories. the principal source of bay scallops for both SF, which has Only 4% of the Florida Gulf individuals possessed haplotypes subpopulations that go extinct intermittently, and, to a lesser in the Atlantic clades (C3-1 and C3-2), and only NC individuals degree, PN, which in some years is genetically indistinguishable possessed Florida Gulf haplotypes. In recently diverged taxa, from CO and other years is quite distinct (Bert et al. in prep.). greater differentiation in mtDNA than in nuclear genes is not This permanent expansion mode could facilitate the retention of uncommon because mtDNA effective population sizes are much mutations in rapidly evolving genes such as mtDNA (Saavedra & lower (in the case of hermaphroditic species, half) (Grant & Pen˜a 2005). Bowen 1998, Fauvelot et al. 2003). POPULATION GENETICS OF EASTERN UNITED STATES BAY SCALLOPS 603

The two sets of populations also differ greatly in population 0% for NJ and NY). Shared haplotypes from different, high-level genetic structure. Atlantic subpopulations are more genetically clades indicate recent or ongoing secondary contact (Excoffier & independent than Florida Gulf populations (except FB). At- Smouse 1994, Carlin et al. 2003). The presence of multiple NC lantic lineages are much older and more complex than Florida haplotypes derived from the H11 lineage, some sequentially, Gulf lineages. Atlantic subpopulations exhibit considerable indicates that H11 has existed in the NC population for consider- evidence of ancient, repeated population declines to low levels able time. Intermittent gene flow between Florida Gulf bay and recoveries to moderate levels (Rogers & Harpending 1992, scallops and the NC population must have a long history. Rogers et al. 1996), whereas Florida Gulf populations exhibit We propose that the NC population subspecies affiliation evidence of a single, large, recent population expansion. has long been dubious because both A. i. irradians and A. i. Many features of the Atlantic subpopulations contribute concentricus genes contribute to that population, perhaps in to the relatively large interpopulation genetic distances: (1) re- different proportions over time; and because, in scallops, mor- peated population depletions resulting from natural and an- phological data vary in ways that might not reflect taxonomic thropogenic causes and subsequent expansions from sometimes affiliation (Heipel et al. 1998). Based on morphology, Clarke few individuals (Chakraborty & Nei 1977, Gruenthal & Burton (1965) first differentiated northern A. i. irradians and southern 2008), (2) substantial geographic distances devoid of bay A. i. concentricus, assigned the NC population to A. i. concentricus, scallops except for infrequent ephemeral populations (Bricelj and noted that Maryland/Virginia waters (probably Chesapeake & Krause 1992, Heffernan et al. 1988, Bologna et al. 2001), (3) Bay; Fig. 1A) had an overlap population where both subspecies occurrences limited to bays and sounds with limited water were found. Soon thereafter, Waller (1969) gave the range of A. i. exchange between subpopulations and between them and the irradians as Massachusetts to NJ and of A. i. concentricus as NJ open ocean (Peterson & Summerson 1992, Marelli et al. 1997a), to Louisiana, and he cited Clarke (1965) as identifying the bays (4) probable sweepstakes recruitment (Hedgecock 1994, Li & of both NJ and Maryland as locations of overlap zones. Thus, in Hedgecock 1998), and (5) generations of supplementation with early morphologically based taxonomic work, NC bay scallops bay scallops from other locations (Bert et al. 2007). In contrast, were deemed to be A. i. concentricus.Wilbur(1995,Wilbur& the Florida Gulf CO population resides in nearshore, open-ocean Gaffney 1997), the first researcher to apply a scientifically con- waters; thus, larval transport from that population to the trolled approach to bay scallop morphologically based taxo- peripheral populations is more likely than it is between Atlantic nomic classification, reared bay scallops from Massachusetts, subpopulations. The critical shallow-water seagrass habitat is NC, and the Florida Gulf in a common garden experiment and distributed more continuously in Florida Gulf waters than in performed a detailed morphological analysis on the F1 progeny Atlantic waters; geographically large intervals devoid of sea- along with wild bay scallops from those 3 locations plus Texas. In grasses are not common. Last, no hatchery-based stock enhance- a principal components analysis (PCA) of F1-progeny morpho- ment of Florida Gulf bay scallop aggregations preceded our metrics and meristics, the Atlantic samples grouped together; sampling. Thus, Atlantic subpopulations have experienced many however, in a PCA of the wild bay scallop data, NC (then still generations of genetic bottlenecking, genetic drift, stochastic considered to be A. i. concentricus) and Texas individuals (A. i. genetic changes, and extinction of local genomes not experienced amplicostatus)overlapped. by Florida Gulf bay scallops at the population level. Atlantic bay In sharp contrast, we maintain that genetics studies have scallops do not exhibit a metapopulation structure but, rather, always shown that NC bay scallops are principally A. i. irradians. comprise a set of nearly independent populations. However, only very recently has that taxonomic association been recognized, and no genetics study has yet recognized the in- Taxonomic Puzzles clusion of A. i. concentricus genes in that population. Despite genetic evidence to the contrary presented in their papers, North Carolina Bay Scallops multiple researchers held to the identification of NC bay scallops The NC population has genetic characteristics indicating as A. i. concentricus for a number of years. Blake and Graves’ that it contains principally A. i. irradians genes, but also possesses (1995) whole-molecule mtDNA RFLP analysis showed that bay A. i. concentricus genes at low frequencies. NC allozyme allele scallops from NC more closely resembled A. i. irradians from frequencies do not differ significantly from those of other Massachusetts than A. i. concentricus from the Florida Gulf. Atlantic populations, and Atlantic allozyme pairwise genetic In separate cluster analyses, one based on the RFLPs of 2 distances with NC as a pair member are about half the values mtDNA gene fragments (Wilbur 1995) and the other on for NJ/NY pairs. The NC population differs from Florida allozyme loci (Marelli et al. 1997b), NC samples grouped with Gulf populations in a manner similar to that of other Atlantic other Atlantic samples of A. i. irradians on branches clearly populations in the number and magnitude of significant allele and sometimes significantly (Wilbur 1995) separated from the frequency differences at specific allozyme loci, in overall allele Florida Gulf A. i. concentricus branch. In another analysis of frequencies, and in its positions in all allozyme and mtDNA RFLP variation in 2 mtDNA gene fragments (Bologna et al. RFLP cluster analyses. However, compared with other Atlantic 2001), nucleotide divergences between NC and Florida Gulf populations, NC’s allozyme–locus pairwise genetic distances samples were nearly 3–10 times greater than those between NC from Florida Gulf populations are significantly (33–50%) and other Atlantic samples. Only very recently, Hemond and smaller, significantly more rare alleles are uniquely shared with Wilbur (2011) recognized NC bay scallops as A. i. irradians,based Florida populations (P < 0.01, R3C and pairwise G-tests), and on allele frequency differences at several microsatellite DNA highly unlikely rare-allele combinations for Florida Gulf alleles (msDNA) loci. (homozygotes, 2 alleles appearing in 1 individual) occur. More- Close examination of the results presented in past studies over, 15% of the NC individuals possessed Florida Gulf also supports our finding that A. i. concentricus genes are pres- haplotypes H11 or H15, or derivatives of those haplotypes (vs. ent in the NC population. In the cluster phenograms presented 604 BERT ET AL. by both Wilbur and Gaffney (1997) and Marelli et al. (1997b), Argopecten irradians taylorae the NC sample was included in the A. i. irradians branch but was FB is uniquely distinct among Florida bay scallop popula- notably more basal, as it is in our UPGMA phenogram. This tions. Although its allozyme diversity was not low compared position could be expected if that sample contained some A. i. with other Florida Gulf populations, its mtDNA diversity concentricus genes, and Wilbur and Gaffney’s (1997) PCA certainly was. Despite its far smaller sample size, FB had the showed a few individuals nested within the Florida Gulf A. i. highest values for all measures of allozyme genetic variability concentricus group. In Campanella et al.’s (2007) analysis of 8 except average number of alleles per locus. In nuclear genes, msDNA loci for Atlantic samples only, all alleles were shared drift-induced changes in allele frequencies can increase hetero- between NY and NJ samples, but the NC sample had unique zygosity in bottlenecked populations (Leberg 1992, Brookes alleles at 3 loci, leading them to conclude that NJ was the point et al. 1997, Planes & Fauvelot 2002). In sharp contrast, FB was of contact between northern A. i. irradians and NC A. i. concentricus. nearly fixed for the most common mtDNA RFLP in Florida However, without including Floridian A. i. concentricus,thegreater Gulf bay scallops, resulting in significantly lower measures of genetic distance of NC bay scallops from the more closely related mtDNA genetic diversity compared with other Florida Gulf northern populations could not be put into its proper perspec- populations. These extremes affected all pairwise comparisons tive. The unique alleles in their NC sample could have been A. with FB as a pair member. i. concentricus alleles embedded in the essentially A. i. irradians Including Atlantic bay scallops in our analyses eliminated population. the possibility that the FB population is related to A. i. irradians. The A. i. concentricus genetic input in NC bay scallops could FB was highly differentiated from all Atlantic samples, including be the product of a former predominance of A. i. concentricus NC. In both allozyme and mtDNA cluster phenograms, FB in NC during a warm climatic period and hybridization clearly grouped with Florida Gulf A. i. concentricus collections; between the 2 subspecies when A. i. irradians invaded, possibly and FB was enriched in rare Florida Gulf allozyme alleles as well during a Pleistocene cool period. When closely related species as in the common Florida Gulf mtDNA haplotype. hybridize, nuclear genes may show little indication of inter- The type of contrast in genetic diversity we observed between breeding, but haplotypes characteristic of one species can occur allozyme loci and mtDNA can result from a brief, severe in comparatively high frequencies in the other species because population bottleneck (Birky 1991, Snowbank & Krajewski mtDNA does not undergo recombination with each genera- 1995, Grant & Bowen 1998). The only indication of a recent tion. The occurrence of root haplotypes H11 and H15 in NC, genetic bottleneck in the FB allozyme data was a low na, but the and the complexity of NC haplotype relationships in clades striking contrast between the low mtDNA diversity of our C1-14 and C2-6 combined, and their derivation from H11, sample (the common Florida Gulf haplotype found in 20 of 21 support this idea. The A. i. concentricus genetic input must have individuals) and the highly diverse sample analyzed by Blake occurred long enough in the past for haplotypes to evolve from and Graves (1995) (17 haplotypes found in 34 individuals), H13. clearly demonstrates that a population collapse and regenera- The presence of a few individuals homozygous for rare tion must have occurred sometime between the collection of Florida Gulf allozyme alleles and the existence of common Blake and Graves’ (1995) sample in 1993 and the collection of Florida Gulf haplotypes also argue for occasional contempo- our sample in 1998. Population expansions and collapses, rary dispersal of individuals with Florida Gulf genomes into the which can eliminate mtDNA lineages (Carlin et al. 2003) and NC population. The probability that those homozygotes would rare haplotypes (Matocq et al. 2000), have repeatedly occurred exist in NC A. i. irradians is remarkably low. The broad dis- in this population at least since 1980 (T. M. Bert and J. M. tributional gap in the Atlantic between east–central Florida Stevely, University of Florida Cooperative Extension Service, and southern NC disrupts gene flow from the most likely source pers. obs.). The FB population principally inhabits nearly of Florida bay scallop alleles in NC—the small A. i. concentricus isolated lagoons with restricted water exchange with the open aggregation inhabiting southeastern Florida. But proximity of ocean and intermittent extreme phytoplankton blooms (Butler the Gulf Stream to both coasts may facilitate larval transport et al. 1995, Phlips et al. 1999, Stevely et al. 2010) that can kill from southeastern Florida to NC, particularly when hurricanes bay scallops. Thus, population crashes of varying magnitude travel northward along the Atlantic coast; or boat traffic (e.g., via are highly likely. Over time, they have rendered the bilge discharge) may occasionally transport larvae. Hurricane FB population as genetically differentiated from other Florida frequency is highest during the late-summer/fall spawning season Gulf populations as is NC. Nevertheless, our genetic analyses (http://www.nhc.noaa.gov), and coastal Atlantic hurricanes show that the FB population is highly differentiated A. i. have been common in some years (e.g., 1972, 1997, 2004; http:// concentricus, confirming the opinion of Blake and Graves www.nhc.noaa.gov.pastall.shtml) just prior to bay scallop pop- (1995) and Marelli et al. (1997a) that A. i. taylorae is not a valid ulation flushes at some locations in the following years (e.g., subspecies. 1973 to 1974, 1998, 2004 to 2005; see Bologna et al. (2001) and Campanella et al. (2007)). In addition, Pleistocene fluctuations Atlantic Bay Scallop Fisheries Management in sea level have, at times, made the Florida peninsula a less formidable barrier to gene flow between the Gulf of Mexico The large genetic distances between Atlantic populations and Atlantic Ocean (Healy 1975). compared with Florida populations may be, in part, the Some, but not all, of the samples analyzed in this study were consequence of both long-term overfishing and repeated bay included in Hemond and Wilbur’s (2011) msDNA study. It scallop stock enhancement in the Atlantic. Both heavy harvest- would be interesting to match their NC msDNA genotypes with ing (Allendorf et al. 2008) and stock enhancement (Bert et al. our genotype/haplotype combinations to investigate further the 2007) can perturb genetic population subdivision, increase context of A. i. concentricus genes in the NC population. genetic distances between populations, and cause evolutionarily POPULATION GENETICS OF EASTERN UNITED STATES BAY SCALLOPS 605 important genetic damage that not only affects the population’s able levels will continue, principally through hatchery-based long-term survivability negatively, but also decreases fishery stock enhancement. In early stock enhancement efforts, the yield. Because all population genetics studies on Atlantic bay number of individuals in and sources of the broodstock, the scallops have been done after years of problematic fisheries effective number of spawners contributing to each brood, and management and of stock enhancement, the natural degree of the number of broods and of released (seeded) individuals in population connectivity can never be known. Today, we see each brood were typically not published and may not have been relatively independent populations that flourish infrequently. recorded. Nor was the genetic diversity of the broodstock or Differences in their genetic diversity, population dynamics, broods estimated before or after release of the broods. Only two degree of geographic and genetic isolation, and ecological assessments of the hatchery contribution to an enhanced stock history justify their independent management. Although some have been done (Krause 1992, Seyoum et al. 2003, Wilbur et al. small amount of gene exchange may occur between bay scallops 2005). Hatchery-based stock enhancement can alter genetic from separate Atlantic coastal bay systems, reliance on gene diversity, decrease fitness, and reduce effective population size, flow between populations to supplement depleted stocks is not principally through reduction of allelic variability and change in a viable management option. allele frequencies of both the recipient population and the Atlantic bay scallop populations have been heavily fished for admixed population if the broodstock or released broods are decades (Bologna et al. 2001). Local fisheries have been allowed genetically differentiated from the recipient population (Bert to harvest local aggregations heavily, even when densities were et al. 2007). Damage can occur in many direct and indirect ways, low (Peterson & Summerson 1992); and harvest seasons may even if care is taken to obtain broodstock that is representative be timed to maximize yield of muscle per scallop (e.g., Geiger of the genetic diversity in the recipient population and to release et al. 2006) or to satisfy other economic interests, regardless of broods that should not alter the genetic diversity of the recipient spawning season. Bay scallops typically undergo natural in- population or admixed population after enhancement. The terannual fluctuations in population abundance (Rhodes 1991). risk of genetic damage to enhanced populations is particu- The abundance of all Atlantic populations is further affected by larly high when the wild individual-to-hatchery individual ratio unpredictable brown and red (Karenia brevis) tides (Tettelbach is low or the enhancement is repeated (Laikre et al. 2010). The & Wenczel 1991, Peterson & Summerson 1992, Campanella genetic diversity of repeatedly enhanced populations can differ et al. 2007), which can deplete or destroy aggregations or whole from that of unenhanced populations as much as that of populations and, less frequently, by population explosions of different species (Bert & Tringali 2001). parasites or predators (Tettelbach & Wenczel 1993), which Two important steps toward minimizing the probability of weaken and deplete local aggregations. In the past, affected inadvertent genetic damage to bay scallop populations are to aggregations refurbished themselves sooner or later, so a liberal ensure that harvest levels do not impede the population’s harvesting strategy seemed reasonable. However, within the reproductive potential and to ensure that stock enhancement past few decades, loss of critical seagrass habitat (Thayer & efforts include a genetic monitoring program. Several models Stuart 1974, Pohle et al. 1991) has also contributed to declines for such a program exist (e.g., Gold 2004, Bert et al. 2007); one in numbers of bay scallops in all areas where they have been has been applied to Florida Gulf bay scallop restoration traditionally harvested (Tettelbach et al. 1990, Peterson & (Wilbur et al. 2005). However, hatchery-based stock enhance- Summerson 1992, Bologna et al. 2001), and recovery of pop- ment has limited potential to remedy the problems that chronic ulations to harvestable densities now can take years (Peterson & overfishing causes. It is, by far, cheaper, easier, and evolution- Summerson 1992) or decades (Bologna et al. 2001, Tettelbach arily safer to manage fisheries sustainably (e.g., Jackson et al. 2009) because local recruitment is limited when bay scallop 2001). density is low (Peterson & Summerson 1992, Arnold et al. 1998), and reliable recruitment from elsewhere is unlikely (this ACKNOWLEDGMENTS study). Therefore, fisheries managers have turned to various types of stock enhancement to bolster or regenerate Atlantic We are grateful to all FWC staff involved with this project for bay scallop aggregations (Peterson & Summerson 1992, assisting with the collection of Florida samples; to S. Tettelbach, Arnold et al. 1998, Goldberg et al. 2000, Tettelbach et al. P. Murphy, and P. Bologna for providing Atlantic samples; to S. 2010; see also Blake and Graves (1995) and Marko and Barr Stephenson for providing the maps for Figure 1; to T. Jones and (2007)). S. Campbell for assisting with the genetic analysis’ and to S. Thus far, stock enhancement efforts of Atlantic bay scallop Tettelbach for reviewing the manuscript. Financial support was aggregations have met with varying degrees of success (Arnold provided by NOAA grant NA76FK0426, project FWC 2234, 2008). Intensive, multiyear efforts seem to be the most success- and the state of Florida. The view expressed herein are those of ful (Tettelbach et al. 2010). The high value of bay scallops as the authors and do not necessarily reflect the views of NOAA or a item ensures that efforts to rebuild stocks to harvest- any of its subagencies.

LITERATURE CITED Abbott, R. T. 1974. American seashells, 2nd edition. New York: Van Allendorf, F. W., P. R. England, G. Luikart, P. A. Ritchie & N. Ryman. Nostrand Reinhold. 663 pp. 2008. Genetic effects of harvest on wild populations. Trends Abele, D., K. Heisel, H. O. Po¨ rtner & S. Puntarulo. 2002. Temperature- Ecol. Evol. 23:327–337. dependence of mitochondrial function and production of reactive Arnold, W. S. 2008. Application of larval release for restocking and oxygen species in the intertidal mud Mya arenaria. J. Exp. Biol. stock enhancement of coastal marine bivalve populations. Rev. Fish. 205:1831–1841. Sci. 16:65–71. 606 BERT ET AL.

Arnold, W. S., N. J. Blake, M. M. Harrison, D. C. Marelli, M. L. Campanella, J. J., P. A. X. Bologna, L. E. J. Kim & J. V. Smalley. 2007. Parker, S. C. Peters & D. E. Sweat. 2005. Restoration of bay scallop Molecular genetic evidence suggests Long Island as the geographic (Argopecten irradians (Lamarck)) populations in Florida coastal origin for the present population of bay scallops in Barnegat Bay, waters: planting techniques and the growth, mortality and repro- New Jersey. J. Shellfish Res. 262:303–306. ductive development of planted scallops. J. Shellfish Res. 24:883– Carlin, J. L., D. R. Robertson & B. W. Bowen. 2003. Ancient 904. divergences and recent connections in two tropical Atlantic reef Arnold, W. S., D. C. Marelli, C. P. Bray & M. M. Harrison. 1998. fishes Epinephelus adscensionis and Rypticus saponaceous (Percoidei: Recruitment of bay scallops Argopecten irradians in Floridan Gulf Serranidae). Mar. Biol. 143:1037–1069. of Mexico waters: scales of coherence. Mar. Ecol. Prog. Ser. Castelloe, J. & A. R. Templeton. 1994. Root probabilities for in- 170:143–157. traspecific gene trees under neutral coalescent theory. Mol. Phylo- Avise, J. C. 2000. Phylogeography: the history and formation of species. genet. Evol. 3:102–113. Cambridge, MA: Harvard University Press. 447 pp. Chakraborty, R. & M. Nei. 1977. Bottleneck effects on average Balco, G. & J. M. Schaefer. 2006. Cosmogenic-nuclide and varve heterozygosity and genetic distance with the stepwise mutation chronologies for the deglaciation of southern New England. Quat. model. Evolution 31:347–356. Geochronol. 1:15–28. Clarke, A. H., Jr. 1965. The scallop superspecies Aequipecten irradians Bargelloni, L., J. A. Alarcon, M. C. Alvarez, E. Penzo, A. Magoulas, (Lamarck). Malacologia 2:161–188. J. Palma & T. Patarnello. 2005. The Atlantic–Mediterranean transi- Clement, M., D. Posada & K. Crandall. 2000. TCS: a computer tion: discordant genetic patterns in two seabream species, Diplodus program to estimate gene genealogies. Mol. Ecol. 99:1657–1660. puntazzo (Cetti) and Diplodus sargus (L.). Mol. Phylogenet. Evol. Cockerham, C. C. 1969. Variance of gene frequencies. Evolution 23:72–84. 36:523–535. Cockerham, C. C. 1973. Analysis of gene frequencies. Genetics 74:679– Bazin, E., S. Gle´ min & N. Galtier. 2006. Population size does not 700. influence mitochondrial genetic diversity in animals. Science Dando, P. R., K. B. Storey, P. W. Hochachka & J. M. Storey. 1981. 312:570–572. Multiple dehydrogenases in marine mollusks: electrophoretic anal- Bert, T. M., C. R. Crawford, M. D. Tringali, S. Seyoum, J. L. Galvin, ysis of alanopine dehydrogenase, strombine dehydrogenase, octo- M. Higham & C. Lund. 2007. Genetic management of hatchery- pine dehydrogenase, and lactate dehydrogenase. Mar. Biol. Lett. based stock enhancement. In: T. M. Bert, editor. Ecological and 2:249–257. genetic implications of aquaculture activities. New York: Springer. Dillon, R. & G. M. Davis. 1980. The Goniobasis of south Virginia and pp. 123–174. northwest North Carolina: genetic and shell morphometric relation- Bert, T. M. & M. D. Tringali. 2001. The effects of various aquacultural ships. Malacologia 20:83–98. breeding strategies on the genetic diversity of successive broods. Dillon, R. T. & J. J. Manzi. 1992. Population genetics of the , J. Biosci. Malaysia 12:13–26. Mercenaria mercenaria, at the northern limit of its range. Can. Birky, C. W. 1991. Evolution and population genetics of organelle J. Fish. Aquat. Sci. 49:2574–2578. genes: mechanisms and models. In: R. K. Selander, A. G. Clark & Edmands, S., P. E. Mobert & R. S. Burton. 1996. Allozyme and T. S. Whittam, editors. Evolution at the molecular level. Sunderland, mitochondrial DAN evidence of population subdivision in the MA: Sinauer. pp. 112–134. purple sea urchin Strongylocentrotus purpuratus. Mar. Biol. Blake, S. G. & J. E. Graves. 1995. Mitochondrial DNA variation in the 126:443–450. bay scallop, Argopecten irradians (Lamarck, 1819), and the Atlantic Excoffier, L. & P. E. Smouse. 1994. Using allele frequencies and calico scallop, Argopecten gibbus (Linnaeus, 1758). J. Shellfish Res. geographic subdivision to reconstruct gene trees within a species: 141:79–85. molecular variance parsimony. Genetics 156:343–359. Bologna, P. A. X., A. E. Wilbur & K. W. Able. 2001. Reproduction, Fauvelot, C., G. Bernardi & S. Planes. 2003. Reductions in the population structure, and recruitment limitation in a bay scallop mitochondrial DNA diversity of coral reef fish provide evidence of (Argopecten irradians Lamarck) population from New Jersey, USA. population bottlenecks resulting from Holocene sea-level change. J. Shellfish Res. 201:89–96. Evolution 57:1571–1583. Boulding, E. G., J. D. G. Boom & A. T. Beckenbach. 1993. Genetic Fu, Y.- X. 1997. Statistical tests of neutrality of mutations against variation in one bottlenecked and two wild populations of the population growth, hitchhiking and background selection. Genetics Japanese scallop (Patinopecten yessoensis): empirical parameters 147:915–925. estimates from coding regions of mitochondrial DNA. Can. J. Fish. Geiger, S. P., J. Cobb & W. S. Arnold. 2006. Variations in growth and Aquat. Sci. 50:1147–1157. reproduction of bay scallops (Argopecten irradians) from six sub- Bowen, B. W. & W. S. Grant. 1997. Phylogeography of the sardines populations in the northeastern Gulf of Mexico. J. Shellfish Res. (Sardinops spp.): assessing biogeographic models and population 25:491–501. histories in temperate upwelling zones. Evolution 51:1601–1610. Gold, J. R. 2004. Stock structure and effective size of red drum Bricelj, V. M. & M. K. Krause. 1992. Resource allocation and (Sciaenops ocellatus) in the northern Gulf of Mexico and implica- population genetics of the bay scallop, Argopecten irradians irradi- tions relative to stock enhancement and recruitment. In: K. M. ans: effects of age and allozyme heterozygosity on reproductive Leber, S. Kitada, H. L. Blankenship & T. Sva˚sand, editors. Stock output. Mar. Biol. 113:253–261. enhancement and sea ranching: developments, pitfalls, and oppor- Brookes, M. I., Y. A. Graneau, P. King, O. C. Rose, C. D. Thomas & tunities. Oxford, UK: Blackwell Publishing. pp. 353–370. J. L. B. Mallet. 1997. Genetic analysis of founder bottlenecks in the Gold, J. R., C. P. Burridge & T. F. Turner. 2001. A modified stepping- rare British butterfly Plebejus argua. Conserv. Biol. 11:648–661. stone model of population structure in red drum from the northern Busack, S. D. & R. Lawson. 2008. Morphological, mitochondrial DNA Gulf of Mexico. Genetica 111:305–317. and allozyme evolution in representative amphibians and reptiles Goldberg, R., J. Pereira & P. Clark. 2000. Strategies for enhancement of inhabiting each side of the Strait of Gibraltar. Biol. J. Linn. Soc. natural bay scallop, Argopecten irradians irradians, populations: a Lond. 94:445–461. case study in the Niantic River estuary, Connecticut, USA. Aquacult. Butler, M. J. IV, J. H. Hunt, W. F. Herrnkind, M. J. Childress, R. Int. 8:139–158. Bertelsen, W. Sharp, T. Matthews, J. M. Field & H. G. Marshall. Grant, W. S. 2005. A second look at mitochondrial DNA variability in 1995. Cascading disturbances in Florida Bay, USA: cyanobacteria European anchovy (Engraulis encrasicolus): assessing models of blooms, sponge mortality and implications for juvenile spiny population structure and the Black Sea isolation hypothesis. lobsters Panulirus argus. Mar. Ecol. Prog. Ser. 129:119–125. Genetica 125:293–309. POPULATION GENETICS OF EASTERN UNITED STATES BAY SCALLOPS 607

Grant, W. S. & B. W. Bowen. 1998. Shallow population histories in deep Li, G. & D. Hedgecock. 1998. Genetic heterogeneity, detected by PCR evolutionary lineages of marine fishes: insights from sardines and SSCP, among samples of larval Pacific ( gigas) anchovies and lessons for conservation. J. Hered. 89:415–426. supports the hypothesis of large variance in reproductive success. Gruenthal, K. M. & R. S. Burton. 2008. Genetic structure of natural Can. J. Fish. Aquat. Sci. 55:1025–1033. populations of the California black ( Marelli, D. C., M. K. Krause, W. S. Arnold & W. G. Lyons. 1997a. Leach, 1814), a candidate for endangered species status. J. Exp. Systematic relationships among Florida populations of Argopecten Mar. Biol. Ecol. 355:47–58. irradians (Lamarck, 1819) (: Pectinidae). 110:31– Haag, C. R., M. Riek, J. W. Hottinger, V. I. Pajunen & D. Ebert. 2005. 41. Genetic diversity and genetic differentiation in Daphnia metapopu- Marelli, D. C., W. G. Lyons, W. S. Arnold & M. K. Krause. 1997b. lations with subpopulations of known age. Genetics 170:1809–1820. Subspecific status of Argopecten irradians concentricus (Say, 1922) Harpending, H. C., S. T. Sherry, A. R. Rogers & M. Stoneking. 1993. and of the bay scallops of Florida. Nautilus 110:42–44. The genetic structure of ancient human populations. Curr. Anthropol. Marko, P. B. & K. R. Barr. 2007. Basin-scale patterns of mtDNA 34:483–496. differentiation and gene flow in the bay scallop Argopecten irradians Harpending H. C. 1994. Signature of ancient population growth in concentricus. Mar. Ecol. Prog. Ser. 349:139–150. a low-resolution mitochondrial DNA mismatch distribution. Hum. Matocq, M. D., J. L. Patton & M. N. F. da Silva. 2000. Population Biol. 66:591–600. genetic structure of two ecologically distinct Amazonian spiny rats: Healy, H. G. 1975. Terraces and shorelines of Florida. U.S. Geological separating history and current ecology. Evolution 54:1423–1432. Survey map series no. 71. http://publicfiles.dep.state.fl.us/FGS/ McElroy, D., P. E. Moran, E. Bermingham & I. Kornfield. 1992. WEB/terraces_300dpi20.pdf. REAP: an integrated environment for the manipulation and phylo- Hedgecock, D. 1994. Does variance in reproductive success limit genetic analysis of restriction data. J. Hered. 83:157–158. effective population sizes of marine organisms? In: A. R. Beaumont, Mousset, S., N. Derome & M. Veuille. 2004. A test of neutrality and editor. Genetics and evolution of marine organisms. New York: constant population size based on the mismatch distribution. Mol. Chapman and Hall. pp. 122–134. Biol. Evol. 21:724–731. Heffernan, P. B., R. L. Walker & D. M. Gillespie. 1988. Biological Murphy, R. W., J. W. Sites, Jr., D. G. Buth & C. H. Haufler. 1990. feasibility of growing the northern bay scallop, Argopecten irradians Proteins I: isozyme electrophoresis. In: D. M. Hollis & C. Moritz, irradians (Lamarck, 1819) in coastal waters of Georgia. J. Shellfish editors. Molecular systematics. Sunderland, MA: Sinauer. pp 45– Res. 7:83–88. 121. Heipel, D. A., J. D. D. Bishop, A. R. Brand & J. P. Thorpe. 1998. Nei, M. 1972. Genetic distance between populations. Am. Nat. 106:283– Population genetic differentiation of the great scallop Pecten 292. maximum in western Britain investigated by randomly amplified Nei, M. 1987. Molecular evolutionary genetics. New York: Columbia polymorphic DNA. Mar. Ecol. Prog. Ser. 162:163–171. University Press. 512 pp. Hellberg, M. E. 2006. Genetic approaches to understanding marine Nei, M., J. C. Stephens & N. Saitou. 1985. Methods for computing the metapopulation dynamics. In: J. P. Kritzer & P. F. Sale, editors. standard errors of branching points in an evolutionary tree and their Marine metapopulations. Amsterdam: Elsevier. pp. 431–455. application to molecular data from humans and apes. Mol. Biol. Hellberg, M. E., D. P. Balch & K. Roy. 2001. Climate-driven range Evol. 2:66–85. expansion and morphological evolution in a marine gastropod. Ota T. 1993. Dispan: Genetic distance and phylogenetic analysis. Science # :1707–1710. Pennsylvania State University, University Park, PA, USA. Avail- Hemond, E. M. & A. E. Wilbur. 2011. Microsatellite loci indicate able at: http://www.bio.psu.edu/People/Faculty/Nei/Lab/Programs. population structure and selection between Atlantic and Gulf of html. Mexico populations of the bay scallop Argopecten irradians. Mar. Palumbi, S. R. 2003. Population genetics, demographic connectivity, Ecol. Prog. Ser. 423:131–142. and the design of marine reserves. Ecol. Appl. 13:S146–S158. Jackson, J. B. C., M. X. Kirby, W. H. Berger, K. A. Bjorndal, L. W. Peterson, C. H. & H. C. Summerson. 1992. Basin-scale coherence of Botsford, B. J. Bourque, R. H. Bradbury, R. Cooke, J. Erlandson, population dynamics of an exploited marine invertebrate, the bay J. A. Estes, T. P. Hughes, S. Kidwell, C. B. Lange, H. S. Lenihan, scallop: implications of recruitment limitation. Mar. Ecol. Prog. Ser. J. M. Pandolfi, C. H. Peterson, R. S. Steneck, M. J. Tegner & R. R. 90:257–272. Warner. 2001. Historical overfishing and the recent collapse of Peterson, C. H., H. C. Summerson & R. A. Luettich, Jr. 1996. Response coastal ecosystems. Science 293:629–638. of bay scallops to spawner transplants: a test of recruitment Jin, L. & J. W. H. Ferguson. 1990. Neighbor-joining tree and UPGMA limitation. Mar. Ecol. Prog. Ser. 132:93–107. Tree software. Houston, TX: Center of Demographic and Popula- Petuch, E. J. 1987. New Caribbean molluscan faunas. Charlottesville, tion Genetics, University of Texas Health Science Center. VA: Coastal Education and Research Foundation. 154 pp. Koehn, R. K., W. J. Diehl & T. M. Scott. 1988. The differential Phlips, E. J., S. Badylak & T. C. Lynch. 1999. Blooms of the contribution by individual enzymes of glycolysis and protein picoplanktonic cyanobacterium Synechococcus in Florida Bay, catabolism to the relationship between heterozygosity and growth a subtropical inner-shelf lagoon. Limnol. Oceanogr. 44:1166– rate of the coot clam, Mulinia lateralis. Genetics 118:121–130. 1175. Krause, M. K. 1992. Phenotypic expression of glucose-6-phosphate Planes, S. & C. Fauvelot. 2002. Isolation by distance and variance drive isomerase genotype in the bay scallop, Argopecten irradians, and the genetic structure of a coral reef fish in the Pacific Ocean. Evolution blue , Mytilus edulis. PhD diss., State University of New York 56:378–389. at Stony Brook. 203 pp. Pohle, D. G., V. M. Bricelj & Z. Garcia-Esquivel. 1991. The eelgrass Laikre, L., M. K. Schwartz, R. S. Waples, N. Ryman & The GeM canopy: an above-bottom refuge from benthic predators for Working Group. 2010. Compromising genetic diversity in the wild: juvenile bay scallops Argopecten irradians. Mar. Ecol. Prog. Ser. unmonitored large-scale release of plants and animals. Trends Ecol. 74:47–59. Evol. 25:520–529. Po¨ rtner, H. O., I. Hardewig & L. S. Peck. 1999. Mitochondrial function Leberg, P. L. 1992. Effects of population bottlenecks on genetic diversity and critical temperature in the Antarctic bivalve, Laternula elliptica. as measured by allozyme electrophoresis. Evolution 46:477–494. Comp. Biochem. Physiol. A 124:179–189. Lessios, H. A., M. J. Garrido & B. D. Kessing. 2001. Demographic Posada, D., K. A. Crandall & A. R. Templeton. 2000. GeoDis: history of Diadema antillarum, a keystone herbivore on Caribbean a program for the cladistic nested analysis of the geographical reefs. Proc. Biol. Sci. 268:2347–2353. distribution of genetic haplotypes. Mol. Biol. 9:487–488. 608 BERT ET AL.

Pruett, C. L., E. Sailliant & J. R. Gold. 2005. Historical population Slatkin, M. & R. R. Hudson. 1991. Pairwise comparisons of mitochon- demography of red snapper (Lutjanus campechanus)fromthe drial DNA sequences in stable and exponentially growing popula- northern Gulf of Mexico based on analysis of sequences or tions. Genetics 129:555–562. mitochondrial DNA. Mar. Biol. 147:593–602. Snowbank, S. A. & C. Krajewski. 1995. Lack of restriction-site Rand, D. M. 1996. Neutrality tests of molecular markers and the variation in mitochondrial-DNA control region of whooping cranes connection between DNA polymorphism, demography, and con- (Grus americana). Auk 112:1045–1049. servation biology. Conserv. Biol. 10:665–671. Sokal, R. R. & F. J. Rohlf. 1995. Biometry. New York: W. H. Freeman. Raymond, M. & F. Rousset. 1995. GENEPOP (version 1.2): population 887 pp. genetics software for exact tests and ecumenicism. J. Hered. 86:248– Sokolova, I. M. & H. O. Po¨ rtner. 2003. Metabolic plasticity and critical 249. temperatures for aerobic scope in a eurythermal marine invertebrate Rhodes, E. W. 1991. Fisheries and aquaculture of the bay scallop, ( saxatilis, : Littorinidae) from different lati- Argopecten irradians, in the eastern United States. In: S. E. Shumway, tudes. J. Exp. Biol. 206:195–207. editor. Scallops: biology, ecology and aquaculture. Developments in Stevely, J. M., D. E. Sweat, T. M. Bert, C. Sim-Smith & M. Kelly. 2010. aquaculture and fisheries science, vol. 21. Amsterdam: Elsevier. pp. Sponge mortality at Marathon and Long Key, Florida: patterns of 913–924. species response and population recovery. Proc. Gulf Caribb. Fish. Rice, W. R. 1989. Analyzing tables of statistical tests. Evolution 43:223– Inst. 63:394–403. 225. Swofford, D. L. & R. M. Selander. 1981. BIOSYS-1: a FORTRAN Rigaa, A., D. Cellos & M. Monnerot. 1997. Mitochondrial DNA from program for the comprehensive analysis of electrophoretic data in the scallop : an unusual polymorphism detected by population genetics and systematics. J. Hered. 72:281–283. restriction fragment length polymorphism analysis. Heredity Tajima, F. 1989. Statistical methods for testing the neutral mutation 78:380–387. hypothesis by DNA polymorphism. Genetics 123:585–595. Rogers, A. R., A. E. Fraley, M. J. Bamshd, W. S. Watkins & L. B. Jorde. Templeton, A. R. 1998. Nested clade analyses of phylogeographic data: 1996. Mitochondrial mismatch analysis is insensitive to the muta- testing hypotheses about gene flow and population history. Mol. tional process. Mol. Biol. Evol. 13:895–902. Ecol. 7:381–397. Rogers, A. R. & H. Harpending. 1992. Population growth makes waves Teske, P. R., H. Hamilton, P. J. Palsbøll, C. K. Choo, H. Gabr, S. A. in the distribution of pairwise genetic differences. Mol. Biol. Evol. Lourie, M. Santos, A. Sreepada, M. I. Cherry & C. A. Matthee. 9:552–569. 2005. Molecular evidence for long-distance colonization in an Indo- Saavedra, C. & J. B. Pen˜a. 2005. Nucleotide diversity and Pleistocene Pacific seahorse lineage. Mar. Ecol. Prog. Ser. 286:249–260. population expansion in Atlantic and Mediterranean scallops Tettelbach, S.T. & P. Wenczel. 1991. Reseeding efforts and the status of (Pecten maximus and P. jacobaeus) as revealed by the mitochondrial bay scallop populations in New York following the appearance of 16s ribosomal RNA gene. J. Exp. Mar. Biol. Ecol. 323:138–150. brown tide. J. Shellfish Res. 12:423–431. Tettelbach, S.T. & P. Wenczel. 1991. Reseeding efforts and the status of Tettelbach, S. 2009. Bay scallop restoration in New York. Ecol. Restor. bay scallop populations in New York following the appearance of 27:20–22. brown tide. J. Shellfish Res. 12:423ndash;431. Tettelbach, S. T., D. Barnes, J. Aldred, G. Rivara, D. Bonal, A. Saavedra, C., C. Zapata, A. Guerra & G. Alvarez. 1993. Allozyme Weinstock, C. Fitzsimons-Diaz, J. Thiel, M. C. Cammarota, A. variation in European populations of the . Mar. Stark, K. Wejnert, R. Ames & J. Carroll. 2010. Utility of high- Biol. 115:85–95. density plantings in bay scallop, Argopecten irradians irradians, Schneider, S. & L. Excoffier. 1999. Estimation of past demographic restoration. Aquacult. Int. DOI: 10.1007/s10499-010-9388-6. parameters from the distribution of pairwise differences when the Tettelbach, S., C. Smith, J. E. Kaldy, T. W. Arroll & M. R. Denson. mutation rates vary among sites: application to human mitochon- 1990. Burial of transplanted bay scallops Argopecten irradians drial DNA. Genetics 152:1079–1089. irradians (Lamarck, 1819) in New York. J. Shellfish Res. 18:47–58. Schneider, S., D. Roessli & L. Excoffier. 2000. Arlequin version 2.0A: Tettelbach, S. T. & P. Wenczel. 1993. Reseeding efforts and the status of software for population genetic data analysis. Geneva: Genetics and bay scallop Argopecten irradians (Lamarck, 1819) populations in Biometry Laboratory, University of Geneva. Available at: http:// New York following the occurrence of ‘‘brown tide’’ algal blooms. cmpg.unibe.ch/software/arlequin3. J. Shellfish Res. 122:423–431. Selander, R. K. 1970. Behavioral and genetic variation in natural Thayer, G. W. & H. H. Stuart. 1974. The bay scallop makes its bed of populations. Am. Zool. 10:53–66. seagrass. Mar. Fish. Rev. 36:27–30. Selander, R. K., R. H. Smith, S. Y. Yang, W. E. Johnson & J. B. Gentry. Wakida-Kusunoki, A. 2009. The bay scallop, Argopecten irradians 1971. Biochemical polymorphism and systematics in the genus amplicostatus, in northeastern Mexico. Mar. Fish. Rev. 71:17–19. Peromyscus. I. Variation in the old-field mouse (Peromyscus polio- Waller, T. R. 1969. The evolution of the Argopecten gibbus stock notus). Stud. Genet. Austin Texas 7103:49–90. (: Bivalvia), with special emphasis on the tertiary and Seyoum, S., T. M. Bert, A. E. Wilbur, W. S. Arnold & C. Crawford. quaternary species of North America. J. Paleontol. 43:1–125. 2003. Development, evaluation, and application of a mitochondrial Weir, B. S. & C. C. Cockerham. 1984. Estimating F-statistics for the DNA genetic tag for the bay scallop (Argopecten irradians). analysis of population structure. Evolution 38:1358–1370. J. Shellfish Res. 22:111–117. Wilbur, A. E. 1995. Population genetics of the bay scallop, Argopecten Shaklee, J. B. & P. Bentzen. 1998. Genetic identification of stocks of irradians (Lamarck): an analysis of geographic variation and the marine fish and shellfish. Bull. Mar. Sci. 652:589–621. consequences of self-fertilization. PhD diss., University of Delaware. Shaw, C. R. & R. Prasad. 1970. Starch gel electrophoresis of enzymes: 128 pp. a compilation of recipes. Biochem. Genet. 4:297–320. Wilbur, A. E. & P. M. Gaffney. 1997. A genetic basis for geographic Slatkin, M. 1977. Gene flow and genetic drift in a species subject to variation in shell morphology in the bay scallop, Argopecten frequent local extinctions. Theor. Popul. Biol. 12:253–262. irradians. Mar. Biol. 128:97–105. Slatkin, M. 1985. Rare alleles as indicators of gene flow. Evolution 39: Wilbur, A. E., S. Seyoum, T. M. Bert & W. S. Arnold. 2005. A genetic 53–65. assessment of bay scallop (Argopecten irradians) restoration efforts Slatkin, M. 1993. Isolation by distance in equilibrium and non- in Florida’s Gulf of Mexico coastal waters (USA). Conserv. Genet. equilibrium populations. Evolution 47:264–279. 6:111–122.