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Estuarine-scale genetic variation in the polychaete Hobsonia florida (Ampharetidae; Annelida) in Long Island Sound and relationships to Pleistocene glaciations.

Introduction

Pleistocene glaciations have been effective forces shaping the historical distribution of species on the Atlantic coast of North America (Pielou, 1991; Hewitt, 2000; Wares, 2002). Massive ice sheets rendered continental areas in the northern latitudes uninhabitable, displacing populations and causing local extinctions. Glacial advances interspersed with warm inter-glacial periods resulted in genetic patterns that are not easily or fully understood (Galbreath and Cook, 2004). Moreover, the associated decline in sea level also intensely impacted estuarine biota. The waning sea limited estuarine connectivity and caused the drying up of isolated lagoons, potentially leading to both the promotion of bottleneck events and population differentiation, as has been found for other coastal and marine systems (Chenoweth et al., 1998; Fauvelot et al., 2003).

The northern temperate coasts and estuaries along the Atlantic ocean were formed [approximately equal to]20,000 years ago after the last glacial advance. Marine and estuarine fauna occurring in these areas are probably descendants of populations inhabiting nonglaciated areas that expanded into the newly created estuarine environments once they became inhabitable. However, present-day population distributions and their genetic characteristics may not be due solely to such historical events, as contemporary processes constrained by local adaptation and life-history characteristics (i.e., salinity requirements, mode of dispersal) may act to isolate populations and create effective barriers to gene flow. We employed a population genetic approach to investigate the relative roles of historical and ecological factors in shaping the distribution of a benthic organism, Hobsonia florida (Hartman, 1951), in the Long Island Sound (LIS), a large estuarine system along the northern Atlantic coast of the United States.

Hobsonia florida is a tube-dwelling ampharetid polychaete with nonplanktonic larvae and is endemic to the Atlantic coast of the United States (Zottoli, 1966, 1974). In addition to its native range, introduced populations of H. florida are now prevalent in the Gulf of Mexico and along the west coast of the United States (Cohen et al., 1998; Hogan, 2002). In LIS, this species is typically found only in the brackish water portion of coastal subestuaries where salinities range from 4 to 18 ppt (Zajac and Whitlatch, 1982; Zajac et al., 2000). Zottoli (1966) has also noted its low-salinity habitat distribution. As such, populations are rather localized in the heads of the small subestuaries and embayments where there is an input of fresh water. Larval development in H, florida occurs in the tubes of females (Zottoli, 1974) where mature eggs are fertilized by spermatozoa that enter the female tube from surrounding waters by ciliary currents. The resulting larvae develop within the tube until reaching two to three chaetigers. The larvae then exit the tube through the anterior opening onto the mud surface and immediately construct their own tubes, where development continues (Zottoli, 1974). These two facets of H. florida's ecology may act as strong isolating mechanisms by limiting gene flow and potentially yielding genetically distinct populations.

Population genetic consequences of H. florida life history and ecology must also be examined in light of historical processes associated with the formation of LIS. Glaciations across North America and Europe eliminated many species over a large part of their northern range (Hewitt, 2000), including coastal and estuarine species, and led to the creation of refugial populations of many northern coastal Atlantic taxa either south of the glacial margin or in nonglaciated coastal regions of the Canadian Maritimes and Iceland (Wares, 2002). It is also likely that there were differences in the pattern and extent of such impacts among intertidal and subtidal, and hard- and soft-sediment dwelling species, which may be reflected in their present-day genetic characteristics (e.g., Jennings et al., 2009).

Long Island Sound was formed following glaciation events at the end of the Pleistocene Epoch approximately 10,000 years ago (Lewis and Stone, 1991; Pielou, 1991). The most recent Wisconsin glaciation reached its southernmost extent approximately 21,000 years ago, and Long Island was created in part from the deposition of glacial debris in the form of terminal and recessional moraines (Lewis and Stone, 1991). The retreat of the Wisconsin glacier resulted in substantial runoff that created glacial Lake Connecticut, which was larger in size but similar in location to the modern LIS (Lewis and Stone, 1991). Lake Connecticut existed for about 2000 years during the time period of 17,500 to 15,500 years ago (Uchupi et al, 2001). Severe erosion eventually allowed Lake Connecticut to drain toward the Atlantic (Lewis and Stone, 1991), and within 500 years an extensive fluvial system was well established throughout the exposed lakebed (Lewis and Stone, 1991). Freshwater channels partitioned the lakebed similarly to a modern-day marsh, creating an intricate channel system (Stone et al., 2005). Marine waters may have entered the basin as early as 15,000 years ago; with the continued rise in sea levels, the basin was eventually engulfed, creating a precursor of LIS. During this period it is likely that the fluvial freshwater system of the drained lakebed slowly changed to a spatially complex estuarine environment with the interface between fresh and marine water migrating westward and northward toward the sources of fresh water, creating a highly dynamic system (Lewis and Stone, 1991).

After the last glacial advance, refugial populations of H. florida could have expanded into the LIS basin as early as 15,000 years ago, entering when Atlantic waters first infringed on the exposed lakebed of Lake Connecticut, via the potentially intricate channel system established from the draining of fresh water toward the Atlantic through the eastern end of LIS. Although present-day LIS has a connection to the New York Bight via the East River in the west, this connection likely was not established until 2000 to 5000 years ago (Ralph Lewis, Marine Sciences Institute, University of Connecticut; pers. comm., see also Adams et al., 2006), well after most of present-day LIS was formed. Brackish conditions in the exposed lakebed may have provided a suitable environment for H. florida. This brackish water environment in the LIS basin lasted for at least 3000 years, before rising sea levels eventually transformed it into the modern-day LIS (Lewis and Stone, 1991). As the basin was converted from brackish to estuarine conditions, some H. florida populations were likely extirpated due to increasing water depth and salinity. However, a slow environmental transformation would have allowed other H. florida populations to migrate with the lower salinity habitats being established in river mouths, becoming ancestral to present-day populations now living in the brackish, estuarine areas of rivers and inlets along the LIS coast. These river drainages along the coast of LIS may constitute distinct islands of low-salinity conditions that, amid otherwise inhospitable environments, are capable of sustaining H. florida populations As larvae are nonpelagic, the retreat of H. florida populations into brackish environments over the last 10,000-12.000 years has likely led to a severe reduction or cessation in gene flow.

To increase our understanding of the interplay of historical and contemporary factors shaping the distribution of benthic organisms along the north Atlantic coast of the United States, we investigated patterns of genetic differentiation in H. florida populations along the Connecticut shore of the LIS and inferred formative processes associated with organismal life history and the recent geologic history of the region.

Materials and Methods

Specimen collection

A minimum of 50 individuals of Hobsonia florida were collected between July and August 2004 from each of four sampling sites spanning about 90 km along the Connecticut coast of LIS (Fig. 1). Wheeler Marsh (WM) is the westernmost site at the mouth of the Housatonic River and is approximately 27 km southwest of the sampling locality at the Branford River (BR). The Hammonasset River (HR) is located in Madison, Connecticut, and flows into central LIS, whereas Bakers Cove (BC) is the easternmost LIS site, located in the upper reaches of the Poquonock River in Groton, Connecticut. In addition to the LIS ingroup sampling, two other locations outside of Connecticut were sampled--one at Childs River (CR) on Cape Cod, Massachusetts (n = 36), and the other at Skagit Flats (SF) in Washington State (n = 40). Specimens of H. florida collected from non-LIS sites served, in a broad sense, as comparative outgroups. In all cases, H. florida tubes were isolated from the surface sediment with the use of 1-2 mm sieves, and the samples were transported to the laboratory where organisms were further isolated and extracted from tubes. Individuals were kept for a minimum of 48 h to allow for digestion of stomach contents (Gibson et al., 1999) prior to being individually stored at -80 [degrees]C until utilized for genetic analyses. Of the total number of individuals collected, 20 were selected from each site for genetic analyses.

[FIGURE 1 OMITTED]

Data collection

Total genomic DNA was isolated using the Qiagen DNeasy Tissue Kit (Qiagen, Inc.), using the whole organism as starting material. Individual worms were brought to room temperature and suspended in 180 [micro]1 of tissue lysis buffer and 20 [micro]1 of proteinase K prior to being ground-up with a modified pipette tip until tissue was no longer visible. Subsequent steps of the extraction procedure followed the manufacturer's protocol.

PCR amplification and sequencing of mtDNA COI were used for population level analyses. The universal primers LCO1490, 5'-GGTCAACAAATCATAAAGATATTGG-3' and HC02198, 5'-TAAACTTCAGGGTGACCAAAAAA-TCA-3' (Folmer et al., 1994) were used to amplify mtDNA COI fragments of 687 base pairs. PCRs were performed using an Eppendorf Mastercycler (Eppendorf North America Inc.) in a 25-[micro]1 volume containing: [approximately equal to]20-50 ng of DNA, 10 mmol [l.sup.-1] Tris-HCl (pH 8.3), 50 mmol [l.sup.-1] KCl, 2.5 mmol [l.sup.-1] [MgCl.sub.2], 200 [micro]mol [l.sup.-1] dNTPs, 5 [micro]g bovine serum albumin (BSA), 0.8 [micro]mol [l.sup.-1] of each primer, and 0.5 U of Amplitaq DNA polymerase (PE Biosystems). Reactions were performed following the protocols of Borda and Siddall (2004) by heating to 94 [degrees]C for 5 min, followed by 15 cycles of 94 [degrees]C (45 s), 46 [degrees]C (45 s), and 72 [degrees]C (45 s), then 25 cycles of 94 [degrees]C (20 s), 45 [degrees]C (20 s) and 72 [degrees]C (30 s) and a final extension at 72 [degrees]C (6 min).

PCR fragments were purified with the QIAquick PCR purifcation kit (Qiagen, Inc.) and sequenced using Big Dye terminators on an ABI 3100 DNA sequencer (Applied Biosystems). The forward and reverse sequences were edited and aligned using Sequencher 4.1 (Gene Codes Corporation).

Population genetic analyses

Haplotypic (h; Nei, 1987) and nucleotide ([pi]; Nei and Li, 1979) diversity estimates were calculated for 20 individuals from each of the six populations as executed in ARLEQUIN (Excoffier et al., 2005). Pairwise genetic distances were calculated in PAUP*4.0b10 (Swofford, 2002) assuming the HKY model of nucleotide substitution as selected according to the Akaike information criterion as implemented in Model-test (Posada and Crandall, 1998). Levels of genetic divergence between samples were calculated with the fixation index (PhiST) (Excoffier et al., 1992) as executed in ARLEQUIN (Excoffier et al., 2005). Because the HKY model is not implemented in ARLEQUIN, the more inclusive Tamura-Nei (TrN) (Tamura and Nei, 1993) model was used for all relevant analyses. Significance of PhiST for all possible pairwise population comparisons was assessed using 2000 permutations. Tests for significant geographic structure among populations sampled across the native range were conducted using analysis of molecular variance (AMOVA; Excoffier et al., 1992). Analyses of correlation between pairwise fixation indices and coastal geographical distances (km) were carried out with the Mantel permutation test (Mantel, 1967), in order to determine the extent to which genetic differentiation among localities could be explained by geographic distance. A simplified minimum coastline distance between all pairs of locations and a genetic distance matrix of PhiST were used for the analysis. All of the above tests were executed using ARLEQUIN 3.11 (Excoffier et al., 1992).

Network analysis

Genealogical relationships among mtDNA COI sequences were examined by constructing a haplotype network using the statistical parsimony method of Templeton et al. (1992) as implemented in TCS software, version 1.06 (Clement et al., 2000). This method estimates the maximum number of substitutions to connect two haplotypes parsimoniously with 95% confidence. This approach is particularly useful for inferring relationships among genes with low levels of divergence. The method also assigns each haplotype in the statistical parsimony network an "outgroup probability" based on coalescent theory (Castelloe and Temple-ton, 1994). The likelihood of rooting is calculated as a function of the position of the haplotype in the network, its frequency, and its number of connections with neighbor haplotypes (Castelloe and Templeton, 1994).

Divergence time estimates

Rate homogeneity among H. florida haplotypes was investigated using the maximum likelihood ratio test as carried out in HYPHY (Pond et al., 2005), to test the appropriateness of applying a molecular clock. Rates of change across all paths were assessed with reference to the outgroup Isolda (another ampharetid polychaete). To our knowledge, only one ampharetid COI mutation rate has been published (0.23% per [10.sup.6] years); however, it was calibrated from hydrothermal vent polychaetes (Cheval-donne et al., 2002). On the basis of evidence suggesting that mutation rates are considerably slower in hydrothermal vent organisms and the fact that the reported ampharetid rate is on the very low end reported for animals (Chevaldonne et al., 2002; Little and Vrijenhoek, 2003), we used a more biologically meaningful COI mutation rate of 2.0% per [10.sup.6] years corresponding to the average of estimates for coastal species (based on 15 sister species pairs of Alpheus shrimp) calibrated according to a well-dated barrier, the Isthmus of Panama (Knowlton et al., 1993; Knowlton and Weigt, 1998). Minimum and maximum values from a HKY distance matrix generated in PAUP*4.0b10 (Swofford, 2002) were used to determine a temporal range of divergence estimates for ingroup taxa.

Results

Haplotypic variation

A total of 15 unique haplotypes were identified within the 120 individuals of Hobsonia florida sampled across 687 bp of mtDNA COI. Overall, 29 polymorphic sites were identified, 86% of which were parsimony informative. The number of haplotypes occurring within each population varied from one (SF) to six (HR), with individuals from the four LIS populations exhibiting the greatest haplotypic diversity (Table 1). Ingroup haplotypic diversities ranged from 0.28 to 0.75, whereas outgroups CR and SF exhibited substantially lower levels, 0.10 and 0.00, respectively. Similarly, nucleotide diversity ([pi]) was also larger in the LIS populations, with an average diversity of 0.0014 compared to 0.00005 for the outgroups. All sampled populations maintained at least one private haplotype ([P.sub.HAP), and the average number of [P.sub.HAP) in LIS populations was 2.75.
Table 1

Hobsonia florida mitochondrial COI genetic variation at each sampling
site

Population    No. of      Haplotypic      Nucleotide       Private
            haplotypes *  diversity,    diversity, [pi]   haplotypes
                          h [dagger]      [dagger]

WM              4         0.60 (0.077)   0.0015 (0.0012)       3
BR              3         0.28 (0.12)    0.0009 (0.0009)       1
HR              6         0.75 (0.076)   0.0022 (0.0015)       5
BC              3         0.53 (0.10)    0.001 (0.0009)        2
CR              2         0.10 (0.10)    0.0001 (0.0003)       1
SF              1         0.00 (0.089)   0.0000 (0.0000)       1

* Results based on 687 base pairs of the mtDNA COI.
[dagger] Standard errors in parentheses for h and [pi] estimates.


Population differentiation

The distribution of haplotype frequencies across populations suggests a high level of genetic distinctiveness among the sampled localities. Of the 15 recovered haplotypes, only two (haplotypes 1 & 6) were shared across sites. Haplotype 1 was found at all LIS sites, occurring as the most common haplotype in three of four localities (Fig. 1). Haplotype 6 was overwhelmingly present in outgroup CR and was also found within a single individual at the BR site. The outgroup SF was the least diverse, maintaining only a single haplotype (haplotype 15); while population HR exhibited the most diversity with six haplotypes, five of which were private.

The AMOVA revealed a highly significant level of genetic heterogeneity among LIS populations (P < 0.001), with 39% of the variance at mtDNA COI attributed to differences among populations (Table 2). These differences became even more pronounced when the analyses included CR (48% among population; data not shown) and both CR and SF (89% among population; Table 2). Pairwise PhiST values ranged from 0.1779 to 0.4891 for LIS sites and were highly significant for all comparisons (P < 0.01) (Table 3).
Table 2

Hierarchical AMOVA between Hobsonia florida populations far mtDNA CO

Population comparisons           Source of  d.f.    % of      P value
                                 variation        variation

LIS sites (WM, BR, HR, BC)         Among      3     38.95     <0.00l
                                   Within    76     61.05     <0.001
                                   Total     79

All sites (including outgroups)    Among      5     89.77     <0.00l
                                   Within   114     10.23     <0.001
                                   Total    119

Table 3

Pairwise PhiST values and corresponding P values, based on mtDNA COI
haplotypes

Population    WM         BR         HR         BC        CR        SF

WM            --
BR          0.2474 **    --
HR          0.4768 **  0.4010 **   --
BC          0.2995 **  0.1779 **  0.4891 **   --
CR          0.6791 **  0.6598 **  0.3418 **  0.7436 **   --
SF          0.9683 **  0.9802 **  0.9552 **  0.9801 **  0.9967 **  --

** Significant at P < 0.01.


Despite the high degree of differentiation among LIS sites, there was no significant correlation with geographical distance (Mantel test, P = 0.638), suggesting that isolation by distance (IBD) does not account for the differences among populations. When the CR population was included, the Mantel test approached significance (P = 0.08), but a model of IBD was still rejected.

In the statistical parsimony analysis of COI variation, haplotypes separated by up to 11 base pair differences had a greater than 95% probability of being connected in a parsimonious fashion. The resulting haplotype network showed a fair amount of phylogeographic structure (Fig. 2). The haplotype network comprises two groups--one consisting of haplotype 15 (outgroup SF), and the other incorporating all the other haplotypes. There were 15 substitutions separating haplotype 15 from its closest relative (haplotype 6), all of which were synonymous changes. The structure of the LIS group is characterized by a high proportion of private haplotypes, radiating from the two most common shared haplotypes (haplotypes 1 and 6). Although haplotype 6 had the highest outgroup probability (0.247), haplotype 1, which was common to all LIS populations, exhibited only a slightly lower value of 0.222.

[FIGURE 2 OMITTED]

Molecular clock

Relative rate constancy of a molecular clock was not rejected at the [alpha] = 0.05 significance level. Using a COI mutation rate of 2% per 106 years, we found that the estimated time of haplotype divergence ranged from approximately 73,000 to 366,000 years.

Discussion

Estuarine-scale genetic differentiation and ecological implications

This study revealed substantial genetic differentiation among relatively proximate populations of Hobsonia folorida in Long Island Sound. Coupled with the low dispersal and habit requirements of the species, this suggests that H. florida potentially exists as a fragmented metapopulation within this estuarine system. Population dynamics and maintenance may be highly localized with effectively little or no interaction among subpopulations. As such, local populations may not be as readily reestablished after severe disturbances or other environmental challenges as are species with higher dispersal potential. Similar results were found by Virgilio et al. (2006) for the nereid polychaete Hediste diversicolor, whose populations along the north Adriatic coast exhibited significant differentiation among sites within estuaries, and by Darling et al. (2004) for populations of the estuarine anemone Nematostella vectensis along the Atlantic coast of the United States. Collectively, these results have implications for our general understanding of biodiversity in estuarine systems specifically, and in the marine environment overall where our understanding of the genetic component of biodiversity is fairly rudimentary and many questions remain (Feral, 2002). Although species richness may be lower in estuaries than in coastal or oceanic environments, the genetic diversity component of biodiversity appears to be significant.

Two additional H. florida populations were sampled as comparative outgroups, both exhibiting substantially lower levels of mtDNA COl variation. The SF outgroup sampled in Puget Sound, Washington, maintained the least diversity, revealing only a single haplotype. This finding is likely due to a founder event, as H. florida was first recorded on the Pacific Coast in the Puget Sound region in 1940 (Cohen et &L, 1998). Our results are consistent with the general expectation that founder populations exhibit lower genetic diversity than anticipated, due to the non-equilibrium state of gene flow and genetic drift (Wares, 2002). In general, H. florida is a successful invasive species, with populations now established in estuaries across all three West Coast states (Cohen et al., 1998; Ray, 2005a, b). Theoretically, potential source populations could be identified by haplotypic comparisons with native populations. The single haplotype identified at SF was not present in any LIS populations, suggesting that the source population was not from the LIS region. The Childs River population also had very little genetic variation relative to the LIS populations, and was dominated by one haplotype that was shared by only BR in LIS (Figs. 1 and 2). The observed pattern of haplotype sharing between CR and LIS populations may have been shaped by a variety of factors including founder events across the newly forming southern New England coast from populations that already had a high degree of spatial genetic variation as a result of repeated glaciations (see below). Differences may have been further compounded by subsequent isolation and drift as conditions became more saline (see below).

Contemporary versus historical processes

Intraspecific patterns of genetic variation can arise from historic or contemporary evolutionary processes (Avise et al., 1987). For example, a strong correlation between genetic and geographical distance might be expected when contemporary gene flow is the main evolutionary force shaping the variation (Durand et al., 2005). In this study, there was no correlation between genetic differentiation and geographical distances among LIS populations (based on the Mantel test), suggesting that patterns of contemporary gene flow were not the predominant factor shaping observed variation within LIS. Our results for H. florida are consistent with those from previous studies that have suggested that isolation by distance may not be strong in organisms that have low dispersal (Miller, 1997; Peterson and Denno. 1998; Darling et al., 2004), particularly when coupled with a narrow habitat niche (also see Kelly et al., 2006). High levels of genetic differentiation among marine and estuarine populations appear to be typically associated with reduced dispersal (e.g., Hess et al., 1988; Hunt, 1993; Hellberg, 1996; Watts and Thorpe, 2006). This life-history factor may be a strong determinant of estuarine and regional population genetic structure via its interaction with historical processes that alter coastal landscapes and seascapes over geologic time, and contemporary events (e.g., changes in salinity regimes or increasing human impacts) in the habitats where the populations reside (e.g., Durand et al., 2005).

We applied a molecular clock to gain additional insights into the relative timing of the observed diversification of H. florida in LIS. Unfortunately, due to the lack of a fossil record, the only ampharetid mtDNA COI clock available was developed in vent species calibrated according to the time that the Farallon-Pacific Ridge subducted under the North-American Plate (Chevaldonne et al., 2002). Given the known bias in mutation rates in hydrothermal vent versus non-vent organisms (Chevaldonne et al., 2002; Little and Vrijenhoek, 2003), we employed a clock for coastal Alpheus shrimp species calibrated based on the well-dated formation of the Isthmus of Panama. Based on an average rate of 2% per million years, the observed mtDNA COI variation in H. florida haplotypes reflects a time period between 73,000 and 366,000 years ago. However, like all clocks not developed for the specific system under study, the employed rate may not necessarily be accurate for LIS H. florida. as there can be considerable variation among even closely related taxa (Thomas et al., 2006). Nevertheless, a minimum timing of 73,000 years may be relatively accurate, as others have found that significant genetic divergence (as observed in H. florida) in Atlantic intertidal, estuarine, and some marine species has likely developed prior to the most recent glacial maximum [approximately equal to]20,000 years ago (Palumbi and Kessing, 1991; Wares and Cunningham, 2001; Wares, 2002; Durand et al., 2005; Jolly et al., 2006).

The present-day genetic patterns found in LIS are consistent with our knowledge of the habitat requirements and life history of H. florida and can be further understood in relation to the geologic history of the region. Estuarine habitats present complex gradients in physical and chemical conditions that can act to isolate populations to various degrees (Bilton et al., 2002). particularly in species such as H. florida that have low dispersal potential and narrow salinity requirements. With repeated glaciations, coastal landscapes along the northwest Atlantic, including their component estuaries and marsh systems, have undergone numerous geomorphologic and geographic changes. Although any specific estuary has an ephemeral geological history, these types of coastal environments have always existed at the margins of glacial and interglacial coastlines, likely resulting in extensive fragmentation and coalescence of populations.

As sea level rose after the last glaciation, southern H. florida populations probably underwent their most recent range expansions, transporting existing genetic variation to previously glaciated areas. When LIS began to develop [approximately equal to] 15,000 years ago, ocean waters entered the extensively channeled basin (Lewis and Stone, 1991) and formed a widespread shallow, brackish environment where H. florida may have existed as a relatively less subdivided population. However, as conditions became more saline, H. florida would have been forced to migrate up the channel system into the present day sub-estuaries along the LIS shore. The retreat inland likely constituted a series of founder events of mixed genetic lineages into individual sub-estuaries. Given the fact that H. florida has nonpelagic larvae, each river basin may constitute an island of habitat conditions suitable for survival. As gene flow would not be able to counteract drift, populations have been free to diverge up to the present. This scenario, consistent with the genetic data, suggests that current population genetic structure of H. florida in LIS has been strongly influenced by a repeated series of glacially mediated vicariance events that likely occurred throughout the Pleistocene, leading to periodic population isolation and mixing, further mediated by low dispersal and specific habitat requirements.

Future work

Prior to advances in genetic techniques, it was generally assumed that genetic divergence and speciation in marine macroinvertebrates was low, but it is becoming increasingly recognized that there are a variety of mechanisms by which variation is generated in such systems (Palumbi, 1994; Kruse et al., 2003). Our study contributes to a growing body of literature suggesting that biological and genetic diversity in marine and estuarine habitats is actually much higher than previously thought. Despite our findings of surprisingly high hapiotypehaplotype variation on such a fine scale, our ability to infer the relative roles for historic and contemporary processes was likely constrained by our choice of molecular marker. The mtDNA COI variation exhibited strong signatures reflecting historical population structure and processes. To further assess the role of contemporary gene flow (or the lack thereof) in shaping the distribution of population genetic variation of H. florida in LIS, future research should include a thorough sampling and analysis of recently developed microsatellite markers for this species (Olson et al., 2006). This work will be instrumental in understanding the extent to which nondispersive reproduction and low salinity requirements potentially isolate populations of H. florida. Furthermore, additional sampling of populations along the south shore of LIS and south of the glacial margin is necessary to directly assess hypotheses related to the role of refugia as potential sources of extant populations in LIS. Sampling of additional populations on and north of Cape Cod would also help in identifying the larger scale geographic patterns of genetic variation in this species, and would provide further insights as to how post-glacial species expansions may have affected these geographic patterns in comparison to current potential isolating mechanisms (see, for example, Jennings et al., 2009). It is becoming increasingly recognized that estuarine fauna possess patterns of genetic variation that reflect complex population histories (Bilton et al., 2002). Consequently, much remains to be discovered about how the distribution of genetic diversity in these systems is related to the ecology of estuarine organisms.

Acknowledgments

This work was supported by grants from the Lerner-Gray Fund for Marine Research, the Scientific Research Society, and the Connecticut Sea Grant College Program. The authors thank Donna Cook for collecting the Childs River samples and for assistance with sample collection in LIS, and David Schull for collecting the Skagit Flat samples. The authors further acknowledge Adalgisa Caccone, who helped facilitate genetic data collection. The comments and suggestions of two anonymous reviewers greatly improved the manuscript.

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MARY ANN OLSON (1), *, ROMAN N. ZAJAC (1), [dagger], AND MICHAEL A. RUSSELLO (2)

(1) Department of Biology and Environmental Science, University of New Haven, 300 Boston Post Road, West Haven, Connecticut 06516; and (2) Department of Biology and Centre for Species at Risk and Habitat Studies, University of British Columbia Okanagan, 3333 University Way, Kelowna, British Columbia V1V 1V7, Canada

Received 5 December 2008; accepted 25 March 2009.

* Present address: Ecoscape Environmental Consultants, 102-450 Neave Court, Kelowna. British Columbia VIV 2M2, Canada.

[dagger] To whom correspondence should be addressed. E-mail: rzajac@newhaven.edu

Abbreviations: AMOVA, analysis of molecular variance; BC. Bakers Cove; BR, Branford River: COI. cytochrome oxidase I; CR, Childs River; HR. Hammonasset River; LIS, Long Island Sound; SF, Skagit Flats; WM, Wheeler Marsh.
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Author:Olson, Mary Ann; Zajac, Roman N.; Russello, Michael A.
Publication:The Biological Bulletin
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Date:Aug 1, 2009
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