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Research Projects

Diversification at a zoogeographic province boundary

Diversification along estuarine gradients

Non-model population genomics in highly polymorphic marine invertebrates

Conservation Genetics:

- Invasive Marine Parasites

- Supportive Breeding of Eastern Oysters in Chesapeake Bay

- Effective Population Size of Eastern Oysters in St. Lucie Estuary, Florida

- Quantifying Hard Clam Larval Abundance in Support of Restoration Efforts in Great South Bay, NY

Diversification at a zoogeographic province boundary

Clustered species range limits in eastern Florida represent a widely recognized boundary between warm-temperate and sub-tropical faunistic provinces. A compression of latitudinal thermoclines along the Florida coast implicates climate as a driving force shaping this community transition, but few studies have directly compared the importance of climate versus hydrographic dispersal barriers or species interactions. The eastern oyster, Crassostrea virginica, is nearly- continuously distributed around Florida, providing an exceptional intraspecific model for distinguishing the relative importance of pre- and postsettlement mechanisms structuring estuarine populations across the province boundary in eastern Florida. A major goal in the Hare lab, currently funded by NSF, is to understand the biotic and abiotic factors that maintain population differentiation in the oyster, and ultimately to test the generality of these influences on other codistributed species.

Hare & Avise 1996 Fig. 1. Eastern oyster allelic frequency clines for three polymorphisms from Massachusetts to Louisiana (far right), an expanded view from Georgia to Miami (middle), and a map of Florida indicating collection localities along eastern Florida.

Much of the eastern oyster genetic cline in Florida has been stable for the past dozen years (~6 generations; K. Cammen senior thesis). Both dispersal barriers and selection could be maintaining this steep genetic cline. Oysters only occur in the hydrographically semi-closed lagoons behind barrier beaches in eastern Florida, where larval dispersal is predicted to be small-scale, bidirectional and follow a stepping-stone pattern. However, if larvae are flushed out and re-enter lagoons through barrier island inlets, then coastal currents may generate a leap-frog pattern of dispersal. Dispersal directionality could result from larval advection by coastal currents and be a particularly important aspect of cline maintenance, and province boundaries in general, because asymmetrical gene flow can steepen clinal variation and truncate species ranges along a selection gradient (Hare et al. 2005). Multilocus assignment tests on newly-settled oysters are being used to measure contemporary dispersal distances and direction without recourse to equilibrium theory. Analysis of thousands of 2007 and 2009 recruits along eastern Florida provide no support for regional dispersal across the step cline (Hare et al. in prep).

The spatial scale of population structure is determined by a balance between dispersal-mediated connectivity, genetic drift, and spatially variable selection. Along the eastern Florida ecotone, oyster dispersal patterns are being interpreted in combination with the spatial pattern and magnitude of selection as measured by post-settlement cohort analyses and reciprocal transplants. Hatchery-related studies involving oyster spawning and culture are being conducted in collaboration with John Scarpa at Harbor Branch Oceanographic Institute. Martha Burford is conducting experiments to distinguish three models by which selection may maintain the oyster cline, (1) hybrid unfitness relative to parentals regardless of habitat, (2) habitat-specific hybrid unfitness, and (3) bounded hybrid superiority, with hybrid success constrained to intermediate habitats. She is measuring fitness-related traits including growth rate, survivorship, rate of reproductive maturation, and infection intensity of a major protozoan parasite, Perkinsus marinus. Reproductive barriers were also investigated by Haibin Zhang with in vitro tests of differential cross-fertilization efficiency (Zhang et al. 2010).

At present, the strongest evidence for divergent selection across the oyster cline comes from a genomic screen of AFLP loci in which locus-specific F_ST estimates were used to calculate a genomic mean F_ST. A null distribution of F_ST was then simulated under neutral drift given the empirical mean F_ST ( Murray and Hare 2006). Nonequilibrium and equilibrium simulations were compared to show that the expected neutral variance in F_ST around the estimated genomic mean was unaffected by secondary contact history. In a comparison of one population each from the Atlantic and Gulf of Mexico (representing the two tails of the eastern Florida oyster cline), 1.4% of polymorphic AFLP loci had F_ST estimates outside the simulated 99th quantile for F_ST. A similar test applied to previously-published codominant oyster loci identified only one outlier locus (3.7%). Thus, our genomic screens suggest that a small portion of the genome is shaped by divergent selection. Additional evidence for divergent selection comes from the reciprocal transplant - Martha Burford found that progeny of NxN and SxS crosses showed significant local adaptation (genetic x environment interaction effects) with respect to juvenile survival, rate of reproductive maturation and pre-spawn wet meat weight (Burford et al. in prep).

Diversification along estuarine gradients

Acartia tonsa is a seasonally dominant species of estuarine copepod along the eastern U.S. coast. Enormous populations of this species graze phytoplankton in the water column and provide important prey for fish. As with all zooplankton with a high potential for long-distance dispersal mediated by hydrographic currents, allopatric differentiation is only expected at the largest of geographic scales. We have found several deeply divergent mitochondrial DNA lineages within A. tonsa sampled from Chesapeake Bay. While deep mitochondrial lineages were previously reported within A. tonsa, Gang Chen has shown that they represent cryptic, reproductively isolated species based on genealogical concordance across mitochondrial and nuclear loci (Chen and Hare 2008).

Cryptic species are not novel or surprising in marine invertebrates any more, but in this case their parapatric distribution along salinity gradients indicates that speciation resulted in ecological niche diversification. Gang has found one lineage primarily at salinities below 11 ppt, while other lineages mostly occur above that threshold. Phylogeographic analyses indicate that the low-salinity lineage is relatively ancient, has a center of origin in subtropical waters, and sequentially colonized low-salinity habitat patches in the eastern U.S. estuaries from south to north (Chen and Hare 2011). The more discontinuous habitat used by the low salinity lineage has promoted allopatric speciation at spatial scales over which the high salinity lineage is only modestly subdivided. Thus, among estuarine species, the apparent propensity for dispersal is far less informative about the potential for diversification than is the discreteness of preferred habitat.

Functional genomic diversification within single estuaries

One mechanism by which marine species with larval dispersal cope with highly heterogeneous and unpredictable environments is through high fecundity and sexually generated zygotic diversity. George Williams (1975) described this life history strategy as the ‘elm/oyster model’ and recognized that meiotic recombination generates abundant zygotic diversity within each family cohort so that, on average, a few genotypes have high fitness in the face of a myriad of selective agents acting during dispersal and after settlement. How much functional diversification results from differential survival at different depths or salinities? What are the implications of this life history strategy for hatchery-based restoration methods that are commonly applied to shellfish? Can hatchery-produced restoration oysters be expected to produce sufficiently diverse cohorts to supplement subsequent recruitment, or are their ‘ecosystem services’ largely somatic on the reef where they are planted, rather than reproductive? Two projects in the Hare Lab are investigating these questions using genomic approaches. Laura Eierman is a PhD student studying the functional genetics of osmoregulatory tolerances in the eurhaline oyster, Crassostrea virginica. Laura is experimentally measuring the salinity tolerance of larvae after reciprocal conditioning broodstock from high- and low-salinity portions of an estuary. She will be examining differentiation of oyster populations at the two salinity extremes in terms of differential expression and divergent allele frequencies. Second, funding is pending for similar functional genomic investigations related to oyster restoration in NY/NJ Harbor Estuary.

Non-model population genomics in highly polymorphic marine invertebrates

Functional genomics will become widely available for ecological and conservation studies when next generation sequencing of representational genomic libraries can efficiently genotype hundreds of individuals for thousands of homologous loci and when these polymorphisms can be accurately scored even in the absence of a reference genome. Highly polymorphic species, such as many marine invertebrates and fishes, offer analytical advantages because even short sequence reads will often provide haplotype data (reveal linked polymorphisms), but high polymorphism also generates bioinformatic challenges when there is no reference genome sequence to guide an assembly. In collaboration with Marie Nydam, we have initiated a sensitivity analysis of methods, at both library preparation and sequence assembly stages, that are potentially advantageous for population genomics without a reference genome. In this case, in order to have the benefit of a reference genome for method development in a highly polymorphic species, we have focused on population samples of the solitary sea squirt, Ciona savignyi.

Invasive Marine Parasites

Macro- and microparasites in marine systems can have strong effects on community structure and devastating consequences for fisheries. Genetic markers are an under-utilized tool for more thoroughly describing parasite diversity, inferring the spatial scale of parasite gene flow, and tracing the circumstances promoting changes in parasite’s geographic range or host spectrum. Work in the Hare lab is currently focused on these goals with two highly-virulent parasites. Eastern oysters suffer severe mortality from epidemic infections by the protist Perkinsus marinus. This parasite was originally known from the Gulf of Mexico north to Chesapeake Bay, but infections have now become common and severe as far north as New England due to an apparent range expansion. The P. marinus genome is being sequenced as a representative of basal apicomplexans, yet very little is known about its population biology, including the relative importance of clonal versus sexual reproduction in nature. Almost all information on this species derives from clonal laboratory cultures. In the Hare lab we assay genetic variation in wild strains by using species-specific primers to PCR amplify P. marinus DNA from genomic DNA of infected oysters. Using microsatellite markers to measure genetic diversity, Peter Thompson has found evidence for a history of sexual recombination within local populations, as well as episodes of recent clonal expansion (Thompson et al 2011). He is testing the generality of this result across the range of P. marinus, analyzing phylogeographic structure from New England to Texas, and collecting DNA sequence data to analyze the evolutionary history of this parasite. Our genetic inferences about P. marinus population processes will inform strategies to control the damage this parasite exerts on natural restored and aquaculture oyster populations.

Research efforts in the Hare lab have also focused on tracing the invasion history of a rhizocephalan (barnacle) parasite that castrates estuarine mud crabs. Loxothylacus panopaei is endemic to the Gulf of Mexico and southeastern Florida, but was introduced to Chesapeake Bay in 1964. Inken Kruse has found that the invasive Chesapeake population is (1) genetically quite distinct from endemic populations in southeastern Florida, (2) infects a different spectrum of broadly-distributed mud crab species than the parasite in the endemic range, and (3) is expanding its range southward into Florida such that contact with the endemic parasite population is expected over the next few years (Kruse and Hare 2007). More extensive sampling of Loxothylacus parasites within the Gulf of Mexico indicates that two cryptic species of Loxothylacus with different preferred hosts occur there, and only one invaded Chesapeake Bay (Kruse et al. in revision). Results of these studies help clarify the source population for the Chesapeake invasion and reject the previous conclusion that the parasite’s preferred hosts had changed after invasion. Thus, flexibility of host spectrum appears not to have contributed to invasion success, whereas tolerance of a temperate climate most likely was key.

Eastern Oysters in Chesapeake Bay

The goal of our research on C. virginica in Chesapeake Bay is to test the effectiveness of current restoration efforts and facilitate those efforts by estimating the spatial scale of larval dispersal. This work is collaborative with Kim Reece and Mark Luckenbach at the Virginia Institute of Marine Science and funded through the NOAA/SeaGrant national Oyster Disease Research Program. Because most Chesapeake oyster restoration efforts in Virginia after 2000 involved the planting of selectively-bred disease tolerant C. virginica oysters, an additional goal of our research is to evaluate the genetic health of Chesapeake oyster populations and the impact of introgression from restoration plantings.

Spatial scale of dispersal: Using microsatellites to test for temporal and spatial subdivision within Chesapeake Bay, Colin Rose found no evidence for temporal heterogeneity and subtle spatial differentiation consistent with isolation by distance (IBD). This IBD pattern could be an evolutionary equilibrium emerging from stepping-stone dispersal after post-Pleistocene colonization of Chesapeake Bay. Alternatively, anthropogenic activities related to fisheries management and restoration may be creating or eroding an IBD pattern. An evolutionary interpretation seems more parsimonious for several reasons (Rose et al. 2006), and work is proceeding to distinguish these alternatives.

Isolation by distance in Chesapeake Bay oysters revealed by plotting pairwise genetic divergence against logarithm of aquatic distance (Rose et al. 2006, population samples shown with black dots in inset map)

Restoration efficacy: One such effort includes our direct estimates of oyster dispersal distances based on genetic tags. The 'tagging' is a consequence of genetic drift and selection during breeding of C. virginica for disease tolerance, and further bottlenecks imposed during each hatchery spawn of selected-strain broodstock needed to produce juveniles for restoration plantings (Hare and Rose in prep.). In a tributary where tagged oysters were planted on a single reef, we have traced dispersal of their progeny by using assignment tests on the multilocus genotypes of newly-settled juveniles collected throughout the tributary. In 2002, a year with the high overall recruitment, almost 1600 oyster juveniles were collected and genotyped for eight microsatellite loci and mitochondrial DNA. We estimated that during that year five to ten percent of juveniles originated from parents at the point source (Hare et al. 2006). Sampling is not yet sufficient to characterize average dispersal distances, but this overall level of population enhancement is both good and bad news. On the good side, substantial recruitment enhancement was accomplished and this would not have been discernable without genetic analysis. On the other hand, the enhancement was much lower than anticipated, precipitating critical evaluation of untested assumptions underlying restoration methods.

Genetic health: Restoration efforts in Virginia waters of Chesapeake Bay have previously focused on supportive breeding using artificially selected, disease tolerant C. virginica brood stock to combat high mortalities from parasitic diseases. Supportive breeding reduces early juvenile mortality in the hatchery in order to release millions of “seed” oysters from relatively few brood stock. This procedure can rapidly increase local census population size but also risks increasing inbreeding and lower genetic diversity in the target populations. Several critical population parameters must be estimated to evaluate these risks. We recently used genetic data to provide the first estimates of genetically effective population size in wild oyster populations as well as in the hatchery brood stock used for restoration, and estimated the annual contribution to recruitment made by restoration seed oysters (Hare et al. 2006; Rose et al. 2006). Based on well known theoretical models, these estimates indicate that overall genetic diversity will be dramatically reduced (50 – 90%), and inbreeding depression is expected to result, from the 5-10% supportive breeding contribution estimated for the Great Wicomico River, Virginia restoration in 2002. The selectively-bred DEBY strain of oyster previously used to produce restoration seed is highly inbred (Hare and Rose in prep.). Even while these restoration efforts might be increasing oyster abundance, the use of inbred brood stock will degrade the genetic health of local populations and limit the prospects for long-term restoration success. Our research helped support a recommendation to stop using artificially selected strains for restoration except under experimental conditions when distinctly “tagged” hatchery seed are needed to test the efficacy of new restoration practices. Selectively-bred disease tolerant oyster strains are expected to find continued profitable use for aquaculture.

Effective Population Size of Eastern Oysters in St. Lucie Estuary, Florida

One goal of the comprehensive Florida everglades restoration plan is to expand the 117 acres of existing oyster reef in St. Lucie Estuary (SLE), in eastern Florida, to 900 acres of functional reef (65% of historic coverage). Oyster habitat is degraded in SLE as elsewhere, but additional severe oyster mortality in the SLE has resulted from flood water releases from Lake Okeechobee. Because each acre of restored reef can cost hundreds of thousands of dollars, and hatchery-based seeding would further elevate costs, it is essential that reef building be optimized to maximize the potential for natural oyster recruitment and population growth. With funding from the U.S. Fish and Wildlife Coastal Program, in collaboration with researchers at the Florida Fish & Wildlife Research Institute, we are initiating studies to determine the origin of new oyster recruits in SLE and test whether they derive from a well-mixed larval pool. We will test for recruitment asymmetries by comparing adult abundance, intensity of recruitment, and the genetically effective size of source populations contributing recruits to the upper, middle and lower estuary. Our study will integrate demographic and genetic measures of population biology to test spatial models of recruitment supply and demand. Our results will help prioritize reef building efforts within the currently defined mesohaline window of optimum oyster habitat, or potentially motivate an expansion of this restoration window to prioritize reef building in the upper or lower estuary if these are found to be important SLE recruitment source populations.

Quantifying Hard Clam Larval Abundance in Support of Restoration Efforts in Great South Bay, NY

Mercenaria mercenaria, also known as hard clams or quahogs, were historically abundant in New York bays and supported large recreational and commercial fisheries on Long Island. New York landings of quahogs in 1976 accounted for 58 percent of total U.S. hard clam harvest, but by 1985 were down 76 percent with a loss of tens of millions of dollars in local economic activity. Recreational harvests in many coastal towns also have been affected. Healthy quahog populations can affect their estuarine environment by increasing water clarity, improving benthic habitat, and facilitating nutrient cycling. These ecosystem services have the potential to synergistically improve the overall health of New York bays and estuaries if a few key species can be restored to large population sizes. Restoration of quahog populations in Great South Bay is uniquely facilitated by an 11,500 acre donation of underwater habitat to The Nature Conservancy – a landmark conservation transaction including over one fifth of the historically productive clam habitat in Great South Bay. Some of the leading theories proposed to explain recent quahog declines focus on aspects of reproduction and larval biology that are very difficult to study. Managers need methods of sampling and analyzing larvae with greater spatial and temporal resolution than is currently possible. The primary obstacle to monitoring larval abundance has been the laborious and slow task of sorting out microscopic larvae from samples and distinguishing various bivalve species. To address this obstacle, we have developed a high-throughput method of measuring hard clam larval abundance using quantitative PCR. With Hatch (USDA) funds and collaborators Carl LoBue (TNC) and Dianna Padilla (Stony Brook Univ.), we are applying the qPCR method to quantify the number of hard clam larvae present in bulk, size-sorted plankton samples from Great South and Peconic Bays, NY.