Genetic diversity across the mitochondrial genome of eastern oysters (Crassostrea virginica) in the northern Gulf of Mexico.

The eastern oyster, Crassostrea virginica, is divided into four populations along the western North Atlantic, however, the only published mitochondrial genome sequence was assembled using one individual in Delaware. This study aimed to (1) assemble C. virginica mitochondrial genomes from Texas with pooled restriction-site-associated DNA sequencing (ezRAD), (2) evaluate the validity of the mitochondrial genome assemblies including comparison with Sanger sequencing data, and (3) evaluate genetic differentiation both between the Delaware and Texas genomes, as well as among three bays in Texas. The pooled-genome-assembled-genomes (PAGs) from Texas exhibited several characteristics indicating that they were valid, including elevated nucleotide diversity in non-coding and the third position of codons, placement as the sister haplotype of the genome from Delaware in a phylogenetic reconstruction of Crassostrea mitochondrial genomes, and a lack of genetic structure in the ND4 gene among the three Texas bays as was found with Sanger amplicons in samples from the same bays several years prior. In the comparison between the Delaware and Texas genome, 27 of 38 coding regions exhibited variability between the two populations, which were differentiated by 273 mutations, versus 1-13 mutations among the Texas samples. Using the full PAGs, there was no additional evidence for population structure among the three Texas bays. While population genetics is rapidly moving towards larger high-density datasets, studies of mitochondrial DNA (and genomes) can be particularly useful for comparing historic data prior to the modern era of genomics. As such, being able to reliably compile mitochondrial genomes from genomic data can improve the ability to compare results across studies.

Genetic and phenotypic variation exhibit both predictable and stochastic patterns across an intertidal fish metapopulation.

Interactions among selection, gene flow, and drift affect the trajectory of adaptive evolution. In natural populations, the direction and magnitude of these processes can be variable across different spatial, temporal, or ontogenetic scales. Consequently, variability in evolutionary processes affects the predictability or stochasticity of microevolutionary outcomes. We studied an intertidal fish, Bathygobius cocosensis (Bleeker, 1854), to understand how space, time, and life stage structure genetic and phenotypic variation in a species with potentially extensive dispersal and a complex life cycle (larval dispersal preceding benthic recruitment). We sampled juvenile and adult life stages, at three sites, over three years. Genome-wide SNPs uncovered a pattern of chaotic genetic patchiness, that is, weak-but-significant patchy spatial genetic structure that was variable through time and between life stages. Outlier locus analyses suggested that targets of spatially divergent selection were mostly temporally variable, though a significant number of spatial outlier loci were shared between life stages. Head shape, a putatively ecologically responsive (adaptive) phenotype in B. cocosensis also exhibited high temporal variability within sites. However, consistent spatial relationships between sites indicated that environmental similarities among sites may generate predictable phenotype distributions across space. Our study highlights the complex microevolutionary dynamics of marine systems, where consideration of multiple ecological dimensions can reveal both predictable and stochastic patterns in the distributions of genetic and phenotypic variation. Such considerations probably apply to species that possess short, complex life cycles, have large dispersal potential and fecundities, and that inhabit heterogeneous environments.

Evidence for population genetic structure in two exploited Mekong River fishes across a natural riverine barrier.

Impacts of urban development on aquatic populations are often complex and difficult to ascertain, but population genetic analysis has allowed researchers to monitor and estimate gene flow in the context of existing and future hydroelectric projects. The Lower Mekong Basin is undergoing rapid hydroelectric development with around 50 completed and under-construction dams and 95 planned dams. The authors investigated the baseline genetic diversity of two exploited migratory fishes, the mud carp Henicorhynchus lobatus (five locations), and the rat-faced pangasiid catfish, Helicophagus leptorhynchus (two locations), in the Lower Mekong Basin using the genomic double digest restriction site-associated DNA (ddRAD) sequencing method. In both species, fish sampled upstream of Khone Falls were differentiated from those collected at other sites, and Ne estimates at the site above the falls were lower than those at other sites. This was the first study to utilize thousands of RAD-generated single nucleotide polymorphisms to indicate that the Mekong's Khone Falls are a potential barrier to gene flow for these two moderately migratory species. The recent completion of the Don Sahong dam across one of the only channels for migratory fishes through Khone Falls may further exacerbate signatures of isolation and continue to disrupt the migration patterns of regionally vital food fishes. In addition, H. lobatus populations downstream of Khone Falls, including the 3S Basin and Tonle Sap system, displayed robust connectivity. Potential obstruction of migration pathways between these river systems resulting from future dam construction may limit dispersal, which has led to elevated inbreeding rates and even local extirpation in other fragmented riverine species.

Cryptic Lineages and a Population Dammed to Incipient Extinction? Insights into the Genetic Structure of a Mekong River Catfish.

An understanding of the genetic composition of populations across management boundaries is vital to developing successful strategies for sustaining biodiversity and food resources. This is especially important in ecosystems where habitat fragmentation has altered baseline patterns of gene flow, dividing natural populations into smaller subpopulations and increasing potential loss of genetic variation through genetic drift. River systems can be highly fragmented by dams built for flow regulation and hydropower. We used reduced-representation sequencing to examine genomic patterns in an exploited catfish, Hemibagrus spilopterus, in a hotspot of biodiversity and hydropower development-the Mekong River basin. Our results revealed the presence of 2 highly divergent coexisting genetic lineages which may be cryptic species. Within the lineage with the greatest sample sizes, pairwise FST values, principal component analysis, and a STRUCTURE analysis all suggest that long-distance migration is not common across the Lower Mekong Basin, even in areas where flood-pulse hydrology has limited genetic divergence. In tributaries, effective population size estimates were at least an order of magnitude lower than in the Mekong mainstream indicating these populations may be more vulnerable to perturbations such as human-induced fragmentation. Fish isolated upstream of several dams in one tributary exhibited particularly low genetic diversity, high amounts of relatedness, and a level of inbreeding (GIS = 0.51) that has been associated with inbreeding depression in other outcrossing species. Our results highlight the importance of assessing genetic structure and diversity in riverine fisheries populations across proposed dam development sites for the preservation of these critically important resources.

Genetic patterns in peripheral marine populations of the fusilier fish Caesio cuning within the Kuroshio Current.

AIM: Mayr's central-peripheral population model (CCPM) describes the marked differences between central and peripheral populations in genetic diversity, gene flow, and census size. When isolation leads to genetic divergence, these peripheral populations have high evolutionary value and can influence biogeographic patterns. In tropical marine species with pelagic larvae, powerful western-boundary currents have great potential to shape the genetic characteristics of peripheral populations at latitudinal extremes. We tested for the genetic patterns expected by the CCPM in peripheral populations that are located within the Kuroshio Current for the Indo-Pacific reef fish, Caesio cuning. METHODS: We used a panel of 2,677 SNPs generated from restriction site-associated DNA (RAD) sequencing to investigate genetic diversity, relatedness, effective population size, and spatial patterns of population connectivity from central to peripheral populations of C. cuning along the Kuroshio Current. RESULTS: Principal component and cluster analyses indicated a genetically distinct lineage at the periphery of the C. cuning species range and examination of SNPs putatively under divergent selection suggested potential for local adaptation in this region. We found signatures of isolation-by-distance and significant genetic differences between nearly all sites. Sites closest to the periphery exhibited increased within-population relatedness and decreased effective population size. MAIN CONCLUSIONS: Despite the potential for homogenizing gene flow along the Kuroshio Current, peripheral populations in C. cuning conform to the predictions of the CCPM. While oceanography, habitat availability, and dispersal ability are all likely to shape the patterns found in C. cuning across this central-peripheral junction, the impacts of genetic drift and natural selection in increasing smaller peripheral populations appear to be probable influences on the lineage divergence found in the Ryukyu Islands.

The Genomic Observatories Metadatabase (GeOMe): A new repository for field and sampling event metadata associated with genetic samples.

The Genomic Observatories Metadatabase (GeOMe, http://www.geome-db.org/) is an open access repository for geographic and ecological metadata associated with biosamples and genetic data. Whereas public databases have served as vital repositories for nucleotide sequences, they do not accession all the metadata required for ecological or evolutionary analyses. GeOMe fills this need, providing a user-friendly, web-based interface for both data contributors and data recipients. The interface allows data contributors to create a customized yet standard-compliant spreadsheet that captures the temporal and geospatial context of each biosample. These metadata are then validated and permanently linked to archived genetic data stored in the National Center for Biotechnology Information's (NCBI's) Sequence Read Archive (SRA) via unique persistent identifiers. By linking ecologically and evolutionarily relevant metadata with publicly archived sequence data in a structured manner, GeOMe sets a gold standard for data management in biodiversity science.

Simulations indicate that scores of lionfish (Pterois volitans) colonized the Atlantic Ocean.

The invasion of the western Atlantic Ocean by the Indo-Pacific red lionfish (Pterois volitans) has had devastating consequences for marine ecosystems. Estimating the number of colonizing lionfish can be useful in identifying the introduction pathway and can inform policy decisions aimed at preventing similar invasions. It is well-established that at least ten lionfish were initially introduced. However, that estimate has not faced probabilistic scrutiny and is based solely on the number of haplotypes in the maternally-inherited mitochondrial control region. To rigorously estimate the number of lionfish that were introduced, we used a forward-time, Wright-Fisher, population genetic model in concert with a demographic, life-history model to simulate the invasion across a range of source population sizes and colonizing population fecundities. Assuming a balanced sex ratio and no Allee effects, the simulations indicate that the Atlantic population was founded by 118 (54-514, 95% HPD) lionfish from the Indo-Pacific, the Caribbean by 84 (22-328, 95% HPD) lionfish from the Atlantic, and the Gulf of Mexico by at least 114 (no upper bound on 95% HPD) lionfish from the Caribbean. Increasing the size, and therefore diversity, of the Indo-Pacific source population and fecundity of the founding population caused the number of colonists to decrease, but with rapidly diminishing returns. When the simulation was parameterized to minimize the number of colonists (high θ and relative fecundity), 96 (48-216, 95% HPD) colonists were most likely. In a more realistic scenario with Allee effects (e.g., 50% reduction in fecundity) plaguing the colonists, the most likely number of lionfish increased to 272 (106-950, 95% HPD). These results, in combination with other published data, support the hypothesis that lionfish were introduced to the Atlantic via the aquarium trade, rather than shipping. When building the model employed here, we made assumptions that minimize the number of colonists, such as the lionfish being introduced in a single event. While we conservatively modelled the introduction pathway as a single release of lionfish in one location, it is more likely that a combination of smaller and larger releases from a variety of aquarium trade stakeholders occurred near Miami, Florida, which could have led to even larger numbers of colonists than simulated here. Efforts to prevent future invasions via the aquarium trade should focus on the education of stakeholders and the prohibition of release, with adequate rewards for compliance and penalties for violations.

Demystifying the RAD fad.

We are writing in response to the population and phylogenomics meeting review by Andrews & Luikart (2014) entitled 'Recent novel approaches for population genomics data analysis'. Restriction-site-associated DNA (RAD) sequencing has become a powerful and useful approach in molecular ecology, with several different published methods now available to molecular ecologists, none of which can be considered the best option in all situations. A&L report that the original RAD protocol of Miller et al. (2007) and Baird et al. (2008) is superior to all other RAD variants because putative PCR duplicates can be identified (see Baxter et al. 2011), thereby reducing the impact of PCR artefacts on allele frequency estimates (Andrews & Luikart 2014). In response, we (i) challenge the assertion that the original RAD protocol minimizes the impact of PCR artefacts relative to that of other RAD protocols, (ii) present additional biases in RADseq that are at least as important as PCR artefacts in selecting a RAD protocol and (iii) highlight the strengths and weaknesses of four different approaches to RADseq which are a representative sample of all RAD variants: the original RAD protocol (mbRAD, Miller et al. 2007; Baird et al. 2008), double digest RAD (ddRAD, Peterson et al. 2012), ezRAD (Toonen et al. 2013) and 2bRAD (Wang et al. 2012). With an understanding of the strengths and weaknesses of different RAD protocols, researchers can make a more informed decision when selecting a RAD protocol.

ezRAD: a simplified method for genomic genotyping in non-model organisms.

Here, we introduce ezRAD, a novel strategy for restriction site-associated DNA (RAD) that requires little technical expertise or investment in laboratory equipment, and demonstrate its utility for ten non-model organisms across a wide taxonomic range. ezRAD differs from other RAD methods primarily through its use of standard Illumina TruSeq library preparation kits, which makes it possible for any laboratory to send out to a commercial genomic core facility for library preparation and next-generation sequencing with virtually no additional investment beyond the cost of the service itself. This simplification opens RADseq to any lab with the ability to extract DNA and perform a restriction digest. ezRAD also differs from others in its flexibility to use any restriction enzyme (or combination of enzymes) that cuts frequently enough to generate fragments of the desired size range, without requiring the purchase of separate adapters for each enzyme or a sonication step, which can further decrease the cost involved in choosing optimal enzymes for particular species and research questions. We apply this method across a wide taxonomic diversity of non-model organisms to demonstrate the utility and flexibility of our approach. The simplicity of ezRAD makes it particularly useful for the discovery of single nucleotide polymorphisms and targeted amplicon sequencing in natural populations of non-model organisms that have been historically understudied because of lack of genomic information.

Between tide and wave marks: a unifying model of physical zonation on littoral shores.

The effects of tides on littoral marine habitats are so ubiquitous that shorelines are commonly described as 'intertidal', whereas waves are considered a secondary factor that simply modifies the intertidal habitat. However mean significant wave height exceeds tidal range at many locations worldwide. Here we construct a simple sinusoidal model of coastal water level based on both tidal range and wave height. From the patterns of emergence and submergence predicted by the model, we derive four vertical shoreline benchmarks which bracket up to three novel, spatially distinct, and physically defined zones. The (1) emergent tidal zone is characterized by tidally driven emergence in air; the (2) wave zone is characterized by constant (not periodic) wave wash; and the (3) submergent tidal zone is characterized by tidally driven submergence. The decoupling of tidally driven emergence and submergence made possible by wave action is a critical prediction of the model. On wave-dominated shores (wave height ≫ tidal range), all three zones are predicted to exist separately, but on tide-dominated shores (tidal range ≫ wave height) the wave zone is absent and the emergent and submergent tidal zones overlap substantially, forming the traditional "intertidal zone". We conclude by incorporating time and space in the model to illustrate variability in the physical conditions and zonation on littoral shores. The wave:tide physical zonation model is a unifying framework that can facilitate our understanding of physical conditions on littoral shores whether tropical or temperate, marine or lentic.

Combined analyses of kinship and FST suggest potential drivers of chaotic genetic patchiness in high gene-flow populations.

We combine kinship estimates with traditional F-statistics to explain contemporary drivers of population genetic differentiation despite high gene flow. We investigate range-wide population genetic structure of the California spiny (or red rock) lobster (Panulirus interruptus) and find slight, but significant global population differentiation in mtDNA (ΦST = 0.006, P = 0.001; D(est_Chao) = 0.025) and seven nuclear microsatellites (F(ST) = 0.004, P < 0.001; D(est_Chao) = 0.03), despite the species' 240- to 330-day pelagic larval duration. Significant population structure does not correlate with distance between sampling locations, and pairwise FST between adjacent sites often exceeds that among geographically distant locations. This result would typically be interpreted as unexplainable, chaotic genetic patchiness. However, kinship levels differ significantly among sites (pseudo-F(16,988) = 1.39, P = 0.001), and ten of 17 sample sites have significantly greater numbers of kin than expected by chance (P < 0.05). Moreover, a higher proportion of kin within sites strongly correlates with greater genetic differentiation among sites (D(est_Chao), R(2) = 0.66, P < 0.005). Sites with elevated mean kinship were geographically proximate to regions of high upwelling intensity (R(2) = 0.41, P = 0.0009). These results indicate that P. interruptus does not maintain a single homogenous population, despite extreme dispersal potential. Instead, these lobsters appear to either have substantial localized recruitment or maintain planktonic larval cohesiveness whereby siblings more likely settle together than disperse across sites. More broadly, our results contribute to a growing number of studies showing that low F(ST) and high family structure across populations can coexist, illuminating the foundations of cryptic genetic patterns and the nature of marine dispersal.

There's no place like home: crown-of-thorns outbreaks in the central pacific are regionally derived and independent events.

One of the most significant biological disturbances on a tropical coral reef is a population outbreak of the fecund, corallivorous crown-of-thorns sea star, Acanthaster planci. Although the factors that trigger an initial outbreak may vary, successive outbreaks within and across regions are assumed to spread via the planktonic larvae released from a primary outbreak. This secondary outbreak hypothesis is predominantly based on the high dispersal potential of A. planci and the assertion that outbreak populations (a rogue subset of the larger population) are genetically more similar to each other than they are to low-density non-outbreak populations. Here we use molecular techniques to evaluate the spatial scale at which A. planci outbreaks can propagate via larval dispersal in the central Pacific Ocean by inferring the location and severity of gene flow restrictions from the analysis of mtDNA control region sequence (656 specimens, 17 non-outbreak and six outbreak locations, six archipelagos, and three regions). Substantial regional, archipelagic, and subarchipelagic-scale genetic structuring of A. planci populations indicate that larvae rarely realize their dispersal potential and outbreaks in the central Pacific do not spread across the expanses of open ocean. On a finer scale, genetic partitioning was detected within two of three islands with multiple sampling sites. The finest spatial structure was detected at Pearl & Hermes Atoll, between the lagoon and forereef habitats (<10 km). Despite using a genetic marker capable of revealing subtle partitioning, we found no evidence that outbreaks were a rogue genetic subset of a greater population. Overall, outbreaks that occur at similar times across population partitions are genetically independent and likely due to nutrient inputs and similar climatic and ecological conditions that conspire to fuel plankton blooms.

Morphological and behavioral evidence for adaptive diversification of sympatric Hawaiian limpets (Cellana spp.).

The endemic Hawaiian limpets (Cellana exarata, Cellana sandwicensis, and Cellana talcosa), reside at different elevations on wave-exposed rocky shores and comprise a monophyletic lineage that diversified within Hawai'i. Here, I report phenotypic differences in shell, soft tissue, and behavioral characters among these limpets and discuss their potential utility in exploiting their respective niches. The high-shore limpet, C. exarata, is characterized by a tall round shell, short mantle tentacles, and long evasion distance when confronted by a predatory gastropod. The mid-shore limpet, C. sandwicensis, is characterized by a shorter oblong shell, long mantle tentacles, and a short evasion distance when confronted by a predatory snail. The low-shore, shallow-subtidal limpet, C. talcosa, is characterized by a flat shell that is thin in juveniles and disproportionately massive in large adults (relative to the other two species), and mantle tentacles of varying lengths (some individuals exhibit short tentacles, some long). These species-specific suites of characters are likely to confer specific fitness advantages on the high shore (C. exarata) where thermal and desiccation stress is severe, on the mid shore (C. sandwicensis) where hydrodynamic forces are severe, and on the low-shallow subtidal shore (C. talcosa) where pelagic predators have free access to the limpets. These data add to the growing body of evidence for adaptive diversification and speciation in the Hawaiian Cellana, and in marine species in general.

Diversification of sympatric broadcast-spawning limpets (Cellana spp.) within the Hawaiian archipelago.

Speciation remains a central enigma in biology, and nowhere is this more apparent than in shallow tropical seas where biodiversity rivals that of tropical rainforests. Obvious barriers to gene flow are few and most marine species have a highly dispersive larval stage, which should greatly decrease opportunities for speciation via geographic isolation. The disparity in the level of geographic isolation for terrestrial and marine species is exemplified in Hawai'i where opportunities for allopatric speciation abound in the terrestrial realm. In contrast, marine colonizers of Hawai'i are believed to produce only a single endemic species or population, due to the lack of isolating barriers. To test the assertion that marine species do not diversify within Hawai'i, we examine the evolutionary origin of three endemic limpets (Cellana exarata, C. sandwicensis and C. talcosa) that are vertically segregated across a steep ecocline on rocky shores. Analyses of three mtDNA loci (12S, 16S, COI; 1565bp) and two nDNA loci (ATPSß, H3; 709bp) in 26 Indo-Pacific Cellana species (N=414) indicates that Hawai'i was colonized once ∼3.4-7.2Ma from the vicinity of Japan. Trait mapping demonstrates that high-shore residence is the ancestral character state, such that mid- and low-shore species are the product of subsequent diversification. The Hawaiian Cellana are the first broadcast-spawners demonstrated to have speciated within any archipelago. The habitat stratification, extensive sympatry, and evolutionary history of these limpets collectively indicate a strong ecological component to speciation and support the growing body of evidence for non-allopatric speciation in the ocean.

Variation in Symbiodinium ITS2 sequence assemblages among coral colonies.

Endosymbiotic dinoflagellates in the genus Symbiodinium are fundamentally important to the biology of scleractinian corals, as well as to a variety of other marine organisms. The genus Symbiodinium is genetically and functionally diverse and the taxonomic nature of the union between Symbiodinium and corals is implicated as a key trait determining the environmental tolerance of the symbiosis. Surprisingly, the question of how Symbiodinium diversity partitions within a species across spatial scales of meters to kilometers has received little attention, but is important to understanding the intrinsic biological scope of a given coral population and adaptations to the local environment. Here we address this gap by describing the Symbiodinium ITS2 sequence assemblages recovered from colonies of the reef building coral Montipora capitata sampled across Kane'ohe Bay, Hawai'i. A total of 52 corals were sampled in a nested design of Coral Colony(Site(Region)) reflecting spatial scales of meters to kilometers. A diversity of Symbiodinium ITS2 sequences was recovered with the majority of variance partitioning at the level of the Coral Colony. To confirm this result, the Symbiodinium ITS2 sequence diversity in six M. capitata colonies were analyzed in much greater depth with 35 to 55 clones per colony. The ITS2 sequences and quantitative composition recovered from these colonies varied significantly, indicating that each coral hosted a different assemblage of Symbiodinium. The diversity of Symbiodinium ITS2 sequence assemblages retrieved from individual colonies of M. capitata here highlights the problems inherent in interpreting multi-copy and intra-genomically variable molecular markers, and serves as a context for discussing the utility and biological relevance of assigning species names based on Symbiodinium ITS2 genotyping.

Defining Boundaries for Ecosystem-Based Management: A Multispecies Case Study of Marine Connectivity across the Hawaiian Archipelago.

Determining the geographic scale at which to apply ecosystem-based management (EBM) has proven to be an obstacle for many marine conservation programs. Generalizations based on geographic proximity, taxonomy, or life history characteristics provide little predictive power in determining overall patterns of connectivity, and therefore offer little in terms of delineating boundaries for marine spatial management areas. Here, we provide a case study of 27 taxonomically and ecologically diverse species (including reef fishes, marine mammals, gastropods, echinoderms, cnidarians, crustaceans, and an elasmobranch) that reveal four concordant barriers to dispersal within the Hawaiian Archipelago which are not detected in single-species exemplar studies. We contend that this multispecies approach to determine concordant patterns of connectivity is an objective and logical way in which to define the minimum number of management units and that EBM in the Hawaiian Archipelago requires at least five spatially managed regions.

Contrasting phylogeography in three endemic Hawaiian limpets (Cellana spp.) with similar life histories.

The marine environment offers few obvious barriers to dispersal for broadcast-spawning species, yet population genetic structure can occur on a scale much smaller than the theoretical limits of larval dispersal. Comparative phylogeographical studies of sympatric sister species can illuminate how differences in life history, behaviour, and habitat affinity influence population partitioning. Here we use a mitochondrial DNA marker (612 bp of cytochrome c oxidase subunit I) to investigate population structure of three endemic Hawaiian broadcast-spawning limpets (Cellana spp.) with planktonic larvae that are competent to settle within 4 days. All three species exhibit significant population structure and isolation by distance, but the spatial scales of partitioning differ among the species. Cellana talcosa (n = 105) exhibits strong population structure between Kauai and the other main Hawaiian Islands (MHI) where the maximum channel width is 117 km, and no shared haplotypes were observed (Phi(CT) = 0.30, P < 0.001). In contrast, populations of Cellana exarata (n = 149) and Cellana sandwicensis (n = 109) exhibit weaker population structure within the MHI (Phi(ST) = 0.03-0.04, P < 0.05), and between the MHI and the Northwestern Hawaiian Islands (Phi(ST) = 0.03-0.09, P < 0.01), where the maximum channel width is 260 km. Biogeographical range and microhabitat use were correlated with estimates of dispersal, while phylogenetic affiliation and minimum pelagic larval duration were poor predictors of population partitioning. Despite similar life histories, these closely related limpets have contrasting patterns of population structure, illustrating the danger of relying on model species in management initiatives to predict population structure and dispersal in the context of marine protected area delineation.