Does a complex life cycle affect adaptation to environmental change? Genome-informed insights for characterizing selection across complex life cycle.

Complex life cycles, in which discrete life stages of the same organism differ in form or function and often occupy different ecological niches, are common in nature. Because stages share the same genome, selective effects on one stage may have cascading consequences through the entire life cycle. Theoretical and empirical studies have not yet generated clear predictions about how life cycle complexity will influence patterns of adaptation in response to rapidly changing environments or tested theoretical predictions for fitness trade-offs (or lack thereof) across life stages. We discuss complex life cycle evolution and outline three hypotheses-ontogenetic decoupling, antagonistic ontogenetic pleiotropy and synergistic ontogenetic pleiotropy-for how selection may operate on organisms with complex life cycles. We suggest a within-generation experimental design that promises significant insight into composite selection across life cycle stages. As part of this design, we conducted simulations to determine the power needed to detect selection across a life cycle using a population genetic framework. This analysis demonstrated that recently published studies reporting within-generation selection were underpowered to detect small allele frequency changes (approx. 0.1). The power analysis indicates challenging but attainable sampling requirements for many systems, though plants and marine invertebrates with high fecundity are excellent systems for exploring how organisms with complex life cycles may adapt to climate change.

Estimating population density and connectivity of American mink using spatial capture-recapture.

Estimating the abundance or density of populations is fundamental to the conservation and management of species, and as landscapes become more fragmented, maintaining landscape connectivity has become one of the most important challenges for biodiversity conservation. Yet these two issues have never been formally integrated together in a model that simultaneously models abundance while accounting for connectivity of a landscape. We demonstrate an application of using capture-recapture to develop a model of animal density using a least-cost path model for individual encounter probability that accounts for non-Euclidean connectivity in a highly structured network. We utilized scat detection dogs (Canis lupus familiaris) as a means of collecting non-invasive genetic samples of American mink (Neovison vison) individuals and used spatial capture-recapture models (SCR) to gain inferences about mink population density and connectivity. Density of mink was not constant across the landscape, but rather increased with increasing distance from city, town, or village centers, and mink activity was associated with water. The SCR model allowed us to estimate the density and spatial distribution of individuals across a 388 km² area. The model was used to investigate patterns of space usage and to evaluate covariate effects on encounter probabilities, including differences between sexes. This study provides an application of capture-recapture models based on ecological distance, allowing us to directly estimate landscape connectivity. This approach should be widely applicable to provide simultaneous direct estimates of density, space usage, and landscape connectivity for many species.

Reef-Specific Patterns of Gene Expression Plasticity in Eastern Oysters (Crassostrea virginica).

Understanding the interaction between phenotypic plasticity and evolutionary processes is important for predicting a species' response to changing environment. Strong recurrent selection each generation may be an important process in highly fecund species with broad dispersal and extensive early mortality. We tested whether selection was associated with spatial divergence in gene expression plasticity for osmoregulation in the eastern oyster (Crassostrea virginica). We collected adult oysters from high and low salinity reefs within a single estuary and after 9 weeks of acclimation at 10 and 30 salinity, measured gene expression in 24 oysters using next-generation RNA sequencing technology. The oysters had significantly different expression (DE) in response to salinity treatments for 7936 (18.9%) transcripts overall, with planned contrasts showing 8× more DE in oysters from the high-salinity reef and 15× more DE between reefs when tested at 10 salinity. The reef-by-treatment interaction was also genomically pervasive (5858 DE transcripts, 13.9%). Inter-reef F ST for transcript SNPs averaged 0.0025 with the top 1% between 0.29 and 0.73. Transcripts containing "outlier" SNPs were significantly enriched for osmoregulatory genes and showed patterns of variation consistent with selection on the low-salinity reef. Both phenotypic plasticity and recurrent selection seem to be important factors determining the realized niche of oysters within estuaries.

Brooktrout Lake case study: biotic recovery from acid deposition 20 years after the 1990 Clean Air Act Amendments.

The Adirondack Mountain region is an extensive geographic area (26,305 km(2)) in upstate New York where acid deposition has negatively affected water resources for decades and caused the extirpation of local fish populations. The water quality decline and loss of an established brook trout (Salvelinus fontinalis [Mitchill]) population in Brooktrout Lake were reconstructed from historical information dating back to the late 1880s. Water quality and biotic recovery were documented in Brooktrout Lake in response to reductions of S deposition during the 1980s, 1990s, and 2000s and provided a unique scientific opportunity to re-introduce fish in 2005 and examine their critical role in the recovery of food webs affected by acid deposition. Using C and N isotope analysis of fish collagen and state hatchery feed as well as Bayesian assignment tests of microsatellite genotypes, we document in situ brook trout reproduction, which is the initial phase in the restoration of a preacidification food web structure in Brooktrout Lake. Combined with sulfur dioxide emissions reductions promulgated by the 1990 Clean Air Act Amendments, our results suggest that other acid-affected Adirondack waters could benefit from careful fish re-introduction protocols to initiate the ecosystem reconstruction of important components of food web dimensionality and functionality.

Transcriptomic analysis of candidate osmoregulatory genes in the eastern oyster Crassostrea virginica.

BACKGROUND: The eastern oyster, Crassostrea virginica, is a euryhaline species that can thrive across a wide range of salinities (5-35). As with all estuarine species, individual oysters must be able to regulate their osmotic balance in response to constant temporal variation in salinity. At the population level, recurrent viability selection may be an additional mechanism shaping adaptive osmoregulatory phenotypes at the margins of oyster salinity tolerance. To identify candidate genes for osmoregulation, we sequenced, assembled, and annotated the transcriptome of wild juvenile eastern oysters from 'high' and 'low' salinity regimes. Annotations and candidates were mostly based on the Pacific oyster (Crassostrea gigas) genome sequence so osmoregulatory relevance in C. virginica was explored by testing functional enrichment of genes showing spatially discrete patterns of expression and by quantifying coding sequence divergence. RESULTS: The assembly of sequence reads and permissive clustering of potentially oversplit alleles resulted in 98,729 reftigs (contigs and singletons). Of these, 50,736 were annotated with 9,307 belonging to a set of candidate osmoregulatory genes identified from the C. gigas genome. A total of 218,777 SNPs (0.0185 SNPs/bp) were identified in annotated reftigs of C. virginica. Amino acid divergence between translations of C. virginica annotated reftigs and C. gigas coding sequence averaged 23.2 % with an average dN/dS ratio of 0.074, suggesting purifying selection on protein sequences. The high and low salinity source oysters each expressed a subset of genes unique to that group, and the functions for these annotated genes were consistent with known molecular mechanisms for osmotic regulation in molluscs. CONCLUSIONS: Most of the osmoregulatory gene candidates experimentally identified in C. gigas are present in this C. virginica transcriptome. In general these congeners show coding sequence divergence too high to make the C. gigas genome a useful reference for C. virginica bioinformatics. However, strong purifying selection is characteristic of the osmoregulatory candidates so functional annotations are likely to correspond. An initial examination of C. virginica presence/absence expression patterns across the salinity gradient in a single estuary suggests that many of these candidates have expression patterns that co-vary with salinity, consistent with osmoregulatory function in C. virginica.

Immune priming prior to pathogen exposure sheds light on the relationship between host, microbiome and pathogen in disease.

Dynamic interactions between host, pathogen and host-associated microbiome dictate infection outcomes. Pathogens including Batrachochytrium dendrobatidis (Bd) threaten global biodiversity, but conservation efforts are hindered by limited understanding of amphibian host, Bd and microbiome interactions. We conducted a vaccination and infection experiment using Eastern hellbender salamanders (Cryptobranchus alleganiensis alleganiensis) challenged with Bd to observe infection, skin microbial communities and gene expression of host skin, pathogen and microbiome throughout the experiment. Most animals survived high Bd loads regardless of their vaccination status and vaccination did not affect pathogen load, but host gene expression differed based on vaccination. Oral vaccination (exposure to killed Bd) stimulated immune gene upregulation while topically and sham-vaccinated animals did not significantly upregulate immune genes. In early infection, topically vaccinated animals upregulated immune genes but orally and sham-vaccinated animals downregulated immune genes. Bd increased pathogenicity-associated gene expression in late infection when Bd loads were highest. The microbiome was altered by Bd, but there was no correlation between anti-Bd microbe abundance or richness and pathogen burden. Our observations suggest that hellbenders initially generate a vigorous immune response to Bd, which is ineffective at controlling disease and is subsequently modulated. Interactions with antifungal skin microbiota did not influence disease progression.

Cryptic diversity and comparative phylogeography of the estuarine copepod Acartia tonsa on the US Atlantic coast.

Unexpectedly strong geographic structures in many cosmopolitan species of marine holoplankton challenge the traditional view of their unrestrained dispersal and presumably high gene flow. We investigated cryptic lineage diversity and comparative phylogeography of a common estuarine copepod, Acartia tonsa, on the US Atlantic coast, using mitochondrial (mtCOI) and nuclear (nITS) gene markers. Three broadly sympatric lineages (F, S, X) were defined by genealogically concordant clades across both gene trees, strongly supporting recognition as reproductively isolated species. Limited dispersal seems to have had a major role in population differentiation of A. tonsa in general, with gene flow propensities rank ordered X > S > F. Geographic structure was found only at large scales (1000-2000 km) in X and S. Phylogeographic patterns in all three lineages were mostly concordant with previously recognized zoogeographic provinces but a large mid-Atlantic gap in the occurrence of lineage X, coupled with its presence in Europe, suggests possible nonindigenous origins. For lineage F, physiological adaptation to low-salinity environments is likely to have accentuated barriers to gene flow and allopatric differentiation at both regional and continental scales. Three allopatric F sublineages inferred a southern centre of origin and a stepwise northward diversification history at the continental scale. The most recently derived F sublineages, in the mid-Atlantic Bight, showed strong phylogeographic patterns at nITS albeit weaker at mtCOI. Applying a crustacean mtCOI molecular clock suggests that A. tonsa lineages diverged pre-Pleistocene but mid-Atlantic F lineage diversification may be post-Pleistocene.

Understanding and estimating effective population size for practical application in marine species management.

Effective population size (N(e)) determines the strength of genetic drift in a population and has long been recognized as an important parameter for evaluating conservation status and threats to genetic health of populations. Specifically, an estimate of N(e) is crucial to management because it integrates genetic effects with the life history of the species, allowing for predictions of a population's current and future viability. Nevertheless, compared with ecological and demographic parameters, N(e) has had limited influence on species management, beyond its application in very small populations. Recent developments have substantially improved N(e) estimation; however, some obstacles remain for the practical application of N(e) estimates. For example, the need to define the spatial and temporal scale of measurement makes the concept complex and sometimes difficult to interpret. We reviewed approaches to estimation of N(e) over both long-term and contemporary time frames, clarifying their interpretations with respect to local populations and the global metapopulation. We describe multiple experimental factors affecting robustness of contemporary N(e) estimates and suggest that different sampling designs can be combined to compare largely independent measures of N(e) for improved confidence in the result. Large populations with moderate gene flow pose the greatest challenges to robust estimation of contemporary N(e) and require careful consideration of sampling and analysis to minimize estimator bias. We emphasize the practical utility of estimating N(e) by highlighting its relevance to the adaptive potential of a population and describing applications in management of marine populations, where the focus is not always on critically endangered populations. Two cases discussed include the mechanisms generating N(e) estimates many orders of magnitude lower than census N in harvested marine fishes and the predicted reduction in N(e) from hatchery-based population supplementation.

Are bivalves susceptible to domestication selection? Using starvation tolerance to test for potential trait changes in eastern oyster larvae.

Conservation efforts are increasingly being challenged by a rapidly changing environment, and for some aquatic species the use of captive rearing or selective breeding is an attractive option. However, captivity itself can impose unintended artificial selection known as domestication selection (adaptation to culture conditions) and is relatively understudied for most marine species. To test for domestication selection in marine bivalves, we focused on a fitness-related trait (larval starvation resistance) that could be altered under artificial selection. Using larvae produced from a wild population of Crassostrea virginica and a selectively bred, disease-resistant line we measured growth and survival during starvation versus standard algal diet conditions. Larvae from both lineages showed a remarkable resilience to food limitation, possibly mediated by an ability to utilize dissolved organic matter for somatic maintenance. Water chemistry analysis showed dissolved organic carbon in filtered tank water to be at concentrations similar to natural river water. We observed that survival in larvae produced from the aquaculture line was significantly lower compared to larvae produced from wild broodstock (8 ± 3% and 21 ± 2%, respectively) near the end of a 10-day period with no food (phytoplankton). All larval cohorts had arrested growth and depressed respiration during the starvation period and took at least two days to recover once food was reintroduced before resuming growth. Respiration rate recovered rapidly and final shell length was similar between the two treatments Phenotypic differences between the wild and aquaculture lines suggest potential differences in the capacity to sustain extended food limitation, but this work requires replication with multiple selection lines and wild populations to make more general inferences about domestication selection. With this contribution we explore the potential for domestication selection in bivalves, discuss the physiological and fitness implications of reduced starvation tolerance, and aim to inspire further research on the topic.

Correction: Restoring oysters to urban estuaries: Redefining habitat quality for eastern oyster performance near New York City.

[This corrects the article DOI: 10.1371/journal.pone.0207368.].

Author Correction: Large-scale variation in density of an aquatic ecosystem indicator species.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cryptic ecological diversification of a planktonic estuarine copepod, Acartia tonsa.

The recent discovery of cryptic species in marine holoplankton, organisms that 'drift' in oceanic currents throughout their life cycle, contrasts with their potential for long-distance passive dispersal and presumably high gene flow. These observations suggest that holoplankton species are adapting to surprisingly small-scale oceanographic features and imply either limited dispersal or strong selection gradients. Acartia tonsa is a widespread and numerically dominant estuarine copepod containing deep mitochondrial lineages within and among populations along the northwestern Atlantic coast. In this study, we intensively investigated A. tonsa populations in Chesapeake Bay with the goals of testing species status for the deep lineages and testing for their association with environmental features over space and time. Phylogenetic analyses of DNA sequences from mitochondrial cytochrome c oxidase I (mtCOI) and the nuclear ribosomal internal transcribed spacer (nITS) resolved two concordant monophyletic clades. Deep divergence between the two clades (13.7% uncorrected sequence divergence for mtCOI and 32.2% for nITS) and genealogical concordance within sympatric populations strongly suggest that the two clades represent reproductively isolated cryptic species. Based on restriction fragment length polymorphisms of mtCOI, representatives from the two clades were found consistently associated with contrasting salinity regimes (oligohaline vs. meso-polyhaline) with an overlap between 2 and 12 PSU in samples from 1995 to 2005. Finding these patterns in one of the best-known estuarine copepods reinforces the conclusion that marine biodiversity is underestimated, not only in terms of species numbers, but also with respect to niche partitioning and the potential importance of ecological divergence in marine holoplankton.

Restoring oysters to urban estuaries: Redefining habitat quality for eastern oyster performance near New York City.

Restoring and conserving coastal resilience faces increasing challenges under current climate change predictions. Oyster restoration, in particular, faces threats from alterations in precipitation, warming water temperatures, and urbanization of coastlines that dramatically change salinity patterns, foster the proliferation and spread disease, and disrupt habitat connectivity, respectively. New York City (NYC) coastal waters, once home to a booming oyster fishery for eastern oysters (Crassostrea virginica), are now nearly devoid of live oyster reefs. Oyster restoration in urban estuaries is motivated by the synergistic ecosystem benefits this native keystone species can deliver. Recent surveys have documented substantial remnant populations of adult oysters in the upper low salinity zone of the Hudson/Raritan Estuary (HRE) near Tarrytown, NY. This study assessed fitness-related performance across the HRE salinity gradient to evaluate habitat suitability on an estuarine scale. Oysters were hatchery-produced from wild, moderate-salinity broodstock, then outplanted for measurement of growth, survival, reproduction and disease prevalence over two years. Survival was generally higher in the lower salinity river sites and in the higher salinity Jamaica Bay sites relative to mesohaline NYC harbor sites. Growth rate was highest in Jamaica Bay and had high variation among other sites. Surprisingly, the highest proportion of individuals with sex-differentiated gametes and the highest average gonad maturation index was found at a low salinity site. Consistent with the advanced gametogenesis measured in experimental animals at low salinity, annual wild recruitment was documented near the low salinity remnant population in each of five monitored years. These results suggest that the remnant HRE oyster population is a robust, self-sustaining population that can be leveraged to support restoration of subpopulations in other parts of the estuary, but further research is required to determine if the mesohaline and near-ocean reaches of the HRE can support the full oyster life cycle.

Large-scale variation in density of an aquatic ecosystem indicator species.

Monitoring indicator species is a pragmatic approach to natural resource assessments, especially when the link between the indicator species and ecosystem state is well justified. However, conducting ecosystem assessments over representative spatial scales that are insensitive to local heterogeneity is challenging. We examine the link between polychlorinated biphenyl (PCB) contamination and population density of an aquatic habitat specialist over a large spatial scale using non-invasive genetic spatial capture-recapture. Using American mink (Neovison vison), a predatory mammal and an indicator of aquatic ecosystems, we compared estimates of density in two major river systems, one with extremely high levels of PCB contamination (Hudson River), and a hydrologically independent river with lower PCB levels (Mohawk River). Our work supports the hypothesis that mink densities are substantially (1.64-1.67 times) lower in the contaminated river system. We demonstrate the value of coupling the indicator species concept with well-conceived and spatially representative monitoring protocols. PCBs have demonstrable detrimental effects on aquatic ecosystems, including mink, and these effects are likely to be profound and long-lasting, manifesting as population-level impacts. Through integrating non-invasive data collection, genetic analysis, and spatial capture-recapture methods, we present a monitoring framework for generating robust density estimates across large spatial scales.

Genetic diversity and expanding nonindigenous range of the rhizocephalan Loxothylacus panopaei parasitizing mud crabs in the western north Atlantic.

Nonindigenous parasite introductions and range expansions have become a major concern because of their potential to restructure communities and impact fisheries. Molecular markers provide an important tool for reconstructing the pattern of introduction. The parasitic castrator Loxothylacus panopaei, a rhizocephalan barnacle, infects estuarine mud crabs in the Gulf of Mexico and southeastern Florida. A similar parasite introduced into Chesapeake Bay before 1964, presumably via infected crabs associated with oysters from the Gulf of Mexico, was identified as L. panopaei. Our samples of this species during 2004 and 2005 show that the introduced range has expanded as far south as Edgewater, Florida, just north of the northern endemic range limit. The nonindigenous range expanded southward at a rate of up to 165 km/yr with relatively high prevalence, ranging from 30 to 93%. Mitochondrial DNA sequences from the cytochrome oxidase I gene showed that these nonindigenous L. panopaei are genetically distinct from the endemic parasites in southeastern Florida and the eastern Gulf of Mexico. The genetic difference was also associated with distinct host spectra. These results are incompatible with an eastern Gulf source population, but suggest that unrecognized genetic and phenotypic population structure may occur among Gulf of Mexico populations of Loxothvlacus.

Accurate recapture identification for genetic mark-recapture studies with error-tolerant likelihood-based match calling and sample clustering.

Error-tolerant likelihood-based match calling presents a promising technique to accurately identify recapture events in genetic mark-recapture studies by combining probabilities of latent genotypes and probabilities of observed genotypes, which may contain genotyping errors. Combined with clustering algorithms to group samples into sets of recaptures based upon pairwise match calls, these tools can be used to reconstruct accurate capture histories for mark-recapture modelling. Here, we assess the performance of a recently introduced error-tolerant likelihood-based match-calling model and sample clustering algorithm for genetic mark-recapture studies. We assessed both biallelic (i.e. single nucleotide polymorphisms; SNP) and multiallelic (i.e. microsatellite; MSAT) markers using a combination of simulation analyses and case study data on Pacific walrus (Odobenus rosmarus divergens) and fishers (Pekania pennanti). A novel two-stage clustering approach is demonstrated for genetic mark-recapture applications. First, repeat captures within a sampling occasion are identified. Subsequently, recaptures across sampling occasions are identified. The likelihood-based matching protocol performed well in simulation trials, demonstrating utility for use in a wide range of genetic mark-recapture studies. Moderately sized SNP (64+) and MSAT (10-15) panels produced accurate match calls for recaptures and accurate non-match calls for samples from closely related individuals in the face of low to moderate genotyping error. Furthermore, matching performance remained stable or increased as the number of genetic markers increased, genotyping error notwithstanding.

Nonrandom larval dispersal can steepen marine clines.

Sharp and stable clinal variation is enigmatic when found in species with high gene flow. Classical population genetic models treat gene flow as a random homogenizing force countering local adaptation across habitat discontinuities. Under this view, dispersal over large spatial scales will lower the effectiveness of adaptation by natural selection at finer spatial scales. Thus, random gene flow will create a shallow phenotypic cline across an ecotone in response to a steep selection gradient. In sedentary marine species that disperse primarily as larvae, nonrandom dispersal patterns are expected due to coastal hydrodynamics. Surprisingly sharp phenotypic and genotypic clines have been documented in marine species with high gene flow. We are interested in the extent to which nonrandom dispersal could accentuate such clines. We model a linear species range in which populations have stable and uniform densities along a selection gradient; in contrast to random dispersal, convergent advection of larvae can amplify phenotypic differentiation if coupled with a semipermeable dispersal barrier in the convergence zone. The migration load caused by directional dispersal pushes the phenotypic mean away from the local trait optimum in downstream populations, that is, near the convergence zone. A dispersal barrier is possible as a result of colliding currents if the water and larvae are mostly displaced offshore, away from suitable settlement habitat. Disjunctions in a quantitative trait were enlarged in the convergence zone by faster current flows or a more complete dispersal barrier. With advection of larvae per generation one-third as far as the average dispersal distance by diffusion, convergence on a dispersal barrier with 40% permeability generated a trait disjunction across the convergence zone of two phenotypic standard deviations. Without directional dispersal, similar clines also developed across a habitat gap, where population density was low, or across dispersal barriers with less than 1% permeability. These findings suggest that the types of hydrographic phenomena often associated with marine transition zones can strongly affect the balance between gene flow and selection and generate surprisingly steep clines given the large-scale gene flow expected from larvae.

Genetic evidence on the demography of speciation in allopatric dolphin species.

Under a neutral model, the stochastic lineage sorting that leads to gene monophyly proceeds slowly in large populations. Therefore, in many recent species with large population size, the genome will have mixed support for monophyly unless historical bottlenecks have accelerated coalescence. We use genealogical patterns in mitochondrial DNA and in introns of four nuclear loci to test for historical bottlenecks during the speciation and divergence of two temperate Lagenorhynchus dolphin species isolated by tropical Pacific waters (an antitropical distribution). Despite distinct morphologies, foraging behaviors, and mitochondrial DNAs, these dolphin species are polyphyletic at all four nuclear loci. The abundance of shared polymorphisms between these sister taxa is most consistent with the maintenance of large effective population sizes (5.09 x 10(4) to 10.9 x 10(4)) during 0.74-1.05 million years of divergence. A variety of population size histories are possible, however. We used gene tree coalescent probabilities to explore the rejection region for historical bottlenecks of different intensity given best estimates of effective population size under a strict isolation model of divergence. In L. obliquidens the data are incompatible with a colonization propagule of an effective size of 10 or fewer individuals. Although the ability to reject less extreme historical bottlenecks will require data from additional loci, the intermixed genealogical patterns observed between these dolphin sister species are highly probable only under an extended history of large population size. If similar demographic histories are inferred for other marine antitropical taxa, a parsimonious model for the Pleistocene origin of these distributions would not involve rare breaches of a constant dispersal barrier by small colonization propagules. Instead, a history of large population size in L. obliquidens and L. obscurus contributes to growing biological and environmental evidence that the equatorial barrier became permeable during glacial/interglacial cycles, leading to vicariant isolation of antitropical populations.

Hybridization between previously isolated ancestors may explain the persistence of exactly two ancient lineages in the genome of the oyster parasite Perkinsus marinus.

Theory predicts that neutral genetic variation accumulates within populations to a level determined by gains through mutation and losses by genetic drift. This balance results in a characteristic distribution of allelic variation with the maximum allelic difference determined by effective population size. Here, we report a striking departure from these expectations in the form of allelic dimorphism, observed at the majority of seven loci examined in Perkinsus marinus, an important oyster parasite that causes Dermo disease. DNA sequences were collected from five loci flanking microsatellite repeats and two loci coding for superoxide dismutase enzymes that may mediate the parasite's interaction with its host. Based on 474 sequences, sampled across 5000 km of the eastern United States coastline, no more than two alleles were observed at each locus (discounting singletons). Depending on the locus, the common allele ranged in overall frequency from 72% to 92%. At each locus the two alleles differed substantially (3.8% sequence difference, on average), and the among-locus variance in divergences was not sufficient to reject a simultaneous origin for all dimorphisms using approximate Bayesian methods. Dimorphic alleles were estimated to have diverged from a common ancestral allele at least 0.9 million years ago. Across these seven loci, only five other alleles were ever observed, always as singletons and differing from the dimorphic alleles by no more than two nucleotides. Free recombination could potentially have shuffled these dimorphisms into as many as 243 multilocus combinations, but the existence of only ten combinations among all samples strongly supports low recombination frequencies and is consistent with the observed absence of intragenic recombination. We consider several demographic and evolutionary hypotheses to explain these patterns. Few can be conclusively rejected with the present data, but we advance a recent hybridization of ancient divergent lineages scenario as the most parsimonious.

Defining loci in restriction-based reduced representation genomic data from nonmodel species: sources of bias and diagnostics for optimal clustering.

Next generation sequencing holds great promise for applications of phylogeography, landscape genetics, and population genomics in wild populations of nonmodel species, but the robustness of inferences hinges on careful experimental design and effective bioinformatic removal of predictable artifacts. Addressing this issue, we use published genomes from a tunicate, stickleback, and soybean to illustrate the potential for bioinformatic artifacts and introduce a protocol to minimize two sources of error expected from similarity-based de-novo clustering of stacked reads: the splitting of alleles into different clusters, which creates false homozygosity, and the grouping of paralogs into the same cluster, which creates false heterozygosity. We present an empirical application focused on Ciona savignyi, a tunicate with very high SNP heterozygosity (~0.05), because high diversity challenges the computational efficiency of most existing nonmodel pipelines while also potentially exacerbating paralog artifacts. The simulated and empirical data illustrate the advantages of using higher sequence difference clustering thresholds than is typical and demonstrate the utility of our protocol for efficiently identifying an optimum threshold from data without prior knowledge of heterozygosity. The empirical Ciona savignyi data also highlight null alleles as a potentially large source of false homozygosity in restriction-based reduced representation genomic data.