Next-generation data filtering in the genomics era.

Genomic data are ubiquitous across disciplines, from agriculture to biodiversity, ecology, evolution and human health. However, these datasets often contain noise or errors and are missing information that can affect the accuracy and reliability of subsequent computational analyses and conclusions. A key step in genomic data analysis is filtering - removing sequencing bases, reads, genetic variants and/or individuals from a dataset - to improve data quality for downstream analyses. Researchers are confronted with a multitude of choices when filtering genomic data; they must choose which filters to apply and select appropriate thresholds. To help usher in the next generation of genomic data filtering, we review and suggest best practices to improve the implementation, reproducibility and reporting standards for filter types and thresholds commonly applied to genomic datasets. We focus mainly on filters for minor allele frequency, missing data per individual or per locus, linkage disequilibrium and Hardy-Weinberg deviations. Using simulated and empirical datasets, we illustrate the large effects of different filtering thresholds on common population genetics statistics, such as Tajima's D value, population differentiation (FST), nucleotide diversity (π) and effective population size (Ne).

Rapid genetic adaptation to a novel ecosystem despite a large founder event.

Introduced and invasive species make excellent natural experiments for investigating rapid evolution. Here, we describe the effects of genetic drift and rapid genetic adaptation in pink salmon (Oncorhynchus gorbuscha) that were accidentally introduced to the Great Lakes via a single introduction event 31 generations ago. Using whole-genome resequencing for 134 fish spanning five sample groups across the native and introduced range, we estimate that the source population's effective population size was 146,886 at the time of introduction, whereas the founding population's effective population size was just 72-a 2040-fold decrease. As expected with a severe founder event, we show reductions in genome-wide measures of genetic diversity, specifically a 37.7% reduction in the number of SNPs and an 8.2% reduction in observed heterozygosity. Despite this decline in genetic diversity, we provide evidence for putative selection at 47 loci across multiple chromosomes in the introduced populations, including missense variants in genes associated with circadian rhythm, immunological response and maturation, which match expected or known phenotypic changes in the Great Lakes. For one of these genes, we use a species-specific agent-based model to rule out genetic drift and conclude our results support a strong response to selection occurring in a period gene (per2) that plays a predominant role in determining an organism's daily clock, matching large day length differences experienced by introduced salmon during important phenological periods. Together, these results inform how populations might evolve rapidly to new environments, even with a small pool of standing genetic variation.

Large genetic divergence underpins cryptic local adaptation across ecological and evolutionary gradients.

Environmentally covarying local adaptation is a form of cryptic local adaptation in which the covariance of the genetic and environmental effects on a phenotype obscures the divergence between locally adapted genotypes. Here, we systematically document the magnitude and drivers of the genetic effect (VG) for two forms of environmentally covarying local adaptation: counter- and cogradient variation. Using a hierarchical Bayesian meta-analysis, we calculated the overall effect size of VG as 1.05 and 2.13 for populations exhibiting countergradient or cogradient variation, respectively. These results indicate that the genetic contribution to phenotypic variation represents a 1.05 to 2.13 s.d. change in trait value between the most disparate populations depending on if populations are expressing counter- or cogradient variation. We also found that while there was substantial variance among abiotic and biotic covariates, the covariates with the largest mean effects were temperature (2.41) and gamete size (2.81). Our results demonstrate the pervasiveness and large genetic effects underlying environmentally covarying local adaptation in wild populations and highlight the importance of accounting for these effects in future studies.

Rapid genetic adaptation to recently colonized environments is driven by genes underlying life history traits.

BACKGROUND: Uncovering the mechanisms underlying rapid genetic adaptation can provide insight into adaptive evolution and shed light on conservation, invasive species control, and natural resource management. However, it can be difficult to experimentally explore rapid adaptation due to the challenges associated with propagating and maintaining species in captive environments for long periods of time. By contrast, many introduced species have experienced strong selection when colonizing environments that differ substantially from their native range and thus provide a "natural experiment" for studying rapid genetic adaptation. One such example occurred when sea lamprey (Petromyzon marinus), native to the northern Atlantic, naturally migrated into Lake Champlain and expanded their range into the Great Lakes via man-made shipping canals. RESULTS: Utilizing 368,886 genome-wide single nucleotide polymorphisms (SNPs), we calculated genome-wide levels of genetic diversity (i.e., heterozygosity and π) for sea lamprey collected from native (Connecticut River), native but recently colonized (Lake Champlain), and invasive (Lake Michigan) populations, assessed genetic differentiation between all populations, and identified candidate genes that responded to selection imposed by the novel environments. We observed a 14 and 24% reduction in genetic diversity in Lake Michigan and Lake Champlain populations, respectively, compared to individuals from the Connecticut River, suggesting that sea lamprey populations underwent a genetic bottleneck during colonization. Additionally, we identified 121 and 43 outlier genes in comparisons between Lake Michigan and Connecticut River and between Lake Champlain and Connecticut River, respectively. Six outlier genes that contained synonymous SNPs in their coding regions and two genes that contained nonsynonymous SNPs may underlie the rapid evolution of growth (i.e., GHR), reproduction (i.e., PGR, TTC25, STARD10), and bioenergetics (i.e., OXCT1, PYGL, DIN4, SLC25A15). CONCLUSIONS: By identifying the genomic basis of rapid adaptation to novel environments, we demonstrate that populations of invasive species can be a useful study system for understanding adaptive evolution. Furthermore, the reduction in genome-wide levels of genetic diversity associated with colonization coupled with the identification of outlier genes underlying key life history traits known to have changed in invasive sea lamprey populations (e.g., growth, reproduction) illustrate the utility in applying genomic approaches for the successful management of introduced species.

Life history variation is maintained by fitness trade-offs and negative frequency-dependent selection.

The maintenance of diverse life history strategies within and among species remains a fundamental question in ecology and evolutionary biology. By using a near-complete 16-year pedigree of 12,579 winter-run steelhead (Oncorhynchus mykiss) from the Hood River, Oregon, we examined the continued maintenance of two life history traits: the number of lifetime spawning events (semelparous vs. iteroparous) and age at first spawning (2-5 years). We found that repeat-spawning fish had more than 2.5 times the lifetime reproductive success of single-spawning fish. However, first-time repeat-spawning fish had significantly lower reproductive success than single-spawning fish of the same age, suggesting that repeat-spawning fish forego early reproduction to devote additional energy to continued survival. For single-spawning fish, we also found evidence for a fitness trade-off for age at spawning: older, larger males had higher reproductive success than younger, smaller males. For females, in contrast, we found that 3-year-old fish had the highest mean lifetime reproductive success despite the observation that 4- and 5-year-old fish were both longer and heavier. This phenomenon was explained by negative frequency-dependent selection: as 4- and 5-year-old fish decreased in frequency on the spawning grounds, their lifetime reproductive success became greater than that of the 3-year-old fish. Using a combination of mathematical and individual-based models parameterized with our empirical estimates, we demonstrate that both fitness trade-offs and negative frequency-dependent selection observed in the empirical data can theoretically maintain the diverse life history strategies found in this population.

Among-family variation in survival and gene expression uncovers adaptive genetic variation in a threatened fish.

Variation in among-family transcriptional responses to different environmental conditions can help to identify adaptive genetic variation, even prior to a selective event. Coupling differential gene expression with formal survival analyses allows for the disentanglement of treatment effects, required for understanding how individuals plastically respond to environmental stressors, from the adaptive genetic variation responsible for differential survival. We combined these two approaches to investigate responses to an emerging conservation issue, thiamine (vitamin B1 ) deficiency, in a threatened population of Atlantic salmon (Salmo salar). Thiamine is an essential vitamin that is increasingly limited in many ecosystems. In Lake Champlain, Atlantic salmon cannot acquire thiamine in sufficient quantities to support natural reproduction; fertilized eggs must be reared in hatcheries and treated with supplemental thiamine. We evaluated transcriptional responses (via RNA sequencing) to thiamine treatment across families and found 3,616 genes differentially expressed between control (no supplemental thiamine) and treatment individuals. Fewer genes changed expression equally across families (i.e., additively) than exhibited genotype × environment interactions in response to thiamine. Differentially expressed genes were related to known physiological effects of thiamine deficiency, including oxidative stress, cardiovascular irregularities and neurological abnormalities. We also identified 1,446 putatively adaptive genes that were strongly associated with among-family survival in the absence of thiamine treatment, many of which related to neurogenesis and visual perception. Our results highlight the utility of coupling RNA sequencing with formal survival analyses to identify candidate genes that underlie the among-family variation in survival required for an adaptive response to natural selection.

Inbreeding load and inbreeding depression estimated from lifetime reproductive success in a small, dispersal-limited population.

The fitness consequences of inbreeding and the individual behaviors that prevent its detrimental effects can be challenging to document in wild populations. Here, we use field and molecular data from a 17-year study of banner-tailed kangaroo rats (Dipodomys spectabilis) to quantify the relationship between inbreeding, mate kinship, and lifetime reproductive success. Using a pedigree that was reconstructed using genetic and field data within a Bayesian framework (median probability of parental assignment = 0.92, mean pedigree depth = 6 generations), we estimated both inbreeding coefficients and kinship between individuals that produced offspring (mean inbreeding coefficient = 0.07, mean mate kinship = 0.08). We also used the pedigree, in combination with census data, to generate a series of fitness estimates, ranging from survival to reproductive maturity to lifetime reproductive success. We found that the population's inbreeding load was low to moderate (0.98-4.66 haploid lethal equivalents) and increased with the time frame over which fitness was estimated (lowest for survival to maturity, highest for adult-to-adult reproductive success). Fitness decreased with increasing inbreeding coefficients. For example, lifetime reproductive success was reduced by 24% for individuals with inbreeding coefficients greater than twice the population mean. Within full sibling pairs, the sibling with less-related mates produced an average of 30% more offspring over its lifetime. These data further illustrate that inbreeding can have a negative effect on lifetime reproductive success.

Long-term demographic and genetic effects of releasing captive-born individuals into the wild.

Because of continued habitat destruction and species extirpations, the need to use captive breeding for conservation purposes has been increasing steadily. However, the long-term demographic and genetic effects associated with releasing captive-born individuals with varied life histories into the wild remain largely unknown. To address this question, we developed forward-time, agent-based models for 4 species with long-running captive-breeding and release programs: coho salmon (Oncorhynchus kisutch), golden lion tamarin (Leontopithecus rosalia), western toad (Anaxyrus boreas), and Whooping Crane (Grus americana). We measured the effects of supplementation by comparing population size and neutral genetic diversity in supplemented populations to the same characteristics in unaltered populations 100 years after supplementation ended. Releasing even slightly less fit captive-born individuals to supplement wild populations typically resulted in reductions in population sizes and genetic diversity over the long term when the fitness reductions were heritable (i.e., due to genetic adaptation to captivity) and populations continued to be regulated by density-dependent mechanisms over time. Negative effects for species with longer life spans and lower rates of population replacement were smaller than for species with shorter life spans and higher rates of population replacement. Programs that released captive-born individuals over fewer years or that avoided breeding individuals with captive ancestry had smaller reductions in population size and genetic diversity over the long term. Relying on selection in the wild to remove individuals with reduced fitness mitigated some negative demographic effects, but at a substantial cost to neutral genetic diversity. Our results suggest that conservation-focused captive-breeding programs should take measures to prevent even small amounts of genetic adaptation to captivity, quantitatively determine the minimum number of captive-born individuals to release each year, and fully account for the interactions among genetic adaptation to captivity, population regulation, and life-history variation.

Efectos Genéticos y Demográficos a Largo Plazo de la Liberación de Individuos Nacidos en Cautiverio Resumen Debido a la continua destrucción de hábitats y a la extracción de especies, la necesidad de utilizar la reproducción en cautiverio con motivos de conservación ha aumentado constantemente. Sin embargo, los efectos genéticos y demográficos a largo plazo asociados con la liberación de individuos nacidos en cautiverio con historias de vida variadas permanecen en gran parte desconocidos. Para responder a la pregunta anterior desarrollamos modelos de tiempo futuro basados en agentes para cuatro especies con programas de reproducción en cautiverio y liberación con una larga duración: el salmón plateado (Oncorhynchus kisutch), la marmoseta dorada (Leontopithecus rosalia), el sapo occidental (Anaxyrus boreas), y la grulla trompetera (Grus americana). Medimos los efectos de la suplementación al comparar el tamaño poblacional y la diversidad genética neutra en las poblaciones suplementadas con las mismas características en poblaciones sin alteraciones 100 años después de la suplementación. La liberación de individuos criados en cautiverio con una mínima aptitud física como suplemento para las poblaciones silvestres resultó típicamente en la reducción del tamaño poblacional y de la diversidad genética a largo plazo cuando la reducción en la aptitud fue heredable (es decir, debido a la adaptación genética al cautiverio) y las poblaciones siguieron reguladas a lo largo del tiempo por los mecanismos dependientes de la densidad. Los efectos negativos para las especies con ciclos de vida mayores y tasas más bajas de reemplazo poblacional fueron menores que para aquellas especies con ciclos de vida más cortos y tasas más altas de reemplazo poblacional. Los programas que liberaron individuos criados en cautiverio durante menos años o que evitaron reproducir individuos con ascendencia en cautiverio tuvieron reducciones menores en el tamaño poblacional y en la diversidad genética a largo plazo. La dependencia de la selección silvestre para extirpar a los individuos con aptitud física reducida mitigó algunos efectos demográficos negativos, pero a un precio sustancial para la diversidad genética neutra. Nuestros resultados sugieren que los programas de reproducción en cautiverio enfocados en la conservación deberían tomar medidas para prevenir las más mínimas cantidades de adaptación genética al cautiverio, determinar cuantitativamente el número mínimo de individuos nacidos en cautiverio para liberar cada año, y compensar totalmente las interacciones entre la adaptación genética al cautiverio, la regulación poblacional, y la variación en historias de vida.

Rapid genetic adaptation to a novel environment despite a genome-wide reduction in genetic diversity.

Introduced species often colonize regions that have vastly different ecological and environmental conditions than those found in their native range. As such, species introductions can provide a deeper understanding into the process of adaptive evolution. In the 1880s, steelhead trout (Oncorhynchus mykiss) from California were introduced into Lake Michigan (Laurentian Great Lakes, North America) where they established naturally reproducing populations. In their native range, steelhead hatch in rivers, migrate to the ocean and return to freshwater to spawn. Steelhead in Lake Michigan continue to swim up rivers to spawn, but now treat the freshwater environment of the Great Lakes as a surrogate ocean. To examine the effects of this introduction, we sequenced the genomes of 264 fish. By comparing steelhead from Lake Michigan to steelhead from their ancestral range, we determined that the introduction led to consistent reductions in genetic diversity across all 29 chromosomes. Despite this reduction in genetic diversity, three chromosomal regions were associated with rapid genetic adaptation to the novel environment. The first region contained functional changes to ceramide kinase, which likely altered metabolic and wound-healing rates in Lake Michigan steelhead. The second and third regions encoded carbonic anhydrases and a solute carrier protein, both of which are critical for osmoregulation, and demonstrate how steelhead physiologically adapted to freshwater. Furthermore, the contemporary release of diverse hatchery strains into the lake increased genetic diversity but reduced the signature of genetic adaptation. This study illustrates that species can rapidly adapt to novel environments despite genome-wide reductions in genetic diversity.

Integrating larval connectivity with local demography reveals regional dynamics of a marine metapopulation.

Many ocean species exist within what are called marine metapopulations: networks of otherwise isolated local populations connected by the exchange of larval offspring. In order to manage these species as effectively as possible (e.g., by designing and implementing effective networks of marine protected areas), we must know how many offspring are produced within each local population (i.e., local demography), and where those offspring disperse (i.e., larval connectivity). Although there is much interest in estimating connectivity in the relatively simple sense of identifying the locations of spawning parents and their settling offspring, true measures of demographic connectivity that account for among-site variation in offspring production have been lacking. We combined detailed studies of local reproductive output and larval dispersal of a coral reef fish to quantify demographic connectivity within a regional metapopulation that included four widely spaced islands in the Bahamas. We present a new method for estimating demographic connectivity when the levels of dispersal among populations are inferred by the collection of genetically "tagged" offspring. We estimated that 13.3% of recruits returned to natal islands, on average (95% CI = 1.1-50.3%), that local retention was high on one of the islands (41%, 95% CI = 6.0-97.0%), and that larval connectivity was appreciable, even between islands 129 km apart (mean = 1.6%, 95% CI = 0.20-8.8%). Our results emphasize the importance of properly integrating measurements of production with measurements of connectivity. Had we not accounted for among-site variation in offspring production, our estimates of connectivity would have been inaccurate by a factor as much as 6.5. At a generational timescale, lifetime offspring production varied substantially (a fivefold difference among islands) and the importance of each island to long-term metapopulation growth was dictated by both larval production and connectivity. At the scale of our study (local populations inhabiting 5-ha reefs), the regional metapopulation could not grow without external input. However, an exploratory analysis simulating a network of four marine protected areas suggested that reserves of >65 ha each would ensure persistence of this network. Thus, integrating studies of larval connectivity and local demography hold promise for both managing and conserving marine metapopulations effectively.

Captive Ancestry Upwardly Biases Estimates of Relative Reproductive Success.

Supplementation programs, which release captive-born individuals into the wild, are commonly used to demographically bolster declining populations. In order to evaluate the effectiveness of these programs, the reproductive success of captive-born individuals released into the wild is often compared to the reproductive success of wild-born individuals in the recipient population (relative reproductive success, RRS). However, if there are heritable reductions in fitness associated with captive breeding, gene flow from captive-born individuals into the wild population can reduce the fitness of the wild population. Here, we show that when captive ancestry in the wild population reduces mean population fitness, estimates of RRS are upwardly biased, meaning that the relative fitness of captive-born individuals is over-estimated. Furthermore, the magnitude of this bias increases with the length of time that a supplementation program has been releasing captive-born individuals. This phenomenon has long-term conservation impacts since management decisions regarding the design of a supplementation program and the number of individuals to release can be based, at least in part, on RRS estimates. Therefore, we urge caution in the interpretation of relative fitness measures when the captive ancestry of the wild population cannot be precisely measured.

Genetic adaptation to captivity can occur in a single generation.

Captive breeding programs are widely used for the conservation and restoration of threatened and endangered species. Nevertheless, captive-born individuals frequently have reduced fitness when reintroduced into the wild. The mechanism for these fitness declines has remained elusive, but hypotheses include environmental effects of captive rearing, inbreeding among close relatives, relaxed natural selection, and unintentional domestication selection (adaptation to captivity). We used a multigenerational pedigree analysis to demonstrate that domestication selection can explain the precipitous decline in fitness observed in hatchery steelhead released into the Hood River in Oregon. After returning from the ocean, wild-born and first-generation hatchery fish were used as broodstock in the hatchery, and their offspring were released into the wild as smolts. First-generation hatchery fish had nearly double the lifetime reproductive success (measured as the number of returning adult offspring) when spawned in captivity compared with wild fish spawned under identical conditions, which is a clear demonstration of adaptation to captivity. We also documented a tradeoff among the wild-born broodstock: Those with the greatest fitness in a captive environment produced offspring that performed the worst in the wild. Specifically, captive-born individuals with five (the median) or more returning siblings (i.e., offspring of successful broodstock) averaged 0.62 returning offspring in the wild, whereas captive-born individuals with less than five siblings averaged 2.05 returning offspring in the wild. These results demonstrate that a single generation in captivity can result in a substantial response to selection on traits that are beneficial in captivity but severely maladaptive in the wild.

Bayesian parentage analysis with systematic accountability of genotyping error, missing data and false matching.

MOTIVATION: The goal of any parentage analysis is to identify as many parent-offspring relationships as possible, while minimizing incorrect assignments. Existing methods can achieve these ends, but they require additional information in the form of demographic data, thousands of markers and/or estimates of genotyping error rates. For many non-model systems, it is simply not practical, cost-effective or logistically feasible to obtain this information. Here, we develop a Bayesian parentage method that only requires the sampled genotypes to account for genotyping error, missing data and false matches. RESULTS: Extensive testing with microsatellite and SNP datasets reveals that our Bayesian parentage method reliably controls for the number of false assignments, irrespective of the genotyping error rate. When the number of loci is limiting, our approach maximizes the number of correct assignments by accounting for the frequencies of shared alleles. Comparisons with exclusion and likelihood-based methods on an empirical salmon dataset revealed that our Bayesian method had the highest ratio of correct to incorrect assignments.

Spatial and temporal patterns of larval dispersal in a coral-reef fish metapopulation: evidence of variable reproductive success.

Many marine organisms can be transported hundreds of kilometres during their pelagic larval stage, yet little is known about spatial and temporal patterns of larval dispersal. Although traditional population-genetic tools can be applied to infer movement of larvae on an evolutionary timescale, large effective population sizes and high rates of gene flow present serious challenges to documenting dispersal patterns over shorter, ecologically relevant, timescales. Here, we address these challenges by combining direct parentage analysis and indirect genetic analyses over a 4-year period to document spatial and temporal patterns of larval dispersal in a common coral-reef fish: the bicolour damselfish (Stegastes partitus). At four island locations surrounding Exuma Sound, Bahamas, including a long-established marine reserve, we collected 3278 individuals and genotyped them at 10 microsatellite loci. Using Bayesian parentage analysis, we identified eight parent-offspring pairs, thereby directly documenting dispersal distances ranging from 0 km (i.e., self-recruitment) to 129 km (i.e., larval connectivity). Despite documenting substantial dispersal and gene flow between islands, we observed more self-recruitment events than expected if the larvae were drawn from a common, well-mixed pool (i.e., a completely open population). Additionally, we detected both spatial and temporal variation in signatures of sweepstakes and Wahlund effects. The high variance in reproductive success (i.e., 'sweepstakes') we observed may be influenced by seasonal mesoscale gyres present in the Exuma Sound, which play a prominent role in shaping local oceanographic patterns. This study documents the complex nature of larval dispersal in a coral-reef fish, and highlights the importance of sampling multiple cohorts and coupling both direct and indirect genetic methods in order disentangle patterns of dispersal, gene flow and variable reproductive success.

How much does inbreeding contribute to the reduced fitness of hatchery-born steelhead (Oncorhynchus mykiss) in the wild?

Many declining populations are supplemented with captive-born individuals that are released directly into the wild. Because captive-born individuals can have lower fitness in the wild than their wild-born counterparts, a comprehensive understanding of the mechanisms responsible for the reduced fitness of these individuals is required for appropriate conservation and management decisions. Inbreeding among captive-born individuals is one plausible mechanism because captive breeding programs frequently use small numbers of breeders to create large numbers of siblings that are subsequently released together into the wild. We tested this hypothesis in a supplementation program for steelhead (Oncorhynchus mykiss) from the Hood River, Oregon, for which first-generation hatchery fish were demonstrated to have lower fitness in the wild than their wild-born counterparts. To determine the contribution of inbreeding to this fitness decline, we first assigned 11 run-years of hatchery steelhead (3005 fish) back to their broodstock parents (462 fish) using 8 polymorphic microsatellite loci. By combining pedigree analyses with species-specific estimates of genetic load, we found that inbreeding could at most account for a 1-4% reduction in the fitness of hatchery fish relative to wild fish. Thus, inbreeding alone cannot adequately explain the 15% average fitness decline observed in first-generation hatchery fish from this population.

Bayesian parentage analysis reliably controls the number of false assignments in natural populations.

Parentage analysis in natural populations is a powerful tool for addressing a wide range of ecological and evolutionary questions. However, identifying parent-offspring pairs in samples collected from natural populations is often more challenging than simply resolving the Mendelian pattern of shared alleles. For example, large numbers of pairwise comparisons and limited numbers of genetic markers can contribute to incorrect assignments, whereby unrelated individuals are falsely identified as parent-offspring pairs. Determining which parentage methods are the least susceptible to making false assignments is an important challenge facing molecular ecologists. In a recent paper, Harrison et al. (2013a) address this challenge by comparing three commonly used parentage methods, including a Bayesian approach, in order to explore the effects of varied proportions of sampled parents on the accuracy of parentage assignments. Unfortunately, Harrison et al. made a simple error in using the Bayesian approach, which led them to incorrectly conclude that this method could not control the rate of false assignment. Here, I briefly outline the basic principles behind the Bayesian approach, identify the error made by Harrison et al., and provide detailed guidelines as to how the method should be correctly applied. Furthermore, using the exact data from Harrison et al., I show that the Bayesian approach actually provides greater control over the number of false assignments than either of the other tested methods. Lastly, I conclude with a brief introduction to solomon, a recently updated version of the Bayesian approach that can account for genotyping error, missing data and false matching.

Negative frequency dependent selection unites ecology and evolution.

From genes to communities, understanding how diversity is maintained remains a fundamental question in biology. One challenging to identify, yet potentially ubiquitous, mechanism for the maintenance of diversity is negative frequency dependent selection (NFDS), which occurs when entities (e.g., genotypes, life history strategies, species) experience a per capita reduction in fitness with increases in relative abundance. Because NFDS allows rare entities to increase in frequency while preventing abundant entities from excluding others, we posit that negative frequency dependent selection plays a central role in the maintenance of diversity. In this review, we relate NFDS to coexistence, identify mechanisms of NFDS (e.g., mutualism, predation, parasitism), review strategies for identifying NFDS, and distinguish NFDS from other mechanisms of coexistence (e.g., storage effects, fluctuating selection). We also emphasize that NFDS is a key place where ecology and evolution intersect. Specifically, there are many examples of frequency dependent processes in ecology, but fewer cases that link this process to selection. Similarly, there are many examples of selection in evolution, but fewer cases that link changes in trait values to negative frequency dependence. Bridging these two well-developed fields of ecology and evolution will allow for mechanistic insights into the maintenance of diversity at multiple levels.

Dispersive currents explain patterns of population connectivity in an ecologically and economically important fish.

How to identify the drivers of population connectivity remains a fundamental question in ecology and evolution. Answering this question can be challenging in aquatic environments where dynamic lake and ocean currents coupled with high levels of dispersal and gene flow can decrease the utility of modern population genetic tools. To address this challenge, we used RAD-Seq to genotype 959 yellow perch (Perca flavescens), a species with an ~40-day pelagic larval duration (PLD), collected from 20 sites circumscribing Lake Michigan. We also developed a novel, integrative approach that couples detailed biophysical models with eco-genetic agent-based models to generate "predictive" values of genetic differentiation. By comparing predictive and empirical values of genetic differentiation, we estimated the relative contributions for known drivers of population connectivity (e.g., currents, behavior, PLD). For the main basin populations (i.e., the largest contiguous portion of the lake), we found that high gene flow led to low overall levels of genetic differentiation among populations (F ST = 0.003). By far the best predictors of genetic differentiation were connectivity matrices that were derived from periods of time when there were strong and highly dispersive currents. Thus, these highly dispersive currents are driving the patterns of population connectivity in the main basin. We also found that populations from the northern and southern main basin are slightly divergent from one another, while those from Green Bay and the main basin are highly divergent (F ST = 0.11). By integrating biophysical and eco-genetic models with genome-wide data, we illustrate that the drivers of population connectivity can be identified in high gene flow systems.

Genomic DNA extraction optimization and validation for genome sequencing using the marine gastropod Kellet's whelk.

Next-generation sequencing technologies, such as Nanopore MinION, Illumina Hiseq and Novaseq, and PacBio Sequel II, hold immense potential for advancing genomic research on non-model organisms, including the vast majority of marine species. However, application of these technologies to marine invertebrate species is often impeded by challenges in extracting and purifying their genomic DNA due to high polysaccharide content and other secondary metabolites. In this study, we help resolve this issue by developing and testing DNA extraction protocols for Kellet's whelk (Kelletia kelletii), a subtidal gastropod with ecological and commercial importance, by comparing four DNA extraction methods commonly used in marine invertebrate studies. In our comparison of extraction methods, the Salting Out protocol was the least expensive, produced the highest DNA yields, produced consistent high DNA quality, and had low toxicity. We validated the protocol using an independent set of tissue samples, then applied it to extract high-molecular-weight (HMW) DNA from over three thousand Kellet's whelk tissue samples. The protocol demonstrated scalability and, with added clean-up, suitability for RAD-seq, GT-seq, as well as whole genome sequencing using both long read (ONT MinION) and short read (Illumina NovaSeq) sequencing platforms. Our findings offer a robust and versatile DNA extraction and clean-up protocol for supporting genomic research on non-model marine organisms, to help mediate the under-representation of invertebrates in genomic studies.

Paternity assignment and demographic closure in the New Zealand southern right whale.

The identification and characterization of reproductively isolated subpopulations or 'stocks' are essential for effective conservation and management decisions. This can be difficult in vagile marine species like marine mammals. We used paternity assignment and 'gametic recapture' to examine the reproductive autonomy of southern right whales (Eubalaena australis) on their New Zealand (NZ) calving grounds. We derived DNA profiles for 34 mother-calf pairs from skin biopsy samples, using sex-specific markers, 13 microsatellite loci and mtDNA haplotypes. We constructed DNA profiles for 314 adult males, representing 30% of the census male abundance of the NZ stock, previously estimated from genotypic mark-recapture modelling to be 1085 (95% CL 855, 1416). Under the hypothesis of demographic closure and the assumption of equal reproductive success among males, we predict: (i) the proportion of paternities assigned will reflect the proportion of the male population sampled and (ii) the gametic mark-recapture (GMR) estimate of male abundance will be equivalent to the census male estimate for the NZ stock. Consistent with these predictions, we found that the proportion of assigned paternities equalled the proportion of the census male population size sampled. Using the sample of males as the initial capture, and paternity assignment as the recapture, the GMR estimate of male abundance was 1001 (95% CL 542, 1469), similar to the male census estimate. These findings suggest that right whales returning to the NZ calving ground are reproductively autonomous on a generational timescale, as well as isolated by maternal fidelity on an evolutionary timescale, from others in the Indo-Pacific region.