A second unveiling: Haplotig masking of the eastern oyster genome improves population-level inference.

Genome assembly can be challenging for species that are characterized by high amounts of polymorphism, heterozygosity, and large effective population sizes. High levels of heterozygosity can result in genome mis-assemblies and a larger than expected genome size due to the haplotig versions of a single locus being assembled as separate loci. Here, we describe the first chromosome-level genome for the eastern oyster, Crassostrea virginica. Publicly released and annotated in 2017, the assembly has a scaffold N50 of 54 mb and is over 97.3% complete based on BUSCO analysis. The genome assembly for the eastern oyster is a critical resource for foundational research into molluscan adaptation to a changing environment and for selective breeding for the aquaculture industry. Subsequent resequencing data suggested the presence of haplotigs in the original assembly, and we developed a post hoc method to break up chimeric contigs and mask haplotigs in published heterozygous genomes and evaluated improvements to the accuracy of downstream analysis. Masking haplotigs had a large impact on SNP discovery and estimates of nucleotide diversity and had more subtle and nuanced effects on estimates of heterozygosity, population structure analysis, and outlier detection. We show that haplotig masking can be a powerful tool for improving genomic inference, and we present an open, reproducible resource for the masking of haplotigs in any published genome.

Development and Evaluation of High-Density SNP Arrays for the Eastern Oyster Crassostrea virginica.

The eastern oyster Crassostrea virginica is a major aquaculture species for the USA. The sustainable development of eastern oyster aquaculture depends upon the continued improvement of cultured stocks through advanced breeding technologies. The Eastern Oyster Breeding Consortium (EOBC) was formed to advance the genetics and breeding of the eastern oyster. To facilitate efficient genotyping needed for genomic studies and selection, the consortium developed two single-nucleotide polymorphism (SNP) arrays for the eastern oyster: one screening array with 566K SNPs and one breeders' array with 66K SNPs. The 566K screening array was developed based on whole-genome resequencing data from 292 oysters from Atlantic and Gulf of Mexico populations; it contains 566,262 SNPs including 47K from protein-coding genes with a marker conversion rate of 48.34%. The 66K array was developed using best-performing SNPs from the screening array, which contained 65,893 oyster SNPs including 22,984 genic markers with a calling rate of 99.34%, a concordance rate of 99.81%, and a much-improved marker conversion rate of 92.04%. Null alleles attributable to large indels were found in 13.1% of the SNPs, suggesting that copy number variation is pervasive. Both arrays provided easy identification and separation of selected stocks from wild progenitor populations. The arrays contain 31 mitochondrial SNPs that allowed unambiguous identification of Gulf mitochondrial genotypes in some Atlantic populations. The arrays also contain 756 probes from 13 oyster and human pathogens for possible detection. Our results show that marker conversion rate is low in high polymorphism species and that the two-step process of array development can greatly improve array performance. The two arrays will advance genomic research and accelerate genetic improvement of the eastern oyster by delineating genetic architecture of production traits and enabling genomic selection. The arrays also may be used to monitor pedigree and inbreeding, identify selected stocks and their introgression into wild populations, and assess the success of oyster restoration.

Invertebrate methylomes provide insight into mechanisms of environmental tolerance and reveal methodological biases.

There is a growing focus on the role of DNA methylation in the ability of marine invertebrates to rapidly respond to changing environmental factors and anthropogenic impacts. However, genome-wide DNA methylation studies in nonmodel organisms are currently hampered by a limited understanding of methodological biases. Here, we compare three methods for quantifying DNA methylation at single base-pair resolution-whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and methyl-CpG binding domain bisulfite sequencing (MBDBS)-using multiple individuals from two reef-building coral species with contrasting environmental sensitivity. All methods reveal substantially greater methylation in Montipora capitata (11.4%) than the more sensitive Pocillopora acuta (2.9%). The majority of CpG methylation in both species occurs in gene bodies and flanking regions. In both species, MBDBS has the greatest capacity for detecting CpGs in coding regions at our sequencing depth, but MBDBS may be influenced by intrasample methylation heterogeneity. RRBS yields robust information for specific loci albeit without enrichment of any particular genome feature and with significantly reduced genome coverage. Relative genome size strongly influences the number and location of CpGs detected by each method when sequencing depth is limited, illuminating nuances in cross-species comparisons. As genome-wide methylation differences, supported by data across bisulfite sequencing methods, may contribute to environmental sensitivity phenotypes in critical marine invertebrate taxa, these data provide a genomic resource for investigating the functional role of DNA methylation in environmental tolerance.

Minor Genetic Consequences of a Major Mass Mortality: Short-Term Effects in Pisaster ochraceus.

AbstractMass mortality events are increasing globally in frequency and magnitude, largely as a result of human-induced change. The effects of these mass mortality events, in both the long and short term, are of imminent concern because of their ecosystem impacts. Genomic data can be used to reveal some of the population-level changes associated with mass mortality events. Here, we use reduced-representation sequencing to identify potential short-term genetic impacts of a mass mortality event associated with a sea star wasting outbreak. We tested for changes in the population for genetic differentiation, diversity, and effective population size between pre-sea star wasting and post-sea star wasting populations of Pisaster ochraceus-a species that suffered high sea star wasting-associated mortality (75%-100% at 80% of sites). We detected no significant population-based genetic differentiation over the spatial scale sampled; however, the post-sea star wasting population tended toward more differentiation across sites than the pre-sea star wasting population. Genetic estimates of effective population size did not detectably change, consistent with theoretical expectations; however, rare alleles were lost. While we were unable to detect significant population-based genetic differentiation or changes in effective population size over this short time period, the genetic burden of this mass mortality event may be borne by future generations, unless widespread recruitment mitigates the population decline. Prior results from P. ochraceus indicated that natural selection played a role in altering allele frequencies following this mass mortality event. In addition to the role of selection found in a previous study on the genomic impacts of sea star wasting on P. ochraceus, our current study highlights the potential role the stochastic loss of many individuals plays in altering how genetic variation is structured across the landscape. Future genetic monitoring is needed to determine long-term genetic impacts in this long-lived species. Given the increased frequency of mass mortality events, it is important to implement demographic and genetic monitoring strategies that capture baselines and background dynamics to better contextualize species' responses to large perturbations.

Extensive genome-wide duplications in the eastern oyster (Crassostrea virginica).

Genomic structural variation is an important source of genetic and phenotypic diversity, playing a critical role in evolution. The recent availability of a high-quality reference genome for the eastern oyster, Crassostrea virginica, and whole-genome sequence data of samples from across the species range in the USA, provides an opportunity to explore structural variation across the genome of this species. Our analysis shows significantly greater individual-level duplications of regions across the genome than that of most model vertebrate species. Duplications are widespread across all ten chromosomes with variation in frequency per chromosome. The eastern oyster shows a large interindividual variation in duplications as well as particular chromosomal regions with a higher density of duplications. A high percentage of duplications seen in C. virginica lie completely within genes and exons, suggesting the potential for impacts on gene function. These results support the hypothesis that structural changes may play a significant role in standing genetic variation in C. virginica, and potentially have a role in their adaptive and evolutionary success. Altogether, these results suggest that copy number variation plays an important role in the genomic variation of C. virginica. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Quantifying dispersal variability among nearshore marine populations.

Dispersal drives diverse processes from population persistence to community dynamics. However, the amount of temporal variation in dispersal and its consequences for metapopulation dynamics is largely unknown for organisms with environmentally driven dispersal (e.g., many marine larvae, arthropods and plant seeds). Here, we used genetic parentage analysis to detect larval dispersal events in a common coral reef fish, Amphiprion clarkii, along 30 km of coastline consisting of 19 reef patches in Ormoc Bay, Leyte, Philippines. We quantified variation in the dispersal kernel across seven years (2012-2018) and monsoon seasons with 71 parentage assignments from 791 recruits and 1,729 adults. Connectivity patterns differed significantly among years and seasons in the scale and shape but not in the direction of dispersal. This interannual variation in dispersal kernels introduced positive temporal covariance among dispersal routes that theory predicts is likely to reduce stochastic metapopulation growth rates below the growth rates expected from only a single or a time-averaged connectivity estimate. The extent of variation in mean dispersal distance observed here among years is comparable in magnitude to the differences across reef fish species. Considering dispersal variation will be an important avenue for further metapopulation and metacommunity research across diverse taxa.

These aren't the loci you'e looking for: Principles of effective SNP filtering for molecular ecologists.

Sequencing reduced-representation libraries of restriction site-associated DNA (RADseq) to identify single nucleotide polymorphisms (SNPs) is quickly becoming a standard methodology for molecular ecologists. Because of the scale of RADseq data sets, putative loci cannot be assessed individually, making the process of filtering noise and correctly identifying biologically meaningful signal more difficult. Artefacts introduced during library preparation and/or bioinformatic processing of SNP data can create patterns that are incorrectly interpreted as indicative of population structure or natural selection. Therefore, it is crucial to carefully consider types of errors that may be introduced during laboratory work and data processing, and how to minimize, detect and remove these errors. Here, we discuss issues inherent to RADseq methodologies that can result in artefacts during library preparation and locus reconstruction resulting in erroneous SNP calls and, ultimately, genotyping error. Further, we describe steps that can be implemented to create a rigorously filtered data set consisting of markers accurately representing independent loci and compare the effect of different combinations of filters on four RAD data sets. At last, we stress the importance of publishing raw sequence data along with final filtered data sets in addition to detailed documentation of filtering steps and quality control measures.

Decimation by sea star wasting disease and rapid genetic change in a keystone species, Pisaster ochraceus.

Standing genetic variation enables or restricts a population's capacity to respond to changing conditions, including the extreme disturbances expected to increase in frequency and intensity with continuing anthropogenic climate change. However, we know little about how populations might respond to extreme events with rapid genetic shifts, or how population dynamics may influence and be influenced by population genomic change. We use a range-wide epizootic, sea star wasting disease, that onset in mid-2013 and caused mass mortality in Pisaster ochraceus to explore how a keystone marine species responded to an extreme perturbation. We integrated field surveys with restriction site-associated DNA sequencing data to (i) describe the population dynamics of mortality and recovery, and (ii) compare allele frequencies in mature P. ochraceus before the disease outbreak with allele frequencies in adults and new juveniles after the outbreak, to identify whether selection may have occurred. We found P. ochraceus suffered 81% mortality in the study region between 2012 and 2015, and experienced a concurrent 74-fold increase in recruitment beginning in late 2013. Comparison of pre- and postoutbreak adults revealed significant allele frequency changes at three loci, which showed consistent changes across the large majority of locations. Allele frequency shifts in juvenile P. ochraceus (spawned from premortality adults) were consistent with those seen in adult survivors. Such parallel shifts suggest detectable signals of selection and highlight the potential for persistence of this change in subsequent generations, which may influence the resilience of this keystone species to future outbreaks.

Expressed exome capture sequencing: A method for cost-effective exome sequencing for all organisms.

Exome capture is an effective tool for surveying the genome for loci under selection. However, traditional methods require annotated genomic resources. Here, we present a method for creating cDNA probes from expressed mRNA, which are then used to enrich and capture genomic DNA for exon regions. This approach, called "EecSeq," eliminates the need for costly probe design and synthesis. We tested EecSeq in the eastern oyster, Crassostrea virginica, using a controlled exposure experiment. Four adult oysters were heat shocked at 36°C for 1 hr along with four control oysters kept at 14°C. Stranded mRNA libraries were prepared for two individuals from each treatment and pooled. Half of the combined library was used for probe synthesis, and half was sequenced to evaluate capture efficiency. Genomic DNA was extracted from all individuals, enriched via captured probes, and sequenced directly. We found that EecSeq had an average capture sensitivity of 86.8% across all known exons and had over 99.4% sensitivity for exons with detectable levels of expression in the mRNA library. For all mapped reads, over 47.9% mapped to exons and 37.0% mapped to expressed targets, which is similar to previously published exon capture studies. EecSeq displayed relatively even coverage within exons (i.e., minor "edge effects") and even coverage across exon GC content. We discovered 5,951 SNPs with a minimum average coverage of 80×, with 3,508 SNPs appearing in exonic regions. We show that EecSeq provides comparable, if not superior, specificity and capture efficiency compared to costly, traditional methods.

Life-history predicts past and present population connectivity in two sympatric sea stars.

Life-history traits, especially the mode and duration of larval development, are expected to strongly influence the population connectivity and phylogeography of marine species. Comparative analysis of sympatric, closely related species with differing life histories provides the opportunity to specifically investigate these mechanisms of evolution but have been equivocal in this regard. Here, we sample two sympatric sea stars across the same geographic range in temperate waters of Australia. Using a combination of mitochondrial DNA sequences, nuclear DNA sequences, and microsatellite genotypes, we show that the benthic-developing sea star, Parvulastra exigua, has lower levels of within- and among-population genetic diversity, more inferred genetic clusters, and higher levels of hierarchical and pairwise population structure than Meridiastra calcar, a species with planktonic development. While both species have populations that have diverged since the middle of the second glacial period of the Pleistocene, most P. exigua populations have origins after the last glacial maxima (LGM), whereas most M. calcar populations diverged long before the LGM. Our results indicate that phylogenetic patterns of these two species are consistent with predicted dispersal abilities; the benthic-developing P. exigua shows a pattern of extirpation during the LGM with subsequent recolonization, whereas the planktonic-developing M. calcar shows a pattern of persistence and isolation during the LGM with subsequent post-Pleistocene introgression.

Haplotyping RAD loci: an efficient method to filter paralogs and account for physical linkage.

Next-generation sequencing of reduced-representation genomic libraries provides a powerful methodology for genotyping thousands of single-nucleotide polymorphisms (SNPs) among individuals of nonmodel species. Utilizing genotype data in the absence of a reference genome, however, presents a number of challenges. One major challenge is the trade-off between splitting alleles at a single locus into separate clusters (loci), creating inflated homozygosity, and lumping multiple loci into a single contig (locus), creating artefacts and inflated heterozygosity. This issue has been addressed primarily through the use of similarity cut-offs in sequence clustering. Here, two commonly employed, postclustering filtering methods (read depth and excess heterozygosity) used to identify incorrectly assembled loci are compared with haplotyping, another postclustering filtering approach. Simulated and empirical data sets were used to demonstrate that each of the three methods separately identified incorrectly assembled loci; more optimal results were achieved when the three methods were applied in combination. The results confirmed that including incorrectly assembled loci in population-genetic data sets inflates estimates of heterozygosity and deflates estimates of population divergence. Additionally, at low levels of population divergence, physical linkage between SNPs within a locus created artificial clustering in analyses that assume markers are independent. Haplotyping SNPs within a locus effectively neutralized the physical linkage issue without having to thin data to a single SNP per locus. We introduce a Perl script that haplotypes polymorphisms, using data from single or paired-end reads, and identifies potentially problematic loci.

Fine-scale partitioning of genomic variation among recruits in an exploited fishery: causes and consequences.

Conservation and management of exploited species depends on accurate knowledge of how genetic variation is partitioned across a fishery, especially as it relates to recruitment. Using double-digest restriction-site associated DNA sequencing, we surveyed variation in 7,382 single nucleotide polymorphisms (SNPs) in red snapper (Lutjanus campechanus) young-of-the-year (YOY) sampled at six localities and in adults sampled at two localities in the northern Gulf of Mexico. Significant genetic heterogeneity was detected between the two adult samples, separated by ~600 km, and at spatial scales less than five kilometers among samples of YOY. Genetic differences between YOY samples and between YOY samples and adult samples were not associated with geographic distance, and a genome scan revealed no evidence of loci under selection. Estimates of the effective number of breeders, allelic richness, and relatedness within YOY samples were not consistent with sweepstakes recruitment. Instead, the data demonstrate, at least within one recruitment season, that multiple pulses of recruits originate from distinct groups of spawning adults, even at small spatial scales. For exploited species with this type of recruitment pattern, protection of spawning adults over wide geographic areas may be critical for ensuring productivity and stability of the fishery by maintaining larval supply and connectivity.

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.

dDocent: a RADseq, variant-calling pipeline designed for population genomics of non-model organisms.

Restriction-site associated DNA sequencing (RADseq) has become a powerful and useful approach for population genomics. Currently, no software exists that utilizes both paired-end reads from RADseq data to efficiently produce population-informative variant calls, especially for non-model organisms with large effective population sizes and high levels of genetic polymorphism. dDocent is an analysis pipeline with a user-friendly, command-line interface designed to process individually barcoded RADseq data (with double cut sites) into informative SNPs/Indels for population-level analyses. The pipeline, written in BASH, uses data reduction techniques and other stand-alone software packages to perform quality trimming and adapter removal, de novo assembly of RAD loci, read mapping, SNP and Indel calling, and baseline data filtering. Double-digest RAD data from population pairings of three different marine fishes were used to compare dDocent with Stacks, the first generally available, widely used pipeline for analysis of RADseq data. dDocent consistently identified more SNPs shared across greater numbers of individuals and with higher levels of coverage. This is due to the fact that dDocent quality trims instead of filtering, incorporates both forward and reverse reads (including reads with INDEL polymorphisms) in assembly, mapping, and SNP calling. The pipeline and a comprehensive user guide can be found at http://dDocent.wordpress.com.

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.

Shallow gene pools in the high intertidal: extreme loss of genetic diversity in viviparous sea stars (Parvulastra).

We document an extreme example of reproductive trait evolution that affects population genetic structure in sister species of Parvulastra cushion stars from Australia. Self-fertilization by hermaphroditic adults and brood protection of benthic larvae causes strong inbreeding and range-wide genetic poverty. Most samples were fixed for a single allele at nearly all nuclear loci; heterozygotes were extremely rare (0.18%); mitochondrial DNA sequences were more variable, but few populations shared haplotypes in common. Isolation-with-migration models suggest that these patterns are caused by population bottlenecks (relative to ancestral population size) and low gene flow. Loss of genetic diversity and low potential for dispersal between high-intertidal habitats may have dire consequences for extinction risk and potential for future adaptive evolution in response to climate and other selective agents.

The evolving male: spinner dolphin (Stenella longirostris) ecotypes are divergent at Y chromosome but not mtDNA or autosomal markers.

The susceptibility of the Y chromosome to sexual selection may make this chromosome an important player in the formation of reproductive isolating barriers, and ultimately speciation. Here, we investigate the role of the Y chromosome in phenotypic divergence and reproductive isolation of spinner dolphin (Stenella longirostris) ecotypes. This species contains six known ecotypes (grouped into four subspecies) that exhibit striking differences in morphology, habitat and mating system, despite having adjacent or overlapping ranges and little genetic divergence at previously studied mtDNA and autosomal markers. We examined the phylogeographic structure for all six ecotypes across the species range (n = 261, 17 geographic locations) using DNA sequences from three Y chromosome markers, two maternally inherited mitochondrial (mtDNA) markers, and a biparentally inherited autosomal intron. mtDNA and autosomal analyses revealed low divergence (most Φ(ST) values <0.1) between ecotypes and geographic regions, concordant with previous studies. In contrast, Y intron analyses revealed fixed differences amongst the three most phenotypically divergent groups: S. l. longirostris vs. S. l. roseiventris vs. combined S. l. orientalis/S. l. centroamericana/Tres Marias ecotypes). Another ecotype (whitebelly), previously postulated to be a hybrid between the two phenotypically most divergent ecotypes, had Y haplotypes from both putative parent ecotypes, supporting a hybrid designation. Reduced introgression of the Y chromosome has previously been observed in other organisms ranging from insects to terrestrial mammals, and here we demonstrate this phenomenon in a marine mammal with high dispersal capabilities. These results indicate that reduced introgression of the Y chromosome occurs in a wide taxonomic range of organisms and support the growing body of evidence that rapid evolution of the Y chromosome is important in evolutionary diversification.

Next-generation sequencing for high-throughput molecular ecology: a step-by-step protocol for targeted multilocus genotyping by pyrosequencing.

Next-generation sequencing technology can now provide population biologists and phylogeographers with information at the genomic scale; however, many pertinent questions in population genetics and phylogeography can be answered effectively with modest levels of genomic information. For the past two decades, most population-level studies have lacked nuclear DNA (nDNA) sequence data due to the complications and cost of amplifying and sequencing diploid loci. However, pyrosequencing of emulsion PCR reactions, amplifying from only one molecule at a time, can generate megabases of clonally amplified loci at high coverage, thereby greatly simplifying allelic sequence determination. Here, we present a step-by-step methodology for utilizing the 454 GS FLX Titanium pyrosequencing platform to simultaneously sequence 16 populations (at 20 individuals per population) at 10 different nDNA loci (3,200 loci in total) in one plate of sequencing for less than the cost of traditional Sanger sequencing.

Extraordinarily rapid life-history divergence between Cryptasterina sea star species.

Life history plays a critical role in governing microevolutionary processes such as gene flow and adaptation, as well as macroevolutionary processes such speciation. Here, we use multilocus phylogeographic analyses to examine a speciation event involving spectacular life-history differences between sister species of sea stars. Cryptasterina hystera has evolved a suite of derived life-history traits (including internal self-fertilization and brood protection) that differ from its sister species Cryptasterina pentagona, a gonochoric broadcast spawner. We show that these species have only been reproductively isolated for approximately 6000 years (95% highest posterior density of 905-22 628), and that this life-history change may be responsible for dramatic genetic consequences, including low nucleotide diversity, zero heterozygosity and no gene flow. The rapid divergence of these species rules out some mechanisms of isolation such as adaptation to microhabitats in sympatry, or slow divergence by genetic drift during prolonged isolation. We hypothesize that the large phenotypic differences between species relative to the short divergence time suggests that the life-history differences observed may be direct responses to disruptive selection between populations. We speculate that local environmental or demographic differences at the southern range margin are possible mechanisms of selection driving one of the fastest known marine speciation events.