Whole-genome sequencing reveals fine-scale environment-associated divergence near the range limits of a temperate reef fish.

Environmental variation is increasingly recognized as an important driver of diversity in marine species despite the lack of physical barriers to dispersal and the presence of pelagic stages in many taxa. A robust understanding of the genomic and ecological processes involved in structuring populations is lacking for most marine species, often hindering management and conservation action. Cunner (Tautogolabrus adspersus) is a temperate reef fish with both pelagic early life-history stages and strong site-associated homing as adults; the species is also of interest for use as a cleaner fish in salmonid aquaculture in Atlantic Canada. We aimed to characterize genomic and geographic differentiation of cunner in the Northwest Atlantic. To achieve this, a chromosome-level genome assembly for cunner was produced and used to characterize spatial population structure throughout Atlantic Canada using whole-genome sequencing. The genome assembly spanned 0.72 Gbp and 24 chromosomes; whole-genome sequencing of 803 individuals from 20 locations from Newfoundland to New Jersey identified approximately 11 million genetic variants. Principal component analysis revealed four regional Atlantic Canadian groups. Pairwise FST and selection scans revealed signals of differentiation and selection at discrete genomic regions, including adjacent peaks on chromosome 10 across multiple pairwise comparisons (i.e. FST 0.5-0.75). Redundancy analysis suggested association of environmental variables related to benthic temperature and oxygen range with genomic structure. Results suggest regional scale diversity in this temperate reef fish and can directly inform the collection and translocation of cunner for aquaculture applications and the conservation of wild populations throughout the Northwest Atlantic.

Variation in the genomic basis of parallel phenotypic and ecological divergence in benthic and pelagic morphs of Icelandic Arctic charr (Salvelinus alpinus).

Sympatric adaptive phenotypic divergence should be underlain by genomic differentiation between subpopulations. When divergence drives similar patterns of phenotypic and ecological variation within species we expect evolution to draw on common allelic variation. We investigated divergence histories and genomic signatures of adaptive divergence between benthic and pelagic morphs of Icelandic Arctic charr. Divergence histories for each of four populations were reconstructed using coalescent modelling and 14,187 single nucleotide polymorphisms. Sympatric divergence with continuous gene flow was supported in two populations while allopatric divergence with secondary contact was supported in one population; we could not differentiate between demographic models in the fourth population. We detected parallel patterns of phenotypic divergence along benthic-pelagic evolutionary trajectories among populations. Patterns of genomic differentiation between benthic and pelagic morphs were characterized by outlier loci in many narrow peaks of differentiation throughout the genome, which may reflect the eroding effects of gene flow on nearby neutral loci. We then used genome-wide association analyses to relate both phenotypic (body shape and size) and ecological (carbon and nitrogen stable isotopes) variation to patterns of genomic differentiation. Many peaks of genomic differentiation were associated with phenotypic and ecological variation in the three highly divergent populations, suggesting a genomic basis for adaptive divergence. We detected little evidence for a parallel genomic basis of differentiation as most regions and outlier loci were not shared among populations. Our results show that adaptive divergence can have varied genomic consequences in populations with relatively recent common origins, similar divergence histories, and parallel phenotypic divergence.

Putative chromosomal rearrangements are associated primarily with ecotype divergence rather than geographic separation in an intertidal, poorly dispersing snail.

Littorina saxatilis is becoming a model system for understanding the genomic basis of ecological speciation. The parallel formation of crab-adapted ecotypes that exhibit partial reproductive isolation from wave-adapted ecotypes has enabled genomic investigation of conspicuous shell traits. Recent genomic studies suggest that chromosomal rearrangements may enable ecotype divergence by reducing gene flow. However, the genomic architecture of traits that are divergent between ecotypes remains poorly understood. Here, we use 11,504 single nucleotide polymorphism (SNP) markers called using the recently released L. saxatilis genome to genotype 462 crab ecotype, wave ecotype and phenotypically intermediate Spanish L. saxatilis individuals with scored phenotypes. We used redundancy analysis to study the genetic architecture of loci associated with shell shape, shape corrected for size, shell size and shell ornamentation, and to compare levels of co-association among different traits. We discovered 341 SNPs associated with shell traits. Loci associated with trait divergence between ecotypes were often located inside putative chromosomal rearrangements recently characterized in Swedish L. saxatilis. In contrast, we found that shell shape corrected for size varied primarily by geographic site rather than by ecotype and showed little association with these putative rearrangements. We conclude that genomic regions of elevated divergence inside putative rearrangements were associated with divergence of L. saxatilis ecotypes along steep environmental axes-consistent with models of adaptation with gene flow-but were not associated with divergence among the three geographical sites. Our findings support predictions from models indicating the importance of genomic regions of reduced recombination allowing co-association of loci during ecological speciation with ongoing gene flow.

Genomic and machine learning-based screening of aquaculture-associated introgression into at-risk wild North American Atlantic salmon (Salmo salar) populations.

The negative genetic impacts of gene flow from domestic to wild populations can be dependent on the degree of domestication and exacerbated by the magnitude of pre-existing genetic differences between wild populations and the domestication source. Recent evidence of European ancestry within North American aquaculture Atlantic salmon (Salmo salar) has elevated the potential impact of escaped farmed salmon on often at-risk wild North American salmon populations. Here, we compare the ability of single nucleotide polymorphism (SNP) and microsatellite (SSR) marker panels of different sizes (7-SSR, 100-SSR and 220K-SNP) to detect introgression of European genetic information into North American wild and aquaculture populations. Linear regression comparing admixture predictions for a set of individuals common to the three datasets showed that the 100-SSR panel and 7-SSR panels replicated the full 220K-SNP-based admixture estimates with low accuracy (r2 of .64 and .49, respectively). Additional tests explored the effects of individual sample size and marker number, which revealed that ~300 randomly selected SNPs could replicate the 220K-SNP admixture predictions with greater than 95% fidelity. We designed a custom SNP panel (301-SNP) for European admixture detection in future monitoring work and then developed and tested a python package, salmoneuadmix (https://github.com/CNuge/SalmonEuAdmix), which uses a deep neural network to make de novo estimates of individuals' European admixture proportion without the need to conduct complete admixture analysis utilizing baseline samples. The results demonstrate the mobilization of targeted SNP panels and machine learning in support of at-risk species conservation and management.

Fine-scale environmentally associated spatial structure of lumpfish (Cyclopterus lumpus) across the Northwest Atlantic.

Lumpfish, Cyclopterus lumpus, have historically been harvested throughout Atlantic Canada and are increasingly in demand as a solution to controlling sea lice in Atlantic salmon farms-a process which involves both the domestication and the transfer of lumpfish between geographic regions. At present, little is known regarding population structure and diversity of wild lumpfish in Atlantic Canada, limiting attempts to assess the potential impacts of escaped lumpfish individuals from salmon pens on currently at-risk wild populations. Here, we characterize the spatial population structure and genomic-environmental associations of wild populations of lumpfish throughout the Northwest Atlantic using both 70K SNP array data and whole-genome re-sequencing data (WGS). At broad spatial scales, our results reveal a large environmentally associated genetic break between the southern populations (Gulf of Maine and Bay of Fundy) and northern populations (Newfoundland and the Gulf of St. Lawrence), linked to variation in ocean temperature and ice cover. At finer spatial scales, evidence of population structure was also evident in a distinct coastal group in Newfoundland and significant isolation by distance across the northern region. Both evidence of consistent environmental associations and elevated genome-wide variation in F ST values among these three regional groups supports their biological relevance. This study represents the first extensive description of population structure of lumpfish in Atlantic Canada, revealing evidence of broad and fine geographic scale environmentally associated genomic diversity. Our results will facilitate the commercial use of lumpfish as a cleaner fish in Atlantic salmon aquaculture, the identification of lumpfish escapees, and the delineation of conservation units of this at-risk species throughout Atlantic Canada.

Reference genome of lumpfish Cyclopterus lumpus Linnaeus provides evidence of male heterogametic sex determination through the AMH pathway.

Teleosts exhibit extensive diversity of sex determination (SD) systems and mechanisms, providing the opportunity to study the evolution of SD and sex chromosomes. Here we sequenced the genome of the common lumpfish (Cyclopterus lumpus Linnaeus), a species of increasing importance to aquaculture, and identified the SD region and master SD locus using a 70 K single nucleotide polymorphism array and tissue-specific expression data. The chromosome-level assembly identified 25 diploid chromosomes with a total size of 572.89 Mb, a scaffold N50 of 23.86 Mb and genome annotation-predicted 21,480 protein-coding genes. Genome-wide association analysis located a highly sex-associated region on chromosome 13, suggesting that anti-Müllerian hormone (AMH) is the putative SD factor. Linkage disequilibrium and heterozygosity across chromosome 13 support a proto-XX/XY system, with an absence of widespread chromosome divergence between sexes. We identified three copies of AMH in the lumpfish primary and alternate haplotype assemblies localized in the SD region. Comparison to sequences from other teleosts suggested a monophyletic relationship and conservation within the Cottioidei. One AMH copy showed similarity to AMH/AMHY in a related species and was also the only copy with expression in testis tissue, suggesting this copy may be the functional copy of AMH in lumpfish. The two other copies arranged in tandem inverted duplication were highly similar, suggesting a recent duplication event. This study provides a resource for the study of early sex chromosome evolution and novel genomic resources that benefits lumpfish conservation management and aquaculture.

The interaction of resource use and gene flow on the phenotypic divergence of benthic and pelagic morphs of Icelandic Arctic charr (Salvelinus alpinus).

Conceptual models of adaptive divergence and ecological speciation in sympatry predict differential resource use, phenotype-environment correlations, and reduced gene flow among diverging phenotypes. While these predictions have been assessed in past studies, connections among them have rarely been assessed collectively. We examined relationships among phenotypic, ecological, and genetic variation in Arctic charr (Salvelinus alpinus) from six Icelandic localities that have undergone varying degrees of divergence into sympatric benthic and pelagic morphs. We characterized morphological variation with geometric morphometrics, tested for differential resource use between morphs using stable isotopes, and inferred the amount of gene flow from single nucleotide polymorphisms. Analysis of stable isotopic signatures indicated that sympatric morphs showed similar difference in resource use across populations, likely arising from the common utilization of niche space within each population. Carbon isotopic signature was also a significant predictor of individual variation in body shape and size, suggesting that variation in benthic and pelagic resource use is associated with phenotypic variation. The estimated percentage of hybrids between sympatric morphs varied across populations (from 0% to 15.6%) but the majority of fish had genotypes (ancestry coefficients) characteristic of pure morphs. Despite evidence of reduced gene flow between sympatric morphs, we did not detect the expected negative relationship between divergence in resource use and gene flow. Three lakes showed the expected pattern, but morphs in the fourth showed no detectable hybridization and had relatively low differences in resource use between them. This coupled with the finding that resource use and genetic differentiation had differential effects on body shape variation across populations suggests that reproductive isolation maintains phenotypic divergence between benthic and pelagic morphs when the effects of resource use are relatively low. Our ability to assess relationships between phenotype, ecology, and genetics deepens our understanding of the processes underlying adaptive divergence in sympatry.

Design and characterization of an 87k SNP genotyping array for Arctic charr (Salvelinus alpinus).

We have generated a high-density, high-throughput genotyping array for characterizing genome-wide variation in Arctic charr (Salvelinus alpinus). Novel single nucleotide polymorphisms (SNPs) were identified in charr from the Fraser, Nauyuk and Tree River aquaculture strains, which originated from northern Canada and fish from Iceland using high coverage sequencing, reduced representation sequencing and RNA-seq datasets. The array was designed to capture genome-wide variation from a diverse suite of Arctic charr populations. Cross validation of SNPs from various sources and comparison with previously published Arctic charr SNP data provided a set of candidate SNPs that generalize across populations. Further candidate SNPs were identified based on minor allele frequency, association with RNA transcripts, even spacing across intergenic regions and association with the sex determining (sdY) gene. The performance of the 86,503 SNP array was assessed by genotyping Fraser, Nauyuk and Tree River strain individuals, as well as wild Icelandic Arctic charr. Overall, 63,060 of the SNPs were polymorphic within at least one group and 36.8% were unique to one of the four groups, suggesting that the array design allows for characterization of both within and across population genetic diversity. The concordance between sdY markers and known phenotypic sex indicated that the array can accurately determine the sex of individuals based on genotype alone. The Salp87k genotyping array provides researchers and breeders the opportunity to analyze genetic variation in Arctic charr at a more detailed level than previously possible.