Multi-genome comparisons reveal gain-and-loss evolution of anti-Mullerian hormone receptor type 2 as a candidate master sex-determining gene in Percidae.

BACKGROUND: The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii, and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. RESULTS: We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex-determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplicates (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been likely lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome 18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variations (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex-determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in the testis than in the ovary. CONCLUSIONS: Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Conserved islands of divergence associated with adaptive variation in sockeye salmon are maintained by multiple mechanisms.

Local adaptation is facilitated by loci clustered in relatively few regions of the genome, termed genomic islands of divergence. The mechanisms that create and maintain these islands and how they contribute to adaptive divergence is an active research topic. Here, we use sockeye salmon as a model to investigate both the mechanisms responsible for creating islands of divergence and the patterns of differentiation at these islands. Previous research suggested that multiple islands contributed to adaptive radiation of sockeye salmon. However, the low-density genomic methods used by these studies made it difficult to fully elucidate the mechanisms responsible for islands and connect genotypes to adaptive variation. We used whole genome resequencing to genotype millions of loci to investigate patterns of genetic variation at islands and the mechanisms that potentially created them. We discovered 64 islands, including 16 clustered in four genomic regions shared between two isolated populations. Characterisation of these four regions suggested that three were likely created by structural variation, while one was created by processes not involving structural variation. All four regions were small (< 600 kb), suggesting low recombination regions do not have to span megabases to be important for adaptive divergence. Differentiation at islands was not consistently associated with established population attributes. In sum, the landscape of adaptive divergence and the mechanisms that create it are complex; this complexity likely helps to facilitate fine-scale local adaptation unique to each population.

Further evidence from common garden rearing experiments of heritable traits separating lean and siscowet lake charr (Salvelinus namaycush) ecotypes.

Genetic evidence of selection for complex and polygenically regulated phenotypes can easily become masked by neutral population genetic structure and phenotypic plasticity. Without direct evidence of genotype-phenotype associations it can be difficult to conclude to what degree a phenotype is heritable or a product of environment. Common garden laboratory studies control for environmental stochasticity and help to determine the mechanism that regulate traits. Here we assess lipid content, growth, weight, and length variation in full and hybrid F1 crosses of deep and shallow water sympatric lake charr ecotypes reared for nine years in a common garden experiment. Redundancy analysis (RDA) and quantitative-trait-loci (QTL) genomic scans are used to identify associations between genotypes at 19,714 single nucleotide polymorphisms (SNPs) aligned to the lake charr genome and individual phenotypes to determine the role that genetic inheritance plays in ecotype phenotypic diversity. Lipid content, growth, length, and weight differed significantly among lake charr crosses throughout the experiment suggesting that pedigree plays a large roll in lake charr development. Polygenic scores of 15 SNPs putatively associated with lipid content and/or condition factor indicated that ecotype distinguishing traits are polygenically regulated and additive. A QTL identified on chromosome 38 contained >200 genes, some of which were associated with lipid metabolism and growth, demonstrating the complex nature of ecotype diversity. The results of our common garden study further indicate that lake charr ecotypes observed in nature are predetermined at birth and that ecotypes differ fundamentally in lipid metabolism and growth.

Multi-genome comparisons reveal gain-and-loss evolution of the anti-Mullerian hormone receptor type 2 gene, an old master sex determining gene, in Percidae.

The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplications (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome-18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variants (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in testis than ovary. Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

A new GTSeq resource to facilitate multijurisdictional research and management of walleye Sander vitreus.

Conservation and management professionals often work across jurisdictional boundaries to identify broad ecological patterns. These collaborations help to protect populations whose distributions span political borders. One common limitation to multijurisdictional collaboration is consistency in data recording and reporting. This limitation can impact genetic research, which relies on data about specific markers in an organism's genome. Incomplete overlap of markers between separate studies can prevent direct comparisons of results. Standardized marker panels can reduce the impact of this issue and provide a common starting place for new research. Genotyping-in-thousands (GTSeq) is one approach used to create standardized marker panels for nonmodel organisms. Here, we describe the development, optimization, and early assessments of a new GTSeq panel for use with walleye (Sander vitreus) from the Great Lakes region of North America. High genome-coverage sequencing conducted using RAD capture provided genotypes for thousands of single nucleotide polymorphisms (SNPs). From these markers, SNP and microhaplotype markers were chosen, which were informative for genetic stock identification (GSI) and kinship analysis. The final GTSeq panel contained 500 markers, including 197 microhaplotypes and 303 SNPs. Leave-one-out GSI simulations indicated that GSI accuracy should be greater than 80% in most jurisdictions. The false-positive rates of parent-offspring and full-sibling kinship identification were found to be low. Finally, genotypes could be consistently scored among separate sequencing runs >94% of the time. Results indicate that the GTSeq panel that we developed should perform well for multijurisdictional walleye research throughout the Great Lakes region.

High-density genomic data reveal fine-scale population structure and pronounced islands of adaptive divergence in lake whitefish (Coregonus clupeaformis) from Lake Michigan.

Understanding patterns of genetic structure and adaptive variation in natural populations is crucial for informing conservation and management. Past genetic research using 11 microsatellite loci identified six genetic stocks of lake whitefish (Coregonus clupeaformis) within Lake Michigan, USA. However, ambiguity in genetic stock assignments suggested those neutral microsatellite markers did not provide adequate power for delineating lake whitefish stocks in this system, prompting calls for a genomics approach to investigate stock structure. Here, we generated a dense genomic dataset to characterize population structure and investigate patterns of neutral and adaptive genetic diversity among lake whitefish populations in Lake Michigan. Using Rapture sequencing, we genotyped 829 individuals collected from 17 baseline populations at 197,588 SNP markers after quality filtering. Although the overall pattern of genetic structure was similar to the previous microsatellite study, our genomic data provided several novel insights. Our results indicated a large genetic break between the northwestern and eastern sides of Lake Michigan, and we found a much greater level of population structure on the eastern side compared to the northwestern side. Collectively, we observed five genomic islands of adaptive divergence on five different chromosomes. Each island displayed a different pattern of population structure, suggesting that combinations of genotypes at these adaptive regions are facilitating local adaptation to spatially heterogenous selection pressures. Additionally, we identified a large linkage disequilibrium block of ~8.5 Mb on chromosome 20 that is suggestive of a putative inversion but with a low frequency of the minor haplotype. Our study provides a comprehensive assessment of population structure and adaptive variation that can help inform the management of Lake Michigan's lake whitefish fishery and highlights the utility of incorporating adaptive loci into fisheries management.

A chromosomal inversion may facilitate adaptation despite periodic gene flow in a freshwater fish.

Differences in genomic architecture between populations, such as chromosomal inversions, may play an important role in facilitating adaptation despite opportunities for gene flow. One system where chromosomal inversions may be important for eco-evolutionary dynamics is in freshwater fishes, which often live in heterogenous environments characterized by varying levels of connectivity and varying opportunities for gene flow. In the present study, reduced representation sequencing was used to study possible adaptation in n = 345 walleye (Sander vitreus) from three North American waterbodies: Cedar Bluff Reservoir (Kansas, USA), Lake Manitoba (Manitoba, Canada), and Lake Winnipeg (Manitoba, Canada). Haplotype and outlier-based tests revealed a putative chromosomal inversion that contained three expressed genes and was nearly fixed in walleye assigned to Lake Winnipeg. These patterns exist despite the potential for high gene flow between these proximate Canadian lakes, suggesting that the inversion may be important for facilitating adaptive divergence between the two lakes despite gene flow. However, a specific adaptive role for the putative inversion could not be tested with the present data. Our study illuminates the importance of genomic architecture consistent with local adaptation in freshwater fishes. Furthermore, our results provide additional evidence that inversions may facilitate local adaptation in many organisms that inhabit connected but heterogenous environments.

The ghosts of propagation past: haplotype information clarifies the relative influence of stocking history and phylogeographic processes on contemporary population structure of walleye (Sander vitreus).

Stocking of fish is an important tool for maintaining fisheries but can also significantly alter population genetic structure and erode the portfolio of within-species diversity that is important for promoting resilience and adaptability. Walleye (Sander vitreus) are a highly valued sportfish in the midwestern United States, a region characterized by postglacial recolonization from multiple lineages and an extensive history of stocking. We leveraged genomic data and recently developed analytical approaches to explore the population structure of walleye from two midwestern states, Minnesota and Wisconsin. We genotyped 954 walleye from 23 populations at ~20,000 loci using genotyping by sequencing and tested for patterns of population structure with single-SNP and microhaplotype data. Populations from Minnesota and Wisconsin were highly differentiated from each other, with additional substructure found in each state. Population structure did not consistently adhere to drainage boundaries, as cases of high intra-drainage and low inter-drainage differentiation were observed. Low genetic structure was observed between populations from the upper Wisconsin and upper Chippewa river watersheds, which are found as few as 50 km apart and were likely homogenized through historical stocking. Nevertheless, we were able to differentiate these populations using microhaplotype-based co-ancestry analysis, providing increased resolution over previous microsatellite studies and our other single SNP-based analyses. Although our results illustrate that walleye population structure has been influenced by past stocking practices, native ancestry still exists in most populations and walleye populations may be able to purge non-native alleles and haplotypes in the absence of stocking. Our study is one of the first to use genomic tools to investigate the influence of stocking on population structure in a nonsalmonid fish and outlines a workflow leveraging recently developed analytical methods to improve resolution of complex population structure that will be highly applicable in many species and systems.

Mixed-stock analysis using Rapture genotyping to evaluate stock-specific exploitation of a walleye population despite weak genetic structure.

Mixed-stock analyses using genetic markers have informed fisheries management in cases where strong genetic differentiation occurs among local spawning populations, yet many fisheries are supported by multiple, weakly differentiated stocks. Freshwater fisheries exemplify this problem, with many populations supported by multiple stocks of young evolutionary age and isolated across small spatial scales. Consequently, attempts to conduct genetic mixed-stock analyses of inland fisheries have often been unsuccessful. Advances in genomic sequencing offer the ability to discriminate among populations with weak population structure, providing the necessary resolution to conduct mixed-stock assignment among previously indistinguishable stocks. We used genomic data to conduct a mixed-stock analysis of eastern Lake Erie's commercial and recreational walleye (Sander vitreus) fisheries and estimate the relative harvest of weakly differentiated stocks (pairwise F ST < 0.01). Using RAD-capture (Rapture), we sequenced and genotyped individuals from western and eastern basin local spawning stocks at 12,081 loci with 95% reassignment accuracy, which was not possible in the past using microsatellite markers. A baseline assessment of 395 walleye from 11 spawning stocks identified three reporting groups and refined previous assessments of gene flow among walleye stocks. Genetic assignment of 1,075 walleye harvested in eastern Lake Erie's recreational and commercial fisheries indicated that western basin stocks constituted the majority of harvest during the peak walleye fishing season (July-September), whereas eastern basin individuals comprised much of the early season harvest (May-June). Clear spatial structure in harvest composition existed; catches in more easterly sites contained more individuals of eastern basin origin than did more westerly sites. Our study provides important stock contribution estimates for Lake Erie fishery management and demonstrates the utility of genomic data to facilitate mixed-stock analysis in exploited fish populations having weak population structure or limited existing genetic resources.

Attack of the PCR clones: Rates of clonality have little effect on RAD-seq genotype calls.

Interpretation of high-throughput sequence data requires an understanding of how decisions made during bioinformatic data processing can influence results. One source of bias that is often cited is PCR clones (or PCR duplicates). PCR clones are common in restriction site-associated sequencing (RAD-seq) data sets, which are increasingly being used for molecular ecology. To determine the influence PCR clones and the bioinformatic handling of clones have on genotyping, we evaluate four RAD-seq data sets. Data sets were compared before and after clones were removed to estimate the number of clones present in RAD-seq data, quantify how often the presence of clones in a data set causes genotype calls to change compared to when clones were removed, investigate the mechanisms that lead to genotype call changes and test whether clones bias heterozygosity estimates. Our RAD-seq data sets contained 30%-60% PCR clones, but 95% of RAD-tags had five or fewer clones. Relatively few genotypes changed once clones were removed (5%-10%), and the vast majority of these changes (98%) were associated with genotypes switching from a called to no-call state or vice versa. PCR clones had a larger influence on genotype calls in individuals with low read depth but appeared to influence genotype calls at all loci similarly. Removal of PCR clones reduced the number of called genotypes by 2% but had almost no influence on estimates of heterozygosity. As such, while steps should be taken to limit PCR clones during library preparation, PCR clones are likely not a substantial source of bias for most RAD-seq studies.

Building a global genomics observatory: Using GEOME (the Genomic Observatories Metadatabase) to expedite and improve deposition and retrieval of genetic data and metadata for biodiversity research.

Genetic data represent a relatively new frontier for our understanding of global biodiversity. Ideally, such data should include both organismal DNA-based genotypes and the ecological context where the organisms were sampled. Yet most tools and standards for data deposition focus exclusively either on genetic or ecological attributes. The Genomic Observatories Metadatabase (GEOME: geome-db.org) provides an intuitive solution for maintaining links between genetic data sets stored by the International Nucleotide Sequence Database Collaboration (INSDC) and their associated ecological metadata. GEOME facilitates the deposition of raw genetic data to INSDCs sequence read archive (SRA) while maintaining persistent links to standards-compliant ecological metadata held in the GEOME database. This approach facilitates findable, accessible, interoperable and reusable data archival practices. Moreover, GEOME enables data management solutions for large collaborative groups and expedites batch retrieval of genetic data from the SRA. The article that follows describes how GEOME can enable genuinely open data workflows for researchers in the field of molecular ecology.

Characterization of a Y-specific duplication/insertion of the anti-Mullerian hormone type II receptor gene based on a chromosome-scale genome assembly of yellow perch, Perca flavescens.

Yellow perch, Perca flavescens, is an ecologically and economically important species native to a large portion of the northern United States and southern Canada and is also a promising candidate species for aquaculture. However, no yellow perch reference genome has been available to facilitate improvements in both fisheries and aquaculture management practices. By combining Oxford Nanopore Technologies long-reads, 10X Genomics Illumina short linked reads and a chromosome contact map produced with Hi-C, we generated a high-continuity chromosome-scale yellow perch genome assembly of 877.4 Mb. It contains, in agreement with the known diploid chromosome yellow perch count, 24 chromosome-size scaffolds covering 98.8% of the complete assembly (N50 = 37.4 Mb, L50 = 11). We also provide a first characterization of the yellow perch sex determination locus that contains a male-specific duplicate of the anti-Mullerian hormone type II receptor gene (amhr2by) inserted at the proximal end of the Y chromosome (chromosome 9). Using this sex-specific information, we developed a simple PCR genotyping assay which accurately differentiates XY genetic males (amhr2by+ ) from XX genetic females (amhr2by- ). Our high-quality genome assembly is an important genomic resource for future studies on yellow perch ecology, toxicology, fisheries and aquaculture research. In addition, characterization of the amhr2by gene as a candidate sex-determining gene in yellow perch provides a new example of the recurrent implication of the transforming growth factor beta pathway in fish sex determination, and highlights gene duplication as an important genomic mechanism for the emergence of new master sex determination genes.