Epigenetic and Genetic Differentiation Between Coregonus Species Pairs.

Phenotypic diversification is classically associated with genetic differentiation and gene expression variation. However, increasing evidence suggests that DNA methylation is involved in evolutionary processes due to its phenotypic and transcriptional effects. Methylation can increase mutagenesis and could lead to increased genetic divergence between populations experiencing different environmental conditions for many generations, though there has been minimal empirical research on epigenetically induced mutagenesis in diversification and speciation. Whitefish, freshwater members of the salmonid family, are excellent systems to study phenotypic diversification and speciation due to the repeated divergence of benthic-limnetic species pairs serving as natural replicates. Here we investigate whole genome genetic and epigenetic differentiation between sympatric benthic-limnetic species pairs in lake and European whitefish (Coregonus clupeaformis and Coregonus lavaretus) from four lakes (N = 64). We found considerable, albeit variable, genetic and epigenetic differences between species pairs. All SNP types were enriched at CpG sites supporting the mutagenic nature of DNA methylation, though C>T SNPs were most common. We also found an enrichment of overlaps between outlier SNPs with the 5% highest FST between species and differentially methylated loci. This could possibly represent differentially methylated sites that have caused divergent genetic mutations between species, or divergent selection leading to both genetic and epigenetic variation at these sites. Our results support the hypothesis that DNA methylation contributes to phenotypic divergence and mutagenesis during whitefish speciation.

Genome-wide DNA methylation predicts environmentally driven life history variation in a marine fish.

Epigenetic modifications are thought to be one of the molecular mechanisms involved in plastic adaptive responses to environmental variation. However, studies reporting associations between genome-wide epigenetic changes and habitat-specific variations in life history traits (e.g., lifespan, reproduction) are still scarce, likely due to the recent application of methylome resequencing methods to non-model species. In this study, we examined associations between whole genome DNA methylation and environmentally driven life history variation in 2 lineages of a marine fish, the capelin (Mallotus villosus), from North America and Europe. In both lineages, capelin harbor 2 contrasting life history tactics (demersal vs. beach-spawning). Performing whole genome and methylome sequencing, we showed that life history tactics are associated with epigenetic changes in both lineages, though the effect was stronger in European capelin. Genetic differentiation between the capelin harboring different life history tactics was negligible, but we found genome-wide methylation changes in both lineages. We identified 9,125 European and 199 North American differentially methylated regions (DMRs) due to life history. Gene ontology (GO) enrichment analysis for both lineages revealed an excess of terms related to neural function. Our results suggest that environmental variation causes important epigenetic changes that are associated with contrasting life history tactics in lineages with divergent genetic backgrounds, with variable importance of genetic variation in driving epigenetic variation. Our study emphasizes the potential role of genome-wide epigenetic variation in adaptation to environmental variation.

Stepping up to genome scan allows stock differentiation in the worldwide distributed blue shark Prionace glauca.

The blue shark Prionace glauca is a top predator with one of the widest geographical distributions of any shark species. It is classified as Critically Endangered in the Mediterranean Sea, and Near Threatened globally. Previous genetic studies did not reject the null hypothesis of a single global population. The blue shark was proposed as a possible archetype of the "grey zone of population differentiation," coined to designate cases where population structure may be too recent or too faint to be detected using a limited set of markers. Here, blue shark samples collected throughout its global range were sequenced using a specific RAD method (DArTseq), which recovered 37,655 genome-wide single nucleotide polymorphisms (SNPs). Two main groups emerged, with Mediterranean Sea and northern Atlantic samples (Northern population) differentiated significantly from the Indo-west Pacific samples (Southern population). Significant pairwise FST values indicated further genetic differentiation within the Atlantic Ocean, and between the Atlantic Ocean and the Mediterranean Sea. Reconstruction of recent demographic history suggested divergence between Northern and Southern populations occurred about 500 generations ago and revealed a drastic reduction in effective population size from a large ancestral population. Our results illustrate the power of genome scans to detect population structure and reconstruct demographic history in highly migratory marine species. Given that the management plans of the blue shark (targeted or bycatch) fisheries currently assume panmictic regional stocks, we strongly recommend that the results presented here be considered in future stock assessments and conservation strategies.

Genome assembly, structural variants, and genetic differentiation between lake whitefish young species pairs (Coregonus sp.) with long and short reads.

Nascent pairs of ecologically differentiated species offer an opportunity to get a better glimpse at the genetic architecture of speciation. Of particular interest is our recent ability to consider a wider range of genomic variants, not only single-nucleotide polymorphisms (SNPs), thanks to long-read sequencing technology. We can now identify structural variants (SVs) such as insertions, deletions and other rearrangements, allowing further insights into the genetic architecture of speciation and how different types of variants are involved in species differentiation. Here, we investigated genomic patterns of differentiation between sympatric species pairs (Dwarf and Normal) belonging to the lake whitefish (Coregonus clupeaformis) species complex. We assembled the first reference genomes for both C. clupeaformis sp. Normal and C. clupeaformis sp. Dwarf, annotated the transposable elements and analysed the genomes in the light of related coregonid species. Next, we used a combination of long- and short-read sequencing to characterize SVs and genotype them at the population scale using genome-graph approaches, showing that SVs cover five times more of the genome than SNPs. We then integrated both SNPs and SVs to investigate the genetic architecture of species differentiation in two different lakes and highlighted an excess of shared outliers of differentiation. In particular, a large fraction of SVs differentiating the two species correspond to insertions or deletions of transposable elements (TEs), suggesting that TE accumulation may represent a key component of genetic divergence between the Dwarf and Normal species. Together, our results suggest that SVs may play an important role in speciation and that, by combining second- and third-generation sequencing, we now have the ability to integrate SVs into speciation genomics.

Evaluating the accuracy of variant calling methods using the frequency of parent-offspring genotype mismatch.

The use of next-generation sequencing (NGS) data sets has increased dramatically over the last decade, but there have been few systematic analyses quantifying the accuracy of the commonly used variant caller programs. Here we used a familial design consisting of diploid tissue from a single lodgepole pine (Pinus contorta) parent and the maternally derived haploid tissue from 106 full-sibling offspring, where mismatches could only arise due to mutation or bioinformatic error. Given the rarity of mutation, we used the rate of mismatches between parent and offspring genotype calls to infer the single nucleotide polymorphism (SNP) genotyping error rates of FreeBayes, HaplotypeCaller, SAMtools, UnifiedGenotyper, and VarScan. With baseline filtering HaplotypeCaller and UnifiedGenotyper yielded more SNPs and higher error rates by one to two orders of magnitude, whereas FreeBayes, SAMtools and VarScan yielded lower numbers of SNPs and more modest error rates. To facilitate comparison between variant callers we standardized each SNP set to the same number of SNPs using additional filtering, where UnifiedGenotyper consistently produced the smallest proportion of genotype errors, followed by HaplotypeCaller, VarScan, SAMtools, and FreeBayes. Additionally, we found that error rates were minimized for SNPs called by more than one variant caller. Finally, we evaluated the performance of various commonly used filtering metrics on SNP calling. Our analysis provides a quantitative assessment of the accuracy of five widely used variant calling programs and offers valuable insights into both the choice of variant caller program and the choice of filtering metrics, especially for researchers using non-model study systems.

Polygenic selection drives the evolution of convergent transcriptomic landscapes across continents within a Nearctic sister species complex.

In contrast to the plethora of studies focusing on the genomic basis of adaptive phenotypic divergence, the role of gene expression during speciation has been much less investigated and consequently less understood. Yet, the convergence of differential gene expression patterns between closely related species-pairs might reflect the role of natural selection during the process of ecological speciation. Here, we test for intercontinental convergence in differential transcriptional signatures between limnetic and benthic sympatric species-pairs of Lake Whitefish (Coregonus clupeaformis) and its sister lineage, the European Whitefish (Coregonus lavaretus), using six replicated sympatric species-pairs (two in North America, two in Norway and two in Switzerland). We characterized both sequence variation in transcribed regions and differential gene expression between sympatric limnetic and benthic species across regions and continents. Our first finding was that differentially expressed genes (DEG) between limnetic and benthic whitefish tend to be enriched in shared polymorphism among sister lineages. We then used both genotypes and covariation in expression in order to infer polygenic selection at the gene level. We identified parallel outliers and DEG involving genes primarily overexpressed in limnetic species relative to the benthic species. Our analysis finally revealed the existence of shared genomic bases underlying parallel differential expression across replicated species-pairs from both continents, such as a cis-eQTL affecting the pyruvate kinase expression level involved in glycolysis. Our results are consistent with a long-standing role of natural selection in maintaining trans-continental diversity at phenotypic traits involved in ecological speciation between limnetic and benthic whitefishes.

Model-based demographic inference of introgression history in European whitefish species pairs'.

Parallel phenotypic differentiation is generally attributed to parallel adaptive divergence as an evolutionary response to similar environmental contrasts. Such parallelism may actually originate from several evolutionary scenarios ranging from repeated parallel divergence caused by divergent selection to a unique divergence event followed by gene flow. Reconstructing the evolutionary history underlying parallel phenotypic differentiation is thus fundamental to understand the relative contribution of demography and selection on genomic divergence during speciation. In this study, we investigate the divergence history of replicate European whitefish (Coregonus lavaretus), limnetic and benthic species pairs from two lakes in Norway and two lakes in Switzerland. Demographic models accounting for semi-permeability and linked selection were fitted to the unfolded joint allele frequency spectrum built from genome-wide SNPs and compared to each other in each species pair. We found strong support for a model of asymmetrical post-glacial secondary contact between glacial lineages in all four lakes. Moreover, our results suggest that heterogeneous genomic differentiation has been shaped by the joint action of linked selection accelerating lineage sorting during allopatry, and heterogeneous migration eroding divergence at different rates along the genome following secondary contact. Our analyses reveal how the interplay between demography, selection and historical contingency has influenced the levels of diversity observed in previous whitefish phylogeographic studies. This study thus provides new insights into the historical demographic and selective processes that shaped the divergence associated with ecological speciation in European whitefish.

Genetic and morphological support for possible sympatric origin of fish from subterranean habitats.

Two blind Iran cave barbs, Garra typhlops and Garra lorestanensis, exist in sympatry in a single subterranean habitat, raising the hypothesis that they may represent a case of sympatric speciation following a colonization event. Their different mental disc forms have prompted some authors to propose the alternative hypothesis of two separate colonization events. In this study, we analysed a genome-wide panel of 11,257 SNPs genotyped by means of genotyping-by-sequencing combined with mitochondrial cytochrome c oxidase sub-unit I sequence data, field observations and morphological traits to test these two hypotheses. Field data suggest some degree of ecological divergence despite some possible niche overlap such that hybridization is possible. According to both nuclear and mtDNA data, the cave barb species are monophyletic with close phylogenetic relationships with Garra gymnothorax from the Karun-Dez and Karkheh river basins. The historical demography analysis revealed that a model of Isolation-with-Migration (IM) best fitted the data, therefore better supporting a scenario of sympatric origin than that of allopatric isolation followed by secondary contact. Overall, our results offer stronger support to the hypothesis that speciation in the subterranean habitat could have occurred in sympatry following a colonization event from the Karun-Dez-Karkheh basins in the Zagros Mountains of Iran.

Modeling the Multiple Facets of Speciation-with-Gene-Flow toward Inferring the Divergence History of Lake Whitefish Species Pairs (Coregonus clupeaformis).

Parallel divergence across replicated species pairs occurring in similar environmental contrasts may arise through distinct evolutionary scenarios. Deciphering whether such parallelism actually reflects repeated parallel divergence driven by divergent selection or a single divergence event with subsequent gene flow needs to be ascertained. Reconstructing historical gene flow is therefore of fundamental interest to understand how demography and selection jointly shaped genomic divergence during speciation. Here, we use an extended modeling framework to explore the multiple facets of speciation-with-gene-flow with demo-genetic divergence models that capture both temporal and genomic variation in effective population size and migration rate. We investigate the divergence history of replicate sympatric species pairs of Lake Whitefish (normal benthic and dwarf limnetic) characterized by variable degrees of ecological divergence and reproductive isolation. Genome-wide SNPs were used to document the extent of genetic differentiation in each species pair, and 26 divergence models were fitted and compared with the unfolded joint allele frequency spectrum of each pair. We found evidence that a recent (circa 3,000-4,000 generations) asymmetrical secondary contact between expanding postglacial populations has accompanied Whitefish diversification. Our results suggest that heterogeneous genomic differentiation has emerged through the combined effects of linked selection generating variable rates of lineage sorting across the genome during geographical isolation, and heterogeneous introgression eroding divergence at different rates across the genome upon secondary contact. This study thus provides a new retrospective insight into the historical demographic and selective processes that shaped a continuum of divergence associated with ecological speciation.

Sex matters in massive parallel sequencing: Evidence for biases in genetic parameter estimation and investigation of sex determination systems.

Using massively parallel sequencing data from two species with different life history traits, American lobster (Homarus americanus) and Arctic Char (Salvelinus alpinus), we highlight how an unbalanced sex ratio in the samples and a few sex-linked markers may lead to false interpretations of population structure and thus to potentially erroneous management recommendations. Here, multivariate analyses revealed two genetic clusters separating samples by sex instead of by expected spatial variation: inshore and offshore locations in lobster, or east and west locations in Arctic Char. To further investigate this, we created several subsamples artificially varying the sex ratio in the inshore/offshore and east/west groups and then demonstrated that significant genetic differentiation could be observed despite panmixia in lobster, and that FST values were overestimated in Arctic Char. This pattern was due to 12 and 94 sex-linked markers driving differentiation for lobster and Arctic Char, respectively. Removing sex-linked markers led to nonsignificant genetic structure in lobster and a more accurate estimation of FST in Arctic Char. The locations of these markers and putative identities of genes containing or nearby the markers were determined using available transcriptomic and genomic data, and this provided new information related to sex determination in both species. Given that only 9.6% of all marine/diadromous population genomic studies to date have reported sex information, we urge researchers to collect and consider individual sex information. Sex information is therefore relevant for avoiding unexpected biases due to sex-linked markers as well as for improving our knowledge of sex determination systems in nonmodel species.

Convergence in organ size but not energy metabolism enzyme activities among wild Lake Whitefish (Coregonus clupeaformis) species pairs.

The repeated evolution of similar phenotypes by similar mechanisms can be indicative of local adaptation, constraints or biases in the evolutionary process. Little is known about the incidence of physiological convergence in natural populations, so here we test whether energy metabolism in 'dwarf' and 'normal' Lake Whitefish evolves by similar mechanisms. Prior genomic and transcriptomic studies have found that divergence in energy metabolism is key to local adaptation in whitefish species pairs, but that distinct genetic and transcriptomic changes often underlie phenotypic evolution among lakes. Here, we predicted that traits at higher levels of biological organization, including the activities of energy metabolism enzymes (the product of enzyme concentration and turnover rate) and the relative proportions of metabolically active tissues (heart, liver, skeletal muscle), would show greater convergence than genetic and transcriptomic variation. We compared four whitefish species pairs and found convergence in organ size whereby all dwarf whitefish populations have a higher proportion of red skeletal muscle, three have relatively larger livers and two have relatively larger ventricles than normal fish. On the other hand, hepatic and muscle enzyme activities showed little convergence and were largely dependent on lake of origin. Only the most genetically divergent species pair (Cliff Lake) displayed white muscle enzyme activities matching results from laboratory-reared normal and dwarf whitefish. Overall, these data show convergence in the evolution of organ size, but not in the activities of candidate enzymes of energy metabolism, which may have evolved mainly as a consequence of demographic or ecological differences among lakes.