Investigating structural variant, indel and single nucleotide polymorphism differentiation between locally adapted Atlantic salmon populations.

Genomic structural variants (SVs) are now recognized as an integral component of intraspecific polymorphism and are known to contribute to evolutionary processes in various organisms. However, they are inherently difficult to detect and genotype from readily available short-read sequencing data, and therefore remain poorly documented in wild populations. Salmonid species displaying strong interpopulation variability in both life history traits and habitat characteristics, such as Atlantic salmon (Salmo salar), offer a prime context for studying adaptive polymorphism, but the contribution of SVs to fine-scale local adaptation has yet to be explored. Here, we performed a comparative analysis of SVs, single nucleotide polymorphisms (SNPs) and small indels (<50 bp) segregating in the Romaine and Puyjalon salmon, two putatively locally adapted populations inhabiting neighboring rivers (Québec, Canada) and showing pronounced variation in life history traits, namely growth, fecundity, and age at maturity and smoltification. We first catalogued polymorphism using a hybrid SV characterization approach pairing both short- (16X) and long-read sequencing (20X) for variant discovery with graph-based genotyping of SVs across 60 salmon genomes, along with characterization of SNPs and small indels from short reads. We thus identified 115,907 SVs, 8,777,832 SNPs and 1,089,321 short indels, with SVs covering 4.8 times more base pairs than SNPs. All three variant types revealed a highly congruent population structure and similar patterns of F ST and density variation along the genome. Finally, we performed outlier detection and redundancy analysis (RDA) to identify variants of interest in the putative local adaptation of Romaine and Puyjalon salmon. Genes located near these variants were enriched for biological processes related to nervous system function, suggesting that observed variation in traits such as age at smoltification could arise from differences in neural development. This study therefore demonstrates the feasibility of large-scale SV characterization and highlights its relevance for salmonid population genomics.

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.

Structural Variants and Speciation: Multiple Processes at Play.

Research on the genomic architecture of speciation has increasingly revealed the importance of structural variants (SVs) that affect the presence, abundance, position, and/or direction of a nucleotide sequence. SVs include large chromosomal rearrangements such as fusion/fissions and inversions and translocations, as well as smaller variants such as duplications, insertions, and deletions (CNVs). Although we have ample evidence that SVs play a key role in speciation, the underlying mechanisms differ depending on the type and length of the SV, as well as the ecological, demographic, and historical context. We review predictions and empirical evidence for classic processes such as underdominance due to meiotic aberrations and the coupling effect of recombination suppression before exploring how recent sequencing methodologies illuminate the prevalence and diversity of SVs. We discuss specific properties of SVs and their impact throughout the genome, highlighting that multiple processes are at play, and possibly interacting, in the relationship between SVs and speciation.

Widespread Deviant Patterns of Heterozygosity in Whole-Genome Sequencing Due to Autopolyploidy, Repeated Elements, and Duplication.

Most population genomic tools rely on accurate single nucleotide polymorphism (SNP) calling and filtering to meet their underlying assumptions. However, genomic complexity, resulting from structural variants, paralogous sequences, and repetitive elements, presents significant challenges in assembling contiguous reference genomes. Consequently, short-read resequencing studies can encounter mismapping issues, leading to SNPs that deviate from Mendelian expected patterns of heterozygosity and allelic ratio. In this study, we employed the ngsParalog software to identify such deviant SNPs in whole-genome sequencing (WGS) data with low (1.5×) to intermediate (4.8×) coverage for four species: Arctic Char (Salvelinus alpinus), Lake Whitefish (Coregonus clupeaformis), Atlantic Salmon (Salmo salar), and the American Eel (Anguilla rostrata). The analyses revealed that deviant SNPs accounted for 22% to 62% of all SNPs in salmonid datasets and approximately 11% in the American Eel dataset. These deviant SNPs were particularly concentrated within repetitive elements and genomic regions that had recently undergone rediploidization in salmonids. Additionally, narrow peaks of elevated coverage were ubiquitous along all four reference genomes, encompassed most deviant SNPs, and could be partially associated with transposons and tandem repeats. Including these deviant SNPs in genomic analyses led to highly distorted site frequency spectra, underestimated pairwise FST values, and overestimated nucleotide diversity. Considering the widespread occurrence of deviant SNPs arising from a variety of sources, their important impact in estimating population parameters, and the availability of effective tools to identify them, we propose that excluding deviant SNPs from WGS datasets is required to improve genomic inferences for a wide range of taxa and sequencing depths.

How chromosomal inversions reorient the evolutionary process.

Inversions are structural mutations that reverse the sequence of a chromosome segment and reduce the effective rate of recombination in the heterozygous state. They play a major role in adaptation, as well as in other evolutionary processes such as speciation. Although inversions have been studied since the 1920s, they remain difficult to investigate because the reduced recombination conferred by them strengthens the effects of drift and hitchhiking, which in turn can obscure signatures of selection. Nonetheless, numerous inversions have been found to be under selection. Given recent advances in population genetic theory and empirical study, here we review how different mechanisms of selection affect the evolution of inversions. A key difference between inversions and other mutations, such as single nucleotide variants, is that the fitness of an inversion may be affected by a larger number of frequently interacting processes. This considerably complicates the analysis of the causes underlying the evolution of inversions. We discuss the extent to which these mechanisms can be disentangled, and by which approach.

A supergene in seaweed flies modulates male traits and female perception.

Supergenes, tightly linked sets of alleles, offer some of the most spectacular examples of polymorphism persisting under long-term balancing selection. However, we still do not understand their evolution and persistence, especially in the face of accumulation of deleterious elements. Here, we show that an overdominant supergene in seaweed flies, Coelopa frigida, modulates male traits, potentially facilitating disassortative mating and promoting intraspecific polymorphism. Across two continents, the Cf-Inv(1) supergene strongly affected the composition of male cuticular hydrocarbons (CHCs) but only weakly affected CHC composition in females. Using gas chromatography-electroantennographic detection, we show that females can sense male CHCs and that there may be differential perception between genotypes. Combining our phenotypic results with RNA-seq data, we show that candidate genes for CHC biosynthesis primarily show differential expression for Cf-Inv(1) in males but not females. Conversely, candidate genes for odorant detection were differentially expressed in both sexes but showed high levels of divergence between supergene haplotypes. We suggest that the reduced recombination between supergene haplotypes may have led to rapid divergence in mate preferences as well as increasing linkage between male traits, and overdominant loci. Together this probably helped to maintain the polymorphism despite deleterious effects in homozygotes.

Searching for intralocus sexual conflicts in the three-spined stickleback (Gasterosteus aculeatus) genome.

Differences between sexes in trait fitness optima can generate intralocus sexual conflicts that have the potential to maintain genetic diversity through balancing selection. However, these differences are unlikely to be associated with strong selective coefficients and are challenging to detect. Additionally, recent studies have highlighted that duplications on sexual chromosomes can create artifactual signals of intralocus sexual conflicts. Thus, testing the relationship between intralocus sexual conflicts and balancing selection requires stringent filtering of duplicated regions, and dedicated methods to detect loci with low levels of intersex differentiation. In this study, we investigated intralocus sexual conflicts in the three-spined stickleback using whole-genome sequencing (mean coverage = 12×) of 50 females and 49 males from an anadromous population in the St. Lawrence River, Québec, Canada. After stringent filtering of duplications from the sex chromosomes, we compared three methods to detect intralocus sexual conflicts. We found only two genomic regions under potential intralocus sexual conflict that also showed signals of balancing selection. Overall, our results suggest that most intralocus sexual conflicts do not drive long-term balancing selection and are most likely transient.

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.

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.

A setback into a success: What can batch effects tell us about best practices in genomics?

The increasing access to high-throughput sequencing is certainly one of the major changes that molecular ecology has gone through over the last decade. With the positive trend towards more open science, most sequencing data sets are now available on public databases, which holds amazing potential, but also risks of introducing batch effects in studies combining data sets. In this issue of Molecular Ecology Resources, Lou and Therkildsen (2022) offer a timely discussion on the matter by analyzing an imperfect low-coverage Whole Genome Sequencing data set, in which they test the effects of differences in sequencing choices, DNA degradation, and read depth on routine population genomics analyses. Through a series of diagnostic tools, they uncover multiple factors producing technical artefacts that can bias estimates of genetic diversity, inference of population structure, and selection scans. For each confounding factor, they demonstrate the effectiveness of mitigation approaches and suggest other avenues to deal with the issue. In this perspective, we highlight considerations regarding (1) effects that arise from differences between batches of sequencing; (2) unavoidable heterogeneity within data sets; and (3) more general concerns around the use of next-generation sequencing in population genomics. Altogether, by exploring what may have appeared at first glimpse as a "failed" sequencing project, Lou and Therkildsen (2022) end up setting a standard of best practices to make the most of heterogeneous whole-genome sequences, opening a promising avenue towards efficient reuse of published data sets.

Evolution: How important is the dimensionality of natural selection in local adaptation?

Organisms adapt to their local environments, which may vary in one aspect, or many. A new study shows that such dimensionality matters, as it impacts the magnitude and dynamics of local adaptation, with broader ecological consequences, such as the evolution of generalists.

Chromosome-level assembly reveals a putative Y-autosomal fusion in the sex determination system of the Greenland Halibut (Reinhardtius hippoglossoides).

Despite the commercial importance of Greenland Halibut (Reinhardtius hippoglossoides), important gaps still persist in our knowledge of this species, including its reproductive biology and sex determination mechanism. Here, we combined single-molecule sequencing of long reads (Pacific Sciences) with chromatin conformation capture sequencing (Hi-C) data to assemble the first chromosome-level reference genome for this species. The high-quality assembly encompassed more than 598 Megabases (Mb) assigned to 1594 scaffolds (scaffold N50 = 25 Mb) with 96% of its total length distributed among 24 chromosomes. Investigation of the syntenic relationship with other economically important flatfish species revealed a high conservation of synteny blocks among members of this phylogenetic clade. Sex determination analysis revealed that similar to other teleost fishes, flatfishes also exhibit a high level of plasticity and turnover in sex determination mechanisms. A low-coverage whole-genome sequence analysis of 198 individuals revealed that Greenland Halibut possesses a male heterogametic XY system and several putative candidate genes implied in the sex determination of this species. Our study also suggests for the first time in flatfishes that a putative Y-autosomal fusion could be associated with a reduction of recombination typical of the early steps of sex chromosome evolution.

A large chromosomal inversion shapes gene expression in seaweed flies (Coelopa frigida).

Inversions often underlie complex adaptive traits, but the genic targets inside them are largely unknown. Gene expression profiling provides a powerful way to link inversions with their phenotypic consequences. We examined the effects of the Cf-Inv(1) inversion in the seaweed fly Coelopa frigida on gene expression variation across sexes and life stages. Our analyses revealed that Cf-Inv(1) shapes global expression patterns, most likely via linked variation, but the extent of this effect is variable, with much stronger effects in adults than larvae. Furthermore, within adults, both common as well as sex-specific patterns were found. The vast majority of these differentially expressed genes mapped to Cf-Inv(1). However, genes that were differentially expressed in a single context (i.e., in males, females, or larvae) were more likely to be located outside of Cf-Inv(1). By combining our findings with genomic scans for environmentally associated SNPs, we were able to pinpoint candidate variants in the inversion that may underlie mechanistic pathways that determine phenotypes. Together the results of this study, combined with previous findings, support the notion that the polymorphic Cf-Inv(1) inversion in this species is a major factor shaping both coding and regulatory variation resulting in highly complex adaptive effects.

Locally Adaptive Inversions Modulate Genetic Variation at Different Geographic Scales in a Seaweed Fly.

Across a species range, multiple sources of environmental heterogeneity, at both small and large scales, create complex landscapes of selection, which may challenge adaptation, particularly when gene flow is high. One key to multidimensional adaptation may reside in the heterogeneity of recombination along the genome. Structural variants, like chromosomal inversions, reduce recombination, increasing linkage disequilibrium among loci at a potentially massive scale. In this study, we examined how chromosomal inversions shape genetic variation across a species range and ask how their contribution to adaptation in the face of gene flow varies across geographic scales. We sampled the seaweed fly Coelopa frigida along a bioclimatic gradient stretching across 10° of latitude, a salinity gradient, and a range of heterogeneous, patchy habitats. We generated a chromosome-level genome assembly to analyze 1,446 low-coverage whole genomes collected along those gradients. We found several large nonrecombining genomic regions, including putative inversions. In contrast to the collinear regions, inversions and low-recombining regions differentiated populations more strongly, either along an ecogeographic cline or at a fine-grained scale. These genomic regions were associated with environmental factors and adaptive phenotypes, albeit with contrasting patterns. Altogether, our results highlight the importance of recombination in shaping adaptation to environmental heterogeneity at local and large scales.

Thermal adaptation rather than demographic history drives genetic structure inferred by copy number variants in a marine fish.

Increasing evidence shows that structural variants represent an overlooked aspect of genetic variation with consequential evolutionary roles. Among those, copy number variants (CNVs), including duplicated genomic regions and transposable elements (TEs), may contribute to local adaptation and/or reproductive isolation among divergent populations. Those mechanisms suppose that CNVs could be used to infer neutral and/or adaptive population genetic structure, whose study has been restricted to microsatellites, mitochondrial DNA and Amplified fragment length polymorphism markers in the past and more recently the use of single nucleotide polymorphisms (SNPs). Taking advantage of recent developments allowing CNV analysis from RAD-seq data, we investigated how variation in fitness-related traits, local environmental conditions and demographic history are associated with CNVs, and how subsequent copy number variation drives population genetic structure in a marine fish, the capelin (Mallotus villosus). We collected 1538 DNA samples from 35 sampling sites in the north Atlantic Ocean and identified 6620 putative CNVs. We found associations between CNVs and the gonadosomatic index, suggesting that six duplicated regions could affect female fitness by modulating oocyte production. We also detected 105 CNV candidates associated with water temperature, among which 20% corresponded to genomic regions located within the sequence of protein-coding genes, suggesting local adaptation to cold water by means of gene sequence amplification. We also identified 175 CNVs associated with the divergence of three previously defined parapatric glacial lineages, of which 24% were located within protein-coding genes, making those loci potential candidates for reproductive isolation. Lastly, our analyses unveiled a hierarchical, complex CNV population structure determined by temperature and local geography, which was in stark contrast to that inferred based on SNPs in a previous study. Our findings underline the complementarity of those two types of genomic variation in population genomics studies.

Copy number variants outperform SNPs to reveal genotype-temperature association in a marine species.

Copy number variants (CNVs) are a major component of genotypic and phenotypic variation in genomes. To date, our knowledge of genotypic variation and evolution has largely been acquired by means of single nucleotide polymorphism (SNPs) analyses. Until recently, the adaptive role of structural variants (SVs) and particularly that of CNVs has been overlooked in wild populations, partly due to their challenging identification. Here, we document the usefulness of Rapture, a derived reduced-representation shotgun sequencing approach, to detect and investigate copy number variants (CNVs) alongside SNPs in American lobster (Homarus americanus) populations. We conducted a comparative study to examine the potential role of SNPs and CNVs in local adaptation by sequencing 1,141 lobsters from 21 sampling sites within the southern Gulf of St. Lawrence, which experiences the highest yearly thermal variance of the Canadian marine coastal waters. Our results demonstrated that CNVs account for higher genetic differentiation than SNP markers. Contrary to SNPs, for which no significant genetic-environment association was found, 48 CNV candidates were significantly associated with the annual variance of sea surface temperature, leading to the genetic clustering of sampling locations despite their geographic separation. Altogether, we provide a strong empirical case that CNVs putatively contribute to local adaptation in marine species and unveil stronger spatial signal of population structure than SNPs. Our study provides the means to study CNVs in nonmodel species and highlights the importance of considering structural variants alongside SNPs to enhance our understanding of ecological and evolutionary processes shaping adaptive population structure.

Making the most of population genomic data to understand the importance of chromosomal inversions for adaptation and speciation.

Chromosomal inversions are increasingly found to differentiate locally adapted populations. This adaptive role is predictable because reduced recombination protects allelic combinations from gene flow. However, we are far from understanding how frequently inversions contribute to local adaptation and how widespread this phenomenon is across species. In a "From the Cover" article in this issue of Molecular Ecology, Huang, Andrew, Owens, Ostevik, and Rieseberg (2020) provide an important step towards this goal not only by finding adaptive inversions in a sunflower ecotype, but also by reversing the approach used to investigate the link between adaptation and inversions. Most studies compare two phenotypes and uncover divergence at a few regions, of which some can subsequently be identified as inversions. In contrast, Huang et al first catalogue putative inversions and then test genotype-environment associations, which allows them to ask systematically whether inversions may be adaptive and in which ecological contexts. They achieve that by revisiting a previous reduced-representation sequencing (RAD-sequencing) data set, demonstrating the suitability of this method to detect inversions in species with limited genomic resources. As such, Huang et al pave the way for a better understanding of the evolutionary role of structural genomic variation and highlight that accounting for inversions in population genomics is now possible, and much needed, in a wider range of organisms.

Shared ancestral polymorphisms and chromosomal rearrangements as potential drivers of local adaptation in a marine fish.

Gene flow has tremendous importance for local adaptation, by influencing the fate of de novo mutations, maintaining standing genetic variation and driving adaptive introgression. Furthermore, structural variation as chromosomal rearrangements may facilitate adaptation despite high gene flow. However, our understanding of the evolutionary mechanisms impending or favouring local adaptation in the presence of gene flow is still limited to a restricted number of study systems. In this study, we examined how demographic history, shared ancestral polymorphism, and gene flow among glacial lineages contribute to local adaptation to sea conditions in a marine fish, the capelin (Mallotus villosus). We first assembled a 490-Mbp draft genome of M. villosus to map our RAD sequence reads. Then, we used a large data set of genome-wide single nucleotide polymorphisms (25,904 filtered SNPs) genotyped in 1,310 individuals collected from 31 spawning sites in the northwest Atlantic. We reconstructed the history of divergence among three glacial lineages and showed that they probably diverged from 3.8 to 1.8 million years ago and experienced secondary contacts. Within each lineage, our analyses provided evidence for large Ne and high gene flow among spawning sites. Within the Northwest Atlantic lineage, we detected a polymorphic chromosomal rearrangement leading to the occurrence of three haplogroups. Genotype-environment associations revealed molecular signatures of local adaptation to environmental conditions prevailing at spawning sites. Our study also suggests that both shared polymorphisms among lineages, resulting from standing genetic variation or introgression, and chromosomal rearrangements may contribute to local adaptation in the presence of high gene flow.

A Roadmap for Understanding the Evolutionary Significance of Structural Genomic Variation.

Structural genomic variants (SVs) are ubiquitous and play a major role in adaptation and speciation. Yet, comparative and population genomics have focused predominantly on gene duplications and large-effect inversions. The lack of a common framework for studying all SVs is hampering progress towards a more systematic assessment of their evolutionary significance. Here we (i) review how different types of SVs affect ecological and evolutionary processes; (ii) suggest unifying definitions and recommendations for future studies; and (iii) provide a roadmap for the integration of SVs in ecoevolutionary studies. In doing so, we lay the foundation for population genomics, theoretical, and experimental approaches to understand how the full spectrum of SVs impacts ecological and evolutionary processes.

Hybridization and transgressive exploration of colour pattern and wing morphology in Heliconius butterflies.

Hybridization can generate novel phenotypes distinct from those of parental lineages, a phenomenon known as transgressive trait variation. Transgressive phenotypes might negatively or positively affect hybrid fitness, and increase available variation. Closely related species of Heliconius butterflies regularly produce hybrids in nature, and hybridization is thought to play a role in the diversification of novel wing colour patterns despite strong stabilizing selection due to interspecific mimicry. Here, we studied wing phenotypes in first- and second-generation hybrids produced by controlled crosses between either two co-mimetic species of Heliconius or between two nonmimetic species. We quantified wing size, shape and colour pattern variation and asked whether hybrids displayed transgressive wing phenotypes. Discrete traits underlain by major-effect loci, such as the presence or absence of colour patches, generate novel phenotypes. For quantitative traits, such as wing shape or subtle colour pattern characters, hybrids only exceed the parental range in specific dimensions of the morphological space. Overall, our study addresses some of the challenges in defining and measuring phenotypic transgression for multivariate traits and our data suggest that the extent to which transgressive trait variation in hybrids contributes to phenotypic diversity depends on the complexity and the genetic architecture of the traits.