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.

Genomics for monitoring and understanding species responses to global climate change.

All life forms across the globe are experiencing drastic changes in environmental conditions as a result of global climate change. These environmental changes are happening rapidly, incur substantial socioeconomic costs, pose threats to biodiversity and diminish a species' potential to adapt to future environments. Understanding and monitoring how organisms respond to human-driven climate change is therefore a major priority for the conservation of biodiversity in a rapidly changing environment. Recent developments in genomic, transcriptomic and epigenomic technologies are enabling unprecedented insights into the evolutionary processes and molecular bases of adaptation. This Review summarizes methods that apply and integrate omics tools to experimentally investigate, monitor and predict how species and communities in the wild cope with global climate change, which is by genetically adapting to new environmental conditions, through range shifts or through phenotypic plasticity. We identify advantages and limitations of each method and discuss future research avenues that would improve our understanding of species' evolutionary responses to global climate change, highlighting the need for holistic, multi-omics approaches to ecosystem monitoring during global climate change.

The Evolutionary Complexities of DNA Methylation in Animals: From Plasticity to Genetic Evolution.

The importance of DNA methylation in plastic responses to environmental change and evolutionary dynamics is increasingly recognized. Here, we provide a Perspective piece on the diverse roles of DNA methylation on broad evolutionary timescales, including (i) short-term transient acclimation, (ii) stable phenotypic evolution, and (iii) genomic evolution. We show that epigenetic responses vary along a continuum, ranging from short-term acclimatory responses in variable environments within a generation to long-term modifications in populations and species. DNA methylation thus unlocks additional potential for organisms to rapidly acclimate to their environment over short timeframes. If these changes affect fitness, they can circumvent the need for adaptive changes at the genome level. However, methylation has a complex reciprocal relationship with genetic variation as it can be genetically controlled, yet it can also induce point mutations and contribute to genomic evolution. When habitats remain constant over many generations, or populations are separated across habitats, initially plastic phenotypes can become hardwired through epigenetically facilitated mutagenesis. It remains unclear under what circumstances plasticity contributes to evolutionary outcomes, and when plastic changes will become permanently encoded into genotype. We highlight how studies investigating the evolution of epigenetic plasticity need to carefully consider how plasticity in methylation state could evolve among different evolutionary scenarios, the possible phenotypic outcomes, its effects on genomic evolution, and the proximate energetic and ultimate fitness costs of methylation. We argue that accumulating evidence suggests that DNA methylation can contribute toward evolution on various timescales, spanning a continuum from acclimatory plasticity to genomic evolution.

Captive rearing effects on the methylome of Atlantic salmon after oceanic migration: Sex-specificity and intergenerational stability.

Captive rearing in salmon hatcheries can have considerable impacts on both fish phenotype and fitness within a single generation, even in the absence of genetic change. Evidence for hatchery-induced changes in DNA methylation is becoming abundant, though questions remain on the sex-specificity of these effects, their persistence until spawning and potential for transmission to future generations. Here we performed whole genome methylation sequencing of fin tissue for 16 hatchery and 16 wild Atlantic salmon (Salmo salar) returning to spawn in the Rimouski River, Québec, Canada. We identified two cohorts of hatchery-reared salmon through methylation analysis, one of which was epigenetically similar to wild fish, suggesting that supplementation efforts may be able to minimize the epigenetic effects of hatchery rearing. We found considerable sex-specific effects of hatchery rearing, with few genomic regions being affected in both males and females. We also analysed the methylome of 32 F1 offspring from four groups (pure wild, pure hatchery origin and reciprocal hybrids). We found that few epigenetic changes due to parental hatchery rearing persisted in the F1 offspring though the patterns of inheritance appear to be complex, involving nonadditive effects. Our results suggest that the epigenetic effects of hatchery rearing can be minimal in F0 . There may also be minimal epigenetic inheritance and rapid loss of epigenetic changes associated with hatchery rearing. However, due to sex-specificity and nonadditive patterns of inheritance, methylation changes due to captive rearing are rather complex and the field would benefit from further research on minimizing the epigenetic effects of captive rearing in conservation efforts.

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.

Thermal regime during parental sexual maturation, but not during offspring rearing, modulates DNA methylation in brook charr (Salvelinus fontinalis).

Epigenetic inheritance can result in plastic responses to changing environments being faithfully transmitted to offspring. However, it remains unclear how epigenetic mechanisms such as DNA methylation can contribute to multigenerational acclimation and adaptation to environmental stressors. Brook charr (Salvelinus fontinalis), an economically important salmonid, is highly sensitive to thermal stress and is of conservation concern in the context of climate change. We studied the effects of temperature during parental sexual maturation and offspring rearing on whole-genome DNA methylation in brook charr juveniles (fry). Parents were split between warm and cold temperatures during sexual maturation, mated in controlled breeding designs, then offspring from each family were split between warm (8°C) and cold (5°C) rearing environments. Using whole-genome bisulfite sequencing, we found 188 differentially methylated regions (DMRs) due to parental maturation temperature after controlling for family structure. By contrast, offspring rearing temperature had a negligible effect on offspring methylation. Stable intergenerational inheritance of DNA methylation and minimal plasticity in progeny could result in the transmission of acclimatory epigenetic states to offspring, priming them for a warming environment. Our findings have implications pertaining to the role of intergenerational epigenetic inheritance in response to ongoing climate change.

Conservation implications of revised genetic structure resulting from new population discovery: the threatened eastern sand darter (Ammocrypta pellucida) in Canada.

Human activity can put non-game fishes at higher risk of extinction because of inappropriate management action. Eastern sand darter (Ammocrypta pellucida), a small benthic fish classified as threatened across much of its northern range, inhabits increasingly fragmented sandy habitats and, as a non-game fish, may be easily overlooked in conservation efforts. In this study, the authors use genotype data from nine microsatellite loci and cytochrome oxidase I (COI) sequencing data across its northern native range to re-assess genetic structure and to characterize a newly discovered, geographically disjunct population. Previous microsatellite marker analyses had identified seven distinct population genetic clusters across the region sampled; the analysis of this study showed that the newly discovered population (West Lake, Ontario) exhibits a divergent structure. COI haplotype analysis suggests that a single haplotype recolonized the Great Lakes and surrounding water bodies after the Wisconsinan glacial period, and subsequent fluctuation in water levels and habitat fragmentation resulted in divergence of genetic clusters. Although the novel West Lake population has a common ancestral source with other populations in the broader region, its divergent genetic signature merits its consideration as a separate conservation unit. The analyses of this study highlight the potential conservation implications of the discovery of new populations, particularly those of at-risk species, even within a region that has been genetically well characterized.

Epigenetic inheritance and reproductive mode in plants and animals.

Epigenetic inheritance is another piece of the puzzle of nongenetic inheritance, although the prevalence, sources, persistence, and phenotypic consequences of heritable epigenetic marks across taxa remain unclear. We systematically reviewed over 500 studies from the past 5 years to identify trends in the frequency of epigenetic inheritance due to differences in reproductive mode and germline development. Genetic, intrinsic (e.g., disease), and extrinsic (e.g., environmental) factors were identified as sources of epigenetic inheritance, with impacts on phenotype and adaptation depending on environmental predictability. Our review shows that multigenerational persistence of epigenomic patterns is common in both plants and animals, but also highlights many knowledge gaps that remain to be filled. We provide a framework to guide future studies towards understanding the generational persistence and eco-evolutionary significance of epigenomic patterns.

Population differences in Chinook salmon (Oncorhynchus tshawytscha) DNA methylation: Genetic drift and environmental factors.

Local adaptation and phenotypic differences among populations have been reported in many species, though most studies focus on either neutral or adaptive genetic differentiation. With the discovery of DNA methylation, questions have arisen about its contribution to individual variation in and among natural populations. Previous studies have identified differences in methylation among populations of organisms, although most to date have been in plants and model animal species. Here we obtained eyed eggs from eight populations of Chinook salmon (Oncorhynchus tshawytscha) and assayed DNA methylation at 23 genes involved in development, immune function, stress response, and metabolism using a gene-targeted PCR-based assay for next-generation sequencing. Evidence for population differences in methylation was found at eight out of 23 gene loci after controlling for developmental timing in each individual. However, we found no correlation between freshwater environmental parameters and methylation variation among populations at those eight genes. A weak correlation was identified between pairwise DNA methylation dissimilarity among populations and pairwise F ST based on 15 microsatellite loci, indicating weak effects of genetic drift or geographic distance on methylation. The weak correlation was primarily driven by two genes, GTIIBS and Nkef. However, single-gene Mantel tests comparing methylation and pairwise F ST were not significant after Bonferroni correction. Thus, population differences in DNA methylation are more likely related to unmeasured oceanic environmental conditions, local adaptation, and/or genetic drift. DNA methylation is an additional mechanism that contributes to among population variation, with potential influences on organism phenotype, adaptive potential, and population resilience.

Rearing environment affects the genetic architecture and plasticity of DNA methylation in Chinook salmon.

Genetic architecture and phenotypic plasticity are important considerations when studying trait variation within and among populations. Since environmental change can induce shifts in the genetic architecture and plasticity of traits, it is important to consider both genetic and environmental sources of phenotypic variation. While there is overwhelming evidence for environmental effects on phenotype, the underlying mechanisms are less clear. Variation in DNA methylation is a potential mechanism mediating environmental effects on phenotype due to its sensitivity to environmental stimuli, transgenerational inheritance, and influences on transcription. To characterize the effect of environment on methylation, we created two 6 × 6 (North Carolina II) Chinook salmon breeding crosses and reared the offspring in two environments: uniform hatchery tanks and seminatural stream channels. We sampled the fish twice during development, at the alevin (larval) and fry (juvenile) stages. We measured DNA methylation at 13 genes using a PCR-based bisulfite sequencing protocol. The genetic architecture of DNA methylation differed between rearing environments, with greater additive and nonadditive genetic variance in hatchery fish and greater maternal effects in seminatural channel fish, though gene-specific variation was evident. We observed plasticity in methylation across all assayed genes, as well as gene-specific effects at two genes in alevin and six genes in fry, indicating developmental stage-specific effects of rearing environment on methylation. Characterizing genetic and environmental influences on methylation is critical for future studies on DNA methylation as a potential mechanism for acclimation and adaptation.

DNA Methylation Profiles Suggest Intergenerational Transfer of Maternal Effects.

The view of maternal effects (nongenetic maternal environmental influence on offspring phenotype) has changed from one of distracting complications in evolutionary genetics to an important evolutionary mechanism for improving offspring fitness. Recent studies have shown that maternal effects act as an adaptive mechanism to prepare offspring for stressful environments. Although research into the magnitude of maternal effects is abundant, the molecular mechanisms of maternal influences on offspring phenotypic variation are not fully understood. Despite recent work identifying DNA methylation as a potential mechanism of nongenetic inheritance, currently proposed links between DNA methylation and parental effects are indirect and primarily involve genomic imprinting. We combined a factorial breeding design and gene-targeted sequencing methods to assess inheritance of methylation during early life stages at 14 genes involved in growth, development, metabolism, stress response, and immune function of Chinook salmon (Oncorhynchus tshawytscha). We found little evidence for additive or nonadditive genetic effects acting on methylation levels during early development; however, we detected significant maternal effects. Consistent with conventional maternal effect data, maternal effects on methylation declined through development and were replaced with nonadditive effects when offspring began exogenous feeding. We mapped methylation at individual CpG sites across the selected candidate genes to test for variation in site-specific methylation profiles and found significant maternal effects at selected CpG sites that also declined with development stage. While intergenerational inheritance of methylated DNA is controversial, we show that CpG-specific methylation may function as an underlying molecular mechanism for maternal effects, with important implications for offspring fitness.

Inbreeding effects on gene-specific DNA methylation among tissues of Chinook salmon.

Inbreeding depression is the loss of fitness resulting from the mating of genetically related individuals. Traditionally, the study of inbreeding depression focused on genetic effects, although recent research has identified DNA methylation as also having a role in inbreeding effects. Since inbreeding depression and DNA methylation change with age and environmental stress, DNA methylation is a likely candidate for the regulation of genes associated with inbreeding depression. Here, we use a targeted, multigene approach to assess methylation at 22 growth-, metabolic-, immune- and stress-related genes. We developed PCR-based DNA methylation assays to test the effects of intense inbreeding on intragenic gene-specific methylation in inbred and outbred Chinook salmon. Inbred fish had altered methylation at three genes, CK-1, GTIIBS and hsp70, suggesting that methylation changes associated with inbreeding depression are targeted to specific genes and are not whole-genome effects. While we did not find a significant inbreeding by age interaction, we found that DNA methylation generally increases with age, although methylation decreased with age in five genes, CK-1, IFN-É£, HNRNPL, hsc71 and FSHb, potentially due to environmental context and sexual maturation. As expected, we found methylation patterns differed among tissue types, highlighting the need for careful selection of target tissue for methylation studies. This study provides insight into the role of epigenetic effects on ageing, environmental response and tissue function in Chinook salmon and shows that methylation is a targeted and regulated cellular process. We provide the first evidence of epigenetically based inbreeding depression in vertebrates.