Molecular Ecology

Living at high density and with low genetic diversity are factors that should both increase the susceptibility of organisms to disease. Therefore, group living organisms, especially those that are inbred, should be especially vulnerable to infection and therefore have particular strategies to cope with infection. Phenotypic plasticity, underpinned by epigenetic changes, could allow group living organisms to rapidly respond to infection challenges. To explore the potential role of epigenetic modifications in the immune response to a group-living species with low genetic diversity, we compared the genome-wide DNA methylation profiles of five colonies of social spiders (Stegodyphus dumicola) in their natural habitat in Namibia at the point just before they succumbed to infection to a point at least six months previously where they were presumably healthier. We found increases in genome- and chromosome-wide methylation levels in the CpG, CHG, and CHH contexts, although the genome-wide changes were not clearly different from zero. These changes were most prominent in the CHG context, especially at a narrow region of chromosome 13, hinting at an as-of-yet unsuspected role of this DNA methylation context in phenotypic plasticity. However, there were few clear patterns of differential methylation at the base level, and genes with a known immune function in spiders had mean methylation changes close to zero. Our results suggest that DNA methylation may change with infection at large genomic scales, but that this type of epigenetic change is not necessarily integral to the immune response of social spiders.

Many wild populations are increasingly stressed by rapid climatic change. While behavioural plasticity can enable limited tactical adaptive responses, standing genetic variation limits the species capacity to respond to climate change velocity. Epigenetic modification may provide a more rapid and plastic adaptive mechanism, but has been little studied in wild-living animals. Here we investigated CpG methylation during the pre-natal and early-life development of 95 European badger cubs between 2003 and 2011). During 10-months of delayed pre-implantation variability in precipitation between previous years February and April was the top determinant of methylation patterns among neonates, followed by mean temperature and temperature variability. Among the 4,641 significant weather-associated CpG sites, most occurred in the 47S rDNA region. Methylation of 47S rDNA was also associated with early-life weight, implying a mechanism that relays environmental stress to phenotypic stress. We also detected evidence for predictive adaptive response. Among the 1,641 CpG sites associated with early-life weight, pathways were associated with early-life growth, immune regulation, and to the development of aggression for competitive access to weather-limited food resources were over-represented. We conclude that a species epigenetics can have an important role in adaptive plasticity to environmental changes with important implications for biodiversity conservation and management.

Interspecific gene flow by hybridization may weaken species barriers and adaptive divergence, but can also initiate reinforcement of reproductive isolation trough natural and sexual selection. The extent of interspecific gene flow and its consequences for the initiation and maintenance of species barriers in natural systems remain poorly understood, however. To assess genome-wide patterns of gene flow between the two closely related European dung fly species Sepsis cynipsea and Sepsis neocynipsea (Diptera: Sepsidae), we tested for historical gene flow with the aid of ABBA-BABA test using whole-genome resequencing data from pooled DNA of male specimens originating from natural and laboratory populations. We contrasted genome-wide variation in DNA sequence differences between samples from sympatric populations of the two species in France and Switzerland with that of interspecific differences between pairs of samples involving allopatric populations from Estonia and Italy. In the French Cevennes, we detected a relative excess of DNA sequence identity, suggesting interspecific gene flow in sympatry. In contrast, at two sites in Switzerland, we observed a relative depletion of DNA sequence identity compatible with reinforcement of species boundaries in sympatry. Our results suggest that the species boundaries between S. cynipsea and S. neocynipsea in Europe depend on the eco-geographic context.

High intra-specific genetic diversity is associated with adaptive potential which is key for resilience to global change. However, high variation may also support deleterious alleles through genetic load, unless purged, thereby increasing the risk of inbreeding depression if population sizes decrease rapidly. Purging of deleterious variation has now been demonstrated in some threatened species. However, less is known about the costs of population declines and inbreeding in species with large population sizes and high genetic diversity even though this encompasses many species globally that have or are expected to undergo rapid population declines. Caribou is a species of ecological and cultural significance in North America with a continental-wide distribution supporting extensive phenotypic variation, but with some populations undergoing significant declines resulting in their at-risk status in Canada. We assessed intra-specific genetic variation, adaptive divergence, inbreeding, and genetic load across populations with different demographic histories using an annotated chromosome-scale reference genome and 66 whole genome sequences. We found high genetic diversity and nine phylogenomic lineages across the continent with adaptive diversification of genes, but also high genetic load among lineages. We also found highly divergent levels of inbreeding across individuals including the loss of alleles by drift (genetic erosion) but not purging, likely due to rapid population declines not allowing time for purging of deleterious alleles. As a result, further inbreeding may need to be mitigated through conservation efforts. Our results highlight the double-edged sword of genetic diversity that may be representative of other species-at-risk affected by anthropogenic activities.

The molecular mechanisms underlying intraspecific variation in life history strategies are still poorly understood, despite the importance of this question for understanding of organisms responses to environmental variability. Theoretical work proposed that epigenetic mechanisms such as DNA methylation might regulate intraspecific variation in life history strategies, however this assumption has rarely been verified empirically in wild populations. We examined associations between genome-wide methylation changes and environmentally-driven life history variation in two lineages of a marine fish that diverged approximatively 2.5 Mya, the capelin (Mallotus villosus), from North America and Europe. In both lineages, capelin harbour two contrasted life history strategies: some are strictly semelparous, experience fast actuarial senescence, but benefit from high hatching success by spawning on demersal sites where water temperature is low and relatively stable. In contrast, others are facultative iteroparous, have slower actuarial senescence, and suffer from lower hatching success by breeding in the intertidal zone where temperature is warmer, thermohaline parameters are less stable, and egg desiccation risk is high. Performing whole genome and epigenome sequencing, we showed that these contrasted life history strategies are more likely governed by epigenetic changes than by differences in DNA sequence. While genetic differentiation between the capelin harbouring different life history strategies was negligible, we detected parallel genome-wide methylation changes across lineages. We identified 1,067 differentially methylated regions (DMRs) comprising 15,818 CpGs, with 22% of them located within 5-kb around genes comprising promotor regions. We found that all DMRs were hypermethylated in demersal-spawning individuals. This striking result suggests that lower water temperature at demersal sites leads to an overall hypermethylation of the genome determined during the epigenetic reprogramming occurring over embryonic development. Our study emphasizes that parallel epigenetics changes in lineages with divergent genetic background could have a functional role in the regulation of intraspecific life history variation.

Repeated adaptive divergence in replicates of phenotypic diversification offers a propitious context to identify the molecular bases associated to adaptive divergence. A currently hotly debated topic pertains to the relative role of genomic vs. epigenomic variation in shaping patterns of phenotypic variation at the gene expression level. Here, we combined genomic, epigenomic and transcriptomic information from 64 individuals in order to quantify the relative role of SNPs and DNA methylation variation in the repeated evolution of four limnetic-benthic whitefish species pairs from Europe and North America. We first found evidence for 149 convergent differentially methylated regions (DMRs) between species across continents, which significantly influenced levels of gene expression. Hyper-methylated DMRs in the limnetic species were globally associated to an expression repression relatively to benthic species, and inversely. Furthermore, we identified 108 convergent genetic variants (eQTLs) associated to gene expression differences between species. Gene expression differences were more pronounced in genes harbouring eQTL compared to those associated with DMRs, thus revealing a greater effect of eQTLs on gene expression. Multivariate analyses allowed partitioning the relative contribution of epi-/genomic changes and their association to gene expression variation. Most of the gene expression variation was significantly explained by genomic (4.1%) and putatively genomic-epigenomic interactive variation (46.7%), while \"pure\" epigenomic variation explained marginally 2.3% of the gene expression variation across continents. This study provides a rare qualitative and quantitative documentation of the relative role of genomic, DNA methylation and their interaction in shaping patterns of convergent gene expression during the process of ecological speciation.

Marine invertebrates with limited dispersal abilities exhibit high levels of genetic divergence among populations. However, the spatial extent of genetic differentiation in these species remains poorly understood because identifying natural barriers to gene flow can be challenging in the marine environment. In this study, we investigated the population genetic structure of the interstitial annelid Olavius algarvensis, a species that lays eggs in its immediate surroundings and does not have an active dispersal phase. We analyzed the mitochondrial and nuclear genome sequences of hundreds to thousands of individuals from eleven sites in the Mediterranean, spanning microgeographic scales of < 5 km to macrogeographic scales of 800 km. Comparisons of single nucleotide polymorphisms (SNPs) in mitochondrial genomes revealed a complex history of introgression events, with as many as six mitochondrial lineages co-occurring in individuals from the same site. In contrast, SNP analyses of nuclear genomes revealed clear genetic differentiation at micro- and macrographic scales, characterised by a significant isolation by distance pattern (IBD). IBD patterns further indicated the presence of a historical physical barrier to gene flow on the east coast of the island of Elba corresponding to the historical shoreline around Elba during the Last Glacial Maximum in the Late Pleistocene, and highlighting the influence of geological forces in shaping population genetic structuring in the species today. Overall, our results provide strong empirical evidence for the high genomic diversification across spatial scales in marine interstitial fauna.

Small, isolated populations are prone to inbreeding, increasing the proportion of homozygous sites across the genome that can be quantified as runs of homozygosity (ROH). Caribou (Rangifer tarandus) are declining across their range in Canada; thus, understanding the effects of inbreeding on genetic potential is pertinent for conserving small, isolated populations. We quantified ROH in high-coverage whole genomes of boreal caribou from small, isolated populations in southern Ontario, Canada, in comparison to caribou from the continuous range of Ontario, other caribou ecotypes in Canada, and western Greenland. Sampled populations presented divergent evolutionary histories, differing population sizes, and extents of isolation. We conducted BLAST searches across regions of elevated heterozygosity to identify genes that have maintained variation despite inbreeding. We found caribou from recently isolated populations in Ontario had a large proportion of their genome in long ROH. We observed even larger proportions but shorter ROH in western Greenland, indicating that inbreeding has occurred over a longer period in comparison to other populations. We observed the least inbreeding in barren-ground and eastern migratory caribou, which occur in larger population sizes than boreal caribou. Despite vastly different inbreeding extents, we found regions of high heterozygosity maintained across all populations. Within these islands of heterozygosity, we identified genes associated with immunity, signaling regulation, nucleotide binding, toxin elimination, and feeding behaviour regulation. In this study, we confirm inbreeding in isolated populations of a species at risk, but also uncover high variation in some genes maintained across divergent populations despite inbreeding, suggesting strong balancing selection.

Genetic variation is critical for evolutionary responses to environmental change. Links between genetic variation and behavioural or life history traits may reveal how varied strategies influence evolutionary trends in speciation and adaptation. Traits associated with movement typically correlate with population genetic structure and could help predict populations vulnerability to geographic processes such as habitat fragmentation and disease spread. With their wide diversity in behaviours and ecologies, bats provide a useful testing ground for hypotheses about population structure related to species-specific movement patterns. We used a global sample of microsatellite data (n=233 sites from 17 bat species) associated with published studies to examine potential links between genetic variation and migration and mating strategies. The genetic measures we tested were population-specific differentiation, gene diversity, and allelic richness. Using Bayesian models that accounted for phylogenetic distances among species, we identified no correlations between migration or mating strategy and genetic variation. Our results do not support long-standing hypotheses about dispersal-mediated genetic structure, and contrast with prior studies on bat genetic diversity and differentiation. We discuss the need for continued research into the complex association of ecological, biogeographical, and behavioural factors that facilitate gene flow among populations, especially in species with diverse movement patterns.

Chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), threatens amphibian populations worldwide. MHC II has been implicated in Bd susceptibility, but the role of MHC in Bd susceptibility remains poorly understood. In this study, we clustered MHC II {beta}1 alleles into functional supertypes and investigated their diversity in amphibian species with differential susceptibility. Despite sharing similar alpha diversity in MHC II supertypes, the hosts exhibited distinct beta diversity. We demonstrated MHC supertypes can predict Bd susceptibility better than individual MHC alleles, with some supertypes conferring protection and others increasing risk. This suggests that MHC alleles function more as part of a complex network than as independent entities. We also quantified MHC allelic-specific expression and observed individual-level variability in expression across both species. Notably, positive and negative associations among MHC alleles were observed. The similarity of these patterns between the two species suggests the presence of conserved regulatory mechanisms across amphibians. Our results provide valuable resources and insights to advance the understanding of adaptive immunological systems and to develop targeted conservation strategies to preserve biodiversity.

Early-life experiences can have profound and long-lasting effects on adult phenotype and thereby Darwinian fitness, though the mechanisms driving these effects remain poorly understood. Epigenetic alterations, especially DNA methylation which affects gene expression, potentially mediate developmental condition effects on adult phenotype. We tested for such effects using captive zebra finches that were reared in either small or large broods, a manipulation that is known to have pleiotropic phenotypic effects. Analyzing whole genome DNA methylation patterns in erythrocytes from 50 individuals sampled in adulthood, we found 0.8% of all CpG sites after filtering to be differentially methylated after correction for multiple testing. We identified 149 non-transiently differentially methylated sites (DMSs) where the DNA methylation difference between treatments was larger than 25%. These DMSs were located in 19 autosomal chromosomes, in or near genes involved in critical biological processes such as cell growth, division, and differentiation, regulation of immune response, muscle contraction, and neuronal signaling. These findings suggest that epigenetic modifications such as DNA methylation potentially mediate long-term effects of early-life adversity via differential gene expression, but follow-up studies are needed to identify the extent to which the observed DMSs are functionally related to the previously observed phenotypic effects.

Anthropized environments are significantly challenging for wild species. Rapid adaptation to such habitats is thus key for their long-term persistence. Deciphering the environmental factors associated with species tolerance to modified habitats is fundamental for understanding the genetic and connectivity patterns of individuals and the local adaptation of their populations. We studied the Giant Toad, Rhinella horribilis, from two landscapes with distinct levels of anthropogenic habitat modification, assessed their genomic diversity, structure and connectivity with ddRAD-seq genomic data, identified potential outlier loci and their relationship with environmental and physicochemical water variables, and evaluated if populations from the two study sites showed signals of parallel adaptation. Both concordant and unique patterns were found regarding landscape factors and genotype-environment associations related with the degree of anthropic modification between landscapes. Genomic structure and connectivity were significantly associated with the presence of temporary water bodies, low vegetation cover, high humidity, solar radiation, and temperature. Notably, we identified both shared and distinct outlier SNPs and annotated functional genes for the two landscapes. Genes were enriched for biological processes and metabolic pathways, which were in turn correlated with environmental and physicochemical water variables. Genes and metabolic pathways were associated mainly with embryonic development, sexual maturation and immune responses. Studies such as this one, in an often-disregarded species, illustrate how parallel and un-parallel adaptive landscape genomic patterns arise in the stressful conditions of anthropized habitats.

Genomic diversity is the fundamental building block of biodiversity and the necessary ingredient for adaptation. Our rapidly increasing ability to quantify functional, compositional, and structural genomic diversity of populations forces the question of how to balance conservation goals - should the focus be on important functional diversity and key life history traits or on maximizing genomic diversity as a whole? Specifically, the intra-specific diversity (biocomplexity) comprised of phenotypic and genetic variation can determine the ability of a population to respond to changing environmental conditions. Here, we explore the biocomplexity of Californias Central Valley Chinook salmon (Oncorhynchus tshawytscha) population complex at the genomic level. Notably, despite apparent gene flow among individuals with the same migration (life history) phenotypes inhabiting different tributaries, each group is characterized by a surprising component of unique genomic diversity. Our results emphasize the importance of formulating conservation goals focused on maintaining biocomplexity at both the phenotypic and genotypic level. Doing so will maintain the species adaptive potential and increase the probability of persistence of the population complex despite changing environmental pressures.

Speciation is a key driver of biodiversity and understanding its genomic underpinnings is important for predicting and managing biodiversity. Structural variants (SVs) are large-scale (>50 bp) changes in the genome and have been implicated in adaptive divergence and reproductive isolation. We investigated the role of SVs in the speciation and divergence of two deer species (Odocoileus spp.) across their North American range. Using multiple long-read and a short-read datasets, our bioinformatics workflow revealed SVs and genomic features that were unique to each species. The majority of species-specific SVs were deletions and insertions suggesting that these variants may show higher likelihoods of fixation within populations. Further, while most SVs were intergenic, some genes found to be impacted by species-specific SVs were under positive selection inferred from dN/dS. We also observed an increased number of enhancer motifs found in SVs compared to the rest of the genome. The SV-affected genes were associated with reproduction, sensory adaptation, and several metabolic pathways. Many of these functions are relevant to fertility and deer biology and therefore may provide insights into potential mechanisms leading to reproductive divergence.

Identifying the molecular mechanisms involved in rapid adaptation to novel environments and determining their predictability are central questions in Evolutionary Biology and pressing issues due to rapid global changes. Complementary to genetic responses to selection, faster epigenetic variations such as modifications of DNA methylation may play a substantial role in rapid adaptation. In the context of rampant urbanization, joint examinations of genomic and epigenomic mechanisms are still lacking. Here, we investigated genomic (SNP) and epigenomic (CpG methylation) responses to urban life in a passerine bird, the Great tit (Parus major). To test whether urban evolution is predictable (i.e parallel) or involves mostly non-parallel molecular processes among cities, we analysed three distinct pairs of city and forest Great tit populations across Europe. Results reveal a polygenic response to urban life, with both many genes putatively under weak divergent selection and multiple differentially methylated regions (DMRs) between forest and city great tits. DMRs mainly overlapped transcription start sites and promotor regions, suggesting their importance in the modulation gene expression. Both genomic and epigenomic outliers were found in genomic regions enriched for genes with biological functions related to nervous system, immunity, behaviour, hormonal and stress responses. Interestingly, comparisons across the three pairs of city-forest populations suggested little parallelism in both genetic and epigenetic responses. Our results confirm, at both the genetic and epigenetic levels, hypotheses of polygenic and largely non-parallel mechanisms of rapid adaptation in new environments such as urbanized areas.

Climate change is causing rapid increases in temperature which drives genomic changes tied to adaptation. However, predicting the outcomes of climate change presents challenges as the anticipated conditions have yet to be experienced by natural populations. Modelling and lab experiments suggest that natural populations will experience shifts in life history, physiology, phenology, and ecology, but the underlying genomic mechanisms involved are unknown. However, some contemporary natural populations experience habitat warming through geothermal activity and can provide valuable insights into evolutionary responses. Geothermally warmed habitats should impose strong selection on ectotherms compared to ambient habitats as they increase metabolic demands, alter developmental processes, and offer novel ecological conditions. We leveraged Icelandic threespine sticklebacks (Gasterosteus aculeatus) from populations that have adaptively diverged along a geothermal/ambient habitat axis. We obtained 173,485 single nucleotide polymorphisms (SNPs) across four independent instances of population divergence using whole genome sequencing. While the majority of genomic differentiation between geothermal/ambient ecotypes was non-parallel, the MAPK signalling pathway appeared across all ecotype pairs. We also identified a putative inversion located on chromosome XXI which appears to drive parallel genomic differentiation between geothermal and ambient ecotypes. Candidate genes within the putative inversion correspond to metabolic adaptations, including regulation of appetite and fat content. Appetite level showed strong heritable divergence between ecotypes, while the rate of weight loss during starvation and fat levels differed between ecotypes. Overall, both polygenic adaptation and parallel structural variation appeared to be key genomic mechanisms for adaptation to geothermally warmed environments. While allelic divergence was largely unique across populations, it resulted in similar functional phenotypic outcomes. Thus, structural and allelic variation both operate to facilitate adaptation to warming environments. Therefore, while management from a genomic perspective will play a role in mitigating the effects of climate change, this study suggests that consideration of functional molecular pathways will be key to conservation but with precise changes being difficult to predict due to the highly polygenic nature of thermal adaptation.

Understanding how small populations cope with loss of genetic diversity and deleterious variation is crucial to address the current biodiversity crisis. Insular populations are particularly interesting as they have often persisted at low population sizes and higher inbreeding than their mainland counterparts. While the genome-wide consequences of inbreeding in threatened insular species have received some attention, comparative genomics between insular and mainland populations of wide-spread and genetically diverse species have rarely been performed. Yet, they are particularly well suited to inform about the consequences of drastic population declines from initially large populations - a phenomenon that is becoming increasingly common. The spot-nosed monkey (Cercopithecus petaurista), the Campbells monkey (Cercopithecus campbelli) and the green monkey (Chlorocebus sabaeus) are common and genetically diverse West African primates. Insular populations can be found at the Bijagos Archipelago, Guinea-Bissau. Here, we assessed the genome-wide diversity, inbreeding, genetic load and adaptive variation using whole genome sequencing data from insular and mainland populations. In the three species, island populations showed lower genome-wide diversity and higher inbreeding. Genetic drift has likely promoted the conversion of masked genetic load into realized load without increased purging of deleterious variation. Additionally, we found no evidence for accumulation of deleterious variation, suggesting that these populations are not yet at risk of extinction by genetic factors and may act as reservoirs of mainland genetic diversity. We highlight, however, that other anthropogenic factors are threatening these insular primates and therefore conservation management should target their immediate threats and safeguard against additional loss of diversity.

Phenotypic traits modulate the fate of species interactions with one another and the environment; thus, traits directly shape the past, present, and future evolutionary trajectories of populations. As such, distinct species-specific responses to a shifting environment are widely documented in the form of distinct genetic signatures, i.e., genetic diversity, reflecting differential responses over time. While the link between genetic diversity and phenotypic traits is seemingly fundamental, it has been challenging to establish unequivocally. Across an exemplar freshwater fish metacommunity, we employ phenotypic traits to test if they are significantly related to observed genetic patterns among species. Associated traits were then used to construct trait-based predictive models of genetic diversity. We collected representative constituents of a freshwater fish community (N=31 species) sampled across 75 sites within the White River Basin (Ozark Plateau, USA). For each species, we derived three genetic diversity indices (=HS/HT/G"ST) from SNP data (N=2,000 loci) and assessed 28 phenotypic traits related to morphology, life history, and ecology. We identified a series of traits (N=2-5, depending upon the index) strongly associated with facets of genetic diversity. These were subsequently applied in predictive models that explained 31-68% of the genetic variability across species, suggesting a potential utility as an imputation tactic for data-deficient species. Our approach effectively linked species-specific traits with genetic diversity within and among populations, thereby further clarifying correlations between contemporary ecological processes, as modulated by species traits, and long-term evolutionary trajectories.

Infectious diseases are a leading cause of biodiversity declines, but the factors shaping the prevalence and intensity of wildlife disease outbreaks remain unresolved. Host-associated microbes are likely a key component of host resistance to infection, but a hosts ability to recruit such protective microbes may be restricted by the availability of microbes present in their immediate environment. Here we investigate the associations between the abiotic environment, skin microbiome, and infection with the Dermocystid pathogen Amphibiothecum meredithae in Palmate newts (Lissotriton helveticus) on the Isle of Rum, Scotland. We found more acidic ponds to be associated with higher skin microbiome diversity and lower pathogen prevalence. Predicted functional analysis of microbiota composition identified bacterially-mediated pathways linked to host immunity upregulated in skin microbiotas associated with more acidic ponds and lower infection prevalence. This study highlights the potential importance of the environment in modulating host- microbiome-pathogen relationships and patterns of infection in the wild.

Genetic variation is essential for adaptation to rapid environmental changes. Identifying genetic variation associated with climate-change related phenotypes is therefore the necessary first step towards predictive models of genomic vulnerability. Here we used a whole-genome scan to identify candidate genetic variants associated with differences in behavioural resilience to ocean acidification in a coral reef fish. We identified three genomic regions that differ between individuals that are behaviourally tolerant compared with behaviourally sensitive to elevated CO2. These include a dopamine receptor (drd4rs), cadherin related family member 5-like (cdhr5l), Synapse-associated protein 1 (syap1), and GRB2 Associated Regulator of MAPK1 Subtype 2 (garem2), which have previously been found to modify behaviour related to boldness, novelty seeking, and learning in other species, and differ between behaviourally tolerant and sensitive individuals. Consequently, the identified genes are promising candidates in the search of the genetic underpinnings and adaptive potential of behavioural resilience to ocean acidification in fishes.