Epigenetic mechanisms, such as DNA methylation, can influence gene regulation and affect phenotypic variation, raising the possibility that they contribute to ecological adaptation. To being to address this issue requires high-resolution sequencing studies of natural populations to pinpoint epigenetic regions of potential ecological and evolutionary significance. However, such studies are still relatively uncommon, especially in insects, and are mainly restricted to a few model organisms. Here, we characterize patterns of DNA methylation for natural populations of Timema cristinae adapted to two host plant species (i.e., ecotypes). By integrating results from sequencing of whole transcriptomes, genomes, and methylomes, we investigate whether environmental, host, and genetic differences of these stick insects are associated with methylation levels of cytosine nucleotides in CpG context. We report an overall genome-wide methylation level for T. cristinae of ~14%, being enriched in gene bodies and impoverished in repetitive elements. Genome-wide DNA methylation variation was strongly positively correlated with genetic distance (relatedness), but also exhibited significant host-plant effects. Using methylome-environment association analysis, we pinpointed specific genomic regions that are differentially methylated between ecotypes, with these regions being enriched for genes with functions in membrane processes. The observed association between methylation variation with genetic relatedness and the ecologically-important variable of host plant suggest a potential role for epigenetic modification in T. cristinae adaptation. To substantiate such adaptive significance, future studies could test if methylation has a heritable component and the extent to which it responds to experimental manipulation in field and laboratory studies.

A species’ demographic history provides important context to contemporary population genetics and a possible insight into past responses to climate change. An individual’s genome provides a window into the evolutionary history of contemporary populations. Pairwise Sequentially Markovian Coalescent (PSMC) analysis uses information from a single genome to derive fluctuations in effective population size change over the last ~5 million years. Here we apply PSMC analysis to two European nightjar (Caprimulgus europaeus) genomes, sampled in Northwest and Southern Europe, with the aim of revealing the demographic history of nightjar in Europe. We successfully reconstructed effective population size over the last 5 million years for two contemporary nightjar populations. Our analysis shows that nightjar are responsive to global climate change, with effective population size broadly increasing under stable warm periods and decreasing during cooler spans and prolonged glacial periods. PSMC analysis on the pseudo-diploid combination of the two genomes revealed fluctuations in gene flow between the populations over time, with gene flow ceasing by the last-glacial maximum. This pattern of differentiation is in line with the species utilising different refugia during glacial maxima. We suggest that nightjar in Europe may show latitudinal (East-West) genetic structuring as a result of reduced gene flow between different glacial refugia. Finally, our results suggest that migratory behaviour in nightjar likely evolved prior to the last-glacial maximum, with long-distance migration seemingly persisting throughout the Pleistocene. However, further genetic structure analysis of nightjar from known breeding sites across the species’ contemporary range is needed to fully understand the extent and origins of range-wide differentiation in the species.