Variations in DNA Methylation Are Landmarks of Freshwater Adaptation in Three-Spined Sticklebacks.

Understanding the phenotypic consequences of epigenetic variation and its role in adaptation remains a central challenge in evolutionary biology. Marine and freshwater sticklebacks provide a powerful system to study the interplay between genetic and epigenetic components of phenotypic plasticity that enables colonization of contrasting salinity habitats. Here, we used whole-genome bisulfite sequencing (WGBS) to characterize DNA methylation entropy-a measure of epigenetic stochasticity-in gill tissue from marine and freshwater ecotypes. We found that freshwater sticklebacks exhibit elevated methylation entropy in divergence islands (DIs), genomic regions known as hotspots of genetic divergence between marine and freshwater populations. Within DIs, we identified a subset of genes exhibiting concurrent increases in methylation entropy and transcriptional variance, including osmoregulatory candidates involved in growth modulation, cytoskeletal reorganization, metabolism, and extracellular matrix remodeling. Their linked variability suggests that they may act as "adaptation capacitors" facilitating phenotypic plasticity during salinity transitions. Exploratory enrichment analysis further revealed overrepresented epigenetic regulators within DIs, such as DNA demethylase TET1 and chromatin remodelers ARID5B and BPTF, indicating a potential regulatory basis by which these factors may convert genetic variation into epigenetic diversity. Collectively, our findings demonstrate that DIs are focal points of both genetic divergence and epigenetic heterogeneity, consistent with a model in which DIs may act as multi-layered genomic regions associated with adaptive responses to salinity change.