Genome-wide and gene-specific DNA methylation across developmental stages in Pogonomyrmex californicus: A socially polymorphic ant.

DNA methylation has been proposed as an epigenetic driver of phenotypic plasticity in social insects, yet experimental evidence remains limited. Even less is known about the role of epigenetic mechanisms underlying behavioural and social polymorphism. We quantified CpG methylation for the socially polymorphic harvester ant Pogonomyrmex californicus across larvae, pupae, workers and queens using Oxford Nanopore Technologies (ONT) sequencing. These results were compared against the current gold standard whole-genome bisulfite sequencing (WGBS). Methylation sites were highly correlated between WGBS and ONT, validating the use of ONT for high-throughput epigenomic profiling. Genome-wide methylation was low (~3%), consistent with findings in other (Hymenoptera: Formicidae), and highly clustered within gene bodies, especially exons, while introns, intergenic DNA, promoters and transposable elements were hypo-methylated. Gene body methylation (GBM) correlated positively with gene expression in queens, corroborating previous reports for other insects, suggesting a conserved regulatory role for DNA methylation in insects. A comparison between developmental stages revealed significant stage-specific differences in GBM frequencies. Workers and queens, although from different populations, shared a substantial core of methylated loci enriched for olfactory-receptor activity and biosynthetic pathways, processes that are central to caste-specific behaviour and physiology. These shared methylation signatures, coupled with stage-dependent variability, highlight DNA methylation as a possible factor in developmental stages and caste differentiation. In the future, it is essential to disentangle the effects of caste and variation between populations. Our study establishes P. californicus as a powerful model for dissecting how epigenetic modifications interface with gene expression to generate developmental and complex social organization, which is largely unexplored.