Phylogenetic shifts in gene body methylation correlate with gene expression and reflect trait conservation

A subset of genes in plant genomes are labeled with DNA methylation specifically at CG residues. These genes, known as gene-body methylated (gbM), have a number of associated characteristics. They tend to have longer sequences, to be enriched for intermediate expression levels, and to be associated with slower rates of molecular evolution. Most importantly, gbM genes tend to maintain their level of DNA methylation between species, suggesting that this trait is under evolutionary constraint. Given the degree of conservation in gbM, we still know surprisingly little about its function in plant genomes or whether gbM is itself a target of selection. To address these questions, we surveyed DNA methylation across eight grass (Poaceae) species that span a gradient of genome sizes. We first established that genome size correlates with genome-wide DNA methylation levels, but less so for genic levels. We then leveraged genomic data to identify a set of 2,982 putative orthologs among the eight species and examined shifts of methylation status for each ortholog in a phylogenetic context. A total of 55% of orthologs exhibited a shift in gbM, but these shifts occurred predominantly on terminal branches, indicating that shifts in gbM are rarely conveyed over time. Finally, we found that the degree of conservation of gbM across species is associated with increased gene length, reduced rates of molecular evolution, and increased gene expression level, but reduced gene expression variation across species. Overall, these observations suggest a basis for evolutionary pressure to maintain gbM status over evolutionary time.