Identification of Conserved Clustered Unmethylated Sites

For each orphan MTase family associated with incomplete methylation of the genome (Fig 4), we identified conserved unmethylated sites based on conservation of flanking gene sequences (Table 1). For every unmethylated site, we took the amino acid sequences of the two flanking genes, and identified best hits in each of the other genomes using BLASTP. Pairs of unmethylated sites across the two genomes were considered conserved if their flanking genes were reciprocal best hits in the respective other genome. For select conserved sites, genomic DNA sequences upstream of the putative target gene were subject to multiple alignment using MAFFT [59] and visualized in Jalview [60].