Five hundred million years of methylation: tracing the mutational origins of vertebrate genome composition

Methylation-associated deamination removes CpG from vertebrate genomes, but how it affects the dinucleotide profile remains unresolved. We analysed 753 vertebrate and 481 invertebrate genomes to test whether CpG loss defines a compositional axis and to identify its strongest signature. CpG depletion was the dominant axis of vertebrate dinucleotide variation. Unexpectedly, its strongest between-genome correlate was AG/CT rather than the direct mutational product TpG/CpA, which showed the expected dataset-wide mass balance but varied little among genomes. A forward-evolution model based on measured seven-nucleotide human germline substitution rates produced neither CpG depletion nor AG/CT enrichment when methylated-CpG mutability was excluded. Adding one CpG-specific mutability term, calibrated only to the mammalian CpG ratio, reproduced both features, identifying AG/CT as a second-order consequence of the context-dependent mutation network. Within genomes, CpG depletion was strongest in transposable elements and weakened with distance from them. Across vertebrates, the axis followed Amniota more closely than endothermy and was associated with an expanded GC-rich isochore compartment. A Machine Learning analysis shows that CpG depletion and AG/CT were the principal features separating vertebrates from invertebrates, in which both were markedly attenuated. Thus, a methylation-associated axis organises vertebrate dinucleotide composition, and its strongest marker is not the immediate product of CpG deamination.