Neutral mutation disequilibrium is common in prokaryotes and animals

Widely used models of sequence evolution assume DNA sequences are at equilibrium with respect to the processes that drive divergence, mutation and natural selection. Violations of this assumption have been shown to affect the conclusions from analyses of genetic variation. However, there is currently a lack of statistical methods to assess whether this phenomenon occurs with sufficient frequency and magnitude to warrant concern. Here, we introduce such techniques and apply them to cases where genetically encoded changes to mutation are expected to induce neutral mutation disequilibrium (NMD) across an entire genome (loss of the hypermutable base 5-methyl-cytosine in Drosophila melanogaster), or in a limited genomic region (translocation of the Fxy gene to the pseudo-autosomal region in Mus musculus). We find that the proportion of 9,237 genes in NMD in the D. melanogaster genome (~0.81) is markedly elevated compared to its sister taxa D. simulans (~0.31) which retains 5-methyl-cytosine. We further establish that the translocation of Fxy has resulted in ~3x increase in the magnitude of NMD. These empirical case studies validate the robustness of our methods. Applying the methods to human evolution, from 613 genes, we estimated the proportion of NMD for the exons as ~0.64 and introns as ~0.22. These results establish NMD as a common phenomenon and suggest that conclusions from studies that assumed equilibrium in order to understand biological mechanisms may misattribute to biology what should be credited to measurement error.