Proteome-wide evolutionary rate covariation reveals the mitonuclear coevolutionary landscape in Squamata

The reciprocal evolutionary adjustment between mitochondrial and nuclear genomes is well characterized at the oxidative phosphorylation (OXPHOS) interface, yet the extent to which mitonuclear coevolution extends beyond mitochondrial pathways remains largely unknown. Evolutionary Rate Covariation (ERC) can detect long-term coevolutionary relationships by identifying protein pairs whose evolutionary rates covary across a phylogeny, but its proteome-wide applications have so far been limited to a few taxa. Here, we scaled ERC to nearly three million protein pairs across 34 squamate proteomes, reconstructing a proteome-wide coevolutionary network. Mitochondrial OXPHOS genes emerge as network hubs, and their coevolutionary neighborhoods extend well beyond the oxidative phosphorylation pathway into calcium signaling, peroxisomes, and pigmentation-related genes, a signal robust even at high correlation stringency. In squamates, coloration plays roles in courtship signaling and UV protection. The coevolutionary association between mitochondrial OXPHOS genes and nuclear genes involved in pigmentation may be consistent with the mitonuclear ecology hypothesis, which posits that ornamental coloration advertises cellular respiratory efficiency. We further exploited the deep mitonuclear phylogenetic discordance of Squamata to test whether coevolution also leaves topological signatures in gene trees. Nuclear genes supporting the mitochondrial topology were short and phylogenetically poorly informative, suggesting stochastic rather than coevolutionary discordance. Together, these results demonstrate that ERC captures functional coevolution that topology-based approaches miss, and reveal that the evolutionary reach of mitochondria extends beyond OXPHOS.