Mitochondrial genome evolution: the influence of partitioning, calibration, and gene heterogeneity on pleurodontan substitution rates

Substitution rate estimates are a key source of information in modern evolutionary biology, underpinning divergence time inference and other evolutionary analyses. Mitochondrial DNA nucleotide substitution rates, in particular, are commonly used for these purposes. However, these rates are typically derived from a small set of genes, closely related species, or from a limited number of model organisms. Such limitations become increasingly problematic at deeper phylogenetic levels, where errors in rate estimates and divergence times tend to accumulate with evolutionary distance. Here, we use nearly complete mitogenomes of 27 pleurodontan (Squamata: Pleurodonta) species to estimate substitution rates for the whole clade, paying special attention to the effect of data partitioning, calibrations and model choices on these estimations. The substitution rate estimates we obtained are consistent with previous findings for specific lineages within the group. Rates across individual genes ranged from approximately 0.004 to 0.02 substitutions/site/million years, with notable differences between coding and non-coding regions, and among codon positions. Calibrations had a less pronounced effect on the analyses than anticipated, although subtle differences were observed. These findings underscore the challenges of estimating targeted nucleotide substitution rates, especially for lineages with limited genomic data, as is the case for several Squamata lineages. Moreover, the results provide valuable insights into the evolutionary dynamics of Pleurodonta and emphasize the importance of incorporating robust data and models to improve accuracy in substitution rates and divergence time estimations.