Conflicting Timelines: Exploring patterns of mito-nuclear discordance in divergence estimates among tetrapods

Phylogenetic studies across a range of tetrapod groups have historically utilised either mitochondrial DNA, concatenated mito-nuclear matrices, or nuclear loci to infer divergence estimates. As such, the discordance between estimates inferred using differing data types has been a topic of interest over the past decade. Although several studies have looked into divergence disparities in smaller taxonomic groups, often with mixed results, a systematic investigation of the pattern of mito-nuclear divergence discordance in tetrapods across deep-time remains to be undertaken. In this study, we aimed to quantify the extent of divergence disparity inferred by the aforementioned data types in each of the four major tetrapod groups, namely primates (Order Primates), birds (Class Aves), squamates (Order Squamata), and anurans (Order Anura), while controlling for calibration strategies, taxon sampling and other a priori distributions. We also calculated substitution saturation for all groups across data types in order to elucidate its role in generating unreliable divergence estimates. Our findings indicate that mitochondrial estimates consistently underestimate basal divergence times and overestimate recent divergences across all groups (apart from anurans) as compared to the nuclear datasets. We also find that divergence times estimated using concatenated matrices skew in favour of the nuclear tree. Furthermore, substitution saturation is substantial in all of the mitochondrial datasets across groups, and interestingly present in the nuclear dataset for Anurans, resulting in a reduction in overall mito-nuclear divergence disparity for the crown ages of the group. These results call for a revisit of divergence dates estimated using mitochondrial DNA, while advocating for saturation testing prior to divergence dating and highlighting the inherent bias in divergence dates estimated using concatenated mito-nuclear matrices.