Large-Scale Comparative Mitogenomics of 170 Turtle Species Reveals Evolutionary Patterns of Codon Aversion and tRNA Structural Diversity

The order Testudines, one of the oldest monophyletic vertebrate lineages, offers an excellent opportunity to investigate mitogenome evolution and deep phylogenetic inference. However, comparative analyses across the order remain limited. To address this gap, we conducted a large-scale comparative mitogenomic analysis of 170 turtle species across 14 families, including 26 newly assembled mitogenomes. Using 13 protein-coding genes (PCGs) and 22 mitochondrial tRNAs, we characterized codon usage patterns, codon aversion motifs (CAMs), and tRNA secondary structures. Our analysis revealed a strong AT bias and significant inter-familial variation in codon usage bias. Multiple lines of evidence consistently indicated that natural selection was the primary force shaping codon usage patterns. We also identified species-specific CAMs and genus-associated tRNA secondary structure features as potential molecular indicators for evolutionary patterns and structural diversity. Phylogenetic analyses using Maximum Likelihood (ML) and Bayesian Inference (BI) recovered the two major suborders and provided insights into historically debated deep-node relationships, including the placement of Chelydridae and generic boundaries within Chelidae. Overall, this study provides a comprehensive perspective on mitogenome evolution in turtles and highlights the potential of codon aversion patterns and tRNA structural diversity as informative molecular evidence for comparative evolutionary genomics and mitochondrial diversity in Testudines.