The Sturnidae family comprises 123 recognized species in 35 genera. The taxa Mimidae and Buphagidae were formerly treated as subfamilies within Sturnidae. The phylogenetic relationships among the Sturnidae and related taxa (Sturnidae sensu lato) remain unresolved due to high rates of morphological change and concomitant morphological homoplasy. This study presents five new mitogenomes of Sturnidae sensu lato and comprehensive mitogenomic analyses. The investigated mitogenomes exhibit an identical gene composition of 37 genes—including 13 protein-coding genes (PCGs), 2 rRNA genes, and 22 tRNA genes—and one control region (CR). The most important finding of this study is drawn from CAM analyses. The surprisingly unique motifs for each species provide a new direction for the molecular species identification of avian. Furthermore, the pervasiveness of the natural selection of PCGs is found in all examined species when analyzing their nucleotide composition and codon usage. We also determine the structures of mt-tRNA, mt-rRNA, and CR structures of Sturnidae sensu lato. Lastly, our phylogenetic analyses not only well support the monophyly of Sturnidae, Mimidae, and Buphagidae, but also define nine stable subclades. Taken together, our findings will enable the further elucidation of the evolutionary relationships within Sturnidae sensu lato.

Papilionoideae is the most species-rich subfamily of the third largest angiosperm family Fabaceae. One constituent large group, the inverted-repeat-lacking clade (IRLC), is well-known for the broad loss of one IR copy. Accumulating observations of massive plastomic disparities have made IRLC a well-suited model for exploring plastome evolution. However, there is still a large amount left to explore. The present study focused on the plastid tRNA (pttRNA) evolution within Papilionoideae, employing the currently densest sampling strategies for both the IRLC (156) and non-IRLC (109) lineages. Strikingly, our results revealed abundant inter-lineage variabilities in both tRNA sequences and structures, including a 3 nt difference in the average size of trnS-UGA, the consensus sequence disparities across 29 tRNAs, the distinct 3 nt indels in trnA-UGC, and an impressive 248 nt intron loss of IRLC trnI-GAU (potential markers). Additionally, there was unequal stability of the atypical secondary structures in trnS-GGA and trnS-UGA, as well as significantly diverse compositions of substitution events in all compared tRNAs (p < 0.05). Ultimately, these findings not only demonstrate the significant differences and unique markers of IRLC pttRNAs compared to other non-IRLC Papilionoideae, but also draw an important conclusion that the large losses of one IR potentially promote highly diverse evolutionary patterns of IRLC, which could partly compensate for the potential IR-lacking impacts.

Mitochondrial codon usage and selective pressures drive adaptive evolution and translation efficiency. This study provides insights into the evolutionary constraints shaping 13 core mitochondrial proteins in Corvides, based on 117 species, including 51 newly assembled genomes. We present evidence linking codon-level sequence architecture, protein structure, and selective pressures. These protein-coding genes (PCGs) are under strong purifying selection, with dN/dS ratios ranging from 0.00779 (MT-CO1) to 0.16214 (MT-ATP8). This conservation is reflected in the 3D model of MT-CO1, where conserved residues cluster within 12 transmembrane helices forming the core of its proton-pumping function. At the sequence level, we identify signatures of selection for translational efficiency, which are critical for accurate synthesis and folding. These signatures include a significant preference for “optimal” codons that perfectly match tRNA anticodons (p < 0.001). We also find lineage-specific features such as codon aversion motifs (CAMs). The MT-ATP6 gene exhibits complete aversion of the CGA codon in Oriolus chinensis and of the ACT codon in O. kundoo. These sequence-level features resolve the deep phylogenetic relationships within the group, demonstrating the effectiveness of our multi-layered analytical framework. Overall, our results link codon-level sequence architecture with protein structural constraints, functional evolutionary signals, and mitochondrial genome evolution in Corvides.

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.