Adaptations of mitochondrial complexes in aquatic mites: A comparative study of Eupodides and Anystides

Aquatic mites have successfully colonized freshwater habitats, providing an excellent model for investigating the molecular basis of adaptive evolution. Because mitochondrial oxidative phosphorylation (OXPHOS) is essential for cellular energy production, adaptive changes in mitochondrial protein-coding genes may reflect evolutionary responses to environmental pressures. In this study, the signatures of episodic diversifying selection in mitochondrial protein-coding genes of representatives from the Eupodides and Anystides lineages were investigated and the potential structural significance of positively selected amino acid residues were assessed. Mitochondrial protein-coding genes were extracted from NCBI mitochondrion RefSeq datasets of Eupodides and Anystides taxa. Evolutionary analyses were performed at nucleotide level using the Mixed Effects Model of Evolution (MEME) implemented in the Datamonkey platform to identify codon sites evolving under episodic diversifying selection. To evaluate the potential functional relevance of these sites, positively selected residues were mapped onto three-dimensional protein models predicted by AlphaFold. The analyses identified multiple codon sites showing significant evidence of episodic diversifying selection, with the strongest signals (p < 0.01) detected in the cox1, cox3, nd5, nd2, cytB, and atp6 genes. Structural mapping revealed that several positively selected residues were located in regions that may influence protein conformation or participate in functional domains associated with electron transport and proton translocation. These observations suggest that adaptive amino acid substitutions may contribute to functional modifications of mitochondrial respiratory complexes and may represent habitat, group or lineage-specific evolutionary responses to ecological conditions experienced by mites especially Trombidiformes. Structural comparisons indicated that most positively selected sites were not associated with major alterations in predicted protein architecture, suggesting that adaptive evolution primarily acts through subtle functional optimization. In contrast, the ATP6 protein exhibited lineage-specific differences in transmembrane helix organization, indicating that prolonged adaptive divergence may have contributed to structural differentiation between aquatic and terrestrial mites.