Mitochondrial Genome Characterization of Six Spiny Crawler Mayflies and Comparative Analysis Within Ephemerellidae (Ephemeroptera: Pannota)

Mitogenomes provide important molecular information for resolving evolutionary relationships in mayflies. However, both the evolutionary patterns of mitogenomes and the phylogenetic structure of Ephemerellidae remain insufficiently understood. In this study, we sequenced six complete mitogenomes representing five ephemerellid genera and performed comprehensive comparative analyses that included all currently available ephemerellid mitogenomes. Our results reveal that <i>trnI</i>-associated inversion and translocation are characteristic features of Ephemerellidae mitogenomes, likely generated through tandem duplication followed by random loss during mitochondrial evolution. All species exhibited strong A + T bias and distinct compositional asymmetry, with codon usage heavily skewed toward A/T at third codon positions. Nucleotide diversity and evolutionary rate analyses indicated that <i>ND6</i> and <i>ND2</i> were the most variable protein-coding genes, whereas <i>COX1</i> was the most conserved among the 13 protein-coding genes. Phylogenetic analyses based on 13 PCGs and two rRNAs using Bayesian inference (BI) and Maximum likelihood (ML) consistently supported the monophyly of <i>Torleya</i>, <i>Cincticostella</i>, and <i>Serratella</i>, and recovered stable relationships among the major lineages within Ephemerellidae. Overall, the six newly sequenced mitogenomes enrich the mitochondrial genomic resources for Ephemerellidae and provide valuable insights into mitogenome evolution and phylogenetic relationships in this family.