Characterization and Phylogenomic Implications of the Mitochondrial Genome of <i>Rhithrogena elasmaris</i> (Ephemeroptera: Heptageniidae)

This study reports the first complete mitochondrial genome of <i>Rhithrogena elasmaris</i> and provides a comprehensive analysis of its structural features and codon usage patterns. Combined with published mitochondrial genomes of 29 Heptageniidae species, we further evaluated genetic diversity, phylogenomic relationships, and differentiation patterns within the family. The mitochondrial genome of <i>R. elasmaris</i> is 15,326 bp in length with a GC content of 36.12%, and comprises the 37 typical mitochondrial genes. It shows a strong AT bias and clear codon usage preferences, with neutrality and PR2 plots indicating natural selection as the dominant evolutionary force. Simple sequence repeats are widely distributed, and tRNA structures are generally conserved despite frequent base mismatches. Comparative analyses demonstrate that gene order in Heptageniidae is highly conserved, although one copy of <i>trnM</i> has been lost in some species. Nucleotide diversity is relatively high (Pi = 0.223), with nad6 being the most variable protein-coding gene and <i>cox1</i> the most conserved. Ka/Ks values < 1 across all 13 protein-coding genes indicate strong purifying selection, with varying intensity reflecting functional constraints. Genetic structure and phylogenomic analyses support distinct subfamily level divergence within Heptageniidae. However, <i>R. elasmaris</i> and <i>Paegniodes cupulatus</i> exhibit mixed mitochondrial signals, suggesting possible incomplete lineage sorting or ancient mitochondrial introgression. The phylogeny supports the subfamily framework (Heptageniinae + [Ecdyonurinae + Rhithrogeninae]), with <i>Rhithrogena</i> forming a basal lineage within Rhithrogeninae. Divergence modeling indicates that Ecdyonurinae and Heptageniinae diverged first, followed by the split of Rhithrogeninae from Heptageniinae. This study enriches the molecular data resources for Heptageniidae and provides a refined framework for studying its systematics, evolutionary history, and ecological adaptation. Future work integrating nuclear genomic datasets will be necessary to further clarify speciation processes and adaptive evolution.