# Characterization and Phylogenomic Implications of the Mitochondrial Genome of Rhithrogena elasmaris (Ephemeroptera: Heptageniidae)

## TL;DR

This study sequenced the mitochondrial genome of Rhithrogena elasmaris and analyzed its structure and evolution in relation to other Heptageniidae species.

## Contribution

The first complete mitochondrial genome of Rhithrogena elasmaris is characterized, providing insights into its phylogenomic relationships and evolutionary patterns.

## Key findings

- The mitochondrial genome of R. elasmaris is 15,326 bp long with 37 typical mitochondrial genes and strong AT bias.
- Phylogenomic analysis supports a subfamily framework of (Heptageniinae + [Ecdyonurinae + Rhithrogeninae]) with Rhithrogena as a basal lineage.
- High nucleotide diversity and strong purifying selection were observed across protein-coding genes.

## Abstract

This study reports the first complete mitochondrial genome of Rhithrogena elasmaris 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 R. elasmaris 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 trnM has been lost in some species. Nucleotide diversity is relatively high (Pi = 0.223), with nad6 being the most variable protein‐coding gene and cox1 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, R. elasmaris and Paegniodes cupulatus exhibit mixed mitochondrial signals, suggesting possible incomplete lineage sorting or ancient mitochondrial introgression. The phylogeny supports the subfamily framework (Heptageniinae + [Ecdyonurinae + Rhithrogeninae]), with Rhithrogena 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.

This study presents the first complete mitochondrial genome of Rhithrogena elasmaris, revealing its structural features and codon usage patterns. Comparative analysis with 29 other Heptageniidae species showed high genetic diversity and strong purifying selection across protein‐coding genes. The phylogenomic results support a subfamilial framework of (Heptageniinae + [Ecdyonurinae + Rhithrogeninae]) and suggest possible mitochondrial introgression or incomplete lineage sorting in some lineages.