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

## TL;DR

This study sequenced six mayfly mitogenomes, revealing evolutionary patterns and phylogenetic relationships within the Ephemerellidae family.

## Contribution

The study provides new insights into mitogenome evolution and phylogeny in Ephemerellidae through comparative analysis of six newly sequenced genomes.

## Key findings

- trnI-associated inversion and translocation are characteristic features of Ephemerellidae mitogenomes.
- ND6 and ND2 are the most variable protein-coding genes, while COX1 is the most conserved.
- Phylogenetic analyses support the monophyly of Torleya, Cincticostella, and Serratella.

## Abstract

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 trnI‐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 ND6 and ND2 were the most variable protein‐coding genes, whereas COX1 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 Torleya, Cincticostella, and Serratella, 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.

Ephemerellidae is a diverse family of mayflies widely distributed in the Holarctic and Oriental regions, comprising more than 300 described species. However, mitochondrial genomic data for this family remain scarce, limiting our understanding of its evolutionary history. In this study, we sequenced and annotated six complete mitogenomes representing five genera of Ephemerellidae. The distinct patterns of tRNA gene rearrangement and duplication revealed here provide new insights into the evolution and diversity of Ephemerellidae and offer valuable molecular resources for phylogenetic and taxonomic studies within Ephemeroptera.