Comparative Mitogenomics Reveals Gene Rearrangement and Phylogenetic Relationships in Siphlonuroidea (Insecta: Ephemeroptera).

Siphlonuroidea is a superfamily within Ephemeroptera, yet the phylogenetic relationships among its constituent families and their placement relative to other mayfly lineages remain unresolved. To address these questions, we generated and analyzed 16 newly assembled mitochondrial genomes from 14 species across Ephemeroptera, including three species of Ameletidae, four of Siphlonuridae, and nine mitochondrial genomes from seven species of Isonychiidae. Comparative mitogenomic analysis revealed two distinct tRNA gene rearrangement patterns within Siphlonuridae, including trnI-trnQ-trnM-trnQ-trnM-trnQ-trnM-trnQ-trnM and trnI-trnM-trnQ-trnM. In contrast, all Ameletidae mitogenomes share an identical rearrangement of trnI-trnQ-trnM-trnM, which constitutes a potential synapomorphy supporting the monophyly of this family. Compositional analysis further showed that Siphlonuridae and Ameletidae exhibit significantly higher and highly similar A+T contents, clustering together in hierarchical analyses. This characteristic contrasts sharply with Isonychiidae, which displays markedly lower A+T content. Phylogenomic inference based on the PCG12 dataset supports a sister-group relationship between Siphlonuridae and Ameletidae, with this clade itself forming the sister group to a well-supported clade of Isonychiidae and Heptageniidae. Divergence time estimation places the origin of the Ameletidae and Siphlonuridae lineage in the Late Jurassic (174.71 Mya), while Isonychiidae diverged in the Early Cretaceous (136.81 Mya). In conclusion, Siphlonuridae and Ameletidae show a closer affinity and belong to Siphlonuroidea. Isonychiidae shares a closer relationship with Heptageniidae and remains outside Siphlonuroidea. Siphluriscidae is recovered as the sister lineage to all other extant Ephemeroptera, confirming its status as the earliest-diverging extant mayfly lineage.