Increasing 28 mitogenomes of Ephemeroptera, Odonata and Plecoptera support the Chiastomyaria hypothesis with three different outgroup combinations.

Background: The phylogenetic relationships of Odonata (dragonflies and damselflies) and Ephemeroptera (mayflies) remain unresolved. Different researchers have supported one of three hypotheses (Palaeoptera, Chiastomyaria or Metapterygota) based on data from different morphological characters and molecular markers, sometimes even re-assessing the same transcriptomes or mitochondrial genomes. The appropriate choice of outgroups and more taxon sampling is thought to eliminate artificial phylogenetic relationships and obtain an accurate phylogeny. Hence, in the current study, we sequenced 28 mt genomes from Ephemeroptera, Odonata and Plecoptera to further investigate phylogenetic relationships, the probability of each of the three hypotheses, and to examine mt gene arrangements in these species. We selected three different combinations of outgroups to analyze how outgroup choice affected the phylogenetic relationships of Odonata and Ephemeroptera.

Methods: Mitochondrial genomes from 28 species of mayflies, dragonflies, damselflies and stoneflies were sequenced. We used Bayesian inference (BI) and Maximum likelihood (ML) analyses for each dataset to reconstruct an accurate phylogeny of these winged insect orders. The effect of outgroup choice was assessed by separate analyses using three outgroups combinations: (a) four bristletails and three silverfish as outgroups, (b) five bristletails and three silverfish as outgroups, or (c) five diplurans as outgroups.

Results: Among these sequenced mitogenomes we found the gene arrangement in Heptageniidae (Ephemeroptera), and an inverted and translocated between the 12S RNA gene and the control region in Ephemerellidae (Ephemeroptera). The gene arrangement in Heptageniidae (Ephemeroptera) can be explained via the tandem-duplication and random loss model, and the transposition and inversion of genes in Ephemerellidae can be explained through the recombination and tandem duplication-random loss (TDRL) model. Our phylogenetic analysis strongly supported the Chiastomyaria hypothesis in three different outgroup combinations in BI analyses. The results also show that suitable outgroups are very important to determining phylogenetic relationships in the rapid evolution of insects especially among Ephemeroptera and Odonata. The mt genome is a suitable marker to investigate the phylogeny of inter-order and inter-family relationships of insects but outgroup choice is very important for deriving these relationships among winged insects. Hence, we must carefully choose the correct outgroup in order to discuss the relationships of Ephemeroptera and Odonata.