Cryptic Species Exist in Vietnamella sinensis Hsu, 1936 (Insecta: Ephemeroptera) from Studies of Complete Mitochondrial Genomes

SIMPLE SUMMARY: The family Vietnamellidae (Ephemeroptera) is one of the oldest insect families in the world. However, there are still controversies about the phylogenetic relationships among Vietnamellidae, Ephemerellidae, and Teloganodidae. The mitochondrial (mt) genome can be used to discuss phylogenetic relationships and cryptic species. We sequenced and annotated three complete mt genomes of Vietnamella sinensis from three different populations, identifying a cryptic species of V. sinensis and discuss the phylogenetic relationships of Vietnamellidae. Based on the genetic distance of the whole mt genomes, the phylogenetic relationship of three populations was uncovered and the divergence time of V. sinensis QY indicated that it was a cryptic species of V. sinensis. ABSTRACT: Ephemeroptera (Insecta: Pterygota) are widely distributed all over the world with more than 3500 species. During the last decade, the phylogenetic relationships within Ephemeroptera have been a hot topic of research, especially regarding the phylogenetic relationships among Vietnamellidae. In this study, three mitochondrial genomes from three populations of Vienamella sinensis collected from Tonglu (V. sinensis TL), Chun’an (V. sinensis CN), and Qingyuan (V. sinensis QY) in Zhejiang Province, China were compared to discuss the potential existence of cryptic species. We also established their phylogenetic relationship by combining the mt genomes of 69 Ephemeroptera downloaded from NCBI. The mt genomes of V. sinensis TL, V. sinensis CN, and V. sinensis QY showed the same gene arrangement with lengths of 15,674 bp, 15,674 bp, and 15,610 bp, respectively. Comprehensive analyses of these three mt genomes revealed significant differences in mt genome organization, genetic distance, and divergence time. Our results showed that the specimens collected from Chun’an and Tonglu in Zhejiang Province, China belonged to V. sinensis, and the specimens collected from Qingyuan, Zhejiang Province, China were a cryptic species of V. sinensis. In maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees, the monophyly of the family Vietnamellidae was supported and Vietnamellidae has a close relationship with Ephemerellidae.