Complete mitochondrial genome sequences facilitate species identification and analyses of phylogenetic relationships. However, the available data are limited to the diverse and widespread insect family Cicadellidae. This study analyzes and summarizes the complete mitochondrial genome structure characteristics of 11 leafhopper subfamilies and two newly sequenced Typhlocybinae species, Empoascanara wengangensis and E. gracilis. Moreover, using 13PCGs and rRNA data to analyze the nucleotide diversity, evolution rate, and the phylogenetic relationship between the subfamilies of 56 species, verifying the taxonomic status analysis of E. wengangensis and E. gracilis. The analysis results show that the genome structures of the subfamilies and the newly sequenced two species are very similar, and the size of the CR region is significantly related to the repeat unit. However, in the entire AT-skews and CG-skews, the AT-skews of other subfamilies are all positive, and CG-skews are negative, while Empoascini of Typhlocybinae and Ledrinae are the opposite. Furthermore, among 13PCGs, the AT-skews of 13 species are all negative while CG-skews are positive, which from Empoascini in Typhlocybinae, Idiocerinae, Cicadellinae, Ledrinae, and Evacanthinae. Phylogenetic analysis shows that ML and PB analysis produce almost consistent topologies between different data sets and models, and some relationships are highly supported and remain unchanged. Mileewinae is a monophyletic group and is a sister group with Typhlocybinae, and the sister group of Evacanthinae is Ledrinae + Cicadellinae. Phylogenetic analysis grouped the two newly sequenced species with other species of Typhlocybinae, which was separated from other subfamilies, and all Erythroneurini insects gathered together. However, E. gracilis grouped into a single group, not grouped with species of the same genus (Empoascanara). This result does not match the traditional classification, and other nuclear genes or transcriptome genes may be needed to verify the result. Nucleotide diversity analysis shows that nad4 and nad5 may be evaluated as potential DNA markers defining the Cicadellidae insect species.
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