2.4　Evolutionary Analysis

The evolutionary relationship was assessed using a combination of phylogenetic analysis，comparison of genetic distances and intergenic spacers.A phylogenetic tree was constructed by the ML method using whole mt genome data.The concatenated nucleotide sequences of 13 PCGs in 17 complete mt genomes were aligned using MAFFT v7.215(Katoh and Standley，2013)，and unreliably aligned regions were removed using Gblocks(Castresana，2000).Eubasilissa regina(Trichoptera)was used as an outgroup.The best model used in the ML phylogenetic tree was determined by using“find best DNA/protein models(ML)”in Mega 7.0.The“GTR+G”model produced the lowest values for both the BIC(Bayesian Information Criterion)and the AICc(corrected Akaike information criterion)，therefore it was chosen for phylogenetic analysis.The phylogenetic tree among the Lepidoptera was constructed using MEGA 7.0(Kumar et al.，2016)with 500 bootstrap replicates.

Genetic distance is generally considered to be an important aspect of species discrimination(Hebert et al.，2003).To further explore the evolutionary development of the O.sinensis host insect with other species，we calculated Kimura's two parameter(K2P)genetic distances among different genes and genome regions using MEGA 7.0(Kumar et al.，2016).Pairwise distances were calculated among all sequenced Hepialidae individuals and compared with that of the O.sinensis host insect.(https://www.daowen.com)

The intergenic regions do not encode genes；as a result，intergenic regions are under more relaxed selection and evolve more quickly than coding genes.Consequently，intergenic regions can be used to analyze the evolutionary relationships of closely related species(Ghikas et al.，2010).We hand-counted and compared the intergenic-spacer sequences of Hepialidae to further evaluate the phylogenetic development of the host insect of O.sinensis.