LAUSR

Abstract We used Illumina next-generation sequencing data to generate the nearly complete mitochondrial genomes (mitogenomes) of three phasmatodeans (Megalophasma granulata, Calvisia medogensis, and Phyllium tibetense) without the aid of additional techniques such as long-range PCR or cloning. The most surprising finding was the presence of a novel gene arrangement “trnR-trnA-trnN-trnSAGN-trnE- trnF ” (genes underlined are encoded by the minority strand) that was detected in M. granulata. The ancestral order of this tRNA gene cluster is typically “trnA-trnR-trnN-trnSAGN-trnE- trnF ”. However, trnA was inserted between trnR and trnN in M. granulata, and this represents a rare case of gene rearrangement in Phasmatodea. Phylogenetic analyses were conducted on the concatenated nucleotide sequences of all protein-coding genes and two ribosomal RNA genes. Both maximum likelihood and Bayesian analyses failed to support the monophyly of Areolatae, Anareolatae, Diapheromeridae, Phasmatidae, Heteropterygidae, Necrosciinae, and Necrosciini. However, the monophyly of several lower taxonomic groups were confirmed in our analysis. For instance, two Ramulus species and Entoria okinawaensis were recovered in the monophyletic Clitumnini, and Phraortes, Phyllium, and Bacillus species formed individual monophyletic clades. Our results support the hypothesis that wing-loss independently occurred several times in Verophasmatodea.