The classification of stick and leaf insects (Order Phasmatodea) is flawed at various taxonomic ranks due to a lack of robust phylogenetic relationships and convergent morphological characteristics. In this study, we sequenced nine new mitogenomes that ranged from 15,011 bp to 17,761 bp in length. In the mitogenome of Carausis sp., we found a translocation of trnR and trnA, which can be explained by the tandem duplication/random loss (TDRL) model. In the Stheneboea repudiosa Brunner von Wattenwyl, 1907, a novel mitochondrial structure of 12S rRNA-CR1-trnI-CR2-trnQ-trnM was found for the first time in Phasmatodea. Due to the low homology of CR1 and CR2, we hypothesized that trnI was inverted through recombination and then translocated into the middle of the control region. Control region repeats were frequently detected in the newly sequenced mitogenomes. To explore phylogenetic relationships in Phasmatodea, mtPCGs from 56 Phasmatodean species (composed of 9 stick insects from this study, 31 GenBank data, and 16 data derived from transcriptome splicing) were used for Bayesian inference (BI), and maximum likelihood (ML) analyses. Both analyses supported the monophyly of Lonchodinae and Necrosciinae, but Lonchodidae was polyphyletic. Phasmatidae was monophyletic, and Clitumninae was paraphyletic. Phyllidae was located at the base of Neophasmatodea and formed a sister group with the remaining Neophasmatodea. Bacillidae and Pseudophasmatidae were recovered as a sister group. Heteroptergidae was monophyletic, and the Heteropteryginae sister to the clade (Obriminae + Dataminae) was supported by BI analysis and ML analysis.

Extreme weather poses huge challenges for animals that must adapt to wide variations in environmental temperature and, in many cases, it can lead to the local extirpation of populations or even the extinction of an entire species. Previous studies have found that one element of amphibian adaptation to environmental stress involves changes in mitochondrial gene expression at low temperatures. However, to date, comparative studies of gene expression in organisms living at extreme temperatures have focused mainly on nuclear genes. This study sequenced the complete mitochondrial genomes of five Asian hylid frog species: Dryophytes japonicus, D. immaculata, Hyla annectans, H. chinensis and H. zhaopingensis. It compared the phylogenetic relationships within the Hylidae family and explored the association between mitochondrial gene expression and evolutionary adaptations to cold stress. The present results showed that in D. immaculata, transcript levels of 12 out of 13 mitochondria genes were significantly reduced under cold exposure (p < 0.05); hence, we put forward the conjecture that D. immaculata adapts by entering a hibernation state at low temperature. In H. annectans, the transcripts of 10 genes (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COX1, COX2 and ATP8) were significantly reduced in response to cold exposure, and five mitochondrial genes in H. chinensis (ND1, ND2, ND3, ND4L and ATP6) also showed significantly reduced expression and transcript levels under cold conditions. By contrast, transcript levels of ND2 and ATP6 in H. zhaopingensis were significantly increased at low temperatures, possibly related to the narrow distribution of this species primarily at low latitudes. Indeed, H. zhaopingensis has little ability to adapt to low temperature (4 °C), or maybe to enter into hibernation, and it shows metabolic disorder in the cold. The present study demonstrates that the regulatory trend of mitochondrial gene expression in amphibians is correlated with their ability to adapt to variable climates in extreme environments. These results can predict which species are more likely to undergo extirpation or extinction with climate change and, thereby, provide new ideas for the study of species extinction in highly variable winter climates.

Stick and leaf insects are a group among the Insecta that are famous for their extraordinary mimicry ability. Since the establishment of the Phasmatodea, their internal classification has been constantly revised. Mitochondrial genes as molecular markers have been widely used for species classification, but the phylogenetic relationships within the Phasmatodea remain to be thoroughly discussed. In the present study, five mitogenomes of Phasmatodea ranging from 15,746 bp to 16,747 bp in length were sequenced. Bayesian inference (BI) and maximum likelihood (ML) analyses were carried out based on a 13 PCGs data matrix (nt123) and a combined matrix of 13 PCGs and two rRNA genes (nt123_rRNA). The present study supports the conclusion that Phylliidae was the basal group of Neophasmatodea and confirms the monophyly of Lonchodinae and Necrosciinae, but it shows that Lonchodidae was polyphyletic. A sister group of Bacillidae and Pseudophasmatidae was also recovered. The phylogenetic tree based on the nt_123 dataset showed higher node support values. The construction of a divergent time tree in this study supported the conclusion that extant Phasmatodea originated in the Jurassic (170 Mya) and most lineages diverged after the Cretaceous–Paleogene extinction event. To explore whether the mitochondrial genes of Phraortes liaoningensis collected from high latitudes where low temperatures occur for eight months of the year are under selection pressure, this study used the branch-site model and the branch model to analyze the selection pressure on the 13 mitochondria protein-coding genes (PCGs). We found that both ND2 and ND4L of Ph. liaoningensis exhibited positive selection sites using the branch-site model. This study shows that a low-temperature environment causes mitochondrial genes to be selected to meet the energy requirements for survival.