Differential Mitochondrial Genome Expression of Four Hylid Frog Species under Low-Temperature Stress and Its Relationship with Amphibian Temperature Adaptation.

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.

How Does Mitochondrial Protein-Coding Gene Expression in Fejervarya kawamurai (Anura: Dicroglossidae) Respond to Extreme Temperatures?

Unusual climates can lead to extreme temperatures. Fejervarya kawamurai, one of the most prevalent anurans in the paddy fields of tropical and subtropical regions in Asia, is sensitive to climate change. The present study focuses primarily on a single question: how do the 13 mitochondrial protein-coding genes (PCGs) respond to extreme temperature change compared with 25 °C controls? Thirty-eight genes including an extra tRNA-Met gene were identified and sequenced from the mitochondrial genome of F. kawamurai. Evolutionary relationships were assessed within the Dicroglossidae and showed that Dicroglossinae is monophyletic and F. kawamurai is a sister group to the clade of (F. multistriata + F. limnocharis). Transcript levels of mitochondrial genes in liver were also evaluated to assess responses to 24 h exposure to low (2 °C and 4 °C) or high (40 °C) temperatures. Under 2 °C, seven genes showed significant changes in liver transcript levels, among which transcript levels of ATP8, ND1, ND2, ND3, ND4, and Cytb increased, respectively, and ND5 decreased. However, exposure to 4 °C for 24 h was very different in that the expressions of ten mitochondrial protein-coding genes, except ND1, ND3, and Cytb, were significantly downregulated. Among them, the transcript level of ND5 was most significantly downregulated, decreasing by 0.28-fold. Exposure to a hot environment at 40 °C for 24 h resulted in a marked difference in transcript responses with strong upregulation of eight genes, ranging from a 1.52-fold increase in ND4L to a 2.18-fold rise in Cytb transcript levels, although COI and ND5 were reduced to 0.56 and 0.67, respectively, compared with the controls. Overall, these results suggest that at 4 °C, F. kawamurai appears to have entered a hypometabolic state of hibernation, whereas its mitochondrial oxidative phosphorylation was affected at both 2 °C and 40 °C. The majority of mitochondrial PCGs exhibited substantial changes at all three temperatures, indicating that frogs such as F. kawamurai that inhabit tropical or subtropical regions are susceptible to ambient temperature changes and can quickly employ compensating adjustments to proteins involved in the mitochondrial electron transport chain.

Nine Mitochondrial Genomes of Phasmatodea with Two Novel Mitochondrial Gene Rearrangements and Phylogeny.

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.

Characterization of Two Mitogenomes of Hyla sanchiangensis (Anura: Hylidae), with Phylogenetic Relationships and Selection Pressure Analyses of Hylidae.

Hyla sanchiangensis (Anura: Hylidae) is endemic to China and is distributed across Anhui, Zhejiang, Fujian, Guangdong, Guangxi, Hunan, and Guizhou provinces. The mitogenomes of H. sanchiangensis from two different sites (Jinxiu, Guangxi, and Wencheng, Zhejiang) were sequenced. Phylogenetic analyses were conducted, including 38 mitogenomes of Hylidae from the NCBI database, and assessed the phylogenetic relationship of H. sanchiangensis within the analyzed dataset. Two mitogenomes of H. sanchiangensis showed the typical mitochondrial gene arrangement with 13 protein-coding genes (PCGs), two ribosomal RNA genes (12S rRNA and 16S rRNA), 22 transfer RNA (tRNA) genes, and one non-coding control region (D-loop). The lengths of the 12S rRNA and 16S rRNA genes from both samples (Jinxiu and Wencheng) were 933 bp and 1604 bp, respectively. The genetic distance (p-distance transformed into percent) on the basis of the mitogenomes (excluding the control region) of the two samples was calculated as 4.4%. Hyla sanchiangensis showed a close phylogenetic relationship with the clade of (H. annectans + H. tsinlingensis), which was supported by ML and BI analyses. In the branch-site model, five positive selection sites were found in the clade of Hyla and Dryophytes: Cytb protein (at position 316), ND3 protein (at position 85), and ND5 protein (at position 400) have one site, respectively, and two sites in ND4 protein (at positions 47 and 200). Based on the results, we hypothesized that the positive selection of Hyla and Dryophytes was due to their experience of cold stress in historical events, but more evidence is needed to support this conclusion.