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Dryophytes versicolor is one of the most extreme freeze-tolerant frogs from eastern North America. In this study, the mitochondrial genome of D. versicolor was sequenced to analyze the phylogenetic relationships among Hylidae and investigate mitochondrial gene expression in response to freezing and anoxia. The total length of the D. versicolor mitogenome is the longest known to date among the available family members of Hylidae. Both maximum likelihood (ML) and Bayesian inference (BI) analyses strongly supported D. versicolor as a sister clade to (D. japonica + D. ussuriensis) + (D. suweonensis + D. immaculata (KP212702)), and indicated that Dryophytes is monophyletic. Using the mitochondrial genome, gene expression analysis was performed using RT-qPCR in skeletal muscle samples, and determined that relative levels of D. versicolor COX2 increased by 2.40 ± 0.23 fold in response to anoxia, but did not change with exposure to freezing. In addition, ND3 transcript levels decreased in response to anoxia but remained constant during freezing. By contrast, COX1 transcript levels decreased with exposure to freezing, but did not change under anoxic conditions. These results suggest that modulations of protein-coding mitochondrial genes of D. versicolor may play a role in the molecular response to freezing and anoxia tolerance. [ABSTRACT FROM AUTHOR]