Two-Fold ND5 Genes, Three-Fold Control Regions, lncRNA, and the "Missing" ATP8 Found in the Mitogenomes of Polypedates megacephalus (Rhacophridae: Polypedates)

Simple Summary: The spot-legged treefrog Polypedates megacephalus (Anura: Rhacophoridae) is widely distributed in Asia. In previous studies, it has been noted that the mitochondrial gene ATP8 has not been confidently annotated in any sequenced Polypedates to date. Duplications of control regions (CRs) are often observed in the tree frog family Rhacophoridae, and in most cases the copied CRs are highly similar to one another. This feature may lead to sequence assembly errors. Our findings are the first to detect two copies of the ND5 genes and three copies of the CRs in P. megacephalus by employing a "primer bridging" approach and allude to the presence of ATP8 via bioinformatic analyses and RT-qPCR. However, the question of whether the ATP8 is functional needs to be addressed in future studies. In prior research on the mitochondrial genome (mitogenome) of Polypedates megacephalus, the one copy of ND5 gene was translocated to the control region (CR) and the ATP8 gene was not found. Gene loss is uncommon among vertebrates. However, in this study, we resequenced the mitogenomes of P. megacephalus from different regions using a "primer bridging" approach with Sanger sequencing technologies, which revealed the "missing" ATP8 gene in P. megacephalus as well as three other previously published Polypedates. The mitogenome of this species was found to contain two copies of the ND5 genes and three copies of the control regions. Furthermore, multiple tandem repeats were identified in the control regions. Notably, we observed that there was no correlation between genetic divergence and geographic distance. However, using the mitogenome, gene expression analysis was performed via RT-qPCR of liver samples and it was thus determined that COIII, ND2, ND4, and ND6 were reduced to 0.64 +/- 0.24, 0.55 +/- 0.34, 0.44 +/- 0.21 and 0.65 +/- 0.17, respectively, under low-temperature stress (8 ffiC) as compared with controls (p < 0.05). Remarkably, the transcript of long non-coding RNA (lncRNA) between positions 8029 and 8612 decreased significantly with exposure to low-temperature stress (8 degrees C). Antisense ND6 gene expression showed a downward trend, but this was not significant. These results reveal that modulations of protein-coding mitochondrial genes and lncRNAs of P. megacephalus play a crucial role in the molecular response to cold stress.