The complete mitochondrial genome of Rana johnsi (Smith, 2009) (Anura: Ranidae) and its phylogeny

Abstract Rana johnsi (Smith 2009) firstly considered as the member of genus Pseudorana, has been moved into the genus Rana. In this study, we sequenced the complete mitochondrial (mt) genome of R. johnsi using the Sanger method. The circular mt genome was 17,873 bp in length and contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosome RNA genes, and one control region. The overall nucleotide composition in majority-strand was 28% A, 29% T, 29% C, and 14% G. We discussed the phylogenetic relationship of R. johnsi in genus Rana using ML and BI analyses based on 13 PCGs. Excluding the clade of subgenus Lithobates, Rana draytonii was the basal clade to all other Rana species, which included R. johnsi as the basal clade. The monophyly of genus Rana was supported, whereas Pseudorana was failed to support.

The phylogenetic relationships of Rana were involved in a heated discussion using mitochondrial (mt) genomes (Dubois 1992;Frost et al. 2006;Che et al. 2007). Rana johnsi (Smith 2009) (Anura: Ranidae), called as John's groove-toed frog (Fei et al. 1999), was distributed in China, Vietnam, Laos, Thailand, and Cambodia (Frost 2021). Whereas R. johnsi was belonging to genus Pseudorana according to the taxonomy of Fei et al. (2010). But now it is considered as genus Rana species. The mitogenome of R. johnsi has not been reported to date. So, in this study, we sequenced and analyzed the mt genome of R. johnsi to discuss its phylogenetic relationship.
The frog sample (No. GXJX20180723-1) collected from Jinxiu, Guangxi Province, China (24.14 N,110.18 E) was identified by JY Zhang and stored at À40 C in the Animal Specimen Museum, College of Life Sciences and Chemistry, Zhejiang Normal University, China. Total genomic DNA (No. YNGZW0723) was extracted from leg muscle using an Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech Company, Shanghai, China) and stored in the Zhang's lab (http://sky.zjnu.edu.cn/2019/0319/c4853a284409/ page.htm, DN Yu, email: ydn@zjnu.cn). The mt genome was amplified by polymerase chain reaction (PCR) with universal primers according to Zhang et al. (2013). The specific primers were designed based on the amplified fragments by Primer Premier version 5.0 (Primer Biosoft International, Palo Alto, CA) to amplify the remaining gaps between sequences. The obtained whole mt genome was deposited in the NCBI with accession number MZ571365.
To explore the phylogenetic relationship of R. johnsi, a total of 28 mitogenomes were analyzed, including 23 mt genomes of Rana downloaded from NCBI (Lin et al. 2014;Ni et al. 2016;Li et al. 2016aLi et al. , 2016bLiu et al. 2017;Chen 2018;Yang et al. 2018;Huang et al. 2019;Jiang et al. 2020;Wang et al. 2020;Suk et al. 2021;Xiong et al. 2021) and four mt genomes of Odorrana schmackeri, O. livida, Amolops hongkongensis, and A. wuyiensis (Zhang et al. 2018) as outgroups (Figure 1). To align the 13 PCGs, we used Clustal W in Mega version 7.0 (Kumar et al. 2016). The optimal partitions and best-fitting models (GTR þ I þ G) were generated by PartitionFinder version 1.1.1 (Lanfear et al. 2012) based on the Bayesian information criterion (BIC) (Schwarz 1978). The phylogenetic relationship was constructed based on 13 PCGs of the 28 species using Bayesian inference (BI) and maximum-likelihood (ML) methods via MrBayes version 3.1.2 (Huelsenbeck and Ronquist 2001) and RAxML version 8.2.0 (Stamatakis 2006), respectively. The BI and ML trees were showed the same topology ( Figure 1). In BI and ML trees, subgenus Lithobates formed the basal clade within all other Rana species (Fouquette and Dubois 2014). Excluding the clade of subgenus Lithobates, Rana draytonii was the basal clade to other Rana species, which included the clade of R. johnsi as the basal clade. The species of R. sangzhiensis and R. johnsi were belonging to the genus Pseudorana (Dubois 1992), but both of them were located in different clades of Rana in this study. R. sangzhiensis had a sister relationship with the clade of R. zhenhaiensis, R. dabieshanensis, and R. omeimontis. In this study, the monophyly of Pseudorana was not supported. The removal of Pseudorana was also supported by the studies of Che et al. (2007) and Pyron and Wiens (2011). The phylogenetic relationships of the other Rana species were similar to the results of Chen (2018), Wang et al. (2020), and Suk et al. (2021). According to the importance of mt taxonomy and phylogenetic inference, our study provided a correct phylogenetic relationship of R. johnsi. The new mt genomes of Rana can give us a further understanding of phylogenetic relationships within Rana.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by the College Students' Innovation and Entrepreneurship Project of China (201910345030 and 202010345026) and the College Students' Innovation and Entrepreneurship Project of Zhejiang Province (202010345R128). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI (https://www.ncbi.nlm.nih.gov/nuccore/MZ571365) under the accession no. MZ571365.