The Complete Mitochondrial Genome of Rhacophorus dennysi (Anura: Rhacophoridae) with Novel Gene Arrangements and its Phylogenetic Implications

Yongmin Li1,2, Huabin Zhang1, Xiaoyou Wu1, Dongwei Li 2, Peng Yan1 and Xiaobing Wu1* 1Anhui Province Key Laboratory for Conservation and Exploitation of Biological Resource, College of Life Sciences, Anhui Normal University, Wuhu, Anhui, China 2College of Biology and Food Engineering, Fuyang Normal University, Fuyang, Anhui, China Article Information Received 01 September 2019 Revised 01 May 2020 Accepted 19 June 2020 Available online 14 October 2020


O n l i n e F i r s t A r t i c l e
mtDNA is an important molecular marker and has been widely used in the studies of genetics, phylogenetics and phylogeography. In the present study, we determined the complete nucleotide sequence of the mitochondrial genome of Rhacophorus dennysi. We performed phylogenetic analyses based on complete mt genomes of the newly sequenced and other reported species of Ranoidea to assess the taxonomic position of Rhacophorus dennysi, and to test the phylogenetic relationship of Rhacophoridae and Ranidae.

Sample collection and PCR
The R. dennysi sample was collected from Qifeng, Guniujiang, Anhui province in China. This frog sample used was stored at -40°C (Sample No. AM12026) in the Conservation Biology Laboratory, College of Life Sciences of Anhui Normal University. Total DNA was extracted from a piece of muscle tissue by proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation (Sambrook et al., 2001).
To determine the complete mitochondrial genomic sequence of R. dennysi, polymerase chain reaction (PCR) was carried out with the primers for the mtDNAs of frogs described in the literatures (Kurabayashi and Sumida, 2009;Zhang et al., 2013). Furthermore, based on the complete mtDNA sequences of R. schlegelii (AB202078) and P. megacephalus (AY458598), we also designed two pairs of primers to amplify mt fragments from the Cytb gene to the ND5 gene. PCR reaction volume of 30 μl contained 21µl sterile double distilled water, 3 µl 10× reaction buffer (with Mg 2+ ), 2.5 µl (2.5 mmol/l) dNTPs, 1 µl each primer (10µmol/l), 0.5µl Taq DNA polymerase (TaKaRa Bio Inc., Otsu, Shiga, Japan) and 1 µl template DNA. Amplification was performed using Applied Biosystems 2720 Thermal Cycler with the following conditions: initial denaturation at 94°C for 4 min, 32 cycles of denaturation at 94°C for 40 s, annealing at 52-58°C for 40 s and elongation at 72°C for 60 s, and a final extension at 72 °C for 10 min. The resulting PCR fragments were separated by electrophoresis in 1.0% agarose gels, then PCR products were purified using TIANquick Midi Purification Kit (TIANGEN Bio Inc., Beijing, China), and then directly sequenced on an automated sequencer (ABI 3730) from both strands.

Sequence assembly and analysis
Nucleotide sequences were checked and assembled using the program SeqMan (DNASTAR Inc., Madison, WI, USA). The 13 protein-coding and two rRNA genes were annotated by comparison with the known complete mtDNA sequences of Rhacophorus schlegelii (Sano et al., 2005), Polypedates megacephalus (Zhang et al., 2005) and Buergeria buergeri (Sano et al., 2004). The 22 tRNA genes were identified by their cloverleaf secondary structure and anticodon sequences using tRNA Scan-SE v.2.0.2 (http://lowelab.ucsc.edu/tRNAscan-SE; Lowe and Chan, 2016). The complete mtDNA sequence of R. dennysi was deposited in GenBank with the accession number KM035412.

Phylogenetic reconstruction
In order to address the phylogenetic relationships among Rhacophoridae, 3 additional, previously published Rhacophoridae mitogenomes were included in the analysis. In addition, mitochondrial genomes from one species in the family Mantellidae, twenty-two species in Ranidae, and thirteen species in Dicroglossidae were retrieved from GenBank to further confirm the phylogenetic position of the family Rhacophoridae among Ranoidea. Additionally, three Microhylidae species were used as the outgroups based on Pyron and Wiens (2011) (Table I).
We constructed the phylogenies using the concatenated 12 mt protein-coding genes and partitioned these genes by codon position. The best fitted substitution model for each partition was estimated using Akaike Information Criterion (AIC) implemented in jModeltest v.2.1.7 (Darriba et al., 2012). The GTR+I+G model was chosen for ML and Bayesian inference (BI) analyses, which were separately performed using RaxML (Kozlov et al., 2019) with 1000 bootstrap replications and MrBayes v.3.2.7 (Ronquist et al., 2012). Besides, the following settings were applied in the BI analysis: 10 million Markov chain Monte Carlo (MCMC) generations, a sampling frequency of 1000, burn-in = 1000.

Genome organization of R. dennysi mtDNA
The R. dennysi mt genome was 18,052 bp in length, containing 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region (Table II). The base composition of the light strand (L-strand) was 31.5.9% A, 31.0% T, 23.2% C, and 14.3% G, which is similar to other vertebrates (Zhang et al., 2015;Li et al., 2016).

O n l i n e F i r s t A r t i c l e
Remarkably, the tree frog R. dennysi possessed a novel mitogenomic gene organization much different from other neobatrachians. In the R. dennysi mt genome, the ND5 gene between tRNA Ser (AGY) and ND6 was translocated to a position between the CR and tRNA Thr . In this new mitogenome, four tRNA genes (tRNA Thr , tRNA Leu(CUN) , tRNA Pro and tRNA Phe ) Formed a TLPF tRNA cluster, different from the neobatrachian-type arrangement (Sumida et al., 2001;Irisarri et al., 2012;Li et al., 2014).
Among the 13 protein-encoding genes in the R. dennysi mitogenome, most of these protein-coding genes started with the common initiation codon ATG except two genes (ND1 and COI) beginning with ATA, one gene (ND2) beginning with ATT. Stop codons were variable for all protein-coding genes. Seven protein genes (ND2, COII, ATP8, ND4L, ND5, ND6 and Cytb) used complete stop codon TAR, and COI ended with AGG, whereas other genes (ND1, ATP6, COIII, ND3 and ND4) ended with incomplete stop codon T.
The noncoding regions in the R. dennysi mtDNA contained the control region and some spacers. The control region was located between the Cytb and ND5 genes with the length of 2,603 bp. The length of the CR in this study is obviously longer than that of R. Dennysi (2,122 bp) in the literature of Huang et al. (2016).

Phylogenetic analyses
The BI and ML analyses of the molecular dataset produced the identical topologies and very similar branch support (Fig. 1). In the phylogeny of Rhacophoridae, Ranidae, Dicroglossidae and Mantellidae, the monophyly of Dicroglossidae, Ranidae and Rhacophoridae are well supported. Fig. 1. The phylogeny of Ranidae sensu lato (Dubois, 2005) inferred from the combined sequences of 12 protein-coding and two rRNA genes. The Bayesian tree was shown here, the ML had an identical tree topology. Numbers of nodes were support values from ML (bootstrap proportions; left) and BI (posterior probabilities; right).

Gene rearrangement and the significance for the phylogeny
In present study, we discovered a novel gene arrangement of R. dennysi mt genome. The ND5 gene and four tRNA genes (tRNA Thr , tRNA Leu(CUN) , tRNA Pro and tRNA Phe forming TLPF tRNA cluster) were located between the CR and the 12S rRNA gene, which differed from the neobatrachian-type arrangement (LTPF tRNA cluster) but shared similarities to those of R. schlegelii (Sano et al., 2005) and P. megacephalus (Zhang et al., 2005), two other species from the same family. Gene rearrangement in animal mtDNA is generally believed to take place through the Tandem Duplication and Random Loss (TDRL) model (San Mauro et al., 2006). According to the TDRL model, a multigene portion of the genome is duplicated, then one copy becomes nonfunctional and is subsequently deleted from the genome.

Phylogenetic analyses of Rhacophoridae
In our phylogenetic trees, two species of the genus Rhacophorus (R. schlegelii and R. dennysi) were clustered together with the representative of the genus Polypedates (P. megacephalus), indicating a very close phylogenetic relationship, meanwhile, the representative of the genus Buergeria (B. buergeri) occupied the basal position in the clade of Rhacophoridae. The phylogenetic relationship among the representatives of the family Rhacophoridae revealed here is congruent with the results of most phylogenetic analyses (Yu et al., 2009;Pyron and Wiens, 2011;Zhang et al., 2013Zhang et al., , 2018Chen et al., 2017;Chan et al., 2018).

CONCLUSIONS
As a whole, this study provides some evidence for the generic classification of Rhacophoridae proposed by Pyron and Wiens (2011), and our phylogenetic analyses supported the sister-group relationship between ((Rhacophoridae + Mantellidae) + Ranidae) and Dicroglossidae.

O n l i n e F i r s t A r t i c l e O n l i n e F i r s t A r t i c l e
Complete Mitochondrial Genome of Rhacophorus dennysi