The mitochondrial genome and phylogenetic analysis of Rhacophorus rhodopus

Abstract

Classification of the genus Rhacophorus has been problematic. In particular there has been considerable controversy surrounding the phylogenetic relationships among Rhacophorus rhodopus, R. bipunctatus, and R. reinwardtii. To examine the relationship among these Rhacophorus species, we assembled the complete mitochondrial genome sequence of R. rhodopus. The R. rhodopus genome is 15,789 bp in length with 12 protein-coding genes (PCGs) (losing ND5), two ribosomal genes, 22 transfer RNA genes, and a control region (D-loop). Base composition of the overall sequence was 60.86% for A + T content and 39.14% for C + G content. Most of the PCGs used ATG as a start codon, except for the COX I gene, which used the ATA start codon. COX I and ND6 used AGG and ATP8 stop codons respectively, while ND3 and ND4L used the TAA stop codon. For the remaining seven genes, the stop codons was incomplete. In addition, both 5' and 3' of the control areas had distinct repeating regions. Based on three datasets and two methods (Bayesian inference (BI) and maximum likelihood (ML)), we reconstructed three phylogenetic trees to explore the taxonomic status of the species and the phylogenetic relationship among R. rhodopus, R. bipunctatus and R. reinwardtii. Our results indicated that these three species are non-monophyletic; thus, the phylogenetic relationship among them is complex and difficult to determine. Further, R. rhodopus is divided into three lineages from different parts of China. The two Rhacophorus samples showed very close phylogenetic relationship with R. rhodopus. Our results add to the mitochondrial genome database of amphibians and will help to disentangle the phylogenetic relationships within the Rhacophoridae.

Figure 1

Rhacophorus species collected from Motuo County in China.

Figure 2

The complete mitochondrial genome sequence of Rhacophorus rhodopus collected from Motuo County in China.

Figure 3

Putative tRNA secondary structures found mitochondrial genome of Rhacophorus rhodopus.

Figure 4

Structures and alignments of identified putative termination-associated sequences (TAS) and conserved sequence blocks (CSB1-3). Alignment gaps and nucleotides identical to the first line are indicated by a dot (·), respectively. Variable nucleotides are marked with corresponding nucleotides.

Figure 5

BI and ML analysis of 11 species complete mitochondrial genome sequence, Bufo gargarizans and Bufo tibetanus as outgroups. Tree topologies produced by BI and ML analyses were equivalent. Bayesian posterior probability (PP) and bootstrap support (BP) values for ML analyses are shown in order on the nodes.

Figure 6

BI and ML analysis of 102 species based on 12S + 16S + CYTB genes sequence. Bufo gargarizans and Bufo tibetanus as outgroups. Tree topologies produced by BI and ML analyses were equivalent. Bayesian posterior probability (PP) and bootstrap support (BP) values for ML analyses are shown in order on the nodes.

Figure 7

BI and ML analysis of Rhacophorus rhodopus, Rhacophorus bipunctatus and Rhacophorus reinwardtii, based on 12S and 16S. Polypedates colletti and Polypedates cruciger as outgroups. Tree topologies produced by BI and ML analyses were equivalent. Bayesian posterior probability (PP) and bootstrap support (BP) values for ML analyses are shown in order on the nodes.