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Molecular Phylogenetic Status of Rhacophorus laoshan and Zhangixalus yinggelingensis (Anura: Rhacophoridae) from China


Molecular Phylogenetic Status of Rhacophorus laoshan and Zhangixalus yinggelingensis (Anura: Rhacophoridae) from China

Limei Yuan1, Kong Yang1* and Dechun Jiang2*

1Institute of Qinghai-Tibetan Plateau, Southwest Minzu University, Chengdu, Sichuan, P.R. China.

2Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan, P.R. China.


Recently, Rhacophorus sensu lato was divided into three genera. In addition, Rhacophorus laoshan and Zhangixalus yinggelingensis were placed in the genus Rhacophorus sensu stricto and Zhangixalus, respectively, only based on morphological characters. The research on the genus Rhacophorus sensu lato faces major challenges due to their complex interspecies relationships, and the phylogenetic status of many species are still unclear, which hampered the taxonomy and protection of these species. In this study, we investigated the molecular phylogenetic status of R. laoshan and Z. yinggelingensis using mitochondrial DNA (12S rRNA, tRNAVal, 16S rRNA and Cyt b) fragments and nuclear DNA (RAG-1, RHOD, TYR) fragments. Our results revealed that Rhacophorus laoshan is closely related to R. verrucopus, R. orlovi and R. calcaneus, and Zhangixalus yinggelingensis should indeed be placed under Zhangixalus. This research clarified the phylogenetic positions of two species within the genus Rhacophorus sensu lato, and it also has an impact on the protection and biogeographic analysis of these species.

Article Information

Received 11 June 2020

Revised 27 September 2020

Accepted 10 November 2020

Available online 30 December 2021

(early access)

Published 15 July 2022

Authors’ Contribution

DCJ and KY conceived the study. LMY performed the analysis and prepared the initial manuscript draft. DCJ approved the final version of the manuscript.

Key words

Rhacophorus, Zhangixalus, Tree frog


* Corresponding author:,

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Old world tree frogs of the family Rhacophoridae include 428 species that are widely distributed across Subsaharan Africa and southern Asia (from Sri Lanka, Nepal, and India, to Japan, the Philippines and Sulwesi) (Frost, 2020). The genus Rhacophorus sensu lato is one of the most diverse genera of the family Rhacophoridae, which contains 94 species and is widely distributed across China, Japan, and India, and from the Philippines to Sulawesi (Frost, 2020).

Many new species of the genus Rhacophorus sensu lato have been discovered recently (Dehling, 2015; Hamidy and Kurniati, 2015; Kropachev et al., 2019; Matsui et al., 2013; Nguyen et al., 2017; Pan et al., 2017b; Streicher et al., 2014; Yu et al., 2019), and these studies have made valuable taxonomic contributions of the genus Rhacophorus sensu lato. In addition, in the last ten years, there have been many reports that have inferred the phylogenetic relationships within the genus Rhacophorus sensu lato (Chen et al., 2019; Li et al., 2008, 2009, 2012a,b, 2013; O’Connell et al., 2018a,b; Pan et al., 2017a; Yu et al., 2008). However, the molecular data of many species of Rhacophorus sensu lato are still poorly known and not publicly available, such as these cryptic species R. laoshan and Z. yinggelingensis from southern China. A brown tree frog species, R. laoshan, which was first described in Mo et al. (2008) based on seven adult male specimens collected from Cenwanglaoshan Nature Reserve, Guangxi, China. This frog can be distinguished from all other Asian Rhacophorus Kuhl and van Hasselt, 1822 by the combination of: skin brown and smooth; Y-shaped cartilage visible dorsally on tips of fingers and toes; outer fingers one-third webbed; distinct dermal ridges present on forearms, above vent, and calcars present on heels; anterior and posterior surface of thighs tangerine in color without distinct dark or light spots; tympanum distinct and large, about 6.6% of SVL; dorsum brown with wide dark cross-shaped mark (Mo et al., 2008). To date, R. laoshan is still known from type series only, the validity of this species is based on morphological evidence only (Mo et al., 2008). Similarly, another species, Z. yinggelingensis, was first described in Chou et al. (2007), and it is known from the type locality in the Yinggeling Mountains of Hainan, China (Frost, 2020). It can be distinguished from other species by the following combination of characteristics: snout rounded, without protruding process; green dorsum with few fine white spots, dorsolateral folds absent; skin on dorsum smooth, flanks and venter granulate; metatarsal skinfolds faint; fingers and toes webbed; front of thigh yellow and red-tinged, rear of thigh and inner side of tibia red; white-tipped tuberculous dermal ridge on cloaca present, not well developped; iris silvery, partly light golden in upper half (Chou et al., 2007). In addition, no further specimens or new distribution record has been reported for these two poorly known tree frogs, to the best of our knowledge (Fei et al., 2009, 2012; Frost, 2020).

Recently, Jiang et al. (2019) divided Rhacophorus sensu lato into three genera: genus Rhacophorus sensu stricto Kuhl and Van Hassalt, 1822, genus Leptomantis Peters, 1867, and a new genus erected, namely Zhangixalus Jiang et al., 2019. Furthermore, R. laoshan and Z. yinggelingensis were placed in the genus Rhacophorus sensu stricto and Zhangixalus, respectively, only based on morphological characters (Jiang et al., 2019). Due to the lack of molecular data for the two species, their phylogenetic status remains unclear. Previous studies suggested that it is essential to clarify species’ phylogenetic status using molecular data. We thus conducted molecular phylogenetic analyses of the two Rhacophorus sensu lato species to clarify their taxonomic status.


Ethical statement

This study was performed in accordance with the approval of Experimental Animal Ethics Committee of Chengdu Institute of Biology, Chinese Academy of Sciences.

Taxon sampling and data collection

Two specimens were collected in this study, including one specimen of Z. yinggelingensis (HN2018002), which was collected in Hainan, China on 2018 (19°3’3.84N, 109°32’20.60″E) at 1,569 m altitude (Fig. 1), and one specimen of R. laoshan (1705014), which was collected from Cenwanglaoshan Nature Reserve (Guangxi, China) (Fig. 1). After euthanization, liver tissues were preserved in 95% ethanol for DNA extraction. Except for the two individuals, sequences of 39 individuals were collected from GenBank in this study, including sequences for eight species of Rhacophorus sensu stricto, one species of Leptomantis, and 18 species of Zhangixalus as ingroups and two species of Polypedates as outgroups according to previous studies (Jiang et al., 2019; Li et al., 2008, 2009). All novel sequences obtained in this study were deposited in GenBank (Accession No. are shown in Supplementary Table S1).

DNA extraction, amplification, and sequencing

Genomic DNA of R. laoshan was extracted from ethanol-preserved liver tissue samples using an Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech, China), according to the protocols of the manufacturer. A total of 1890 bp of the 12S rRNA, tRNAVal, and 16S rRNA was targeted and amplified using the primers that are listed in Table I. Polymerase chain reaction (PCR) were performed at 25 μL volume, including 12.5 µl Taq PCR Master Mix (2 X blue dye), 9.5 µl ddH2O, 1µl FS01, 1µl Rend (C=10µl/L), and 1 µl DNA template. PCR cycles consisted of initial denaturation at 95°C for 3 min followed by 35 cycles: denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 1 min. A final elongation step of 10 min at 72°C was performed.


Table I. Primers used in this study.


Primer sequence




Wilkinson et al., 2002



Wilkinson et al., 2002



Wilkinson et al., 2002



Yang, 2018



Yang, 2018


In order to conduct further research, we used high-throughput sequencing technology to extract genes of Z. yinggelingensis. A total of 0.88 μg DNA extracted from Z. yinggelingensis muscle tissue was sheared into short fragments (<500bp) using the Covaris system to construct pair-end DNA libraries. DNA libraries were constructed and sequenced using Illumina-Hi-Seq 2500. The sample was sequenced to a target 10×depth (30Gbp) of its genome. Reads containing more low-quality bases (phred score <30) than 20% were filtered out. Gene fragments of other species in this study were aligned using MEGA 7.0 (Kumar et al., 2016) and adjusted manually. Then, we used the MEANGS ( to get the purpose genes. The process are as follows: The alignments of genes that aligned using MEGA 7.0 were used in construction profile HMM(s) with hmmer-build in HMMER V3.16 (Eddy, 2008). Hereafter, nhmmer were used to search reads that fitting the hmm profiler for each gene from high-throughout sequencing data. Finally, we assembled these matched reads with the de novo assembler SSAKE v2.2.1 (Warren et al., 2007), and the assembled scaffolds were identified by blastting to the purpose DNA fragments, including four mitochondrial fragments, 12S rRNA, tRNAVal, 16S rRNA, Cytochrome b (Cyt b), and three nuclear genes, Recombination activating gene 1 (RAG-1), Exon 1 of tyrosinase (RHOD) and exon 1 of tyrosinase (TYR). In order to evaluate the accuracy of the assembled genes of Z. yinggelingensis, we mapped all reads back to the assembled genes, and statistic the mapping quality and average depth of these genes.

Phylogenetic analyses

Phylogenetic trees were reconstructed by only mitochondrial fragments (12S rRNA, tRNAVal, 16S rRNA) and combined mitochondrial and nuclear fragments (12S rRNA, tRNAVal, 16S rRNA, Cyt b, RAG-1, RHOD, TYR), respectively. Sequences were aligned using MEGA 7.0 (Kumar et al., 2016) with default settings and rechecked in MAFFT version 7 (Katoh and Standley, 2013), then adjusted manually. Different gene fragments of each individual were concatenated by using Phylosuite (Zhang et al., 2020), and positions of indels were treated as missing data for all datasets. The most optimum substitution model of evolution (GTR+I+G for mitochondrial fragments, HKY+I for RAG-1 gene and RHOD gene, and SYM+G for TYR gene) was calculated by Partition Finder v2.1.1 (Lanfear et al., 2017) under Akaike Information Criterion (AIC) (lnL= -18812.57, AICc= 37858.23). We then reconstructed phylogenetic trees using the Bayesian inference (BI) and maximum likelihood (ML) methods, respectively.

The Bayesian phylogenetic tree was constructed using MrBayes 3.2.0 (Ronquist et al., 2012). Runs of Markov chains for 5,000,000 generations were summarized and sampled every 100 generations. The first 25% of samples as burn-in, and convergence was investigated using the parameter of average standard deviation of split frequencies ≤ 0.01 (Ronquist et al., 2012). Nodes were considered strongly supported when BPP ≥ 0.95. Software RAxML v8.2.10 (Stamatakis, 2014) was used to implement Maximum Likelihood (ML) analysis under the best-fit model of evolution (GTRGAMMA) based on the AIC criterion. And bootstrap proportions (BSP) were evaluated with 1000 nonparametric bootstrap pseudoreplications, and it were considered significantly supported when the node’s BSP ≥ 70. In addition, uncorrected pairwise distance (p-distances) of the four mitochondrial fragments (12S rRNA, tRNAVal, 16S rRNA and Cyt b) and three nuclear genes (RAG-1, RHOD and TYR) dataset among species in Rhacophorus sensu lato were calculated using MEGA 7 (Kumar et al., 2016), with the “P-distance” model and “Transitions + Transversions” substitutions to include.


The mapping average depth of 12S rRNA, tRNAVal, and 16S rRNA, Cyt-b, RAG-1, RHOD, and TYR for the Z. yinggelingensis is 3331, 2315, 13.95, 5.847, 9.681, respectively. Simultaneously, the mitochondrial fragments consisted of 1890 bp, and the combined mitochondrial and nuclear fragments consisted of 3746 bp, including 1890 bp for 12S rRNA, tRNAVal, and 16S rRNA, 546 bp for Cyt-b, 507 bp for RAG-1, 279 bp for RHOD, 524 bp for TYR. The present molecular phylogeny is concordant with previous molecular studies regarding the monophyly of Rhacophorus sensu lato (Chan et al., 2018, 2019; Li et al., 2008, 2009, 2012a, 2013; O’Connell et al., 2018b; Pan et al., 2017a). Both mitochondrial fragments (Supplementary Fig. S1) and combined mitochondrial and nuclear fragments (Fig. 2 and Supplementary Fig. S2) resulted in virtually identical topology. Although interspecific relationships were not fully resolved, the phylogenetic trees from both the BI and ML methods showed that all species contained in Rhacophorus sensu lato were clustered to three clades as monophyletic groups and each clade consisted of the same species. According to phylogenetic trees from combined mitochondrial and nuclear fragments, clade A was congruent with genus Zhangixalus and was strongly supported by a bootstrap proportion of 83 and a posterior probability of 0.96. Clade B was congruent with genus Leptomantis and it was sister group to Zhangixalus that supported by a bootstrap proportion of 80 and a posterior probability of 0.96, which was consistent with Jiang et al. (2019), but was different from Chen et al. (2019). Chen et al. (2019) indicated that Leptomantis was sister group to Rhacophorus sensu stricto. They used anchored hybrid enrichment (AHE) targeted sequencing and their sequences were long larger than this study. Clade C was congruent with genus Rhacophorus sensu stricto and supported by a bootstrap proportion of 100 and a posterior probability of 1.00.

The phylogeny (Fig. 2 and Supplementary Fig. S2) depicted that R. laoshan belongs to Rhacophorus sensu stricto and Z. yinggelingensis was nested within Zhangixalus. Jiang et al. (2019) propose the morphological diagnosis and difference of each genus of Rhacophorus sensu lato. They shed light on Zhangixalus differs from Rhacophorus sensu stricto by the absence of dermal folds along limbs and tarsal projections (vs. present), the absence of supracloacal fold (vs. present or not). And we found that R. laoshan and Z. yinggeligensis corresponds with the morphological diagnosis of Rhacophorus sensu stricto and Zhangixalus, respectively. On the one hand, the morphological characteristics of R. laoshan similar to the characteristics diagnosis of Rhacophorus sensu lato in the presence of dermal folds along limbs (Mo et al., 2008; Jiang et al., 2019). On the other hand, the morphological characteristics of Z. yinggelingensis similar to the following combination of characteristics diagnosis of Zhangixalus: Dermal folds along limbs absent; tarsal projections absent; body size relatively moderate or large, SVL 30–120 mm (mostly above 50 mm); dorsal coloration mostly green (Chou et al., 2007; Jiang et al., 2019). Consequently, based on our molecular results and published morphological data, we agree with Jiang et al. (2019), place R. laoshan and Z. yinggelingensis into the genera Rhacophorus and Zhangixalus, respectively.

Furthermore, in the clade Rhacophorus sensu stricto, R. laoshan was the sister-taxon to the assembly of three species (BPP = 1.00; BSP = 99), including R. verrucopus (6254 RAO), R. orlovi (AMNH A161405) and R. calcaneus (FMNH 256465; Fig. 2). However, we cannot confirm the sister species of R. laoshan, and it may be related to R. calcaneus, which was recovered as polyphyletic and formed two distinct clades. Specifically, between the two R. calcaneus clades, one taxon (FMNH 256465, from Laos) was sister to R. orlovi (AMNH A161405; BPP = 1.00; BSP = 100) possessed a genetic divergence of 0.2% (Supplementary Table S2). Additionally, the second R. calcaneus (AMNH A163749, from Vietnam) and R. robertingeri (VNMN:4123, from Vietnam) formed a well-supported clade with short branch length (BPP = 1.00; BSP = 100), and this clade possessed a genetic divergence of 0.3% (Supplementary Table S2). However, on the one hand, the localities of the species R. calcaneus were certainty only from three places (Frost, 2020), and the localities of the two specimens of R. calcaneus (FMNH 256465, from Laos; AMNH A163749, from Quang Nam, Vietnam) didn’t fall within the known distribution sites of R. calcaneus. On the other hand, some species are poorly diagnosed yet and characters highly convergent in previous studies (David et al., 2007, 2015a; Guo et al., 2014). Consequently, we suspect that the two specimens (FMNH 256465, AMNH A163749) of R. calcaneus were misidentifications. Due to we do not have the two specimens, it is need to confirm whether the two specimens were misidentified in future study. Moreover, it may be that some sequences of the species R. calcaneus from the NCBI have problems, because taxonomic identification problems of existing Genbank sequences are very common (Guo et al., 2014; Ren et al., 2018).


For Z. yinggelingensis, the p-distances varied from 3.0% to 7.0% between it and other species of Zhangixalus that collected in this study [minimum distance to Z. duboisi; maximum distance to Z. achantharhena (Supplementary Table S2)]. Additionally, Chou et al. (2007) suggested that Z. yinggelingensis seems to be closely related to Z. arvalis, Z. aurantiventris, Z. chenfui, Z. dorsoviridis, Z. hungfuensis, Z. taipeianus, and Z. yaoshanensis, Z. moltrechti, Z. nigropunctatus on the basis of morphological evidences. However, there are many morphologically similar and conservative species in Zhangixalus (Li et al., 2012), and we can’t distinguish them well. Thus, we suggest that it not only use morphological and molecular datasets, but also their distributions when we identified the species, which can reduce the probability of misidentifications.

Although phylogenetic status of Z. yinggelingensis is not resolved well, we recognize the validity of it using the molecular data for the first time. However, why phylogenetic status of Z. yinggelingensis is not resolved well? The possible reasons are as follows: (1) The sequence of Z. yinggelingensis in this study is short; (2) There are only some species of Zhangixalus in this study; (3) the genus Zhangixalus evolution history is complex. As genomic data have become more common in phylogenetic studies, many studies have been shown that it can use genomic data resolving both shallow and deep-scale evolutionary relationships (Brandrud et al., 2019; Burbrink and Gehara, 2018; Chen et al., 2019; Godefroid et al., 2019; Kuntner et al., 2019; Spriggs et al., 2019). Thus, it seems possible that we can use genomic data to resolve the phylogenetic status of Z. yinggelinensis in future study. In addition, the research on amphibians in Southern China was scarce, and there have been some cryptic species were discovered in the past three years (Liu et al., 2017; Pan et al., 2017b; Yu et al., 2019). Finally, we suggest that we can strengthen the investigation of amphibian diversity in southern China, which lay a foundation for the study of biogeography in southern China.


In this study, we have clarified the phylogenetic status of R. laoshan and Z. yinggelingensis using mitochondrial fragments (12S rRNA, tRNAVal, 16S rRNA and Cyt b) and nuclear DNA (RAG-1, RHOD, TYR) fragments. Our results suggest that R. laoshan and Z. yinggelingensis belongs to Rhacophorus sensu stricto and Zhangixalus, respectively. Finally, this research play an important role in the further studies, such as protection, biogeographical patterns and the evolution of life history strategies of these species.


This work was supported by the National Natural Science Foundation of China (31900322; 32070410); Sichuan Science and Technology Bureau (2020YFH0005); and Innovative Research Project for Postgraduates of Southwest Minzu university (CX2019SZ89).

Supplementary material

There is supplementary material associated with this article. Access the material online at:

Statement of conflict of interest

The authors have declared no conflict of interest.


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Pakistan Journal of Zoology


Vol. 54, Iss. 5, Pages 2003-2500


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