a new species of the genus rhacophorus (anura ......kit (transgene) was used to purify each dna...

Asian Herpetological Research 2017, 8(1): 1–13DOI: 10.16373/j.cnki.ahr.160064

1. Introduction

Currently, the ranoid treefrog genus Rhacophorus contains 88 species, the largest genus in the Rhacophoridae, which are widely distributed across India, China, Japan, mainland South-east Asia, the Greater Sunda Islands and Philippines (Frost, 2016). The most obvious characteristics of the species of the genus Rhacophorus are the presence of intercalary cartilage between terminal and penultimate phalanges of digits, terminal phalanges of fingers and toes Y-shaped, tips of the digits expanded into large disks bearing circum-marginal grooves, webbed fingers, horizontal pupil, and the skin not co-ossified to

A New Species of the Genus Rhacophorus (Anura: Rhacophoridae)from Dabie Mountains in East China

Tao PAN1#, Yanan ZHANG1#, Hui WANG1, Jun WU2, Xing KANG1, Lifu QIAN1,4, Kai LI1, Yu ZHANG1, Jinyun CHEN3, Dingqi RAO4, Jianping JIANG5 and Baowei ZHANG1,6*

# Both authors contributed equally to this paper.* Corresponding author: Prof. Baowei ZHANG, from the College of Life Sciences, Anhui University, Hefei, China, with his research focusing onmolecular ecology of amphibians.E-mail: [email protected]: 5 December 2016 Accepted: 1 March 2017

Abstract A new species of rhacophorid of the genus Rhacophorus is described from the Dabie Mountains of west Anhui, east China. The new species, Rhacophorus zhoukaiyae sp. nov. is distinguished from its congeners by a combination of the following characters: 1) the ventral surface and front-and-rear of the femur is paler yellowish and decorated with irregular grayish blotching, and without obvious spots on the dorsum of the hand and foot webbing; 2) the outer metatarsal tubercle is small; 3) outer fingers are half-webbed and outer toes two third webbed; 4) the skin on the dorsum is smooth and without compressed warts; 5) the throat, chest and belly are pure paler yellowish; 6) the dorsal part of the fingers and toes are grayish-white; 7) the iris is golden-yellow. In addition, the phylogenetic tree showed that all the individuals of R. zhoukaiyae sp. nov. clustered into one distinct clade which suggested the validity of this species. This results could also be used to the support of species delimitation. Currently, this species is known only from mid-elevation montane evergreen forest in the Dabie Mountains of west Anhui, China.

skull (Brown and Alcala, 1994; Duellman and Trueb, 1986; Hamidy and Kurniati, 2015; Inger and Stuebing, 1997; Liem, 1970; Malkmus et al., 2002). Generally, species in Rhacophorus have stunning coloration and distinct morphology, and interesting breeding behavior (Davis, 1965; Harvey et al., 2002). Therefore, previous studies on the taxonomic classifications and species identities of ranoid frogs were focused on overall morphological similarities and differences (Dubois, 1992; Fei et al., 2005; Liem, 1969; Rao et al., 2006). However, another opinion suggested that the morphological characters were unstable because of extensive homoplasy in both larvae and adults (Bossuyt and Milinkovitch, 2000; Li et al., 2012b; Stuart, 2008). In recent years, to fill the gaps in previous morphological research, a growing number of studies, based on molecular data, have appeared (Delorme et al., 2005; Frost et al., 2006; Li et al., 2008; Li et al., 2009; Li et al., 2013; Li et al., 2012a; Li et al., 2012b; Orlov et al., 2013; Wilkinson et al.,

Keywords Rhacophorus, Rhacophorus zhoukaiyae sp. nov., phylogeny, Rhacophoridae, Dabie Mountains

1 Anhui Key Laboratory of Eco-engineering and Bio-technique, School of Life Sciences, Anhui University, Hefei 230601, Anhui, China

2 Ministry of Environmental Protection, Nanjing Institute of Environmental Sciences, Nanjing 210042, Jiangsu, China 3 Department of Life Science, Huainan Normal University, Huainan 232001, Anhui, China 4 Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China5 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, Sichuan, China6 School of Biosciences, Cardiff University, Cardiff, CF103AX, United Kingdom

ORIGINAL ARTICLE

http://dict.cn/morphological differences

Asian Herpetological Research Vol. 82

2002; Yu et al., 2008; Yu et al., 2009). Phylogenetic reconstructions and species delimitation based on genetic data provide new opportunities to reveal cryptic species (de Luna Sales et al., 2013; Mo et al., 2016; Newman et al., 2012; Rannala and Yang, 2003; Yang and Rannala, 2010), which are indistinguishable by morphological characteristics (Li et al., 2012b; Rao et al., 2006; Stuart et al., 2006).

Dabie Mountains, southeast China, is an isolated mountain system, remote from the Tsinling Mountains and Wannan Mountains region. It stretches from east to west for over 200 km and covers an area of 50 000 km2. The mountains consist of low-mid elevation hills, lower than 1800 m a.s.l. According to the vegetation type of China, Dabie Mountains is located in the ecotone of subtropical evergreen broad-leaved forest and warm-temperate deciduous broad-leaved forest zone. The climate type of this mountain is the transitional zone from subtropical region to temperate zone, where the transitional climate characteristics are pronounced. In addition, annual average temperature in this area is 12.5°C and the mean annual precipitation is 1 832.8 mm (Fang et al., 2007). The characteristics of the Palaearctic and the Oriental region can be found in Dabie Mountains. In addition, some endemic species had been found in those mountains, like Moschus anhuiensis (Pan et al., 2015), Protobothrops dabieshanensis (Huang et al., 2012). During the fieldwork in Dabie Mountains from 2013 to 2015, we collected several specimens of unknown species of Rhacophorus. We have referred to the reference and identify them as R. nigropunctatus (Fei et al., 2012). However, based on our morphological analyses, we concluded that this unknown

species own apparent difference morphological characters with R. nigropunctatus. In addition, we got the support the status of this unknown species from genetic data. Thus, we conducted morphological and molecular genetic analyses to resolve the taxonomic status and affinities of these taxa. Based on these data, we describe those specimens as a new species from Dabie Mountains in Anhui, China.

2. Materials and Methods

2.1 Data collection and taxa sampling During 2012 and 2015, nine specimens of Rhacophorus were collected from the Dabie Mountains, China (Figure 1). They were euthanized and fixed in 10% formalin and subsequently transferred to 75% ethanol for storage. Before being fixed in formalin, muscle tissues from all individuals were sampled and preserved in 100% ethanol for DNA extraction. All specimens and tissue samples were deposited at the museum of Anhui Key Laboratory of Eco-engineering and Bio-technique, Anhui University, China.

2.2 DNA Extraction, PCR Amplification, and Sequencing Total DNA was extracted from the samples using a standard proteinase K/phenol-chloroform protocol (Sambrook et al., 1989). An EasyPure PCR Purification Kit (TransGene) was used to purify each DNA extraction. Two pairs of primers were designed for amplifying 12S and 16S ribosome RNA (rRNA) of the mtDNA genome using Primer Premier 5.0 (Clarke and Gorley, 2001) based on R. schlegelii (NC007178) and Buergeria buergeri (NC008975) (Table 1). In addition, we also amplified and

Figure 1 Sampled sites (Green stars) of R. zhoukaiyae in Dabie Mountains. The red triangle reprensent distribution map of R. dorsoviridis; The blue square reprensent distribution map of R. nigropuncatatus. The distribution of species was collected from http://research.amnh.org/vz/herpetology/amphibia/index.php/.

Tao PAN et al. A New Species of Rhacophorus from Dabie MountainsNo. 1 3

sequenced five nuclear gene fragments with the indicated primer pairs, including brain-derived neurotrophic factor (BDNF), proopiomelanocortin (POMC), recombination activating gene 1 (Rag 1), rhodopsin exon 1 (RHOD) and tyrosinase exon 1 (TYR) (Bossuyt and Milinkovitch, 2000; Li et al., 2009; van der Meijden et al., 2007; Vieites et al., 2007; Wiens et al., 2005). Polymerase chain reactions (PCR) were performed using a reaction mixture (25 μL) containing 1 μL genomic DNA (concentration 10-50 ng/μL), 2.5 μL 10 × buffer, 1 μL of 2.5 mol/L MgSO4, 2 μL of 2 mol/L dNTPs, 1 U Taq polymerase (Meridian Bioscience, Singapore) and 0.3 mol/L of each of the primers. Pure molecular biology grade water was added to reach the appropriate volume. The amplification protocol of partial 12S and 16S ribosomal genes and complete valine t-RNA included an initial denaturation step of 95°C for 5 min; this was followed by 32 cycles of denaturation at 95°C for 30 s, primer annealing at 55°C for 30 s, and an extension at 72°C for 80 s, with a final extension at 72°C for 10 min. For Rag 1 cycle conditions included an initial denaturation step of 95°C for 5 min; this was followed by 32 cycles of denaturation at 95°C for 30 s, primer annealing at 54°C for 30 s, and an extension at 72°C for 80 s, with a final extension at 72°C for 10 min. For POMC cycle conditions included an initial denaturation step of 95°C for 5 min; this was followed by 32 cycles of denaturation at 95°C for 30 s, primer annealing at 57°C for 30 s, and an extension at 72°C for 40 s, with a final extension at 72°C for 10 min. For

BDNF cycle conditions included an initial denaturation step of 95°C for 5 min; this was followed by 32 cycles of denaturation at 95°C for 30 s, primer annealing at 51°C for 30 s, and an extension at 72°C for 40 s, with a final extension at 72°C for 10 min. For RHOD and TYR cycle conditions included an initial denaturation step of 95°C for 5 min; this was followed by 32 cycles of denaturation at 95°C for 30 s, primer annealing at 56°C for 30 s, and an extension at 72°C for 40 s, with a final extension at 72°C for 10 min. PCR products were purified using an EasyPure PCR Purification Kit (TransGene), and sequenced using previous primers and the BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems) following the manufacturer’s instructions on an ABI Prism 3730 automated sequencer. All the sequences obtained in this study were deposited into GenBank (Table S1). In analysis, these sequences were trimmed to match data downloaded from GenBank, then all the sequences were aligned automatically using Clustal X version 1.83 with default parameter (Thompson et al., 1997), followed by visual confirmation and manual adjustments. Nucleotide sites with ambiguous alignments were removed from the analyses, and gaps were analyzed as missing data.

2.3 Phylogenetic Analyses For the phylogenetic analyses, sequences of 36 species of Rhacophorus were used in combination with sequences of two outgroup species, Polypedates megacephalus (Rhacophoridae, Polypedates) and Spinomantis peraccae (Mantellidae,

Table 1 Primers used in PCR and sequencing.

Locus Primer Primer sequence Size (bp) Cited source

Recombination activating gene 1 L6300 5'-CTG GTC GTC AGA TCT TTC AGC-3' 1164 Li et al. (2009)

H6301 5'-GCA AAA CGT TGA GAG TGA TAA C-3' Li et al. (2009)

Proopiomelanocortin R7120 5'-TAY TGR CCC TTY TTG TGG GCR TT-3' 601 Wiens et al. (2005)

L7121 5'-GGA RCA CTT YCG ATG GGG YAA ACC-3' Wiens et al. (2005)

Brain-derived neurotrophic factor R7151 5'-CTA TCT TCC CCT TTT AAT GGT C-3 614 Vieites et al. (2007)

L7152 5'-ACC ATC CTT TTC CTT ACT ATG G-3' Van der Meijden et al. (2007)

Exon 1 of rhodopsin L2903 5'-ACC ATG AAC GGA ACA GAA GGY CC-3' 315 Bossuyt and Milinkovitch (2000)

H2904 5'-GTA GCG AAG AAR CCT TCA AMG TA-3' Bossuyt and Milinkovitch (2000)

Exon 1 of tyrosinase L2976 5'-TGC TGG GCR TCT CTC CAR TCC CA-3' 531 Bossuyt and Milinkovitch (2000)

H2977 5'-AGG TCC TCY TRA GGA AGG AAT G-3' Bossuyt and Milinkovitch (2000)

Partial 12S and 16S ribosomal genes Rha1-F 5'-GAT ATT AGT CGG CGT TAA GCG TG-3' 2100 This study

and complete valine t-RNA Rha1-R 5'- GCT GCC TTC AGG CCC AC-3' This study

Rha2-F 5'-AAA AGA GCT AAC CCG TCT CTG T -3' This study

Rha2-R 5'- CGG TCT GAA CTC AGA TCA CG -3' This study


Spinomantis) (Li et al., 2013; Li et al., 2012b). Sequence data were obtained from GenBank (Table S1). In total, there were 36 of 89 species in Rhacophorus involved in our phylogeny study (Table S1). Two different datasets were generated for different analyses. Dataset 1, used for phylogenetic analysis of Rhacophorus by Maximum Likelihood (ML) and Bayesian methods, which was composed of the 12S and 16S rRNA genes together with the complete t-RNA for the valine sequence of the Rhacophorus species and the outgroups (Table S1). Dataset 2 contained 8 gene fragments (12S, 16S, Val, BDNF, POMC, Rag 1, RHOD, TYR) (Table S1). However, it was only used to calculate a Bayesian consensus tree. The best-fit model of evolution was calculated by MrModeltest 1.0 b under the AIC criterion (Table 2) (Nylander, 2003). ML analyses were performed in RAxML version 8 (Stamatakis, 2014) and a general time reversible model of nucleotide substitution under the Gamma model of rate heterogeneity (i.e., GTRCAT). Support for the internal branches for the best-scoring tree was evaluated via the bootstrap test with 1000 iterations. A Bayesian inference of phylogeny was performed using the MrBayes 3.1.2 software program (Huelsenbeck and Ronquist, 2005), using the best-fit substitution model. Two Markov Chain Monte Carlo (MCMC) models were runs to provide additional confirmation of the convergence of posterior probability distributions. Analyses were run for 3 000 000 generations. Chains were sampled every 1000 generations. The first 25% of the total trees were discarded as “burn-in” and the remaining trees were used to generate a majority-rule consensus tree and to calculate Bayesian posterior probabilities. In addition, the uncorrected p-distance pairwise sequences divergence among species in Rhacophorus using mtDNA gene fragment was calculated using MEGA version 6 (Tamura et al., 2013).

2.4 Species delimitation The species delimitation of the new species and relative species was implemented in BPP v. 3.0 (Rannala and Yang, 2003; Yang and Rannala, 2010) based on the reversible-jump Markov Chain Monte Carlo (rjMCMC) model sampling with the guide tree. We assessed the stability of the status of our new species by four different prior scheme (Table 3). Low values for priors generally infer large population sizes and deep divergence, whereas higher values infer small population sizes and shallow divergence for θ and τ respectively. Each analysis runs three independent chains of 500 000 steps, sampling every fifth step, with 100 000 burn-in

steps.

2.5 Morphological analysis Morphological analysis was also taken on the new species with its closely related species, R. dorsoviridis, using measurements obtained with dial calipers to the nearest 0.1 mm. The measurements and their abbreviations include: SVL (snout-vent length); HL (head length, from posterior corner of mandible to tip of snout); HW (head width, at the angle of jaws); SL (snout length, the distance from tip of the snout to the anterior end of the eye); IN (internarial distance); ED (horizontal eye diameter); IO (interorbital distance, least distance between upper eyelids); UE (maximum width of upper eyelid); HTD (horizontal tympanic diameter); LAHL (length of lower arm and hand); HAL (hand length, from proximal end of outer palmar tubercle to tip of the third finger); LAD (diameter of lower arm); HLL (hindlimb length or leg length); TL [tibia (shank) length]; TW (tibia width); FOL (foot length, from proximal end of inner metatarsal tubercle to tip of fourth toe); TFL (length of tarsus and foot, from proximal end of tarsus to tip of the fourth toe); FDW III (the disk width of the third finger III) and TDW IV (the disk width of the fourth toe IV). Sex was determined mainly by three characteristics: 1) generally, body size of females is larger than the males; 2) for males of the new species an external single subgular vocal sac is present and the vocal slits on both sides are near the base of the tongue; 3) eggs inside the abdomen of a specimen obviously indicates it as female.

3. Results

3.1 Molecular phylogenetic analyses The aligned mtDNA gene fragments from Rhacophorus consisted of 1935 bp nucleotide positions before trimming (Dataset 1). In total, 1844 nucleotide positions were retained for genealogical reconstructions. The fragments contained 934 constant and 917 potentially phylogenetic informative characters. The nuclear genes of the new species were all trimmed to the maximum length of the sequences from GenBank (Table S1). The phylogeny, based on the combined mitochondrial sequence and nuclear gene sequences, are largely consistent with the results from mitochondrial data alone (Figure 2 and Figure S1). The ML or BI phylogenetic approaches based on different datasets resulted on virtually identical topologies, and all terminal clades obtained relatively high-supporting values. The genus Rhacophorus formed a monophyletic group, containing three major clades, Clades A, B and C. Clade A contained six subclades, subclade A1 to


Figure 2 Bayesian phylogenetic tree of the genus Rhacophorus based on the combined nuclear and mitochondrial dataset with posterior probabilities for branches (>80% retained).


subclade A6. Among them, both bootstraps support level and posterior probabilities for the subclade A6 got high value (100% for bootstrap proportions and 1.0 for Bayesian posterior probability, respectively; Figure 2 and Figure S1). Two species in subclade A6 were a sister group, which formed into a monophyletic clade. In addition, the species delimitation based on the data set of subclade A6 showed that the probability values of two separate species exceeded 0.99, no matter which scheme was used. All data supported that both of these species in subclade A6 could be regarded as a valid species (Table 3). The average genetic distance between two species was 0.032 (Table S2), it was larger than the genetic distance between R. wui and R. hungfuensis (0.022, Table S2), and close to that of R. hongchibaensis and R. hungfuensis (0.031, Table S2). Among those three species, R. wui and R. hongchibaensis were recently described by Li et al. (2012) based on the molecular and morphological data. In addition, some genetic distances between species were smaller than that of R. zhoukaiyae sp. nov. and R. dorsoviridis (Table S2), such as R. hui and R. dugritei (0.005), R. omeimontis and R. duboisi (0.012), R. burmanus and R. duboisi (0.031). Phylogenetic trees and genetic variation imply those specimens collected from

Dabie Mountains represent a new species.

3.2 Taxon descriptionRhacophorus zhoukaiyae sp. nov.urn:lsid:zoobank.org:act: 9135258E-0841-443C-8D4D-907BB37F6858Tao PAN, Yanan ZHANG, Baowei ZHANGHolotype. AHU-RhaDb-150420-01, an adult male (Figure 3A–B, E–F) from Qianping, Jinzhai, Anhui, China (31.2953°N, 115.7252°E; Elevation, 781 m a.s.l.), collected by Tao PAN, Lifu QIAN and Xing KANG on 20 April 2015 on the Dabie Mountains.Paratypes AHU-RhaDb-150418-01 and AHU-R h a D b - 1 5 0 4 1 8 - 0 4 , t w o a d u l t m a l e s , A H U -RhaDb-150418-02 and AHU-RhaDb-150418-03, two adult females, collected from Puandonggou, Jinzhai, Anhui, China (31.2256°N, 115.6726°E; Elevation, 773 m a.s.l.) on 18 April 2015 by Baowei ZHANG, Tao PAN, Lifu QIAN, Zhonglou SUN, Zhaojie PENG, Wenliang ZHOU, Xing KANG, Xiaonan SUN, Yanan ZHANG; AHU-RhaDb-150419, AHU-RhaDb-150420-01, AHU-RhaDb-150420-02, AHU-RhaDb-150420-03, four adult males, collected from Qianping, Jinzhai, Anhui, China (31.2953° N, 115.7252° E) on 19 April 2015 to 20 April 2015 by Baowei ZHNAG, Tao PAN, Lifu QIAN,

Table 2 Summary information for the gene locus used in this study.

Locus Alignment length (bp) Number of taxa Number of variable sites Best-fitted model

Partial 12S and 16S and complete t-RNA Val 1844 91 917 GTR + I + G

BDNF 608 15 26 GTR + I

POMC 272 14 38 GTR + G

Rag 1 1130 16 124 GTR + I

RHOD 278 33 50 HKY + G

TYR 532 29 86 SYM + I + G

Table 3 The species delimitation results of subclade A6 based on the mitochondrial gene in BPP.

SchemePrior distribution Posterior probabilities

θ τ P PR. zhoukaiyae R. dorsoviridis

Scheme 1 G (2, 1000) G (2, 2000) 0.9998 0.9998

Scheme 2 G (2, 100) G (2, 200) 0.9999 0.9999

Scheme 3 G (2, 1000) G (2, 200) 1 1

Scheme 4 G (2, 100) G (2, 2000) 0.9995 0.9995


Figure 3 R. zhoukaiyae sp. nov. (A) Dorsolateral view of the live adult male R. zhoukaiyae sp. nov. holotype AHU-RhaDb-150420-01; (B) Ventral view of the live holotype; (C) Dorsolateral view of the live adult female R. zhoukaiyae sp. nov. paratypes AHU-RhaDb-150418-03; (D)Dorsal views of the live adult female paratype AHU-RhaDb-150418-03; (E) Ventral view of the right hand of the live adult male R. zhoukaiyae sp. nov. holotype; (F) Ventral view of the right foot of the live adult male R. zhoukaiyae sp. nov. holotype; (G) Dorsal views of the live R. zhoukaiyae sp. nov. tadpole; (H) The habitat of R. zhoukaiyae sp. nov. in Dabie Mountains, Anhui, China.


Zhonglou SUN, Zhaojie PENG, Wenliang ZHOU, Xing KANG, Xiaonan SUN, Yanan ZHANG; AHU-RhaDb-120428, an adult female, collected from Qianping, Jinzhai, Anhui, China (31.2953° N, 115.7252° E) on 28 April 2012 by Baowei ZHANG, Jinyun CHEN and Tao PAN.Etymology The new species is named after Professor Kaiya ZHOU (School of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu, China). The suggested English name is the Anhui tree frog.Diagnosis R. zhoukaiyae sp. nov. is associated with Rhacophorus by the following characteristics: the presence of intercalary cartilage between terminal and penultimate phalanges of digits, terminal phalanges of fingers and toes Y-shaped, tips of the digits expanded into large disks bearing circum-marginal grooves, webbed fingers, horizontal pupil, and the skin is not co-ossified to skull (Brown and Alcala, 1994; Duellman and Trueb, 1986; Hamidy and Kurniati, 2015; Inger and Stuebing, 1997; Liem, 1970; Malkmus et al., 2002).

R. zhoukaiyae sp. nov. is distinguished from all other Rhacophorus species by the following combination of morphological characters: 1) the ventral surface and front- and -rear of the femur is paler yellowish with irregular grayish blotching, and without obvious spots on the dorsum of the hand and foot webbing; 2) the outer metatarsal tubercle is small; 3) outer fingers are half-webbed and outer toes two third webbed; 4) the skin on the dorsum is smooth and without compressed warts; 5) the throat, chest and belly are pure paler yellowish; 6) the dorsal part of the fingers and toes are grayish-white; 7) the iris is golden-yellow (Figure 3).Description of the Holotype Adult male (Figure 3A–B, E–F), SVL 32.71 mm. Further measurements provided in Table 4. Body moderate; head shorter than width (HL/HW = 0.83); snout pointed, distinct protruding, projecting beyond jaw slightly; canthus rostralis rounded; loreal region oblique and slightly concave; tympanum distinct and medium-sized, slightly larger than half eye width (HTD/ED1/2 =1.06); vomerine teeth present and arranged into a “\ /” shape; tongue moderately large and oval, with a small notch at the posterior tip; dorsal skin smooth without light yellow spots; skin on chest, abdomen and ventral side of femur granulated.

Length of lower arm and hand approximately equal to half of snout-vent length. “Y”-shaped phalanx in the tips of fingers and toes. Fingers slender, with interdigital web; feeble dermal fringe along fingers; relative length of fingers I < II < IV < III; tips of fingers expanded into round disks with distinct circum-marginal grooves;

fingers moderately webbed, webbing formula: I 1-–1- II 1+–1+ III 2-–2- IV; subarticular tubercles prominent; thenar tubercle elliptical and prominent (Figure 3E); palmar tubercle small and flat; nuptial pads indistinct. Hind limbs moderately short; TBL 15.73 mm accounting for 48.09% of SVL; relative toe length I < II < III < V < IV; tips of toes expanded into round disks with distinct circum marginal grooves and the rounded discs on toes smaller than on fingers; toes moderately webbed, webbing formula: I 1+–1- II 1+–2- III 2+–2 IV 2–2+ V (Figure 3F); dermal fringe along toes feeble; inner metatarsal tubercle prominent and distinctively larger than outer metatarsal tubercle.Coloration of holotype in life The dorsal and lateral sides/surfaces of the body are bright green without spots; the ventral surface and the front- and -rear of the femur are yellowish and decorated with irregular grayish blotching; the dorsal part of the fingers and the toes are grayish-white. The throat, chest and belly are an immaculate yellowish-white. Pupils are horizontal with dark charcoal gray, and iris is golden-yellow.Coloration of holotype in preservative In general, the bright green fades and tends to blue-gray. The ventral part of the limbs and belly are yellowish-gray, the grayish blotching being obvious.Variation The variation in measurements is given in Table 4. In general females are larger than males, SVL 42.12–44.67 mm in females (n = 3) and 27.90–37.12 mm in males (n = 6, including the holotype). The lateral tubercles of males are more evident than for females. Individuals are relatively uniform in body coloration.Secondary sexual characters An external single subgular vocal sac is present in male, with vocal slits on both sides near the base of the tongue.Comparison Many studies focused on whether R. nigropunctatus and R. dorsoviridis are conspecific, but it is still controversial (Fei et al., 2012; Li et al., 2012a; Orlov et al., 2001). In our study, both are regarded as valid species and are compared with our new species separately. According to the phylogenetic study, the sister branch of R. zhoukaiyae sp. nov. is R. dorsoviridis. Based on the photos, we can concluded that R. zhoukaiyae sp. nov. differs from R. dorsoviridis by the relatively small, irregular and grayish blotches on the ventral surface and the front- and -rear of the femur (blotches relatively large, rounded and black for R. dorsoviridis), dorsal part of the inner toes are without obvious spots (obvious spots on the dorsum of the inner toes for R. dorsoviridis) and golden-yellow iris of eye (reddish-white iris for R. dorsoviridis) (Figure 4). In addition, we also compared


the morphorlogical difference with the description of R. dorsoviridis in other reference (Bourret, 1937; Fei et al., 2012; Orlov et al., 2001; Zhang et al., 2011). R. dorsoviridis differs from this new species by lacking nuptial pads (indistinct nuptial pads for new species), having a single medial vocal sac that is not distended (an external single subgular vocal sac for new species); flanks white with variable black spots (paler yellowish and decorated with irregular grayish blotching for new species); venter white with a few small black spots below shank (paler yellowish skin on chest, abdomen and ventral side of femur granulated for new species); feet and hand yellowish-white (the ventral surface and the front- and -rear of the femur are yellowish and decorated with irregular grayish blotching for new species; the

dorsal part of the fingers and the toes are grayish-white for new species); the posterior surface and occasionally the dorsomedial surface of tibiotarsus with large black spots (vs. irregular grayish blotching), iris red or yellow above, pale below (iris golden-yellow for new species) (Bourret, 1937; Fei et al., 2012; Orlov et al., 2001; Zhang et al., 2011).

According to the previous studies, R. zhoukaiyae sp. nov. was once thought to be R. nigropunctatus, based on their similar morphological characteristics. However, in our study, R. zhoukaiyae sp. nov. differs from R. nigropunctatus by having grayish blotching (the blotching are black for R. nigropunctatus), white throat (the black throat for R. nigropunctatus) and fingers half webbed (rudimentary webbed in R. nigropunctatus) (Liu

Table 4 Measurements (in mm) of R. zhoukaiyae sp. nov., collected from Dabie Mountains in Anhui province, and R. dorsoviridis, collected from Pingbian in Yunnan,China, by Dingqi Rao.

Rhacophorus zhoukaiyae sp. nov. R. dorsoviridis

HolotypeParatypes

Males Females Males

AHU-RhaDb-

AHU-RhaDb-

AHU-RhaDb-

AHU-RhaDb-

AHU-RhaDb-

AHU-RhaDb-

AHU-RhaDb-

AHU-RhaDb-

AHU-RhaDb- KIZ001 KIZ002

150420-01 150418-01 150418-04 150419 150420-03 150420-02 150418-03 150418-02 120428

SVL 32.71 27.90 36.88 34.91 34.21 37.12 42.12 44.67 43.67 35.12 36.48

HL 11.25 9.49 11.40 11.39 12.66 12.62 14.65 14.53 14.19 13.88 13.34

HW 13.62 11.56 12.96 14.44 13.30 14.37 16.29 17.94 14.80 14.02 14.04

SL 5.23 3.80 4.74 5.39 5.67 5.16 5.83 5.79 5.60 5.70 6.22

IN 3.90 3.07 3.78 4.49 3.85 4.40 4.68 4.74 4.74 5.28 5.06

ED 4.77 3.26 4.14 4.52 4.06 4.45 5.25 5.20 4.58 3.72 4.52

IO 5.28 5.01 4.97 5.33 5.07 5.38 4.41 6.41 5.83 5.12 5.24

UE 2.16 1.27 2.38 2.16 1.94 2.57 2.14 1.72 3.18 4.66 4.92

HTD 2.54 2.16 2.51 2.21 2.20 2.44 2.73 3.12 2.87 2.64 3.04

LAHL 17.68 14.65 18.82 17.17 16.87 18.63 21.77 23.16 21.42 19.64 19.28

HAL 10.91 8.16 11.49 12.00 10.13 12.73 13.35 13.59 13.24 11.70 11.56

LAD 2.82 2.42 4.35 4.02 3.26 3.19 3.85 3.70 3.14 3.72 4.32

HLL 56.53 41.93 53.42 55.38 51.03 60.56 69.82 73.75 68.04 51.62 50.02

TBL 15.73 12.43 14.88 15.12 15.25 16.66 19.72 20.36 18.97 16.26 16.06

TW 3.25 2.48 4.76 4.44 3.45 3.58 4.53 4.19 4.45 4.32 4.26

FOL 15.55 11.96 14.56 15.87 14.00 17.10 20.74 20.59 18.27 16.48 17.08

TFL 24.37 18.26 23.07 24.47 22.29 26.56 31.18 32.94 29.21 37.14 37.18

FDW III 2.27 1.43 2.42 2.34 2.07 2.39 2.42 2.42 2.39 2.42 2.56

TDW IV 2.10 1.48 1.76 1.91 1.78 1.98 2.22 2.22 2.38 2.04 2.28


et al., 1962). To rule out with a clear conscience that none of the described species are conspecific with the type specimens of our new species, we also extend their comparisons enormously. The other nine Rhacophorus species in China did not provide any genetic data for the present study, such as R. arvalis, R. aurantiventris, R. laoshan , R. leucofasciatus , R. prasinatus , R. taipeianus, R. viridimaculatus, R. yaoshanensis and R. yinggelingensis. R. zhoukaiyae sp. nov. differs from R. laoshan by the dorsum bright green (dorsum chocolate brown, with green spots for R. laoshan) and fingers half webbed and toes two thirds webbed (fingers rudimentary webbed and toes half webbed for R. laoshan). R. zhoukaiyae sp. nov. differs from R. leucofasciatus by having blotches on its flanks and front and rear of thighs (absent in R. leucofasciatus), fingers half webbed and toes two thirds webbed (fingers and toes fully webbed for R. leucofasciatus). R. zhoukaiyae sp. nov. differs from R. yaoshanensis by the grayish blotches on the ventral surface and the front and rear of the femur (without blotches for R. yaoshanensis), and the dorsal part of the toes grayish-white (yellowish for R. yaoshanensis). The front-and-rear of the thigh yellowish and fingers half webbed for R. zhoukaiyae sp. nov., which distinguishes it from R. yinggelingensis (yellowish-

orange for the front and rear of the femora and fingers one third webbed). R. zhoukaiyae sp. nov. differs from R. viridimaculatus by dorsal surface bright green and fingers and toes grayish-white (dorsal surface grown or gray-brown, with small light green spots and dark patterns of irregular shape, fingers and toes brightly-brown for R. viridimaculatus). R. zhoukaiyae sp. nov. differs from R. aurantiventris and R. arvalis by the irregular and grayish blotches on the ventral surface and the front and rear of the femora (absent in R. aurantiventris and R. arvalis); differs from R. taipeianus and R. prasinatus by toes two thirds webbed (toes fully webbed for R. taipeianus and R. prasinatus) and dorsal part of the fingers and toes grayish-white (yellowish for R. taipeianus and lavender for R. prasinatus).Distribution This species is currently only distributed in the Dabie Mountains area in Jinzhai County, Anhui Province, China. Currently, the natural habitats of this species are the freshwater marshes, intermittent freshwater marshes, ponds, and irrigated land.

4. Discussion

Accurate taxonomic recognition is a prerequisite for preserving biodiversity, especially given the background

Figure 4 The main difference between R. zhoukaiyae sp. nov. and R. dorsoviridis. (A) Eye: golden-yellow iris for R. zhoukaiyae sp. nov. and reddish-white iris for R. dorsoviridis; (B) Toes: dorsal part of the inner toes without obvious spots for R. zhoukaiyae sp. nov. and obvious spots on the dorsum of the inner toes for R. dorsoviridis; (C) Blotches: relatively small, irregular and grayish blotches on the ventral surface and the front and rear of the femora for R. zhoukaiyae sp.nov. and blotches relatively large, rounded and black for R. dorsoviridis.


of the decline of species and extinctions worldwide (Stuart et al., 2004). However, this fundamental task is challenged by the existence of cryptic species complexes (Pfenninger and Schwenk, 2007; Vences et al., 2005), a group consisting of two or more species that are reproductively isolated from each other but virtually identical in morphology (Bickford et al., 2007). The genus Rhacophorus contains more than 80 species and is the largest genus in the Rhacophoridae (Frost, 2016). However, until today, the taxonomic status of some species remains unresolved (Dehling, 2015; Li et al., 2012b; Rowley et al., 2012; Streicher et al., 2014), because most cryptic congeners in the genus Rhacophorus are difficult to distinguish from each other (Fei et al., 2009). According to the phylogenetic analysis, all the individuals of R. zhoukaiyae sp. nov. formed into a distinct monophyletic clade which was the sister group of R. dorsoviridis (Figure 2 and Figure S1). The genetic distance between them, based on the Kimura 2-parameter model, was close to genetic distance ranges among different species in Rhacophorus (Table S2). The species delimitation to these two species, based on the Bayesian Phylogenetics and Phylogeography (BPP) method, showed that the probability values in which R. zhoukaiyae sp. nov. and R. dorsoviridis of two separate species exceeded 0.99, which indicated that R. zhoukaiyae sp. nov. should be a valid species (Table 3). In morphological analyses, obvious morphological character differentiations were found between R. zhoukaiyae sp. nov. and R. dorsoviridis (Figure 4). R. nigropunctatus had been recorded in the Dabie Mountains (Chen, 1991). Unfortunately, there are no voucher specimens to verify the record. In a recent field survey (Pan et al., 2014), just one individual of Rhacophorus was observed, which is specimen AHU-RhaDb-120428 in this study. Therefore, we conclude that the records of R. nigropunctatus are based on misidentified R. zhoukaiyae sp. nov. (Chen, 1991). Until now, R. zhoukaiyae sp. nov. has been found only in the Dabie Mountains area, whereas its closely related species, R. dorsoviridis, is endemic to Yunnan Province, China, and Vietnam (Li et al., 2013; Li et al., 2012a). The obvious morphological character differentiations are also found between R. zhoukaiyae sp. nov. and R. dorsoviridis, mainly includes the shape, color, size and location of the spots and color of iris (Figure 4). On the basis of the species concept (Coyne and Orr, 2004; Van Valen, 1976), the discontinuous habitat of these two sibling species also demonstrated that R. zhoukaiyae sp. nov. should be an independent species. On the basis of the morphologically or genetically distinguishable lineage

which occupies an adaptive zone minimally different from that of any other lineage, we conclude that R. zhoukaiyae sp. nov. is a valid species. So far, females and larval stages are unknown. More breeding ecology of this new species needs to be done to understand them.

Acknowledgements We thank Wenliang ZHOU, Zhonglou SUN, Zhaojie PENG and Xiaonan SUN for their helps in sample collecting, thank Feng CHENG for his help in morphological data collecting, thank Martin BURROWS for inputing concerning the quality of the writing as regards the English language, thank Jiatang LI (Chengdu Institute of Biology, Chinese Academy of Sciences) for the study designing, thank Cheng LI (Chengdu Institute of Biology, Chinese Academy of Sciences) for the morphological identification, and thank the reviewers for their suggestions. This work was supported by the National Natural Science Foundation of China (Grant Nos. 31272332, 31071894, 30911120031, 30670243), Anhui Province Higher Education Revitalization Plan, 2014 Colleges and Universities Outstanding Youth Talent Support Program, Foundation for Graduate Student Academic Innovation Research Project of Anhui University (yqh100081), the Foundation for College Student Innovation & Venture Project of Anhui University (201610357026).

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Table S1 Samples, with sampling site, museum voucher nos., and GenBank accession nos. of corresponding sequences. “---” represens no molecular data. * Sequences new to this study.

Taxon Locality Voucher Number

Accession Number

Partial 12S and 16S and

complete t-RNA Val

BDNF POMC RAG-1 RHOD TYR

Sub-order AnuraFamily MantellidaeI Spinomantis

S. peraccae Madagascar: Fianarantsoa, Ivohibe, Andringitra Volotsangana River UMMZ 213278 DQ283036 --- --- --- --- ---

RhacophoridaaeII PolypedatesP. megacephalus China Genbank AF458141 --- --- --- --- ---III RhacophorusR. annamensis Vietnam: Bu Gia Map National ParkKIZ 64 JX219448 --- --- --- --- ---

R. annamensis Vietnam: Ta Kou Mountain Natural Reserve KIZ 1196 JX219446 --- --- --- --- ---

R. annamensis Vietnam AMNH A161414 DQ283047 --- --- --- --- ---R. arboreus Japan TTU-R-11748 AF458142 --- --- --- --- ---R. bipunctatus China: Hainan SN 030035 EU215529 --- --- --- --- ---R. bipunctatus Vietnam Genbank AF458144 --- --- --- --- ---

R. bipunctatus Myanmar: Bee Hoe village, Chin State CAS 235303 JX219444 --- --- --- --- ---

R. bipunctatus Myanmar: Putao District, Kachin State CAS 229913 JX219445 --- --- --- --- ---

R. bipunctatus Vietnam: Ha Tinh, Huong Son District AMNH-A 161418 AY843750 --- --- --- --- ---

R. burmanus China: Motuo, Xizang RAO 6239 JX219422 --- --- --- --- ---R. burmanus China: Mt. Gaoligong, Yunnan SCUM 060614L EU215537 --- --- --- --- ---

R. calcaneus Laos FMNH 256465 GQ204770 GQ204719 --- --- GQ204600 GQ204655 ---

R. chenfui China: Zhaotong, Yunnan RAO ZT 0806013 JX219431 --- --- --- --- ---R. chenfui China: Mt. Omei, Sichuan Li05 JX219432 --- --- --- --- ---R. chenfui China: Mt. Omei, Sichuan SCUM 060404L EU215534 --- --- --- --- ---R. chuyangsinensis Vietnam: Bi Doup National Park KIZ 528 JX219450 --- --- --- --- ---R. chuyangsinensis Vietnam: Bi Doup National Park KIZ 746 JX219451 --- --- --- --- ---R. dennysi China: Shaoguan, Guangdong SCUM 060401L EU215545 --- --- --- --- ---R. dennysi China: Taoyuan, Hunan Li06 JX219433 --- --- --- --- ---R. dorsoviridis China: Pingbian, Yunnan RAO060821199 JX219426 --- --- --- --- ---R. dorsoviridis China: Jinping, Yunnan RAO060821200 JX219424 --- --- --- --- ---R. dorsoviridis China: Pingbian, Yunnan YN080446 JX219425 --- --- --- --- ---R. dorsoviridis Vietnam: Sa Pa, Lao Cai ROM38015 JX219423 --- --- --- --- ---R. duboisi Vietnam: Sa Pa, Lao Cai ROM 38758 JX219414 --- --- --- --- ---R. duboisi China:Lvchun County, Yunnan LC0805088 JX219417 --- --- --- --- ---R. duboisi Vietnam: Sa Pa, Lao Cai ROM 38771 JX219413 --- --- --- --- ---R. duboisi China: Jinping, Yunnan RAOL060821289 JX219415 --- --- --- --- ---R. duboisi China: Lvchun County, Yunnan LC0805089 JX219416 --- --- --- --- ---R. duboisi China: Jinxiu, Guangxi RAOYN080492 JX219412 --- --- --- --- ---R. duboisi China: Mt. Dawei, Yunnan YN080484 JX219418 --- --- --- --- ---R. duboisi China: Mt. Dawei, Yunnan SCUM 061104L EU215536 --- --- --- --- ---R. dugritei China: Hongya, Sichuan SCUM 051017L EU215540 --- --- --- --- ---R. dugritei China: Baoxing, Sichuan SCUM 051001L EU215541 --- --- --- --- ---R. dulitensis Malaysia RAO081201 JX219434 --- --- --- --- ---R. feae China: Hekou, Yunnnan SCUM 050642WXJ EU215544 --- --- --- --- ---R. gauni Malaysia: Sarawak, Bintulu DivisionFMNH273928 JX219456 --- --- --- --- ---R. hui China: Zhaojue, Sichuan, Li01 JN688878 --- --- --- --- ---R. hungfuensis China: Wenchuan, Sichuan SCUM 060425L EU215538 --- --- --- --- ---R. kio China: Xishuangbanna, Yunnan SCUM 37941C EU215532 GQ285703 GQ285734 GQ285766 EU215562 EU215592R. kio Vietnam Genbank AF458147 --- --- --- --- ---R. maximus China: Motuo, Xizang RAO 6241 JX219411 --- --- --- --- ---

Taxon Locality Voucher Number

Accession NumberPartial 12S and 16S and

complete t-RNA Val

BDNF POMC RAG-1 RHOD TYR

R. minimus China: Mt. Dayao, Guangxi KIZ 061214YP EU215539 --- --- --- --- ---R. moltrechti China: Lianhuachi, Taiwan SCUM 061106L EU215543 --- --- --- --- ---R. monticola Indonesia: Sulawesi RMB 1236 AY326060 --- --- --- --- ---R. nigropunctatus Vietnam: Sa Pa, Lao Cai ROM 38011 JX219427 --- --- --- --- ---R. nigropunctatus China: Weining, Guizhou GZ 070658 JX219430 --- --- --- --- ---R. nigropunctatus China: Longling, Yunnan RAO 3496 JX219428 --- --- --- --- ---R. nigropalmatus Malaysia RAO081203 JX219438 --- --- --- --- ---R. nigropalmatus Malaysia RAO 081204 JX219437 --- --- --- --- ---R. nigropunctatus China: Longling, Yunnan RAO 3494 JX219429 --- --- --- --- ---R. nigropunctatus China: Weining, Guizhou SCUM 070657L EU215533 GQ285704 GQ285735 GQ285767 EU215563 EU215593R. omeimontis China: Mt. Omei, Sichuan SC 080505 JX219421 --- --- --- --- ---R. omeimontis China: Yaan, Sichuan Li02 JX219420 --- --- --- --- ---R. omeimontis China: Pengxian, Sichuan SCUM 0606137L EU215535 --- --- --- --- ---R. omeimontis China: Zhanotong, Yunnan ZT 0806010 JX219419 --- --- --- --- ---R. orlovi China: Maguan, Yunan RAO 03309 JX219435 --- --- --- --- ---R. orlovi Vietnam AMNH A161405 DQ283049 --- --- --- --- ---R. pardalis Malaysia: Bintulu Division, SarawakFMNH 273243 JX219454 --- --- --- --- ---R. pardalis Malaysia: Bintulu Division, SarawakFMNH 273245 JX219453 --- --- --- --- ---

R. pardalis Malaysia: Kota Marudu District, Sabah FMNH 235741 JX219452 --- --- --- --- ---

R. puerensis Vietnam: Sa Pa, Lao Cai ROM 37996 JN688891 --- --- --- --- ---R. puerensis China: Puer, Yunnan SCUM 060649L EU215542 --- --- --- --- ---R. reinwardtii Malaysia RAO081205 JX219443 --- --- --- --- ---R. rhodopus China: Motuo, Xizang RAO 06245 JX219441 --- --- --- --- ---R. rhodopus China: Lvchun, Yunnan Lc0805109 JX219440 --- --- --- --- ---R. rhodopus China: Mengyang, Jinghong SCUM 060692L EU215531 --- --- --- --- ---R. rhodopus China: Longchuan, Yunnan Loc 08007018 JX219439 --- --- --- --- ---R. rhodopus China: Motuo, Xizang RAO L062456 JX219442 --- --- --- --- ---R. rufipes Malaysia: Bintulu Division, SarawakFMNH 272858 JX219455 --- --- --- --- ---R. schlegelii Japan:Hiroshima Genbank AB202078 --- --- --- --- ---R. sp. China: Malipo, Wenshan, Yunnan RAO 03308 GQ285680 --- --- --- --- ---R. sp. China: Malipo, Wenshan, Yunnan RAO 03324 JX219408 --- --- --- --- ---R. sp. China: Malipo, Wenshan, Yunnan RAO 03326 JX219409 --- --- --- --- ---R. sp. China: Malipo, Wenshan, Yunnan RAO 03321 JX219410 --- --- --- --- ---R. translineatus China: Motuo, Xizang RAO 6237 JX219449 --- --- --- --- ---R. verrucopus China: Motuo, Xizang 6254 RAO JX219436 --- --- --- --- ---R. hongchibaensis China: Wuxi, Chongqing CIB 097687 JN688883 --- --- --- JN688897 JN688906R. hongchibaensis China: Wuxi, Chongqing CIB 097696 JN688882 --- --- --- JN688898 JN688907R. wui China: Lichuan, Hubei CIB 097685 JN688881 --- --- --- JN688896 JN688910R. wui China: Lichuan, Hubei CIB 097690 JN688880 --- --- --- JN688900 JN688909Rhacophorus zhoukaiyae sp. nov.* China: Jinzhai Country, Anhui AHU-

RhaDb-150418-01 KU601494 KU601449 KU601458 KU601467 KU601476KU601485Rhacophorus zhoukaiyae sp. nov.* China: Jinzhai Country, Anhui AHU-



RhaDb-150418-04 KU601497 KU601452 KU601461 KU601470 KU601479KU601488Rhacophorus zhoukaiyae sp. nov.* China: Jinzhai Country, Anhui AHU-RhaDb-150419 KU601498 KU601453 KU601462 KU601471 KU601480KU601489Rhacophorus zhoukaiyae sp. nov.* China: Jinzhai Country, Anhui AHU-



RhaDb-150420-02 KU601501 KU601456 KU601465 KU601474 KU601483KU601492Rhacophorus zhoukaiyaes p. nov.* China: Jinzhai Country, Anhui AHU-RhaDb-120428 KU601502 KU601457 KU601466 KU601475 KU601484KU601493

(Continued Table S1)

Tab

le S

2 U

ncor

rect

ed p

airw

ise

dist

ance

s (P-

dist

ance

) am

ong

spec

ies i

n Rhacophorus i

nclu

ded

in o

ur p

hylo

gene

tic a

naly

ses (

belo

w th

e di

agon

al) a

nd S

tand

ard

Erro

r Est

imat

es (a

bove

the

diag

onal

).

1 2

3 4

5 6

7 8

9 10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

1

R. d

uboi

si

0.00

2 0.

004

0.00

4 0.

004

0.00

5 0.

005

0.00

5 0.

005

0.00

5 0.

005

0.00

5 0.

005

0.00

5 0.

005

0.00

6 0.

006

0.00

6 0.

006

0.00

7 0.

006

0.00

6 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

9 0.

008

0.00

8 0.

008

0.00

8 0.

009

2 R.

om

eim

ontis

0.

012

0.00

4 0.

004

0.00

4 0.

005

0.00

5 0.

005

0.00

5 0.

005

0.00

4 0.

004

0.00

4 0.

005

0.00

5 0.

006

0.00

6 0.

006

0.00

6 0.

007

0.00

6 0.

006

0.00

7 0.

008

0.00

9 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

009

0.00

8 0.

008

0.00

8 0.

009

0.00

8 3

R. b

urm

anus

0.

031

0.03

2 0.

004

0.00

4 0.

005

0.00

5 0.

005

0.00

5 0.

005

0.00

4 0.

004

0.00

4 0.

005

0.00

5 0.

006

0.00

6 0.

006

0.00

6 0.

006

0.00

6 0.

007

0.00

7 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

009

0.00

8 4

R. zh

ouka

iyae

sp. n

ov.

0.04

2 0.

042

0.04

2 0.

004

0.00

5 0.

005

0.00

6 0.

006

0.00

5 0.

005

0.00

5 0.

005

0.00

5 0.

006

0.00

6 0.

006

0.00

7 0.

006

0.00

6 0.

006

0.00

7 0.

007

0.00

8 0.

008

0.00

8 0.

009

0.00

8 0.

008

0.00

8 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

9 0.

008

5 R.

dor

sovi

ridi

s 0.

044

0.04

5 0.

045

0.03

2 0.

006

0.00

6 0.

006

0.00

5 0.

005

0.00

5 0.

005

0.00

5 0.

006

0.00

6 0.

006

0.00

6 0.

007

0.00

6 0.

006

0.00

6 0.

007

0.00

7 0.

008

0.00

9 0.

008

0.00

9 0.

008

0.00

8 0.

009

0.00

8 0.

008

0.00

8 0.

009

0.00

8 0.

008

0.00

8 0.

009

0.00

8 6

R. sc

hleg

elii

0.05

7 0.

058

0.05

9 0.

066

0.07

2 0.

006

0.00

6 0.

006

0.00

6 0.

006

0.00

5 0.

005

0.00

6 0.

006

0.00

6 0.

007

0.00

7 0.

007

0.00

7 0.

007

0.00

7 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

9 0.

008

0.00

9 0.

008

0.00

9 0.

009

7 R.

hon

gchi

baen

sis

0.05

1 0.

052

0.05

0 0.

062

0.06

3 0.

065

0.00

4 0.

004

0.00

4 0.

004

0.00

4 0.

004

0.00

5 0.

006

0.00

6 0.

006

0.00

7 0.

007

0.00

7 0.

006

0.00

7 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

007

0.00

8 0.

008

0.00

9 0.

008

0.00

8 0.

008

0.00

8 0.

009

8 R.

min

imus

0.

054

0.05

5 0.

050

0.06

2 0.

062

0.07

2 0.

033

0.00

4 0.

004

0.00

5 0.

005

0.00

5 0.

006

0.00

6 0.

006

0.00

6 0.

007

0.00

7 0.

007

0.00

7 0.

007

0.00

7 0.

008

0.00

8 0.

008

0.00

9 0.

008

0.00

8 0.

008

0.00

7 0.

008

0.00

8 0.

009

0.00

8 0.

008

0.00

8 0.

008

0.00

8 9

R. w

ui

0.05

4 0.

053

0.05

4 0.

066

0.06

6 0.

071

0.03

5 0.

037

0.00

3 0.

005

0.00

5 0.

004

0.00

6 0.

005

0.00

6 0.

006

0.00

7 0.

006

0.00

7 0.

006

0.00

6 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

007

0.00

8 0.

008

0.00

9 0.

008

0.00

9 0.

008

0.00

9 0.

008

10

R. h

ungf

uens

is

0.05

5 0.

053

0.05

4 0.

060

0.06

2 0.

068

0.03

1 0.

035

0.02

2 0.

005

0.00

5 0.

004

0.00

6 0.

005

0.00

6 0.

006

0.00

7 0.

006

0.00

6 0.

006

0.00

6 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

9 0.

008

11

R. d

ugri

tei

0.04

7 0.

047

0.04

3 0.

056

0.05

8 0.

065

0.03

3 0.

035

0.03

9 0.

039

0.00

2 0.

004

0.00

5 0.

005

0.00

6 0.

006

0.00

6 0.

006

0.00

6 0.

006

0.00

6 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

007

0.00

8 0.

008

0.00

9 0.

008

0.00

8 0.

008

0.00

8 0.

009

12

R. h

ui

0.04

6 0.

045

0.04

1 0.

053

0.05

7 0.

061

0.03

2 0.

033

0.03

6 0.

036

0.00

5 0.

004

0.00

5 0.

005

0.00

6 0.

006

0.00

6 0.

006

0.00

7 0.

006

0.00

6 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

007

0.00

7 0.

008

0.00

9 0.

008

0.00

8 0.

008

0.00

8 0.

008

13

R. p

uere

nsis

0.05

1 0.

049

0.04

9 0.

059

0.06

1 0.

062

0.04

1 0.

046

0.04

6 0.

042

0.03

6 0.

034

0.00

5 0.

005

0.00

6 0.

006

0.00

6 0.

006

0.00

6 0.

006

0.00

6 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

14

R. a

rbor

eus

0.04

9 0.

049

0.04

8 0.

055

0.05

8 0.

063

0.06

1 0.

063

0.06

4 0.

063

0.05

2 0.

050

0.05

3 0.

005

0.00

6 0.

006

0.00

6 0.

006

0.00

7 0.

006

0.00

7 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

009

0.00

8 0.

008

0.00

8 0.

008

0.00

9 0.

008

0.00

8 0.

008

0.00

9 0.

009

15

R. m

oltr

echt

i 0.

054

0.05

5 0.

055

0.06

4 0.

067

0.06

6 0.

062

0.06

4 0.

061

0.06

2 0.

050

0.04

7 0.

057

0.05

3 0.

006

0.00

6 0.

007

0.00

6 0.

006

0.00

6 0.

007

0.00

8 0.

008

0.00

8 0.

009

0.00

8 0.

008

0.00

8 0.

008

0.00

7 0.

008

0.00

8 0.

009

0.00

8 0.

009

0.00

8 0.

009

0.00

9 16

R.

nig

ropu

ncta

tus

0.09

1 0.

089

0.08

8 0.

091

0.09

2 0.

095

0.10

0 0.

101

0.10

0 0.

099

0.09

0 0.

090

0.09

2 0.

089

0.09

0 0.

005

0.00

5 0.

007

0.00

7 0.

006

0.00

7 0.

007

0.00

7 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

007

0.00

7 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

009

17

R. n

igro

punc

tatu

s (JX

2194

27)

0.08

5 0.

086

0.08

2 0.

083

0.08

7 0.

093

0.09

5 0.

092

0.09

4 0.

091

0.08

4 0.

085

0.08

9 0.

084

0.08

6 0.

057

0.00

6 0.

007

0.00

7 0.

007

0.00

7 0.

007

0.00

8 0.

008

0.00

8 0.

009

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

9 0.

008

0.00

8 0.

008

0.00

9 0.

009

18

R. c

henf

ui

0.09

8 0.

097

0.09

0 0.

097

0.09

9 0.

103

0.10

6 0.

109

0.10

4 0.

104

0.09

5 0.

095

0.09

4 0.

093

0.09

9 0.

075

0.07

9 0.

007

0.00

7 0.

007

0.00

7 0.

008

0.00

8 0.

009

0.00

9 0.

009

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

9 0.

009

0.00

8 0.

009

0.00

9 0.

009

19

R. d

enny

si

0.08

1 0.

082

0.07

8 0.

079

0.08

4 0.

095

0.09

0 0.

084

0.09

0 0.

085

0.08

4 0.

082

0.08

2 0.

090

0.08

9 0.

108

0.10

5 0.

115

0.00

7 0.

006

0.00

7 0.

007

0.00

7 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

007

0.00

7 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

20

R. m

axim

us

0.09

1 0.

092

0.08

9 0.

088

0.09

6 0.

095

0.09

7 0.

103

0.09

9 0.

095

0.09

1 0.

090

0.09

2 0.

090

0.08

8 0.

107

0.10

5 0.

116

0.09

6 0.

007

0.00

7 0.

008

0.00

8 0.

008

0.00

8 0.

007

0.00

7 0.

008

0.00

8 0.

007

0.00

8 0.

007

0.00

9 0.

008

0.00

8 0.

008

0.00

9 0.

008

21

R. fe

ae

0.07

6 0.

075

0.07

5 0.

076

0.08

1 0.

081

0.08

1 0.

090

0.08

8 0.

085

0.08

0 0.

079

0.07

6 0.

071

0.07

5 0.

091

0.09

1 0.

099

0.08

2 0.

086

0.00

7 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

7 0.

007

0.00

7 0.

007

0.00

7 0.

007

0.00

8 0.

008

0.00

8 0.

008

0.00

8 0.

008

22

R. d

ulite

nsis

0.

114

0.11

3 0.

117

0.11

8 0.

121

0.13

2 0.

126

0.12

6 0.

124

0.12

5 0.

115

0.11

3 0.

121

0.12

1 0.

121

0.13

5 0.

126

0.14

0 0.

128

0.13

5 0.

117

0.00

7 0.

007

0.00

8 0.

008

0.00

8 0.

007

0.00

7 0.

007

0.00

7 0.

007

0.00

7 0.

008

0.00

7 0.

008

0.00

8 0.

007

0.00

8 23

R.

gaun

i 0.

141

0.13

9 0.

137

0.14

2 0.

142

0.14

2 0.

140

0.14

3 0.

142

0.14

1 0.

138

0.13

8 0.

140

0.14

3 0.

140

0.15

7 0.

152

0.15

6 0.

146

0.15

3 0.

132

0.14

0 0.

007

0.00

8 0.

008

0.00

8 0.

007

0.00

8 0.

008

0.00

7 0.

007

0.00

7 0.

008

0.00

8 0.

008

0.00

7 0.

008

0.00

8 24

R.

mon

ticol

a 0.

129

0.12

8 0.

126

0.12

7 0.

129

0.13

0 0.

133

0.13

1 0.

130

0.12

7 0.

125

0.12

5 0.

130

0.12

7 0.

124

0.14

6 0.

146

0.15

1 0.

132

0.14

0 0.

122

0.13

0 0.

118

0.00

8 0.

007

0.00

7 0.

008

0.00

7 0.

008

0.00

7 0.

008

0.00

7 0.

008

0.00

7 0.

007

0.00

7 0.

008

0.00

8 25

R.

orl

ovi

0.15

3 0.

152

0.15

3 0.

151

0.14

9 0.

160

0.15

7 0.

152

0.15

7 0.

154

0.14

9 0.

150

0.15

3 0.

146

0.14

8 0.

166

0.16

6 0.

180

0.16

2 0.

153

0.14

7 0.

158

0.14

7 0.

141

0.00

6 0.

006

0.00

7 0.

006

0.00

7 0.

007

0.00

8 0.

007

0.00

8 0.

007

0.00

8 0.

008

0.00

8 0.

008

26

R. sp

0.

155

0.15

5 0.

152

0.15

2 0.

151

0.15

9 0.

161

0.15

8 0.

159

0.15

6 0.

153

0.15

4 0.

154

0.14

6 0.

155

0.16

9 0.

169

0.17

5 0.

163

0.15

8 0.

154

0.15

5 0.

148

0.13

8 0.

076

0.00

5 0.

007

0.00

6 0.

007

0.00

7 0.

007

0.00

7 0.

008

0.00

8 0.

008

0.00

8 0.

007

0.00

8 27

R.

ver

ruco

pus

0.15

3 0.

154

0.15

1 0.

153

0.15

1 0.

157

0.15

6 0.

156

0.15

3 0.

151

0.14

9 0.

152

0.15

2 0.

142

0.15

0 0.

164

0.16

9 0.

174

0.16

1 0.

155

0.15

1 0.

158

0.14

4 0.

142

0.08

3 0.

049

0.00

7 0.

006

0.00

7 0.

007

0.00

7 0.

007

0.00

8 0.

008

0.00

7 0.

008

0.00

7 0.

008

28

R. c

alca

neus

0.

142

0.13

8 0.

144

0.13

9 0.

139

0.14

6 0.

144

0.14

7 0.

149

0.14

4 0.

141

0.13

9 0.

138

0.13

2 0.

137

0.15

6 0.

151

0.15

8 0.

144

0.14

2 0.

129

0.14

9 0.

149

0.14

0 0.

112

0.11

9 0.

113

0.00

7 0.

007

0.00

7 0.

007

0.00

7 0.

008

0.00

8 0.

008

0.00

8 0.

008

0.00

8 29

R.

chu

yang

sine

nsis

0.

138

0.13

9 0.

138

0.14

2 0.

141

0.15

1 0.

147

0.13

9 0.

143

0.14

3 0.

137

0.13

6 0.

142

0.13

4 0.

138

0.15

4 0.

153

0.16

1 0.

144

0.15

1 0.

134

0.14

1 0.

143

0.12

9 0.

119

0.11

5 0.

117

0.11

4 0.

007

0.00

6 0.

007

0.00

7 0.

007

0.00

7 0.

007

0.00

8 0.

007

0.00

8 30

R.

tran

slin

eatu

s 0.

141

0.14

1 0.

143

0.14

1 0.

143

0.14

9 0.

141

0.14

2 0.

143

0.14

3 0.

139

0.14

1 0.

139

0.13

6 0.

144

0.15

6 0.

156

0.16

2 0.

149

0.14

8 0.

136

0.13

8 0.

143

0.13

7 0.

127

0.12

5 0.

123

0.12

1 0.

097

0.00

7 0.

007

0.00

7 0.

008

0.00

7 0.

008

0.00

8 0.

008

0.00

8 31

R.

rhod

opus

0.

145

0.14

5 0.

144

0.14

6 0.

147

0.15

6 0.

150

0.14

8 0.

152

0.15

1 0.

146

0.14

7 0.

148

0.14

4 0.

145

0.15

7 0.

158

0.17

1 0.

150

0.15

2 0.

139

0.15

1 0.

143

0.14

2 0.

131

0.13

2 0.

125

0.12

7 0.

117

0.11

6 0.

004

0.00

4 0.

006

0.00

5 0.

007

0.00

7 0.

007

0.00

8 32

R.

rein

war

dtii

0.14

6 0.

145

0.14

3 0.

146

0.14

3 0.

158

0.15

3 0.

147

0.15

2 0.

154

0.14

3 0.

142

0.14

9 0.

140

0.14

2 0.

155

0.15

9 0.

167

0.15

3 0.

153

0.13

9 0.

146

0.13

8 0.

142

0.12

5 0.

131

0.12

0 0.

123

0.11

9 0.

115

0.07

0 0.

005

0.00

6 0.

006

0.00

7 0.

007

0.00

7 0.

008

33

R. b

ipun

ctat

usB

0.14

3 0.

142

0.14

3 0.

142

0.14

5 0.

154

0.14

9 0.

144

0.14

6 0.

146

0.14

3 0.

141

0.14

7 0.

141

0.14

4 0.

159

0.15

8 0.

171

0.14

9 0.

146

0.13

7 0.

145

0.14

4 0.

141

0.12

6 0.

128

0.12

0 0.

119

0.10

9 0.

112

0.07

3 0.

074

0.00

6 0.

006

0.00

7 0.

007

0.00

7 0.

008

34

R. k

io

0.14

6 0.

143

0.14

2 0.

148

0.15

1 0.

154

0.14

4 0.

143

0.14

6 0.

143

0.14

5 0.

143

0.14

4 0.

141

0.14

5 0.

159

0.16

0 0.

168

0.14

8 0.

149

0.14

0 0.

150

0.15

1 0.

133

0.12

9 0.

128

0.12

4 0.

124

0.11

7 0.

116

0.08

9 0.

090

0.08

1 0.

006

0.00

7 0.

008

0.00

7 0.

008

35

R. b

ipun

ctat

usA

0.13

8 0.

138

0.13

2 0.

141

0.14

5 0.

146

0.14

2 0.

143

0.13

9 0.

138

0.13

4 0.

133

0.13

9 0.

137

0.13

8 0.

153

0.14

9 0.

166

0.14

2 0.

143

0.13

6 0.

147

0.14

7 0.

138

0.13

0 0.

130

0.12

4 0.

127

0.11

5 0.

111

0.08

9 0.

090

0.08

4 0.

087

0.00

8 0.

008

0.00

7 0.

009

36

R. a

nnam

ensi

s 0.

166

0.16

5 0.

162

0.16

5 0.

168

0.17

7 0.

170

0.16

8 0.

170

0.16

5 0.

169

0.16

8 0.

168

0.16

5 0.

172

0.17

8 0.

174

0.18

0 0.

172

0.17

7 0.

164

0.17

5 0.

168

0.16

2 0.

160

0.15

4 0.

150

0.15

3 0.

141

0.14

0 0.

144

0.14

3 0.

140

0.13

6 0.

138

0.00

8 0.

008

0.00

9 37

R.

par

dalis

0.

164

0.16

2 0.

161

0.15

7 0.

164

0.16

2 0.

158

0.15

7 0.

160

0.16

1 0.

158

0.15

7 0.

160

0.15

2 0.

156

0.16

4 0.

165

0.17

9 0.

164

0.16

4 0.

146

0.16

4 0.

159

0.14

4 0.

146

0.15

4 0.

148

0.14

5 0.

142

0.13

4 0.

146

0.14

5 0.

141

0.14

5 0.

149

0.16

5 0.

008

0.00

8 38

R.

nig

ropa

lmat

us

0.14

0 0.

137

0.14

0 0.

136

0.14

1 0.

151

0.14

2 0.

140

0.14

0 0.

139

0.14

1 0.

137

0.14

1 0.

139

0.14

3 0.

164

0.15

6 0.

167

0.14

2 0.

150

0.13

4 0.

142

0.15

8 0.

145

0.13

8 0.

135

0.13

9 0.

131

0.12

7 0.

126

0.13

2 0.

135

0.12

6 0.

133

0.13

8 0.

158

0.15

5 0.

008

39

R. ru

fipes

0.

163

0.16

4 0.

166

0.16

7 0.

166

0.16

6 0.

163

0.16

6 0.

164

0.16

3 0.

164

0.16

2 0.

162

0.16

9 0.

166

0.17

9 0.

179

0.18

3 0.

168

0.17

0 0.

154

0.17

2 0.

161

0.14

5 0.

166

0.17

2 0.

162

0.16

8 0.

162

0.15

8 0.

170

0.17

1 0.

165

0.17

0 0.

171

0.19

0 0.

173

0.17

5

a new species of the genus rhacophorus (anura ......kit (transgene) was used to purify each dna...

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