Taxonomic Revision of Raorchestes menglaensis (Kou,1990) (Amphibia:Anura),with Descriptions of Two New Species from Yunnan,China

Ke JIANG,Jinlong REN,5,Jian WANG,Junfeng GUO,Zeng WANG,5,Yonghong LIU,Dechun JIANG and Jiatang LI,6＊

1 CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province,Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,Sichuan,China

2 Southeast Asia Biodiversity Research Institute,Chinese Academy of Sciences,Yezin,Nay Pyi Taw 05282,Myanmar

3 College of Life Science and Technology,Honghe University,Mengzi 661199,Yunnan,China

4 Xishuangbanna Tropical Botanical Garden,Chinese Academy of Sciences,Xishuangbanna 666303,Yunnan,China

5 University of Chinese Academy of Sciences,Beijing 100049,China

6 CAS Center for Excellence in Animal Evolution and Genetics,Chinese Academy of Sciences,Kunming 650223,Yunnan,China

Abstract Raorchestes is a group of Bush frogs mainly distributed in South and Southeast Asia that are poorly recognized by morphological criteria.We recognize the true R.menglaensis (Kou) based on 10 specimens newly collected from the type locality.An expanded description is also provided,and we delist the recently reported record,R.parvulus (Boulenger) from China.Combing with morphological and molecular data,two new species are respectively described from Menghai and Lvchun,Yunnan,China.Raorchestes hillisi sp.nov. is distinguished from all other congeners by the following combination of morphological characters:(1) small body sized (males 15.9-17.7 mm,n =7; female 17.5 mm,n =1); (2) head longer than wide; (3) snout longer than eye diameter; (4) the tip of upper jaws slightly notched; (5) internasal space slightly less than interorbital space; (6) tympanum distinct; (7)fingers lacking lateral dermal fringe; (8) outside of toe I and both sides of toe II lacking lateral dermal fringe,other toes having weak lateral dermal fringes; (9) rudimentary web on toes; (10) lacking a series of tubercles along the outer sides of forearm and foot; (11) discs of fingers and toes not orange.Raorchestes huanglianshan sp.nov. is distinguished from all other congeners by the following combination of morphological characters:(1) small body size (males 17.0-19.6 mm,n =12; female 21.5 mm,n =1); (2) head slightly wider than long or equal; (3) snout longer than eye diameter; (4)the tip of upper jaws slightly notched; (5) internasal space distinctly less than interorbital space (6) tympanum distinct;(7) fingers lacking lateral dermal fringe; (8) toes lacking lateral dermal fringe; (9) rudimentary web on toes; (10)lacking a series of tubercles along the outer sides of forearm and foot; (11) discs of all fingers and toes orange or parts of them orange in life.

Keywords bush frog,distribution,Raorchestes hillisi sp.nov.,Raorchestes huanglianshan sp.nov.,Raorchestes parvulus,revision,taxonomy

1.Introduction

The Bush frogs in the genusRaorchestesBiju,Shouche,Dubois,Dutta,and Bossuyt,2010 are largely nocturnal species,having small body size (SVL 15 to 45 mm) and large transparent gular pouch while calling,lacking vomerine teeth,which are holding direct development (Bijuet al.,2010).Sixty-two species are known inRaorchestesand mainly distributed in South and Southeast Asia,including southern India to Nepal,Myanmar,Thailand,Laos,southern China,Vietnam,and West Malaysia.More than 80% known species distributed in Western Ghats of southern India (Frost,2020).

Four species,R.longchuanensis(Yang and Li,1979),R.menglaensis(Kou,1990),R.parvulus(Boulenger,1893),andR.cangyuanensisWu,Suwannapoon,Xu,Murphy,and Che,2019 are currently recorded from China (Wuet al.,2019; Yuet al.,2019; AmphibiaChina,2020).However,Raorchestesspecies were barely understand from China,for example,bothR.menglaensisandR.longchuanensiswere only known from their original descriptions decades ago (Yang and Li,1979; Kou,1990).No detailed morphological,phylogenetic,and ecological data reported,manyRaorchestesspecies still remain poorly diagnosed,and the limited available information hampered the identification of these small-sized tree frogs.

In addition,the taxonomic status ofR.menglaensisseems to be questionable.Originally described by Kou (1990) from“Zhushihe,Mengla County,Yunnan Province”,China,the morphology ofR.menglaensishas been known from type series and a single male specimen only (Feiet al.,2009).Moreover,although the phylogenetic position ofR.menglaensiswas provided by Liet al.(2009) and Yuet al.(2009) based on the same specimen (060821286Rao) from “Lvchun,Yunnan”,it cannot represent trueR.menglaensisfrom type locality.With the lack of trueR.parvulus,Yuet al.(2019) recently reportedR.parvulusas a new country record to China based on specimens collected from Mengla County,however,the locality of their new record is close to the type locality ofR.menglaensis.Given the conservative evolution of the morphology and high frequency of variation in the genusRaorchestes(Orlovet al.,2012;Yuet al.,2019),the taxonomic identity ofR.menglaensisis needed to be clarified.

In the present study,the phylogenetic relationships amongRaorchestesspecies were reconstructed,including all species and some unidentified populations from China and southeastern Asia,and the samples ofR.menglaensis,R.longchuanensis,andR.cangyuanensiswere from their type localities.We confirmed the phylogenetic position and morphology of trueR.menglaensis,and both the phylogenetic and morphological evidence revealed that the record ofR.‘parvulus’from Chinaby Yuet al.(2019) should be misidentification ofR.menglaensis.In addition,populations respectively from Menghai,southern Yunnan and Lvchun,southeastern Yunnan formed two distinct clades,which represent two new species,and we describe them based on molecular and morphological differences herein.

2.Materials and Methods

2.1.SamplingA total of 38 specimens were collected from Yunnan,China,including 10 specimens (9 males,one female)from Zhushihe,Mengla; six specimens (five males,one female)from Menglun,Mengla; eight specimens (seven males,one female) from Xiding,Menghai; 14 specimens (13 males,one female) from Mt.Huanglian,Lvchun.Following euthanasia,liver tissues was taken and preserved in 95% ethanol,and the specimen was fixed in 10% formalin solution and was transferred to 75% ethanol after fieldwork.All specimens including types were deposited in the Herpetological Museum,Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu,China.

2.2.Morphological dataMorphological comparisons:All measurements were made with slide calipers to the nearest 0.1 mm.Morphological characters used and their measurement methods followed Feiet al.(2009),webbing formula followed Savage and Heyer (1997).The morphological characters and their abbreviations as:SVL,snout-vent length; HL,head length;HW,head width; SL,snout length; INS,internarial distance;IOS,interorbital distance; EHD,eye horizontal diameter; UEW,maximum width of upper eyelid; TD,tympanum diameter;FAHL,forelimb and hand length; LAW,width of lower arm;HAL,hand length; FML,femur length; TBL,tibia length; TFL,length of tarsus and foot; FOL,foot length.

Morphological data of congeners were obtained from vouchered specimens (Appendix) as well as from literatures(Kou,1990; Boulenger,1893; Smith,1924; Bossuyt and Dubois,2001; Feiet al.,2009).

2.3.Molecular data and analysesGenomic DNA was extracted from liver tissue preserved in 95% ethanol using an Ezup Column Animal Genomic DNA Purification Kit(Sangon Biotech,China).For each specimen in this study,the mitochondrial gene 16S ribosomal RNA (16S rRNA) gene was sequenced.The fragments of 16S rRNA were amplified using primers 16Sar-L (5'-CGCCTGTTTATCAAAAACAT-3')and 16Sbr-H (5'-CCGGTCTGAACTCAGATCACGT-3')(Vijayakumaret al.,2014).Polymerase chain reactions (PCR)amplifications were performed in a 25 μl reaction volume with an initial denaturation at 94 °C for 3 min,followed by 40 cycles of 94 °C for 1 min,51 °C for 1 min,72 °C for 1 min,and a final extension at 72 °C for 10 min.The PCR products were purified and then sequenced in both forward and reverse directions using an ABI 3730xL sequencer (Applied Biosystems,Foster City,CA,USA) by Sangon Biotech Co.,Ltd (Chengdu,China).All sequences were submitted and deposited in GenBank(Table 1).Additional 68 sequences ofRaorchestesspecies and three outgroup taxa were obtained from GenBank; outgroup taxa,Beddomixalus bijui(Zachariah,Dinesh,Radhakrishnan,Kunhikrishnan,Palot,and Vishnudas),Nasutixalus medogensis(Jiang,Wang,Yan,and Che),andPseudophilautus asankai(Manamendra-Arachchi and Pethiyagoda),were selected based on previous phylogenetic studies (Jianget al.,2016; Chanet al.,2018).

The dataset was aligned and edited using MEGA 6.0 withdefault settings (Tamuraet al.,2013),and the alignment was checked by eye and adjusted manually.Bayesian inference (BI)analyses were implemented using MrBayes v3.1.2 (Ronquist and Huelsenbeck,2003).The best-fit model was selected in PartitionFinder 2 (Lanfearet al.,2017) under Akaike Information Criterion (AIC),which is a GTR+I+G model for all the three codon positions.Four Markov chains were executed and the data set was run for 10 million generations to allow adequate time for convergence,sampling the Markov chains at intervals of 100 generations.Bayesian posterior probability(BPP) was determined to test the confidence of tree topology,nodes in the trees were considered strongly supported when BPP≥ 0.95.Convergence was investigated in Tracer 1.6 (Rambautet al.,2013),and the first 25% trees were discarded as burn-in.Maximum likelihood (ML) were conducted in RAxML 8.2.10(Stamatakis,2014) under the best-fit model of evolution based

on the AIC (GTRGAMMA) criterion.Tree searches were performed 100 times and bootstrap proportions (BSP) were assessed using the rapid-bootstrapping algorithm (1000 nonparametric bootstrap replicates) to test the node support,where nodes with BSP ≥ 70 were significantly supported.Uncorrected pairwise distances (p-distances) of the 16s dataset among species of targetedRaorchestestaxa were calculated using MEGA 6(Tamuraet al.,2013).

Table 1 Information on Voucher No.,GenBank No.,and localities of specimens used in this study,collections and their abbreviations see material and methods.

(Continued Table 1)

2.4.AbbreviationsCIB,the Herpetological Museum,Chengdu Institute of Biology,Chinese Academy of Sciences;KIZ,Kunming Institute of Zoology,Chinese Academy of Sciences; CESF,Centre for Ecological Sciences-Frogs,Indian Institute of Science; BNHS,Bombay Natural History Society;AGCZRL,Abasaheb Garware College-Zoology Research Laboratory; ROM,Royal Ontario Museum; Rao,Dr.Dingqi Rao’s lab collections in KIZ; KUHE,Graduate School of Human and Environmental Studies,Kyoto University; TNHM (H),Trivandrum Natural History Museum; LSUHC,La Sierra University Herpetological Collection; WHT,Wildlife Heritage Trust of Sri Lanka.K and Y,unknown resource.

3.Results

3.1.Phylogenetic analysesPhylogenetic analyses showed that our newly collectedRaorchestesspecimens were recovered as three major clades (Figure 1):

i) the first clade from Menghai,Yunnan,China formed a distinct clade with strong support (Figure 1,clade B; BSP=100;BPP=1.00),which represents a new species,Raorchestes hillisisp.nov.,as described herein,individuals within clade B possessed no genetic divergence;

ii) the second clade from Mt.Huanglian,Lvchun,Yunnan,China,and together withRaorchestes ‘menglaensis’(060821286Rao,from Lvchun,Yunnan,China) (Liet al.,2009)formed a monophyletic clade with short internal branch lengths (clade C; BSP=98; BPP=1.00),which represents another new species,Raorchestes huanglianshansp.nov.,the uncorrected geneticp-distances varied from 0-1.74% within clade C;

iii) the third clade included specimens from Zhushihe,Mengla,Yunnan,China (type locality ofR.menglaensis),Menglun,Mengla and some specimens stated to beRaorchestes‘parvulus’ by Yuet al.(2019) (clade D; BSP=54),the uncorrected geneticp-distances varied from 0-0.25% within clade D (Table 2).

Although the trueR.menglaensis(asR.menglaensis sensu stricto) seemed to be nested within theR.parvuluslineages,R.parvuluswas recovered as paraphyletic,which were recovered as several highly diverged,strongly supported,monophyletic matrilines that correspond with geographic distribution.However,we cannot evaluate the taxonomic statuses of these unexamined clades but only assess the phylogenetic position ofR.menglaensissensu stricto(see below).Based on both morphological and genetic data obtained from topotypes ofR.menglaensis,we provided a re-description and revised previous misidentifications of this highly variable species herein.

3.2.Morphological comparisons

i) our newly collected specimens from Mengla,Yunnan are similar to the original description ofR.menglaensisin having:head longer than wide; snout longer than eye diameter; fingers and toes lacking lateral dermal fringe; discs of fingers and toes not orange.

ii)R.menglaensisdiffers fromR.parvulusin having:fingers and toes lacking lateral dermal fringes.

iii) morphological descriptions provided by Yuet al.(2019)differ fromR.parvulusin having:fingers and toes lacking lateral dermal fringes.

3.3.Systematic account

Re-description of Raorchestes menglaensis (Kou)(Figures 2,3)

Philautus menglaensisKou,1990,in Zhao (ed.),From Water onto Land:210.Type locality:Zhushihe,Mengla,Yunnan,China; alt.900 m.Holotype:(YU) A845090,♂,by original designation.

Philautus(Philautus)menglaensis:Bossuyt and Dubois,2001,Zeylanica,6:59.

Aquixalus menglaensis:Fei,Hu,Ye,and Huang,2009,Fauna Sinica,Amph.2:722.

Raorchestes menglaensis:Biju,Shouche,Dubois,Dutta,and Bossuyt,2010,Curr.Sci.,Bangalore,98:1120,by implication.

Liuixalus menglaensis:Fei,Ye,and Jiang,2012,Colored Atlas Chinese Amph.Distr.:513.

Raorchestesparvulus:Yu,Liu,Hou,Li,and Yang,2019,Zootaxa,4577 (2):386.

Referred specimens:nine male specimens and one female specimen from Zhushihe (type locality),Mengla,Yunnan;five male specimens and one female specimen from Menglun,Mengla,Yunnan.Specimens collected by Ke JIANG,Junfeng GUO,Wenbo YU,Yonghong LIU,Zhongxiong FU,Hanliu AI.

Remark:the specific epithet is named after its type locality Mengla (Yunnan,China).We suggest “Mengla Bush Frog” as its English common name,and “Meng La Guan Shu Wa (勐腊灌树蛙)” as its Chinese common name.

Diagnosis:(1) relatively small body sized (males 16.6-21.6 mm,n=15; female 18.9-20.5 mm,n=2); (2) head longer than wide; (3)snout longer than eye diameter; (4) the tip of upper jaws slightly notched; (5) internasal space sub-equal to interorbital space; (6)tympanum distinct; (7) fingers lacking lateral dermal fringe; (8)toes lacking lateral dermal fringe; (9) rudimentary web on toes;(10) lacking a series of tubercles along the outer sides of forearm and foot; (11) discs of fingers and toes not orange in life.

Figure 1 BI phylogenetic tree estimated from mtDNA sequences depicting phylogenetic relationships of Raorchestes,supportive values are showed above branches as BPP/BSP,values in parentheses after clade labels represent uncorrected p-distances of the corresponding clades.

Description:small body sized (males 16.6-21.6 mm,n=15;female 18.9-20.5 mm,n=2).Head longer than wide; snout rounded,upper jaw protruding slightly in ventral view; the tip of upper jaws slightly notched; canthus rostralis distinct; loreal region concave; snout slightly longer than eye diameter; nostrils oval,lacking flap,closer to snout tip than to eye; internasal space near equal to interorbital space; interorbital space 1.50 times larger than upper eyelid width; tongue notched at the tip; lingual papillae absent; vomerine teeth absent; tympanum distinct; supratympanic fold distinct; eyes small,pupil horizontal.

Forelimb length near equal to half of body length; fingers lacking lateral dermal fringe and web; subarticular tubercles prominent (fingers:I=1,II=1,III=2,IV=1); relative lengths of fingers:I ＜ II ＜ IV ＜ III; discs rounded; circummarginal grooves present on all fingers; nuptial pad present on the dorsal surface of first finger.

Hindlimbs short,heels just touch when folded at right angles to the body; tibiotarsal articulation reaching the eye when adpressed; supernumerary tubercles and subarticular tubercle present (toes:I=1,II=1,III=2,IV=3,V=2); relative lengths of toes:I ＜ II ＜ III ＜ V ＜ IV; disks rounded,the size of disks equal to these on fingers; circummarginal grooves present on all toes; no web between toe I and toe II,web rudimentary between other toes,webbing formula:II 1-2 III 1-21/2IV 21/2-1 V; toes lacking lateral dermal fringe; subarticular tubercles prominent,rounded; inner metatarsal tubercle distinct,oval,outer metatarsal tubercle absent.

Skin on dorsal surface smooth,few small tubercles scattered on shoulder and towards the vent.Distinct tubercles scattered along the temporal region and lateral body.Ventral surface covered with relatively large flat tubercles.

Coloration in life:dorsal color pale brown or dark brown,with a dark triangular blotch on interorbital space,two or more patches of yellow pigmentation on dorsal head; a “X”shaped dark brown or black blotch on back,from posterior eyes to each side of lumbar,sometimes indistinct; a dark brown or black blotch on each crotch; dorsal limbs with more or less distinct dark brown or black bars; dorsal surface usually covered by small white warts; some individuals having a pale line along the middle of back and hind limbs; dorsal side of discs pale brown or yellow.Ventral sides of head and body grayishwhite,with small dark gray spots,the tubercles white; ventral side of limbs gray.Iris brown.

Coloration in preservative:dorsal color changed to gray,the blotches or spots brownish-gray,warts white.Ventral color white,spots brownish-gray.

Sexual dimorphism:in males,white nuptial pad present on the dorsal surface of the first finger; a large external single subgular vocal sac present (vocal sac of some individuals not expanded),a pair of slit-like openings near the inner corners of mouth; white lineae masculinae visible on ventral body.

Variation:The measurements are given in Table 3 and 4,and the variation are as follows: Vocal sac of four specimens(CIB116337-38,CIB116340,CIB116343) from type locality and five specimens from Menglun (CIB116347-51) are distinctly expended,other five specimens (CIB116339,CIB116341-42,CIB116344-45) from type locality are not expanded.

Distribution:known from Zhushihe (type locality) (Figure 4) and Menglun,Yunnan,China; based on the phylogenetic relationships,it is probably also from Lan Son,Thailand;Louangphrabang,Laos; and Cambodia.

Habitat:this species was found calling on bushes near evergreen broad-leaved forest at night,at altitude from 520 m to 903 m.Megophrys parva(Boulenger),Leptobrachium chapaense(Bourret),Limnonectes liui(Yang),Polypedates impresusYang,andTheloderma albopunctatum(Liu and Hu) were found.

Raorchestes hillisi sp.nov.Jiang,Ren,Guo,Wang,and Li(Figures 5,6,7).

Figure 3 Photographs of a male individual of Raorchestes menglaensis from Menglun,Mengla,Yunnan,showing (A) dorsal view;(B) ventral view.Photographs by Jinlong REN.

Holotype:CIB 116329,an adult male (field No.BN2018006),collected from Xiding (21.935607°N,100.149527°E,altitude 1719 m),Menghai,Yunnan,China on 10 May 2018 by Ke JIANG.

Paratypes:six adult males (CIB 116330-35) and an adult female CIB 116336,as same locality and date as holotype,collected by Ke JIANG,Junfeng GUO,Lingui He,Kai WANG and Zhongxiong FU.

Etymology:the specific epithet “hillisi” is named after Dr.David M.Hillis (University of Texas at Austin),for his excellent contributions to the evolutionary studies,and we acknowledge his support to us.We suggest “Hillis’s Bush Frog” as its English common name,and “Meng Hai Guan Shu Wa (勐海灌树蛙)”as its Chinese common name.

Diagnosis:this new species is identified to the genusRaorchestesby its molecular phylogenetic position and the following morphological characters:(1) having small body size (SVL 15.0 to 45.0 mm); (2) large gular pouch transparent while calling; (3)lacking vomerine teeth.

Raorchestes hillisisp.nov.is distinguished from all othercongeners by the following combination of morphological characters:(1) small body sized (males 15.9-17.7 mm,n=7; female 17.5 mm,n=1); (2) head longer than wide; (3) snout longer than eye diameter; (4) the tip of upper jaws slightly notched; (5)internasal space sub-equal to interorbital space; (6) tympanum distinct; (7) fingers lacking lateral dermal fringe; (8) outside of toe I and both sides of toe II lacking lateral dermal fringe,other toes having weak lateral dermal fringes; (9) rudimentary web on toes; (10) lacking a series of tubercles along the outer sides of forearm and foot; (11) discs of fingers and toes not orange in life.

Table 3 Measurements of Raorchestes menglaensis from type locality Zhushihe,Mengla,Yunnan (in mm).

Table 4 Measurements of Raorchestes menglaensis from Menglun,Mengla,Yunnan (in mm).

Description of holotype:small body size (SVL=17.3 mm).Head longer than wide (HL/HW=1.12); snout rounded,upper jaw protruding slightly in ventral view; the tip of upper jaws slightly notched; canthus rostralis distinct; loreal region concave;snout slightly longer eye diameter (SL/ED=1.13); nostrils oval,lacking flap,closer to snout tip than to eye; internasal space near equal to interorbital space (IOS/INS = 1.05); interorbital space 1.40 times larger than upper eyelid width (IOS=2.1 mm; UEW=1.4 mm); tongue notched at the tip; lingual papillae absent;vomerine teeth absent; tympanum distinct; supratympanic fold distinct; eyes small,pupil horizontal; a large external single subgular vocal sac present,a pair of slit-like openings near the inner corners of mouth.

Forelimb length near equal to half of body length (FAHL/SVL=0.52); fingers lacking lateral dermal fringe and web;subarticular tubercles prominent (fingers:I=1,II=1,III=2,IV=1); relative lengths of fingers:I ＜ II ＜ IV ＜ III; discs rounded;circummarginal grooves present on all fingers; nuptial pad present on the dorsal surface of first finger.

Hindlimbs short,heels just touch when folded at right angles to the body (TBL=8.3 mm,FML=8.5 mm); tibiotarsal articulation reaching the eye when adpressed; supernumerary tubercles and subarticular tubercle present (toes:I=1,II=1,III=2,IV=3,V=2); relative lengths of toes:I ＜ II ＜ III ＜ V ＜IV; disks rounded,the size of disks equal to these on fingers;circummarginal grooves present on all toes; no web between toe I and toe II,web rudimentary between other toes,webbing formula:II 1-2 III 1-21/2IV 21/2-1 V; outside of toe I and both sides of toe II lacking lateral dermal fringe,other toes having weak lateral dermal fringe; subarticular tubercles prominent,rounded; inner metatarsal tubercle distinct,oval,outer metatarsal tubercle absent.

Skin on dorsal surface smooth,few indistinct tubercles scattered on shoulder and towards the vent.Distinct tubercles scattered along the temporal region and lateral body.ventral surface covered with relatively large flat tubercles.

Figure 4 The type localities of Raorchestes species related to this study,Raorchestes menglaensis (brown triangle),Raorchestes hillisi sp.nov.(green square),Raorchestes huanglianshan sp.nov.(blue star),R.longchuanensis (orange circle,No.1),R.cangyuanensis (orange circle,No.2),and R.parvulus (orange circle,No.3).

Coloration in life:dorsal and lateral sides brown,with a dark brown triangular blotch on interorbital space,one parch of yellowish-brown pigmentation on forehead,two patches of yellowish-brown pigmentation on dorsal head; an indistinct“)(”shaped dark brown blotch on fore part of back; an distinct dark brown blotch on each crotch; dorsal limbs with more or less distinct dark brown bars; dorsal surface without white warts; dorsal side of discs yellowish-brown.A dark brown stripe under the supratympanic fold,the posterior end of jaws and upper arm with yellowish-brown pigmentations.Ventral sides of head and body grayish-white,with small dark gray spots,the tubercles white; ventral side of limbs gray,with white spots.Iris brown.

Coloration in preservative:dorsal color changed to grayishbrown,the blotches or spots blackish-brown; yellowish-brown pigmentations changed to pale brown.Ventral color whitishgray,spots grayish-brown.

Variation:The measurements are given in Table 5 and the variation are as follows: CIB 116334 having a pale line along the middle of back and hind limb; three males (CIB 116332-33,115335) having lighter ground color on dorsal side.

Sexual dimorphism:in males,white nuptial pad present on the dorsal surface of the first finger; a large external single subgular vocal sac present,extended to the chest,a pair of slitlike openings near the inner corners of mouth; white lineae masculinae visible on ventral body.

Distribution:currently known only from the type locality,Xiding (Figure 4),Menghai,Yunnan.

Habitat:this new species was found in bushes surrounded by moist subtropical secondary evergreen broad-leaved forest(Figure 8) near Xiding Village at night (10 pm.to 12 pm.) on 10 May 2018,the altitude is 1719 m a.s.l.,the weather is light rain.All male individuals were found on the bush leaves while calling,eggs were not observed.Polypedates impresusandMicrohyla fissipesBoulenger were also found in bushes,andPseudocalotes microlepis(Boulenger) was found on trees near the bushes.

Figure 5 Photographs of the holotype of Raorchestes hillisi sp.nov.in life from the type locality,Xiding,Menghai,Yunnan,showing (A) dorsolateral view;(B) dorsal view; (C) ventral view; (D) ventral view of hand; (E) ventral view of foot.Photographs by Jinlong REN.

Table 5 Measurements of Raorchestes hillisi sp.nov.from Xiding,Menghai,Yunnan (in mm).

Figure 7 Photographs of holotype of Raorchestes hillisi sp.nov.,showing (A) dorsal view; (B) ventral view; (C) ventral view of hand; (D) ventral view of foot.Photographs by Junfeng GUO.

Comparison:only five species of the genusRaorchestesknown from Southeast Asia and southern China except for this new species,includingR.cangyuanensis,R.gryllus(Smith,1924),R.longchuanensis,R.menglaensis,andR.parvulus(Frost,2019; Yuet al.,2019).The new speciesdiffers fromR.cangyuanensisby smaller body size (males SVL 15.9-17.7 mm,n=7 vs.16.1-20.0 mm,n=3),head longer than wide,tympanum distinct,outer metatarsal tubercle present,discs of fingers and toes not orange(vs.males SVL 16.1-20.0 mm,n=3,head wider than long,tympanum indistinct,outer metatarsal tubercle absent,discs of fingers and toes orange); differs fromR.gryllusby having a head longer than wide,tympanum distinct,toe webbing rudimentary,and lacking a series of tubercles along the outer sides of forearm and foot (vs.head wider than long,tympanum indistinct,toe webbing more than half,and having a series of tubercles along the outer sides of forearm and foot); differs fromR.longchuanensisby head longer than wide,fingers lacking lateral dermal fringe,discs of fingers and toes not orange (vs.head length equal to width,fingers I and II having lateral dermal fringes,discs of finger and toes orange); differs fromR.menglaensisby head longer than wide,tympanum distinct,dorsal surface smooth,without white tubercles (vs.head length equal to width,tympanum indistinct,dorsal surface rough,with white tubercles); differs fromR.parvulusby head longer than wide,snout longer than eye diameter,canthus rostralis distinct,and fingers lacking lateral dermal fringe (vs.head wider than long,snout shorter than eye diameter,canthus rostralis rounded,and inside of all fingers having lateral dermal fringe).

Raorchestes huanglianshan sp.nov.Jiang,Wang,Ren,and Li(Figures 9，10).

Kirtixalus menglaensis:Yu,Rao,Zhang,and Yang,2009,Mol.Phylogenet.Evol.,50:578.

Pseudophilautus menglaensis:Li,Che,Murphy,Zhao,Zhao,Rao,and Zhang,2009,Mol.Phylogenet.Evol.,53:519.

Raorchestesmenglaensis:Yu,Liu,Hou,Li,and Yang,2019,Zootaxa,4577 (2):386.

Figure 8 Habitat of (A) Raorchestes hillisi sp.nov.from the type locality,Xiding,Menghai,Yunnan,altitude 1719 m,and (B)Raorchestes huanglianshan sp.nov.from the type locality,Mt.Huanglian,Lvchun,Yunnan,altitude 1823 m.Photographs by Ke JIANG and Jian WANG.

Table 6 Measurements of Raorchestes huanglianshan sp.nov.from Mt.Huanglian,Lvchun,Yunnan (in mm). P:Paratype,H:Holotype.

Figure 10 Photographs of holotype of Raorchestes huanglianshan sp.nov.,showing (A) dorsal view; (B) ventral view; (C) ventral view of hand; (D) ventral view of foot.Photographs by Ke JIANG.

Holotype:an adult male CIB 116353 (field no.YN2018303),collected from Mt.Huanglian (22.932255°N,102.288264°E,altitude 1823 m),Lvchun,Yunnan,China on May 2018 by Jian WANG.

Paratypes:11 adult males (CIB 116354-364),and an adult female(CIB 116365),as same locality and date as holotype,collected by Jian WANG.

Etymology:the specific epithet “huanglianshan” is named after the type locality Mt.Huanglian,“shan” means “mountain” in Chinese.We suggest “Huanglianshan Bush Frogs” as its English common name,and “Huang Lian Shan Guan Shu Wa (黄连山灌树蛙)” as its Chinese common name.

Diagnosis:this new species is identified to the genusRaorchestesby its molecular phylogenetic position and the following morphological characters:(1) having small body size (SVL 15.0 to 45.0 mm); (2) large gular pouch transparent while calling; (3)lacking vomerine teeth.

Raorchestes huanglianshansp.nov.is distinguished from all other congeners by the following combination of morphological characters:(1) small body sized (males 17.0-19.6 mm,n=12;female 21.5 mm,n=1); (2) head slightly wider than long or equal; (3) snout longer than eye diameter; (4) the tip of upper jaws slightly notched; (5) internasal space distinctly less than interorbital space (6) tympanum distinct; (7) fingers lacking lateral dermal fringe; (8) toes lacking lateral dermal fringe; (9)rudimentary web on toes; (10) lacking a series of tubercles along the outer sides of forearm and foot; (11) discs of all fingers and toes orange or parts of them orange in life.

Description of holotype:small body size (SVL=19.6).Head width near equal to head length (HW/HL=1.01); snout rounded,upper jaw protruding slightly in ventral view; the tip of upper jaws slightly notched; canthus rostralis distinct; loreal region concave; snout slightly longer eye diameter (SL/ED=1.29); nostrils oval,lacking flap,closer to snout tip than to eye;internasal space less than interorbital space (IOS/INS=1.24);interorbital space 1.63 times larger than upper eyelid width (IOS=2.6 mm; UEW=1.6 mm); tongue notched at the tip; lingual papillae absent; vomerine teeth absent; tympanum distinct;supratympanic fold distinct; eyes small,pupil horizontal; a large external single subgular vocal sac present,a pair of slit-like openings near the inner corners of mouth.

Forelimb length near equal to half of body length (FAHL/SVL=0.52); fingers lacking lateral dermal fringe and web;subarticular tubercles prominent (fingers:I=1,II=1,III=2,IV=1); relative lengths of fingers:I ＜ II ＜ IV ＜ III; discs rounded;circummarginal grooves present on all fingers; nuptial pad present on the dorsal surface of first finger.

Hindlimbs short,heels just touch when folded at right angles to the body (TBL=8.6 mm,FML=8.5 mm); tibiotarsal articulation reaching the eye when adpressed; supernumerary tubercles and subarticular tubercle present (toes:I=1,II=1,III=2,IV=3,V=2); relative lengths of toes:I ＜ II ＜ III ＜ V ＜IV; disks rounded,the size of disks equal to these on fingers;circummarginal grooves present on all toes; no web between toe I and toe II,web rudimentary between other toes,webbing formula:II 1-2 III 1-2-IV 2--1 V; toes lacking lateral dermal fringe;subarticular tubercles prominent,rounded; inner metatarsal tubercle distinct,oval,outer metatarsal tubercle absent.

Skin on dorsal surface smooth,large tubercles scattered on back.Large tubercles scattered on the posterior temporal region and lateral body; supratympanic fold distinct.Ventral surface covered with large flat tubercles.

Coloration in life:dorsal color brownish-gray,with a brown triangular blotch on interorbital space,two patches of orange pigmentation on dorsal head; an indistinct “X” shaped brown blotch on back,from posterior eyes to middle of back; a brown blotch on each crotch; each side of dorsal thigh and tibia with one distinct brown bars; both dorsal and ventral side of discs orange,rather distinct on ventral side.Three brown spots under the eye,and a brown stripe under the supratympanic fold.Ventral sides of head and body grayish-white,with small gray spots,the tubercles white; ventral side of limbs gray,with white spots.Iris brown.

Coloration in preservative:dorsal color changed to pale brownish-gray,the blotches or spots brownish-gray.Ventral color yellowish-white,spots brownish-gray.

Variation:The measurements are given in Table 6 and the variation are as follows:CIB 116359 having a pale line along the middle of back and hind limb; two males (CIB 116363-64)having distinctly darker ground color on dorsal side.

Sexual dimorphism:in males,white nuptial pad present on the dorsal surface of the first finger; a large external single subgular vocal sac present,a pair of slit-like openings near the inner corners of mouth; white lineae masculinae visible on ventral body; the body size of males less than the female.

Distribution:currently known from the type locality,Mt.Huanglian (Figure 4),Lvchun,Yunnan,China.Based on the phylogenetic relationships,it is probably also from Chiangmai,Thailand.

Habitat:this species was found calling from April to October on low plants near evergreen broad-leaved forest in Huanglianshan Nature Reserve,at relatively high altitude 1600-1900 m (Figure 8).Large numbers of individuals were observed on the leaves ofAgeratina adenophora(Spreng.)in October after raining,and courtship behavior was also observed,the temperature was 14-20℃.In the habitat,more than ten amphibian species were found,such as:Tylototriton shanjingNussbaum,Brodie,and Yang,Leptobrachella ventripunctatus(Fei,Ye,and Li),Megophrys jingdongensisFei and Ye,Megophrys rubrimeraTapley,Cutajar,Mahony,Chung,Dau,Nguyen,Luong,and Rowley,Hyla gongshanensisLi and Yang,Nidirana lini(Chou),Odorrana jingdongensisFei,Ye,and Li,Nanorana yunnanensis(Anderson),N.unculuanus(Liu,Hu,and Yang),Feihyla fuhuaFei,Ye,and Yang,Gracixalus yunnanensisYu,Hui,Wang,Rao,Wu,and Yang,Zhangixalus feae(Boulenger),Rhacophorus rhodopusLiu and Hu.

Comparison:Raorchestes hillisisp.nov.differs fromR.cangyuanensisby snout longer than eye diameter,tympanum distinct,outer metatarsal tubercle present,discs of fingers and toes not orange (snout shorter than eye diameter or equal,tympanum indistinct,outer metatarsal tubercle absent,discs of fingers and toes orange); differs fromR.gryllusby tympanum distinct,toe webbing rudimentary,and lacking a series of tubercles along the outer sides of forearm and foot (vs.tympanum indistinct,toe webbing more than half,and having a series of tubercles along the outer sides of forearm and foot);differs fromR.longchuanensisby wider than long,fingers lacking lateral dermal fringe,dorsal body having a distinct “X” shaped black blotch (vs.head length equal to width,fingers I and II having lateral dermal fringes,dorsal body lacking a “X” shaped black blotch or indistinct); differs fromR.menglaensisby head wider than long,tympanum distinct,dorsal surface smooth,without white tubercles,discs of fingers and toes orange (vs.head length equal to width,tympanum indistinct,dorsal surface rough,with white tubercles,discs not orange); differs fromR.parvulusby snout longer than eye diameter,canthus rostralis distinct,and fingers and toes lacking lateral dermal fringe (vs.snout shorter than eye diameter,canthus rostralis rounded,and inside of all fingers having lateral dermal fringe,toes having lateral dermal fringe except for toe V); differs fromR.hillisiby head wider than long,toes lacking lateral dermal fringe,discs of fingers and toes orange (vs.head longer than wide,toes having lateral dermal fringe except outside of toe I and both sides of toe II,discs not orange).

4.Discussion

4.1.Taxonomic confusions in Raorchestes menglaensisTaxonomic studies ofRaorchestesare difficult to be conducted due to their morphological conservativeness and remarkably similar characters,which may cause ambiguities in taxonomy and distributions.The record ofR.parvulusfrom China was reported by Yuet al.(2019) based on specimens collected from Menglun,Mengla,southern Yunnan,but these samples clustered with ourR.menglaensisfrom the type locality (Zhushihe,Mengla,Yunnan) without distinct divergence (Figure 1; Table 2).Furthermore,the important morphological character of the specimens provided by Yuet al.(2019) is distinctively different fromR.parvulus(Boulenger,1893; Bossuyt and Dubois,2001) in having:both fingers and toes lacking lateral dermal fringes (vs.fingers and toes having fringes,but not evident on toe V inR.parvulus) whereas similar toR.menglaensis.Thus,based on our results,we conclude that the record ofR.parvulusfrom China that reported by Yuet al.(2019) is actually misidentification ofR.menglaensis,and it should be revised toR.menglaensis.

Moreover,Yuet al.(2019) used a sequence ofR.menglaensisfrom GenBank (GenBank accession no.GQ285676) in the phylogenetic tree and the locality was given as “Mengla,Yunnan”.However,according to the GenBank accession no.(GQ285676),this sequence was submitted by Liet al.(2009) and the locality was actually provided as “Lvchun,Yunnan”,and this sequence clustered with our samples ofR.huanglianshanfrom Lvchun,Yunnan.Therefore,the sequence ofR.‘menglaensis’(GQ285676) used by Yuet al.(2019) should be revised toR.huanglianshan.

4.2.Taxonomic identity of Raorchestes parvulusRaorchestesparvuluswas originally described by Boulenger (1893) from“Karin Bia-po”,eastern Myanmar (Figure 4; orange circle,no.3).This species has been widely reported across northern Indochina,including Myanmar,China,Thailand,Vietnam,Laos,Cambodia,Bangladesh and peninsular Malaysia (Frost,2020),however,most of the country records lacking detailed comparison with the type specimen and original description(Bourret,1942; Taylor,1962; Nutphund,2001; Ohleret al.,2002;Stuartet al.,2005; Grismeret al.,2006; Ghose and Bhuiyan,2012).Additionally,recent phylogenetic analysis failed to support the monophyly ofR.parvulusclade (Chanet al.,2018; Wuet al.,2019),and none of the available sequences ofR.parvulusin GenBank is come from its type locality,which means the phylogenetic position ofR.parvuluscannot be confirmed at present.

According to the original description ofR.parvulusprovided by Boulenger (1893) and description of lectotype (MSNG 29838.A,adult female) by Bossuyt and Dubois (2001),it can be distinguished from all Chinese congeners by the following combination of morphological characters:(1) head broader than long; (2) snout length shorter than eye diameter; (3) interorbital space larger than upper eyelid,equal to internarial distance; (4)tympanum distinct; (5) dermal fringe on in side of all fingers; (6)dermal fringe on all toes,but not evident along toe V.Therefore,R.parvulusis currently unknown from China.Consequently,the record ofR.parvulusshould be delisted from Chinese herpetofauna.

4.3.Underestimated Raorchestes diversity from ChinaYet despite the fact that fewRaorchestesspecies have been known from China,the recent studies continuously uncoveredRaorchestesdiversity from China (Wuet al.,2019; this study).Along with the two new species described herein,the number of the genusRaorchestesis increased to five,includingR.longchuanensis,R.menglaensis,R.cangyuanensis,R.hillisi,andR.huanglianshan.Due to the small body size and limited dispersal ability,Raorchestesspecies could be easily affected by biogeographic isolation,which may generate considerable biodiversity.These recent findings suggest that the trueRaorchestesbiodiversity from China has been underestimated,and future herpetofauna survey will likely continue to yield new discoveries from the region.

AcknowledgementsWe thank Mr.Kai WANG(University of Oklahoma,USA),Mr.Lingui HE,Mr.Wenbo YU,Mr.Zhongxiong FU,Mr.Hanliu AI,Mr.Guangxu HUANG,and Mr.Wen ZHANG for the fieldwork,Mr.Zhian ZHANG (Huanglianshan National Nature Reserve)for supporting our research,Mr.Mian HOU (Sichuan Normal University) for proving some information.We also thank the reviewers for their suggestions.This work was supported by the Strategic Priority Research Program of Chinese Academy of Sciences (CAS) (XDB31000000); the National Natural Science Foundation of China (31722049,31900322,31401983); the CAS“Light of West China” Program (2018XBZG_JCTD_001)and Talent Program from Organization Department of Sichuan Provincial Committee; Biological Resources Programme,Chinese Academy of Sciences (KFJ-BRP-017-14),the International Partnership Program of Chinese Academy of Sciences (151751KYSB20190024),and Biodiversity Survey,Observation and Assessment Program of Ministry of Ecology and Environment of China and Honghe University (14bs10,2015HB0302) to WJ.