A peer-reviewed open-access journal Zookeys 1 151:47—65 (2023) doi: 10.3897/zookeys.1151.95616 RESEARCH ARTICLE #ZooKey S https:/ / ZOO keys. pensoft.net Launched to accelerate biodiversity research A new species of Bush frog (Anura, Rhacophoridae, Raorchestes) from southeastern Yunnan, China Junkai Huang'?’, Xiao Long Liu?’, Lingyun Du'”’,, Justin M. Bernstein’, Shuo Liu®, Yun Yang®, Guohua Yu'”, Zhengjun Wu!” | Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education, Guilin 541004, China 2 Guangxi Key Laboratory of Rare and Endangered Animal Ecology, Guangxi Normal University, Guilin 541006, Guangxi, China 3 Key Laboratory for Conserving Wildlife with Small Populations in Yunnan, Southwest Forestry University, Kunming 650224, Yunnan, China 4 Department of Biological Sciences, Rutgers University Newark, 195 University Ave Newark, NJ 07102, USA 5 Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China 6 The Management Bureau of Wenshan National Nature Reserve, Wenshan 663000, Yunnan, China Corresponding authors: Guohua Yu (yugh2018@126.com), Zhengjun Wu (wu_zhengjun@aliyun.com) Academic editor: A. Ohler | Received 24 September 2022 | Accepted 20 December 2022 | Published 28 February 2023 Attps://z00 bank. org/4AACBOA6-2800-465 D-A 1 0F-E69B4CEE3E05 Citation: Huang J, Liu XL, Du L, Bernstein JM, Liu S, Yang Y, Yu G, Wu Z (2023) A new species of Bush frog (Anura, Rhacophoridae, Raorchestes) from southeastern Yunnan, China. ZooKeys 1151: 47-65. https://doi.org/10.3897/ zookeys.1151.95616 Abstract In this study, based on morphological and molecular data, a new bush frog species is described from Yunnan, China. Eleven samples of Raorchestes malipoensis sp. nov. were collected from Malipo County, southeastern Yunnan. This species can be distinguished from other congeners by a combination of 13 morphological characters. Phylogenetic analyses based on the 16S rRNA gene indicate that these indi- viduals form a monophyletic group, and genetic divergence between this clade and its closest relatives is higher than 3.1%, which is comparable to the divergence between recognized Raorchestes species. The discovery of this new species suggests that additional extensive surveys in the southeastern Yunnan would yield more amphibian lineages yet unknown to science. Keywords Kurixalus gryllus, Raorchestes gryllus, Raorchestes malipoensis sp. nov., taxonomy * These authors contributed equally. Copyright Junkai Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 48 Junkai Huang et al. / ZooKeys 1151: 47-65 (2023) Introduction The genus Raorchestes Biju, Shouche, Dubois, Dutta & Bossuyt, 2010 belongs to the family Rhacophoridae Hoffman, 1932. It includes bush frogs with adult size ranging from 10.0 mm to 50.5 mm (Priti et al. 2016). They are distinguished by the presence of a transparent/translucent vocal sac, the absence of vomerine teeth, and direct development without free swimming tadpoles (Seshadri et al. 2012). The genus Raorchestes currently contains 74 species, ranging from the southern tip of the Indian Peninsula to northeastern India, Indo-China, and southwestern China (Frost 2021): most are from south and Southeast Asia including southern India to Nepal, Myanmar, Thailand, Laos, southern China, Vietnam, and West Malaysia. Of the 74 recognized species, seven species have been originally described from China: Raorchestes longchuanensis (Yang & Li, 1978), R. menglaensis (Kou, 1990), R. andersoni (Anderson, 1927), R. cangyuanensis (Wu et al., 2019), R. dulongensis (Wu et al., 2021), R. hillisi Jiang et al., 2020), and Rk. huanglianshan (Jiang et al., 2020). Detailed ecological data is not available for the species reported in China except for R. longchuanensis, for which Yan et al. (2021) reported the breeding mode. Many Raorchestes species from the region were described with few diagnostic char- acters and limited morphological data, which hampers the identification of these small- sized bush frogs (Jiang et al. 2020). In addition, the taxonomy of Raorchestes gryllus is under dispute. It was originally described as Philautus gryllus Smith, 1924, from Lang- bian Peaks, southern Vietnam. Biju et al. (2010) classified this species into Raorchestes according to the 16S sequences from Pac Ban, Tuyen Quang, northern Vietnam, and recently Poyarkov et al. (2021) suggested a transfer to Kurixalus based on morphological and molecular data of specimens from the type locality (Langbian, southern Vietnam). In this work we studied specimens allocated to Raorchestes from Malipo County. This county is located in the southeast of Yunnan Province, and lies on the China- Vietnam border where few herpetological investigations have been conducted. During the fieldwork, we collected 11 specimens of a small-sized bush frog that could be as- signed to the genus Raorchestes based on morphological and molecular evidence. Phy- logenetically, these specimens were grouped together with a misidentified “R. gryllus” from Pac Ban, Tuyen Quang, northern Vietnam. However, considering that the type locality of Philautus gryllus, Langbian Plateau, is 1200 km far from the China-Vietnam border and that obvious morphological differences exist between Philautus gryllus and the lineage consisting of individuals from China-Vietnam border region, we consider that these specimens represent a new species that we formally describe here. Materials and methods Sampling Fieldwork was conducted at Malipo County, Yunnan Province, China (23.182°N, 104.78°E, elevation 1496 m). Six specimens were collected on 7 May 2019 (Figs 1, 2) and another five specimens were collected on 22 July 2020. Specimens were collected by hand A new species of Bush frog 49 110°0'O"E 10°0'0"N (*) Raorchestes malipoensis sp. nov. @) Kurixalus gryllus O Raorchestes tuberohumerus O Raorchestes parvulus O Raorchestes menglaensis 1 @ Raorchestes longchuanensis @ Raorchestes huanglianshan @ Raorchestes hillisi 0°00" : ; a idl O Raorchestes ghatei @ Raorchestes dulongensis oO Raorchestes cangyuanensis @ Raorchestes bombayensis 80°0'0"E 90°0'0"E 100°0'0"E 110°0'0"E Figure |. The type locality of Raorchestes malipoensis sp. nov., its closest relatives, and also Kurixalus gryllus (previously Raorchestes gryllus). and subsequently euthanized with 20% ethanol following standard euthanasia protocols for amphibians. Liver or muscle tissues were taken from the specimens and preserved in 95% ethanol before fixing them in 75% ethanol. Voucher specimens SWFU 3110, SWFU 3113, SWFU 3114, SWFU 3116, SWFU 3111, and SWFU 3112 were deposited at Southwest Forestry University (SWFU). GXNU 000338, GXNU 000339, GXNU 000340, GXNU 000341, GXNU 000342 were deposited at Guangxi Normal University (GXNU). Morphology and morphometrics All the measurements were made with slide calipers to the nearest 0.1 mm. Morpho- logical terminology and measurement methods followed Fei et al. (2009). The mor- phological characters include: snout-vent length (SVL); head length (HL); head width 50 Junkai Huang et al. / ZooKeys 1151: 47-65 (2023) Province, 23.182°N, 104.78°E, elevation 1496 m, China. (HW); snout length (SL); internarial distance (INS); interorbital distance (IOS); eye horizontal diameter (EHD); maximum width of upper eyelid (UEW); tympanum di- ameter (TD); forelimb and hand length (FAHL); width of lower arm (LAW); hand length (HAL); femur length (FML); tibia length (TBL); length of tarsus and foot (TFL); foot length (FOL); tibia width (TBW); and femur width (FMW). Morpho- logical measurements of the specimens are given in Table 1. Males and females (breed- ing individuals) were identified based on the presence or absence of an external single subgular vocal sac. Comparative morphological data of congeneric species were taken from previous studies and are presented in Table 2. DNA sequencing and analyses of sequences Total DNA was extracted using a commercial tissue DNA isolation kit (Chenlu Biotech, China). For seven specimens in this study, the mitochondrial gene 16S ribo- somal RNA (16S rRNA) gene was sequenced. ‘The fragments of 16S rRNA were am- plified using primers 16Sar-L (5°-CGCCTGTTTATCAAAAACAT-3’) and 16Sbr- H (5’-CCGGTCTGAACTCAGATCACGT-~-3’) (Palumbi et al. 1991). Polymerase chain reactions (PCR) amplifications were performed in a 25 ul reaction volume with an initial denaturation at 94 °C for 5 min, followed by 35 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 A new species of Bush frog 51 Table I. Measurements (mm) of adult specimens in the type series of Raorchestes malipoensis sp. nov. Abbreviations defined in the Materials and methods. Sex Males (n = 4) Females (n = 2) Males (n = 3) Females (n = 2) Catalog No. SWFU SWFU SWFU SWFU SWFU SWFU GXNU GXNU GXNU GXNU- GXNU 3110 3113 3114 3116 3111 3112 000338 000339 000341 000340 000342 SVL 17.1 17.0 16.5 14.7 19.3 19.0 17.5 17.7 17.3 18.7 18.3 HL 5.7 5.8 6.3 5.2 6.5 79 5.9 6.4 Def, 6.3 6.7 HW RA 75 8.2 5.5 8.2 7.9 6.5 6.8 6.4 Pp 2 SL 2.5 2.6 1.8 D5 2.9 2.6 2 2.6 2.4 25 2.2 INS 1.5 2.2 2.2 2.0 2:2. 2.1 2.2 2.1 2.3 2:2, 2 IOS 2.7 2.7 2.9 2.9 BD 2.9 2.9 2.6 2.9 227: 2.9 UEW 1.2 1.2 1.9 1.4 1.6 1.7 1.6 1.3 1.4 1.4 1.7 EHD vip 2.1 2.1 2.6 2.6 2.8 2.4 2.6 2.4 2:5 DD TD 1.4 1.4 LS 1.3 1.1 1.5 1.2 1.5 1.4 1.6 1.4 FAHL 9.3 9.3 8.8 7.0 8.6 9.6 7.8 8.2 Were, 7.3 fea HAL 5.6 5.6 5.3 4.2 5:2 5.6 5.3 52. 553 4.6 5.4 LAW 1.7 2.0 1.6 1.3 1.4 1.6 1.5 1.2 1.6 1.3 1.3 TBL 9.1 8.7 8.4 75 9.1 9.2 9 8.8 8 8.7 8.8 FML View) 9.2 8.3 7.1 8.0 10.2 Te 8 75 8.2 7.9 TBW 2.1 2.6 1.8 1.5 2.0 2.8 1.7 1.7 1.8 1.8 1.9 TFL 9.8 10.9 10.6 8.8 10.0 11.8 10.8 11 10.1 9.6 10.7 Table 2. The source of morphological data for Raorchestes species used in this study. ID Raorchestes species Literature 3 Raorchestes ghatei Padhye, Sayyed, Jadhav, & Dahanukar, 2013 Padhye et al. 2013 4 Raorchestes parvulus (Boulenger, 1893) Bossuyt and Dubois 2001 5 Raorchestes cangyuanensis Wu, Suwannapoom, Xu, Murphy, & Che, 2019 Wau et al. 2019 6 Raorchestes longchuanensis (Yang & Li, 1978) Al-Razi et al. 2020 b; Yang and Li 1978 Wi Raorchestes menglaensis (Kou, 1990) Jiang et al. 2020 8 Raorchestes hillisi Jiang Ren, Guo, Wang & Li, 2020 Jiang et al. 2020 g Raorchestes huanglianshan Jiang, Wang, Ren, & Li, 2020 Jiang et al. 2020 10 = Raorchestes dulongensis Wu, Liu, Gao, Wang, Li, Zhou, Yuan, & Che, 2021 Wu et al. 2021 11 Raorchestes andersoni (Ahl, 1927) Bossuyt and Dubois 2001 12. Raorchestes rezakhani Al-Razi, Maria, & Muzaffar, 2020 Al-Razi et al. 2020 a 13 Raorchestes annandalii (Boulenger, 1906) Che et al. 2020 PCR products were sequenced using an ABI 3730 automated sequencer. To study the phylogenetic relationships among Raorchestes species, matrilineal genealogies were reconstructed based on the 16S fragment. Fifty-two sequences of Raorchestes and repre- sentative outgroups (Jiang et al. 2020) were downloaded from GenBank (Table 3). The dataset was checked by eye and manually adjusted using MEGA 6.0 with default set- tings (Tamura et al. 2013), and the alignment was checked by eye and adjusted manu- ally. JMODELTEST v. 2.1.7 (Darriba et al. 2012) was used to select an appropriate nucleotide substitution model for Bayesian Inference (BI). The GTR+G+I model was chosen as the best-fit model following the Bayesian information criterion (BIC; Posada 2008). Bayesian analysis was performed using MrBayes 3.2 (Ronquist et al. 2012). For BI analyses, the Monte Carlo Markov chain length was run for 120,000,000 genera- 52 Junkai Huang et al. / ZooKeys 1151: 47-65 (2023) SWFU 3111 R. malipoensis sp. nov. GXNU 000341 R. malipoensis sp. nov. GXNU 000338 R. malipoensis sp. nov. GXNU 000339 R. malipoensis sp. nov. SWFU 3110 A. malipoensis sp. nov. (GXNU 000340 R. malipoensis sp. nov. (GQ285674 “R. gryllus" GXNU 000342 R. malipoensis sp. nov. MN475870 R. hillisi MT488411 R. Jongchuanensis MW537814 R. dulongensis MH590201 R. parvulus MT488410 R. menglaensis MT488414 R. huanglianshan MN475866 R. cangyuanensis JX092657 R. bombayensis JX092697 R. tuberohumerus KF366391 R. ghater KM596557 R. indigo 0.99/94 —————._. JX092651 R. ponmudi ___ KM596556 R. emerald JX092654 R. griet JX092716 R. coonoorensis JX092691 R. charius JX092719 R. marki KMS596540 R. aureus JX092715 R. tinniens KMS596552 R. montanus KM596575 R. primarrumfi AY141841 R. signatus JX092667 R. chromasynchysi JX092649 R. graminirupes JX092677 R. crustai JX092679 R. johnceei 0.98/88 JX092665 R. glandulosus JX092686 R. jayarami JX092680 R. bobingeri JX092659 R. Juteolus JX092721 R. travancoricus (092701 FR. kadalarensis JX092723 R. agasthyaensis EU450017 R. chiorosomma JX092656 R. chotta JX092661 R. nerostagona JX092650 R. akroparaliagi JX092653 R. beddomii JX092693 R. theverkaufi JX092655 R. munnarensis JX092708 R. anilf JX092683 R. resplendens JX092668 R. dubois JX092674 R. manohar JX092722 R. uthamani JxX092718 R. kaikatti KM596558 R. kakachi JX092684 R. sushili a- JX092666 R. ochlandrae 1 kis96549 R. flaviocularis GQ285673 Philautus abditus | Outgroup 0.04 Figure 3. Phylogram of Raorchestes derived from analyses of concatenated DNA fragments of the mi- tochondrial 16S rRNA. Nodal support values with Bayesian posterior probability (BPP) > 0.95 / ML inferences (ML-BS) > 70 are shown near the respective nodes. A “-” denotes a Bayesian posterior prob- ability < 0.95 and bootstrap support < 70. The scale bar represents 0.1 nucleotide substitutions per site. tions and sampled every 100 generations with a burn-in of 25%. Convergence was assessed by the average standard deviation of split frequencies (below 0.01) and ESS values (greater than or equal to 200) in TRACER 1.5 (Rambaut and Drummond. 2009). Maximum likelihood (ML) analyses were performed using RAxML v. 8.2.10 (Stamatakis 2014) with 1000 rapid bootstrap replicates under GTR+I+G nucleotide substitution model for the concatenated dataset (Stamatakis, 2014). Mean genetic dis- tances (uncorrected p-distance) between and within species were calculated in MEGA v. 6.0.6 (Tamura et al. 2013) based on 16S sequences. Results The final DNA sequence dataset is consisted of 59 sequences and the length of the sequence alignment is 542 base pairs (bp) (Table 3), of which 194 sites are vari- able and 135 are parsimony informative. The BI and ML trees had almost identical topologies (Fig. 3). The samples from Malipo County, Yunnan Province form a monophyletic group and the sample from Pac Ban, Tuyen Quang (northern Vi- etnam) previously identified as R. gryllus was also nested in the clade with strong support (Fig. 3). Genetic distances between the samples from Malipo County and the other species of Raorchestes varied from 3.1% (R. longchuanensis) to 6.0% (R. huanglianshan) (Table 4). A new species of Bush frog 53 Table 3. Information on voucher numbers, GenBank accession numbers, and localities of specimens used in this study; for collections and their abbreviations see Material and methods. Species Voucher No. GenBank No. Locality Resource Ingroup Raorchestes malipoensis sp. nov. SWFU 3110 ON128247 Malipo, Yunnan, China This study Raorchestes malipoensis sp. nov. SWFU 3111 ON128241 Malipo, Yunnan, China This study Raorchestes malipoensis sp. nov. ROM 30288 GQ285674 Pac Ban, Tuyen Quang, Vietnam Li et al. 2009 Raorchestes malipoensis sp. nov. | GXNU 000338 ON128246 Malipo, Yunnan, China ‘This study Raorchestes malipoensis sp. nov. | GXNU 000339 ON128245 Malipo, Yunnan, China ‘This study Raorchestes malipoensis sp. nov. | GXNU 000340 ON128244 Malipo, Yunnan, China ‘This study Raorchestes malipoensis sp. nov. | GXNU 000341 ON128243 Malipo, Yunnan, China This study Raorchestes malipoensis sp. nov. | GXNU 000342 ON128242 Malipo, Yunnan, China This study Raorchestes dulongensis KIZ 035082 MW537814 Qinlangdang, Yunnan, China We et al. 2021 Raorchestes hillisi CIB 116331 MT488411 Xiding, Yunnan, China Jiang et al. 2020 Raorchestes longchuanensis KIZ 048468 MN475870 Unknown Wau et al. 2019 Raorchestes parvulus LSUHC:11118 = MH590201 Gunung Stong, Kelantan, Malaysia Chan et al. 2018 Raorchestes menglaensis CIB 116349 MT488410 Menglun, Yunnan, China Jiang et al. 2020 Raorchestes huanglianshan CIB 116365 MT488414 Lvchun, Yunnan, China Jiang et al. 2020 Raorchestes cangyuanensis KIZ 015855 MN475866 Cangyuanensis, Yunnan, China Wau et al. 2019 Raorchestes tuberohumerus CESF 148 JX092697 Western Ghats, India Vijayakumar et al. 2014 Raorchestes bombayensis CESF 1010 JX092657 Western Ghats, India Vijayakumar et al. 2014 Raorchestes ghatei AGCZRL KF366391 Western Ghats, India Padhye et al. 2013 Amphibia 128 Raorchestes griet CESF 073 JX092654 Western Ghats, India Vijayakumar et al. 2014 Raorchestes coonoorensis CESF 439 JX092716 Western Ghats, India Vijayakumar et al. 2014 Raorchestes charius CESF 132 JX092691 Western Ghats, India Vijayakumar et al. 2014 Raorchestes marki CESF 467 JX092719 Western Ghats, India Vijayakumar et al. 2014 Raorchestes indigo CESF 138 KM596557_ = Kudremukh Massif, Western Ghats, Vijayakumar et al. 2014 India Raorchestes emeraldi CESF 1365 KM596556 _ Valparai plateau, Western Ghats, India Vijayakumar et al. 2014 Raorchestes ponmudi CESF 063 JX092651 Western Ghats, India Vijayakumar et al. 2014 Raorchestes aureus CESF 1164 KM596540 Malabar, Western Ghats, India Vijayakumar et al. 2014 Raorchestes montanus CESF 130 KM596552 Western Ghats, India Vijayakumar et al. 2014 Raorchestes tinniens CESF 438 Jx092715 Western Ghats, India Vijayakumar et al. 2014 Raorchestes primarrumfi CESF 442 KM596575 _ Nilgiri Massif} Western Ghats,India Vijayakumar et al. 2014 Raorchestes signatus Unknow AY141841 Sri Lanka Meegaskumbura et al. 2002 Raorchestes chromasynchysi CESF 1127 JX092667 Western Ghats, India Vijayakumar et al. 2014 Raorchestes chotta CESF 1003 JX092656 Western Ghats, India Vijayakumar et al. 2014 Raorchestes nerostagona CESF 1061 JX092661 Western Ghats, India Vijayakumar et al. 2014 Raorchestes kadalarensis CESF 1766 Jx092701 Western Ghats, India Vijayakumar et al. 2014 Raorchestes agasthyaensis CESF 492 JX092723 Western Ghats, India Vijayakumar et al. 2014 Raorchestes travancoricus CESF 473 JX092721 Western Ghats, India Vijayakumar et al. 2014 Raorchestes luteolus CESF 1012 JX092659 Western Ghats, India Vijayakumar et al. 2014 Raorchestes beddomii CESF 072 JX092653 Western Ghats, India Vijayakumar et al. 2014 Raorchestes theuerkauft CESF 1342 JX092693 Western Ghats, India Vijayakumar et al. 2014 Raorchestes munnarensis CESF 094 JX092655 Western Ghats, India Vijayakumar et al. 2014 Raorchestes anili CESF 386 JX092708 Western Ghats, India Vijayakumar et al. 2014 Raorchestes resplendens CESF 1258 JX092683 Western Ghats, India Vijayakumar et al. 2014 Raorchestes dubois CESF 114 JX092668 Western Ghats, India Vijayakumar et al. 2014 Raorchestes kakachi CESF 1385 KM596558 Western Ghats, India Vijayakumar et al. 2014 Raorchestes kaikatti CESF 444 JX092718 Western Ghats, India Vijayakumar et al. 2014 Raorchestes sushili CESF 1259 JX092684 Western Ghats, India Vijayakumar et al. 2014 Raorchestes flaviocularis CESF 1252 KM596549 — Manalar Plateau, Western Ghats, India Vijayakumar et al. 2014 Raorchestes ochlandrae CESF 1111 JX092666 Western Ghats, India Vijayakumar et al. 2014 Raorchestes manohari CESF 1187 JX092674 Western Ghats, India Vijayakumar et al. 2014 54 Junkai Huang et al. / ZooKeys 1151: 47-65 (2023) Species Voucher No. GenBank No. Locality Resource Raorchestes uthamani CESF 483 JX092722 Western Ghats, India Vijayakumar et al. 2014 Raorchestes chlorosomma FB-2008c EU450017 Munnar, Idukki, Kerala, India Biju and Bossuyt 2009 Raorchestes crustai CESF 1199 JX092677 Western Ghats, India Vijayakumar et al. 2014 Raorchestes graminirupes CESF 044 JX092649 Western Ghats, India Vijayakumar et al. 2014 Raorchestes johneeei CESF 1236 JX092679 Western Ghats, India Vijayakumar et al. 2014 Raorchestes glandulosus CESF 1080 JX092665 Western Ghats, India Vijayakumar et al. 2014 Raorchestes jayarami CESF 1260 JX092686 Western Ghats, India Vijayakumar et al. 2014 Raorchestes bobingeri CESF 1238 JX092680 Western Ghats, India Vijayakumar et al. 2014 Raorchestes akroparallagi CESF 061 JX092650 Western Ghats, India Vijayakumar et al. 2014 Outgroup Philautus abditus ROM 33145 GQ285673 Krong Pa, Gia Lai, Vietnam Liet al. 2009 Table 4. Uncorrected pairwise sequence divergence (%) among 16S ribosomal RNA mtDNA sequences, including R. malipoensis sp. nov., R. dulongensis, R. hillisi, R. longchuanensis, R. menglaensis, R. huanglianshan, R. cangyuanensis, R. parvulus, R. bombayensis, R. tuberohumerus, and R. ghatei as shown in phylogenetic tree presented in Fig. 3. Species 1 2 3 4 5 6 7 8 9 10 R. malipoensis sp. nov. R. bombayensis 4.5 R. tuberohumerus 4.8 2.0 R. ghatei 4.5 4.2 4.0 R. parvulus 5.9 5.1 5.9 5.4 R. cangyuanensis 5.7 6.6 6.8 5.6 7.1 R. longchuanensis 3.1 4.7 4.9 4.0 4.0 5.6 R. menglaensis 532. 4.2 4.9 527. 2.0 5.9 4.7 R. hillisi 4.3 4.9 512 43 61 6.3 4.2 5.4 R. huanglianshan 6.0 6.1 6.4 5:2 45 6.8 5.4 4.5 5.4 R. dulongensis 5,2 6.1 6.4 5.0 6.6 6.6 4.2 6.2 33 ees Taxonomic account Raorchestes malipoensis sp. nov. https://zoobank.org/ODCF253A-45E1-4354-9C6B-AA44E7C6C309 Fig. 4, Table 1 “Pseudophilautus gryllus” (Li et al. 2009). “Raorchestes gryllus” (Biju et al. 2010). Holotype. GXNU 000339, adult male, collected from Malipo County, Yunnan Province (23.182°N, 104.78°E, elevation 1496 m) on 22 July 2020 by Shuo Liu. Paratypes. SWFU 3110, SWFU 3113, SWFU 3114, SWFU 3116, GXNU 000338, GXNU 000341 (six adult males), SWFU 3111, SWFU 3112, GXNU 000340, GXNU 000342 (four adult females), collected at the same locality as the holotype on 22 July 2020 by Xiaolong Liu and Shuo Liu. A new species of Bush frog 2» Diagnosis. The genus Raorchestes is a group of small frogs, diagnosed primar- ily on the basis of an adult snout-vent length between 15 and 45 mm; vomerine teeth absent; large gular pouch transparent while calling; nocturnally active; direct development without free-swimming tadpoles in all species for which the develop- ment is known (Biju et al. 2010). Although the mode of development in the new species remains unknown, R. malipoensis sp. nov. is placed in the genus Raorchestes due to the combination of following characters: small body size, vomerine teeth absent, single translucent external subgular vocal sac present, and tips of all fingers and toes expanded into discs with circum-marginal grooves. The new species is dis- tinguished from geographically and molecularly relevant congeners by the following combination of characters: (1) very small body size (males SVL 14.6-17.7 mm, n = 7; females SVL 18.3-19.3 mm, 7 = 4); (2) head wider than long; (3) tympanum small, supratympanic fold distinct; (4) tips of all fingers and toes yellow; (5) web- bing formula (I 2-2 I] 2—2 12-3 IV 3-2 V); (6) inner and outer metacarpal tubercle indistinct; (7) heels not meeting when limbs held at right angles to body; (8) tibiotarsal articulation reaching anterior border of eye when hindlimb is stretched alongside of body; (9) iris golden brown; (10) nuptial pad small and milky white; (11) inner metatarsal tubercle rounded, outer metatarsal tubercle absent; (12) fingers and toes having lateral dermal fringe; and (13) interorbital distance larger than eye horizontal diameter. Description of the holotype. Adult male (Fig. 4), body size small (SVL 17.7 mm); head wider than long (HL 6.4 mm; HW 6.8 mm); top of head rela- tively flat; snout rounded in profile, projecting beyond lower jaw; snout length al- most equal to interorbital distance at narrowest point (SL 2.6 mm; IOS 2.6 mm); the canthus rostralis rounded, loreal region slightly concave; tympanum small (TD 1.5 mm); internarial distance wider than maximum width of upper eyelid (INS 2.1 mm; UEW 1.3 mm); nostril slightly closer to tip of snout than to anterior corner of eyes; tongue pyriform, with a deep notch at posterior tip; vomerine teeth absent; pineal ocellus absent; eyes moderately large (EHD 2.6 mm) and protrud- ing, pupil horizontal; supratympanic fold distinct, from posterior corner of eye to above insertion of arm. Forelimbs fairly robust (FAHL 8.2 mm); relative finger lengths: I< I< IV < IU, tips of all four fingers expanded into discs with circum-marginal grooves; all fingers with lat- eral dermal fringes on both sides; subarticular tubercles distinct, rounded; supernumer- ary tubercles absent; no webbing between fingers; inner and outer metacarpal tubercle indistinct; nuptial pad is small and milky white on dorsal surface of the first finger. Foot long and relatively robust (TFL 11 mm), longer than tibia length (TBL 8.8 mm); relative toe lengths: I < II < V < III < IV; tips of toes with discs having circum-marginal grooves, toe discs smaller than finger discs; all toes with lateral dermal fringes on both sides; subarticular tubercles distinct, rounded; supernumerary tuber- cles absent; webbing formula (I 2 — 2 I] 2-2 HI 2-3 1V 3-2 V); inner metatarsal tubercle rounded, outer metatarsal tubercle absent. 56 Junkai Huang et al. / ZooKeys 1151: 47-65 (2023) Sao . _ Figure 4. Holotype (GXNU 000339) of Raorchestes malipoensis sp. nov. in life. Dorsal surfaces of head, body, forelimbs, thighs, and tibia rough with small gran- ules; upper eyelid with several small granules; throat, chest, and ventral surfaces of forelimbs smooth; abdomen, ventral side of thigh, and area around vent with granules; dorsolateral folds absent. Coloration of holotype in life. For coloration of the holotype in life see Fig. 4. Dorsal surface beige, with pale brown band between eyes; dorsal surface with a dark brown X-shaped marking; pale brown interorbital rectangle between eyes; upper and lower lips with white and black dots; supratympanic fold pale brown; iris golden brown; dorsal parts of arms and legs with dark brown crossbars that align; crotch with a distinct black patch bordering large creamy white plaque below the black patch near the groin; dorsal thigh beige with one brown crossbar when leg is bent in resting posi- tion; ventral surface body and beige, and area around vent with small black spots; discs of fingers and toes yellow. Coloration in alcohol. After preservation in alcohol, the general pattern did not change. Dorsal color changed to grayish brown, the blotches or spots blackish brown, discs on the fingers become pale gray similar to the body color, ventral side become whiter (Fig. 5). Etymology. The specific epithet is named for the type locality, Malipo County, Yunnan Province, China. We suggest “Malipo Bush Frog” as its English common name, and “Ma Li Po Guan Shu Wa (JR 52 HE DY IEE)” as its Chinese common name. A new species of Bush frog 57 Figure 5. Holotype (GXNU 000339) of Raorchestes malipoensis sp. nov. in preservative, showing A dor- sal view B ventral view C ventral view of hand D ventral view of foot. Distribution. Currently known from the type locality, Malipo County (Fig. 1), Yunnan Province, China and Pac Ban, Tuyen Quang, in north of Vietnam. Variation. The measurements are given in Table 1. GXNU 000338 has large black spots on dorsal side and GXNU000342 has distinctly darker ground color on dorsal side. Comparisons. Rather than comparing Rk. malipoensis sp. nov. to all known Raorchestes, we focus on our morphological comparison with phylogenetically closely related taxa and species without genetic data in adjacent countries (Table 5). The new species differs from R. menglaensis by 1) tubercles absent along the outer side of the forearm and foot; (2) head wider than long; (3) tympanum distinct (TD 1.1-1.6 mm, 7 = 11); (4) webbing formula (12-2 12-2 II12-—3IV3-2V); (5) lateral dermal fringe present (6) inner and outer metacarpal tubercle indistinct; (7) outer metatarsal tubercle absent; and (8) relative toe lengths: I < I< V < Ill < IV (vs. a series of tubercles along the outer side of the forearm and foot; head length and head width are approximately the same; tympanum indistinct; webbing formula (1 1 —2 UI 1 - 2'* IV 2'7_1 V); lateral dermal fringe present; inner and outer metatarsal tubercle present; outer metatarsal tubercle present; relative toe lengths: III = V, or V > III). Junkai Huang et al. / ZooKeys 1151: 47-65 (2023) 58 Pury ‘ueuun, ‘uendsued APIT? A? IP 1 quasqy punoy asurIO quasaigy qom AreyuowIpmy ped jendnu ystppoy jouNSsIpUy 'IGH < AGH CHa > SOI € = 4 ‘wu 061-19 € = 4 ‘wu 061-191 sisuauvntsuva “y “uUMOUWUN sueow Pak Ge 92: Pury ‘ueuuny ‘UNYSA'T APAPIIPIPI quasqy punoy JOUTISIPUT JOUTISIPUT asueiC quasqy AIl~Cé AI-~C-TUIC-Tu quasaig yunsiq 'IGH < AGH CH4a=SOl Jo “Ha < SOI Bo USEC LT | ere col WS eS else] uvgsuvysuvng “Yy POR ‘ueuUny ‘Teysusj APA? ITIP TI quasqy punoy jouNSsIpUy jounSsIpUy quasaig Alrame Al aic-l IWC - TO quasaig youUnIsIq 'IGH 7 AGH CHa 7 SOI y=4'LLI-GCFL [Witla |t €=4 ‘LS /I-SFL 2871129 “UY une) ‘ueuuny ‘ueyssuor) Al? I? A? If? 1 qjuasqy punoy quasalg quasaig asurso Jo ystharry qom AreyUsuTIpMy quasqy yuUTIsIq TCH 7 ACH CHu 7 SOI € = 4% ‘0'6I-0'S1 €=4 ‘0610'S sisuasuopnp "yy vIskeyey sepnsutued pur ryNsuruag euTypopuy Al? UI? A? Il? 1 quasqy quasal qUasa J quasaig wmnrpow uasaid Suiqqay\, y~unsiq TGH < AGH CHa * SOI LH ueoce ees 4 snjnasvd "y ule) ‘ueUuUN, ‘epsuay/y III AGH ‘'IGH < ACH CHa = CHa < SOI SOI 1° “CHa < SOI SL = AEF 1-9 91 IL = 4% ‘€6I-9F1 7 = 4 *G'07-6'81 F=4 C'6I-E 81 4 as ae Ma 9 | L=4‘L 1-991 sisuavpjsuau “Yy ‘sou ‘ds szsuaodypu “y asury sy Zug] 907 aane]ay gpsaqni yesreejoU 19INC apsaqny yesreyeqou JOUUT gposoqn) yedseoejou JoInNC gposeqn) yedseoejour Jouuy JOJOO ISIC] asuLy [eULop [esole'T] qoM 207, ped jendny umuedurdAT, IQH/AGH CH4/SOI (uruu UT) IJNpe Jo TAS (Wruu UT) sayeuay YNpe Jo TAS (Wrud UT) sayeur IyNpe JO TAS soodg ‘So1IUNOD SUTPUNOIINS UT eIep NIUIS OU YIM dsoy} JO exe) parejas Ajasoy ATTeonauasoyAyd yam ‘aou ‘ds sesuaodyyu ‘y Jo uosireduroyr *g¢ sjqeL aD) A new species of Bush frog Pony) ‘ueuuny pur I9qIT, Teuruedyy Yon ‘erpuy ley pue eury ‘ueuuny AI? A=I07 171 quasqy quasat] quasat quasai gy asurio SuIqqem ¢/T yunsIq 'IGH < AGH CHd > SOI 7 = 4 “WU ('7—-C'ET 7 = 4 WU ('F7—-G'ET 1uossapuv “Yy weujor A ‘Ney A* II quasqy Wasoig Wuasoig quasaig ysnrys Jo ‘a8ueIO “YsIPpry Surqqam ¥/T quasar] j~unsiq IGH * ACH CHd < SOI G = 4 “WU 6'E7-F IT G = 4 “WU 6 'E7-F'1T sisuauvngIsuo] “Y eIPUy ‘syeYyt) UIOISA/\ AI> I> ASIL>1 quasqy Juasoi qom AreyusuTpNy JOUTISIPUT ‘IGH < AGH CHi = SOI 2° ‘GH < SOI ¢= 4 WwW 7'8I-H'LZT ¢=u4 WW 7'8[-H'ZT snAgUngo1aqny Y PIPUy ‘sIeYyL) UTOISa\\ Surqqam ¢/T younNsIpuy —= U4 “WW QE —= U4 WU OE sisuakvquiog "Y RIPU] UsJoyseay ou pure sedepeurnpy Al? HI=A? Il? 1 quasqy quasqy quasaig quasaig quasaig qom AreyusuTIpNy yuUTIsIq TGH 7 AGH CH4 = SOI 7° “GH4 < SOI T= 4% ‘wu (°/T [= 4 ‘wu ("/T Hppunuuv y ysopepsueg usoIseayIION Al? I> A? IL? 1 juasqy quasqy quasqy quasqy Ysniy 10 YsIppry quasqy AT— KT ALE — “AlLUIC- *I We - Cl quasqy JOUNSIPUyT TGH < ACH CHa 7 SOI y= U4 “WU (61-881 y = u “WU ()'6[-8'81 TUvgYVZIL Y eIPU] ‘s¥eYyr) UIDISOA\ APIIFAP IPI quasqy punoy quasaig AT-Y%TALE-Z WaAc-CuUCc- TI quasqy jOUNsSIpuy IGH < ACH ZZ = 4 B6CH'S1 EL =4 B67-H'S1 6 = 4 “CCT-T'6I pags “y asuey syISug] 902 aanyEjay gposaqn) yesreyeJoU J3INC posaqn) yesreyejour JUUT gpseqn yedresejowl JoInNC 9progny jedreoeiour JOUUT TOTO IST] asury [eULop [eIoIe_] gomM 907, ped jendny umnuedurAT, ‘IQH/AGH CHa /SOI (urud UT) INpe Jo TAS (uruu UT) saTeuaz YNpe Jo TAS (Wrud UT) sayeur I[Npe JO TAS soodg ‘ponunuo’y *g ajqey 60 Junkai Huang et al. / ZooKeys 1151: 47-65 (2023) The new species differs from R. parvulus by (1) smaller female body size (females 18.3-19.3 mm, nv = 4); (2) interorbital distance larger than eye horizontal diameter; and (3) inner and outer metacarpal tubercle indistinct; (vs. female 23.6 mm, n = 1; interorbital distance smaller than eye horizontal diameter; inner and outer meta- carpal tubercle present). The new species differs from Rk. dulongensis by (1) head wider than long; (2) inter- orbital distance larger than eye horizontal diameter; (3) nuptial pad present; (4) yellow disc; and (5) inner and outer metacarpal tubercle indistinct (vs. head smaller than long; interorbital distance smaller than eye horizontal diameter; nuptial pad absent; greyish or orange disc; inner and outer metacarpal tubercle indistinct present). The new species differs from R. Aillisi by (1) larger female body size (females 18.3-19.3 mm, 7 = 4); (2) head wider than long; (3) interorbital distance larger than eye horizontal diameter; (4) webbing formula (I 2-2 112-2 H12-31IV3-2V); and (5) and relative toe lengths: I< I] < V < II < IV (vs. female 17.5 mm, 7 = 1; head longer than wider; interorbital distance smaller than eye horizontal diameter; webbing formula (II 1-2 II 1-2'? IV 2-1 V); relative toe lengths: I < I< I< V