Zoosyst. Evol. 100 (3) 2024, 1041-1052 | DOI 10.3897/zse.100.127635 yee BERLIN A field survey on the genus Xenophrys (Amphibia, Megophryidae) confirms underestimated diversity in the Gaoligong Mountains, with the description of a new species Yun-He Wu! 2", Zhong-Bin Yu!", Jin-Min Chen!, Felista Kasyoka Kilunda!?, Ding-Can Zhang’, Chang-Sheng Zuo*, An-Ru Zuo*, Zheng-Pan Duan’, Jing Che! 1 Key Laboratory of Genetic Evolution and Animal Models, and Yunnan Key Laboratory of Biodiversity and Ecological Conservation of Gaoligong Mountain, Kunming Institute of Zoology, Chinese Academy of Sciences, 650223, Kunming, Yunnan, China 2 Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, 05282, Yezin, Nay Pyi Taw, Myanmar 3 Kunming College of Life Science, University of the Chinese Academy of Sciences, 650204, Kunming, Yunnan, China 4 Administrative Bureau of Tongbiguan Provincial Nature Reserve, 679300, Dehong, Yunnan, China https://zoobank. org/0A DBE 147-7D99-45EC-A77D-9520BBIB7A9A Corresponding author: Jing Che (chej@mail.kiz.ac.cn) Academic editor: Umilaela Arifin # Received 16 May 2024 Accepted 9 July 2024 Published 25 July 2024 Abstract The Gaoligong Mountains, located in the western part of China’s Yunnan Province adjoining northern Myanmar, harbor a striking diversity of species and endemism. Previous studies have shown that amphibian diversity in this region remains underestimated. A field survey carried out in 2023 oversaw a collection of eight Xenophrys specimens from the Tongbiguan Provincial Nature Reserve, Yunnan Province, China. Subsequent molecular analyses revealed two distinct and previously undescribed lineages. Based on mor- phological evidence, we formally describe one of the lineages as a new species and tentatively assign the other lineage to _X. sp. due to the absence of adult specimens for examination. Our results bring the total number of Xenophrys species to 29 and the number of Xenophrys species known to occur in China to 11. Furthermore, our study reveals that five species and putative species of Xenophrys (X. dehongensis, X. glandulosa, X. periosa, X. yingjiangensis sp. nov., and_X. sp.) exhibit sympatric distribution. These findings highlight the need for future research to investigate the mechanisms of sympatric coexistence in Xenophrys. In addition, our study confirms that the amphibian diversity of the Gaoligong Mountains is undoubtedly underestimated. As a result, continued exploration of amphibians in the future is necessary to obtain a clearer understanding of the overall biodiversity in this region. Key Words Biodiversity, cryptic species, frog, sympatric distribution, Tongbiguan Provincial Nature Reserve, Xenophrys yingjiangensis sp. Nov. Introduction The Asian horned toads of the subfamily Megophryinae (Bonaparte, 1850) are widely distributed in tropical Asia, from India and Bhutan to China and south to the Sundas and the Philippines (Frost 2024). It currently includes 136 recognized species, with more than half of the species * These authors contributed equally to this work. described in the last 10 years (Frost 2024). As a conse- quence of both morphological similarity among species and the complex patterns of genetic divergence, the ge- neric classification of the subfamily Megophryinae has been constantly under debate (e.g., Delorme et al. 2006; Fei et al. 2009; Chen et al. 2017; Mahony et al. 2017; Lyu et al. 2023). In this study, we followed the classification Copyright Wu, Y.-H. 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. 1042 system outlined in Frost (2024) and Lyu et al. (2023), in which Megophryidae was divided into 10 genera for the convenience of our comparisons. The genus Xenophrys Gunther, 1864, of the subfamily Megophryinae is distributed widely throughout southern China to the Indochina Peninsula and currently includes 28 recognized species (Frost 2024). These species inhabit primarily montane forests. To date, 10 species have been recorded in China (Frost 2024), with nearly half of the recognized species described in the last 10 years (e.g., Mahony et al. 2018; Lyu et al. 2023; Shu et al. 2023). Moreover, five species of Xenophrys have been recorded in Yunnan (AmphibiaChina 2024; Frost 2024). The Gaoligong Mountains, located in the western part of China’s Yunnan Province bordering northern Myan- mar, form a long, narrow mountain range. It stretches 600 km from the Tibetan Plateau to Myanmar across a 5° latitude with a large elevation range of 210 m to 5000 m. Renowned as one of the world’s most significant biodiversity hotspots outside of the tropics, its complex geography, hydrology, and climate have fostered many distinct habitat types that support diverse biotic com- ponents (Chaplin 2005). Several cryptic and novel am- phibian species have been described from this mountain ecosystem in recent years (AmphibiaChina 2024), indi- cating that amphibian diversity in the region may still be diverse and largely underestimated. A recent herpetological survey conducted at the Gaol- igong Mountains, Yunnan Province, China, saw a collec- tion of some Xenophrys specimens. Subsequent studies, including molecular data and morphological compar- isons, revealed that these specimens represent five dis- tinct evolutionary lineages, two of which could not be assigned to any known Xenophrys species. Therefore, we herein describe one of the two lineages as a new species. 99°E 99°E Figure 1. Known distribution of Xenophrys yingjiangensis sp. yingjlangensis Sp. NOV. zse.pensoft.net Wu, Y.-H. et al.: A new species of Xenophrys (Amphibia, Megophryidae) from Yunnan, China Materials and methods Sampling Field surveys were conducted in August 2023. A total of eight Xenophrys specimens were collected in Tongbiguan Town, Yingjiang County, Yunnan, China (Fig. 1). After taking photographs, the toads were euthanized using ben- zocaine. Liver tissue was taken from the specimens and preserved in 95% ethanol at -80 °C. The specimens were then fixed in 10% formalin and subsequently stored in 75% ethanol after 24 hours. All the newly collected spec- imens were deposited in the herpetological collection of the Museum of the Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). Molecular data and phylogenetic analyses Total genomic DNA was extracted using the standard phe- nol-chloroform extraction protocol (Sambrook et al. 1989). The mitochondrial fragment 16S ribosomal RNA gene (16S rRNA) was amplified and sequenced using the primer pairs (5’-3’) 16SAR (CGCCTGTTTAYCAAAAACAT) and 16SBR (CCGGTYTGAACTCAGATCAYGT) (Koch- er et al. 1989). The polymerase chain reaction (PCR) was performed in a 25 ul volume reaction with the following conditions: an initial denaturing step at 94 °C for 4 min; 35 cycles of denaturing at 94 °C for 40 s; annealing at 55 °C for AO s; and extending at 72 °C for 1 min; and a final extend- ing step of 72 °C for 10 min. PCR products were sequenced using the same forward and reverse primers as those used in PCR. Sequencing was conducted using an ABI 3730xl DNA automated sequencer (Applied Biosystems, UK). All sequences were assembled from forward and reverse reads 102°E 105°E 102°E 105°E nov. The red pentagram indicates the type locality of Xenophrys Zoosyst. Evol. 100 (3) 2024, 1041-1052 and edited manually using DNASTAR LASERGENE 7.1. New sequences were deposited in GenBank (Table 1). Maximum likelihood (ML) and Bayesian inference (BI) were used to infer phylogenetic trees. Fifty-eight homolo- gous sequences of Xenophrys and representative outgroups (Brachytarsophrys feae, Leptobrachella ventripunctata, and Leptobrachium huashen) were downloaded from the GenBank (Table 1). The dataset was aligned using MUS- CLE v3.8 (Edgar 2004), checked by eye, and trimmed to minimize missing characters in MEGA v6.0.6 (Tamura et al. 2013). BI and ML analyses were performed on the CIP- RES web server (Miller et al. 2010) using MrBayes v3.2.4 (Ronquist et al. 2012). For BI analyses, the best-fit model of evolution was determined using the Bayesian information criterion (BIC; Posada 2008) implemented in jModelTest 1043 2.1.7 (Darriba et al. 2012). GTR+G was the best-fit model of evolution for 16S rRNA. The Monte Carlo Markov chain length was run for 10 million generations and sampled ev- ery 1000 generations, with a burn-in of 25%. Convergence was assessed by the average standard deviation of split fre- quencies (below 0.01) and ESS values (greater than or equal to 200) in TRACER 1.6 (Rambaut et al. 2014). ML analyses were performed using RAxML-HPC BlackBox 8.2.10 (Sta- matakis 2014) with 1,000 bootstrap replicates and using the standard bootstrap search (random seed value 12,345) un- der the GTR+gamma nucleotide substitution model. Mean genetic distances between and within species were calcu- lated using uncorrected pairwise distances (p-distance) by 16S rRNA implemented in MEGA v6.0.6, with complete deletion of missing data and gaps (Tamura et al. 2013). Table 1. Localities, voucher information, and Genbank accession numbers for all specimens used in this study. Species Voucher Locality Accession No. Reference Xenophrys ancrae SDBDU 2009. 727 India, Arunachal, Changlang KY022318 Mahony et al. 2018 Xenophrys ancrae ZS| A 11606 India, Arunachal, Changlang MN734391 Mahony et al. 2020 Xenophrys auralensis NCSM 79599 Cambodia, Kampong Speu, Aural KX811807 Chen et al. 2017 Xenophrys awuh SDBDU 2007.192 India, Nagaland state, Kohima district, above New Ministers’ Hill, _MN734399 Mahony et al. 2020 Aradurah Forest Xenophrys awuh MZUHC 314 India, Mizoram MT793046 — Lalronunga et al. 2020 Xenophrys dehongensis SYS a005823 China, Yunnan, Yingjiang 0Q180993 Lyu et al. 2023 Xenophrys dehongensis KIZ 053847 China, Yunnan, Tongbiguan PP989323 This study Xenophrys dzukou SDBDU 2007.106 India, Nagaland, Kohima KY022324 Mahony et al. 2017 Xenophrys flavipunctata SDBDU 2009.298 India, East Khasi Hills district, Meghalaya MH647517 Mahony et al. 2018 Xenophrys flavipunctata SDBDU 2007.134 India, Kohima district, Nagaland MH647518 Mahony etal. 2018 Xenophrys glandulosa KIZ 013609 China, Yunnan, Wenlong KX811761 Chen et al. 2017 Xenophrys glandulosa SYS a003757 China, Yunnan, Mt Gaoligong MH406754 Liu et al. 2018 Xenophrys glandulosa KIZ 053845 China, Yunnan, Tongbiguan PP989322 This study Xenophrys himalayana SDBDU 2009.1227 India, West Kameng district, Arunachal Pradesh MH647526 ~=Mahony et al. 2018 Xenophrys himalayana SDBDU 2009.1206 India, West Kameng district, Arunachal Pradesh MH647527 ~=Mahony et al. 2018 Xenophrys lancangica SYS a007794 China, Yunnan, Simao 0Q180994 Lyu et al. 2023 Xenophrys lancangica SYS a007825 China, Yunnan, Jinghong 0Q180997 Lyu et al. 2023 Xenophrys lekaguli FMNH 265955 Thailand, Sa Kaeo, Mueang Sa Kaeo KY022214 Mahony et al. 2017 Xenophrys major RGK 0089 India, Manipur, Tamenglong KY022308 Mahony et al. 2017 Xenophrys major SDBDU 2007.229 India, Nagaland, Kohima MH647514 Mahony et al. 2018 Xenophrys mangshanensis KIZ 021786 China, Guangdong, Mt Nanling KX811790 Chen et al. 2017 Xenophrys mangshanensis SYS a002177 China, Guangdong, Huaiji MH406666 Liu et al. 2018 Xenophrys maosonensis KIZ 016045 China, Yunnan, Xichou KX811780 Chen et al. 2017 Xenophrys maosonensis SYS a008748 China, Guangxi, Mt Shiwandashan 0Q181000 Lyu et al. 2023 Xenophrys maosonensis SYS a008766 China, Guangxi, Mt Shiwandashan 0Q181002 Lyu et al. 2023 Xenophrys medogensis KIZ 06657 China, Xizang, Beibeng KX811768 Chen et al. 2017 Xenophrys medogensis SYS a002932 China, Xizang, Medog MH406725 Liu et al. 2018 Xenophrys megacephala ZSIA 11213 India, Meghalaya, Ri Bhoi KY022315 Mahony et al. 2018 Xenophrys montana SDBDU 2011.1047 India, West Bengal, Darjeeling KY022312 Mahony et al. 2017 Xenophrys montana SDBDU 2011.1049 India, West Bengal, Darjeeling MH647506 = Mahony et al. 2018 Xenophrys numhbumaeng SDBDU 2007.041 India, Manipur, Tamenglong KY022316 Mahony et al. 2017 Xenophrys numhbumaeng BNHS 6076 India, Manipur, Tamenglong MN734393 Mahony et al. 2020 Xenophrys oreocrypta SDBDU 2008.1400 India, West Garo Hills district, Meghalaya MH647520 Mahony et al. 2018 Xenophrys oreocrypta SDBDU 2009.1108 India, West Garo Hills district, Meghalaya MH647521 Mahony etal. 2018 Xenophrys oropedion ZS| A 11601 India, Meghalaya state, East Khasi Hills district, Shillong, Malki Forest MN734394 Mahony et al. 2020 Xenophrys oropedion ZSI A 11603 India, Meghalaya state, East Khasi Hills district, Shillong, Malki Forest MN734395 Mahony et al. 2020 Xenophrys pangdaensis YBU 21248 China, Xizang, Yadong County, Pangda Village ORO26569 Shu et al. 2023 Xenophrys pangdaensis YBU 21261 China, Xizang, Yadong County, Pangda Village ORO26572 Shu et al. 2023 Xenophrys periosa SDBDU 2009.793 India, East Siang district, Arunachal Pradesh MH647522 Mahony etal. 2018 Xenophrys periosa SDBDU 2009.1285 India, West Kameng district, Arunachal Pradesh MH647524 = Mahony et al. 2018 Xenophrys periosa CIB YN201909160 China, Yunnan, Gongshan, Dulongjiang MT225581 Shi et al. 2020 Xenophrys periosa KIZ 053849 China, Yunnan, Tongbiguan PP989324 This study Xenophrys robusta K5207/ZS111404 India, Sikkim, North Sikkim KX894674 Deuti et al. 2017 Xenophrys robusta SDBDU 2011.1057 India, West Bengal, Darjeeling KY022314 Mahony et al. 2018 Xenophrys serchhipii SDBDU 2009.612 India, Tripura, North Tripura KY022323 Mahony et al. 2018 Xenophrys serchhipil SDBDU 2008.1492 India, Manipur, Tamenglong MN734405 = Mahony et al. 2020 Xenophrys “sp.17” KIZ 011940 Myanmar, Myitkyina KX811792 Chen et al. 2017 Xenophrys “sp.17” KIZ 048503 China, Yunnan, Tongbiguan Provincial Nature Reserve KX811793 Chen et al. 2017 zse.pensoft.net 1044 Wu, Y.-H. et al.: A new species of Xenophrys (Amphibia, Megophryidae) from Yunnan, China Species Voucher Locality Accession No. Reference Xenophrys “sp.17” KIZ 048504 China, Yunnan, Tongbiguan Provincial Nature Reserve KX811794 Chen et al. 2017 Xenophrys “sp.17” KIZ 048505 China, Yunnan, Tongbiguan Provincial Nature Reserve KX811795 Chen et al. 2017 Xenophrys yingjiangensis sp. nov. KIZ 053814 China, Yunnan, Tongbiguan Provincial Nature Reserve PP989318 This study Xenophrys yingjiangensis sp. nov. KIZ 053815 China, Yunnan, Tongbiguan Provincial Nature Reserve PP989320 This study Xenophrys yingjiangensis sp. nov. KIZ 053828 China, Yunnan, Tongbiguan Provincial Nature Reserve PP989319 This study Xenophrys yingjiangensis sp. nov. KIZ 053848 China, Yunnan, Tongbiguan Provincial Nature Reserve PP989317 This study Xenophrys sp. KIZ 053846 China, Yunnan, Tongbiguan Provincial Nature Reserve PP989321 This study Xenophrys takensis FMNH 261711 Thailand, Kampaeng, Khlong Lan KY022215 Mahony et al. 2017 Xenophrys truongsonensis IEBRA 4943 Vietnam, Dak Lak ON146200 Luong et al. 2022 Xenophrys truongsonensis IEBRA 4948 Vietnam, Lam Dong ON146201 Luong et al. 2022 Xenophrys truongsonensis IEBRA 4952 Vietnam, Ninh Thuan ON146202 Luong et al. 2022 Xenophrys zhang KIZ 014278 China, Xizang, Nyalam KX811765 Chen et al. 2017 Xenophrys zhangi SYS a008204 China, Xizang, Nyalam 0Q180998 Lyu et al. 2023 Xenophrys zunhebotoensis RGK41 India, Nagaland, Zunheboto KY022322 Mahony et al. 2017 Xenophrys zunhebotoensis SDBDU 2009.374 India, Nagaland, Kohima MN734418 = Mahony et al. 2020 Outgroups Brachytarsophrys feae KIZ YNO70570 China, Yunnan, Longchuan KX811809 Chen et al. 2017 Leptobrachella ventripunctata KIZ 046940 China, Yunnan, Wenlong KX811929 Chen et al. 2017 Leptobrachium huashen KIZ 049025 China, Yunnan, Mengyang KX811931 Chen et al. 2017 Morphology and morphometrics All the measurements were recorded with digital cali- pers to the nearest 0.1 mm. Morphological terminology followed Fei et al. (2009). Twenty-five measurements included the following: SVL: snout-vent length (mea- sured from tip of snout to vent); HDL: head length (measured from tip of snout to rear of jaw); HDW: max- imum head width (measured width of head at its widest point); SNT: snout length (measured from tip of snout to anterior corner of ocular aperture); ED: eye diameter (diameter of exposed portion of eyeball); UEW: width of upper eyelid (maximum width of upper eyelid); TD: tympanum diameter (measured as maximal diame- ter of tympanum); DNE: distance from nostril to eye (distance from the front of the eye to the center of the nostril); SN: distance from the center of the nostril to the tip of the snout; IND: internarial distance (distance between nares); TEY: distance from anterior edge of tympanum to posterior corner of eye; IOD: interorbital distance (measured at narrowest point between eyes on top of the head); FAL: forearm length (measured from the elbow to the wrist); LAD: (diameter of lower arm); FHL: forearm and hand length (distance from elbow to the tip of the third finger); TL: tibia length (distance from knee to heel); HL: hand length (distance from the posterior end of the inner metacarpal tubercle to tip of third finger); HLL: hindlimb length; FL: foot length (distance from the proximal end of inner metatarsal tubercle to the tip of fourth toe); THL: thigh length (from the cloaca to the knee); TAL: tarsus length (measured as the distance from knee to heel); FLI-IV: first to fourth finger length. Results The aligned sequence matrix of the 16S gene contained 490 bp, among which 215 sites were variable and 159 were par- simony-informative (including outgroups). Both BI and ML trees had almost identical topologies with relatively robust zse.pensoft.net support for most nodes, differing mainly at terminal nodes identified as weakly supported or collapsed. The genus Xe- nophrys was recovered as monophyletic with strong support from both analyses (BPP=1; BS=96; Fig. 2). The newly col- lected sympatric samples from Tongbiguan Nature Reserve, Yunnan, China, were divided into five highly divergent clades with strong nodal supports: three were nested within a clade containing recognized species, while the other two formed their own previously unknown lineages (Fig. 2). The newly collected specimen (KIZ 053849) and X. periosa (including the type specimens) nested into a single clade with strong support (BPP=1; BS=81; clade A), and the within-group mean genetic p-distance was 1.7% (0%-3.4%, Suppl. material 1). The newly collected specimen (KIZ 053845) strongly clustered with X. glan- dulosa (BPP=1; BS=100; clade B), with maximal uncor- rected pairwise 16S distances of merely 0.0%. The newly collected specimen (KIZ 053847) formed a monophylet- ic clade with XY. dehongensis from the paratype (BPP=1; BS=99; clade E), with shallow within-group genetic dif- ferentiation (0.7%, Suppl. material 1). For the two new distinct clades, the newly collected sam- ples and samples proposed as Megophrys sp. 17 in Chen et al. (2017) clustered into a monophyletic clade with a strong nodal support (BPP=1; BS=99; clade D). This clade was recovered as a sister taxon to _X. dehongensis. In addition, the remaining sample (KIZ 053846) formed an indepen- dent monophyletic clade, which clustered with X. awuh, X. zunhebotoensis, X. serchhipii, X. numhbumaeng, X. orope- dion, X. ancrae, X. megacephala, and_X. dzukou with mod- erate support (BPP=0.96; BS=81; clade C). The two puta- tive new species showed obvious genetic divergence from the other congeners. The genetic distance between the new populations and the other congeneric species ranged from 5.3% (with X. megacephala) to 12.0% (with X. lancangi- ca) for clade D, 7.1% (with X. ancrae), and 12.2% (with X. robusta) for clade C (Suppl. material 1). It 1s compa- rable to the divergences among the nearest neighbor ge- netic distances of the described Xenophrys species, which ranged from 2.1% (X. mangshanensis and X. maosonensis) to 15.7% (X. dzukou and R. awuh). In addition, these levels Zoosyst. Evol. 100 (3) 2024, 1041-1052 1/100 Leptobrachium huashen KIZ 049025 1045 Xenophrys periosa CIB YN201909160 181g Xenophrys periosa SDBDU 2009.1285 A Xenophrys periosa SDBDU 2009.793 Xenophrys periosa K1Z 053849 1/100 p-Xenophrys himalayana SDBDU 2009.1227 Xenophrys himalayana SDBDU 2009.1206 1/100 - Xenophrys medogensis SYS a002932 1/95 Xenophrys medogensis K1Z 06657 ‘100 pXenophrys robusta SDBDU 2011.1057 ‘Xenophrys robusta K5207/ZS11 1404 ‘ Xenophrys glandulosa SY S$ a003757 1/10 Xenophrys glandulosa K1Z, 053845 B “Xenophrys glandulosa KIZ 013609 1/100 -—Xenophrys flavipunctata SDBDU 2007.134 Xenophrys flavipunctata SDBDU 2009.298 ‘Xenophrys truongsonensis TEBRA4943 Xenophrys truongsonensis TEBRA4952 ‘Xenophrys truongsonensis IEBRA4948 97 | ~26¢Xenophrys maosonensis SYS a008748 Aenophrys maosonensis SYS a008766 Xenophrys maosonensis K1Z 016045 1/100 - Xenophrys mangshanensis KIZ 021786 Xenophrys mangshanensis SYS a002177 1/100 ¢ Xenophrys pangdaensis YBU21248 1/84 Xenophrys pangdaensis YBU21261 1/100 -Xenophrys lancangica SYS a007825 Xenophrys lancangica SYS a007794 1/100 -Xenophrys major RGK 0089 Xenophrys major SDBDU 2007.229 1/98_pXenophrys montana SDBDU 2011.1049 Xenophrys montana SDBDU 2011.1047 1/97 _¢ Xenophrys zhangi KIZ 014278 -Xenophrys zhangi SYS a008204 1/100 pXenophrys orepedion ZSI A 11603 Xenophrys oropedion ZS1 A 11601 1/97 -Xenophrys awuh SDBDU 2007.192 -/81 -Xenophrys awuh MZUHC 314 1/100 - Xenophrys zunhebotoensis SDBDU 2009.374 ‘Xenophrys zunhebotoensis RGK 0041 1 1/100_-Xenophrys serchhipii SDBDU 2008.1492 ‘Xenophrys serchhipii SDBDU 2009.612 1/100 ¢Xenophrys numhbumaeng BNHS 6076 1/98 ‘Xenophrys numhbumaeng SDBDU 2007.041 1/100 -Xenophrys oropedion ZS1 A 11601 -Xenophrys oropedion ZSI A 11603 1/100 _pXenophrys ancrae SDBDU 2009.727 0.96/81 aenaphr ancrae ZSI A 11606 enophrys megacephala ZSI A 11213 Cc Xenophrys sp. K1Z 053846 ‘Xenophrys dzukou SDBDU 2007.106 Xenophrys yingjiangensis sp. nov. KIZ 053848 Xenophrys yingjiangensis sp. noy. KLZ 053814 99 | Xenophrys yingjiangensis sp. nov. KIZ 053828 Xenophrys yingjiangensis sp. nov. KIZ 053815 Xenophrys "sp.17" KIZ 048503 Xenophrys "sp.17" KIZ 048504 Xenophrys "sp.17" K1Z 048505 ‘Xenophrys "sp.17" KIZ 011940 1/99 == Xenophrys gahonsetae SYS a005823 E Xenophrys dehongensis K1Z 053847 Xenophrys auralensis NCSM 79599 Xenophrys lekaguii FMNH 265955 Xenophrys takensis FMNH 261711 0.96/- 1/100 1/99, 0.96/76 D ‘Brachytarsophrys feae KIZ YNO70570 Leptobrachella ventripunctata KIZ 046940 0.05 Figure 2. Phylogram of Xenophrys based on the mitochondrial 16S rRNA gene. Node values with Bayesian posterior probabilities (BPP) < 0.95 and Bootstrap support (BS) < 70 are not shown. A “-” denotes Bayesian posterior probabilities (BPP) < 0.95 and boot- strap support (BS) < 70. New samples for the present study are indicated in bold font. of pairwise divergence of the 16S rRNA gene exceeded the acceptable threshold (3%) of species-level genetic diver- gence in anurans (Vences et al. 2005). Morphologically, these specimens from Clade D dif- fered from recognized congeners; therefore, we formally describe them as new. However, Clade C contains only one subadult specimen, and further surveys are needed to retrieve adult specimens. Taxonomic account Xenophrys yingjiangensis Wu, Yu, Chen & Che, sp. nov. https://zoobank.org/C2E61BB7-426B-448F-8A 9C-66B993E7201A Figs 3, 4, Table 2 Chresonymy. Megophrys sp17., Chen et al. 2017. Type material. Holotype: KIZ 053848, an adult male collected from Tongbiguan Provincial Nature Reserve, Yingjiang County, Yunnan, China (24.563°N, 97.639°E; el- evation 1478 ma.s.l.), collected by Zhong-Bin Yu, Dong An, Tian-En Chen, and Xian-Kun Huang on 12 August 2023. Paratypes: KIZ 048503-KIZ 048505, three adult males, from Tongbiguan Provincial Nature Reserve, Yingjiang County, Yunnan, China (24.546°N, 97.759°E; elevation 809 m as.l.), collected by Jin-Min Chen and Mian Hou on 11 August 2013; KIZ 053828, one adult male, collected at the same locality and with the same collection information as the holotype. Etymology. The specific epithet “yingjiang” is a Latinized adjective derived from the name of Yingjiang County, Yunnan Province, China, where the new species occurs. We propose the English common name “Yingji- ang horned toad” and the Chinese common name “Ying Jiang Jido Chan (4470 FAHEY”. Diagnosis. Xenophrys yingjiangensis sp. nov. differs from its congeners by a combination of the following morphological characters: (1) medium adult size, adult male SVL 44.6—49.8 mm (N=5); (2) head slightly lon- ger than wide; (3) tympanum distinct, narrow anteriorly, slightly widening posteriorly; (4) pupil vertically ellip- tical; (5) vomerine ridges and vomerine teeth present; (6) tongue large, oval-shaped, feebly notched posterior- ly; (7) relative finger lengths: II < IV < I < II; (8) the heels slightly overlapping when the tibias are positioned at right angles to the body axis; (9) tibio-tarsal articula- tion of straightened limb reaching the nostril; (10) lateral dermal fringes on toes distinct, narrow; (11) toes with ru- dimentary webbing; (12) inner metatarsal tubercle large, elongate; (13) a distinct narrow ‘\ /’-shaped parietoscapu- lar ridge present; (14) flesh pink ventral surface of thighs. Description of the holotype (measurements in Table 2). KIZ 053848, mature male, sized medium body (SVL 45.0 mm); head moderate (HDL/SVL 39.6%, HDW/ SVL 38.9%), slightly longer than wide (HDW/HDL 98.3%); snout obtusely rounded in dorsal view, obtusely projecting beyond the lower jaw in profile, without rostral appendage; triangular in dorsal view; top of head flat; loreal zse.pensoft.net 1046 Wu, Y.-H. et al.: A new species of Xenophrys (Amphibia, Megophryidae) from Yunnan, China D. Ventral view; E. Ventral view of hand, and F. Ventral view of foot. Photos by Zhong-Bin Yu. region vertical and concave; canthus rostralis angular; eyes large (ED/HDL 31.5%); eye less than twice as long as max- imum tympanum diameter (ED/TD 207.4%) and shorter than snout length (SNT 6.8 mm, ED/SNT 82.4%); tympa- num distinct, circular in shape, relatively small (TD/HDL 15.2%), with upper border concealed by supratympanic ridge; eye-tympanum distance (TYE 3.3 mm) longer than tympanum diameter (TD 2.7 mm); nostril rounded, laterally positioned, nostril closer to the tip of snout than to the ante- rior corner of the eye (SN/DNE 81.6%); internarial distance greater than interorbital distance IND/IOD 109.4%) and width of upper eyelid (IND/UEW 126.1%); pineal ocellus absent; vomerine teeth in two oblique series, positioned be- tween choanae, separated from each other by distance equal to distance from choanae; maxillary teeth present; choanae oval; tongue large, oval-shaped, feebly notched posteriorly; single internal vocal sac, with a sac slit opening on floor of mouth at each corner; pupil vertically elliptical (Fig. 3B). zse.pensoft.net Forelimbs moderately long and thin; forearm not en- larged relative to the upper arm, its length shorter than the hand length (FAL/HL 86.4%); fingers long and narrow, lateral fringes on fingers absent, relative finger lengths: IT