104 urn:lsid:arphahub.com:pub:f2cd1fff-21e4-581f-a7fa-850997197b7f urn:lsid:zoobank.org:pub:B1C81912-2D17-4CD8-8D2C-EFEAAAB2EF75 Vertebrate Zoology VZ 1864-5755 2625-8498 Senckenberg Gesellschaft für Naturforschung 10.3897/vz.73.e100889 100889 Research Article Amphibia Anura Megophryidae Taxonomy A new species of Boulenophrys from central Hunan Province, China (Anura: Megophryidae) Qian Tianyu 1 Hu Ke 1 Mo Xiaoyang 2 Gao Zhiwei 1 Zhang Na 2 Yang Daode csfuyydd@126.com 1 Institute of Wildlife Conservation, Central South University of Forestry and Technology, Changsha 410004, China Central South University of Forestry and Technology Changsha China College of Life Science, Hunan Normal University, Changsha 410081, China Hunan Normal University Changsha China

Corresponding author: Daode Yang (csfuyydd@126.com)

Academic editors

Raffael Ernst

 2023 03 10 2023 73 915 930 A6E3F6E2-927A-58BD-BA58-406167EE6371 73D439BE-596C-4A69-BCA3-7E4B35440C11 22 01 2023 18 09 2023 Tianyu Qian, Ke Hu, Xiaoyang Mo, Zhiwei Gao, Na Zhang, Daode Yang 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. http://zoobank.org/73D439BE-596C-4A69-BCA3-7E4B35440C11

Abstract

We re-examined the megophryinid population from Mt. Hengshan, Hunan Province, China previously identified as Boulenophrys brachykolos (under the name Megophrys brachykolos). Based on newly obtained molecular data, this population appears to be an independent lineage with a relatively distant phylogenetic relationship to B. brachykolos sensu stricto. Furthermore, this population exhibits distinct morphological characteristics that distinguish it from all its congeners. Therefore, we propose to recognize the Hengshan population of B. brachykolos as a new species, Boulenophrys hengshanensissp. nov. described herein.

 Frog diversity morphology phylogeny taxonomy Introduction

The genus Boulenophrys Fei, Ye and Jiang, 2016 is the largest branch of the Asian horned frog subfamily Megophryinae Bonaparte, 1850, contains 65 recognized species, distributed throughout southern China, and south to the Indochina Peninsula (Frost 2023; Lyu et al. 2023). Due to the conserved morphological characters, a large number of cryptic species from southern China were unrecognized in the past, and have been revealed based on large-scale molecular sampling efforts (i.e. Chen et al. 2017; Liu et al. 2018). This also resulted in taxonomic uncertainty in many species that were generally considered widespread. For example, Boulenophrys brachykolos (Inger and Romer, 1961) was previously regarded as widespread throughout southern China and northern Vietnam (Fei et al. 2009; Orlov et al. 2002). However, only specimens from its type locality Hong Kong and adjacent Shenzhen, Guangdong Province were confirmed by molecular and morphological data (Lyu et al. 2023). The record of B. brachykolos from Mt. Dayaoshan, Guangxi Province was recently revised and identified as an independent species B. yaoshanensisQi et al., 2021. Additionally, the record from Mt. Mangshan, Hunan Province was cast into doubt following random tadpole sampling (Qian et al. 2023). As a result, Lyu et al. (2023) concluded that B. brachykolos is restricted to Hong Kong and Shenzhen and suggested to remove all records from other localities.

Mo et al. (2009) first reported the presence of B. brachykolos on Mt. Hengshan, Hunan Province, providing measurements of five specimens (sex unmentioned). Shen et al. (2014) subsequently provided a detailed description and identified four vouchered female specimens from Hunan Province as B. brachykolos, two of which were collected from Mt. Hengshan. All specimens examined by Shen et al. (2014) displayed a series of morphological characteristics, including relatively smooth dorsum skin, a small horn-like tubercle on the upper eyelid, short hindlimbs with heels not touching each other when hindlimbs held at right angles to body, the absence of vomerine teeth, toes with rudimentary webbing and lacking lateral fringes, and an interorbital triangle dorsum pattern. These characteristics were consistent with the original description of B. brachykolos by Inger and Romer (1961), as well as the revised description based on the examination of topotype specimens by Fei et al. (2009). However, the absence of molecular data for this population called for a re-examination of its identification as B. brachykolos.

To clarify the taxonomic status of the Boulenophrys population from Mt. Hengshan, a field survey was carried out in June 2021, during which a series of specimens was collected. To determine the phylogenetic position of this population, DNA sequences were extracted from the newly collected specimens and compared to those of its congeners. The Hengshan population and both the topotypical B. brachykolos specimens and all known Boulenophrys congeners were also morphologically examined and compared. Our results revealed that the specimens from Mt. Hengshan represent an independent lineage and can be separated from all known congeners by a series of morphological characteristics. Thus, we propose to recognize the B. brachykolos population from Mt. Hengshan as a new species, which we describe below.

 Materials and Methods

A total of 20 newly collected specimens from Mt. Hengshan, Nanyue District, Hengyang Prefecture, Hunan Province, China were preserved in 75% alcohol and deposited in the Institute of Wildlife Conservation, Central South University of Forestry and Technology (CSUFT). For DNA extraction, tissue samples (liver) were preserved in 95% ethanol. Two museum specimens of the Hengshan population were preserved in formalin and deposited in the College of Life Sciences, Hunan Normal University (HNNU). Comparative specimens were examined from CSUFT and the Museum of Biology, Sun Yat-sen University (SYS) as listed in Appendix I.

Molecular data

We selected six newly collected specimens that exhibited several variable morphological features (see Variation) for DNA extraction. Two mitochondrial genes, namely the genes for 16S ribosomal RNA (16S) and cytochrome C oxidase subunit I (CO1) were selected for molecular analysis following Qi et al. (2021). Genomic DNA was extracted from 95%-ethanol-preserved tissues using a Tsingke TSP201-200 DNA extraction kit. Primer sequences were retrieved from the literature: L3975 and H4551 for 16S following Simon et al. (1994) and dgLCO and dgHCO for CO1 following Meyer et al. (2005). PCR amplification was carried out in 50 µl reaction volumes under the following conditions: 98°C for 2 min; 30 cycles of 98°C for 10 sec, 55°C for 10 sec, 72°C for 10 sec, with a final extension step at 72°C for 5 min. PCR purification and sequencing were performed by Biomarker Technologies Co. (Beijing, China).

For reconstructing the phylogenetic tree, the corresponding sequences of 64 known Boulenophrys species and an undescribed species from Hunan Province (“Megophrys” sp. 27 of Liu et al. (2018)) were downloaded from GenBank. We selected Xenophrys glandulosa and X. mangshanensis as outgroups, because these taxa performed well in several Boulenophrys studies (e.g. Lyu et al. 2020, 2021; Qi et al. 2021; Wang et al. 2022). All samples used in the molecular analysis are listed in Table 1. Sequences of two genes were aligned respectively in MEGA 6 (Tamura et al. 2013) by the MUSCLE algorithm (Edgar 2004) with default parameters. Finally, the alignment of 545 base pairs (bp) of 16S and 627 bp of CO1, were concatenated into an 1172-bp-long dataset. Phylogenetic trees were constructed using Bayesian inference (BI) implemented in MrBayes 3.2.5 (Ronquist et al. 2012). The best-fitting models and the best partitioning scheme were selected using the corrected Akaike information criterion (AICc) in PartitionFinder 2 (Lanfear et al. 2017). The selected partitions and models were GTR + I + G for 16S, SYM + I for CO1 first codon position, HKY for CO1 second codon position, and GTR + G for CO1 third codon position. Two independent runs were conducted during the BI analyses with 10,000,000 generations each, and they were sampled every 1000 generations with the first 25% of samples discarded as burn-in, resulting in a potential scale reduction factor (PSRF) of <0.005.

Specimens, localities, and GenBank accession numbers of all samples used in the molecular analysis.

ID Species Voucher Locality 16S CO1 Reference
1 Boulenophrys hengshanensis sp. nov. CSUFT HS210603 Mt. Hengshan, Nanyue Dist., Hengyang, Hunan, China ON209287 --- This study
2 B. hengshanensis sp. nov. CSUFT HS210606 Mt. Hengshan, Nanyue Dist., Hengyang, Hunan, China ON209292 --- This study
3 B. hengshanensis sp. nov. CSUFT HS210608 Mt. Hengshan, Nanyue Dist., Hengyang, Hunan, China ON209289 OQ910104 This study
4 B. hengshanensis sp. nov. CSUFT HS210610 Mt. Hengshan, Nanyue Dist., Hengyang, Hunan, China ON209293 OQ910105 This study
5 B. hengshanensis sp. nov. CSUFT HS210612 Mt. Hengshan, Nanyue Dist., Hengyang, Hunan, China ON209288 OQ910106 This study
6 B. hengshanensis sp. nov. CSUFT HS210614 Mt. Hengshan, Nanyue Dist., Hengyang, Hunan, China ON209291 OQ910107 This study
7 B. hengshanensis sp. nov. CSUFT HS210617 Mt. Hengshan, Nanyue Dist., Hengyang, Hunan, China ON209294 OQ910108 This study
8 B. hengshanensis sp. nov. CSUFT HS210621 Mt. Hengshan, Nanyue Dist., Hengyang, Hunan, China ON209290 OQ910109 This study
9 B. xuefengmontis SYS a004364 Wugang County, Hunan, China MH406813 MH406275 Lyu et al. (2023)
10 B. xuefengmontis SYS a004365 Wugang County, Hunan, China MH406814 MH406276 Lyu et al. (2023)
11 “Megophrys” sp. 27 SYS a004279 Taoyuan County, Hunan, China MH406793 MH406255 Liu et al. (2018)
12 “Megophrys” sp. 27 SYS a004280 Taoyuan County, Hunan, China MH406794 MH406256 Liu et al. (2018)
13 B. acuta SYS a002159 Heishiding Nature Reserve, Guangdong, China MF667869 MH406099 Liu et al. (2018)
14 B. angka KIZ 040592 Kiew Mae Pan Nature Trail, Doi Inthanon, Chiang Mai, Thailand MN508048 --- Wu et al. (2019)
15 B. anlongensis CIB AL21090531017 Anlong County, Guizhou, China MT823185 MT823262 Li et al. (2020)
16 B. baishanzuensis CIB QY20200719001 Baishanzu National Park, Qingyuan, Zhejiang, China MW001150 MT998291 Wu et al. (2020)
17 B. baolongensis KIZ 019216 Baolong Town, Chongqing, China KX811813 KX812093 Chen et al. (2017)
18 B. binchuanensis KIZ 019442 Mt. Jizu, Yunnan, China KX811850 KX812113 Chen et al. (2017)
19 B. binlingensis SYS a005313 Mt. Wawu, Sichuan, China MH406892 MH406354 Liu et al. (2018)
20 B. boettgeri SYS a004149 Mt. Wuyi, Fujian, China MF667878 MH406247 Liu et al. (2018)
21 B. brachykolos SYS a002258 Hong Kong, China KJ560403 MH406120 Liu et al. (2018)
22 B. caobangensis IBER 4385 Nguyen Binh, Cao Bang, Vietnam LC483945 --- Nguyen et al. (2020)
23 B. caudoprocta SYS a004281 Zhangjiajie, Hunan, China MH406795 MH406257 Liu et al. (2018)
24 B. cheni SYS a002142 Taoyuandong, Hunan, China KJ560398 MH406098 Liu et al. (2018)
25 B. chishuiensis CIB CS20190518019 Chishui County, Guizhou, China MN954708 MN928959 Xu et al. (2020)
26 B. congjiangensis GZNU 20200706003 Yueliangshan Nature Reserve, Congjiang, Guizhou, China MW959773 MW959761 Luo et al. (2021)
27 B. daiyunensis SYS a001733 Mt. Daiyun, Fujian, China MH406643 MH406079 Liu et al. (2018)
28 B. daoji SYS a004088 Fenghua, Zhejiang, China MH406782 MH406242 Liu et al. (2018)
29 B. daweimontis KIZ 048938 Mt. Dawei, Yunnan, China KX811870 KX812126 Chen et al. (2017)
30 B. dongguanensis SYS a001973 Mt. Yinping, Guangdong, China MH406647 MH406083 Liu et al. (2018)
31 B. fansipanensis AMS R186115 Sa Pa, Lao Cai, Vietnam MH514887 MW086548 Tapley et al. (2021)
32 B. fengshunensis SYS a004724 Fengshun County, Guangdong, China MH406848 MH406310 Liu et al. (2018)
33 B. frigida AMS R186131 Mount Ky Quan San, Bat Xat District, Lao Cai, Vietnam MT364279 MW086550 Tapley et al. (2021)
34 B. hoanglienensis VNMN 2018.02 Sa Pa, Lao Cai, Vietnam MH514889 MW086551 Tapley et al. (2021)
35 B. boettgeri SYS a002703 Mt. Huangshan, Anhui, China MF667883 MH406161 Lyu et al. (2023)
36 B. hungtai SYS a007575 Jiexi County, Guangdong, China OL635592 OL634859 Wang et al. (2022)
37 B. insularis SYS a002168 Nan’ao Island, Guangdong, China MF667886 MF667923 Liu et al. (2018)
38 B. jiangi CIB KKS20180426001 Kuankuoshui Nature Reserve, Guizhou, China MN107744 MN107749 Liu et al. (2020)
39 B. jingdongensis SYS a003928 Mt. Wuliang, Yunnan, China MH406773 MH406232 Liu et al. (2018)
40 B. jinggangensis SYS a004028 Mt. Jinggang, Jiangxi, China MH406780 MH406239 Liu et al. (2018)
41 B. jiulianensis SYS a004219 Mt. Jiulian, Jiangxi, China MH406791 MH406253 Liu et al. (2018)
42 B. kuatunensis SYS a003449 Guadun, Fujian, China MF667881 MH406206 Liu et al. (2018)
43 B. leishanensis SYS a002213 Mt. Leigong, Guizhou, China MH406673 MH406113 Liu et al. (2018)
44 B. liboensis GNUG 20150813001 Libo, Guizhou, China MF285253 --- Zhang et al. (2017)
45 B. lini SYS a002381 Mt. Jinggang, Jiangxi, China MF667874 MH406135 Liu et al. (2018)
46 B. lishuiensis CIB WYF00169 Lishui, Zhejiang, China KY021418 --- Wang et al. (2017b)
47 B. lushuiensis CIB YN201909288 Lushui County, Yunnan, China MW001225 MW000912 Shi et al. (2021)
48 B. minor SYS a003209 Dujiangyan, Sichuan, China MF667862 MH406194 Liu et al. (2018)
49 B. mirabilis SYS a002192 Huaping Nature Reserve, Guangxi, China MH406669 MH406109 Liu et al. (2018)
50 B. mufumontana SYS a006391 Mt. Mufu, Pingjiang County, Hunan, China MK524105 MK524136 Wang et al. (2019a)
51 B. nankunensis SYS a004501 Mt. Nankun, Guangdong, China MH406822 MH406284 Liu et al. (2018)
52 B. nanlingensis SYS a001964 Nanling Nature Reserve, Guangdong, China MH406646 MH406082 Liu et al. (2018)
53 B. obesa SYS a002272 Heishiding Nature Reserve, Guangdong, China KJ579122 MH406124 Liu et al. (2018)
54 B. omeimontis SYS a005301 Mt. Le, Sichuan, China MH406887 MH406349 Liu et al. (2018)
55 B. palpebralespinosa KIZ 011650 Pu Hu Nature Reserve, Thanh Hoa, Vietnam KX811889 KX812138 Chen et al. (2017)
56 B. puningensis SYS a005770 Puning City, Guangdong, China OL635585 OL634853 Wang et al. (2022)
57 B. qianbeiensis CIB TZ20190608017 Huanglian Nature Reserve, Guizhou, China MT651554 MT654521 Su et al. (2020)
58 B. rubrimera AMS R177676 Sa Pa, Lao Cai, Vietnam MF536419 MW086542 Tapley et al. (2021)
59 B. sangzhiensis SYS a004306 Zhangjiajie, Hunan, China MH406797 MH406259 Liu et al. (2018)
60 B. sanmingensis SYS a002486 Mt. Emeifeng, Fujian, China MH406697 MH406145 Liu et al. (2018)
61 B. shimentaina SYS a002077 Shimentai Nature Reserve, Guangdong, China MH406655 MH406092 Liu et al. (2018)
62 B. shunhuangensis HNNU 18NS01 Nanshan Forest Park, Hunan, China MK836023 MK977594 Wang et al. (2019b)
63 B. spinata SYS a002226 Mt. Leigong, Guizhou, China MH406675 MH406115 Liu et al. (2018)
64 B. tongboensis SYS a002415 Mt. Tongbo, Jiangxi, China MH406690 MH406138 Liu et al. (2018)
65 B. tuberogranulata SYS a004310 Badagongshan Nature Reserve, Hunan, China MH406801 MH406263 Liu et al. (2018)
66 B. wugongensis SYS a004800 Mt. Wugong, Jiangxi, China MH406853 MH406315 Liu et al. (2018)
67 B. wuliangshanensis SYS a003924 Mt. Wuliang, Yunnan, China MH406771 MH406230 Liu et al. (2018)
68 B. wushanensis SYS a003008 Mt. Wu, Hubei, China MH406732 MH406184 Liu et al. (2018)
69 B. xiangnanensis SYS a002874 Mt. Yangming, Hunan, China MH406713 MH406165 Liu et al. (2018)
70 B. xianjuensis CIB XJ190505 Xianju County, Zhejiang, China MN563753 MN563769 Wang et al. (2020)
71 B. yangmingensis SYS a002877 Mt. Yangming, Hunan, China MH406716 MH406168 Liu et al. (2018)
72 B. yaoshanensis SYS a002189 Dayaoshan Nature Reserve, Guangxi, China MH406667 MH406107 Liu et al. (2018)
73 B. yingdeensis SYS a002100 Shimentai Nature Reserve, Guangdong, China MH406658 MH406095 Liu et al. (2018)
74 B. yunkaiensis SYS a004637 Yukaishan Nature Reserve, Guangdong, China MH406843 MH406305 Liu et al. (2018)
75 Xenophrys glandulosa SYS a003758 Mt. Gaoligong, Yunnan, China MH406755 MH406214 Liu et al. (2018)
76 X. mangshanensis SYS a002177 Mt. Sanyue, Guangdong, China MH406666 MH406106 Liu et al. (2018)
Morphological data

Morphological measurements from preserved specimens were conducted as described by Lyu et al. (2021) using a digital caliper (to the nearest 0.1 mm). Definitions were as follows: Snout-vent length from tip of snout to vent (SVL); head length from the point behind the angle of the jaw to tip of snout (HDL); head width at the widest point (HDW); snout length from the tip of snout to the anterior corner of the eye (SNT); internasal distance (IND); interorbital distance (IOD), the minimum distance between upper eyelids; eye diameter (ED); tympanum diameter (TD); tympanum-eye distance from the anterior edge of tympanum to posterior corner of the eye (TED); hand length from the proximal border of the outer palmar tubercle to the tip of finger III (HND); length of lower arm and hand, from the flexed elbow to the tip of finger III (LAH); tibia length from the outer surface of the flexed knee to the heel (TIB); foot-tarsus length from the distal end of the shank to the tip of digit IV (FTL). Sex was determined by the presence of nuptial pads/spines in males and/or gonadal inspection.

Morphological data for genera delimitation was based on information that summarized by Lyu et al. (2023). Comparative data of Boulenophrys species were based on the examination of museum specimens listed in Appendix I and on information available in the literature (Inger and Romer 1961; Tian et al. 2000; Ye et al. 2007; Jiang et al. 2008; Mo et al. 2010; Wang et al. 2012, 2014, 2017a, 2017b, 2019a, 2019b, 2020, 2022; Liao et al. 2013; Li et al. 2014, 2018, 2020; Shen et al. 2014; Fei and Ye 2016; Tapley et al. 2017, 2018, 2021; Zhang et al. 2017; Messenger et al. 2019; Wu et al. 2019, 2020; Liu et al. 2020; Lyu et al. 2020, 2021, 2023; Nguyen et al. 2020; Su et al. 2020; Xu et al. 2020; Luo et al. 2021; Shi et al. 2021; Qi et al. 2021).

 Acoustic data

Advertisement calls were recorded using a TASCAM DR05X digital recorder (96 kHz, 24-bit) with a built-in microphone from a male paratype specimen on 4 June 2021 at about 20:48 h. Calls were recorded at approximately 0.2 m with the frog. The ambient temperature was not recorded. Calls were analyzed with Raven Pro 1.6 software (Cornell Lab of Ornithology). Spectrograms were produced applying a Hann window with 512 samples window size, and overlap of 50%, and a hop size of 256 samples. Call parameters were measured following Tapley et al. (2021): Call duration (ms), inter-call interval (ms), number of calls per call groups, call repetition rate (calls/s), number of pulses per call, and dominant frequency (kHz).

 Mapping

The software Quantum GIS (QGIS v.3.16, Hannover) was used to make a topographic map (Fig. 1) using the 30 m spatial resolution Advanced Spaceborne Thermal Emission and Reflection (ASTER) Global Digital Elevation Model (GDEM) layer available from Geospatial Data Cloud (http://gscloud.cn). GPS coordinates were retrieved from the literature (Wang et al. 2019a; Lyu et al. 2020, 2023; Shang et al. 2023; Qian et al. 2023) or from information recorded with museum specimens. Localities reported in literature without coordinates (Shen et al. 2014; Liu et al. 2018; Gao et al. 2022) were estimated using TIANDITU (https://maps.tianditu.gov.cn, accessed on 23 December 2022).

10.3897/vz.73.e100889.figure1 17DB8C8A-2172-52B2-8688-0C8B90882D3F

Distribution map of Boulenophrys species in Hunan Province (A). The green spot in (B) indicates the collection site of Boulenophrys hengshanensissp. nov. in China. The map in (C) represents a zoomed-in topographic map of Mt. Hengshan.

https://binary.pensoft.net/fig/915471 Results

The reconstructed Bayesian topology is shown in Fig. 2. The samples from Mt. Hengshan clustered into a poorly supported clade (Bayesian Posterior Probability [BPP] value 0.68) with B. shunhuangensis, B. mirabilis, B. xuefengmontis and “Megophrys” sp. 27 in Liu et al. (2018). Within this clade, each lineage is strongly supported with a BPP value of 1.00, except for B. shunhuangensis and B. mirabilis. The Hengshan population is sister to “Megophrys” sp. 27 from Taoyuan County, about 200 km northwest of Mt. Hengshan (see Fig. 1). In addition, the undescribed species shows a relatively distant phylogenetic relationship to B. brachykolos.

10.3897/vz.73.e100889.figure2 230B776E-949C-5C04-854B-376D80375930

Bayesian topology of Boulenophrys based on the 16S and CO1 genes. Numbers on nodes are Bayesian posterior probabilities (BPP) greater than or equal to 0.60.

https://binary.pensoft.net/fig/915472

Based on examination of topotype specimens of B. brachykolos and the revised diagnosis provided by Lyu et al. (2023), our undescribed species from Mt. Hengshan is distinguished from B. brachykolos by the tongue not notched posteriorly (vs. notched posteriorly), the absence of vomerine teeth and vomerine ridges (vs. presence of vomerine ridges and weakly developed vomerine teeth), and a relative finger length of III>I>II≥IV (vs. III>I>IV>II). Furthermore, this undescribed species is separated from all known congeners by a series of diagnostic morphological traits (see Comparisons) and described herein as a new species.

Family: Megrophyidae

Subfamily: Megophryinae

Animalia Anura Megophryidae B3D3552F-814A-57A1-AC7D-C3D6787225C3 Boulenophrys hengshanensis https://zoobank.org/A03130D7-3C1E-4DAB-8AFC-A0FABD61BB28 sp. nov. Figures 3 , 4 , 5 , Table 2 Chresonymy.

Megophrys brachykolos – Mo et al. (2009), Shen et al. (2014), Gao et al. (2022)

 Holotype.

HNNU 0706000A, adult female, collected by Youhui Shen from Nanyue Hengshan, Hengyang, Hunan Province, China in June 2007.

 Paratypes.

HNNU 1997:6-17, adult female, collected by Youhui Shen from Laotage, Nanyue Hengshan, Hengyang, Hunan Province, China on 17 June 1997; CSUFT HS210602–603, 605–610, 612–616, and 618–620, sixteen adult males; CSUFT HS210604, 611, 617, and 621, four adult females, collected by KH and ZG from Nanyue Hengshan Scenic Area (112.714N, 27.277E, ca. 450 m a.s.l.), Nanyue District, Hengyang Prefecture, Hunan Province, China on 4 June 2021.

10.3897/vz.73.e100889.figure3 68D07CA6-488A-5A61-B5E7-07F3914729BE

Holotype of Boulenophrys hengshanensissp. nov. (HNNU 0706000A, adult female). A Dorsal, B ventral views of body; C lateral view of head; D volar view of hand; E plantar view of foot. Scale bars equal to 10 mm.

https://binary.pensoft.net/fig/915473 Etymology.

The specific epithet “hengshanensis” refers to Mt. Hengshan, the type locality of the new species.

 Diagnosis.

Among the ten genera of the subfamily Megophryinae, the new species is assigned to Boulenophrys based on its slender body (vs. thickset and stocky in Brachytarsophrys), a distinct tympanum visible (vs. concealed and invisible in Brachytarsophrys), a narrower and non-depressed head (vs. enlarged and depressed in Brachytarsophrys, Grillitschia, Pelobatrachus, and Megophrys), head without a transverse groove behind (vs. present in Brachytarsophrys, Pelobatrachus, and Megophrys), the short temporal region being relatively closer to the posterior corner of eye (vs. elongated and far from posterior corner of eye in Grillitschia, Megophrys, and Pelobatrachus), maxillary teeth present (vs. absent in Ophryophryne), a small horn-like tubercle on upper eyelid present (vs. margin of eyelid smooth in Atympanophrys), supratympanic fold distinctly curved over tympanum (vs. slightly curved or not curved in Atympanophrys), asperities on lower jaw absent (vs. present in Xenophrys), subarticular tubercles on fingers present (vs. absent in Jingophrys, Xenophrys, and Sarawakiphrys), nuptial pad present on fingers I and II in breeding males (vs. absent in Sarawakiphrys, and absent or only present on finger I in Jingophrys), and the absence of ventrolateral tubercles (vs. present in Jingophrys).

Boulenophrys hengshanensissp. nov. can be further distinguished from its congeners by the combination of: (1) body of moderate size of SVL 35.7–41.2 mm (n = 16) in males, and 37.5–50.2 mm (n = 6) in females; (2) hindlimbs short, heels not meeting each other when hindlimbs held at right angles to body, TIB/SVL 0.38–0.46; (3) tympanum rounded, upper margin concealed by supratympanic fold, TD/ED 0.48–0.63; (4) absence of vomerine teeth and vomerine ridges; (5) posterior margin of tongue not notched; (6) dorsum skin rough with several granules and not continuous X-shaped skin ridges in males, relatively smooth in females; (7) ventral surface smooth with rounded tubercles; (8) two metacarpal tubercles present; (9) distinct subarticular tubercles on bases of each finger; (10) fingers lacking webs and lateral fringes; (11) relative finger length III > I > II ≥ IV; (12) rudimentary toe webs, absent lateral fringes; (13) the presence of dense tiny spines on surface of temporal region, upper and lower lips, and from the loreal region to the tip of snout in males; and (14) in breeding males, nuptial pads with small dense spines on fingers I–II.

 Description of the holotype.

Adult female, moderate body size, SVL 37.5 mm; head wider than long, HDL/HDW 0.97; snout short, with a bluntly rounded tip, SNT/HDL 0.33; eye large, with vertical pupils, ED/HDL 0.32; tympanum distinct and round, with upper border concealed by supratympanum fold, TD/ED 0.53; oval nostrils, orientated laterally, narial rim slightly prominent; absence of vomerine teeth and vomerine ridge; maxillary teeth present; rounded tongue with no notch at the posterior margin.

Fingers without webbing and lateral fringes; relative finger length III > I > II > IV; distinct subarticular tubercles present at the bases of each finger; large inner metacarpal tubercle, relatively small outer metacarpal tubercle, both distinctly visible.

Hindlimbs short, TIB/SVL 0.45; heels not touching each other when hindlimbs held at right angles to the body; toes with rudimentary webs and lacking lateral fringes; relative toe length IV > III > V > II > I; subarticular tubercles distinct at bases of toes I, II, and III, absent at base of toe IV, and very small and flat at base of toe V; inner metatarsal tubercle elongated and large, outer metatarsal tubercle absent.

Dorsal skin rough, with several posterior enlarged tubercles on dorsum and flanks; weakly X-shaped skin ridge and dorsolateral skin ridges on dorsum; small horn-like tubercle at the edge of each upper eyelid; supratympanic fold distinct and narrow, extending from posterior eye borders, curves down broadly through upper tympanum and gradually widen above insertion of arm; flanks with sparse tubercles; ventral surface of head and body granular; thigh smooth anteriorly, with dense tubercles posteriorly; pectoral and femoral glands prominent, the latter closer to knee than to vent.

 Coloration of holotype.

In preservation, dorsally light brown without distinct markings; several brown narrow stripes present on lower arms, hands, thighs and shanks; dark brown pigmentation present on hindlimbs posteriorly where meets the venter. Ventrally, skin beige with light brown pigmentation; several dark brown blotches present on lower lip; a distinct, short, dark brown stripe longitudinally on throat; a dark brown, wide band present on ventrolateral margin of trunk; several brown spots scattered on belly; inner thigh almost clear proximally, with several brown spots on distal half; shanks and foots scattered with brown spots; pectoral and femoral glands clear, unpigmented; palm and sole brown with pale tubercles.

 Coloration of paratype in life.

A color photograph of the holotype in life is available in Shen et al. (2014), but with a low resolution. The detailed description of the coloration in life was based on newly collected paratype specimens. — MALE. The following description is based on a male paratype CSUFT HS210605 (Fig. 4A–B). The dorsal surface is light brown with dark brown markings; reddish small tubercles cover the dorsal surface; a wide dark brown band is present on each upper eyelid, and extends posteriorly where the two bands meet each other; the dorsal pattern shows several transverse bars; a distinct large X-shaped pattern is present on the middle of the dorsum; the intersection of the X-shape is elongated longitudinally; wide dark brown bars are present on dorsal surface of lower arms and hindlimbs, and form large blotches on elbows. The background coloration on ventral surface is dark purple; the skin on chin and chest is rather dark, with several white blotches and stripes that are embellished with red pigmentation; four distinct longitudinal stripes on throat; on the chest, the background skin color is gradually faded; dense red spots are distinctly visible, and extend laterally to flanks; two white pectoral glands are distinctly prominent; white blotches cover the most of the belly, which is scattered with grey and dark brown spots, and sparse red spots; the ventral surface of the thigh is purple, with white tubercles proximally and white blotches distally; two white femoral glands are distinctly prominent; the palm and sole are dark purple; the digital discs, palm tubercles are pale whitish-purple; the groin is pigmented red. Laterally, the enlarged portion of supratympanic fold above insertion of arm is beige; the iris is reddish-white; the sclera is golden; the tympanum is golden with dark brown pigmentation, with a distinct dark spot in the center. — FEMALE. A female paratype CSUFT HS210604 (Fig. 4C–D) has a similar dorsal pattern to the description above; the ventral surface is paler, and with fewer reddish pigmentation; the inner aspect of thigh lacks white blotches, but with pale gray pigmentation and several black dots; the tip of toe is pink, with pale orange pigmentation.

10.3897/vz.73.e100889.figure4 06B8FE45-89A1-5A40-8A32-932576BA6BE6

Paratypes of Boulenophrys hengshanensissp. nov. in life. A, B Male paratype CSUFT HS210605; C, D Female paratype CSUFT HS210604.

https://binary.pensoft.net/fig/915474 Sexual secondary characteristics.

In male specimens, tubercles with tiny central spines are present on the surface of the temporal region, on the upper and lower lip, and from the loreal region to the tip of snout; similar spines are also present on the tubercles around the cloacal region, the inner aspects of thigh, shank, and tarsus. Several individuals show these spines on the outer aspect of the hindlimbs and lower arms, and alongside the ridge of supratympanic fold (CSUFT HS210602–603, 613, 616, 618–620). Nuptial pads are present on fingers I and II. Additionally, male specimens have strong arms during the breeding season, and show a rough dorsum with granules, while the dorsum of female specimens is relatively smooth.

 Variation.

Measurements of all type specimens are shown in Table 2. The relative finger length is III>I>II=IV in several specimens (CSUFT HS210601–603, 609, 611, 613–616). The extension of supratympanic fold above insertion of arm forms a depressed supraaxillary gland in two specimens (CSUFT HS210604, 620), and is slightly swollen in five specimens (CSUFT HS210606, 611–14), and is very swollen in several specimens (CSUFT HS210602–603, 605, 607, 609–610, 615–619, 621). In life, several individuals (CSUFT HS210608, 15, 18, 21) show an extremely dark skin on the venter and an absence of red pigmentation, sometimes rendering the longitudinal stripes on throat indistinguishable (CSUFT HS210615).

Measurements of the type series of Boulenophrys hengshanensissp. nov. The voucher number of the holotype is highlighted by an asterisk. For abbreviations, see Materials and Methods.

Voucher Sex SVL HDL HDW SNT IND IOD ED TD TED HND RAH TIB FTL FL
CSUFT HS210602 M 36.5 13.6 14.1 4.3 4.0 3.8 5.0 2.5 1.7 9.8 16.9 16.4 24.2 14.9
CSUFT HS210603 M 36.0 13.4 14.0 3.7 4.3 3.4 5.4 2.7 1.5 9.1 16.9 16.3 24.1 14.6
CSUFT HS210605 M 36.8 13.3 14.0 3.9 4.5 3.5 5.2 2.5 1.7 10.0 17.7 17.1 24.9 15.6
CSUFT HS210606 M 38.0 13.8 14.1 4.0 3.9 3.5 5.0 2.6 1.6 9.5 17.2 16.1 23.1 14.2
CSUFT HS210607 M 39.1 14.2 14.7 4.5 5.1 4.1 6.3 3.0 1.6 9.6 17.2 16.9 24.6 14.9
CSUFT HS210608 M 39.0 13.5 13.9 4.1 4.3 3.5 5.2 2.8 1.5 9.4 16.3 16.3 23.2 14.5
CSUFT HS210609 M 39.4 14.9 15.1 4.5 4.7 4.2 5.5 3.1 1.7 10.8 18.1 18.1 25.8 16.2
CSUFT HS210610 M 41.2 15.1 16.3 4.5 4.7 4.0 5.6 2.8 1.8 10.4 18.7 18.2 26.5 16.8
CSUFT HS210612 M 39.7 14.4 15.0 4.2 4.6 3.8 5.4 2.7 2.1 9.4 17.3 16.7 23.9 14.6
CSUFT HS210613 M 39.6 13.7 13.8 4.0 4.6 3.7 5.5 3.2 1.5 9.9 17.4 16.5 23.5 14.6
CSUFT HS210614 M 39.1 13.9 14.2 4.2 5.0 3.8 5.4 2.8 1.8 10.2 17.6 17.5 24.6 15.4
CSUFT HS210615 M 35.8 13.0 13.0 4.1 4.4 3.8 5.1 2.6 1.6 9.4 16.5 16.0 21.8 13.9
CSUFT HS210616 M 38.3 13.6 13.9 4.1 4.2 3.5 5.4 2.8 1.7 9.8 17.8 17.7 24.9 15.4
CSUFT HS210618 M 40.4 13.4 13.8 4.2 4.1 4.0 5.2 2.7 1.8 9.9 17.9 18.2 25.0 15.3
CSUFT HS210619 M 40.3 13.6 14.1 4.3 4.6 3.6 5.5 3.1 1.9 10.0 17.4 15.9 24.2 15.4
CSUFT HS210620 M 35.7 13.2 13.5 4.1 4.4 3.7 5.2 2.7 1.7 9.6 17.5 16.3 24.0 15.4
HNNU 0706000A* F 37.5 14.1 14.5 4.7 4.3 3.6 4.5 2.4 2.8 10.1 17.4 16.7 23.8 16.4
HNNU 1997:6-17 F 47.9 16.4 17.1 5.4 4.7 4.0 5.7 3.6 3.4 11.9 20.8 18.2 27.4 18.6
CSUFT HS210604 F 50.2 16.4 17.3 5.2 4.8 4.6 6.2 3.2 2.6 12.2 22.7 21.4 29.0 19.1
CSUFT HS210611 F 41.8 14.4 15.0 4.1 4.1 3.5 5.3 2.7 1.9 10.1 19.2 18.8 26.2 16.3
CSUFT HS210617 F 47.6 16.4 17.3 5.1 5.1 4.0 6.2 3.7 2.1 11.0 19.9 20.0 28.8 18.9
CSUFT HS210621 F 43.8 15.1 15.3 4.1 5.0 4.8 5.9 3.3 2.1 11.0 20.5 19.3 27.8 17.6
Advertisement call.

Call descriptions are based on the calls of male paratype CSUFT HS210614 (SVL 39.1 mm). Spectrograms and waveforms are shown in Fig. 5. Six call groups with a total of 135 calls were analyzed. Each call group contains an average of 22.5±2.9 (20–28) calls. Calls were an average of 100±6.8 (87–123) ms in duration, with an average inter-call interval of 168±48.7 (95–492) ms. Calls were repeated at a rate of 3.7±0.1 (3.6–3.9) calls/s per call group. Each call consisted of 18±1.6 (14–24) pulses. The dominant frequency was 2.7±0.1 (2.5–2.8) kHz, no evident harmonics.

10.3897/vz.73.e100889.figure5 AE43FC84-72F5-5D10-9D6D-F28A39540D75

Advertisement call of Boulenophrys hengshanensissp. nov. A 60 s waveform showing five call groups; B 2 s waveform; C respective spectrogram showing seven calls form male paratype CSUFT HS210614.

https://binary.pensoft.net/fig/915475 Comparisons.

By having a moderate body size of SVL 35.7–41.2 mm (n = 17) in males, and 37.5–50.2 mm (n = 5) in females, B. hengshanensissp. nov. differs from the larger species B. binlingensis (vs. 45.1–51.0 mm [n = 3] in males), B. caudoprocta (vs. 70.8–81.3 mm [n = 3] in males, and 77.8 mm [n = 1] in female), B. chishuiensis (vs. 43.1–44.1 mm [n = 3] in males), B. jingdongensis (vs. 53.0–56.5 mm [n = 3] in males, and 63.5 mm [n = 1] in female), B. liboensis (vs. 60.8–70.6 mm [n = 8] in females), B. mirabilis (vs. 55.8–61.4 mm [n = 2] in males, and 68.5–74.8 mm [n = 2] in females), B. omeimontis (vs. 56.0–59.5 mm [n = 10] in males, and 58.0–72.5 mm [n = 3] in females), B. qianbeiensis (vs. 49.3–58.2 mm [n = 6] in males), B. sangzhiensis (vs. 53.0–60.8 mm [n = 7] in males, and 73.0 mm [n = 1] in female), B. shuichengensis (vs. 102.0–118.3 mm [n = 8] in males, and 99.8–115.6 mm [n = 7] in females), B. spinata (vs. 47.2–54.4 mm [n = 18] in males, and 54.0–55.0 mm [n = 2] in females); and the smaller species B. acuta (vs. 27.1–33.0 mm [n = 10] in males, and 34.4 mm [n = 1] in female), B. angka (vs. 31.2–32.1 mm [n = 2] in males), B. baishanzuensis (vs. 28.4–32.4 mm [n = 6] in males), B. cheni (vs. 26.2–29.5 mm [n = 15] in males, and 31.8–34.1 [n = 3] in females), B. congjiangensis (vs. 28.6–33.4 mm [n = 15] in males), B. daiyunensis (vs. 27.6–28.7 mm [n = 4] in males, and 33.7–35.6 mm [n = 3] in females), B. daoji (vs. 32.6–33.6 mm [n = 4] in males), B. frigida (vs. 30.3–31.8 mm [n = 4] in males), B. jiangi (vs. 39.5–40.4 mm [n = 2] in females), B. jiulianensis (vs. 30.4–33.9 mm [n = 9] in males), B. kuatunensis (vs. 26.2–31.4 mm [n = 24] in males), B. lishuiensis (vs. 31.0–34.8 mm [n = 2] in males), B. mufumontana (vs. 30.1–30.8 mm [n = 2] in males, and 36.3 mm [n = 2] in females), B. nankunensis (vs. 29.9–34.9 mm [n = 11] in males), B. ombrophila (vs. 27.4–34.5 mm [n = 5] in males, and 32.8–35.0 mm [n = 4] in females), B. rubrimera (vs. 26.7–30.5 mm [n = 8] in males), B. sanmingensis (vs. 28.0–30.6 mm [n = 12] in males, and 32.9 mm [n = 1] in female), B. shimentaina (vs. 28.0–30.6 mm [n = 12] in males), B. shunhuangensis (vs. 30.3–33.7 mm [n = 10] in males), B. tongboensis (vs. 26.5–31.5 mm [n = 5] in males), B. wugongensis (vs. 31.0–34.1 mm [n = 4] in males), B. wuliangshanensis (vs. 27.3–31.6 mm [n = 10] in males), B. wushanensis (vs. 30.4–35.5 mm [n = 10] in males), B. yingdeensis (vs. 33.2–35.3 mm [n = 4] in males), B. puningensis (vs. 31.7–34.6 mm [n = 4] in males), and B. hungtai (vs. 25.8–33.3 mm [n = 12] in males).

Boulenophrys hengshanensissp. nov. differs from its phylogenetically closest congeners B. xuefengmontis, B. shunhuangensis, and B. mirabilis by heels not meeting each other when hindlimbs are held at right angles to the body (vs. just meeting in B. xuefengmontis, and overlapping in B. shunhuangensis and B. mirabilis), the presence of dense tiny spines on the surface of the tympanic region, upper and lower lip, and from the loreal region to the tip of snout in males (vs. absent in B. xuefengmontis), a relative finger length of III>I>II≥IV (vs. III>IV>II>I in B. shunhuangensis, and III>IV>I>II in B. xuefengmontis and B. mirabilis), the upper margin of the tympanum concealed by a supratympanic fold (vs. tympanum with clear boundary in B. mirabilis), a small horn-like tubercle on the upper eyelid (vs. large in B. mirabilis), the absence of lateral fringes on the fingers (vs. presence in B. mirabilis), an internasal distance larger than the interorbital distance (vs. smaller in B. mirabilis), and the presence of distinct subarticular tubercles on bases of each finger (vs. indistinct in B. mirabilis).

Compared to other congeners occurring in Hunan Province, B. hengshanensissp. nov. differs by the absence of vomerine teeth and vomerine ridges (vs. presence of vomerine teeth in B. caudoprocta, B. jiulianensis, B. nanlingensis, and B. shimentaina; B. xiangnanensis and B. yangmingensis lack vomerine teeth, but have weak or strong vomerine ridges), the unnotched posterior margin of tongue (vs. notched in B. cheni, B. nanlingensis, B. jiulianensis, and B. sangzhiensis), by a small horn-like tubercle on the upper eyelid (vs. a large tubercle in B. caudoprocta, and B. jinggangensis). Boulenophrys hengshanensissp. nov. differs from B. cheni, B. lini, B. sangzhiensis [male only], and B. xiangnanensis by lateral fringes on toes absent (vs. moderate or wide fringes), by having only rudimentary webbing on toes (vs. 1/4 on each side of toe IV in B. sangzhiensis), the absence of lateral fringes on fingers (vs. present in B. shimentaina), the presence of distinct subarticular tubercles on bases of each finger (vs. absent or indistinct in B. shimentaina, and B. jiulianensis), heels not meeting each other when hindlimbs are held at right angles to the body (vs. just touching or overlapping in B. cheni, B. jinggangensis, B. jiulianensis, B. lini, B. mufumontana, B. nanlingensis, B. sangzhiensis, B. shimentaina, B. tuberogranulata, and B. yangmingensis), the upper margin of tympanum concealed by a supratympanic fold (vs. tympanum with clear boundary in B. jinggangensis, B. jiulianensis, B. lini, B. ombrophila, B. shimentaina, B. wugongensis, B. xiangnanensis, and B. yangmingensis), the presence of dense tiny spines on the surface of the tympanic region, upper and lower lip, and from the loreal region to the tip of snout in males (vs. absent in B. tuberogranulata), nuptial pads with small, dense spines present on fingers I–II in breeding males (vs. absence of nuptial pads in breeding males in B. nanlingensis, and nuptial pads with large spines in B. sangzhiensis), a relative finger length of III>I>II≥IV (vs. II>IV>I>II in B. jinggangensis, II>IV>I≥II in B. lini, III>IV>II>I in B. cheni, II>IV=I>II in B. dongguanensis, III>IV=I>II in B. wugongensis, and III>I>IV=II in B. mufumontana), and the presence of vocal sacs in males (vs. absent in B. caudoprocta).

Furthermore, B. hengshanensissp. nov. differs by the absence of vomerine teeth and vomerine ridges from B. daiyunensis, B. daweimontis, B. dongguanensis, B. fansipanensis, B. fengshunensis, B. frigida, B. hoanglienensis, B. insularis, B. jingdongensis, B. liboensis, B. lushuiensis, B. nankunensis, B. palpebralespinosa, B. puningensis, B. qianbeiensis, B. rubrimera, B. tongboensis, and B. yingdeensis, all of which have vomerine teeth, and it further differs from B. obesa, B. yaoshanensis, and B. xianjuensis, all of which lack vomerine teeth but have weak or strong vomerine ridges. Boulenophrys hengshanensissp. nov. differs by the posterior margin of the tongue being not notche, from B. binlingensis, B. boettgeri, B. jingdongensis, B. kuatunensis, B. liboensis, B. minor, B. omeimontis, B. sanmingensis, B. spinata, and B. tongboensis, all of which have a notch on the posterior tongue. Furthermore, B. hengshanensissp. nov. differs from B. baolongensis, B. hoanglienensis, B. insularis, B. lushuiensis, B. qianbeiensis, and B. shuichengensis, all of which have a weakly or feebly notched tongue on posterior margin, by having a small horn-like tubercle on upper eyelid. Boulenophrys hengshanensissp. nov. differs from B. acuta, B. liboensis, B. palpebralespinosa, and B. shuichengensis, all of which have a large horn-like tubercle on upper eyelid, by lateral fringes on toes being absent. Boulenophrys hengshanensissp. nov. differs from B. binchuanensis, B. boettgeri, B. jingdongensis, B. liboensis, B. qianbeiensis, B. sanmingensis, B. shuichengensis, B. spinata, and B. wushanensis (male only), all of which have moderate or wide lateral fringes on toes, by having only rudimentary webbing on toes, B. hengshanensissp. nov. further differs from B. jingdongensis (vs. half-webbed), B. qianbeiensis (vs. one-third webbing), B. shuichengensis (vs. 1/3 one each side of toe IV), and B. spinata (vs. half-webbed). By the absence of lateral fringes on fingers, B. hengshanensissp. nov. differs from B. daiyunensis, B. daoji, and B. tongboensis, all of which have lateral fringes on fingers. By the presence of distinct subarticular tubercles on bases of each finger, B. hengshanensissp. nov. differs from B. wuliangshanensis, B. nankunensis, B. fansipanensis, B. hoanglienensis, B. frigida, and B. rubrimera, in all of which subarticular tubercles are absent or indistinct; it also differs in this trait from B. angka (subarticular tubercles only present on the base of fingers I–II). Boulenophrys hengshanensissp. nov. differs by heels not meeting each other when the hindlimbs are held at right angles to the body from B. acuta, B. anlongensis, B. baishanzuensis, B. baolongensis, B. binlingensis, B. boettgeri, B. congjiangensis, B. chishuiensis, B. daoji, B. jiangi, B. jingdongensis, B. leishanensis, B. liboensis, B. lishuiensis, B. jiangi, B. obesa, B. omeimontis, B. palpebralespinosa, B. qianbeiensis, B. sanmingensis, B. spinata, B. tongboensis, B. wuliangshanensis, B. xianjuensis, B. yunkaiensis, and B. yaoshanensis, in all of which the heels are touching or overlapping. Furthermore, B. hengshanensissp. nov. differs by the upper margin of the tympanum concealed by a supratympanic fold from B. acuta, B. boettgeri, B. dongguanensis, B. fengshunensis, B. hungtai, B. insularis, B. kuatunensis, B. leishanensis, B. lishuiensis, B. nankunensis, B. obesa, B. puningensis, B. rubrimera, B. xianjuensis, B. yingdeensis, B. yaoshanensis, and B. yunkaiensis, all of which have a tympanum with clear boundary. Boulenophrys hengshanensissp. nov. differs by the presence of dense tiny spines on the surface of the tympanic region, the upper and lower lip, and from the loreal region to the tip of the snout in males from B. hungtai, B. puningensis, B. yaoshanensis, and B. yingdeensis, all of which are lacking these spines. Boulenophrys hengshanensissp. nov. differs by nuptial pads with small, dense spines present on fingers I–II in breeding males from B. daiyunensis having no nuptial pads in breeding males; it further differs from B. daoji, B. sanmingensis, and B. angka, all of which nuptial pad only present on finger I, and from B. qianbeiensis, and B. spinata, both have large spines on nuptial pads. By having a relative finger length of III > I > II ≥ IV, B. hengshanensissp nov. differs from B. daiyunensis (vs. III>IV>II=I), B. sanmingensis (vs. III>IV>II>I), B. tongboensis (vs. III>IV>I>II), B. dongguanensis (III>IV≥I>II), B. nankunensis (vs. III>IV>I>II), B. xianjuensis (vs. III>IV>I>II), B. puningensis (vs. III>IV>II=I), B. hungtai (vs. III>IV>II>I), and B. baishanzuensis (vs. III>IV>II>I). By the presence of outer metacarpal tubercle, B. hengshanensissp. nov. differs from B. fansipanensis and B. hoanglienensis, in which the outer metacarpal tubercle is absent.

 Distribution and Habitat.

The new species is currently only known from Mt. Hengshan, Nanyue District, Hengyang Prefecture, Hunan Province, China. The amphibian fauna in this area was first reported by Shen (1965), with a total of 14 amphibians recorded: Bufo gargarizans Cantor, 1842, Fejervarya multistriata (Hallowell, 1860), Hoplobatrachus chinensis (Osbeck, 1765), Quasipaa boulengeri (Günther, 1889), Rana hanluica Shen, Jiang & Yang, 2007 (reported as Rana japonica japonica Boulenger, 1879), Pelophylax hubeiensis (Fei & Ye, 1982), Pelophylax nigromaculatus (Hallowell, 1860), Hylarana latouchii (Boulenger, 1899), Hylarana guentheri (Boulenger, 1882), Polypedates megacephalus Hallowell, 1861, Microhyla heymonsi Vogt, 1911, M. pulchra (Hallowell, 1860), and M. fissipes Boulenger, 1884. Due to the following checklist reported by Shen (1983), Andrias davidianus (Blanchard, 1871), Amolops sinensis Lyu, Wang & Wang, 2019 (reported as Amolops ricketii [Boulenger, 1899]), Zhangixalus dennysi (Blanford, 1881) were also recorded in Mt. Hengshan. A relatively comprehensive amphibian survey in Mt. Hengshan was conducted from 2006 to 2007 by Mo et al. (2009). These authors reported the new species (identified as B. brachykolos) from the forest road between 744–900 m elevation during night surveys from 19:00 to 23: 00 h. They also recorded H. guentheri, F. multistriata, and M. heymonsi occurred between 200–300 m elevation; P. megacephalus and M. heymonsi between 600–700 m elevation; and Bu. gargarizans, R. hanluica between 1000–1100 m elevation. During our rapid survey conducted in June 2021, we observed Bu. gargarizans, Quasipaa exilispinosa (Liu & Hu, 1975), P. megacephalus, and A. sinensis to co-exist with B. hengshanensissp. nov. at 450 m elevation. The calling activities of male B. hengshanensissp. nov. started at about 18:00 h and did not decline until 23:00 h. Calling male frogs were found sitting in a roadside ditch, with shallow flowing water attaining the feet, or hide in the bush, or perched on low plants (Fig. 6).

10.3897/vz.73.e100889.figure6 B65F8575-0953-57D2-9991-2B32417A0981

Unvouchered calling male frog of Boulenophrys hengshanensissp. nov. under a bush.

https://binary.pensoft.net/fig/915476 Conservation status.

Mt. Hengshan is a relatively isolated mountain in Hunan Province (Fig. 1). Unsuitable habitat (e.g. farmland, country, rivers, and roads) and large regions with low altitude areas around Hengshan could be barriers to the dispersal of the new species. As noticed by Mo et al. (2009), Mt. Hengshan is protected by the Hunan Nanyuehengshan National Nature Reserve, but it is also a famous tourist destination (known as Nanyuehengshan Scenic Area). The potential threat to all amphibians in Mt. Hengshan is mainly human disturbance from tourist areas (Mo et al. 2009).

 Discussion

The discovery of the new species sheds light on the previously underestimated diversity within central Hunan Province, where mountains are isolated and sporadic. The related species (B. shunhuangensis, B. xuefengmontis, and “Megophrys” sp. 27) were all found in hilly areas in western Hunan. Thus, the herpetofauna of the isolated Mt. Hengshan in central Hunan is similar to the herpetofauna of the western hills (Xuefeng Mountains), but not the herpetofauna of the southern hills (Nanling Mountains) or eastern hills (Luoxiao Mountains).

Before the description of B. hengshanensissp. nov., 16 species had been reported from Hunan Province either as the type locality or with confirmed species identity using molecular data, which are: B. caudoprocta, B. cheni, B. jinggangensis, B. jiulianensis, B. lini, B. mufumontana, B. nanlingensis, B. ombrophila, B. sangzhiensis, B. shimentaina, B. shunhuangensis, B. tuberogranulata, B. wugongensis, B. xiangnanensis, B. xufengmontis, and B. yangmingensis (Lyu et al. 2023; Shang et al. 2023). The description of B. hengshanensissp. nov. increased the total number of species of Boulenophrys from Hunan Province to 17, highlighting the high species richness of Boulenophrys in this region. However, some records of Boulenophrys species in Hunan Province, such as B. kuatunensis and B. boettgeri, have been denied by Lyu et al. (2023) due to taxonomic uncertainties. Further investigation of Boulenophrys species in Hunan Province is warranted to better understand the diversity and evolutionary history of this genus in this biologically important region.

 Acknowledgements

We thank Jian Wang and an anonymous reviewer for their helpful comments on the previous version of this manuscript. We thank Yingyong Wang and Shuo Qi for access to museum specimen collections and associated support. We also thank Yuan Li for the help in molecular data analysis, Dejia Hou for generating DEM files, Leqiang Zhu for the assistance during specimen examination. TQ thanks the Cornell Lab of Ornithology for providing license support for Raven Pro software. This work was supported by the National Natural Science Foundation of China (Grant No. 31472021), the Project for Wildlife Conservation and Management of the National Forestry and Grassland Administration of China (Grant No. 2022-HN-001), and the Wildlife Conservation Project of Hunan Province (Grant No. HNYB2022-001).

 References BeddardFE (1907) Contributions to the knowledge of the anatomy of the batrachian family Pelobatidae.Proceedings of the Zoological Society of London77: 871–911. https://doi.org/10.1111/j.1469-7998.1907.tb06963.x BonaparteC-L (1850) Conspectus Systematum Herpetologiae et Amphibiologiae. Editio altera reformata [Lugduni Batavorum]. E. J. Brill, Leiden, 1 p. BoulengerGA (1903) Descriptions of three new batrachians from Tonkin. Annals and Magazine of Natural History, Series 7, 12: 186–188. https://doi.org/10.1080/00222930308678835 BoulengerGA (1899) On a collection of reptiles and batrachians made by Mr. J. D. La Touche in N.W. Fokien, China. Proceedings of the Zoological Society of London 67: 159–172, pls. XVI–XIX. http://dx.doi.org/10.1111/j.1469-7998.1899.tb06855.x ChenJMZhouWWPoyarkovNA JrStuartBLBrownRMLathropAWangYYYuanZYJiangKHouMChenHMSuwannapoomCNguyenSNDuongTVPapenfussTJMurphyRWZhangYPCheJ (2017) A novel multilocus phylogenetic estimation reveals unrecognized diversity in Asian horned toads, genus Megophrys sensu lato (Anura: Megophryidae).Molecular Phylogenetics and Evolution106: 28–43. https://doi.org/10.1016/j.ympev.2016.09.004 DelormeMDuboisAGrosjeanSOhlerA (2006) Une nouvelle ergotaxinomie des Megophryidae (Amhpibia, Anura).Alytes24: 6–21. FeiLHuSQYeCYHuangYZ (2009) Fauna Sinica, Amphibia, Volume 2, Anura.Science Press, Beijing, 957 pp. FeiLYeCY (2016) Amphibians of China, Volume I.Science Press, Beijing, 1040 pp. FrostDR (2023) Amphibian Species of the World: An Online Reference. Version 6.1. Electronic Database. American Museum of Natural History, New York, NY. https://amphibiansoftheworld.amnh.org/index.php [accessed: 2 May 2023] GaoZWQianTYJiangJPHouDJDengXJYangDD (2022) Species diversity and distribution of amphibians and reptiles in Hunan Province, China. Biodiversity Science 30: 21290. https://doi.org/10.17520/biods.2021290 IngerRFRomerJD (1961) A new pelobatid frog of the genus Megophrys from Hong Kong.Fieldiana39: 533–538. https://doi.org/10.5962/bhl.title.3373 IngerRFStuebingRBTanFL (1995) New species and new records of anurans from Borneo.The Raffles Bulletin of Zoology43: 115–131. IUCN SSC Amphibian Specialist Group (2022) Megophrys brachykolos. The IUCN Red List of Threatened Species 2022: e.T57633A63856967. https://doi.org/10.2305/IUCN.UK.2022-2.RLTS.T57633A63856967.en [accessed 22 Jan 2023] JiangJPYeCYFeiL (2008) A new horn toad Megophrys sangzhiensis from Hunan, China (Amphibia, Anura).Zoological Research29: 219–222. https://zoores.ac.cn/en/article/doi/10.3724/SP.J.1141.2008.02219 KuhlHvan HasseltJC (1822) Uittreksels uit brieven van de Heeren Kuhl en van Hasselt, aan de Heeren C. J. Temminck, Th. van Swinderen en W. de Haan, D. J. van Ewyck, H. Boie en Z E. den Minister voor het openhaar Onerdijs, de Nationale Nijverheid en de Kolonien.Algemeene konst-en Letterbode7: 99–104. LanfearR.FrandsenPBWrightAMSenfeldTCalcottB (2017) PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses.Molecular Biology and Evolution34: 772–773. https://doi.org/10.1093/molbev/msw260 LiSZXuNLiuJJiangJPWeiGWangB (2018) A new species of the Asian toad genus Megophrys sensu lato (Amphibia: Anura: Megophryidae) from Guizhou Province, China.Asian Herpetological Research9: 224–239. https://doi.org/10.16373/j.cnki.ahr.180072 LiYZhangDDLyuZTWangJLiYLLiuZYChenHHRaoDQJinZFZhangCYWangYY (2020) Review of the genus Brachytarsophrys (Anura: Megophryidae), with revalidation of Brachytarsophrys platyparietus and description of a new species from China.Zoological Research41: 105–122. https://doi.org/10.24272/j.issn.2095-8137.2020.033 LiYLJinMJZhaoJLiuZYWangYYPangH (2014) Description of two new species of the genus Megophrys (Amphibia: Anura: Megophryidae) from Heishiding Nature Reserve, Fengkai, Guangdong, China, based on molecular and morphological data.Zootaxa3795: 449–471. https://doi.org/10.11646/zootaxa.3795.4.5 LiaoCLLiangQQWangYJiangJP (2013) Additional notes on Megophrys sangzhiensis Jiang, Ye et Fei, 2008 (Amphibia, Anura, Megophryidae).Sichuan Journal of Zoology32: 217–218. LiuJLiSWeiGXuNChengYWangBWuJ (2020) A new species of the Asian toad genus Megophrys sensu lato (Anura: Megophryidae) from Guizhou Province, China.Asian Herpetological Research11: 1–18. https://doi.org/10.16373/j.cnki.ahr.190041 LiuZYChenGLZhuTQZengZCLyuZTWangJMessengerKGreenbergAJGuoZXYangZHShiSHWangYY (2018) Prevalence of cryptic species in morphologically uniform taxa – Fast speciation and evolutionary radiation in Asian frogs.Molecular Phylogenetics and Evolution127: 723–731. https://doi.org/10.1016/j.ympev.2018.06.020 LuoTWangYLWangSWLuXLWangWFDengHQZhouJ (2021) A species of the genus Panophrys (Anura, Megophryidae) from southeastern Guizhou Province, China.ZooKeys1047: 27–60. https://doi.org/10.3897/zookeys.1047.61097 LyuZTLiYQZengZCZhaoJLiuZYGuoGXWangYY (2020) Four new species of Asian horned toads (Anura, Megophryidae, Megophrys) from southern China.ZooKeys942: 105–140. https://doi.org/10.3897/zookeys.942.47983 LyuZTZengZCWangJLiuZYHuangYQLiWZWangYY (2021) Four new species of Panophrys (Anura, Megophryidae) from eastern China, with discussion on the recognition of Panophrys as a distinct genus.Zootaxa4927: 9–40. https://doi.org/10.11646/zootaxa.4927.1.2 MahonySFoleyNMBijuSDTeelingEC (2017) Evolutionary history of the Asian Horned Frogs (Megophryinae): Integrative approaches to timetree dating in the absence of a fossil record.Molecular Biology and Evolution34: 744–771. https://doi.org/10.1093/molbev/msw267 MahonySTeelingECBijuSD (2013) Three new species of horned frogs, Megophrys (Amphibia: Megophryidae), from northeast India, with a resolution to the identity of Megophrys boettgeri populations reported from the region.Zootaxa3722: 143–169. https://doi.org/10.11646/zootaxa.3722.2.2 MalkmusRMantheyUVogelGHoffmannPKosuchJ (2002) Amphibians & Reptiles of Mount Kinabalu (North Borneo).Fuldaer Verlagsanstalt, Fulda, 424 pp. MathewRSenN (2010) Pictorial Guide to the Amphibians of North East India.Zoological Survey of India, Kolkata, 144 pp. MessengerKRDahnHALiangYXiePWangYLuC (2019) A new species of the genus Megophrys Günther, 1864 (Amphibia: Anura: Megophryidae) from Mount Wuyi, China.Zootaxa4554: 561–583. https://doi.org/10.11646/zootaxa.4554.2.9 MeyerCPGellerJBPaulayG (2005) Fine scale endemism on coral reefs: Archipelagic differentiation in turbinid gastropods.Evolution59: 113–125. https://doi.org/10.1111/j.0014-3820.2005.tb00899.x MoJWYangDDLiuS (2009) Vertical distribution of amphibian resources of Nanyue Hengshan National Nature Reserve of Hunan Province.Sichuan Journal of Zoology28: 145–146. MoXYShenYHLiHHWuXS (2010) A new species of Megophrys (Amphibia: Anura: Megophryidae) from the northwestern Hunan Province, China.Current Zoology56: 432–436. https://doi.org/10.1093/czoolo/56.4.432 MunirMHamidyAFarajallahASmithEN (2018) A new Megophrys Kuhl and van Hasselt (Amphibia: Megophryidae) from southwestern Sumatra, Indonesia.Zootaxa4442: 389–412. https://doi.org/10.11646/zootaxa.4442.3.3 MunirMHamidyAMatsuiMIskandarDTSidikIShimadaT (2019) A new species of Megophrys Kuhl & Van Hasselt (Amphibia: Megophryidae) from Borneo allied to M. nasuta (Schlegel, 1858).Zootaxa4679: 1–24. https://doi.org/10.11646/zootaxa.4679.1.1 NguyenTQPhamCTNguyenTTLuongAMZieglerT (2020) A new species of Megophrys (Amphibia: Anura: Megophryidae) from Vietnam.Zootaxa4722: 401–422. https://doi.org/10.11646/zootaxa.4722.5.1 PopeCH (1929) Four new frogs from Fukien Province, China.American Museum Novitates352: 1–5. http://digitallibrary.amnh.org/handle/2246/4057 PoyarkovNA JrDuongTVOrlovNLGogolevaSSVassilievaABNguyenLTNguyenVDHNguyenSNCheJMahonyS (2017) Molecular, morphological and acoustic assessment of the genus Ophryophryne (Anura, Megophryidae) from Langbian Plateau, southern Vietnam, with description of a new species.ZooKeys672: 49–120. https://doi.org/10.3897/zookeys.672.10624 QiSLyuZTWangJMoYMZengZCZengYJDaiKYLiYQGrismerLLWangYY (2021) Three new species of the genus Boulenophrys (Anura, Megophryidae) from southern China.Zootaxa5072: 401–438. https://doi.org/10.11646/zootaxa.5072.5.1 QianTYLiYHChenJLiPPYangDD (2023) Tadpoles of four sympatric megophryinid frogs (Anura, Megophryidae, Megophryinae) from Mangshan in southern China.ZooKeys1139: 1–32. https://doi.org/10.3897/zookeys.1139.81641 RonquistFTeslenkoMMarkPAyresDLDarlingAHöhnaSLargetBLiuLSuchardMAHuelsenbeckJP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space.Systematic Biology61: 539–542. https://doi.org/10.1093/sysbio/sys029 ShangYLBLuoYQianTYGaoZWLiYHDengGXYangDD (2023) Taxonomic discussion of Boulenophrys in Mangshan, with three new provincial records of Hunan, China. Life Science Research. https://doi.org/10.16605/j.cnki.1007-7847.2022.12.0235 ShenYH (1994) A new pelobatid toad of the genus Megophrys from China (Anura: Pelobatidae). In: China Zoological Society (Ed.) Proceeding of the 60th Anniversary of the Founding of the China Zoological Society.China Science & Technology Press, Beijing, 603–606. ShenYH [沈猷慧] (1965) A preliminary study on anurans from Nanyue Mountain, Hunan.Chinese Journal of Zoology2: 76–79. [湖南南岳山无尾两栖类的初步研究] https://doi.org/10.13859/j.cjz.1965.02.013 ShenYH [沈猷慧] (1983) Amphibian survey and fauna analysis in Hunan Province.Acta Herpetologica Sinica2: 49–58. [湖南省两栖动物调查及区系分析] ShenYHYangDDMoXYLiHHChenD (2014) Fauna Hunan, Amphibia.Hunan Science and Technology Press, Changsha, 390 pp., 1–40 plates. ShiSCLiDHZhuWBJiangWJiangJPWangB (2021) Description of a new toad of Megophrys Kuhl & Van Hasselt, 1822 (Amphibia: Anura: Megophryidae) from western Yunnan Province, China.Zootaxa4942: 351–381. https://doi.org/10.11646/zootaxa.4942.3.3 SimonCFratiFBeckenbachACrespiBLiuHFlookP (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers.Annals of the Entomological Society of America87: 651–701. https://doi.org/10.1093/aesa/87.6.651 SuHJShiSCWuYQLiGRYaoXGWangBLiSZ (2020) Description of a new horned toad of Megophrys Kuhl & Van Hasselt, 1822 (Anura, Megophryidae) from southwest China.ZooKeys974: 131–159. https://doi.org/10.3897/zookeys.974.56070 TamuraKStecherGPetersonDFilipskiAKumarS (2013) MEGA6: Molecular Evolutionary Genetics Analysis, version 6.0.Molecular Biology and Evolution30: 2725–2729. https://doi.org/10.1093/molbev/mst197 TapleyBCutajarTMahonySNguyenCTDauVQLuongAMLeDTNguyenTTNguyenTQPortwayCLuongHVRowleyJJL (2018) Two new and potentially highly threatened Megophrys Horned frogs (Amphibia: Megophryidae) from Indochina’s highest mountains.Zootaxa4508: 301–333. https://doi.org/10.11646/zootaxa.4508.3.1 TapleyBCutajarTMahonySNguyenCTDauVQNguyenTTLuongHVRowleyJJL (2017) The Vietnamese population of Megophrys kuatunensis (Amphibia: Megophryidae) represents a new species of Asian horned frog from Vietnam and southern China.Zootaxa4344: 465–492. https://doi.org/10.11646/zootaxa.4344.3.3 TapleyBCutajarTNguyenLTPortwayCMahonySNguyenCTHardingLLuongHVRowleyJJL (2021) A new potentially endangered species of Megophrys (Amphibia: Megophryidae) from Mount Ky Quan San, north-west Vietnam.Journal of Natural History54: 2543–2575. https://doi.org/10.1080/00222933.2020.1856952 TianWSHuQX (1983) Taxonomic study on genus Megophrys, with descriptions of two new genera.Acta Herpetologica Sinica, New Series,2: 41–48. TianYZGuXMSunAQ (2000) A new species of Xenophrys in China (Amphibia: Pelobatidae).Acta Zootaxonomica Sinica25: 462–466. WangJLyuZTLiuZYLiaoCKZengZCZhaoJLiYLWangYY (2019a) Description of six new species of the subgenus Panophrys within the genus Megophrys (Anura, Megophryidae) from southeastern China based on molecular and morphological data.ZooKeys851: 113–164. https://doi.org/10.3897/zookeys.851.29107 WangJZengZCLyuZTQiSLiuZYChenHHLuYHXiaoHWLinCRChenKWangYY (2022) Description of three new Boulenophrys species from eastern Guangdong, China, emphasizing the urgency of ecological conservation in this region (Anura, Megophryidae).Zootaxa5099: 91–119. https://doi.org/10.11646/zootaxa.5099.1.4 WangJLiuZYLyuZTZengZCWangYY (2017a) A new species of the genus Xenophrys (Amphibia: Anura: Megophryidae) from an offshore island in Guangdong Province, southeastern China.Zootaxa4324: 541–556. https://doi.org/10.11646/zootaxa.4324.3.8 WangYFLiuBQJiangKJinWXuJNWuCH (2017b) A new species of the horn toad of the genus Xenophrys from Zhejiang, China (Amphibia: Megophryidae).Chinese Journal of Zoology52: 19–29. https://doi.org/10.13859/j.cjz.201701003 WangLDengXJLiuYWuQQLiuZ (2019b) A new species of the genus Megophrys (Amphibia: Anura: Megophryidae) from Hunan, China.Zootaxa4695: 301–330. https://doi.org/10.11646/zootaxa.4695.4.1 WangYYZhangTDZhaoJSungYHYangJHPangHZhangZ (2012) Description of a new species of the genus Xenophrys Günther, 1864 (Amphibia: Anura: Megophryidae) from Mount Jinggang, China, based on molecular and morphological data.Zootaxa3546: 53–67. https://doi.org/10.11646/zootaxa.3546.1.4 WangYYZhaoJYangJHZhouZXChenGLLiuY (2014) Morphology, molecular genetics, and bioacoustics support two new sympatric Xenophrys toads (Amphibia: Anura: Megophryidae) in southeast China. PLoS One 9: e93075. https://doi.org/10.1371/journal.pone.0093075 WuYQLiSZLiuWWangBWuJ (2020) Description of a new horned toad of Megophrys Kuhl & Van Hasselt, 1822 (Amphibia, Megophryidae) from Zhejiang Province, China.ZooKeys1005: 73–102. https://doi.org/10.3897/zookeys.1005.58629 WuYHSuwannapoomCPoyarkovNA JrPawangkhanantPXuKJinJQMurphyRWCheJ (2019) A new species of the genus Xenophrys (Anura: Megophryidae) from northern Thailand.Zoological Research40: 564–574. http://doi.org/10.24272/j.issn.2095-8137.2019.032 XuNLiSZLiuJWeiGWangB (2020) A new species of the horned toad Megophrys Kuhl & Van Hasselt, 1822 (Anura, Megophryidae) from southwest China.ZooKeys943: 119–144. https://doi.org/10.3897/zookeys.943.50343 YangJHWangJWangYY (2018) A new species of the genus Megophrys (Anura: Megophryidae) from Yunnan Province, China.Zootaxa4413: 325–338. https://doi.org/10.11646/zootaxa.4413.2.5 YeCYFeiLXieF (2007) A new species of MegophryidaeMegophrys baolongensis from China (Amphibia: Anura). In: Ji X (Eds) Herpetologica Sinica, Series 11. Southeast University Press, Nanjing, 38–41, pl. 11. ZhangYNLiGXiaoNLiJQPanTWangHZhangBWZhouJ (2017) A new species of the genus Xenophrys (Amphibia: Anura: Megophryidae) from Libo County, Guizhou, China.Asian Herpetological Research8: 75–85. https://doi.org/10.16373/j.cnki.ahr.160041 Appendix 1

Specimens examined

B. brachykolos (13): China: Hong Kong (type locality): SYS a001502–1503; Guangdong: Shenzhen: Mt. Yangtaishan: SYS a002051–2056, 2453–2454; Dapeng Peninsula: SYS a002406–2408.

B. tuberogranulata (12): China: Hunan: Zhangjiajie: Mt. Tianzishan: CSUFT 091–099, 206–208.

B. sangzhiensis (9): China: Hunan: Sangzhi: Mt. Tianpingshan: CSUFT 2006006–2006014.