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Research Article
A new species of Xenophrys (Amphibia, Anura, Megophryidae) from southern Tibet, China
expand article infoGuocheng Shu‡§, Ke Li‡, Yayong Wu‡, Qin Liu‡, Zhongping He‡, Ling Li‡|, He Zhang‡|, Peng Guo‡
‡ Yibin University, Yibin, China
§ Key Lab of Aromatic Plant Resources Exploitation and Utilization in Sichuan Higher Education, Yibin, China
| Shenyang Normal University, Shenyang, China

Corresponding author: Peng Guo ( ybguop@163.com )

Academic editor: Annemarie Ohler
© 2023 Guocheng Shu, Ke Li, Yayong Wu, Qin Liu, Zhongping He, Ling Li, He Zhang, Peng Guo.
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.
Citation: Shu G, Li K, Wu Y, Liu Q, He Z, Li L, Zhang H, Guo P (2023) A new species of Xenophrys (Amphibia, Anura, Megophryidae) from southern Tibet, China. ZooKeys 1182: 307-329. https://doi.org/10.3897/zookeys.1182.106828
ZooBank: urn:lsid:zoobank.org:pub:64598C08-BB92-49D8-B852-E4CE7B01FD6F
Open Access

Abstract

A new species of Xenophrys is described from Yadong County, Tibet Autonomous Region, China based on morphological and molecular evidence. Phylogenetic analyses based on the mitochondrial genes 16S rRNA and COI indicated that this new species represents an independent lineage and the minimum p-distance based on 16S rRNA between this species and its congeners is 4.4%. Additionally, the new species is distinguished from its congeners by a combination of the following morphological characters: (1) small body size, SVL 17.9–22.2 mm in adult males and SVL 23.4 mm in the single adult female; (2) tympanum indistinct, supratympanic fold distinct; (3) canthus rostralis well-developed, snout tip far beyond the margin of the lower lip; (5) pupil vertical; (6) vomerine teeth present, maxillary teeth present; (7) tongue notched posteriorly; (8) supernumerary tubercles absent, subarticular, metacarpal and metatarsal tubercles indistinct; (9) relative finger lengths I < II < IV < III, finger tips rounded, slightly expanded relative to digit widths; (10) toes with narrow lateral fringes and tarsal folds; (11) a dark triangular marking with light edge between eyes, a dark “)(”-shaped marking, with light edge, present on center of dorsum, pectoral glands on sides of the breast.

Key words

Morphology, phylogenetic analyses, taxonomy, Xenophrys pangdaensis sp. nov., Yadong County

Introduction

The Asian horned toad Megophryinae are widely distributed from northern India (west of Nepal) to eastern China and south to the Sundas and the Philippines (Frost 2023). Currently, 132 species have been described until July 2023, more than half of which have been named since the turn of the century (Frost 2023). There has been controversy about the generic classification of this group for a long time (e.g., Huang and Fei 1981; Tian and Hu 1983; Dubois 1987; Lathrop 1997; Rao and Yang 1997; Jiang et al. 2003; Delorme et al. 2006; Fei et al. 2009; Fei and Ye 2016; Chen et al. 2017; Mahony et al. 2017; Liu et al. 2018; Dubois et al. 2021; Lyu et al. 2021, 2023; Frost 2023). So far, the Megophryinae were defined as comprising ten clades by recent multilocus phylogenetic studies, including Atympanophrys, Brachytarsophrys, Megophrys, Ophryophryne, Boulenophrys, Pelobatrachus, Grillitschia, Jingophrys, Sarawakiphrys, and Xenophrys (Lyu et al. 2023). Previously, most authors regarded these clades as five or seven genera (Chen et al. 2017; Liu et al. 2018), while few researchers held a conservative attitude and regarded them as seven subgenera (Mahony et al. 2017; Shi et al. 2020). The genus Panophrys was established by Rao and Yang (1997); however, Dubois et al. (2021) noted that the generic name was preoccupied by Panophrys Dujardin, 1840 (Protozoa), so they chose Boulenophrys to replace it under the Principle of Homonymy.

In this study, we followed the classification system in Dubois et al. (2021) and Lyu et al. (2023) that Megophryidae contains ten genera (Atympanophrys, Brachytarsophrys, Megophrys, Ophryophryne, Boulenophrys, Pelobatrachus, Grillitschia, Jingophrys, Sarawakiphrys, and Xenophrys). Currently, the genus Xenophrys contains 28 recognized species in the world, which are distributed in Nepal, Bhutan, Bangladesh, India, Myanmar, Thailand, Laos, Vietnam, Cambodia, China, and Malaysia, of which ten are recorded in China (Frost 2023). In fact, nearly one third of the species in the genus were described in the last five years (Mahony et al. 2018, 2020; Shi et al. 2020; Luong et al. 2022), so the species diversity of the genus may have been underestimated. The eastern Himalaya is one of the 36 global biodiversity hotspots (Basnet et al. 2019), many new species have been discovered and named in this region in recent years (e.g., Jiang et al. 2016a, b, c; Shi et al. 2020). Over the past two years, several field surveys were conducted in this area and specimens of the family Megophryidae were collected. Also, a new bush frog, Raorchestes yadongensis Zhang, Shu, Liu, Dong, & Guo, 2022, was recently found and described (Zhang et al. 2022). Based on morphological comparison and molecular phylogenetic analyses, some specimens were identified a new member of Xenophrys.

Materials and methods

Sampling

Field surveys were conducted in August 2020 and July 2021. In total, seven adult specimens of Xenophrys were collected from two sites in Yadong County, Tibet Autonomous Region, China (Fig. 1). Thirteen tadpoles of the new taxon were also collected in a puddle where the new taxon was found. In the field, after taking photographs, the toads were euthanized using isoflurane, and then specimens were fixed in 75% ethanol. Tissue samples were taken and preserved separately in 95% ethanol prior to fixation. Specimens collected in this work were deposited in Yibin University. The Animal Care and Use Committee of Yibin University provided full approval for this research (No. 202003).

Figure 1. 

Type locality of the new species Xenophrys in Yadong County, Tibet Autonomous Region, China. The red star indicates the provincial capital, the black triangle indicates Medog County, the blue and black spots indicate the type locality and collection site of some tadpoles, respectively.

Molecular phylogenetic analysis

Total genomic DNA was extracted using TIANamp Genomic DNA Purification Kit (TIANGEN Bio-tech Co., Ltd., Beijing, China), following manufacturer instructions. Two fragments of mitochondrial genes, 16S ribosomal RNA gene (16S) and the cytochrome C oxidase 1 gene (COI), were amplified and sequenced. Primer sequences were retrieved from the literature for 16S (Simon et al. 1994) and COI (Che et al. 2011), respectively. PCR amplifications were performed in a 25 μl volume reaction with the following conditions: an initial denaturing step at 95 °C for 4 min; 36 cycles of denaturing at 95 °C for 40 s, annealing at 55 °C (for 16S)/52 °C (for COI) for 40 s and extending at 72 °C for 70 s, and a final extending step of 72 °C for 10 min. PCR products were sequenced with both forward and reverse primers same as used in PCR. Sequencing was conducted using an ABI3730 automated DNA sequencer in Sangon Biotechnologies Co., Ltd. (Shanghai, China). New sequences were uploaded to GenBank (for accession numbers see Table 1).

Table 1.
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Information of samples used in the molecular analyses.

Species Voucher ID 16S COI References
A. gigantica SYS a003934 MH406766 MH406225 Liu et al. 2018
A. shapingensis SYS a005310; KIZ YPX37515 MH406890 MH406352 Liu et al. 2018
A. wawuensis SYS a005311 MH406891 MH406353 Liu et al. 2018
Bo. binglingensis SYS a005313; KIZ 025807; FMNH 232874 MH406892 MH406354 Liu et al. 2018; Chen et al. 2017; Mahony et al. 2017
Bo. boettgeri SYS a004149; KIZ YPXJK033 MF667878 MH406247 Liu et al. 2018; Chen et al. 2017
Bo. brachykolos SYS a002258; ROM 16634 KJ560403 MH406120 Liu et al. 2018; Chen et al. 2017
Bo. cheni SYS a004050 MF667873 MH406241 Liu et al. 2018
Bo. chishuiensis SYS a005307; KIZ 025788 MH406888 MH406350 Liu et al. 2018; Chen et al. 2017
Bo. daoji SYS a004089 MH406783 MH406243 Liu et al. 2018
Bo. fansipanensis VNMN 2018.01 MH514886 MW086544 Tapley et al. 2021
Bo. frigida AMS R186131 MT364279 MW086550 Tapley et al. 2021
Bo. huangshanensis SYS a002702; KIZ 022004 MF667882 MH406160 Liu et al. 2018; Chen et al. 2017
Bo. kuatunensis SYS a003449 MF667881 MH406206 Liu et al. 2018
Bo. minor SYS a003212; KIZ YPX37545 MF667865 MH406197 Liu et al. 2018; Chen et al. 2017
Bo. nanlingensis SYS a001962 MH406645 MH406081 Liu et al. 2018
Bo. sangzhiensis SYS a004313; KIZ YPX11006 MH406802 MH406264 Liu et al. 2018; Chen et al. 2017
Bo. spinata SYS a002226; KIZ 016100 MH406675 MH406115 Liu et al. 2018; Chen et al. 2017
Bo. wushanensis SYS a003009; KIZ 045469 MH406733 MH406185 Liu et al. 2018; Chen et al. 2017
Br. chuannanensis SYS a004927 MH406902 MH406365 Liu et al. 2018
Br. feae SYS a003913; KIZ 046706 MH406900 MH406363 Liu et al. 2018; Chen et al. 2017
Br. orientalis SYS a004225 OQ180989 MT162625 Li et al. 2020; Lyu et al.2023
Br. platyparietus SYS a005919 OQ180990 MT162633 Li et al. 2020; Lyu et al. 2023
Br. popei SYS a001864 KM504256 MH406361 Liu et al. 2018
G. aceras LSUHC 7038 GQ995534 N Chen et al. 2017
G. longipes IABHU 21101 AB530656 N Hasan et al. 2014
J. cf. pachyproctus CIB022017061805 MN963228 MN964303 Shi et al. 2020
J. vegrandis Z11605 HT KY022305 MH647530 Mahony et al. 2020
J. yeae CIB201706MT02 MN963216 MN964313 Shi et al.2020
J. zhoui CIBMT171053 MN963207 MN964322 Shi et al.2020
J. feii SYS a003876 OQ181007 OQ180893 Lyu et al. 2023
Lep. alpina SYS a003927 MH406905 MH406368 Liu et al. 2018
Lep. laui SYS a003471 MH406903 MH406366 Liu et al. 2018
M. acehensis MZB Amph 26098 MT710708 N Munir et al. 2021
M. lancip ENS 7577 KX773567 N Mahony et al. 2017
M. montana LSUMZ 81916; UTA A-53736, ENS 7381 KX811927 KX812163 Chen et al. 2017; Mahony et al. 2017
M. parallela RMAS 022 KY679898 N Munir et al. 2018
M. selatanensis MZB Amph 22411 MT710704 N Munir et al. 2021
O. hansi AMNH 169285 KY022204 KX812155 Mahony et al. 2017; Chen et al. 2017
O. microstoma AMNH 168682 KY022199 N Mahony et al. 2017
O. poilani AMNH 169287 KY022202 N Mahony et al. 2017
P. baluensis IRSNB 15926 DQ642121 N Mahony et al. 2017
P. edwardinae FMNH 273694 KX811918 KX812050 Chen et al. 2017
P. kalimantanensis KUHE 53577 AB719248 N Hamidy et al. 2012
P. kobayashii UNIMAS 8148 KJ831313 N Oberhummer et al. 2014
P. ligayae KUKUH309095; ZMMUNAP-05015 KY022192 KX812051 Mahony et al. 2017; Chen et al. 2017
P. stejnegeri FMNH 250842; KU 314303 KY022190 KX812052 Mahony et al. 2017; Chen et al. 2017
S. dringi UNIMAS 8948 KJ831316 N Oberhummer et al. 2014
X. ancrae Z11606 [S2011.307] HT MN734391 N Mahony et al. 2020
X. auralensis NCSM 79599 KX811807 N Mahony et al. 2018
X. awuh BN6069 PT KY022319 N Mahony et al. 2020
X. dzukou BN6072 HT KY022324 N Mahony et al. 2020
X. flavipunctata SDBDU 2009.297 TT KY022307 MH647536 Mahony et al. 2018
X. glandulosa SYSa003795 MH406760 MH406219 Shi et al. 2021
X. himalayana BNHS 6050 MH647526 N Mahony et al. 2018
X. lekaguli FMNH 265955 PT KY022214 N Mahony et al. 2017
X. major SDBDU 2007.229 MH647514 N Mahony et al. 2018
X. mangshanensis KIZ021786 KX811790 KX812079 Shi et al. 2020
X. maosonensis ROM 16679 KX811784 KX812081 Shi et al.2020
X. medogensis CIB022017062002 MN963219 MN964310 Shi et al.2020
X. megacephala ZSIC A 11213 HT KY022315 MH647533 Mahony et al. 2018
X. monticola SDBDU 2011.1047 KY022312 N Mahony et al. 2017
X. numhbumaeng BN6076 PT MN734393 N Mahony et al. 2020
X. oreocrypta BN6046 PT KY022306 N Mahony et al. 2020
X. oropedion SDBDU 2009.299 KY022317 MH647534 Mahony et al. 2018
X. pangdaensis sp. nov. YBU21248 HT OR026569 OR026034 This study
X. pangdaensis sp. nov. YBU21259 PT OR026570 OR026035 This study
X. pangdaensis sp. nov. YBU21260 PT OR026571 OR026036 This study
X. pangdaensis sp. nov. YBU21261 PT OR026572 OR026037 This study
X. pangdaensis sp. nov. YBU21262 PT OR026573 OR026038 This study
X. pangdaensis sp. nov. YBU21269 PT OR026574 OR026039 This study
X. periosa BNHS 6061 PT KY022309 MH647528 Mahony et al. 2018
X. robusta SDBDU 2011.1057 TT KY022314 MH647535 Mahony et al. 2018
X. serchhipii SDBDU 2009.612 KY022323 MH647532 Mahony et al. 2018
X. takensis FMNH 261711 KY022215 N Mahony et al. 2017
X. truongsonensis IEBR A.4952 ON146202 N Luong et al. 2022
X. zhangi KIZ014278 KX811765 KX812084 Mahony et al. 2018
X. zunhebotoensis RGK 0041 TT KY022322 N Mahony et al. 2018,
X. dehongensis SYS a003443; KIZ 048507 MH406746 MH406204 Liu et al. 2018; Chen et al. 2017
X. katabhako K5204/ZSI 11401 HT KX894667 N Deuti et al. 2017
X. lancangica SYS a002961; KIZ01464; AMNH168679 MH406728 MH406180 Liu et al. 2018; Chen et al. 2017; Mahony et al. 2017
X. parva SYS a003042; KIZ YPX27643 MH406737 MH406189 Liu et al. 2018; Chen et al. 2017
X. sanu K5197/ZSI 11392 HT KX894678 N Deuti et al. 2017

Specimen status: HT, holotype; PT, paratype; TT, topotype.

For molecular analyses, the available sequences of Xenophrys species were downloaded from GenBank, especially for their holotypes and/or topotypes for which comparable sequences were available (Table 1). Representative species sequences for all recognized megophryid genera were also downloaded for phylogenetic analysis (also including two controversial species X. katabhako comb. nov. and X. sanu comb. nov.). Sequences were assembled and aligned using the Clustalw module in BioEdit 7.0.9.0 (Hall 1999) with default settings. Alignments were checked by eye and revised manually if necessary. PartitionFinder v. 2.1.1 (Lanfear et al. 2017) was used to select the corresponding best-fit nucleotide substitution models for 16S gene/each codon position of COI gene under the Akaike Information Criteria (AIC). Phylogenetic analyses of the concatenated-sequence matrix were conducted in MrBayes v. 3.2.4 (Ronquist et al. 2012). Two independent runs were conducted in the BI analysis, and each run consisted of 5 × 107 generations, sampled every 1000 generations. Runs were considered to have converged when the average standard deviation of split frequencies (ASDSF) was less than 0.01. The first 25% of generations were removed as the “burn-in” stage followed by calculation of Bayesian posterior probabilities (BPP) and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity. The phylogenetic trees were visualized using FigTree 1.4.3 (Rambaut 2016). Mean genetic distances between Xenophrys species were calculated in MEGA 7 (Kumar et al. 2016) using the uncorrected p-distance model based on 16S gene (some species lack of COI gene).

Morphological analysis

A total of seven adult specimens were measured. The terminology and methods followed Mahony (2011). Measurements were taken with a dial caliper to the nearest 0.1 mm. Thirty characters of adult specimens were measured:

EL eye length (horizontal distance between the anterior and posterior borders of orbit);

EN eye-nostril length (distance from front of eye to the center of nostril);

FAL forearm length (distance from elbow to wrist);

FIL first finger length (distance from the tip of the first digit to its base where it joins the second digit);

FIIL second finger length (distance from the tip of the second digit to its base where it joins the first digit);

FIIIL third finger length (distance from the tip of the third digit to its base where it joins the second digit);

FIVL fourth finger length (measured from the tip of the fourth digit to its base where it joins the third digit);

FIIIW minimum third finger width (taken at the base of the terminal portion of the digit, which is expanded on some species);

FIIIDW maximum width of the third fingertip;

FOL foot length (distance from the proximal end of the inner metatarsal tubercle to the tip of the fourth digit);

HAL hand length (distance from wrist to tip of third digit);

HL head length (distance from the rear of the mandible to the tip of the snout);

HLL hindlimb length;

HW head width (distance between the posterior angles of jaw);

IBE internal back of eyes (the shortest distance between the posterior borders of the orbits);

IFE internal front of eyes (shortest distance between the anterior borders of orbits);

IMT length of the inner metatarsal tubercle;

IN internarial distance (shortest distance between two nostrils);

IUE inter upper eyelid width (shortest distance between upper eyelids);

SHL shank length (distance from knee to ankle);

SL snout length (distance from tip of snout to anterior border of the orbit);

SN nostril-snout length (distance from center of the nostril to tip of the snout);

SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent);

TFOL tarsal-foot length (distance from heel to the tip of the fourth digit);

TIVW minimum fourth toe width (taken at the base of the terminal portion of the digit, which is expanded on some species);

TIVDW maximum width of the fourth toe tip;

TL thigh length (distance from cloaca to knee);

TYD largest tympanum diameter;

TYE tympanum-eye distance (distance from the anterior border of the tympanum to the posterior orbital border);

UEW maximum upper eyelid width.

Thirteen tadpoles of the new taxon were measured. The stages of tadpoles were identified following Gosner (1960). Seventeen morphometric characters of tadpoles were measured:

BH maximum body height;

BL body length (distance from tip of snout to trunk-tail junction);

BW maximum body width;

ED maximum eye diameter;

IND internasal distance (distance between center of two naris);

LF maximum height of lower tail fin;

NE naris-eye distance (distance from center of naris to anterior corner of eye);

ODW oral disc width (largest width of oral disc);

PP interpupilar distance;

RN rostro-narial distance (distance from tip of snout to center of naris);

SS snout-spiracle distance (distance from tip of snout to opening of spiracle);

SU snout-upper fin distance (distance from snout to beginning of upper tail fin);

TAL tail length (distance between posterior side of opening of cloaca to tip of tail);

TMH maximum tail muscle height;

TMW maximum tail muscle width;

TOL total length;

UF maximum height of upper tail fin.

Sex and maturity of the specimens were confirmed by direct examination of secondary sexual characters, including vocal sacs, nuptial pads, and the gonadal inspection (Fei and Ye 2016; Mahony et al. 2020). For webbing description, we followed Glaw and Vences (2007). We compared the morphological characters of the new species with literature data for 28 other species of Xenophrys.

Results

Phylogenetic analyses

The aligned sequence matrix of 16S and COI genes contained 574 bps and 663 bps, respectively. Except X. damrei, all other species of Xenophrys were included in the phylogenetic analysis. The model selection suggested that GTR+I+G as the best model for 16S rRNA fragment, and GTR+I+G, GTR+I+G, and HKY+I as the best model for the first, second and third codon position of COI gene, respectively. The BI phylogenetic tree is shown in Fig. 2 with Bayesian posterior probabilities (BPP) for major nodes. The phylogenetic tree showed that all Xenophrys species formed a monophyletic lineage containing 11 independent clades, though some relationships were not resolved in the tree. Those clades correspond exactly to the ten genera previously recognized, including Pelobatrachus (clade A), Sarawakiphrys (clade B), Megophrys (clade C), Brachytarsophrys (clade D), Atympanophrys (clade E), Grillitschia (clade F), Ophryophryne (clade G), Boulenophrys (clade H). Jingophrys (clade I), Xenophrys (clade J), and clade K. Significantly, J. cf. pachyproctus alone formed a clade. All samples from Yadong were strongly supported to be a monophyletic group and formed sister relationships with X. flavipunctata, X. glandulosa, X. himalayana, X. periosa, X. robusta, X. mangshanensis, X. maosonensis, X. truongsonensis, X. medogensis, X. megdogensis, X. monticola, and X. zhangi. X. katabhako comb. nov. and X. monticola formed a clade. X. sanu comb. nov. and X. zhangi clustered into another clade.

Figure 2. 

Phylogenetic tree of the genus Xenophrys inferred from two mitochondrial gene fragments by Bayesian inference.

Interspecific uncorrected p-distance of the Xenophrys species ranged from 0.0 (X. zhangi and X. sanu comb. nov.) to 18.7% (X. awuh and X. dzukou) (Table 2). The minimum p-distance between the unidentified specimens and any other species of Xenophrys was 4.4% (with X. glandulosa) (Table 2).

Table 2.
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Uncorrected pairwise sequence divergences (p-distance, in %) based on 16S ribosomal RNA sequences.

No. Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
1 X. ancrae
2 X. auralensis 9.4
3 X. awuh 9.9 14.7
4 X. dzukou 6.9 9 18.7
5 X. flavipunctata 8.5 9.4 13.6 10.2
6 X. glandulosa 7.7 7.1 12.1 7.5 4.6
7 X. himalayana 7.9 6.7 10.5 8.6 5.2 3.3
8 X. legkaguli 8.4 4 14.8 8.9 9.6 7.8 7.6
9 X. major 7.9 7.7 11.4 8.9 6.6 5.5 4.7 8.8
10 X. mangshanensis 8.2 8 11.5 9.7 5.7 5.6 5.7 9.3 6.9
11 X. maosonensis 7.9 8.4 12.4 9.7 6.1 5.6 5.2 9 6.5 1.9
12 X. medogensis 7 7.4 11.7 8.2 5 3.8 3.3 7.8 5.2 5.4 5
13 X. monticola 9.7 7.5 13.3 10.2 7 5.2 5.6 9 7.4 6.3 6.9 6.7
14 X. megacephala 7.1 9.5 12.1 8.2 9.8 9.1 8.6 8.6 8.4 8.7 8.9 9 10.2
15 X. numhbumaeng 8.2 10.2 10.7 12.2 10 9.8 10 11.5 9.8 10.7 10.9 9.5 11 7.8
16 X. oreocrypta 9.1 8.6 14.5 10.2 7.4 6.9 6.6 8.4 7 8.4 7.8 7.5 8.5 9.4 10.5
17 X. oropedion 8.5 11.1 13.4 11.8 11.3 10.7 10.5 11.2 10.5 10.1 10.7 10.5 11.3 7.8 5.1 11.1
18 X. periosa 8.7 7.3 12.2 8.9 5.6 3.1 2.5 7.8 6 6.1 5.9 3.3 6.2 8.6 10.2 7.2 10.7
19 X. robusta 9.9 8.8 13.3 11.5 6.2 4.4 4.6 9.2 6.8 6.9 6.9 4.8 6.2 10.4 10.6 7.8 11.9 4.1
20 X. serchhipii 8.3 11.4 8.7 11.5 10.2 10.6 10 10.9 10 9.4 10.2 10.4 12.4 8.5 9.2 11.6 10.3 10.4 11.6
21 X. takensis 9.5 4 14.2 9.9 9.4 7.2 6.8 4.7 8.4 8.2 8.4 6.7 8.6 8.8 10.9 9 11.4 7.4 8.6 10.8
22 X. truongsonensis 8 7.7 12.4 10.6 7.2 6.3 6.5 7.9 6.5 3.6 3.3 5.2 7.8 9.1 10.7 7.7 10.4 7.2 7.2 9.9 9
23 X. zhangi 7 6.5 11.4 9 5.6 3.8 4 7.4 4.8 5.2 5 3.6 4.4 9.3 8.9 7.3 10.5 4.4 4.8 10.6 6.7 5
24 X. zunhebotoensis 7.2 12.2 8.1 10.2 10.7 9.9 10.4 12.2 10.6 10.8 11 9.7 11.7 9.2 8.3 11.6 10 10.9 11.9 8.3 11.4 10.9 10.3
25 X. katabhako 8.4 9.5 12.9 9.6 7.3 4.9 5.9 9.3 7.1 5.7 6.3 5.9 2.7 10.1 11.9 8.9 12.5 6.8 6.2 12.2 8.7 7.4 4.3 11
26 X. lancangica 7 7.8 13.6 8.2 7.7 5.9 6.7 7.6 6.9 8.1 7.7 6.7 9.2 8.8 10 7.7 11.3 7.7 7.5 10.8 8.2 7.5 7.1 10.5 8.1
27 X. sanu 8.1 8.6 13.3 9.6 6.8 4.9 4.6 8.7 5.7 6.8 6.5 4.3 5.1 10.5 10.8 8.7 12.8 5.4 5.7 12.2 8.2 6.3 0 11.5 4.3 8.9
28 X. dehongensis 9.9 6.4 16.3 10.1 9.7 7.1 8.2 7.4 9.1 9.3 9.9 8.3 9.7 10.2 11.6 8.5 12.7 8.4 8.4 13.5 7.2 9.5 7.3 13.2 10.4 7.9 9.3
29 X. parva 10.5 6.7 15.9 10.8 10.1 7.5 7.8 7.4 8 8.9 9.1 7.7 9.2 10.2 12 8.6 12.3 8 8.2 12.2 7.4 8.4 7.5 12.7 9.9 7.5 9 6.8
30 X. pangdaensis sp.nov. 7.3 6.5 12.5 9.5 6 4.4 5.6 7.4 6.2 6.1 5.7 4.6 6.2 9.8 10 7.4 11.3 5 5.2 11 7.6 6.1 4.6 10 6.5 5.9 6.3 8.3 7.7

Morphological analysis

All samples from Yadong shared many morphological characters with Xenophrys species, including dorsal skin texture basically smooth, vomerine teeth present, ventral colorations, lateral fringes and webbings on toes, tongues notched posteriorly, maxillary teeth present, and tympanum indistinct. However, they can be distinguished from all recognized congeners by a combination of distinctive morphological characters (see taxonomic accounts below) and these specimens are therefore described as a new species based on the phylogenetic analyses and morphological comparisons.

Taxonomic accounts

Xenophrys pangdaensis sp. nov.

https://zoobank.org/262319B9-D690-4FDB-9A44-621FD390956E
Fig. 3

Type material

Holotype. YBU21248, adult male, collected by Ke Li and He Zhang on 28 August 2021 from Pangda Village (27°17.25'N, 89°0.42'E; ca. 2000 m a.s.l.), Yadong Town, Yadong County, Tibet Autonomous Region, China.

Paratypes. Six adult specimens (males: YBU21258, YBU21259, YBU21260, YBU21261, YBU21269; female: YBU21262) were collected from two very close sites in Yadong Town by Ke Li and He Zhang on 28 August 2021.

Other specimens examined

Thirteen tadpoles were collected by Ke Li and He Zhang on 4 September 2021. Five tadpoles were collected from Pangda Village (17 km, 27°18.18'N, 89°0.34'E), Yadong Town. The other tadpoles were collected from Pangda Village (27°17.25'N, 89°0.42'E) together with the holotype.

Etymology

The species name pangdaensis indicates the type locality of Pangda Village, Yadong County, Tibet Autonomous Region, China.

Suggested name

Pangda Horned Toad (English), and Pang Da Jiao Chan (庞达角蟾, Chinese).

Diagnoses

(1) Small body size, SVL 17.9–22.2 mm (20.5±1.8, n = 6) in adult males and SVL 23.4 mm (n = 1) in the adult female (Table 3); (2) tympanum indistinct, supratympanic fold distinct; (3) canthus rostralis well-developed, snout tip far beyond the margin of the lower lip; (5) pupil vertical; (6) vomerine teeth present, maxillary teeth present; (7) tongue notched terminally; (8) supernumerary tubercles absent, subarticular, metacarpal and metatarsal tubercles indistinct; (9) relative finger lengths I < II < IV < III, finger tips rounded, slightly expanded relative to digit widths; (10) toes with narrow lateral fringes and tarsal folds; (11) a dark triangular marking with light edge between eyes, a dark)(-shaped marking, with light edge, present on center of dorsum, pectoral glands on sides of the breast.

Table 3.
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Measurements (in mm) of the type series of Xenophrys pangdaensis sp. nov.

YBU21248 (holotype) YBU21262 (paratype) YBU21261 (paratype) YBU21259 (paratype) YBU21260 (paratype) YBU21269 (paratype) YBU21258 (paratype)
Sex male female male male male male male
SVL 21.5 23.4 21.7 20.9 18.6 17.9 22.2
FAL 3.4 5.9 7.5 5.5 4.3 3.6 4.6
HAL 6.5 6.3 7.2 5.2 5.1 5.8 8.0
HLL 30.4 33.5 30.5 34.9 30.4 26.6 35.4
SHL 10.9 11.4 11.3 9.8 9.1 9.2 11.6
SL 3.3 3.0 3.1 2.6 2.7 1.8 3.0
FOL 10.8 10.1 11.2 9.7 6.5 8.9 10.9
IN 2.5 2.9 2.1 2.4 2.9 3.1 2.7
IUE 2.9 2.3 2.4 2.1 3.3 2.5 3.4
UEW 1.9 2.0 1.6 1.3 2.11 2.5 1.9
TYD 1.8 1.1 1.7 1.7 1.8 1.6 1.5
HL 7.6 6.7 7.6 6.3 5.4 6.2 7.4
HW 6.9 7.1 7.0 6.2 6.2 6.5 8.5
FIL 2.6 2.6 3.3 2.3 2.2 2.5 3.2
FIIL 3.6 3.9 3.9 3.4 2.6 2.8 3.3
FIIIL 6.0 5.9 5.4 4.8 4.8 5.1 4.9
FIVL 4.4 3.9 4.6 3.7 3.1 3.8 3.3
SN 1.5 1.3 1.8 1.4 1.3 1.4 1.8
EN 1.6 1.4 2.4 1.5 1.5 1.8 1.9
EL 2.7 2.5 2.9 2.6 2.7 2.2 3.1
IFE 2.8 2.9 3.0 3.1 2.9 2.9 3.4
IBE 3.7 3.4 3.3 3.4 3.1 3.1 4.1
TYE 1.7 1.9 1.7 1.4 1.2 1.2 1.9
FIIIW 0.2 0.2 0.9 0.2 0.3 0.2 0.4
FIIIDW 0.3 0.3 0.3 0.3 0.3 0.3 0.4
TIVW 0.4 0.4 0.5 0.4 0.4 0.4 0.5
TIVDW 0.3 0.4 0.4 0.3 0.3 0.3 0.4
TL 9.1 8.9 8.6 9.1 8.9 9.2 10.1
TFOL 14.2 14.4 15.0 13.6 11.9 12.2 16.3
IMT 1.0 0.8 1.1 0.7 0.6 0.7 1.1

Holotype description

Measurements in mm. Mature male, body slender, extremely small (SVL 21.5) (Fig. 3); small protuberance beyond cloaca from dorsal view, not visible from ventral view, not swollen; head moderate, longer than wide (HW 7.0, HL 7.6, IFE 2.8, IBE 3.7); snout nearly rounded in dorsal view, slightly protruding beyond lower jaw, angular in anterior and lateral view; loreal region acute, concave; canthus rostralis angular; dorsal surface of snout slightly concave; nostril oval, closer to eye than tip of snout (SN 1.5, EN 1.6); internarial distance greater than eyelid width, and shorter than narrowest point between upper eyelids (IN 2.5, UEW 1.9, IUE 2.9); eye diameter 1.5 × maximum diameter of visible portion of tympanum, and shorter than snout (EL 2.7, TYD 1.8, SL 3.3); eye-tympanum distance (TYE 1.7) shorter than diameter of visible portion of tympanum; tympanum oval-shaped, slightly oblique, upper border concealed by supratympanic ridge; pupil in life vertically elliptical; pineal ocellus not visible externally; vomerine ridges medium sized, orientated acutely, positioned between to slightly posterior to choanae, vomerine teeth small; maxillary teeth present; tongue moderately large, feebly notched posteriorly, medial lingual process absent.

Figure 3. 

Xenophrys pangdaensis sp. nov. in life. A dorsal view of body B ventral view of body C ventral view of hand D ventral view of foot E lateral view of body F lateral view of head.

Forelimbs moderately long and thin; forearm slender, shorter than hand (FAL 3.4, HAL 6.5); fingers moderately long, with narrow lateral fringes and rudimentary webbing (Fig. 2); finger length formula I < II < IV < III (FIL 2.6, FIIL 3.6, FIIIL 6.0, FIVL 4.4); supernumerary, thenar and metacarpal tubercles absent, with slightly visible subarticular tubercles; finger tips rounded, with subcircular pads, without terminal grooves, slightly expanded relative to digit widths (FIIIW 0.2, FIIIDW 0.3).

Hindlimbs long and thin, heels overlapping when hindlimbs held at right angles to body; thighs slightly shorter than shanks and feet (TL 9.1, SHL 10.9, FOL 10.8); toes with narrow lateral fringes, rudimentary webbing; relative toe lengths I < II < V < III < IV; toe tips rounded, with subcircular pads, terminal grooves absent; supernumerary, subarticular and outer metatarsal tubercles absent; inner metatarsal tubercles indistinct.

Skin of dorsal and ventral surfaces of head, body and limbs basically smooth; dorsal skin with very small densely-distributed granules; tympanum smooth with borders slightly raised; supratympanic ridges thin before and above departure with tympanum and gradually expanding beyond posterior edge of tympanum; skin ridges formed by small disconnected tubercles;)(-shaped skin ridge on center of dorsum, its anterior ends extending posteriorly from above tympanum; flanks with two slender skin ridges, started at the shoulder and ended on both sides of the back of the cloaca; two small pectoral glands positioned on level with axilla; femoral glands moderate, positioned posterior surface of thigh, sub-equally distant from knee and cloaca.

Coloration in life

Dorsal surface basically saddle brown, darker on anterior and hindlimbs than on posterior; orange-red granules scattered on surface; dark brown) (-shaped marking with orange edge on central dorsum; ventrolateral trunk with white spots and orange dots; dark brown triangular pattern with orange edges presents between eyes, and dark brown rod-like pattern positioned in front of triangular pattern; supratympanic fold white mingled with orange flecks; temporal region under supratympanic ridge black; two dark brown patches present on upper lips under eye and nostril on side of head; eight relatively large white patches present on lower lip, symmetrically distributed; two white symmetrically curved lines on both sides of throat; many orange-red dots scattered on surface of throat; iris orange-red; two dark transverse bands on each forearm; finger tips orange-red; large white blotches on belly and ventral surfaces of hindlimbs; three dark transverse bands on anterior surface of thigh and shank; femoral glands white on thigh.

Coloration in preservative

After preservation in ethanol, dorsal surface primarily brown; dark brown triangular pattern with white edges presents between eyes; brown) (-shaped marking with white edge on central dorsum; two white slender skin ridges in flanks; two dark transverse bands on each forearm; dark brown band with white dots in middle of thigh and shank; throat pale brownish grey, two white symmetrically curved lines distinct; eight distinct white patches on lower lip; chest brown with two white pectoral glands; belly pale gray-white with large black-brown blotches on sides; posterior ventral body surface, thigh, and upper part of tibia pale brown with scattered white spots; ventral surfaces of fingers and toes dark brown with white blotches.

Variations

Paratypes generally resemble the holotype but with some differences. For example, a few specimens (YBU21258, YBU21262 and YBU21269) have the head width greater than the head length; YBU21258 had more and larger maxillary teeth, the tongue thinner; rod-like patterns on the top of head different between specimens. Coloration varied on ventral body, with some specimens being darker. The tips of the fingers in some specimens were not orange-red.

Sexual dimorphism

Males: external vocal sac indistinct; internal vocal slit present on floor of mouth near rear of mandible, one on each side; vocal sac, vocal slits, and enlarged forearms all absent in female.

Tadpole

Gosner stages 25–36. Body length range from 6.3–13.8 mm (Table 4); oral disk funnel like, positioned anterior-dorsal, large, width average 1.5× (1.1–1.7, n = 13) maximum body width, rice-like submarginal papillae scattered on lower and upper lips and pointed towards oral cavity; nares oval and closer to eye than to snout (RN 1.0, NE 0.4); internarial distance nearly equal to interorbital distance (IND 2.8, PP 2.7); eyes dorsolateral, pupils rounded; spiracle opens left of body in dorsal view, spiracular tube positioned equidistant between tip of the snout and trunk-tail junction; the tail accounts for 0.7 of the total length (TOL 37.0, TAL 26.6); dorsal fin arise near middle of tail, upper tail fin higher than lower tail, and approximately half of tail muscle height (UF 1.5, LF 1.1, TMW 2.8)(Fig. 4).

Table 4.
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Measurements (in mm) of the tadpoles of Xenophrys pangdaensis sp. nov. N indicates missing data. Character abbreviations are provided in the text.

Characters 1 2 3 4 5 6 7 8 9 10 11 12 13
Stage 36 31 31 31 33 31 35 32 31 27 25 29 36
BH 4.8 4.7 4.5 4.0 4.4 4.3 4.7 6.6 6.1 4.6 2.6 6.2 4.8
BL 11.4 10.3 10.8 10.4 11.0 11.5 10.9 13.8 11.8 10.4 6.3 11.9 10.6
BW 5.5 4.8 4.7 4.3 4.8 4.8 4.9 7.6 6.5 4.8 2.9 7.1 4.5
ED 0.9 1.1 1.0 1.0 1.0 1.0 1.0 1.2 1.0 0.9 0.8 1.0 1.3
IND 2.6 2.7 2.5 2.7 2.8 2.8 2.8 3.5 3.2 2.5 2.0 3.3 N
LF 1.0 1.2 1.1 1.2 1.2 1.0 1.2 1.2 1.2 1.1 0.7 1.2 1.1
NE 0.5 0.4 0.3 0.4 0.6 0.5 0.4 0.5 0.7 0.3 0.3 0.6 0.3
ODW 7.9 7.4 8.1 7.1 7.9 7.2 7.7 8.7 8.3 7.9 4.8 10.1 5.6
PP 3.1 2.7 2.5 2.8 2.8 2.5 2.9 3.3 2.9 2.6 1.6 3.3 N
RN 1.1 0.8 0.9 0.5 0.9 1.0 0.9 1.8 1.2 1.0 0.8 1.4 0.9
SS 5.8 5.8 5.6 5.7 5.7 5.4 5.7 7.4 6.8 5.4 3.7 6.4 5.3
SU 9.0 8.7 9.9 9.0 10.2 9.8 9.8 11.2 10.9 11.1 5.7 12.2 10.3
TAL 30.4 30.6 27.2 27.2 27.1 23.5 28.1 N 31.6 23.4 15.6 30.5 23.7
TMH 1.1 1.3 1.1 1.5 1.5 1.5 1.7 2.7 2.1 1.3 1.1 2.2 2.2
TMW 3.2 2.1 2.1 1.9 2.4 2.9 2.7 4.8 3.5 2.4 1.5 4.6 2.7
TOL 41.4 39.2 37.7 37.7 37.5 34.4 38.6 N 43.0 34.1 22.6 41.7 36.4
UF 1.9 1.4 1.6 1.3 1.4 1.4 1.3 2.1 1.8 1.3 0.9 1.5 1.3
TH 5.5 5.0 5.1 4.4 4.8 5.4 5.2 7.8 5.8 5.0 3.1 7.1 5.1
Figure 4. 

Tadpole of Xenophrys pangdaensis sp. nov. (Gonser stage 29) from Yadong County, Tibet Autonomous Region, China. A dorsal view of the tadpole B ventral view of the tadpole C lateral view of the tadpole. All photographs of just- preserved specimens.

Coloration in preservative. Dorsal and lateral parts of body greyish white, mixed with brown patches; lateral tail semi-transparent brown, muscle scattered with many distinct brown patches; no pigment on upper and lower fins; ventral body semi-transparent white, with tiny gray pigment scattered on it, the viscera can almost be seen; lips semi-transparent white, papillae brown. Coloration in life were not noted.

Distribution and ecology

Xenophrys pangdaensis sp. nov. is only known from the type locality, Yadong Town, Yadong County, Tibet Autonomous Region, China at elevations of 2003–2972 m. All calling males were recorded in August and September on ferns near or on a small stream in the tropical forest (Fig. 5). The tadpoles collected from near the type locality were from Gosner stages 25–36. The habitat is located in the small gully, both sides covered with ferns and other vegetation. None of the adults or tadpoles were found in July, and all specimens were found in late August and early September, implying that the breeding season included August and September. The sympatric species, Raorchestes yadongensis Zhang, Shu, Liu, Dong & Guo, 2022, Nanorana blanfordii (Boulenger, 1882), Duttaphrynus himalayanus (Günther, 1864), and Nanorana liebigii (Günther, 1860) were also recorded.

Figure 5. 

Habitat of Xenophrys pangdaensis sp. nov. in the type locality, Pangda Village, Yadong County, Tibet Autonomous Region, China.

Comparisons

Xenophrys pangdaensis sp. nov. is here compared with all 28 recognized species of the Xenophrys (Table 5). The smallest recognized species of Xenophrys is X. zunhebotoensis (male 28.4–33.9, females 37–39.5). So, by having small body size (males 18.0–22.2 mm, n = 6; female 23.4 mm, n = 1), Xenophrys pangdaensis sp. nov. differs from all congeners from Xenophrys.

Table 5.
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Morphological comparison between Xenophrys pangdaensis sp. nov. and 28 recognized species: 1. SVL in males (mm); 2. SVL in females (mm); 3. ratio TYD/EL in males; 4. ratio TYD/EL in females; 5. ratio SHL/SVL in males; 6. ratio SHL/SVL in females; 7. Horn-like tubercle at edge of upper eyelid absent (0), small (1), slightly larger (2), long point (3); 8. Vomerine teeth absent (0), present (1); 9. Vocal sac absent (0), present (1); 10. Tongue not notched (0), feebly notched(1), notched (2); 11. Lateral fringes on toes absent (0), narrow (1), wide (2); 12. Toes without webbing (0), with rudiment of webbing (1), at most one-fourth webbed (2), at least one-fourth webbed (3); 13. Subarticular tubercles on toes absent (0), indistinct (1), distinct (2); 14. Relative finger lengths; 15. Nuptial pads on finger absent (0), present (1). 16. Dorsal skin texture: smooth (0), smooth with small tubercles (1), rough (2). ? = data not available. ts= this study. Data sources: (a) Boulenger (1908), (b) Bourret (1937), (c) Bourret (1942), (d)Fei at al. (1983), (e) Ye and Fei (1992),(f) Fei et al. (1992), ((g) Huang et al. (1998), (h) Ohler et al. (2002), (i) Stuart et al. (2006), (j)Mathew and Sen (2007), (k) Fei et al. (2009), (l) Fei et al. (2012), (m) Mahony (2011), (n) Mahony et al. (2011), (o) Mahony et al. (2013), (p) Neang et al. (2013), (q)Fei and Ye (2016), (r) Deuti et al.(2017), (s) Mahony et al. (2018), (t) Shi et al. (2020), (u) Mahony et al. (2020), (v) Luong et al. (2022), (w) Lyu et al. (2023).

Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 References
X. pangdaensis sp. nov. 18.0–22.2 21.7–23.4 0.50–0.69 0.48–0.58 0.47–0.52 0.49–0.52 1 1 1 2 1 1 1 I<II<IV<III 0 1 t, s
X. ancrae 39.1–45.0 48.9 0.5–0.63 0.5–0.63 0.46–0.53 0.49 1 1 ? 1 0 1 0 I<II<IV<III 1 1 o
X. awuh 35.7–41.1 43.7–48 0.56–0.64 0.61–0.63 0.49–0.55 0.50–0.53 0 0 1 1 0 1 0 I=II<IV<III 1 0 u
X. auralensis 60.1–76.7 ? 0.6 ? 0.51 ? 1 0/1 1 0 0 1 1 II<I<IV<III 0 1 h, p
X. damrei 47.7–57.1 69.1 0.58 0.58 0.51 0.50 0 1 1 2 0 1 0 IV<I<II<III 0 0 n, p
X. dzukou 34.2–35.3 ? 0.45–0.59 ? 0.47–0.53 ? 0 1 1 1 1 0 0 I=II<IV<III 1 0 u
X. flavipunctata 56.9–68.4 68–74.6 0.41–0.51 0.46–0.51 0.54–0.61 0.54–0.58 0 1 1 2 1 2 0 IV<I=II<III 1 1 s
X. glandulosa 76.3–81 76.5–99.5 0.51–0.65 ?77 0.58–0.60 0.5–0.55 0 1 1 1 2 1 0 II=I<IV<III/ IV<II<I<III 0 0 f, g, k, l, q, s
X. himalayana 68–73.5 83.9 0.54 0.48 0.50–0.54 0.54 1 1 1 2 1 1 0 I<II<V<III<IV 1 1 s
X. lekaguli 40.1–66.6 58.6–94 0.59–0.74 0.58–0.64 0.47–0.52 0.44–0.52 1 1 0 0 0 1 0 IV<II<I<III 0 0 i, p
X. major 71.6–87.5 85.6–98.2 0.33–0.48 0.40–0.41 0.50–0.58 0.50–0.57 1 1 1 1 1 1 0 IV<II=I<III 1 1 q, u
X. mangshanensis 62.5 73 ? ? 0.52 0.54 1 1 1 1 0 0 0 II<I<IV<III 0 0 f, g, k, l, q
X. maosonensis 58–76 68–93.5 0.5 ? 0.5–0.6 ? 1 1 ? 1 0 2 0 ? ? 0 b, c
X. medogensis 57.2–68 75.7–85.5 ? ? 0.56 ? 1 1 1 1 0 1/0 0 I<II<IV<III 0 1 d, k, l, q, t
X. megacephala 48.4–53.4 49.3–64.4 0.54–0.71 0.56–0.82 0.41–0.49 0.41–0.47 0 1 1 0 0 1 0 IV<II<I<III 1 1 m, u
X. monticola 37.8–49.1 40.5–51.1 0.38–0.59 0.44–0.71 0.45–0.51 0.46–0.51 1 0 1 1 1 1 0 I<II<IV<III 1 1 r, s
X. numhbumaeng 33.8–34.6 ? 0.45–0.46 ? 0.52–0.58 ? 0 1 1 1 0 1 0 I=II<IV<III 1 0 u
X. oreocrypta ? 94.9 ? 0.52 ? 0.51 1 1 ? ? 0 1 0 IV<II<I<III ? 1 s
X. oropedion 32.8–39.2 44.1–48.7 0.54–0.65 0.62–0.69 0.43–0.48 0.42–0.43 0 1 1 1 0 0 0 I=II=IV<III 1 1 o
X. periosa 71.3–93.8 112 0.44–0.58 0.45 0.51–0.58 0.51 1 1 1 ? 0 1 0 IV<II<I<III 1 0 s
X. robusta 73.5–83.1 81.3–114 0.38–0.52 0.40–0.74 0.51–0.57 0.46–0.54 1 0 1 0 0 1 0 IV<I=II<III 1 1 a, s
X. serchhipii 36.1–46.7 46.1–53 0.47–0.66 0.50–0.55 0.46–0.53 0.47–0.51 0 1 1 ? 0 1 0 IV<I=II<III 1 1 j, u
X. takensis 47.3–53 72.9 0.42–0.48 0.53 0.41–0.49 0.45 0 1 1 0 0 1 0 IV≤II<I<III/ IV=I<II<III 1 1 s, u
X. truongsonensis 58.8–71.4 65.6–87.3 0.51–0.67 0.53–0.59 0.55–0.64 0.54–0.58 0 1 0 1 1 1 0 I<II<IV<III 0 0 v
X. zhangi 32.5–37.2 ? 0.5 ? 0.49 ? 0 1 1 1 1 0 0 II=I <IV<III 1 1 e, k, l, q
X. zunhebotoensis 28.4–33.9 37–39.5 0.43–0.58 0.48–0.59 0.45–0.54 0.47–0.50 0 0 1 1 0 0 0 IV<I<II≤III
I≤II<IV<III		 1 1 j, u
X. pava 36.6–42.9 41.4–52.1 0.40–0.49 0.44–0.55 0.46–0.48 0.43–0.44 0 1 1 0/1 0 0 0 II<IV<I<III 1 1 u, w
X. dehongensis 34.8–36.7 45.7–46.8 0.34–0.45 0.43–0.44 0.43–0.50 0.46–0.50 0 1 1 0 0 1 0 II<I<IV<III 1 2 w
X. lancangica 64.0–65.4 75.0–88.6 0.63–0.74 0.59–0.77 0.54–0.57 0.56–0.58 0 1 1 1 1 1 0 II<IV<I<III 1 1 w

Xenophrys pangdaensis sp. nov. differs from X. awuh, X. damrei, X. dzukou, X. flavipunctata, X. glandulosa, X. megacephala, X. numhbumaeng, X. oropedion, X. serchhipii, X. takensis, X. truongsonensis, X. zhangi, X. zunhebotoensis, X. pava, X. dehongensis, and X. lancangica by the presence of a horn-like tubercle laterally on the upper eyelid (vs absence of horn-like tubercle at edge of upper eyelid).

Xenophrys pangdaensis sp. nov. differs from X. awuh, X. monticola, X. robusta, and X. zunhebotoensis by presence of vomerine teeth (vs absence of vomerine teeth).

Xenophrys pangdaensis sp. nov. differs from X. lekaguli, X. truongsonensis by presence of vocal sac (vs absence of vocal sac).

Xenophrys pangdaensis sp. nov. differs from X. ancrae, X. awuh, X. dzukou, X. flavipunctata, X. himalayana, X. major, X. megacephala, X. monticola, X. numhbumaeng, X. oropedion, X. periosa, X. robusta, X. serchhipii, X. takensis, X. zhangi, X. zunhebotoensis, X. pava, X. dehongensis and X. lancangica by absence of nuptial pads on fingers (vs presence of nuptial pads on fingers).

Xenophrys pangdaensis sp. nov. differs from X. ancrae, X. awuh, X. dzukou, X. glandulosa, X. major, X. mangshanensis, X. maosonensis, X. medogensis, X. monticola, X. numhbumaeng, X. oropedion, X. truongsonensis, X. zhangi, X. zunhebotoensis, X. pava, X. lancangica (vs tongue feebly notched), X. auralensis, X. lekaguli, X. megacephala, X. robusta, X. takensis, X. dehongensis by tongue distinctly notched (vs tongue not notched).

Xenophrys pangdaensis sp. nov. differs from X. ancrae, X. awuh, X. auralensis, X. damrei, X. lekaguli, X. mangshanensis, X. maosonensis, X. medogensis, X. megacephala, X. numhbumaeng, X. oreocrypta, X. oropedion, X. periosa, X. robusta, X. serchhipii, X. takensis, X. zunhebotoensis, X. pava, and X. dehongensis by having narrow lateral fringes on toes (vs lateral fringes on toes absent), and X. glandulosa (vs lateral fringes on toes wide).

Xenophrys pangdaensis sp. nov. differs from X. dzukou, X. mangshanensis, X. oropedion, X. zhangi, X. pava, and X. zunhebotoensis by toes with rudiment of webbing (vs toes without webbing), X. flavipunctata, and X. maosonensis (vs at most one-fourth webbed).

Xenophrys pangdaensis sp. nov. differs from X. ancrae, X. awuh, X. damrei, X. dzukou, X. flavipunctata, X. glandulosa, X. himalayana, X. lekaguli, X. major, X. mangshanensis, X. maosonensis, X. medogensis, X. megacephala, X. monticola, X. numhbumaeng, X. oreocrypta, X. oropedion, X. periosa, X. robusta, X. serchhipii, X. takensis, X. truongsonensis, X. zhangi, X. zunhebotoensis, X. pava, X. lancangica and X. dehongensis by presence of indistinct subarticular tubercles on toes (vs absence of subarticular tubercles on toes).

Xenophrys pangdaensis sp. nov. differs from X. awuh, X. damrei, X. dzukou, X. glandulosa, X. lekaguli, X. mangshanensis, X. maosonensis, X. numhbumaeng, X. periosa, and X. truongsonensis by dorsal skin texture smooth with small tubercles (vs dorsal skin texture smooth), X. dehongensis (vs dorsal skin rough).

Discussion

The genus Megophrys sensu lato is a large group with extremely high species diversity. With the description of this new species, the members of the group will be 133. Xenophrys pangdaensis sp. nov. represents the 29th known species of Xenophrys in China and the ninth known species of the Asian horned toads from Tibet, China (Shi et al. 2020; Frost 2023). In fact, there are still some pending species whose taxonomic status needs further confirmation between Northeast India and adjacent China. Deuti et al. (2017) who described two small sized new species, X. katabhako and X. sanu, based on morphological and molecular sampling. However, Mahony et al. (2018) found that X. katabhako and X. sanu are nested within the concept of X. monticola. Also, similar results were obtained in this study. X. katabhako and X. monticola formed a clade. X. sanu and X. zhangi clustered into another clade. Finally, they all formed monophyletic group, but the two clades diverged considerably. For the moment, it is necessary to further investigate the taxonomic affinities of these populations by integrating more evidence. Thus, there may be some cryptic species in this group and it should be continuously paid more attentions on the species diversity of the Megophrys sensu lato. Southern Tibet is located in the eastern of Himalayas, which is one of 36 biodiversity hotspots in the world (Basnet et al. 2019). This region is extremely unique and deserves our continued attention. In recent years, many new species have been gradually discovered in this area (Jiang et al. 2012, 2016a, 2016b, 2016c; Shi et al. 2020; Che et al. 2020; Zhang et al. 2022). There is also high species diversity in the middle and lower reaches of the Yarlung Zangbo Grand Canyon, for example, Mahony et al. (2018) revealed cryptic diversity within the Megophrys major species group, which suggests that the species diversity in this area may have been previously underestimated and therefore needs to be further investigated.

The body length (SVL) of the new species ranges from 18.0 to 22.4 mm in males and from 23.4 mm in female, however, the minimum SVL of the other recognized congeners is 28.4–33.9 mm (X. zunhebotoensis) in males and 37–39.5 mm (X. zunhebotoensis) in females. Thus, X. pangdaensis sp. nov. is likely to be the smallest member of all recognized species in Xenophrys. In addition, the members of the Xenophrys are very variable in body length; for example, X. glandulosa reaches approximately 80 mm in males and 76.5–99.5 mm in females, and the body length of X. robusta are even more than 100 mm in females (81.3–114.0 mm). So, it indicates that species of the same genus have a large span in body length. This also reflects the strong morphological plasticity of the Asian horned toads.

It has always been difficult to identify horned toads, especially the species with similar body length. Liu et al. (2018) recognized one sample SYSa002934 from Medog County as X. cf. pachyproctus; however, this sample clustered in a clade with X. medogensis in the results of Shi et al. (2020). Additionally, the evolutionary branch length in the phylogenetic tree between the two species was much shorter than between any other species, and further analysis showed that the genetic divergence between them is only 0.57% based on 16S gene. It is probable that this specimen may be misidentified previously and should be reexamined (Shi et al. 2020; this study). Furthermore, another specimen (CIB022017061805) from Bari, Medog, Tibet, China formed an independent clade in our analysis. Shi et al. (2020) treated it as M. cf. pachyproctus. So far, there is only a morphological description and no available molecular evidence from samples of the type locality (Gelin) for J. pachyproctus. Thus, we suggest that further sampling at Gelin would help to resolve the taxonomic problem of J. pachyproctus in the future.

Acknowledgements

We thank Forest Department of Yadong County for permission for field work and specimen collection. We are grateful to Dr. J. Che (Kunming Institute of Zoology, Chinese Academy of Sciences) and Dr. W. Y. Duan (Kunming Institute of Botany, Chinese Academy of Sciences) for their assistance.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research is supported by the Second Tibetan Plateau Scientific Expedition and Research Program (STEP) (No. 2019QZKK0501), Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA 20050201), National Natural Science Foundation of Sichuan (No. 2023NSFSC1157), Sailing Project of Yibin University (No. 2020QH07), and Key Lab of Aromatic Plant Resources Exploitation and Utilization in Sichuan Higher Education (No.21XLY03).

Author contributions

Guocheng Shu, Conceptualization- Equal, Data curation-Lead, Formal analysis-Lead, Methodology-Lead, Resources-Equal, Software-Equal, Validation- Lead, Visualization-Lead, Writing-original draft-Lead, Writing-review and editing-Lead; Ke Li, Data curation-Supporting, Formal analysis-Equal, Investigation-Lead; Yayong Wu, Writing-review & editing-Equal, Formal analysis-Equal, Methodology-Equal; Qin Liu, Data curation-Supporting, Formal analysis-Supporting, Writing-review & editing-Equal; Zhongping He, Data curation-Supporting, Formal analysis-Supporting, Writing-review and editing-Equal; Lin Li, Formal analysis-Supporting, Visualization-Equal; He Zhang, Data curation-Supporting, Investigation-Equal; Peng Guo, Conceptualization-Equal, Data curation-Equal, Funding acquisition-Lead, Investigation-Equal, Project administration-Lead, Resources-Equal, Supervision-Lead, Validation-Equal, Writing-review and editing-Lead.

Author ORCIDs

Guocheng Shu https://orcid.org/0000-0003-1590-6510

Ke Li https://orcid.org/0000-0001-9695-4236

Yayong Wu https://orcid.org/0000-0003-2752-4085

Qin Liu https://orcid.org/0000-0001-8402-6491

Zhongping He https://orcid.org/0009-0005-7077-8074

Lin Li https://orcid.org/0000-0002-7650-2280

He Zhang https://orcid.org/0000-0002-9511-2552

Peng Guo https://orcid.org/0000-0001-5585-292X

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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Supplementary material

Supplementary material 1 

References utilized for morphological characters of congeners of the genus Xenophrys

Guocheng Shu, Ke Li, Yayong Wu, Qin Liu, Zhongping He, Ling Li, He Zhang, Peng Guo

Data type: xlsx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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