﻿A new species of Xenophrys (Amphibia, Anura, Megophryidae) from southern Tibet, China

﻿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.

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, Cambodia, 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(Mahony et al. , 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.

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).

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 vol-ume 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).
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.Partition-Finder v. 2.1.1 (Lanfear et al. 2017) was used to select the corresponding bestfit 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 × 10 7 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  Specimen status: HT, holotype; PT, paratype; TT, topotype.
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) 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.

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 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.

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.
Etymology.The species name pangdaensis indicates the type locality of Pangda Village, Yadong County, Tibet Autonomous Region, China.
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,  X. X X num mhbum maeng X. X X ser rchhip pii X. X X tak kensis X. X X par rva X. X X zun nhebo otoensis X. X X leg gkagul li X. X X maj jor X. X X med dogen nsis X. X X maosone ensis YBU2 21269 9 YBU2 21248 8 X. X X aur ralens sis X. X X per riosa X. X X zha angi X. X X monticol la YBU2 21259 9 X.X X flav vipunctata X. X X awu uh X. X X truo ongso onensis X. X X dzu ukou X. san nu X. X X kat tabhak ko X. X X oro opedio on X. X X ore eocryp pta X. X X meg gacep phala X. X X him malaya ana YBU2 21262 2 X.X X gla andulo osa X. X X lan ncangica X. X X anc crae X. X X mangsha anensis 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.

P P P. l lig g ga a ay ya ae e e e e P P P. k k ka a al li im m ma a an n nt nta a a an ne en n n ns si i is s s P P P. s s st te ej jn ne e eg g g g ge ge er ri i i P P P. e e ed d dw w wa a ar rd d d d di in na a ae e e P P P. k k ko o ob bay y ya a a a a ash hi ii P P P. b b ba a al lu ue e en n ns s si sis s
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 6.3-13.8mm (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).
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.

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 29 th 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(Jiang et al. , 2016a(Jiang et al. , 2016b(Jiang et al. , 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 28.4-33.9mm (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 (81.3-114.0mm).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.

Figure 1 .
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.
a a aui i i L L. a a alp p pin n na a a 1 huan a gsha a anensis Bo. m mino n r Bo. ch chen eni Bo. br brac achyko o olos Bo. na nanl n ingen nsis Bo. bo boet e tgeri i i Bo. da daoj oji Bo. bi bing glinge e ensis Bo. ch chis shuien n nsis Bo. sa sang ngzhien nsis Bo. fr frig gida Bo. sp spin nata Bo. wu wush s anen nsis Bo. ku kuat a unen n nsis Bo. fa fans sipane e ensis

Figure 2 .
Figure 2. Phylogenetic tree of the genus Xenophrys inferred from two mitochondrial gene fragments by Bayesian inference.

Figure 3 .
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.

Figure 4 .
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 justpreserved specimens.

Table 1 .
Information of samples used in the molecular analyses.
. Seventeen morphometric characters of tadpoles were measured: NE naris-eye distance (distance from center of naris to anterior corner of eye); ODW oral disc width (largest width of oral disc); BHmaximum 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;

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

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