Description of a new horned toad of Megophrys Kuhl & Van Hasselt, 1822 (Amphibia, Megophryidae) from Zhejiang Province, China

Abstract A new species of the Asian horned toad genus Megophrys is described from Zhejiang Province, China, based on multiple data. Molecular phylogenetic analyses based on mitochondrial DNA indicated the new species as an independent clade deeply clustered into the Megophrys clade. The new species is identified from its congeners by a combination of the following characters: body size small (SVL 28.4–32.4 mm in males); vomerine teeth absent; tongue not notched behind; tympanum distinctly visible, oval; a small horn-like tubercle present at the edge of each upper eyelid; two metacarpal tubercles distinctly visible in hand; toes without webbing; heels overlapped when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level to middle of eye when leg stretched forward; an internal single subgular vocal sac in male; in breeding male, the nuptial pads present on the dorsal base of the first two fingers.


Introduction
The Asian horned toad Megophrys Kuhl & Van Hasselt, 1822(Anura: Megophryidae Bonaparte, 1850 is widely distributed in eastern and central China, throughout southeastern Asia, and extending to the islands of the Sunda Shelf and the Philippines (Frost 2020). The generic assignment of species in the group has been controversial for decades (e.g., Tian and Hu 1983;Dubois 1987;Rao and Yang 1997;Lathrop 1997;Jiang et al. 2003;Delorme et al. 2006;Fei et al. 2009;Fei and Ye 2016;Chen et al. 2017;Deuti et al. 2017;Mahony et al. 2017;. Recent molecular phylogenetic studies proposed this group as a monophyletic group (Chen et al. 2017;Mahony et al. 2017;Li et al. 2018a;Liu et al. 2018;Liu et al. 2020;Wang et al. 2020), which was recognized as a big genus Megophrys sensu lato Li et al. 2018b;Liu et al. 2018;Liu et al. 2018;Liu et al. 2020;Lyu et al. 2020;Xu et al. 2020;Wang et al. 2020), though some studies still divided the taxa of the group into different genera and/ or subgenera (Fei and Ye 2016;Chen et al. 2017;Deuti et al. 2017;Liu et al. 2018). The genus Megophrys currently contains 106 species, of which 52 species were described over the last decade (Frost 2020). A number of cryptic species were still indicated in the genus by molecular phylogenetic analyses (e.g., Chen et al. 2017;Liu et al. 2018).
Wuyi Mountain region, located in northern Fujian, southeastern Jiangxi and south Zhejiang provinces of China, is a biodiversity hotspot. In this region, four Megophrys species have been recorded, i.e., M. boettgeri (Boulenger, 1899), M. kuatunensis Pope, 1929, M. ombrophila Messenger & Dahn, 2019, and M. lishuiensis Wang, Liu & Jiang, 2017. However, many mountains in this region, especially in south Zhejiang Province, have been poorly investigated.
During field surveys in Qingyuan County, Zhejiang Province, China, we collected Megophrys specimens. Molecular phylogenetic analyses and morphological comparisons supported some of these specimens as an undescribed taxon that we describe herein as a new species.

Sampling
A total of 15 specimens were sampled in this study: six adult males and one tadpole of the undescribed species and two adult males of M. boettgeri from Qingyuan County, Zhejiang Province, China, and one adult male of M. ombrophila and six adult males of M. kuatunensis from Wuyi Mountain, Fujian Province, China (Table 1; Fig. 1). The developmental stage of tadpole was identified following Gosner (1960). In the field, the toad and tadpole were euthanized using isoflurane, and the specimens were fixed in 75% ethanol. Tissue samples were taken and preserved separately in 95% ethanol prior to fixation. The specimens were deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS).

Molecular data and phylogenetic analyses
Six adult males and one tadpole of the undescribed species, two M. kuatunensis, one M. ombrophila, and two M. boettgeri were included in the molecular analyses (Table 1). Total DNA was extracted using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). Two fragments of the mitochondrial 16S rRNA (16S) and cytochromeoxidase subunit I (COI) genes were amplified. For 16S, the primers P7 (5'-CGC-CTGTTTACCAAAAACAT-3') and P8 (5'-CCGGTCTGAACTCAGATCACGT-3') were used following Simon et al. (1994), and for COI, Chmf4 (5'-TYTCWACWAAY-CAYAAAGAYATCGG-3') and Chmr4 (5'-ACYTCRGGRTGRCCRAARAATCA-3') were used following Che et al. (2012). Gene fragments were amplified under the following conditions: an initial denaturing step at 95 °C for 4 min; 36 cycles of denaturing at 95 °C for 30 s, annealing at 52 °C (for 16S)/47 °C (for COI) for 40 s and extending at 72 °C for 70 s. Sequencing was conducted using an ABI3730 automated DNA sequencer in Shanghai DNA BioTechnologies Co., Ltd. (Shanghai, China). New sequences were deposited in GenBank (for GenBank accession numbers see Table 1). For molecular analyses, the available sequences for congeners of Megophrys were downloaded from GenBank (Table 1), primarily from previous studies (Chen et al. 2017;Liu et al. 2018). For phylogenetic analyses, corresponding sequences of one Leptobrachella oshanensis (Liu, 1950) and one Leptobrachium boringii (Liu, 1945) were also downloaded (Table 1), and used as outgroups following Mahony et al. (2017). Sequences were assembled and aligned using the Clustalw module in BioEdit v.7.0.9.0 (Hall 1999) with default settings. Alignments were checked by eye and revised manually if necessary. For phylogenetic analyses of mitochondrial DNA, the dataset concatenated with 16S and COI gene sequences. To avoid under-or over-parameterization (Lemmon and Moriarty 2004;McGuire et al. 2007), the best partition scheme and the best evolutionary model for each partition were chosen for the phylogenetic analyses using PARTITIONFINDER v. 1.1.1 (Robert et al. 2012). In this analysis, 16S gene and each codon position of COI gene were defined, and Bayesian Inference Criteria was used. As a result, the analysis suggested that the best partition scheme is 16S gene/ each codon position of COI gene, and selected GTR + G + I model as the best model for each partition. Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian Inference (BI) methods, implemented in PhyML v. 3.0 (Guindon et al. 2010) and MrBayes v. 3.12 (Ronquist and Huelsenbeck 2003), respectively. For the ML tree, branch supports were drawn from 10,000 nonparametric bootstrap replicates. In BI, two runs each with four Markov chains were simultaneously run for 50 million generations with sampling every 1,000 generations. The first 25% trees were removed as the "burn-in" stage followed by calculations of Bayesian posterior probabilities (BPP) and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity. Finally, mean genetic distance between Megophrys species based on uncorrected p-distance model was estimated respectively on 16S and COI genes using MEGA v. 6.06 (Tamura et al. 2013).

Morphological comparisons
Six adult males and one tadpole of the undescribed species were measured (Table 1 and Suppl. material 1). For comparisons, six adult male specimens of M. kuatunensis were also measured (Supp. material 1). The terminology and methodology followed Fei et al. (2009). Measurements were taken with a dial caliper to 0.1 mm. Twenty-two morphometric characters of adult specimens were measured:

ED
eye diameter (distance from the anterior corner to the posterior corner of the eye); FIL first finger length (distance from base to tip of finger I); FIIL second finger length (distance from base to tip of finger II); FIIIL third finger length (distance from base to tip of finger III); FIVL fourth finger length (distance from base to tip of finger IV); FL foot length (distance from tarsus to the tip of fourth toe); HDL head length (distance from the tip of the snout to the articulation of jaw); HDW maximum head width (greatest width between the left and right articulations of jaw); HAL hand length (distance from tip of third digit to proximal edge of inner palmar tubercle); IND internasal distance (minimum distance between the inner margins of the external nares); IOD interorbital distance (minimum distance between the inner edges of the upper eyelids); LAL length of lower arm and hand (distance from the elbow to the distal end of the Finger IV); LW lower arm width (maximum width of the lower arm); SNT distance between the nasal the posterior edge of the vent; SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent); SL snout length (distance from the tip of the snout to the anterior corner of the eye); TFL length of foot and tarsus (distance from the tibiotarsal articulation to the distal end of the Toe IV); THL thigh length (distance from vent to knee); TL tibia length (distance from knee to tarsus); TW maximal tibia width; TYD maximal tympanum diameter; UEW upper eyelid width (greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis).
For the single tadpole of the undescribed species, eleven morphometric characters were measured:

BH
maximum body height; BW maximum body width; IOS interocular distance (minimum distance between eye); MW mouth width (distance between two corners of mouth); SL snout length (distance from the tip of the snout to the anterior corner of the eye); SS snout to spiraculum (distance from spiraculum to the tip of the snout); SVL snout-vent length; TAH tail height (maximum height between upper and lower edges of tail); TAL tail length (distance from base of vent to the tip of tail); TBW maximum width of tail base; TOL total length (distance from the tip of the snout to the tip of tail).
To reduce the impact of allometry, the correct value from the ratio of each character to SVL was calculated, and then was log-transformed for the following morphometric analyses. Mann-Whitney U tests were conducted to test the significance of differences on morphometric characters between the undescribed species and M. kuatunensis. The significance level was set at 0.05. Furthermore, principal component analyses (PCA) were conducted to highlight whether the different species were separated in morphometric space.
The new species was also compared with all other Megophrys species on morphology. Comparative data were obtained for related species as described in literature (Table 2).

Bioacoustics analyses
The advertisement calls of the undescribed species were recorded from the holotype specimen CIBQY20200726001 in the field on 26 July 2020 from Qingyuan County, Zhejiang Province, China. When registrating the male in the stream the ambient air temperature was 21.5 °C and there was air humidity of 87%. For comparisons, the advertisement calls of M. kuatunensis from Wuyi Mountain, Fujian Province, China were recorded from the specimens CIBWY18082410, CIBWY18082411 and CIB-WY18082412 at an ambient air temperature of 22.0 °C and air humidity of 88% on 24 August 2018. SONY PCM-D50 digital sound recorder was used to record within 20 cm of the calling individual. The sound files in wave format were resampled at 48 kHz with sampling depth 24 bits. The sonograms and waveforms were generated by WaveSurfer software (Sjöander and Beskow 2000) from which all parameters and characters were measured. Ambient temperature was taken by a digital hygrothermograph.

Phylogenetic analyses
Aligned sequence matrix of 16S+COI contains 1104 bp. ML and BI trees of the mitochondrial DNA dataset presented almost consistent topology, and as well, though relationships of many clades were unresolved (Fig. 2). In mitochondrial DNA trees, all samples of the undescribed species were clustered into one clade which was deeply clustered into the Megophrys clade. The species is likely sister to M. kuatunensis (bootstrap supports < 50% and BPP = 0.51) though the relationships between the two species and most other congeners were not resolved (all bootstrap supports < 50% and many BPP < 0.95).
Based on the molecular phylogenetic analyses, morphological comparisons (Supp. material 4), and bioacoustics differences, the specimens from Qiangyuan County, Zhejiang Province, China represent a new species which is described as follows.   Paratype. Five adult males collected from the same place as holotype collected by Bin Wang. CIBQY20200719001-CIBQY20200719004 collected on 19 July 2020 by Bin Wang, and CIBQY20200726002 collected by Zhonghao Luo on 26 July 2020.
Diagnosis. Megophrys baishanzuensis sp. nov. is assigned to the genus Megophrys based on molecular phylogenetic analyses and the following generic diagnostic characters: snout shield-like; projecting beyond the lower jaw; canthus rostralis distinct; chest glands small and round, closer to the axilla than to midventral line; femoral glands on rear part of thigh; vertical pupils (Fei et al. 2009).
Megophrys baishanzuensis sp. nov. could be distinguished from its congeners by a combination of the following morphological characters: body size small (SVL 28.4-32.4 mm in males); vomerine teeth absent; tongue not notched behind; tympanum distinctly visible, oval; a small horn-like tubercle at the edge of each upper eyelid; two metacarpal tubercles distinctly visible in hand; toes without webbing; heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level to the middle of eye when leg stretched forward.
Description of holotype. (Figs 4A, B, E, G, H, 5). SVL 28.5 mm; head width larger than head length (HDW/HDL ratio ca. 1.3); snout obtusely pointed, protruding well beyond the margin of the lower jaw in ventral view; loreal region vertical and concave; canthus rostralis well-developed; top of head flat in dorsal view; eye large, eye diameter 46.0% of head length; pupils vertical; nostril orientated laterally, closer to snout than eye; tympanum distinct, 55.8% of eye diameter; vomerine ridges present and vomerine teeth absent; margin of tongue smooth, not notched behind.
Forelimbs slender, the length of lower arm and hand 47.0% of SVL; fingers slender, relative finger lengths: I < II < IV < III; tips of digits globular, without lateral fringes; subarticular tubercle distinct at the base of each finger; two metacarpal tubercles, prominent, oval-shaped, the inner one bigger than the outer one.
Hindlimbs slender, tibia length 46.5% times of SVL; heels overlapping when thighs are positioned at right angles to the body, tibiotarsal articulation reaching the middle of eye when leg stretched forward; tibia length longer than thigh length; relative toe lengths I < II < V < III < IV; tips of toes round, slightly dilated; subarticular tubercles absent on each toes; toes without webbing but with narrow lateral fringe; inner metatarsal tubercle oval-shaped; outer metatarsal tubercle absent.
Dorsal skin rough, several large warts scattered on flanks; a small horn-like tubercle at the edge of each upper eyelid; tubercles on the dorsum forming a X-shaped ridge, two dorsolateral parallel ridges on either side of the X-shaped ridges; an inverted triangular brown speckle between two upper eyelidsseveral tubercles scattered on dorsal, flanks and dorsal surface of thighs and tibias; supratympanic fold distinct.
Numerous granules scattered on ventrum; pectoral and femoral glands distinct; numerous white granules on outer thighs.
Coloration of holotype in life. (Fig. 5). Dorsal brown, several pink tubercles scattered on dorsal, an inverted triangular brown speckle between the eyes; X-shaped ridges  on the dorsum brown, four dark transverse bands on the dorsal surface of the thigh and shank; ventral surface of body white with brown spots; two dark brown dark bars on the flanks, throat brown; white vertical bars on lower and upper lip; ventral surface of anterior limb dark reddish purple, posterior limb orange with numerous white granules; tip of digits pale grey; inner metatarsal tubercle and two metacarpal tubercles pinkish; soles uniform dark reddish purple; pectoral glands white.
Coloration of holotype in preservation. (Fig. 4A, B, E, G, H). Color of dorsal surface fades to taupe; the inverted triangular brown speckle between the eyes and brown X-shaped ridges on dorsum are more distinct; ventral surface greyish white; creamy-white substitutes the purple grey on tip of digits; the posterior of ventral surface of body, inner of thigh and upper of tibia fades to creamy-white.
Variation. Fig. 6. Measurements and basic statistics of adult specimens are presented in Tables 3 and Supp. material 1. All specimens were similar in morphology but some individuals different from the holotype in color pattern. In CIBQY2020200719001 the tubercles on the dorsum forming two ≻ shaped, disconnected ridges (Fig. 6A); in CIBQY2020200719004 the tubercles on the dorsum forming a big and distinct Xshaped speckle (Fig. 6B); in CIBQY2020200719003 ventral surface of body grey with brown spots (Fig. 6C); in CIBQY2020200726002 ventral surface of body and limbs brownish red (Fig. 6D). Tadpole description. Fig. 7. The tadpole CIBQY20200719006 (Fig. 7) was confirmed as Megophrys baishanzuensis sp. nov by molecular phylogenetic analyses. Measurements in mm. Stage 31. Body slender, body brownish black and tail pale brown, body height greater than tail height; dorsal fin arising behind the origin of the tail, the highest fin near mid-length, tapering gradually to the narrowly pointed tip; tail approximately 1.9 times as long as snout-vent length; tail height 13.6% of tail length; body width longer than body height (BW/BH1.2); eyes large, lateral, nostril near eyes; spiracle on the left side of the body and distinct; oral disk terminal, lips expanded and directed upwardly into a umbelliform oral disk; flank of body brownish black with some white spots, tail fins lightly colored, with small white and black spots. TOL 22.7; SVL 8.7; BW 3.0; BH 2.7; SL 2.0; SS 4.0; IOS 1.8; TAL 14.7; TAH 2.2; TBD 1.5; MW 1.3. Advertisement call. Fig. 4. The call description is based on recordings of the holotype CIBQY20200726001 (Fig. 4; Table 4) from a shrub leaf near the streamlet. Call duration was 151.0-170.0 ms (mean 162.4 ± 5.7). Inter-call interval was 682.0-1869.0 ms (mean 936.8 ± 349.0). Pulse/call was 23.0-30.0 (mean 26.0 ± 2.4); pulse duration was 3.0-6.0 (mean 4.9 ± 6.0) and call repetition rate was 0.79 call/s. Amplitude modulation within note was apparent, beginning with moderately high energy pulses, increasing to the maximum by approximately quarter, and then decreasing towards the end. The average dominant frequency was 3.36 ± 0.06 (3.19-3.38 kHz).
Secondary sexual characters. A single subgular vocal sac present in male. In breeding season, nuptial pads are present on the dorsal base of the first two fingers in males.
By having an internal single subgular vocal sac in male, Megophrys baishanzuensis sp. nov. differs from M. caudoprocta, M. shapingensis, and M. shuichengensis (vs. vocal sac absent in the latter).
The congeners M. boettgeri, M. lishuiensis, M. ombrophila, and M. xianjuensis all occur in Wuyi Mountains, Fujian Province and/or Zhejiang Province, China, and probably have sympatric distribution with Megophrys baishanzuensis sp. nov. (Fei et al. 2012;Wang et al. 2017b;Messenger et al. 2019;Wang et al. 2020). The new species can be distinguished from these species by a series of morphological characters as follows. The new species differs from M. boettgeri by body size smaller (adult males with 28.4-32.4 mm vs. adult males with 34.5-37.8 mm), and in breeding male nuptial pads present on the dorsal base of the first two fingers (vs. nuptial pad only on the first finger). The new species differs from M. lishuiensis by vomerine ridges present (vs. absent), toes with narrow lateral fringe (vs. without), and tibiotarsal articulation reaching the middle of eye when leg stretched forward (vs. reaching the range from tympanum to eye). The new species differs from M. ombrophila by heels overlapping when thighs are positioned at right angles to the body (vs. not meeting), vomerine ridges present (vs. absent), and toes with narrow lateral fringe (vs. without). The new species differs from M. xianjuensis by tibiotarsal articulation reaching the middle of eye when leg stretched forward (vs. reaching the range from tympanum to eye), and toes with narrow lateral fringe (vs. without).
Megophrys baishanzuensis sp. nov. is phylogenetically closest to M. kuatunensis. Megophrys baishanzuensis sp. nov. could be identified from M. kuatunensis distinctly by tibiotarsal articulation reaching the middle of eye when leg stretched forward (vs. reaching the range from tympanum to eye), heels overlapping when thighs are positioned at right angles to the body (vs. not meeting), tongue not notched behind (vs. notched feebly), the supratympanic fold more expanded in dorsal view and tympanum protruding (vs. concave), and having significantly lower ratios of UEW and TFL to SVL in males (all p-values < 0.05; Table 3). On call characters, the new species has slower call repetition rate (0.79 call/s in the new species vs. 1.18 call/s in M. kuatunensis), and has lower dominant frequency (3.19-3.38 kHz in the new species vs. 3.38-3.75 kHz in M. kuatunensis).
Distribution and habitat. Megophrys baishanzuensis sp. nov. is known from the type locality, Baishanzu National Park, Qingyuan County Zhejiang Province, China, at elevations between 1400-1600 m. The individuals of the new species were frequently found in the stream surrounded by evergreen broadleaved forests (Fig. 9). M. boettgeri was also found in the same stream.
Etymology. The specific name baishanzuensis refers to the distribution of this species, Baishanzu National Park, Qingyuan County, Zhejiang Province, China. We propose the common name "Baishanzu horned toad" (English) and Bai Shan Zu Jiao Chan (百山祖角蟾, Chinese).

Discussion
Although Megophrys baishanzuensis sp. nov. superficially resembles M. kuatunensis, molecular phylogenetic analyses, detailed morphological comparisons and call datas all proposed the distinct differences between them. Moreover, the breeding seasons of them are different. According to our surveys, the breeding season of M. kuatunensis is in April to May in Wuyi Mountain, Fujian Province, China. But in this season, we did not find any individual of Megophrys baishanzuensis sp. nov. in Qingyuan County, Zhejiang Province, China. And, the breeding season of the new species should be later than June because in June, we only listened to the calls of one male in the type locality (< 10 °C), and, in late July, the males of the species started to call when the temperature was just higher than 18 °C (but we did not find any female individual and egg of it). Different call characteristics and breeding ecology most probably promoted separation of the two species.
During our several and extensive surveys, we only found fewer than 15 adult males of Megophrys baishanzuensis sp. nov., only in a small stream near the top of the mountain in Baishanzu National Park, Zhejiang Province, China, and even then, we did not find any female, and only found four tadpoles of this species. Obviously, the population of the new species is very endemic and small. Fortunately, this population is in a preserved area in Baishanzu National Park. Of course, we still should make a reinforced plan to preserve this area for this toad species.