Description of a new horned toad of Megophrys Kuhl & Van Hasselt, 1822 (Anura, Megophryidae) from southwest China

Abstract A new species of the genus Megophrys is described from Guizhou Province, China. Molecular phylogenetic analyses supported the new species as an independent clade nested into the Megophrys. The new species could be distinguished from its congeners by a combination of the following characters: body size moderate (SVL 49.3–58.2 mm in males); vomerine ridges present distinctly, vomerine teeth present; tongue feebly notched behind; tympanum distinctly visible, oval; two metacarpal tubercles in hand; toes with one-third webbing and wide lateral fringes; heels overlapped when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level between tympanum and eye when leg stretched forward; an internal single subgular vocal sac present in male; in breeding male, the nuptial pads with large and sparse black nuptial spines present on the dorsal bases of the first two fingers.

For molecular analyses, the available sequence data 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 according to 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 is16S 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 and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity.

Morphological comparisons
In total, 16 specimens including six males of the undescribed species, five males of M. sangzhiensis, and five males of M. spinata were measured (for voucher information see Table 2). The terminology and methods followed Fei et al. (2009). Measurements were taken with a dial caliper to 0.1 mm. Twenty-one morphometric characters of adult specimens were measured: ED eye diameter (distance from the anterior corner to the posterior corner of the eye); 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); HLL hindlimb length (maximum length from the vent to the distal tip of the Toe IV); IAE distance between posterior corner of eyes; IFE distance between anterior corner of eyes; 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); NED nasal to eye distance (distance between the nasal and the anterior corner of the eye); NSD nasal to snout distance (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).  Inger, Stuebing & Tan, 1995Inger et al. 1995M. edwardinae Inger, 1989Inger 1989M. elfina Poyarkov, Duong, Orlov, Gogoleva, Vassilieva, Nguyen, Nguyen, Nguyen, Che & Mahony, 2017Poyarkov et al. 2017 (Ohler, 2003) Ohler 2003 M. gigantica Liu, Hu & Yang, 1960Liu et al. 1960M. glandulosa Fei, Ye & Huang, 1990Fei et al. 1990M. hansi (Ohler, 2003 Ohler 2003 Malkmus &Matsui, 1997 Malkmus andMatsui 1997 In order 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 subsequent morphometric analyses. One-way analysis of variance (ANOVA) was used to test the significance of differences on morphometric characters between different species. The significance level was set at 0.05. To show the spatial distribution of different species on the morphometric characters, principal component analyses (PCA) were performed. These analyses were carried out in the R (R Development Core Team 2008). The new species was also compared with all other Megophrys species on morphology. Comparative data were obtained from related species as described in literature (Table 3).

Bioacoustics analyses
The advertisement calls of the undescribed species were recorded from the holotype specimen CIBTZ20190608017 in a stream at ambient air temperature of 20.5 °C and air humidity of 87 % in the field on 8 June 2019 in Huanglian Nature Reserve, Tongzi County, Guizhou Province, China. The advertisement calls of M. sangzhiensis were recorded from the specimen CIBSZ2012062005 in a stream at ambient air temperature of 18.5 °C and air humidity of 85 % in the field on 20 June 2012 in Sangzhi County, Hunan Province, China. The advertisement calls of M. spinata were recorded from the specimen CIBLS20190801001 in a stream at ambient air temperature of 19.0 °C and air humidity of 85 % in the field on 1 August 2019 in Leishan County, Guizhou Province, China. 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 had almost consistent topology though relationships of some lineages were unresolved (Fig. 2).
In trees, the undescribed species was clustered as an independent clade and sister to a clade in comprising of M. sangzhiensis and M. spinata (Fig. 2). Genetic distances on COI gene with uncorrected p-distance model between all samples of the undescribed species were below 0.2%. The genetic distance between the undescribed species and its closest related species M. sangzhiensis was 4.3 % on COI gene, which was higher or at the same level with those among many pairs of congeners, for example, 3.6 % between M. spinata and M. sangzhiensis, 1.8% between M. huangshanensis and M. boettgeri, and 4.3 % between M. maosonensis and M. mangshanensis (Suppl. material 1: Table S1).

Morphological comparisons
In PCA for males, the total variation of the first two principal components was 63.2 %. In males on the two-dimensional plots of PC1 vs. PC2, the undescribed species could be distinctly separated from M. sangzhiensis and M. spinata (Fig. 3). The results of one-way ANOVA indicated that in males, the undescribed species was significantly different from M. sangzhiensis and M. spinata on many morphometric characters (all p-values < 0.05; Table 4). More detailed descriptions of results from morphological comparisons between the undescribed species and its congeners were presented in the following sections for describing the new species.

Bioacoustics comparisons
There were many differences in sonograms and waveforms of calls between the undescribed species, M. sangzhiensis, and M. spinata ( Fig. 4; Table 5). Firstly, in the note interval, the undescribed species were shorter than those of both M. sangzhiensis and M. spinata. Secondly, the dominant frequency of call in the undescribed species was lower than both of M. sangzhiensis and M. spinata. Thirdly, the amplitude of the undescribed species beginning with moderately high energy pulses, increasing slightly to a maximum by approximately mid note, and then decreasing towards the end of each note, in M. sangzhiensis beginning with maximum energy pulses and then decreasing towards the end of note, and in M. spinata beginning with lower energy pulses, then    Diagnosis. Megophrys qianbeiensis 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.
Megophrys qianbeiensis sp. nov. could be distinguished from its congeners by a combination of the following morphological characters: body size moderate (SVL 49.3-58.2 mm in males); vomerine ridges present distinctly, vomerine teeth present; tongue feebly notched behind; tympanum distinctly visible, oval; two metacarpal tubercles in hand; toes with one-third webbing and wide lateral fringes; heels overlapped when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level between tympanum and eye when leg stretched forward; an internal single subgular vocal sac present in male; in breeding male, the nuptial pads with large and sparse black nuptial spines present on the dorsal bases of the first two fingers.
Description of holotype. (Figs 5, 6). SVL 56.3 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 44.5 % of head length; pupils vertical; nostril orientated laterally, closer to snout than eye; tympanum distinct, TYP/EYE ratio 0.49; vomerine ridges present distinctly as V-shape, vomerine teeth present; margin of tongue smooth, feebly notched behind.
Forelimbs slender, the length of lower arm and hand 42.6 % of SVL; fingers burly, relative finger lengths: II < I < V < 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, 1.54 times of SVL; heels overlapping when thighs are positioned at right angles to the body, tibiotarsal articulation reaching tympanum to 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 present on the base of each toes ; toes with one-third webbing and relative wide lateral fringe; inner metatarsal tubercle oval-shaped; outer metatarsal tubercle absent.
Dorsal skin rough, with numerous granules with black spins; several large warts scattered on flanks; tubercles on the dorsum forming a weak V-shaped ridge; two discontinuous dorsolateral parallel ridges on either side of the V-shaped ridges; an inverted triangular brown speckle between two upper eyelids; several tubercles on the flanks and dorsal surface of thighs and tibias; supratympanic fold distinct.
Ventral surface smooth with numerous white granules; glands on chest indistinct; femoral glands on rear of thighs, numerous white granules on outer thighs; posterior end of the body distinctly protruding and forming an arc-shaped swelling above the anal region.
Coloration of holotype in life (Fig. 5). An inverted triangular brown speckle between the eyes; V-shaped ridges on the dorsum with brown speckle, on transverse bands on the dorsal surface of the thigh and shank; several dark brown and white vertical bars on the lower and upper lip; belly whitish grey with dark brown marbling; ventral surface of posterior limb orange with numerous granules; palms, soles and tip of digits uniform purple grey; femoral glands white.
Coloration of holotype in preservation (Fig. 6). Color of dorsal surface fades to brownness; the inverted triangular brown speckle between the eyes and V-shaped ridges on dorsum indistinct; 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. In CIBTZ20160715003 the dorsolateral parallel ridges are short, just a little bit above the shoulder (Fig. 7A); in CIBTZ20190608015 the X-shaped marking on back of trunk consists of a ridge with brown spots (Fig. 7B) and the posterior belly are orange with black spots on the flank belly (Fig. 7C); in CIBKKS20180722001 the belly is grey brown with some white spots (Fig. 7D).
Advertisement call. The call description is based on recordings of the holotype CIBTZ20190608017 (Fig. 4) from the shrub leaf near the streamlet, and the ambient air temperature was 20.5 °C. Each call consists of 14-26 (mean 22.5 ± 4.4, N = 6) notes. Call duration was 2832-5621 ms (mean 4413 ± 972, N = 6). Call interval was 6812-14387 ms (mean 10878 ± 2701, N = 5). Each note had a duration of 129-211 ms (mean 167 ± 0.02, N = 135) and the intervals between notes 34-94 ms (mean 57 ± 0.01, N = 128). Amplitude modulation within note was apparent, beginning with moderately high energy pulses, increasing slightly to a maximum by approximately mid note, and then decreasing towards the end of each note. The average dominant frequency was 2469 ± 197.47 (2250-3000 Hz, N = 6). Secondary sexual characters. Adult males have a single subgular vocal sac. In breeding males, brownish red nuptial pads are present on the dorsal bases of the first two fingers with big and sparse black nuptial spines (Fig. 5A).
By the tibiotarsal articulation reaching to the level between tympanum and eye when leg stretched forward, Megophrys qianbeiensis sp. nov. differs from M. daweimontis, M. glandulosa, M. lini, M. major, M. medogensis, and M. obesa (vs. reaching the anterior corner of the eye or beyond eye or nostril and tip of snout).
The congeners M. carinense and M. jiangi have sympatric distribution with Megophrys qianbeiensis sp. nov. (Fei et al. 2012). The new species can be distinguished from these species by a series of morphological characters as follows. The new species differs from M. carinense in the smaller body size in the new species (adult males with 49.3-58.2 mm vs. adult males with 92-123 mm in the latter), a horn-like tubercle at the edge of each upper eyelid absent (vs. prominent in the latter), the tongue feebly notched behind (vs. notched behind in the latter). The new species differs from M. jiangi by a larger body size (49.3-58.2 mm in males in the new species vs. 34.4-39.2 mm in the latter), a horn-like tubercle at the edge of each upper eyelid absent (vs. present in the latter), the tongue feebly notched behind (vs. notched behind in the latter), presence of wide lateral fringes on the toes (vs. lacking in the latter), and toes with one-third webbing (vs. rudimentary webbing in the latter).
Megophrys qianbeiensis sp. nov. is phylogenetically closest to M. sangzhiensis and M. spinata. The new species differs from M. sangzhiensis by the following characters: horn-like tubercle absent at the edge of each upper eyelid (vs. present in the latter), toes with one-third webs (vs. with rudimentary webbing in the latter), vomerine ridges present distinctly as V-shape and vomerine teeth present (vs. vomerine ridges separated and weak, vomerine teeth absent in the latter), tibiotarsal articulation reaching to the level between tympanum and eye when leg stretched forward (vs. reaching the anterior corner of eye in the latter), spines on nuptial pads on the first two fingers larger and sparser (vs. finer and thicker in the latter), and having significantly higher ratios of HDL, LAL, HLL, TL, and IAE to SVL. On bioacoustics, the new species differs from M. sangzhiensis in the following characters: lower dominant frequency (2250-3000 Hz in the new species vs. 10380 -13200 Hz in the latter), the amplitude beginning with moderately high energy pulses, increasing slightly to a maximum by approximately mid note, and then decreasing towards the end of each note (vs. beginning with maximum energy pulses and then decreasing towards the end of note in the latter).
The new species differs from M. spinata by the following characters: tibiotarsal articulation reaching the level between tympanum to eye when leg stretched forward (vs. reaching the anterior corner of eye in the latter), present distinctly as V-shape and vomerine teeth present (vs. vomerine ridges separated and weak, vomerine teeth absent in the latter), spines on nuptial pads on the first two fingers little weaker (vs. spines larger in the latter), and having significantly higher ratios of HDW, ED, LAL, TYD and IAE to SVL. On bioacoustics, the new species differs from M. spinata in the following characters: lower dominant frequency (2250-3000 Hz in the new species vs. 4260-4589 Hz in the latter), the amplitude beginning with moderately high energy pulses, increasing slightly to a maximum by approximately mid note, and then decreasing towards the end of each note (vs. beginning with lower energy pulses, then increasing to the maximum by approximately one-four note and then decreasing to the mid note then increasing to the second highest energy pulses and then decreasing towards the end of note in the latter).
Distribution and habitats. Megophrys qianbeiensis sp. nov. is known from Huanglian Nature Reserve, Tongzi County and Kuankuoshui National Nature Reserve, Suiyang County, Guizhou Province, China at elevations between 1400-1600 m. The individuals of the new species were frequently found on stone in the streams surrounded by evergreen broadleaved forests (Fig. 8), and three sympatric amphibian species were found, i.e., Megophrys jiangi, Odorrana margaratae (Liu, 1950), and Zhangixalus omeimontis (Stejneger, 1924). Etymology. The specific epithet qianbeiensis refers to northern part of Guizhou, also called "黔", the province where the type locality of the species belongs to. We propose the common English name "Qianbei horned toad" and Chinese name "Qian Bei Jiao Chan (黔北角蟾)".

Discussion
The new species Megophrys qianbeiensis sp. nov. resembles M. spinata and M. sangzhiensis, and detailed comparisons with different data are important for recognizing them. Our molecular phylogenetic data on mitochondrial DNA and morphological comparisons both separated the new species from the two closely related species. Megophrys spinata were reported to be distributed widely through the provinces of Sichuan, Guizhou, Hunan, Chongqing, Yunnan, and Guangxi (Fei et al. 2012), but detailed investigations with multiple data suggested that several populations of the species should contain cryptic species (including Megophrys qianbeiensis sp. nov. and M. sangzhiensis). In recent years, many new species of the genus Megophrys have been gradually described, of which a large part of number was found in China (Frost 2020). To date, among the 106 species of Megophrys, 56 species were discovered in China. Even so, many cryptic species still need to be described just in southern China (Chen et al. 2017;Liu et al. 2018).
South-western China has long been proposed as biodiversity hotspot (Myers et al. 2000). Guizhou Province is an important part of southwest China, especially with the particular environments of karst rocky desertifcation, and knowledge of biodiversity levels and/or patterns are still seriously lacking. Recently, a series of new amphibian species were described from this province Li et al. 2018aLi et al. , b, 2019aLyu et al. 2019;Wang et al. 2019c;Luo et al. 2020;Liu et al. 2020;Wei et al. 2020;Xu et al. 2020), indicating that species diversity of amphibians in this region is highly underestimated. It is urgent for herpetologists to conduct comprehensive and in-depth surveys to explore the level of amphibian species diversity in this region under accelerating climate changes. Obviously, more work should be conducted on detecting the differentiation of the populations and further describe the cryptic species in this region.