A new species of the horned toad 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 based on mitochondrial DNA and nuclear DNA sequences all strongly supported the new species as an independent clade sister to M. minor and M. jiangi. The new species could be distinguished from its congeners by a combination of the following characters: body size moderate (SVL 43.4–44.1 mm in males, and 44.8–49.8 mm in females; vomerine teeth absent; tongue not notched behind; a small horn-like tubercle at the edge of each upper eyelid; tympanum distinctly visible, rounded; two metacarpal tubercles on palm; relative finger lengths II < I < V < III; toes without webbing; heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level between tympanum and eye when leg stretched forward; in breeding males, an internal single subgular vocal sac in male, and the nuptial pads with black spines on dorsal surface of bases 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 taxonomic arrangements especially on generic assignments of the group have been controversial for a long time (e.g., Tian and Hu 1983;Dubois 1987;Lathrop 1997;Rao and Yang 1997;Jiang et al. 2003;Delorme et al. 2006;Fei et al. 2009;Chen et al. 2016;Fei and Ye 2016;Deuti et al. 2017;Mahony et al. 2017;Frost 2019). Nevertheless, all molecular phylogenetic studies revealed this group as a monophyletic group which corresponds to the family (Chen et al. 2016;Mahony et al. 2017;Liu et al. 2018;Li et al. 2018;Liu et al. 2020;Wang et al. 2020), and thus many researchers considered it as a large genus Megophrys sensu lato Li et al. 2018;Liu et al. 2018Liu et al. , 2020Frost 2020;Wang et al. 2020) although several studies divided the taxa of the group into different genera and subgenera, thus introducing better resolution of relationships within the family (Chen et al. 2016;Fei and Ye 2016;Deuti et al. 2017;Liu et al. 2018).
The large genus Megophrys currently contains 98 species, of which 41 species were described in the last decade (Frost 2020;Liu et al. 2020). Many cryptic species in the genus are indicated by molecular phylogenetic analyses (Chen et al. 2016;Liu et al. 2018) of which several have been described recently (e.g., Liu et al. 2020). Obviously, more cryptic species need to be verified and described in detail.
During field surveys in the Chishui National Nature Reserve, Chishui City, Guizhou Province, China, we collected a series of Megophrys specimens. Our molecular phylogenetic analyses and morphological comparisons support it as an undescribed species, and it is described herein as a new species.

Sampling
Three adult males and five adult females of the undescribed species were collected in Chishui National Nature Reserve, Chishui City, Guizhou Province, China (Suppl. material 1: Table S1; Fig. 1). In the field, the toads were euthanized using isoflurane, and the specimens were fixed in 75% ethanol. Tissue samples were taken and preserved separately in 99% 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 specimens of the undescribed species were included in the molecular analyses (Suppl. material 2: Table S2). Total DNA was extracted using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). Two fragments of the mitochondrial genes encoding16S rRNA and cytochrome oxidase subunit I (COI) were amplified using the primers in Simon et al. (1994) and Che et al. (2012), respectively. PCR were under the following conditions: 37 cycles at 94 °C for 4 min, 95 °C for 1 min, 53 °C (for 16S rRNA)/47 °C (for COI) for 30 sec, and 72 °C for 1 min followed by a 8-min extension at 72 °C. The nuclear gene sequences encoding brain-derived neurotrophic factor (BDNF) and recombination activating gene 1 (RAG1) were amplified using the primers and protocols in Vieites et al. (2007) and Shen et al. (2013), respectively (Suppl. material 3: Table S3). All PCR products were purified with spin columns, and then were sequenced with primers same as used in PCR. Sequencing was conducted using an ABI3730 automated DNA sequencer in Shanghai DNA BioTechnologies Co., Ltd. (Shanghai, China). All sequences were deposited in GenBank (for accession numbers see Suppl. material 2: Table S2).
For molecular analyses, the available sequence data for congeners of Megophrys were downloaded from GenBank (Suppl. material 2: Table S2), 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 (Suppl. material 2: Table S2), and used as outgroups according to Mahony et al. (2017). Sequences were assembled and aligned in BioEdit v. 7.0.9.0 (Hall 1999) with default settings. Alignments were checked by eye and revised manually if necessary. To avoid bias in alignments, GBLOCKS v. 0.91.b (Castresana 2000) with default settings was used to extract regions of defined sequence conservation from the length-variable 16S gene fragments. Non-sequenced fragments were defined as missing loci. For phylogenetic analyses, two datasets were obtained, i.e., two-mitochondrial genes concatenated dataset of 16S+COI and twonuclear genes concatenated dataset of RAG1+BDNF.
Phylogenetic relationships were reconstructed based on the mitochondrial DNA data and nuclear DNA data, respectively. 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. 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 PARTITION-FINDER v. 1.1.1 (Robert et al. 2012). In the analyses, 16S, each codon position of the protein-coding genes (COI, RAG1 and BDNF) were defined, and Bayesian Inference Criteria (BIC) was used. As a result, the analyses selected the best partition scheme (i.e., 16S gene/each codon position of COI gene) and the GTR+ G + I model for each partition for mitochondrial DNA dataset, and as well, selected the best partition scheme (i.e., each codon position of RAG1 and BDNF genes) and the GTR+ G + I as the best model for all codon position of RAG1 and BDNF genes. For the ML tree, branch supports were drawn from 10000 non-parametric bootstrap replicates. In BI analyses, two runs each with four Markov chains were run for 40 million generations with sampling every 1000 generations. The first 25% of generations were removed as the "burn-in" stage followed by calculation of Bayesian posterior probabilities and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity. Finally, genetic distance between species under uncorrected p-distance model was estimated on 16S gene sequences using MEGA v. 6.06 (Tamura et al. 2011).

Morphological comparisons
All adult specimens of the undescribed species were measured. The terminology and methods followed Fei et al. (2009). Measurements were taken with a dial caliper to 0.1 mm. Seventeen 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); 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); SL snout length (distance from the tip of the snout to the anterior corner of the eye); SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent); 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); TYD maximal tympanum diameter; TW maximal tibia width; UEW upper eyelid width (greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis).
We compared morphological characters of the undescribed species with Megophrys congeners. Comparative data were obtained from related species as described in literature (Table 2).

Bioacoustics notes
Ten advertisement calls from two individuals of the new species were recorded on 18 May 2018 between 21:00-23:00 in Chishui City, Guizhou Province, China in the field. SONY PCM-D50 digital sound recorder was used to record within 20 cm of the calling individuals. 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.   (Mathew & Sen, 2007) Mathew and Sen 2007 M. shapingensis Liu, 1950Fei et al. 2009M. shuichengensis Tian & Sun, 1995Fei et al. 2009M. shunhuangensis Wang, Deng, Liu, Wu & Liu, 2019 Wang et al. 2019a M. spinata Liu & Hu, 1973Fei et al. 2009M. stejnegeri Taylor, 1920 Taylor 1920 M. synoria (Stuart, Sok & Neang, 2006) Stuart  (Mathew & Sen, 2007) Mathew and Sen 2007

Phylogenetic analyses
Aligned sequence matrix of 16S+COI and RAG1+BDNF contains 1104 bp and 1582 bp, respectively. ML and BI trees of the mitochondrial DNA dataset presented almost consistent topology (Fig. 2), and as well, ML and BI trees of the nuclear DNA dataset showed almost identical topology (Fig. 3), though relationships of many lineages were unresolved (Figs 2, 3). In mitochondrial DNA trees, the undescribed species was clustered as an independent clade sister to a clade in comprising of M. minor Stejneger, 1926 andM. jiangi Liu, Li, Wei, Xu, Cheng, Wang &Wu, 2020, but in nuclear DNA trees, the undescribed species clade was sister to M. jiangi, and then was clustered together with M. minor. Genetic distances on16S gene with uncorrected p-distance model between samples of the undescribed species were below 0.2%. The genetic distance between the undescribed species and its closest related species M. minor was 2.2% on 16S gene, which was higher or at the same level with those among many pairs of congeners, for  Table S2. example, 1.7% between M. spinata Liu & Hu, 1973and M. sangzhiensis Jiang, Ye & Fei, 2008, 2.1% between M. omeimontis Liu, 1950and M. binlingensis Jiang, Fei & Ye, 2009.2% between M. cheni     Diagnosis. Megophrys chishuiensis 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 chishuiensis sp. nov. could be distinguished from its congeners by a combination of the following morphological characters: (1) body size moderate (SVL 43.4-44.1 mm in males, and 44.8-49.8 mm in females; (2) vomerine teeth absent; (3) tongue not notched behind; (4) a small horn-like tubercle at the edge of each upper eyelid; (5) tympanum distinctly visible, rounded; (6) two metacarpal tubercles on palm; (7) relative finger lengths II < I < V < III; (8) toes without webbing; (9) heels overlapping when thighs are positioned at right angles to the body; (10) tibiotarsal articulation reaching the level between tympanum and eye when leg stretched forward. In breeding male, (11) an internal single subgular vocal sac; (12) nuptial pads with black spines on dorsal surface of bases of the first two fingers.
Description of holotype. (Figs 4, 5). SVL 43.4 mm; head width larger than head length (HDW/HDL ratio about 1.2); 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; a small horn-like tubercle at the edge of the upper eyelid; eye large, eye diameter 43.9% of head length; pupils vertical; nostril orientated laterally, closer to snout than eye; tympanum distinct, TYP/EYE ratio 0.64; vomerine ridges and vomerine teeth absent; margin of tongue smooth, not notched behind.
Forelimbs slender, the length of lower arm and hand 42.4% of SVL; fingers slender, 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, the outer one long and thin, the inner one oval-shaped.
Hindlimbs slender, 1.48 times 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 absent; toes without webbing; no lateral fringe; inner metatarsal tubercle oval-shaped; outer metatarsal tubercle absent.
Dorsal skin rough, with numerous granules; several large warts scattered on flanks; a small horn-like tubercle at the edge of each upper eyelid; tubercles on the dorsum forming a weak X-shaped ridge, the V-shaped ridges disconnect; two discontinuous dorsolateral parallel ridges on either side of the X-shaped ridges; an inverted triangular brown speckle between two upper eyelids; several tubercles on the flanks and dorsal  surface of thighs and tibias and forming four transverse tubercle rows; supratympanic fold distinct.
Ventral surface smooth; chest with small and round glands, closer to the axilla than to midventral line; 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. 4). An inverted triangular brown speckle between the eyes; X-shaped ridges on the dorsum, four transverse bands on the dorsal surface of the thigh and shank; several dark brown and white vertical bars on the lower and upper lip; venter purple grey, some white spots on the ventral surface of body and limbs; palms and soles uniform purple grey, tip of digits pinkish; pectoral and femoral glands white.
Coloration of holotype in preservation. (Fig. 5). Color of dorsal surface fades to olive; the inverted triangular brown speckle between the eyes, X-shaped ridges on dorsum and transverse bands on limbs and digits distinct; ventral surface greyish white; creamy-white substitutes the pinkish on tip of digits; the posterior of ventral surface of body, inner of thigh and upper of tibia light red.
Variations. In CIBCS20190518027, the back is brown with some brick-red granules (Fig. 6A); in CIBCS20190518030, the X-shaped marking on back of trunk consists of a ridge with brown spots (Fig. 6B), and the throat and anterior belly are purplish, with grey spots on the posterior belly and black spots on the flank belly (Fig. 6E); in CIBCS20190518025, the marking on the back consists of a V-shaped ridge (Fig. 6C), and the anterior belly is brownish with some black spots on flank and belly, and posterior belly is beige (Fig. 6F); in CIBCS20190518019, the whole ventrum is purplish except the posterior belly that shows white blotches (Fig. 6D).
Advertisement call. The call description is based on recordings of the holotype CIBCS20190518031 (Fig. 7) from the shrub leaf near the streamlet, and the ambient air temperature was 24.5 °C. Each call consists of 14-20 (mean 16.14 ± 1.95, N = 10) notes. Call duration was 2.10-3.18 second (mean 2.51 ± 0.33, N = 7). Call interval was 0.92-1.32 seconds (mean 1.13 ± 0.15, N = 6). Each note had a duration of 0.07-0.12 seconds (mean 0.98 ± 0.01, N = 113) and the intervals between notes 0.038-0.085 seconds (mean 0.056 ± 0.011, N = 106). 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 5859 ± 118.02.61 (5733-6064 Hz, N = 7).
Secondary sexual characters. Adult females with SVL 44.8-49.8 mm, larger than adult males with 43.4-44.1 mm. Adult males have a single subgular vocal sac. In breeding males, brownish red nuptial pads are present on dorsal surface of the bases of the first and second fingers with black spines obvious under microscope.
With tibiotarsal articulation reaching to the level between tympanum and eye when leg is stretched forward, Megophrys chishuiensis sp. nov. differs from M. baolongensis, M. nankiangensis, M. pachyproctus, M. shuichengensis and M. tuberogranulata Shen, Mo & Li, 2010 (vs. just reaching posterior corner of the eye in the latter); differs from M. daweimontis, M. glandulosa, M. lini, M. major, M. medongensis, M. obesa, and M. sangzhiensis (vs. reaching the anterior corner of the eye or beyond eye or nostril and tip of snout in the latter); differs from M. leishanensis Li, Xu, Liu, Jiang, Wei & Wang, 2018 (vs. reaching (Fei et al. 2012;Zhang et al. 2017;Li et al. 2018;Liu et al. 2020  Megophrys chishuiensis sp. nov. is phylogenetically closest to M. minor, and this new species could be identified from the latter distinctly by having larger body size (SVL 43.4-44.1 mm in males vs. 34.5-41.2 mm in males of M. minor), having a small horn-like tubercle at the edge of each upper eyelid (vs. absent in the latter), tongue not notched behind (vs. notched in the latter), tibiotarsal articulation reaching the level between tympanum to eye when leg stretched forward (vs. reaching the level between eye and tip of snout in the latter), and having two metatarsal tubercles in each hand (vs. absent in the latter).

Discussion
The new species, Megophrys chishuiensis sp. nov., resembles M. minor and M. jiangi, and detailed comparison with different data sets are important for recognizing them. Our molecular phylogenetic data on mitochondrial DNA and nuclear DNA, and morphological comparisons both separated the new species from the two closely related species. Megophrys minor were reported to be distributed widely through the provinces of Sichuan, Guizhou, Chongqing, Yunnan, Guangxi, Jiangxi and north of Vietnam (Fei et al. 2012), but detailed investigations with multiple data suggested that several populations of the species should contain cryptic species (including Megophrys chishuiensis sp. nov. and M. jiangi). In recent years, a lot of new species of the genus Megophrys have been gradually described, of which, a large part of number of species were found in China (Frost 2020). To now, among the 97 species of Megophrys, 51 species were discovered in China. Even so, dozens of cryptic species need to be described (Chen et al. 2016;Liu et al. 2018) just in China. Obviously, we should conduct more investigations on the differentiation of the populations and explore the species identity in the wide range.
Megophrys chishuiensis sp. nov. with a narrow distribution also fits the "micro-endemism" model like many other congeners . Besides, the new species is likely to be threatened by several factors, i.e., developing tourism in Chishui National Nature Reserve, constructions in this area and increasing pollution from tourists. Reasonable managements of tourism in this area may probably facilitate the protection of the populations of the toad and other animal species.