A new Megophrys Kuhl & Van Hasselt (Amphibia, Megophryidae) from southeastern China

Abstract A new species of the genus Megophrys from Zhejiang Province, China is described. Molecular phylogenetic analyses supported the new taxon as an independent clade nested into the Megophrys clade and sister to M. lishuiensis. The new species could be distinguished from its congeners by a combination of the following morphological characteristics: (1) small size (SVL 31.0–36.3 mm in male and 41.6 mm in female); (2) vomerine ridge present and 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 in hand; (7) relative finger lengths: II < I < IV < III; (8) toes with rudimentary webbing at bases; (9) heels overlapping when thighs are positioned at right angles to the body; (10) tibiotarsal articulation reaching tympanum to eye when leg stretched forward; (11) an internal single subgular vocal sac in male; (12) in breeding male, the nuptial pads with black nuptial spines on the dorsal bases of the first and second fingers.

The genus Megophrys is widely distributed from eastern China through the eastern and southern Himalayas, throughout mainland Indochina and the islands of the Sunda shelf in Indonesia and parts of the Philippines (Frost 2019). It currently contains 92 recognised species (Frost 2019), and noticeably, 37 species were described in the last decade (See species list of Megophrys in Frost 2019). The genus is suggested to harbour both cryptic diversity and highly localised species diversification (Chen et al. 2017;Mahony et al. 2017Mahony et al. , 2018Liu et al. 2018). For example, in the genus, just in China, 41 cryptic species were suggested by molecular phylogenetic analyses in , and six of them were subsequently described (Li et al. 2018;Wang et al. 2019). To present, 48 Megophrys species have been described in China. Obviously, we still need to verify that undescribed taxa and conduct deep investigations for exploring underestimated diversity in this group especially based on detailed morphological comparisons, molecular phylogenetics and bioacoustics data.
During our field surveys in the Xianju County, Zhejiang Province, China, some Megophrys specimens were collected from the montane forests. Molecular phylogenetic analyses and morphological comparisons support that it is distinctly different from its Megophrys congeners. Therefore, we describe it herein as a new species.

Specimens
Seven adult males, two adult females, and six tadpoles of the new taxon (for voucher information see Suppl. material 1: Table S1 and Suppl. material 2: Table S2) were collected from the mountain streams of Xianju County, Zhejiang Province, China (Fig. 1). The stages of tadpoles were identified following Gosner (1960). After taking photographs, they were euthanised using isoflurane, and the specimens were then fixed in 75% ethanol. Tissue samples were taken and preserved separately in 95% ethanol prior to fixation. Specimens were deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS).

Molecular data and phylogenetic analyses
Four male, two female, and three tadpole specimens of the new taxon were included in the molecular analyses (for voucher information see Table 1). For phylogenetic analyses, two topotypes of M. kuatunensis and two topotypes of M. boettgeri from Wuyi Mountain, Fujian Province, China (the common type locality of these species; Fig. 1) were also collected and sequenced (for voucher information see Table 1).
For molecular analyses, the available sequence data for all related species of the genus Megophrys were downloaded from GenBank, mainly from previous studies (Chen et al. 2017;Mahony et al. 2017;Liu et al. 2018;Wang et al. 2019; for GenBank accession number see Table 1). For phylogenetic analyses, corresponding sequences of one Leptobrachella oshanensis and one Leptobrachium boringii were downloaded (for GenBank accession number see Table 1) and used as outgroups according to Chen 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. 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.
Phylogenetic trees were reconstructed for the concatenated data of the mitochondrial genes. 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 andHuelsenbeck 2003), respectively. To avoid underor over-parameterisation (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). For this analysis, 16S rRNA and COI genes were defined, and Bayesian Inference Criteria (BIC) was used. As a result, the analyses suggested that the best partition scheme was 16S/COI gene, and selected GTR+I+G model as the best model for all partitions. For the ML tree, branch supports were drawn from 10,000 non-parametric bootstrap replicates. In BI analyses, two runs each with four Markov chains were run for 60 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 was calculated with the pairwise uncorrected p-distance model between the new taxon and its congeners by 16S rRNA gene using MEGA v. 6.06 (Tamura et al. 2013).

Morphological comparisons
Nine adult specimens of the new taxon and the holotype and seven paratypes of M. lishuiensis were measured (for voucher information see Suppl. material 1: Table S1). The terminology and methods followed Fei et al. (2009). Measurements were taken with a dial calliper to 0.1 mm. In total, 22 morphometric characters of adult specimens were measured:

ED
eye diameter (distance from the anterior corner to the posterior corner of the eye); FIIIL third finger length (distance from base to tip of finger III); FIIL second finger length (distance from base to tip of finger II); FIL first finger length (distance from base to tip of finger I); FIVL fourth finger length (distance from base to tip of finger IV); FL foot length (distance from tarsus to the tip of fourth toe); HAL hand length (distance from the posterior end of the inner metacarpal tubercle to the distal tip of finger III); 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); 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 III); 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); SNT distance from the tip of the snout to the naris; 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); 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 seven tadpoles of the new taxon (for voucher information see Suppl. material 2: Table S2), eleven morphometric characters were measured:

BH
maximum body height; BW maximum body width; IOD 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).
In order to reduce the impact of allometry, a size-corrected value from the ratio of each character to SVL was calculated and then log-transformed for the following morphometric analyses. One-way analysis of variance (ANOVA) was used to test the significance of differences on morphometric characters between different sexes as well as between different species. The significance level was set at 0.05. Furthermore, to show the spatial distribution of each species on the morphometric characters, principal component analyses (PCA) were performed. These analyses were carried out in R (R Development Core Team 2008).

Bioacoustics notes
The advertisement calls of the new taxon from Xianju County, Zhejiang Province, China were recorded 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.

Phylogenetic analyses
Aligned sequence matrix of 16S+COI contained 1104 bps. ML and BI trees of the mitochondrial DNA dataset presented almost consistent topology, though relationships of some lineages were unresolved (Fig. 2). All individuals of the new taxon were clustered into one clade which was independently nested into the Megophrys clade and sister to the M. lishuiensis clade. Genetic distances on 16S gene with uncorrected p-distance model between specimens of the new taxon were less than 0.7% (ranging from 0.0% to 0.6%), being lower than most interspecific genetic distances in the genus Megophrys (0.4%-19.3%; Suppl. material 3: Table S3). Genetic distance between the new taxon and its closely-related species M. lishuiensis is 2.8%, higher than that between many substantial species, for example, M. wushanensis vs. M. baolongensis

Morphometric analyses
The results of one-way ANOVA showed that in the new taxon, the male group was significantly different from the female group on SVL and the ratio of ED and IND to SVL  Table 1. (p-value < 0.05). Therefore, morphometric analyses between the new taxon and M. lishuiensis were conducted separately for male and female. In PCA, the total variation of the first two principal components was 56.2% in male and 74.9% in female respectively. In both male and female, the new taxon could be distinctly separated from its phylogenetically sister species M. lishuiensis on the two-dimensional plots of PC1 vs. PC2 (Fig. 3).
The results of one-way ANOVA indicated that the new taxon was significantly different from M. lishuiensis on many morphometric characters (all p-values < 0.05; Table 3). More detailed descriptions of results from morphological comparisons between the new taxon and its congeners were presented in the following sections for describing the new species.
Diagnosis. Megophrys xianjuensis 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 gland small and round, closer to the axilla than to midventral line; femoral gland on rear of thigh; vertical pupils.
The new species could be identified from its congeners by a combination of the following morphological characters: (1) small size (SVL 31.0-36.3 mm in males and 41.6 mm in female); (2) vomerine ridge present and 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 in hand; (7) relative finger lengths: II < I < IV < III; (8) toes with rudimentary webbing at bases; (9) heels overlapping when thighs are positioned at right angles to the body; (10) tibiotarsal articulation reaching tympanum to eye when leg stretched forward; (11) an internal single subgular vocal sac in male; (12) in breeding male, the nuptial pads with black nuptial spines on the dorsal bases of the first and second fingers.
Description of holotype. SVL 34.4 mm; head wider than long (HDW/HDL ratio 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 on in dorsal view; an small horn-like tubercle at the edge of the upper eyelid; eye large and convex, eye diameter 40.4% of head length; pupils vertical; nostril orientated laterally, closer to snout than eye; tympanum distinct, TYP/EYE ratio 0.56; vomerine ridges present and vomerine teeth absent; margin of tongue smooth, not notched behind (Fig. 4A, B, E).
Forelimbs slender, the length of lower arm and hand 44.9% of SVL; fingers slender, relative finger lengths: II < I < IV < III; tips of digits globular, without lateral fringes; subarticular tubercle distinct at the base of each fingers; two metacarpal tubercles, prominent, oval-shaped, the inner one bigger than the outer (Fig. 4G).
Hindlimbs slender, heels overlapping when thighs are positioned at right angles to the body, tibiotarsal articulation reaching tympanum to eye when leg stretched forward; tibia length slightly longer than thigh length; relative toe lengths I < II < V < III < IV; tips of toes round, slightly dilated; subarticular tubercle absent; toes with rudimentary webbing at bases; lateral fringe narrow; inner metatarsal tubercle oval-shaped; outer metatarsal tubercle absent (Fig. 4I).
Dorsal rough, with numerous granules; several large warts scattered on flanks; an small horn-like tubercle at the edge of each upper eyelid; a dark brown inverted triangular pattern between anterior corner of eyes, tubercles on the dorsum forming a weak X-shaped ridge and two discontinuous dorsolateral parallel ridges on either side of the X-shaped ridge; several tubercles on the flanks and dorsal surface of thighs and tibias, and limbs barred with dark brown forming four transverse rows; supratympanic fold distinct (Fig. 4A).
Ventral surface with numerous white granules; chest gland distinct and round, closer to the axilla than to midventral line; femoral gland on rear of thigh; posterior end of the body protrudes distinct and appears as an arc-shaped swelling, upper the anal region (Fig. 4B).
Colouration of holotype in life. An inverted triangular brown speckle between the eyes; an X-shaped ridges on the dorsum of body, four transverse bands on the dorsal surface of the hindlimb; several dark brown and white vertical bars on the lower and upper lip; the venter purple and the colour of throat is deeper than belly, flank and middle of throat with black brown spots, numerous white granules on the ventral surface and limbs; palms and soles purple and the metacarpal tubercles are orange, tip of digits greyish white; pectoral and femoral glands white (Fig. 5).
Preserved holotype colouration. Dorsal surface fade to olive; the inverted triangular brown speckle between the eyes, X-shaped ridges on dorsum and transverse bands on limbs and digits and brown spots on flank and middle throat are more distinct; ventral surface greyish white; creamy-white substitutes the orange in metacarpal tubercles; the posterior of ventral surface of body, inner of thigh and upper of tibia creamy-white (Fig. 4).
Variation. In some adult individuals a brown Y-shaped marking on the dorsum of head and disconnected with a X-shaped marking on back (Fig. 6A); an inverted triangular brown speckle between two upper eyelids with Y-shaped marking on back of trunk, the colouration of dorsum is brown with brick-red and the caudal vertebra is pointed in the adult female (Fig. 6B); in some adult individuals the metacarpal tubercles in palm is grey-white but the tip of fingers is orange and the black spots in flank is smaller (Fig. 6C); in some adult individuals the white granules in ventral surface are more intensive (Fig. 6D).
Advertisement calls. Ten advertisement calls from two individuals of the new species were recorded in the Xianju County, Zhejiang Province, China between 21:00-23:00 on 7 May 2019. The call description is based on recordings of the holotype CIBXJ090503 (Fig. 7) from the stone near the streamlet, and the ambient air temperature was 24.5 °C. Each call consists of 22-62 (mean 49.33 ± 14.50, N = 6) notes. Call duration was 4.90-14.22 second (mean10.95 ± 3.42, N = 6). Call interval was 4.12-7.64 second (mean 6.23 ± 1.56, N = 5). Each note had a duration of 0.06-0.90 second (mean 0.10 ± 0.05, N = 290) and the intervals between notes 0.07-0.88 second (mean 0.13 ± 0.05, N = 284). Amplitude modulation within note was apparent, beginning with lower 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 6400 ± 465.79 (5520-6840 Hz, N = 6).
Secondary sexual characteristics. Adult females with SVL 41.6 mm, larger than adult males with 31.0-36.3 mm. Adult males have a single subgular vocal sac (Fig. 5E). In breeding males, the brownish red nuptial pads on the dorsal bases of the first finger and second fingers with black nuptial spines under microscope.
By By having an internal single subgular vocal sac in male, Megophrys xianjuensis sp. nov. differs from M. caudoprocta, M. shapingensis, and M. shuichengensis (vs. vocal sac absent in the latter).
By having nuptial pads and nuptial spines on the dorsal base of the first and second fingers in breeding male, Megophrys xianjuensis sp. nov. differs from M. acuta, M. feii, M. shapingensis, and M. shuichengensis (vs. nuptial pads and nuptial spines lacking in the latter); differs from M. boettgeri, and M. elfina (vs. nuptial pads and nuptial spines only on the first finger in the latter).
Megophrys boettgeri and M. kuatunensis were suggested to be distributed in Zhejiang Province, China and might be sympatric with Megophrys xianjuensis sp. nov. (Fei et al. 2016;Wang et al. 2017b). The new species can be distinguished from these species by a series of morphological characters as follows. The new species vs. M. boettgeri: vomerine ridges present vs. vomerine ridges absent, tibiotarsal articulation reaching forward to the region between tympanum and eye when hindlimb is stretched along the side of the body vs. tibiotarsal articulation reaching forward to eye, having nuptial pads and nuptial spines on the dorsal base of the first and second fingers in breeding male vs. nuptial pads and nuptial spines only on the first finger, heels overlapping when thighs are positioned at right angles to the body vs. heels just meeting, light round patches on the shoulder absent vs. present. The new species vs. M. kuatunensis: heels overlapping when thighs are positioned at right angles to the body vs. heels not meeting, toes with rudimentary webbing at bases vs. toes without webbing.
Molecular phylogenetic analyses revealed that the new species was genetically closer to M. lishuiensis. The new species can be distinguished from M. lishuiensis by a series of morphological characters as follows. Vomerine ridges present vs. vomerine ridges absent; heels overlapping when thighs are positioned at right angles to the body vs. heels just meeting or not meeting; light round patches on the shoulder absent vs. present; having significantly lower ratios of UEW, ED, TYD, LW and TW to SVL in males; and having significantly higher ratios of UEW, ED and HAL to SVL in females (all p-values < 0.05; Table 3).
Etymology. The specific epithet xianjuensis refers to Xianju County, Zhejiang Province, China, where the type locality of the species is located. We propose the common name "Xianju horned toad" in English and Xian ju Jiao Chan in Chinese.

Discussion
Although Megophrys xianjuensis sp. nov. superficially resembles M. lishuiensis, our integrative comparisons with morphological and molecular data can clearly identify the new species from the latter. This indicates that conserved morphology could hamper species delineation, requiring the incorporation of detailed morphological, genetic, and bioacoustic data to recognise cryptic species. The discovery of Megophrys xianjuensis sp. nov. brings the total number of species in the genus to 93, with 49 of them recorded in China (Fei et al. 2016;Frost 2019). Yet, there remain still dozens of undescribed species just in China (Chen et al. 2017;Liu et al. 2018). As noted, the new species was a newly-found clade which has not been reported in previous phylogenetic works although it belongs to the subgenus Panophrys according to our phylogenetic frameworks and previous classifications Liu et al. 2018).
According to records in Fei et al. (2016) and Wang et al. (2017b), M. boettgeri is widely distributed in Zhejiang Province, China. However, with two-years' field surveys and detailed comparisons with many Megophrys specimens from Xianju County, Zhejiang Province, we have not found M. boettgeri in this region. Similarly, in this county, we have also not found M. kuatunensis and M. lishuiensis even though they were reported to be distributed in western and southern parts of Zhejiang Province, China A B (Fei et al. 2016;Wang et al. 2017b); therefore, we suggest that probably in Xianju County, Zhejiang Province, China, there is no M. boettgeri, M. kuatunensis or M. lishuiensis. Considering the high underestimated species level and localised diversification in Megophrys (Chen et al. 2017;Liu et al. 2018;Wang et al. 2019), wider and in-depth surveys should be conducted in the eastern part of Zhejiang Province and adjacent areas for detecting distributional range of the new species and for finding cryptic species.
Many Megophrys species have narrow distributions fitting the "micro-endemism" model of Liu et al. (2018) and Wang et al. (2019). Similarly, according to our surveys, Megophrys xianjuensis sp. nov. is probably only distributed in a narrow range in Xianju County and/or adjacent regions. Hence, this species is likely to be threatened as scenic sites in Xianju County harbouring its habitats (Fig. 9) are increasingly impacted by tourists and considerable developments in tourist infrastructure easily leading to habitat loss. Additionally, an increased frequency of extreme weather events in recent years also influenced the species; for example, the super typhoon "Lekima" in August 2019 causing heavy rain seriously broke many channels and forests where the species occurs in Xianju County. It is urgent to conduct more surveys to understand the population status of the new species and to develop appropriate protection strategies.