Description of six new species of the subgenus Panophrys within the genus Megophrys (Anura, Megophryidae) from southeastern China based on molecular and morphological data

Abstract The diversity of the subgenus Panophrys within the genus Megophrys has been revealed to be extremely underestimated from southeastern China. Herpetological surveys coupled with extensive sampling in a longitudinal mountain belt located in southeastern China resulted in the discoveries of six new species of the subgenus Panophrys. Furthermore, the new discoveries support the findings of “micro-endemism”, “sympatric phenomenon” and “sympatric but distant phylogenetically” which appear to be common among Panophrys species, and also indicates that the Asian horned toads would be good candidates for studies on speciation and biogeography, and additionally emphasizes the conservation difficulties of these toads.


Introduction
The Asian horned toads (Megophrys) comprise 85 recognized species which were previously classified in the subfamily Megophryinae (Frost 2019). They are widespread in montane forest area in tropical and subtropical Asia, including southern mainland

Sampling
For molecular analysis, a total of 42 samples (17 were attained from GenBank and 25 were new materials in this study) from the collection of unnamed specimens of the subgenus Panophrys, together with 39 samples (37 from GenBank and two new materials) from 21 recognized species of Panophrys were used as in-groups in this study. In addition, four samples (all from GenBank) from two recognized species of the subgenus Atympanophrys, four samples (three from GenBank and one new materials) from two recognized species of the subgenus Brachytarsophrys, three samples (one from GenBank and two new materials) from two recognized species of the subgenus Ophryophryne, two samples (all from GenBank) from two recognized species of the subgenus Pelobatrachus, and six samples (five from GenBank and one new materials) of three recognized species of the subgenus Xenophrys were incorporated into our dataset and used as out-groups. Details see Table 1. All muscle samples were preserved in 95% ethanol and stored at -40 °C.

DNA Extraction, PCR and sequencing
Genomic DNA was extracted from muscular tissue using a DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. All samples were sequenced for two mitochondrial genes, i.e., partial 16S ribosomal RNA gene (16S) and complete cytochrome C oxidase 1 gene (CO1). Primers used for 16S were L3975 (5'-CGCCTGTTTAC-CAAAAACAT-3') and H4551 (5'-CCGGTCTGAACTCAGATCACGT-3') following Simon et al. (1994), and for CO1 were Chmf4 (5'-TYTCWACWAAYCAYAAA-GAYATCGG-3') and Chmr4 (5'-ACYTCRGGRTGRCCRAARAATCA-3') following Meyer et al. (2005). PCR amplifications were processed in a 20-reaction volume with the cycling conditions that initial denaturing step at 95 °C for 4 min, 35 cycles of denaturing at 94 °C for 40 s, annealing at 53 °C for 40 s and extending at 72 °C for 1 min, and final extending step of 72 °C for 10 min. PCR products were purified with spin columns. The purified products were sequenced with both forward and reverse primers using BigDye Terminator Cycle Sequencing Kit per the guidelines, on an ABI Prism 3730 automated DNA sequencer by Shanghai Majorbio Bio-pharm Technology Co., Ltd and Beijing Genomics Institute. All sequences have been deposited in GenBank (Table 1).

Phylogenetic analyses
DNA sequences were aligned in MEGA 6 (Tamura et al. 2013) by the Clustal W algorithm with default parameters (Thompson et al. 1997). Two gene segments, 535 base pairs (bp) of 16S and 645 bp of CO1, were concatenated seriatim into a 1180-bp single sequence. The dataset was partitioned according to the genes and codon positions, and then tested respectively in jmodeltest v2.1.2 with Akaike and Bayesian information criteria, all resulting in the best-fitting nucleotide substitution models of GTR + I + G. Sequenced data was analyzed using Bayesian inference (BI) in MrBayes 3.2.4 (Ronquist et al. 2012). Three independent runs were conducted in BI analysis, each of which was performed for 2,000,000 generations and sampled every 1000 generations with the first 25% samples were discarded as burn-in, resulting a potential scale reduction factor (PSRF) of < 0.01. Pairwise distances (p-distance) were calculated in MEGA 6 using the uncorrected p-distance model.

Morphometrics
All specimens were fixed in 10 % buffered formalin and later transferred to 70% ethanol for preservation, and deposited at the Museum of Biology, Sun Yat-sen University (SYS) and Chengdu Institute of Biology, the Chinese Academy of Sciences (CIB), China. Measurements follow Fei et al. (2009), and were taken with digital calipers to the nearest 0.1 mm. These measurements were as follows: SVL snout-vent length (from tip of snout to vent); HDL head length (from tip of snout to rear of jaws); HDW head width (head width at commissure of jaws); SNT snout length (from tip of snout to anterior corner of eye); ED eye diameter (diameter of exposed portion of eyeball); IOD interorbital distance (minimum distance between upper eyelids); IND internasal distance (distance between nares); TD tympanum diameter (horizontal diameter of tympanum); TED tympanum-eye distance (distance from anterior edge of tympanum to posterior corner of eye); HND hand length (distance from distal end of radioulna to tip of phalanx of finger III); RAD radioulna length; TIB tibia length (distance from knee to heel); FTL foot length (distance from distal end of tibia to tip of distal phalanx of toe IV).
Sex was determined by direct observation of calls, the presence of internal vocal sac openings and the presence of testicles observed through dissection for males, as well as the presence of eggs and ovaries on the abdomen through anatomise for females. Presence or absence of nuptial pads/spines was examined with a microscope.
Comparative morphological data of Megophrys species allocated to the subgenus Panophrys (currently contains 32 species) Tapley et al. 2017;Wang et al. 2017a;Wang et al. 2017b;Zhang et al. 2017;Li et al. 2018;Tapley et al. 2018), and a small-sized species M. feii (incertae sedis), were obtained from examination of museum specimens (see Appendix 1) and from the literature ( Table 2). The order of the new species accounts follows the distributions of the new species that located in the longitudinal mountain belt from the south to the north.

Phylogenetics
The Bayesian inference (BI) phylogenetic tree was integrated in Figure 2; the p-distances at the mitochondrial 16S rRNA gene fragment among all samples of the subgenus Panophrys were given in Table 3.
In our phylogenic tree, all sequences of the genus Megophrys grouped into six clades with strong node support values, which were consistent with the results from Mahony et al. (2017) and Liu et al. (2018), and corresponded to the six subgenera: Panophrys, Ophryophryne, Xenophrys, Atympanophrys, Brachytarsophrys and Pelobatrachus. The subgenus Panophrys is further divided into three subclades, named western subclade A, western subclade B and eastern subclade.   boettgeri, M. huangshanensis, M. kuatunensis, M. brachykolos, M. insularis, M. cheni, M. lini, M. jinggangensis, M. obesa, M. ombrophila, M. acuta, M. sangzhiensis, M. caudoprocta, M. tuberogranulatus and wushanensis, and other six lineages made up of samples from the aforementioned longitudinal mountain belt in the middle of southeastern China with significant genetic differences (Table 3).                        Table 1) clustered into a basal lineage of an eastern subclade with strong node supports and almost have no molecular differences; further, this population can be distinguished from all known species and other undescribed lineages by distinctive morphological characters and significant molecular differences with a lowest p-distance of 2.8%. Therefore, the population from Mt. Mufu represented a separately evolving lineage, and is described as a new species, Megophrys (Panophrys) mufumontana sp. nov., below.
All samples from Mt. Wugong, Jiangxi (samples 30-42 from Yangshimu Scenic Area and Wugongshan Scenic Area) clustered into a lineage with strong node supporting values and almost no genetic differences, which was defined as a species and recognized as M. sp12 by Liu et al. (2018); further, the population from Mt. Wugong can be distinguished from all known species and other undescribed lineages by distinctive morphological differences and significant molecular differences with a lowest p-distance of 4%. Therefore, the population from Mt. Wugong represented a separately evolving lineage and is described as a new species, Megophrys (Panophrys) wugongensis sp. nov., below.
All samples from Mt. Yinping, Guangdong (samples 1-6) clustered into a lineage with strong node supportg values and small genetic differences (highest p-distance 0.2%), which was defined as a species and recognized as M. sp11 by Liu et al. (2018); samples 17-22 from Mt. Nankun, Guangdong clustered into a lineage with strong node support values and small genetic differences (highest p-distance 0.7%), which was defined as a species and recognized as M. sp10 by Liu et al. (2018); these two populations are sister taxa to each other with significant genetic differences (p-distances 2.6-2.8%), and can be further distinguished from all known species and other undescribed lineages by distinctive morphological differences and significant molecular differences. Therefore, the populations from Mt. Yinping and Mt. Nankun represented two separately evolving lineages, and are described as new species, Megophrys (Panophrys) dongguanensis sp. nov. and Megophrys (Panophrys) nankunensis sp. nov., below.
Samples 7-10 from Mt. Jiulian, Jiangxi and samples 11-12 from Mt. Nankun, Guangdong clustered into a lineage with small genetic differences (highest p-distance 0.7%), which is a sister subclade to M. boettgeri and M. huangshanensis with large genetic differences (lowest p-distance 2.3%); therefore, these samples represented a separately evolving lineage, which was defined as a species and recognized as M. sp30 by Liu et al. (2018), and is described as a new species, Megophrys (Panophrys) jiulianensis sp. nov., below.
Samples 26-29 from Mt. Qiyun, Jiangxi were defined as a species and recognized as M. sp6 by Liu et al. (2018) and the samples 23-25 from Nanling Nature Reserve, Guangdong were defined as a species and recognized as M. sp7 by Liu et al. (2018). Although the populations from two locations are divided into two branches, the highest p-distance is only 0.7%. Moreover, there are no distinct morphological characters that can distinguish them from each other. Herein, we considered these two populations as one taxon, which is the sister taxon to M. lini with large genetic differences (p-distances 3.7-4.4%), representing a new species and described as, Megophrys (Panophrys) nanlingensis sp. nov., below. (2) head width slightly larger than head length, HDW/HDL ratio 1.04-1.09; (3) snout pointed in dorsal view; (4) tympanum distinct, moderate-sized, TD/ ED ratio 0.42-0.60; (5) strong vomerine ridge bearing vomerine teeth; (6) margin of tongue not notched behind; (7) hindlimbs short, heels not meeting, tibio-tarsal articulation reaching the region between tympanum and eye; (8) presence of subarticular tubercles and absence of lateral fringes on fingers, relative finger lengths II < I ≤ IV < III; (9) toes with rudiment of webbing at their bases and without lateral fringes, subarticular tubercles only present at the base of each toe; (10) numerous granules present on dorsal surface of body, several large tubercles present on surface of flanks; (11) presence of a barely visible reddish horn-like tubercle at the edge of the upper eyelid; (12) supratympanic fold distinct, whitish; (13) yellowish brown dorsally, with an incomplete dark triangular marking between eyes and usually an X-shaped marking on back of trunk; (14) ventral surface black brown, with white spots on posterior surface of abandon; (15) males with a single subgular vocal sac; (16) presence of nuptial pads with darker nuptial spines on dorsal surface of the first and second fingers in adult males during breeding season, respectively.

Megophrys
Comparisons. Comparative data of Megophrys dongguanensis sp. nov. with M. feii and the 33 recognized members of Megophrys s.l. allocated to the subgenus Panophrys are listed in Table 5.
Description of holotype. Adult male. Body moderate-sized, SVL 38.0 mm; head width slightly larger than head length, HWD/HDL 1.09; snout pointed in dorsal view, projecting, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eye large, ED/HDL 0.40, pupil vertical; nostril oblique ovoid; canthus rostralis well developed, forming the beginning of a fleshy, protruding ridge, that continues over the upper eyelid, and transitions into a supratympanic fold that terminates in the scapular region; loreal region slightly oblique; internasal distance slightly larger than interorbital distance; tympanum distinct, moderate-sized, TD/ED 0.54; large ovoid choanae at the base of the maxilla; presence of vomerine ridge bearing vomerine teeth; margin of tongue not notched posteriorly; internal vocal slits present near the rear of the lower mandible.
Radioulna length and hand length 0.24 of SVL; fingers without webbing and lateral fringes, relative finger length II < I < IV < III; tips of fingers slightly dilated, round; presence of subarticular tubercles on finger III, and one subarticular tubercle at the bases of each finger; outer metacarpal tubercles indistinct, inner metacarpal tubercles distinct and observably enlarged. Hindlimbs short, tibio-tarsal articulation reaching the region between tympanum and eye when hindlimb is stretched along the side of the body; heels not meeting when the flexed hindlimbs are held at right angles to the body axis; tibia length 0.41 of SVL and foot length 0.61 of SVL; relative toe length I < II < V < III < IV; tips of toes round and slightly dilated; presence of rudimentary webbing on toes but absence of lateral fringes and tarsal folds; one subarticular tubercle at the bases of each toe; presence of a long ovoid inner metatarsal tubercle and absence of outer metatarsal tubercle.
Dorsal skin texture rough with dense granules; granules forming discontinuous X-shaped ridge with two discontinuous dorsolateral ridges on both sides at the central trunk; several large tubercles present on dorsal surface of flanks, thighs, shanks and forearms; four small tubercles present on the edge of upper eyelid, one of which is more prominent; distinct narrow supratympanic fold curving posteroventrally from posterior corner of eye to a level above insertion of arm; ventral skin texture smooth, several granules present on surface of abandon, ventral and posterior surface of thighs; pectoral gland small, closer to axilla; single femoral gland on rear of thigh.
Coloration of holotype in life. (Fig. 3A-E) Yellowish brown dorsally, with a dark triangular marking between eyes. A wide oblique black band present on forearm. Dorsal surface of fingers and hindlimbs with dark grey transverse bands. Point of snout dark brown, presence of a vertical dark brown band below the eye. Tubercles on the edge of upper eyelid reddish. Supratympanic fold whitish tan. Ventral surface dark brown, with a black longitudinal band on surface of throat, several white spots present on ventral surface of limbs. Digits, inner and outer metacarpal tubercles greyish white, inner metatarsal tubercle greyish brown. Pectoral glands and femoral glands white. Iris yellowish brown.
Coloration of holotype in preservative. Yellowish brown fades to greyish brown dorsally. Triangular marking between eyes, oblique bands on dorsal forearms, transverse bands on dorsal fingers and hindlimbs become indistinct. Color of ventral surface fades, all bands and spots become indistinct.
Variation. Measurements of type series are listed in Table 4. All paratypes are very similar to holotype in morphology and color pattern. However, one male (SYS a001492) has more distinct skin ridges, granules and tubercles on dorsal surface of body (Fig. 3, F).
Etymology. The specific epithet "dongguanensis" is in reference to the type locality, Dongguan City of the new species. We propose the common English name "Dongguan Horned Toad" and Chinese name "Dong Guan Jiao Chan (东莞角蟾)".
Distribution and natural history. Currently, Megophrys dongguanensis sp. nov. is only known from Mt. Yinping, Guangdong Province, China. It inhabits flowing montane streams and the nearby forest floor and leaf litter at elevations between 100-300 m. Advertisement calls of males were noticed from mid-December until April of the next year just before the rainy season. Males were found calling on rocks in the flowing streams. Tadpoles could be found in this period.  Table 4 Holotype. SYS a004498, adult male, collected by Jian Wang and Hai-Long He on 20 October 2015 from Mt. Nankun (23°38'19"N, 113°53'24"E; 400 m a.s.l.), Longmen County, Huizhou City, Guangdong Province, China. Diagnosis.
(1) Body size small, SVL 29.9-34.9 mm in 11 adult males, 39.4-41.9 mm in two adult females; (2) head width slightly larger than head length, HDW/ HDL ratio 1.00-1.20; (3) snout rounded in dorsal view, tip of snout slightly sharpened; (4) tympanum distinct, moderate-sized, TD/ED ratio 0.43-0.61; (5) strong vomerine ridge bearing vomerine teeth; (6) margin of tongue not notched behind; (7) shanks short, heels not meeting when the flexed hindlimbs are held at right angles to the body axis; tibia-tarsal articulation reaching forward to the region between tympanum and eye when hindlimb is stretched along the side of the body; (8) TIB/SVL ratio 0.35-0.42, FTL/SVL ratio 0.53-0.62; (9) absence of lateral fringes on fingers, presence of an indistinct subarticular tubercle on the bases of each finger, relative finger lengths II < I < IV < III; (10) toes with rudimentary webbing at their bases and without lateral fringes, subarticular tubercles only present on the bases of each toes; (11) dorsal surface with dense granules, surface of flanks and dorsal surface of limbs with large tubercles; (12) edge of eye lid with a small reddish horn-like tubercle; (13) supratympanic fold distinct, forming a depressed supraaxillary gland above insertion of arm; (14) dorsum beige to dark brown, with indistinct light brown patches, with an incomplete dark triangular marking between eyes; (15) males with a single subgular vocal sac, and dense dark villiform nuptial spines present on dorsal surface of first and second fingers during breeding season, respectively; (16) gravid females bear creamy yellow oocytes.
Comparisons. Comparative data of Megophrys nankunensis sp. nov. with M. dongduanensis sp. nov., M. feii and the 33 recognized members of Megophrys s.l. allocated to the subgenus Panophrys are listed in Table 5.
In the ML and BI phylogenetic trees (Fig. 2), Megophrys nankunensis sp. nov. is a sister taxon to M. dongguanensis sp. nov. (p=4.6-5.0%) with high node-supporting value (0.1 in BI, 100% in ML%), and differs from the later by the snout rounded in dorsal view, tip of snout slightly sharpened (vs. snout pointed in dorsal view, tip of snout not sharpened), supratympanic fold forming a depressed supraaxillary gland above insertion of arm (vs. supraaxillary gland absent).
With Megophrys nankunensis sp. nov. further differs from M. feii, for which molecular data are lacking and cannot be allocated to any subgenus base on morphology only ) by the larger body size, SVL 29.9-34.9 mm in males and 39.4-41.9 mm in females (VS. 24.3-25.1 mm in males, 28.2-28.9 mm in females), presence of nuptial pad with nuptial spines in males during breeding season (vs. absent), presence of vomerine teeth (vs. absent), absence of lateral fringes on toes (vs. moderate or wide), heels not meeting when the flexed hindlimbs are held at right angles to the body axis (vs. heels overlapping).
Description of holotype. Adult male. Habitus small, SVL 31.3 mm; head width slightly larger than head length, HDW/HDL 1.12; snout rounded in dorsal view, tip of snout slightly sharpened, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eye large, ED/HDL 0.38; nostril oblique ovoid; pupil vertical; canthus rostralis well developed, forming the beginning of a fleshy, protruding ridge, that continues over the upper eyelid, and transitions into a supratympanic fold that terminates in the scapular region; loreal region vertical; internasal distance slightly larger than interorbital distance; tympanum distinct, moderatesized, TD/ED 0.44; large ovoid choanae at the base of the maxilla; strong vomerine ridge bearing vomerine teeth; margin of tongue weakly notched posteriorly; internal vocal slits present near the rear of the lower mandible.
RAD/SVL 0.22, HND/SVL 0.22; absence of lateral fringes and webbing on fingers, relative finger lengths II < I < IV < III; tip of finger rounded, slightly swollen; presence of a distinct subarticular tubercle on the base of each finger; outer metacarpal tubercles indistinct, inner metacarpal tubercles distinct and observably enlarged. Hindlimbs short, tibio-tarsal articulation reaching forward the anterior margin of tympanum when hindlimb is stretched along the side of the body; heels not meeting when the flexed hindlimbs are held at right angles to the body axis; TIB/ SVL 0.37 and FTL/SVL 0.55; relative toe lengths I < II < V < III < IV; tips of toes round and slightly dilated; presence of rudimentary webbing on toes but absence of lateral fringes and tarsal folds; presence of a subarticular tubercle only at the bases of each toes; presence of a long ovoid inner metatarsal tubercle and absence of outer metatarsal tubercle.
Dorsal skin texture smooth with dense granules, some of which forming a weak X-shaped skin ridge on center of trunk; surface of flanks with large tubercles; presence of a small horn-like tubercle at the edge of eyelid; distinct supratympanic fold curving posteroventrally from posterior corner of eye to a level above insertion of arm, forming a swollen supraaxillary gland above insertion of arm; ventral skin texture smooth with granules on the surface of abdomen; pectoral gland large, equal size to tip of fingers, closer to axilla; single large femoral gland on rear of thigh.
Coloration of holotype in life. (Fig. 4A-D) Dorsal surface beige with obscure darker patches, with a distinct and incomplete dark triangular marking between eyes, unconnected with an incomplete X-shaped marking on center of trunk. Forearm with dark bands dorsally; hindlimb with broad black transverse bands. Tip of snout dark brown. A dark brown vertical band below the eye. Supratympanic fold white. Hornlike tubercle at the edge of the upper eyelid orange. Surface of throat and chest dark brown, with scarlet spots. Posterior region of abdomen white, with dark brown and scarlet spots. Ventral surface of limbs white with brown patches. Ventral surface of hand and foot light brown, subarticular tubercle at the base of each fingers and toes, outer metacarpal tubercle, inner metatarsal tubercle and inner metacarpal tubercle pink. Pectoral and femoral glands white. Iris white.
Coloration of holotype in preservative. On dorsal surface the beige fades to dark grey. Dark interorbital triangular marking becomes more indistinct. Ventral surface pale in color, grey-brownish grounding, markings and mottling more distinct, all scarlet spots absent.
Variation. Measurements and body proportions of type series are given in Table 4. All paratype specimens were very similar in morphology and color pattern. However, the holotype has the dorsal surface beige (vs. reddish brown in paratypes SYS a002033, 4501, and dark brown in paratypes SYS a004502-4506, 4507 (Fig. 4E-F)), dorsal skin texture smooth, granules and tubercles weak (vs. dorsal skin texture relatively rough with more distinct granules and tubercles in paratypes SYS a004502, Etymology. The specific epithet "nankunensis" is in reference to the type locality of the new species: Mt. Nankun. We propose the common English name "Nankunshan Horned Toad" and Chinese name "Nan Kun Shan Jiao Chan (南昆山角蟾)".
Distribution and habits. Currently, Megophrys nankunensis sp. nov. is known only from the type locality, Mt. Nankun in Longmen County, Guangdong Province, China. It inhabits forest floor, leaf litter and the nearby undergrowth rocky mountainous streams (2-3 m wide) surrounded by moist subtropical evergreen broadleaved forests at elevations between 300-600 m. Breeding season of M. nankunensis sp. nov. is from October to the following March, males were found calling under the leaf litter or rocks (Fig. 5A) on the ground in the flowing streams, besides, a pair were observed exposed on the floor in a flowing stream, about 2.5 m wide, prior to amplexus (Fig. 5B)  Diagnosis.
(1) Body slender and small-sized, SVL 30.4-33.9 mm in nine adult males, 34.1-37.5 mm in two adult females; (2) head width slightly larger than head length, HDW/HDL ratio 1.04-1.06; (3) snout rounded in dorsal view; (4) eye large, tympanum distinct, moderate-sized, TD/ED ratio 0.50-0.59; (5) weak vomerine ridge bearing vomerine teeth; (6) tongue weakly notched posteriorly; (7) hindlimbs slender, heels overlapping when the flexed hindlimbs are held at right angles to the body axis, tibia-tarsal articulation reaching forward to the middle of eye when hindlimb is stretched along the side of the body; (8) absence of lateral fringes on fingers, presence of an indistinct subarticular tubercle on the bases of each finger, relative finger lengths II < I < IV < III; (9) toes with rudimentary webbing at their bases and without lateral fringes, subarticular tubercles only present at the base of toe I and II; (10) dorsal skin rough, presence of black spines on granules of dorsal skin, and occasionally present on canthus rostralis and margin of tympanum, presence of large tubercles on flanks, dorsal body and limbs; (11) four prominent parallel dorsolateral ridges with granules bearing black spines on back of trunk, the middle two ridges forming a X-shaped ridge occasionally; (12) a reddish horn-like tubercle bearing a black spine at its tip at the edge of eye lid; (13) distinct supratympanic fold bearing black spines; (14) beige to brownish red above, with an hollow dark triangle between eyes and a rectangular dark marking on the center of the back of trunk; (15) males with a single subgular vocal sac, and presence of nuptial pads bear-ing darker nuptial spines on dorsal surface of the first and second fingers in adult males during breeding season, respectively; (16) gravid females bear creamy yellow oocytes.
Comparisons. Comparative data of Megophrys jiulianensis sp. nov. with M. dongduanensis sp. nov., M. nankunensis sp. nov., M. feii and the 33 recognized members of the Megophrys s.l. allocated to the subgenus Panophrys are listed in Table 5.
Megophrys jiulianensis sp. nov. is sympatric with M. nankunensis sp. nov. in Mt. Nankun, but it can be easily distinguished from the later by heels overlapping when the flexed hindlimbs are held at right angles to the body axis (vs. heels not meeting), TIB/SVL ratio 0.61-0.68 (vs. TIB/SVL ratio 0.35-0.42), supratympanic fold not forming a supraaxillary gland above insertion of arm (vs. supratympanic fold forming a depressed supraaxil- Megophrys jiulianensis sp. nov. further differs from M. feii, for which molecular data are lacking and cannot be allocated to any subgenus base on morphology only ) by the larger body size, SVL 30.4-33.9 mm in males and 34.1-37.5 mm in females (VS. 24.3-25.1 mm in males, 28.2-28.9 mm in females), presence of nuptial pad with nuptial spines in males during breeding season (vs. absent), presence of vomerine teeth (vs. absent), absence of lateral fringes on toes (vs. moderate or wide).
Description of holotype. Adult male. Habitus slender and small, SVL 32.0 mm; head width slightly larger than head length, HDW/HWL 1.04; snout rounded in dorsal view, projecting, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eye large, ED/HDL 0.39; nostril oblique ovoid; pupil vertical; canthus rostralis well developed, forming the beginning of a fleshy, protruding ridge, that continues over the upper eyelid, and transitions into a supratympanic fold that terminates in the scapular region; loreal region vertical; internasal distance slightly larger than interorbital distance; tympanum distinct, moderate-sized, TD/ED 0.52; large ovoid choanae at the base of the maxilla; weak vomerine ridge bearing vomerine teeth; margin of tongue weakly notched posteriorly; internal vocal slits present near the rear of the lower mandible.. RAD/SVL 0.25; absence of lateral fringes and webbing on fingers, relative finger lengths II < I < IV < III; tip of finger rounded, slightly swollen; presence of an indistinct subarticular tubercle on the base of each finger; outer metacarpal tubercles indistinct, inner metacarpal tubercles distinct and observably enlarged. Hindlimbs long, tibio-tarsal articulation reaching forward to the middle of eye when hindlimb is stretched along the side of the body; heels overlapping when the flexed hindlimbs are held at right angles to the body axis; TIB/SVL 0.46 and FTL/SVL 0.62; relative toe lengths I < II < V < III < IV; tips of toes round and slightly dilated; presence of rudimentary webbing on toes but absence of lateral fringes and tarsal folds; presence of a subarticular tubercle only at the bases of the first and second toes; presence of a long ovoid inner metatarsal tubercle and absence of outer metatarsal tubercle.
Dorsum rough with dense granules bearing spines; canthus rostralis, margin of tympanum, supratympanic fold and upper lip with dense spines; presence of large tubercles bearing spines on dorsal surface of body, surface of flanks and dorsal and posterolateral surface of limbs; prominent parallel dorsolateral ridges with granules bearing spines on back of trunk; presence of a horn-like tubercle bearing a spine at its tip at the edge of eye lid; distinct supratympanic fold curving posteroventrally from posterior corner of eye to a level above insertion of arm; ventral skin texture smooth, the lower lip bears spines; sides of belly with large tubercles; ventral skin texture of thighs smooth with a few small tubercles, posterior surface and surface around anus with large tubercles bearing spines; surface of tibia-tarsal with a few tubercles bearing spines; presence of spines on lateral sides of fingers and toes; pectoral gland moderate-sized, closer to axilla; single femoral gland on rear of thigh, distinctly smaller than pectoral gland.
Measurements of holotype (in mm). SVL 32.2, HDL 11.5, HDW 11.4, SNT 3.6, IND 3.5, IOD 3.3, ED 4.2, TD 2.3, TED 1.7, HND 8.0, RAD 8.1, FTL 20.5, TIB 14.7. Coloration of holotype in life. (Fig. 6A-D) Dorsal surface yellowish brown, with an incomplete dark triangular marking between eyes. Spines on dorsal surface, granules and tubercles black. Forearm with a distinct, black oblique band. Transverse bands on hindlimb indistinct. Tip of snout grayish brown. A grayish-brown vertical band below the eye. Tubercle at the edge of the upper eyelid red. Ventral surface yellow, scattered with dense dark gray spots and black scarlet blotches; ventral surface of limbs flesh colored with pink and black spots. Palms and soles dark brown, inner metatarsal tubercle, outer metacarpal tubercle and inner metacarpal tubercle orange red, tip of digits orange-red. Pectoral glands and femoral glands white. Iris white.
Coloration of holotype in preservative. Dorsum yellowish brown fades to greyish brown, scattered with black spots. Greyish black triangular marking between the eyes become more distinct. Ventral surface paled in color, brown grounding, markings and mottling become more distinct.
Variation. Measurements and body proportions of type series are given in Table 6.  All paratype specimens were very similar in morphology and color pattern. However, dorsal skin texture is more rough with well-developed spines in the female specimen SYS a002111 (Fig. 6E-F), dorsal surface yellowish brown in the other female specimen SYS a002110, and the middle two ridges on dorsum forming an X-shape skin ridge in the male specimen SYS a002108.
Etymology. The specific epithet "jiulianensis" is in reference to the known localities of the new species: Mt. Jiulian and Nankunshan Natuire Reserve located in the Jiulian Mountains range. We propose the common English name "Jiulianshan Horned Toad" and Chinese name "Jiu Lian Shan Jiao Chan (九连山角蟾)".
Distribution and natural history. Currently, Megophrys jiulianensis sp. nov. is known from Mt. Nankun in Guangdong Province and the type locality, Jiulian Nature Reserve in Jiangxi Province, China. It inhabits forest floor, leaf litter and the nearby undergrowth mountainous streams surrounded by moist subtropical evergreen broadleaved forests at elevations between 500-800 m. Breeding season of M. jiulianensis sp. nov. is from March to July, males were usually found staying while calling on leaves (Fig. 7A), about 0.1-0.3 m above the ground. After the rain, numerous individuals can be easily found on the road, and a female individual from Mt. Nankun was observed feeding on an earthworm (Fig. 7B)  (2) snout rounded in dorsal view; (3) tympanum distinct, moderate-sized, TD/ED ratio 0.43-0.57; (4) vomerine ridge and vomerine teeth present; (5) tongue notched posteriorly; (6) absence of lateral fringes and webbing on fingers, presence of narrow lateral fringes and rudimentary webbing on toes; (7) presence of a subarticular tubercle at the base of each finger and toe; (8) hindlimbs slender, heels overlapping, tibio-tarsal articulation reaching between the posterior corner to the center of eye; (9) TIB/SVL ratio 0.45-0.51 and FTL/SVL ratio 0.61-0.73; (10) dense conical granules present on surface of temporal region, upper lip, and from loreal region to the tip of snout; (11) granules and tubercles on dorsal surface forming a discontinuous X-shaped ridge and a pair of discontinuous dorsolateral ridges on back of trunk; (12) supratympanic fold distinct, whitish tan; (13) brown dorsally, with a dark triangular marking with light yellow edge between eyes, and an X-shaped or V-shaped marking with light yellow edge on the center of the back of trunk; (14) presence of a single subgular vocal sac in males; (15) nuptial pads and nuptial spines invisible in males during breeding season.
Megophrys Megophrys nanlingensis sp. nov. further differs from M. feii, for which molecular data are lacking and cannot be allocated to any subgenus based on morphology only ) by the larger body size, SVL 30.5-37.3 mm in males (VS. 24.3-25.1 mm in males), presence of nuptial pad with nuptial spines in males during breeding season (vs. absent), presence of vomerine teeth (vs. absent), presence of narrow lateral fringes on toes (vs. moderate or wide).
Description of holotype. Adult male. Body size small, SVL 32.5 mm; head length and head width almost isometric, HDW/HDL 0.99; snout rounded in dorsal view, projecting, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eye large, ED/HDL 0.37, pupil vertical; nostril oblique ovoid; canthus rostralis well developed; loreal region slightly oblique; internasal distance slightly larger than interorbital distance; tympanum distinct, moderate-sized, TD/ED 0.48; large ovoid choanae at the base of the maxilla; presence of vomerine ridge bearing vomerine teeth; margin of tongue notched posteriorly; internal vocal slits present near the rear of the lower mandible.
RAD/SVL 0.25, HND/SVL 0.24; fingers without webbing and lateral fringes, relative finger length II < I < IV < III; tips of fingers slightly dilated, round; one subarticular tubercle at the bases of each finger; outer and inner metacarpal tubercles distinct, and the inner one observably enlarged. Hindlimbs slender, tibio-tarsal articulation reaching forward to the center of the eye when hindlimb is stretched along the side of the body; heels overlapping when the flexed hindlimbs are held at right angles to the body axis; TIB/SVL 0.49 and FTL/SVL 0.69; relative toe length I < II < V < III < IV; tips of toes round and slightly dilated; toes with narrow lateral fringes, rudimentary webbing; one subarticular tubercle at the bases of each toes; presence of a long ovoid inner metatarsal tubercle and absence of outer metatarsal tubercle. Dorsal skin texture rough; head surface rough, with small tapered granules densely covering from temporal region, upper lip, loreal region to tip of snout; granules forming discontinuous X-shaped ridge with two discontinuous dorsolateral ridges on both sides at the central trunk; large tubercles on flanks; a horn-like prominent tubercle on the edge of the upper eyelid; distinct supratympanic fold curving posteroventrally from posterior corner of eye to a level above insertion of arm; ventral skin texture smooth, with several large granules and tubercles on two sides; ventral skin texture of thighs smooth, with a few small tubercles; pectoral gland larger, closer to axilla; single femoral gland on rear of thigh.
Coloration of holotype in life. (Fig. 8A-D) Brown dorsally, with a dark triangular marking with light yellow edge between eyes, and an X-shaped marking with light yellow edge on the center of the back of trunk. Dark brown transverse bands dorsally on lower arms and hindlimbs. Surface of snout brown. Black brown vertical band below the eye on each side. Temporal region brown, supratympanic fold white. Ventral surface pale grey, an indistinct longitudinal stripe on surface of throat. Scarlet spots on surface of chest. Belly whitish grey with dark brown marbling. A pair of black longitudinal stripes scattered with several white tubercles on surface of lateroventral flanks. Ventral surface of limbs light red and scattered with white spots. Ventral surface of hands and feet dark brown, tips of digits pale-grey. Metacarpal tubercle and metatarsal tubercle light red. Pectoral glands and femoral glands white. Iris reddish brown.
Coloration of holotype in preservative. Coloration of dorsal and ventral surface turned pale; transverse bands on limbs, dark longitudinal stripe on surface of throat and black patches on surface of lateroventral flanks became more distinct; scarlet spots on surface of chest faded.
Variation. Measurement data of type series are listed in Table 7. All paratypes are very similar to holotype in morphology and color pattern. However, the male specimen SYS a001963 (Fig. 8E, F) is obviously large in snout-vent  Table 8 Holotype. SYS a002625, adult male, collected by Guo-Ling Chen and Jian Zhao on 9 May 2014 from Yangshimu Scenic Area (27°34'47.93"N, 114°15'7.34  Megophrys wugongensis sp. nov. further differs from M. feii, for which molecular data are lacking and cannot be allocated to any subgenus base on morphology only ) by the larger body size, SVL 31.0-34.1 mm in males and 38.5-42.8 mm in females (VS. 24.3-25.1 mm in males, 28.2-28.9 mm in females), presence of nuptial pad with nuptial spines in males during breeding season (vs. absent), absence of lateral fringes on toes (vs. moderate or wide).
Description of holotype. Adult male. Habitus small, SVL 31.0 mm; head width slightly larger than head length, HDW/HWL 1.03; snout rounded in dorsal view, tip of snout slightly sharpened, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eye large, ED/HDL 0.41; nostril oblique ovoid; pupil vertical; canthus rostralis well developed; loreal region vertical; internasal distance slightly larger than interorbital distance; tympanum distinct, moderate-sized, TD/ED 0.47; large ovoid choanae at the base of the maxilla; weak vomerine ridge present, vomerine teeth absent; margin of tongue not notched posteriorly; internal vocal slits present near the rear of the lower mandible.
RAD/SVL 0.24, HND/SVL 0.22; absence of lateral fringes and webbing on fingers, relative finger lengths II < I = IV < III; tip of finger rounded, slightly swollen; presence of a distinct subarticular tubercle on the base of each finger; outer metacarpal tubercles indistinct, inner metacarpal tubercles distinct and observably enlarged. Hindlimbs short, tibio-tarsal articulation reaching forward the posterior corner of eye when hindlimb is stretched along the side of the body; heels not meeting when the flexed hindlimbs are held at right angles to the body axis; TIB/SVL 0.43 and FTL/SVL 0.61; relative toe lengths I < II < V < III < IV; tips of toes round and slightly dilated; presence of rudimentary webbing on toes but absence of lateral fringes and tarsal folds; presence of a subarticular tubercle only at the bases of each toes; presence of a long ovoid inner metatarsal tubercle and absence of outer metatarsal tubercle.
Dorsal skin texture rough with dense granules, some of which forming an Xshaped skin ridge on center of trunk; surface of flanks with large tubercles; presence of a small horn-like tubercle at the edge of eye lid; distinct supratympanic fold curving posteroventrally from posterior corner of eye to a level above insertion of arm; superior margin of tympanum in connect with supratympanic fold; ventral skin texture smooth with granules on the surface of abdomen; pectoral gland large, closer to axilla; single large femoral gland on rear of thigh.
Measurements of holotype (in mm). SVL 30.8, HDL 11.9, HDW 11.7, SNT 3.5, IND 3.0, IOD 2.8, ED 3.5, TD 1.8, TED 1.7, HND 8.5, RAD 7.2, FTL 21.8, TIB 15.1 Coloration of holotype in life. (Fig. 9A-C) Dorsal surface reddish brown, with a distinct and dark triangular marking with yellow edges between eyes. Hindlimb with broad black transverse bands. A dark brown vertical band below the eye. Canthus rostralis and supratympanic fold white. Horn-like tubercle at the edge of the upper eyelid yellow. Surface of throat and chest dark brown, with scarlet marbling, posterior region of abdomen white. Ventral surface of limbs brown with white spots and patches. Ventral surface of hand and foot brown, inner and outer metatarsal tubercles and inner metacarpal tubercle pink. Pectoral and femoral glands white. Iris reddish brown.
Coloration of holotype in preservative. Dorsum dark brown, markings on dorsal surface became indistinct, transverse bands on limbs became dark grey and became more 1050-1080 m a.s.l., all located in the Luoxiao Mountains in eastern China. All specimens were collected on leaf litter near a stream in the bamboo forest, males were not heard calling. In consideration of the invisible nuptial pad and nuptial spines in all male specimens and the undeveloped fallopian tubes in all female specimens, the breeding season of M. wugongensis sp. nov. still remains unknown. Tadpoles were not observed. Megophrys wugongensis sp. nov. is sympatric with M. jinggangensis in all localities.  Table 9 Holotype. SYS a006391, adult male, collected by Zhi-Tong Lyu on 3 August 2017 from Mt. Mufu (28°58'18.45"N, 113°48'58.53"E; 1300 m a.s.l.), Pingjiang County, Yueyang City, Hunan Province, China.

Megophrys
Paratypes (one male & two females). Adult females, SYS a006390/CIB110012, SYS a006419, and the other adult male, SYS a006392, all collected by Zhi-Ting Lyu on 3 August 2017 from the same locality as the holotype. Diagnosis.
(1) Body size small, SVL 30.1-30.8 mm in two adult males and SVL 36.3 mm in two adult females; (2) head length slightly larger than head width, HDW/ HDL ratio 0.98-0.99; (3) tympanum distinct, moderate-sized, TD/ED ratio 0.51-0.58, upper 1/4 part of the tympanum concealed by supratympanic fold; (4) vomerine teeth absent; (5) margin of tongue not notched posteriorly; (6) heels overlapping, tibia-tarsal articulation reach forward to the tympanum in males and to the eye in females; (7) TIB/SVL ratio 0.47-0.53, FTL/SVL ratio 0.68-0.74; (8) fingers without lateral fringes, presence of a subarticular tubercle at the bases of each finger, relative finger lengths II = IV < I < III; (9) toes with rudimentary webbing at their bases and narrow lateral fringes, subarticular tubercles only present at the base of each toe; (10) numerous granules scattered with tubercles present on dorsal surface of body, limbs and surface of flanks, some of which forming a V-shaped, \ /-shaped or X-shaped skin ridge on central back of trunk; (11) presence of a small horn-like tubercle at the edge of the upper eyelid; (12) supratympanic fold distinct; (13) light brown to dark brown dorsally, with a dark triangular marking between eyes; (14) a pair of dark longitudinal and irregular marking with white edges on its upper side on ventrolateral surface of flanks; (15) surface of throat and chest greyish brown with dark brown patches and creamy white spots, surface of abdomen greyish white with creamy white and orange spots; (16) ventral surface of thighs with dense small whitish tubercles.
Comparisons  rostralis well developed; loreal region vertical; internasal distance slightly larger than interorbital distance; tympanum distinct, moderate-sized, TD/ED 0.56; large ovoid choanae at the base of the maxilla; weak vomerine ridge present, vomerine teeth absent; margin of tongue not notched posteriorly.
RAD/SVL 0.25, HND/SVL 0.30; absence of lateral fringes and webbing on fingers, relative finger lengths II = IV < I < III; tip of finger rounded, slightly swollen; presence of a distinct subarticular tubercle on the base of each finger; outer metacarpal tubercles indistinct, inner metacarpal tubercles distinct and observably enlarged. Hindlimbs long, tibio-tarsal articulation reaching forward to the tympanum when hindlimb is stretched along the side of the body; heels overlapping when the flexed hindlimbs are held at right angles to the body axis; TIB/SVL 0.53 and FTL/SVL 0.74; relative toe lengths I < II < V < III < IV; tips of toes round and slightly dilated; presence of rudimentary webbing and narrow lateral fringes on toes but absence of tarsal folds; presence of a subarticular tubercle only at the bases of each toes; presence of a long ovoid inner metatarsal tubercle and absence of outer metatarsal tubercle. Table 9. Measurements (in mm; minimum-maximum, mean ± SD) of the type series of Megophrys mufumontana sp. nov.
Coloration of holotype in life. (Fig. 10A-D) Dorsal surface brown, with a distinct and incomplete dark triangular marking between eyes. Hindlimb with black transverse bands. A dark brown vertical band below the eye. Horn-like tubercle at the edge of the upper eyelid red. Surface of throat and chest greyish brown with dark brown patches. Surface of abdomen greyish white with creamy white and orange spots. Ventral surface of limbs pink with white spots and light-yellow patches. Ventral surface of hand and foot brown, inner and outer metatarsal tubercles and inner metacarpal tubercle pink. Pectoral and femoral glands white. Iris white.
Coloration of holotype in preservative. Coloration of dorsum dark brown, markings on dorsal surface and transverse bands on limbs became indistinct. Ventral surface of throat, chest and abdomen dark grey. All patches on ventral surface indistinct, all colored spots absent. Ventral surface of limbs light yellow.
Variation. Measurement data of type series are listed in Table 9. All paratypes are very similar to holotype SYS a006391 in morphology and color pattern. However, tibia-tarsal articulation reaching forward to the eye when hindlimb is stretched along the side of the body in all females, and granules and tubercles forming a \ /-shaped skin ridge on central back of trunk in the holotype (vs. X-shaped in SYS a006390, 6419; V-shaped in SYS a006392 (Fig. 10E-F)).
Etymology. The specific epithet "mufumontana" is in reference to the type locality of the new species, Mt. Mufu. We propose the common English name "Mufushan Horned Toad" and Chinese name "Mu Fu Shan Jiao Chan (幕阜山角蟾)".
Distribution and habits. Currently, Megophrys mufumontana sp. nov. is known only from Mt. Mufu, Pingjiang County, Yueyang City, Hunan Province, China at approximate 1300 m a.s.l.. All specimens were collected on leaf litter near a stream (about 5 m wide) surrounded by moist subtropical evergreen broadleaved forests, males were not heard calling. Tadpoles were not observed. Because none of the males have nuptial pads developed and none of the females have fallopian tubes and eggs developed, the breeding season of M. mufumontana sp. nov. remains unknown.

Discussion
Megophrys dongguanensis sp. nov. is easily confused with M. brachykolos because of the relatively short shanks. In addition, the type locality of the new species is at a straightline distance of approximately 72 km from the type locality (Hongkong Island), and at a straight-line distance of approximately 32 km from the closest locality (Sanzhoutian of Shenzhen City) of M. brachykolos. Currently, eight Megophrys species in the subgenus Panophrys were found to have comparatively short shanks with heels not meeting when thighs are adpressed at right angles with respect to the body axis: M. dongguanensis sp. nov., M. nankunensis sp. nov., M. wugongensis sp. nov., M. acuta, M. brachykolos, M. insularis, M. megacephala and M. obesa. In our previous study , 41 cryptic species within the subgenus Panophrys were revealed, and one of them was recently described as Megophrys leishanensis by Li et al. (2018). Moreover, except for M. mufumontana sp. nov. (not mentioned in ), five of them are described in this study. Currently, the total number of recognized species of the subgenus Panophrys rises to 39, which makes it the most species-rich subgenus of Megophrys (≈46.4%). It's worth noting that there remain still 33 undescribed species according to Liu et al. (2018), and 27 of them are found in southeastern China, which further reveals the unusually high level of species diversity in this region.
As the diversity of the subgenus Panophrys was confirmed to be extremely underestimated (Chen et al. 2017;Mahony et al. 2017;Liu et al. 2018), a number of new Panophrys species have been described since 2017 (i.e. Megophrys lishuiensis, M. insularis, M. rubrimera, M. liboensis, and six new species in this study). However, all of these species have narrow distributions. For example, M. insularis is currently known only from an offshore island in Guangdong (Wang et al. 2017b), and M. liboensis is currently known only from a cave in Libo, Guizhou . For the six new species in this study, M. dongguanensis sp. nov., M. nankunensis sp. nov., M. wugongensis sp. nov. and M. mufumontana sp. nov. are currently only found in their type localities. This situation of "micro-endemism" ) has brought great challenges for the protection of these unique toads.
Among the six new species described in this paper, M. jiulianensis sp. nov. is sympatric with M. nankunensis sp. nov. in Mt. Nankun while also being sympatric with M. hongshanensis sp. nov. in Mt. Jiulian. Further, M. mufumontana sp. nov. is sympatric with a known congener M. jinggangensis in Mt. Mufu and M. wugongensis sp. nov. is sympatric with M. jinggangensis in Mt. Wugong. By combining the localities of these species in our phylogenetic trees (Fig. 2), our results also support the conclusion of "sympatric but distant phylogenetically" , that is, sympatric distribution is very common in horned toads within the subgenus Panophrys while they are distantly related in the phylogeny (Fei et al. 2012;Li et al. 2014;Wang et al. 2014;Liu et al. 2018). These geographical patterns of "sympatric but distant phylogenetically" and "micro-endemism" indicate that the Asian horned toads would be good candidates for studies on speciation and biogeography.