A new species of Leptobrachella (Anura, Megophryidae) from Guizhou Province, China

Abstract This study describes a new species of the genus Leptobrachella, Leptobrachella suiyangensissp. nov. from the Huoqiuba Nature Reserve, Suiyang County, Guizhou Province, China, based on morphological data and phylogenetic analyses (16S rRNA mtDNA). The new species can be distinguished from other congeners by the molecular divergence and by a combination of morphological characters, including body size, dorsal and ventral patterns, dorsal skin texture, size of the pectoral and femoral glands, degree of webbing and fringing on the toes and fingers, dorsum coloration, and iris coloration in life. Currently, the genus Leptobrachella contains 75 species, 21 of which are found in China, including seven species reported from Guizhou Province. The uncorrected sequence divergence percentage between Leptobrachella suiyangensissp. nov. and all homologous DNA sequences available for the 16S rRNA gene was found to be >4.7%. The new record of the species and its relationships with others in the same genus imply that species distribution, habitat variation, environmental adaptation, and diversity of the genus Leptobrachella in southwest China need to be further investigated.


Sampling
Eight specimens collected from the aforementioned area ( Fig. 1) were euthanized with chlorobutanol solution and fixed in 10% formalin for 24 h, and then stored in 75% ethanol. Liver and muscular tissues were taken before fixing and preserved in 95% alcohol at -20 °C. All of the specimens are kept at the College of Life Sciences, Guizhou Normal University (GZNU), Guiyang City, Guizhou Province, China.

DNA Extraction, PCR and sequencing
DNA samples were extracted from muscular tissues with a DNA extraction kit (Tiangen Biotech (Beijing) Co. Ltd). The mitochondrial gene and 16S ribosomal RNA gene (16S rRNA) were sequenced (951bp). The fragmented genes were amplified with primer pairs L3975 (5'-CGCCTGTTTACCAAAAACAT-3') and H4551 (5'-CCGGTCT-GAACTCAGATCACGT-3') for 16S rRNA (Simon et al. 1994). PCR amplifications were performed in a 20 μl reaction volume with the following cycling conditions: an initial denaturing step at 95 °C for five min; 35 cycles of denaturing at 95 °C for 40 s, annealing at 53 °C for 40 s and extending at 72 °C for 1 min, followed by a 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 a BigDye Terminator Cycle Sequencing Kit according to the guidelines of the manufacturer. The products were sequenced on an ABI Prism 3730 automated DNA sequencer at Shanghai Majorbio Bio-pharm Technology Co. Ltd. All sequences have been deposited in GenBank (Table  1). For molecular analyses, a total of 77 sequences (74 sequences downloaded from GenBank and three our new sequences) from 55 species of the genus Leptobrachella were used, including one undescribed species from C hina, that is, the populations from Huoqiuba Nature Reserve, Suiyang County, and Guizhou Province. Three species which sequences downloaded from GenBank are used as outgroups (Leptobrachium huashen Fei & Ye, 2005, Leptobrachium cf. chapaense (Bourret, 1937) and Megophrys major Boulenger, 1908Wang et al. 2019; Table 1).

Phylogenetic analyses
All sequences were aligned by MUSCLE v. 3.6 with the default settings (Edgar 2004). Trimming with the gaps partially deleted was performed in MEGA 7.0 (Kumar et al. 2016), while within high variable regions, all gaps were removed. Phylogenetic trees were constructed with both Maximum Likelihood (ML) and Bayesian Inference (BI). The ML was conducted in IQ-TREE  with 2000 ultrafast bootstrapping (Hoang et al. 2018) and was performed until a correlation coefficient of at least 0.99 was reached. The BI was performed in MrBayes v. 3.2.1 (Ronquist et al. 2012), and the best-fit model was obtained by the Akaike Information Criterion (AIC) computed with PartitionFinder 2 (Lanfear et al. 2016), resulting in the best-fitting nucleotide substitution models of GTR + I + G with for BI and ML analysis. Two independent processes were conducted for 10 million generations, sampling every 1000, with four independent chains and a burn-in of 25%. Convergence was assessed referring to the criteria of all parameters having reached stationarity and having obtained satisfactory effective sample sizes (>200) using Tracer v. 1.6. (Rambaut et al. 2014). Nodes in the trees were considered well supported when Bayesian posterior probabilities (BPP) were ≥0.95 and ML ultrafast bootstrap values (UFB) was ≥95 % Hoang et al. 2018). Uncorrected p-distances based on 16S rRNA were calculated in MEGA v. 7.0 (Kumar et al. 2016).

Morphological and morphometric analyses
Morphometric data were taken from eight of most well-preserved adult specimens. Measurements were recorded to the nearest 0.1 mm (Watters et al. 2016) with digital calipers following the methods of Fei et al. (2009) and Rowley et al. (2013). 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 the anterior corner of the eye) EYE eye diameter (diameter of the exposed portion of the eyeballs) IOD interorbital distance (minimum distance between upper eyelids) IND internasal distance (distance between nares) UEW upper eyelid width (measured as the greatest width of the upper eyelid) NEL nostril-eyelid length (distance from nostril to eyelid) NSL nostril-snout length (distance from nostril to snout) TMP tympanum diameter (horizontal diameter of tympanum) TEY tympanum-eye distance (distance from anterior edge of tympanum to posterior corner of eye) TIB tibia length (distance from knee to heel) ML manus length (distance from tip of third digit to proximal edge of inner palmar tubercle) LAHL length of the lower arm and hand (distance from tip of the third finger to elbow) HLL hindlimb length (distance from tip of fourth toe to vent) FOT foot length (from proximal edge of the inner metatarsal tubercle to the tip of the fourth toe) Sex was determined by direct observation of calls in life, the presence of internal vocal sac openings, and the presence of eggs in the abdomen through external inspection. Comparative morphological data of Leptobrachella species were obtained from the references listed in Table 2. Due to the high likelihood of undiagnosed diversity within the genus Yang et al. 2016), where available, we relied on examination of topotypic material and/or original species descriptions.

Results
Phylogenetic trees from Maximum likelihood (ML) and Bayesian inference (BI) were constructed based on DNA sequences of the mitochondrial 16S rRNA gene with a length of 500 bp. The trees present identical topologies (Fig. 2) with the clustered population of Leptobrachella from Huoqiuba Nature Reserve, in which L. alpina + L. purpurus and the population of Leptobrachella from Huoqiuba Nature Reserve show relatively high node supporting values (0.68 in BI and 71% in ML) and exhibit a separate evolving lineage. The smallest pairwise genetic divergence between the population from Suiyang County and all other species of the genus Leptobrachella is 4.71%. This indicates that there is substantial genetic divergence between the species in Leptobrachella and the specimens from Suiyang County, indicating that this new population can be regarded to be a separate lineage and is valid to be described as a new species as below.
Etymology. The specific epithet "suiyangensis" refers to the name of the holotype locality, Taibai Town in Suiyang County of Guizohu Province, China. We suggest as its English name "Suiyang Leaf-litter Toad," and its Chinese name as "Sui Yang Zhang Tu Chan (绥阳掌突蟾)".
Diagnosis. The specimens were assigned to the genus Leptobrachella on the basis of the following characters: (1) small body size; (2) having an elevated inner metacarpal tubercle; (3) having macro-glands on body (including supra-axillary, femoral and ventrolateral glands); (4) lacking vomerine teeth; (5) having small tubercles on eyelids; (6) anterior tip of snout with whitish vertical bar (Dubois 1983;Lathrop et al. 1998;Delorme et al. 2006;Das et al. 2010). Leptobrachella suiyangensis sp. nov. can be distinguished from its congeners by referring to the following characters: (1) small body size (SVL 28.7-29.7 mm in males, 30.5-33.5 mm in females); (2) dorsal skin shagreened, with some of the granules forming longitudinal short skin ridges; (3) tympanum distinctly discernible, slightly concave, with a deep, black, supratympanic line; (4) ventrolateral glands are distinct, forming a dotted line; (5) dorsal surface shagreened and granular, lacking enlarged tubercles or warts, with some of the granules forming short longitudinal folds; (6) flanks with several distinct and large dark blotches; (7) ventral surface of throat grey-white, and surface of chest and belly yellowish creamy-white with marbled texture or with irregular light brown speckling; (8) supra-axillary, femoral, pectoral and ventrolateral glands are distinctly visible; (9) absence of webbing and lateral fringes on fingers, and toes feature rudimentary webbing and a weak lateral fringes; (10) relatively short hindlimbs (TIB/SVL ratio in males 0.46-0.47); (11) longitudinal ridges under the toes are interrupted at the articulations; (12) relative finger lengths I <II < IV < III, relative toe lengths I < II < V < III < IV; (13) dorsum greyish-brown, with small light-orange granules and distinct darker brown markings scattered with irregular light-orange pigmentation, and bicolored iris, coppery orange on the upper half and silver grey on the lower half.
Description of the holotype. GZNU20180606007 (adult male), small body size (SVL 28.7 mm); the head length is slightly larger than the head width (HDL/HDW ratio 1.06); the snout is slightly protruding, projecting beyond the margin of the lower jaw; the nostril is between the snout and the eye (NSL/NEL ratio 0.39); the canthus rostralis is gently rounded; the loreal region is slightly concave; the interorbital space is flat; larger (IOD 2.9 mm) than the upper eyelid (1.6 mm in width), and the internarial distance is 2.8 mm; with vertical pupil; snout length is slightly larger than eye diameter (SNT/EYE ratio 1.71); tympanum is distinct and rounded, its diameter (TMP 2.1 mm) is smaller than that of the eye diameter (EYE 2.4 mm) and longer than the tympanum-eye distance (TMP/TEY ratio 1.91); deep black supratympanic line is present; weakly black supratympanic line exists (Fig. 3C); tympanic rim is distinctly elevated relative to the skin of the temporal region; supratympanic ridge is distinct, extending from the eye to the supra-axillary gland; a few indistinct tubercles present on supratympanic ridge; absent vomerine teeth; vocal sac openings is slit-like, located posterior-laterally on the floor of the mouth close to the margins of the mandible; long and wide tongue, with a small shallow notch at the posterior tip.
The tips of the fingers are rounded, slightly swollen; relative finger lengths are presented as: I <II < IV < III; nuptial pad is absent; absent subarticular tubercles (Fig. 3F); a large, round inner palmar tubercle is distinctly separated from a small, round outer palmar tubercle; finger webbing and dermal fringes absent. Toe tips are similar to those of the fingers; the relative toe length is presented as: I <II <V<III <IV; absent subarticular tubercles; distinct dermal ridges present under the 3 rd to 5 th toes; pronounced larger, oval inner metatarsal tubercle, outer metatarsal tubercle is absent; rudimentary toe webbing; weak lateral fringes present on all toes. Tibia is slightly shorter than half of the snoutvent length (TIB/SVL ratio 0.46); tibiotarsal articulation reaches to the anterior eye; heels meet each other when thighs are appressed at right angles referring to the body.
Dorsal skin is shagreened and scattered with fine and rounded granules, some of the granules forming short longitudinal folds; ventral skin smooth; large pectoral gland, elongated oval, 1.5 mm in length; small femoral gland, rounded, 0.7 mm in diameter, situated on the posteroventral surface of the thigh, closer to tibiotarsal articulation than to the vent; risen supra-axillary gland, 1.3 mm in diameter; ventrolateral gland is distinct as small white dots forming an incomplete line (Fig. 3D).
Coloration of holotype in life. Dorsal skin purple-brown; brown-purplish with dark-brown marks between the eyes and the scapular region, which are scattered with some deep yellow-orange granules more concentrated on the upper eyelid (Fig. 3C). A dark brown ϒ-pattern exists between eyes, linked with dark brown W-shaped marks between axillae. Tympanum is light brown-grey; black-brown tubercles present on dorsum of the body and the limb; those on dorsal side are much more distinct and dense; anterior upper lip features distinct blackish-brown patches; transverse dark-brown bars exist on dorsal surface of the limbs two or three (elbow and upper arms are an exception); indistinct black or brown blotches present on the flanks from groin to axilla; elbow and upper arms have no dark bars but with distinct dark-orange coloration; fingers and toes show indistinct brown blotches; a black spot is present on the loreal region; lower edge of the upper drum ridge is prominently black; ventral surface of the throat is grey-white, and surface of chest and belly is yellowish creamy-white, ventral part with distinct or indistinct light brown speckling mixed with marble texture; ventral surface of the thighs is dark grey and scattered with small light white spots. Supra-axillary gland milky yellow; iris is bicolored, coppery orange on the upper half and silver grey on the lower half.
Coloration of holotype in preservative. In preservation, there are dark brown marks on the dorsum and flanks; dorsum of the body and hindlimbs are dark brown, while dorsum of the forelimbs is yellowish brown; transverse bars on the limbs become more distinct, and dark-brown patterns, marks and spots on the back are indistinct; ventral surface of the body is yellowish brown with brown marbling on the sides and chest; orange supra-axillary, femoral, pectoral and ventrolateral glands fade to greyish white.
Variations. Measurements of the type series are shown in Table 4. Females (mean of SVL (32.0 ± 1.5 mm, n = 3) have larger body size than males (mean of SVL 29.2 ± 0.4 mm, n = 5) (Table 4). In life (Fig. 5), all paratypes match overall characters of the holotype, except the surface of the belly that is scattered with brown speckling   Table 4. Measurements (in mm), and body proportions of Leptobrachella suiyangensis sp.nov. from Suiyang County, Guizhou Province, China.

Distribution and habitats.
Currently, Leptobrachella suiyangensis sp. nov. is known only from its holotype locality, Huoqiuba Nature Reserve, Suiyang County, Guizhou Province, China (Fig. 1). The specimens were collected in a stream (ca 1.5 m in width and ca 10 cm in depth) and from nearby well-preserved bamboo forests (1501 m a.s.l.). During June, males were calling from under bamboo leaves; others perch on or under rocks by the side of the stream.

Comparisons
Leptobrachella suiyangensis sp. nov. differs from all other species of Leptobrachella based on morphological and molecular evidence. Phylogenetically, L. suiyangensis sp. nov., L. alpina and L. purpurus form a clade. Genetically, among this clade, the smallest genetic distance, at 5.49%, is between L. suiyangensis sp. nov. and L. alpina, and the largest genetic distance is 6.27% (L. suiyangensis sp. nov. and L. purpurus). Morphologically, the new species can be distinguished from L. alpina by having a larger body size of males (28.7-29.7 mm vs 24.0-26.4 mm); having narrower lateral fringes on the toes of the male (vs wide in males); dorsum purple-brown to dark purple-brown or grey-purple ground colour; ventral yellowish creamy-white with marbled texture on the chest and belly or with irregular light-brown speckling (vs almost uniformly graybrown on dorsal part, ventral nearly immaculately creamy white, brown specking on margins); ventrolateral glands are characterized by small white dots forming an incomplete line (vs small white dots forming a complete line longitudinally); shoulder-gland is orange-yellow (vs white, around gland); head length greater than head width, HDL/ HDW ratio 1.12 (vs head length equal to head width, HDL/HDW ratio 1.00). The new species can be distinguished from L. purpurus by body size of males (28.7-29.7 mm vs 25.0-27.5 mm); having narrow lateral fringes on the toes of males (vs wide in males); dorsum purple-brown to dark purple-brown or grey-purple ground color, ventral yellowish creamy-white with marbled texture on the chest and belly or with irregular light-brown speckling (vs dorsum coloration purplish brown, ventral side dull white with an indistinct grey dusting); throat immaculate gray (vs throat immaculate pinkish; almost dark orange-yellow on the upper arm (vs upper arms with distinct coppery orange coloration); dark bars on dorsal surface of tibia and tarsus very narrow, especially those on dorsal skin of tarsus (vs relatively broader dark bars on dorsal surface of tibia and tarsus); tibiotarsal articulation reaches to the anterior eye (vs tibiotarsal articulation reaches to posterior corner of the eye); relative length of fingers I <II < IV < III (vs I = II = IV < III).

Discussion
Phylogenetic analyses based on mitochondrial DNA and nuclear DNA all suggested that the new species belongs to Leptobrachella but is separate from its congeners. Genetic distance of the 16S rRNA gene between the new species and its closely related species (L. bijie, L. purpuraventra, L. alpina and L. purpurus) was 4.71-6.27%, within the expected range of interspecific divergences in amphibians (Fouquet et al. 2007), and this genetic distance is much higher than between many sister species, of which, most species have been completely recognized as valid species. For example, in Leptobrachella, the p-distance = 2.35% between L. purpurus and L. alpina. Finally, a series of morphological characters were found to be different between the new species and its congenerson. All in all, multiple pieces of evidence support the validity of the new species.
The new species described in this study increases the number of species of Leptobrachella to 75, with 21 recorded from China (Fei et al. 2012;Sung et al. 2014;Yang et al. 2016Yang et al. , 2018Yuan et al. 2017;Hou et al. 2018;Wang et al. 2018Wang et al. , 2019Chen et al. , 2019Frost 2019). Before the description of the new species herein, only 12 species were recorded from southwest China. This highlights the underestimation of the species diversity of the genus Leptobrachella. Additional field surveys are required to understand the true diversity of amphibians in this genus, which will be useful for conservation strategies.
Studies on the taxonomy and phylogeny of the genus Leptobrachella were difficult to perform because of the morphological conservativeness of the species; in the field, many species appear to be very similar morphologically, and there exist sympatric species. This likely hinders our understanding of these cryptic species (Ohler et al. 2010;Sung et al. 2014;Yang et al. 2016Yang et al. , 2018Yuan et al. 2017;Hou et al. 2018;Wang et al. 2018Wang et al. , 2019Chen et al. 2019). The high species diversity and the degree of endemism indicated that the speciation pattern and sympatry mechanism of species in the genus Leptobrachella also need additional investigation.
Currently, to our knowledge, L. suiyangensis sp. nov. is restricted to rocky streams in bamboo forests. However, the type locality of L. suiyangensis sp. nov. has faced habitat loss and human disturbance, such as artificial grazing and herb collection, which could possibly threaten this species. Leptobrachella suiyangensis sp. nov. is range-restricted to Kuankuoshui National Nature Reserve, which borders the nearby Huoqiuba Nature Reserve and is in the eastern Ta-lou Mountains. These areas feature subtropical evergreen broad-leaved forest and evergreen deciduous broad-leaved mixed forest. Thus, it is likely that other populations of L. suiyangensis sp. nov. may be discovered in the Kuankuoshui Nature Reserve in the near future.