Morphology and molecular genetics reveal two new Leptobrachella species in southern China (Anura, Megophryidae)

Abstract Based on morphological and phylogenetic analyses (16S rRNA mtDNA), two new species of the genus Leptobrachella are described from southern China, namely L.yunkaiensis Wang, Li, Lyu & Wang, sp. n. from Dawuling Forest Station of Guangdong Province and L.wuhuangmontis Wang, Yang & Wang, sp. n. from Mt. Wuhuang of Guangxi Province. To date, the genus Leptobrachella contains 68 species, among which 13 species are known from China. The descriptions of the two new species further emphasize that the species diversity of the genus Leptobrachella from China is still highly underestimated and requires further investigations.


Introduction
The genus Leptolalax Dubois, 1983 within the family Megophryidae Bonaparte, 1850 was currently found to be non-monophyletic with Leptobrachella Smith, 1925, and was assigned as a junior synonym of Leptobrachella based on a large-scale molecular analysis (Chen et al. 2018).Their results also rejected the hypothesis that Leptolalax consists of two subgenera as proposed by Delorme et al. (2006) and Dubois et al. (2010). At present, the genus Leptobrachella contains sixty-six species, widely distributed from southern China west to northeastern India and Myanmar, through mainland Indochina to peninsular Malaysia and the island of Borneo (Frost 2017;Nguyen et al. 2018;Rowley et al. 2016Rowley et al. , 2017Yang et al. 2016;Yuan et al. 2017). They are commonly known as Asian leaf litter frogs. Currently, eleven species of this genus are known from China, i.e., L. alpinus from Yunnan and Guangxi provinces, L. laui from southern Guangdong including Hong Kong, L. liui from Fujian, Jiangxi, Guangdong, Guangxi, Hunan and Guizhou provinces, L. oshanensis from Gansu, Sichuan, Chongqing, Guizhou and Hubei provinces, L. cf. pelodytoides (which may represents a undescribed taxon), L. purpura, L. tengchongensis, L. ventripuntatus, and L. yingjiangensis from Yunnan Province, and L. sungi and L. maoershanensis from Guangxi Province (Sung et al. 2014;Yang et al. 2016;Yuan et al. 2017;Yang et al. 2018).
During field surveys in southern China from 2009 to 2016, a number of specimens were collected from Dawuling Forest Station of Guangdong Province and Mt. Wuhuang of Guangxi Province, respectively ( Fig. 1), that can all be morphologically assigned to the genus Leptobrachella, based on the following characters: (1) comparatively small size, snout-vent length no overlap than 60.0 mm, (2) rounded finger tips, the presence of an elevated inner palmar tubercle not continuous to the thumb, (3) presence of macroglands on body including supra-axillary, pectoral, femoral and ventrolateral glands, (4) vomerine teeth absent, (5) tubercles on eyelids present, and (6) anterior tip of snout with whitish vertical bar (Dubois 1983;Matsui 1997Matsui , 2006Lathrop et al. 1998;Delorme et al. 2006;Das et al. 2010). Subsequent molecular studies on 16S rRNA mtDNA sequences revealed that this collection represents two different undescribed species which can be distinguished from each other and from all other recognized congeners by a combination of morphological characters and molecular divergences; they are described herein as two new species.

Materials and methods
Sampling. For molecular analyses, a total of 65 samples (19 muscle tissues and 46 sequences downloaded from Genbank) from 29 species of the genus Leptobrachella were sequenced, in addition to two undescribed species from China, i.e., the population from Dawuling Forest Station of Guangdong Province and Mt. Wuhuang of Guangxi Province. Additionally, four sequences were downloaded from GenBank as the outgroups (see Table 1; Pelobates syriacus, Pelobates varaldii, Leptobrachium cf. chapaense and Megophrys major). All specimens were previous to fixation 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; tissue samples were preserved in 95% ethanol for molecular studies.
DNA Extraction, PCR and sequencing. DNA was extracted from muscle tissue using a DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. The mitochondrial gene 16S ribosomal RNA gene (16S rRNA) from each sample was sequenced. Fragments of the genes were amplified using primer pairs L3975 (5'-CGCCTGTT-TACCAAAAACAT-3') and H4551 (5'-CCGGTCTGAACTCAGATCACGT-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 one min, and 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 BigDye Terminator Cycle Sequencing Kit according to the guidelines of the manufacturer. The products were sequenced on an ABI Prism 3730 automated DNA sequencer in Shanghai Majorbio Bio-pharm Technology Co., Ltd. All sequences have been deposited in GenBank (Table 1).
Phylogenetic analyses. Sequence alignments were first conducted using Clustal X 2.0 (Thompson et al. 1997), with default parameters and the alignment being checked  (Darriba et al. 2012) with Akaike and Akaike information criteria, the best-fitting nucleotide substitution models are GTR + I + G. Phylogenetic trees were analyzed using maximum likelihood (ML) implemented in RaxmlGUI 1.3 (Silvestro and Michalak 2012), and Bayesian inference (BI) using MrBayes 3.2.4 (Ronquist et al. 2012). For ML analysis, the maximum likelihood tree inferred from 1000 replicates was used to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 60% of bootstrap replicates were collapsed. For BI analysis, two independent runs with four Markov Chain Monte Carlo simulations were performed for ten million iterations and sampled every 1000 th iteration. The first 25% of samples were discarded as burn-in. Convergence of the markov Chain monte carlo simulations was assessed with PSRF ≤ 0.01 and ESS (effective sample size) value > 200 using Tracer v.1.4 (http://tree.bio.ed.ac.uk/software/tracer/). We also calculated pairwise sequence divergence based on uncorrected p-distance using MEGA 6.06 (Tamura et al. 2013).
Morphometrics. Measurements followed Fei et al. (2009) and Rowley et al. (2013), 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 of commissure of jaws); SNT snout length (from tip of snout to anterior corner of eye); EYE eye diameter (diameter of exposed portion of eyeball); IOD interorbital distance (minimum distance between upper eyelids); INDY internasal distance (distance between nares); 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 lower arm and hand (distance from tip of the third finger to elbow); PL pes length (distance from tip of fourth toe to proximal edge of the inner metatarsal tubercle); HLL hindlimb length (distance from tip of fourth toe to vent).
Sex was determined by direct observation of calls in life, the presence of internal vocal sac openings, and the presence of eggs in abdomen through external inspection. Comparative morphological data of Leptobrachella species were obtained from examination of museum specimens (see Appendix 1) and from the references listed in Table 2. Due to the high likelihood of undiagnosed diversity within the genus (Rowley et al. 2016;Yang et al. 2016), where available, we relied on examination of topotypic material and/or original species descriptions.

Results
Bayesian inference (BI) and Maximum likelihood (ML) phylogenetic tree were constructed based on DNA sequences of the mitochondrial 16S gene with a total length of 476 bp. The two analyses resulted in essentially identical topologies ( Fig.  2) with clustered the population of Leptobrachella from Dawuling Forest Station with L. laui, L. liui, and L. maoershanensis with very high node supporting values (1.00 in BI and 91% in ML) and represented a separately evolving lineage. Besides, the population from Mt. Wuhuang was a distinct separately evolving lineage with high node supporting values (1.00/100% in BI and ML). The smallest pairwise genetic divergences between the population from Dawuling Forest Station and all other species of the genus Leptobrachella for which comparable sequences were included was 6.0-6.7% (with L. liui), and between population from Mt. Wuhuang and all other species was 7.4% (with L. aerea) ( Table 3). These values were significantly larger than observed pairwise genetic distances between recognized species Figure 2. Bayesian inference tree derived from partial DNA sequences of the mitochondrial 16S r RNA gene. Numbers before slashes indicate Bayesian posterior probabilities (>60 retained) and numbers after slashes are bootstrap support for maximum likelihood (1000 replicates) analyses (>60 retained). The symbol "-" represents bootstrap value below 0.60/60%.
(p-distance = 2.6%, between L. bourreti and L. oshanensis). Given that the two populations both can be morphologically distinguished with each other, and from all known congeners, we herein describe these specimens as two new species, respectively. Diagnosis.
(1) small size (SVL 25.9-29.3 mm in males, 34.0-35.3 mm in females), (2) dorsal skin shagreened with short skin ridges and raised warts, (3) iris bicolored, coppery orange on upper half and silver on lower half, (4) tympanum distinctly discernible, slightly concave, weakly black supratympanic line present, (5) dorsal surface yellowishbrown grounding, with distinct darker brown markings and rounded spots and scattered with irregular orange patches, (6) flanks with several dark blotches, (7) surface of belly pinkish, with distinct or indistinct light dark brown speckling, (8) supra-axillary, femoral, pectoral and ventrolateral glands distinctly visible, (9) absence of webbing and presence of distinct lateral fringes on fingers, toes with rudimentary webbing and wide lateral fringes, (10) longitudinal ridges under toes not interrupted at the articulations, and (11) dense conical spines present on lateral and ventral surface of tarsus, surface of tibia-tarsal, inner-side surface of shank and surface around cloacal region.
Comparisons. Comparative morphological data of Leptobrachella yunkaiensis sp. n. with 66 recognized Leptobrachella species were obtained from examination of museum specimens (see Appendix 1) and from the references listed in Table 2. All comparative data were shown in Tables 4, 5 (Fig. 4), belly pinkish with distinct or indistinct speckling (vs. belly creamy white with dark brown dusting on margins in L. laui; belly creamy white with dark brown spots on chest and margins in L. liui).
Tips of fingers rounded, slightly swollen; relative finger lengths I = II = IV < III; nuptial pad absent; subarticular tubercles absent; a large, rounded inner palmar tubercle distinctly separated from small, round outer palmar tubercle; absence of webbing and presence of distinct lateral fringes on fingers. Tips of toes like fingers; relative toe length I < II < V < III < IV; subarticular tubercles absent; distinct dermal ridges present under the 3 rd to 5 th toes; large, oval inner metatarsal tubercle present, outer metatarsal tubercle absent; toes webbing rudimentary; wide lateral fringes present on all toes. Tibia 43% of snout-vent length; tibiotarsal articulation reaches to middle of eye; heels just meeting each other when thighs are appressed at right angles with respect to body.
Skin on dorsum shagreened and scattered with fine, round tubercles; short skin ridges and raised warts on dorsum surface present; ventral skin smooth; pectoral gland and femoral gland large, oval; pectoral glands greater than tips of fingers and femoral glands; femoral gland situated on posteroventral surface of thigh, closer to knee than to vent; supra-axillary gland raised. Ventrolateral gland distinctly visible, forming an incomplete line. Dense conical spines on lateral and ventral surface of tarsus, surface of tibia-tarsal, inner-side surface of shank and surface around cloacal region present.
Coloration of holotype in life. Dorsal surface orange-brown with distinct dark brown blotches edged distinct light orange pigmentation. A dark brown triangular pattern between eyes, connected to the dark brown W-shaped marking between axillae. Tympanum black. Orange-brown tubercles present on dorsum of body and limb, those on flanks much distinct and dense; anterior upper lip with distinct blackish brown patches; transverse dark brown bars on dorsal surface of limbs; indistinct dark brown blotches on flanks from groin to axilla; elbow and upper arms without dark bars but with distinct coppery orange coloration; fingers and toes with indistinct dark brown blotches.
Surface of throat creamy white and scattered with small whitish dots; belly pinkish and scattered with small brown speckling; ventral surface of thighs pinkish and scattered with small light orange-brown spots. Supra-axillary coppery orange; femoral, pectoral and ventrolateral glands whitish orange. Iris bicolored, coppery orange on upper half and silver on lower half.
Coloration of holotype in preservative. Dorsum of body and hindlimbs dark brown while dorsum of forelimbs yellowish brown; transverse bars on limbs become more distinct, dark brown patterns, markings and spots on back become indistinct. Ventral surface of body yellowish brown, with brown marbling on sides and chest. Orange supra-axillary, femoral, pectoral and ventrolateral glands fade to greyish white ( Fig. 4C1-C3).

Sexual dimorphism.
Females with a larger body size than males, SVL 34.0-35.3 mm (34.7 ± 0.9) (vs. SVL 25.9-29.3 mm (27.6 ± 1.4) in males); presence of a single vocal sac in males (vs. absent in females); dense conical spines on lateral and ventral surface of tarsus, surface of tibia-tarsal, inner-side surface of shank, surface of thighs and surface around cloacal region distinct in males, and barely visible in females.
Variations. All paratypes match the overall characters of the holotype except that: the heels just meeting each other when thighs are appressed at right angles with respect to body, tibiotarsal articulation reaches to middle of eye in holotype SYS a004665 (vs. tibiotarsal articulation reaches to anterior corner of eye in SYS a004666, reaches the posterior corner of eye in SYS a004669). Surface of belly scattered with distinctly dark brown speckling in holotype (vs. such speckling indistinct in female paratypes SYS a004663, 4690. Tympanum black in the holotype (vs. tympanum black grounding with orange speckling in SYS a004667-4668). Distinct black spots present on dorsum in the female paraype SYS a004690 (Fig. 3).
Etymology. The specific epithet, yunkaiensis, is in reference to the type locality, DWL of Guangdong, China located in the Yunkai Mountains Range. For the common name, we suggest "Yunkai Mountain's Leaf Litter Toad", and Chinese name "Yun Kai Zhang Tu Chan (云开掌突蟾)".
Distribution and habits. Currently, Leptobrachella yunkaiensis sp. n. is known only from its type locality DWL of Guangdong Province (Fig. 1). The new species was found along a clear-water rocky stream (ca. 2-3 m in width and ca. 20-30 cm in depth) and small nearby seeps in well-preserved montane evergreen broadleaf forest (1600 m a.s.l.) (Fig. 5). During April and June, males were found calling mainly hidden under leaf litter, and some were found calling perching on the rocks or under rocks by the side of the stream. Females collected on April bear pure white oocytes. Leptobrachella wuhuangmontis sp. n. significantly differs from L. yunkaiensns sp. n. by a large genetic divergence (p=10.2-11.1%), lateral fringes on toes narrow (vs. wide), black supratympanic line distinct (vs. weak), dorsal surface of body rough and scattered with dense conical tubercles (vs. shagreened with short skin ridges and raised warts), belly greyish white mixed by tiny white and black dots (vs. belly pink with distinct or indistinct speckling).
Ventral surface greyish-white mixed with tiny white and black dots. Supra-axillary, femoral, and ventrolateral glands white, pectoral gland greyish white as the color of ventral surface. Iris bicolored, coppery yellow on upper half and silver on lower half.
Coloration of holotype in preservative. Dorsal of body dark with greyish white dots on flanks, while dorsal of limbs dark brown, transverse bars on dorsal of forelimbs become more distinct, and indistinct on dorsal of hindlimbs, dark brown patterns, markings and spots on back become indistinct. Ventral surface light yellow with brown speckling. Supra-axillary, femoral, ventrolateral and pectoral glands light yellow (Fig. 7).
Sexual dimorphism. Females with a larger body size than males, SVL 33.0-36.0 mm (34.9 ± 1.4) (vs. SVL 25.6-30.0 mm (28.5 ±1.5) in males); presence of a single vocal sac in males (vs. absent in females); dense conical spines on lateral and ventral surface of tarsus, surface of tibia-tarsal, inner-side surface of shank and surface around cloacal region distinct in males (vs. barely visible in females); pectoral gland and femoral gland large, oval, slightly elevated in males (vs. indistinct in females).
Variations. All paratypes match the overall characters of the holotype except that: tibiotarsal articulation reaches to posterior corner of eye in female paratypes SYS a003499, 3504 and reaches to anterior corner of eye in male paratypes SYS a003487 and SYSa 003500 / CIB 107274; pectoral gland large, oval, slightly elevated in all individuals in life, and become indistinct in preservation. Yellow blotches and white speckling present on dorsum in the holotype (vs. indistinct in the female paratype SYS a003499). Elbow and upper arms coppery orange and with distinct dark bars in the holotype (vs. elbow and upper arms light orange, dark bars indistinct in the male paratypes SYS a003488, 3505 and the female paratype SYS a003499) (Fig. 6).
Etymology. The specific epithet, wuhuangmontis, is in reference to the type locality, Mt. Wuhuang of Guangxi Province, China. For the common name, we suggest "Mt. Wuhuang's Leaf Litter Toad", and for the Chinese name "Wu Huang Shan Zhang Tu Chan (五皇山掌突蟾)".
Distribution and habits. Currently, Leptobrachella wuhuangmontis sp. n. is only known from its type locality MWH from Guangxi Province of China (Fig. 1). The new species was found along a clear-water rocky streams and small steep rocky streams in well-preserved montane evergreen broadleaf forest (500 m a.s.l.) (Fig.  8). During field surveys in March, males were found calling exposed on the rocks or hiding in the rock seams; gravid female collected on March and April bear pure white oocytes.

Discussion
Studies of the taxonomy and phylogeny of Leptobrachella are difficult to perform because of the morphological conservativeness and very similar characters (for example, the coloration and the texture of skin) in different environments, which may cause misidentifications (Ohler et al. 2010;Sung et al. 2014). With the evidence of both morphological and phylogenetic analyses, 15 cryptic species of the genus Leptobrachella have been discovered and described since 2010 (Frost 2017;Rowley et al. 2016Rowley et al. , 2017Yang et al. 2016;Yuan et al. 2017). With the description of L. yunkaiensis sp. n. and L. wuhuangmontis sp. n. based on an taxonomical approach, the number of the genus Leptobrachella herein is increased to 68, indicating the underestimated diversity.
During our examination, it was observed that the dense tiny conical spines present on the surface of the lateral and ventral aspects of the tarsus, surface of tibiatarsal, the inner surface of the shank and surface around cloacal region (distinct in males and barely visible in females) in the two new Leptobrachella species described in this study are also present in examined specimens of L. alpinus, L. laui, L. liui, and L. tengchongensis as well as in other cryptic taxa (Wang et al. unpublished data). Thus, this neglected morphological character may be common among congeners of the genus Leptobrachella, and further morphological studies are needed to study this in more detail.
Mt. Wuhuang of Guangxi Province in southern China is known for the extraordinarily high biodiversity, with some new national records discovered in recent years, for example, the national records of Opisthotropis maculosa and Sphenomorphus tonkinensis from Mt. Wuhuang were recorded (Wang et al. 2013;Yang et al. 2011). Except for the new species (Leptobrachella yunkaiensis sp. n.) described in this study, several new species of amphibians and reptiles have been discovered from Dawuling Forest Station during field surveys in the last two years (Wang et al. unpublished data;Wang et al. 2018;Lyu et al. 2018), which suggests a high herpetofaunal biodiversity of Dawuling Forest Station localized in western Guangdong Province, China. Recently, these areas have been subjected to tourism development; thus, conservation strategies and measures for references and enforcements are urgently needed.