Morphology, molecular genetics, and acoustics reveal two new species of the genus Leptobrachella from northwestern Guizhou Province, China (Anura, Megophryidae)

Abstract Two new species of the genus Leptobrachella Smith, 1925, L.bijie J. Wang, Y.L. Li, Y. Li, H.H. Chen & Y.Y. Wang, sp. nov. and L.purpuraventra J. Wang, Y.L. Li, Y. Li, H.H. Chen & Y.Y. Wang, sp. nov., were described from northwestern Guizhou Province, China based on a combination of acoustic, molecular, and morphological data. The new discoveries bring the total number of this genus to 73, with 16 congeners recorded in China, and represent the second and third species of the genus reported from Guizhou Province.


Introduction
The Asian leaf litter toad genus Leptobrachella Smith, 1925 currently contains seventy-one 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). Currently, 14 species of this genus are known from China, i.e., L. alpinus from Yunnan and Guangxi provinces, L. laui from southern Guangdong Province including Hong Kong, L. liui from Fujian, Jiangxi, Guangdong, Guangxi, Hunan and Guizhou provinces, L. mangshanensis from southern Hunan Province, L. oshanensis from Gansu, Sichuan, Chongqing, Guizhou and Hubei provinces, L. cf. pelodytoides (which may be a population of L. eos (Ohler et al. 2011)), L. purpura, L. tengchongensis, L. ventripuntatus and L. yingjiangensis from Yunnan Province, L. wuhuangmontis from southern Guangxi Province, L. yunkaiensis from western GuangdongProvince, and L. sungi and L. maoershanensis from Guangxi Province (Hou et al. 2018;Sung et al. 2014;Yang et al. 2016;Yuan et al. 2017. During recent field surveys in northwestern Guizhou Province of China in 2018, a number of specimens were collected from Zhaozishan Nature Reserve and Wujing Nature Reserve in Qixingguan District of Bijie City, respectively (Figure 1), which can be morphologically assigned to the genus Leptobrachella, based on the following characters: (1) small or moderate size, snout-vent length not greater 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 supraaxillary, 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 1997Lathrop et al. 1998;Delorme et al. 2006;Das et al. 2010). Subsequent 16S rRNA sequences from these specimens revealed that these collections represent two distinct evolving lineages. Combine of morphological characters, acoustic data, and molecular divergences; they are described herein as two new species.

Sampling
For molecular analyses, a total of 71 sequences (23 muscle tissues were sequenced and 48 sequences downloaded from GenBank) from 32 species of the genus Leptobrachella were used, including two undescribed species from China, i.e., the populations from Zhaozishan Nature Reserve and Wujing Nature Reserve of Guizhou Province. And four sequences were downloaded from GenBank as outgroups (see Table 1; Pelobates syriacus, P. varaldii, Leptobrachium cf. chapaense, and Megophrys major).
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; 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) fragment from each sample was sequenced. Fragments were amplified using primer pairs L3975 (5'-CGCCTGTTTACCAAAAACAT-3') and H4551 (5'-CCG-GTCTGAACTCAGATCACGT-3') (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
Sequences were first aligned in Clustal X 2.0 (Thompson et al. 1997), with default. The alignment was then checked and manually revised, if necessary. Trimmed with the gaps were partially deleted in MEGA 6.06 (Tamura et al. 2013 (Tamura et al. 2013).

Morphometrics
Measurements followed Fei et al. (2009) and Rowley et al. (2013), and were taken with a digital caliper 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); EYE eye diameter (diameter of exposed portion of eyeball); IOD interorbital distance (minimum distance between upper eyelids); IND 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 calling in life, the presence of internal vocal sac openings, and the presence of eggs in abdomen seen via 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 Yang et al. 2016), where available, we rely on examination of topotypic material and/or original species descriptions.

Acoustic analyses
We compared the advertisement calls from three localities. One was in Wujing Nature Reserve and two were in Zhaozishan Nature Reserve. Advertisement calls were recorded

ID
Leptobrachella species Literature obtained 1 L. aereus (Rowley, Stuart, Richards, Phimmachak & Sivongxay, 2010) Rowley et al. 2010c between 20:00-24:00 h on 2-6 July 2018, using a Sony PCM-D100 digital sound recorder held within 20 cm of the calling individuals. The ambient temperature of the type locality was obtained using a Volt TP-2200 Humidity & Temperature Logger. The sound files in wave format were sampled at 44.1 kHz with sampling depth 24 bits. Praat 6.0.27 (Boersma 2001) was used to obtain the oscillograms, sonograms and power spectrums (window length = 0.005s). Raven pro 1.5 software (Bioacoustics Research Program 2013) was used to quantify the acoustic properties (window size = 256 points, fast Fourier transform, Hanning window). The measurements taken were as follows: Call Duration: the time between onset of the first pulse and offset of the last pulse in a call; IQR (Inter-quartile Range): Duration of the difference between the 1 st and 3 rd quartile times which divides the selection into four time intervals containing equal energy in the selection; Dominant Frequency: the frequency at which max power occurs within the selection; IQR (Inter-Quartile Range): Bandwidth of the difference between the 1 st and 3 rd quartile frequencies which divides the selection into four frequency intervals containing equal energy in the selection; fNote Pulses: the number of pulses for the first note in a call; sNote Pulses: the number of pulses for the second note in a call; Note Rise Time: the time between onset of the first pulse and pulse of max amplitude; Note Interval: the interval between the first note and the second note in a call; fNote Duration: the duration of the first note in a call; sNote Duration: the duration of the second note in a call.
Mean and standard deviation (SD) were calculated. We used median and interquartile range instead of mean and SD when calculating the undivided properties, like fNote Pulses and sNote Pulses. To identify different groups on acoustic properties, a hierarchical clustering using Mahalanobis distance was conducted (Mahalanobis 1936). The dendrogram was constructed based on Ward's method (Ward Jr 1963). All statistical analyses were conducted in R 3.3.2 (R Core Team 2016).

Molecular results
Bayesian inference (BI) and maximum likelihood (ML) phylogenetic trees were constructed based on DNA sequences of the mitochondrial 16S rRNA gene with a total length of 481-bp. The two analyses resulted in essentially identical topologies ( Figure  2) which clustered the population of Leptobrachella from Jinjiazhai Village (JV) from Wujing Nature Reserve and those from Baimashan Forest Station (BFS) of Zhaozishan Nature Reserve together with very high node supporting values (0.97 in BI and 100% in ML) and represented a separately evolving lineage (Clade A). The population from Qingshan Village (QV) of Zhaozishan Nature Reserve (Clade B) was a sister taxon to Clade A with high node support values (0.99 in BI and 82% in ML). There was almost no genetic divergence between the two populations in Clade A even though the specimens were collected in two different sites with a straight-line distance at approximately 65 kilometers, and the smallest genetic divergence among individuals in Clade B was only 0.3%. The pairwise genetic divergence between Clade A and Clade B was 3.9-4.2%, and between Clade A and all other species of the genus Leptobrachella for which comparable sequences were included was 3.2% (between Clade A and L. bourreti), and between Clade B and all other species was 5.2-5.6% (between Clade B and L. purpura). However, these values were larger than or equal to observed pairwise genetic distances between recognized species (2.2% between L. liui and L. mangshanensis; 3.2% between L. eos and L. purpura) ( Table 3).

Acoustic results
Calling from nine male individuals were measured, respectively. They were recorded in Jinjiazhai Village (two males), Baimashan Forest Station (three males), and Qingshan Village (four males) at an ambient temperature approximately of 18.8 °C, 19.3 °C, and 18.6 °C, respectively. The result of hierarchical clustering analysis was consistent with the molecular result ( Figure 3). Nine calling males were clustered into two clades based  Figure 2. Bayesian inference tree of Leptobrachella species and out-groups derived from partial DNA sequences of the mitochondrial 16S r RNA gene. Numbers before slashes indicate Bayesian posterior probabilities (>0.6 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%. on acoustic properties of advertisement calls. All JV males and BFS males were clustered into Clade A, and all the QV males were clustered into Clade B. In Clade A, there were some differences in the advertisement calls between JV and BFS in Clade B. Measurements of the advertisement calls of the three localities are listed in Table 4. All advertisement calls contain two notes, each of which consists of repeated pulses (Figure 4). Clade A had more fNote pulses in second type of advertisement calls than those of Clade B (3 ± 1 vs. 2 ± 1), more sNote pulses in first type of advertisement calls (4 ± 1 vs. 3 ± 1), and less sNote pulses in the second type of advertisement calls (17 ± 3 vs. 21.5 ± 4). Accordingly, the sNote duration of Clade A was greater than those of  Combining morphological, molecular genetics, and acoustic evidence, we herein describe these specimens as two new species.  Paratypes. Seven adult males, SYS a007313/CIB 110002, SYS a007314-7315, 7317-7320, collected by Honghiu Chen (HHC hereafter), Yongyou Zhao (YYZ hereafter) and Jiahe Li (JHL), the same collection data as the holotype.
(1) small size (SVL 29.0-30.4 mm in eight adult males), (2) dorsal skin shagreened, some of the granules forming longitudinal short skin ridges, (3) iris bicolored, coppery orange on upper half and silver on lower half, (4) tympanum distinctly discernible, slightly concave, distinct black supratympanic line present, (5) internasal distance equal to interorbital distance, (6) supra-axillary, femoral, pectoral and ventrolateral glands distinctly visible, (7) absence of webbing and lateral fringes on fingers, toes with rudimentary webbing and narrow lateral fringes, (8) longitudinal ridges under toes not interrupted at the articulations, (9) relative finger lengths I = II = IV < III, relative toe length I < II < V = III < IV, (10) heels just meeting, tibia-tarsal articulation reaches the region between middle of eye to anterior corner of eye, (11) dorsal surface shagreened and granular, lacking enlarge tubercles or warts, some of the granules forming short longitudinal folds, (12) dorsum greyish-brown grounding, with small light orange granules, distinct darker brown markings scattered with irregular light orange pigmentations, (13) flanks with several dark blotches, longitudinally in two rows, (14) ventral surface white, with distinct nebulous greyish speckling on chest and ventrolateral flanks, (15) dorsal limbs including fingers and toes with dark bars, and (16) dense tiny conical spines present on surface of chest in males during breeding season.
Comparisons. Comparative morphological data of Leptobrachella bijie sp. nov. and 45 recognized Leptobrachella species occurring north of the Isthmus of Kra were listed in Table 5.  Description of holotype. Adult male. Body size small, SVL in 29.3 mm. Head length slightly larger than head width, HDL/HDW 1.03; snout slightly protruding, projecting slightly beyond margin of the lower jaw; nostril closer to snout than eye; canthus rostralis gently rounded; loreal region slightly concave; interorbital space flat, internarial distance equal to interorbital distance, IND/IOD 1.00; pineal ocellus absent; vertical pupil; snout length larger than eye diameter, SNT/EYE 1.11; tympanum distinct, rounded, and slightly concave, diameter smaller than that of the eye and larger than tympanum-eye distance, TMP/EYE 0.53 and TEY/TMP 0.47; upper margin of tympanum incontact with supratympanic ridge; distinct black supratympanic line present; vomerine teeth absent; vocal sac openings slit-like, paired, located posterolaterally on floor of mouth in close proximity to the margins of the mandible; tongue deeply notched behind; supratympanic ridge distinct, extending from posterior corner of eye to supra-axillary gland.
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 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, not interrupted; large, oval inner metatarsal tubercle present, outer metatarsal tubercle absent; toes webbing rudimentary; narrow lateral fringes present on all toes. Tibia 47% 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.
Dorsal surface shagreened and granular, lacking enlarge tubercles or warts, some of the granules forming short longitudinal folds; ventral skin smooth; dense tiny conical spines present on surface of chest; pectoral gland and femoral gland 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.
Coloration of holotype in life. Dorsum greyish-brown grounding, with small reddish granules, distinct darker brown markings and rounded spots and scattered with irregular light orange pigmentation. A dark brown inverted triangular pattern between anterior corner of eyes, in connected to the dark brown W-shaped marking on interorbital region, and the W-shaped marking in connected to the other W-shaped marking between axillae. Tympanum brown. Small light orange granules present on dorsum of body and limb; a dark brown vertical bar under the eye; transverse dark brown bars on dorsal surface of limbs; distinct dark brown blotches on flanks from groin to axilla, longitudinally in two rows; elbow and upper arms with dark bars and distinct coppery orange coloration; fingers and toes with distinct dark bars.
Ventral surface of throat, chest, and belly white, presence of distinct nebulous greyish speckling on chest and ventrolateral flanks; ventral surface of limbs grey purple. Supra-axillary gland coppery orange; femoral, pectoral and ventrolateral glands greyish white. Iris bicolored, coppery orange on upper half and silver on lower half.
Coloration of holotype in preservative. Dorsum of body and limbs dark brown; transverse bars on limbs become more distinct; dark brown patterns, markings and spots on back become indistinct, orange pigmentations become greyish white. Ventral surface of body and limbs greyish white, nebulous speckling on chest and flanks balck brown. Supra-axillary, femoral, pectoral and ventrolateral glands greyish white.  Table 6. All paratypes match the overall characters of the holotype except that: coloration of tympanum brown in the holotype SYS a007316 (vs. black in paratypes SYS a007313/CIB 110002 ( Figure 5E), SYS a007315, 7317 (Figure 5F)); heels just meeting, tibia-tarsal articulation reaching the middle of eye in the holotype (vs. heels slightly overlapping in paratypes SYS a007315, 7317, 7319-7320; tibia-tarsal articulation reaching the anterior corner of eye in paratypes SYS a007315, 7317, 7319); W-shaped marking on interorbital region in connected to the other W-shaped marking between axillae in the holotype (vs. such markings not in connected with each other in paratypes SYS a007313/CIB 110002, SYS a007320); a dark brown inverted triangular pattern between anterior corner of eyes in the holotype (vs. a V-shaped pattern between anterior corner of eyes instead in paratype SYS a007317, 7320); relatively larger black spots on flanks (vs. black spots distinctly small in paratypes SYS a007313/CIB 110002, SYS a007317).

Variations. Measurements and body proportions were listed in
Etymology. The specific epithet bijie is in reference to the type locality, Qingshan Village in Bijie City of Guizohu Province, China. For the common name, we suggest "Bijie Leaf Litter Toad", and for the Chinese name "Bi Jie Zhang Tu Chan (毕节掌突蟾)".
Distribution and habits. Currently, Leptobrachella bijie sp. nov. is known only from its type locality Qingshan Village in Zhaozishan Nature Reserve, Linkou County, Qixingguan District, Bijie City, Guizhou Province, China (Figure 1). The new species was found along a clear-water rocky stream (ca. 2 m in width and ca. 20-30 cm in depth; 1670-1750 m a.s.l.) in karst landforms. The stream was surrounded by broadleaved forest at an altitude below 1700 m, and by coniferous forest at an altitude above 1700 m (Figure 6, 1700 m a.s.l.). On 6 July 2018 at 22:00-23:30 P.M., a large number of males were found calling on leaves of plants ( Figure 10A), and some were found calling perching on the rocks or under rocks by the side of the stream.  Diagnosis.
(1) small size .8 mm in males, 33.0-35.3 mm in females), (2) dorsal skin shagreened, some of the granules forming longitudinal short skin ridges, (3) iris bicolored, coppery orange on upper half and silver on lower half, (4) tympanum distinctly discernible, slightly concave, distinct black supratympanic line present, (5) internasal distance smaller than interorbital distance, IND/IOD ratio 1.03-1.10, (6) supra-axillary, femoral, pectoral and ventrolateral glands distinctly visible, (7) absence of webbing and lateral fringes on fingers, toes with rudimentary webbing and narrow lateral fringes, (8) longitudinal ridges under toes not interrupted at the articulations, (9) heels just meeting or slightly overlapping, tibia-tarsal articulation reaching to the middle of eye, (10) relative finger lengths I = II = IV < III, relative toe length I < II < V < III < IV, (11) dorsal surface shagreened and granular, lacking enlarge tubercles or warts, some of the granules forming short longitudinal folds, (12) dorsum purple brown to dark purple brown or grey purple grounding, with small light orange granules, distinct darker brown markings scattered with irregular light orange pigmentations, (13) flanks with several dark blotches, longitudinally in two rows, (14) ventral surface grey purple, with distinct or indistinct nebulous greyish speckling on chest and ventrolateral flanks, without black spots (seldom present), (15) dorsal limbs including fingers and toes with dark bars, those on forearms indistinct, and (16) dense tiny conical spines present on surface of chest extending to anterior region of abdomen in males, and absent in females during breeding season.
Comparisons. Comparative morphological data of Leptobrachella purpuraventra sp. nov., L. bijie sp. nov., and 45 recognized Leptobrachella species occurring north of the Isthmus of Kra were listed in Table 5.
In the phylogenetic trees (Figure 2), Leptobrachella purpuraventra sp. nov. is a sister taxon to L. bijie sp. nov. with a high support value (99% in BI, 0.82 in ML), and it can be distinguished from the later by a genetic divergence (p=3.9-4.2%). Morphologically, it differs from the later by the coloration of dorsum and ventral, dorsum purple brown to dark purple brown or grey purple grounding, ventral grey purple grounding (vs. dorsum greyish-brown grounding, ventral white grounding); dark bars on dorsal limbs indistinct (vs. distinctly visible); dark bars on dorsal surface of tibia and tarsus much broader, especially those on dorsal skin of tarsus (vs. relatively narrow dark bars on dorsal surface of tibia and tarsus); internasal distance smaller than interorbital distance, IND/IOD ratio 1.03-1.10 (vs. internasal distance equal to interorbital distance, IND/IOD ratio 1.00); larger TEY value, TEY/TMP ratio 0.60-0.76 (vs. TEY/TMP ratio 0.45-0.53); dense tiny conical spines present on surface of chest extending to anterior region of abdomen (vs. such spines less developed, present on surface of chest, not extending to anterior region of abdomen); lateral fringes on toes narrow but more developed and distinct (vs. less developed); length of toe V < III (length of toe V = III).
Compared with the 26 known congeners of the genus Leptobrachella occurring south of the Isthmus of Kra, by the presence of supra-axillary and ventrolateral glands, L. purpuraventra sp. nov. can be easily distinguished from L. arayai, L. dringi, L. fritinniens, L. gracilis, L. hamidi, L. heteropus, L. kajangensis, L. kecil, L. marmorata, L. melanoleuca, L. maura, L. picta, L. platycephala, L. sabahmontana, and L. sola and larger than tympanum-eye distance, TMP/EYE 0.54 and TEY/TMP 0.68; upper margin of tympanum incontact with supratympanic ridge; distinct black supratympanic line present; vomerine teeth absent; vocal sac openings slit-like, paired, located posterolaterally on floor of mouth in close proximity to the margins of the mandible; tongue deeply notched behind; supratympanic ridge distinct, extending from posterior corner of eye to supra-axillary gland.
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 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, not interrupted; large, oval inner metatarsal tubercle present, outer metatarsal tubercle absent; toes webbing rudimentary; narrow lateral fringes present on all toes. Tibia 45% 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.
Dorsal surface shagreened and granular, lacking enlarge tubercles or warts, some of the granules forming short longitudinal folds; ventral skin smooth; dense tiny conical spines present on surface of chest and extending to anterior region of abdomen; pectoral gland and femoral gland 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.
Coloration of holotype in life. Dorsum dark purple brown grounding, with small light orange granules, distinct darker brown markings and rounded spots and scattered with irregular light orange pigmentations. A dark brown V-shaped pattern between anterior corner of eyes, in connected to the dark brown W-shaped marking on interorbital region, and the W-shaped marking in connected to the other Wshaped marking between axillae. Tympanum brown. A dark brown vertical bar under the eye; transverse dark brown bars on dorsal surface of limbs; distinct dark brown blotches on flanks from groin to axilla, longitudinally in two rows; elbow and upper arms with dark bars and distinct coppery orange coloration; fingers and toes with distinct dark bars.
Ventral surface grey purple, with distinct nebulous greyish speckling scattered with white spots on chest and ventrolateral flanks. Supra-axillary gland coppery orange with dark brown speckling; femoral, pectoral and ventrolateral glands greyish white. Iris bicolored, coppery orange on upper half and silver on lower half.
Coloration of holotype in preservative. Dorsum of body and limbs dark brown; transverse bars on limbs become more distinct; dark brown patterns, markings and spots on back become indistinct, orange pigmentations become greyish white. Ventral surface of body and limbs greyish white, nebulous speckling on chest and flanks balck brown. Supra-axillary, femoral, pectoral and ventrolateral glands greyish white.
Distribution and habits. Currently, Leptobrachella purpuraventra sp. nov. is known from its type locality Jinjiazhai Village in Wujing Nature Reserve, Chahe County, and Baimashan Forest Station in Zhaozishan Nature Reserve, both in Qixingguan District, Bijie City, Guizhou Province, China (Figure 1). The new species was found along a clear-water rocky stream (ca. 3 m in width and ca. 10-20 cm in depth) surrounded by a broad-leaved forest in karst landforms (Figure 9, 1600-1900 m a.s.l.). From 2 July to 4 July in 2018 at 21:00-23:50 P.M., a large number of males were found calling on leaves of plants ( Figure 10B), and some were found calling perching on the rocks or under rocks by the side of the stream.

Discussion
The discoveries of Leptobrachella bijie sp. nov. and L. purpuraventra sp. nov. bring the total number of this genus to 73, with 16 of them recorded in China (Fei et al. 2012;Frost 2017;. Before the descriptions of the two new species from northwest- ern Guizhou Province in this study, only L. oshanensis was recorded in northeastern and southern Guizhou Province, which further highlights the underestimated of the species diversity of the genus. Further investigation of the genus in adjacent regions is required.
Studies of the taxonomy and phylogeny of Leptobrachella were difficult to perform because of the morphological conservativeness of the species (for example, the two new species appeared very similar morphologically in the field (Figure 10)), which likely to hinder our understanding of these cryptic species (Ohler et al. 2010;Sung et al. 2014;. Leptobrachella bijie sp. nov. and L. purpuraventra sp. nov. were both found in Zhaozishan Nature Reserve, only approximately seven kilometers apart, straight-line distance, but they possessed a significant genetic divergence (p=3.9-4.2%). This compares to the two populations of L. purpuraventra sp. nov. from Zhaozishan Nature Reserve and Wujing Nature Reserve, which were approximately 65 kilometers apart, but displayed almost no genetic divergence. Without phylogenetic, morphological, and acoustic analyses, it would be difficult to determine the taxonomic status of these two species. Thus, specimen, acoustic data, and tissue sample collection play important roles in discovering the high species diversity of the genus Leptobrachella.
Leptobrachella bijie sp. nov. and L. purpuraventra sp. nov. were found along clearwater rocky streams, and such environments are very limited in the karst landforms. At present, little is known about the ecology and behavior of the two new species, however, the known habitat of the two new species is under threat of degradation, particularly as a result of grazing. Thus, further research on the true distribution, population size and trends, and conservation actions required, are urgently needed.