﻿A new species of the genus Leptobrachella Smith 1925 (Amphibia, Anura, Megophryidae) from Guangxi, China

﻿Abstract A new species of Leptobrachella, L.wumingensissp. nov., was described from the Damingshan National Nature Reserve, Wuming District, Nanning City, Guangxi, China based on morphological, molecular and bioacoustic data. Phylogenetic analysis of 16S mtDNA fragments revealed that the new species is closely related to L.damingshanensis. Uncorrected p-distances between the new species and all homologous DNA sequences available for the 16S gene of Leptobrachella are greater than 7.1%. Morphologically, L.wumingensissp. nov. differs from its congeners in several ways, including a medium body size (SVL 26.0–26.7 mm in males, 30.6–34.8 mm in females), lack of toe webbing and lateral fringes, shagreened and granular dorsal surface, pale brown dorsum with darker brown markings, iris bicolored, with the upper half copper and fading to silver in the lower half, and the presence of small irregular black spots and tangerine tubercles on the flanks. Furthermore, we found the new species to have two types of advertisement calls and relatively high dominant frequencies, making it distinct from its congeners.


Introduction
The genus Leptobrachella Smith, 1925 is distributed in northeastern India, Southeast Asia, Vietnam, Thailand, Myanmar, Malaya, Borneo and Natuna Island (Frost 2023).The species diversity of Leptobrachella has been drastically underestimated due to its small body size, conserved morphology and high degree of sympatry (Chen et al. 2018;Chen et al. 2021aChen et al. , 2021b)).However, by utilizing morphological, molecular and bioacoustic data, numerous new Leptobrachella species have been described in recent decades (Frost 2023).
The genus now comprises 99 species, of which 37 were described in the last five years (Frost 2023).In China, there are 39 Leptobrachella species, and 27 of them were described in the past five years, mostly occurring in southern and western China (AmphibiaChina 2023), indicating an underestimation of the diversity of Leptobrachella.
Between 2019 and 2023, we conducted surveys at the Damingshan National Nature Reserve (DMS), Wuming District, Nanning City, Guangxi, China.We collected nine specimens (hereafter DMS specimens) of Leptobrachella that significantly differed from the sympatric species, L. damingshanensis Chen, Yu, Cheng, Meng, Wei, Zhou & Lu, 2021 and other congeners in morphological characters, including body size and color pattern as well as molecular data.The uncorrected p-distances between 16S mtDNA fragments of DMS specimens and all homologous sequences of Leptobrachella available in GenBank were greater than 7.1%.Additionally, the bioacoustic data of the DMS specimens distinguished them from L. damingshanensis and other advertisement calls available in the genus Leptobrachella.Given the unique morphological characters, the relatively high degree of mtDNA divergence, and the distinct bioacoustics data, we describe the DMS specimens as a new species of Leptobrachella.

Material and methods
Nine Leptobrachella specimens were collected from the Damingshan National Nature Reserve, Wuming District, Nanning City, Guangxi, China (Fig. 1).After fixing the specimens in 10% formalin, they were stored in 75% ethanol and deposited at Nanning Normal University (NNU).Muscle tissue was taken before fixing and stored in 100% ethanol for DNA isolation.We measured all specimens to the nearest 0.1 mm using a digital caliper, following Fei et al. (2009) and Rowley et al. (2016), which included the snout-vent length (SVL), head length from tip of snout to rear of jaws (HL), head width at commissure of jaws (HW), snout length from tip of snout to the anterior eye (SNT), diameter of the exposed portion of eye (ED), interorbital distance (IOD), internarial distance (IN), horizontal diameter of tympanum (TD), distance from anterior edge of the tympanum to posterior corner of eye (TED), manus length from tip of third finger to base of inner palmar tubercle (ML), forelimb length from elbow to the tip of third finger (FLL), thigh length from vent to knee (THL), tibia length with flexed hindlimb (TIB), pes length from tip of fourth toe to base of inner metatarsal tubercle (PL), and maximum diameter of femoral gland (FEM).Sex was determined by the calls of males or gonadal inspection.Comparative morphological data were collected from the literature (Suppl.material 1: table S1) and from the collected specimens (Suppl.material 1: table S2).
Genomic DNA was isolated from muscle tissue using Tiangen Biotech Co. Ltd. tissue extraction kits (Beijing, China).The primer pairs 16Sar_L 5'-CG-CCTGTTTAC CAAAAACAT-3' and 16Sbr_H 5'-CCGGTCTGAACTCAGATCAC-GT-3' (Palumbi et al. 1991) were used to amplify and sequence mitochondrial 16S rRNA gene fragments (~530 bp).The polymerase chain reaction (PCR) amplification was carried out in a 20 μl reaction volume involving the following steps: an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 35 s, annealing at 58 °C for 40 s, and extension at 72 °C for 40 s, and a final extension step at 72 °C for 10 min.The fragments were sequenced using an ABI Prism 3730 automated DNA sequencer (Applied Biosystems, USA), and the newly obtained sequences were submitted to GenBank (accession numbers OM935575-OM935578, OR194551-OR194553).Phylogenetic relationships were inferred using 16S mtDNA fragments through maximum likelihood (ML) and Bayesian inference (BI) analyses.Table 1 provides detailed information on the sequences used in the molecular analyses, which were aligned using the Clustalw module in MEGA v. 7 (Kumar et al. 2016) with default settings.The best-fit models of evolution (GTR+I+G) were determined using MrModeltest v. 2.3 (Nylander 2004) with Akaike and Bayesian information criteria.ML analyses were conducted on the CIPRES science gateway, estimating the proportion of invariable sites from the data and performing 1000 bootstrap pseudo replicates (https://www.phylo.org/portal2)(Miller et al. 2010).BI was performed using MrBayes v. 3.2 (Ronquist et al. 2012), with two independent runs and four Markov Chain Monte Carlo simulations for 30 million iterations.Trees were sampled every 1000 generations, with the first 25% of trees discarded as burn-in.Node values were considered well supported if ML bootstrap supports (BS) were ≥ 70% and Bayesian posterior probabilities (PP) were ≥ 0.95.Outgroups used were Leptobrachium huashen and Xenophrys glandulosa following Chen et al. (2018).Uncorrected p-distances were calculated based on 16S gene fragments using Mega v. 7 with default settings.
Two male advertisement calls were recorded using a SONY ICD-TX50 recorder at an ambient temperature of approximately 21 °C.Acoustic analysis was performed using Raven Pro 1.6 (Cornell Laboratory of Ornithology, Ithaca, NY, USA) following the method outlined by Köhler et al. (2017).The protocol involved calculating audio-spectrograms with a fast-Fourier transform (FFT) of 512 points, 50% overlap, and 172 Hz grid-spacing, using Hanning windows.Refer to Suppl.material 1: table S3 for comparison data on bioacoustics.

Molecular analyses
The molecular analyses conducted using BI and ML methods produced similar topologies, as depicted in Fig. 2.However, the phylogenetic relationships of Leptobrachella species showed weak internal node support and remained unresolved (Fig. 2).The phylogenetic trees indicated that the DMS specimens are closely related to L. damingshanensis, L. nahangensis and L. nyx.Furthermore, the genetic divergence of the 16S gene fragment between the DMS specimens and all other available homologous sequences of Leptobrachella species was found to be greater than 7.1% for the analyzed fragment (Suppl.material 1: table S4).

Bioacoustics
The calls of two individuals were recorded in the field (NNU 01058 and NNU 01086).Two types of advertisement calls (Type A and Type B; Fig. 3) were detected.The call durations of Type A ranged from 0.2454 s to 0.4306 s, with an average of 0.3407 ± 0.0423 s, while the call intervals of Type A ranged from 0.1784 s to 0.3756 s, with an average of 0.2364 ± 0.0333 s.For Type B, the call durations ranged from 0.0416 s to 0.0903 s, with an average of 0.0605 ± 0.0094 s, and the call intervals ranged from 0.3700 s to 1.7740 s, with an average of 0.8932 ± 0.3810 s.The dominant frequencies were observed to be between 6.0-7.5 kHz (at 21.0 °C).The DMS specimens exhibited two types of calling models and relatively high dominant frequencies, which distinguishes them from the known advertisement callings in the genus Leptobrachella (Suppl.material 1: table S3).

Morphological characters
The DMS specimens can be readily differentiated from other species in the same phylogenetic clade.For instance, adult males of L. damingshanensis have a significantly larger body size (SVL 33.6-34.4mm) and possess a pair of distinct tangerine glands on the proximal thigh area, as well as rudimentary webbing and narrow lateral fringes on their toes.In contrast, the DMS specimens have indistinct tangerine glands on the proximal thigh area, and no toe webbing or lateral fringes.
Taken together, molecular data, acoustic analyses and morphological characters support the conclusion that the DMS specimens represent a distinct and previously unrecognized species of Leptobrachella, which is described below.Etymology.The specific name 'wumingensis' is derived from the type locality, Wuming District, Nanning City, Guangxi, China.The proposed common name in English is Wuming Leaf Litter Toad, and in Chinese, it is called Wu Ming Zhang Tu Chan (武鸣掌突蟾).
Diagnosis.Leptobrachella wumingensis sp.nov. is classified under Leptobrachella based on specific morphological features, including its relatively small body size, presence of an inner metacarpal tubercle, macro-glands on the supra-axillary and femoral glands, lack of vomerine teeth, and a whitish vertical bar on the anterior tip of the snout, according to previous studies (Dubois 1983;Lathrop et al. 1998;Delorme et al. 2006;Matsui 2006).Leptobrachella wumingensis sp.nov.can be differentiated from other species in its genus by a combination of the following characters: (1) medium size 8 mm in females); (2) absence of toe webbing and lateral fringes; (3) shagreened and granular dorsal surface; (4) pale brown dorsum with darker brown markings; (5) iris bicolored, with the upper half copper and fading to silver in the lower half; (6) presence of small irregular black spots and tangerine tubercles on the flanks; and (7) two types of advertisement callings and high dominant frequencies.
Description of holotype.Head length almost equal to width (HW/HL = 1.02); snout bluntly rounded in profile and dorsal view, projecting slightly over lower jaw; nostril oval-shaped, closer to tip of snout than eye; canthus rostralis distinct; loreal region distinctly sloping, slightly concave; pupil vertical; eye diameter less than snout length (ED/SNT = 0.96); tympanum distinct and rounded, diameter about 49% that of eye; vomerine teeth absent; tongue with a deep notch at posterior tip; supratympanic fold distinctly raised from corner of eye to the posterior of tympanum (Fig. 4A-D).
Tips of fingers slightly swollen; relative finger lengths I < II < IV < III; subarticular tubercles absent; prominent inner palmar tubercle, small outer palmar tubercle; finger webbing and dermal fringes absent; nuptial pad on fingers absent.Tips of toes rounded, slightly swollen; relative toe lengths I < II < V = III < IV; subarticular tubercles absent, replaced by dermal ridges; prominent and elongated inner metatarsal tubercle; outer metatarsal tubercle absent; toe webbing and lateral fringes absent.Tibia 48% of SVL; tibiotarsal articulation reaching middle of eye; heels meeting when hindlimbs flexed at right angles with respect to body (Fig. 4E, F).Dorsal surface shagreened and granular; upper eyelid with small tubercles; ventral surface without tubercles; flanks with sparse tubercles; pectoral glands elongated, approximately 2.1 mm in diameter; small femoral glands oval, approximately 0.6 mm in diameter, closer to knee than to vent; supra-axillary glands oval, approximately 0.8 mm in diameter; ventrolateral glandular line discrete; ventral surface of thigh with some tubercles (Fig. 4A-D).
Color in life.Dorsum light brown with brown markings, an inverted triangle marking between eyes, an irregular brown marking on scapular region, a brown 'Λ' marking on rear of dorsum; tympanum pale brown; supratympanic fold black from the posterior corner of eye to the anterior of supra-axillary glands; two wide black bars on upper lip; five irregular black spots and several small light tangerine tubercles on flanks; transverse brown bars on dorsal surface of hindlimbs; upper arms light orange; belly with tiny creamy white spots; throat creamy white with tiny light brown spots; lower jaw with creamy white tubercles; ventral surfaces of limbs with sparse creamy white tubercles; pectoral and femoral glands creamy white, supra-axillary glands light orange; pupil black; iris bicolored, upper half copper, fading to silver in lower half (Fig. 4A-D).
Color in preservative.The dorsum and limb surfaces are faded to uniform light brown.Irregular black spots on flanks and bars on limbs are darkish brown.The throat, chest, and belly are creamy white, and pectoral, femoral, supra-axillary, and ventrolateral glands are also creamy white.
Variation.The measurements of the type series are shown in Table 2.The holotype and paratypes exhibit similar color patterns.Females are larger than males in terms of body size.The number of black spots on the flanks varies, ranging from five to eight (Fig. 5A, B).Some individuals have more tubercles on their flanks and their dorsum are rougher compared to the holotype (Fig. 5C).
Ecology and distribution.Leptobrachella wumingensis sp.nov.inhabits evergreen forests in the DMS at elevations of 1000-1200 m.All specimens were discovered over 10 m away from rocky streams (Fig. 5D, E).The species' advertisement calls were heard on the rocks in mid-April, and creamy white eggs were found on females' abdomens in April (Fig. 5F).We dissected four females collected in June and found no eggs in their abdomens.We speculate that the breeding period of the new species is between April and May.Since 2019, we have surveyed over ten rocky streams similar to the type locality but failed to find more sites where the new species occurs.The population of the new species is very rare; we have visited the type locality more than ten times since 2019, but only encountered nine adults.Although both L. wumingensis sp.nov.and L. damingshanensis occur in the DMS, the two species do not inhabit the same streams, and the closest site where both species are found is 10 km away.Currently, L. wumingensis sp.nov. is only known to occur in the DMS (Fig. 1).Comparisons.To begin with, L. wumingensis sp.nov. is distinguished from Leptobrachella species found south of the Isthmus of Kra, Malay Peninsula by the presence of supra-axillary and ventrolateral glands (vs absent in the latter).Furthermore, Leptobrachella wumingensis sp.nov.and L. damingshanensis are sympatric.Leptobrachella wumingensis sp.nov.can be differentiated from L. damingshanensis by several characters, including smaller body size in males L. eos, L. firthi, L. isos, L. pallida, L. petrops, and L. tuberosa by the presence of black spots on its flanks (vs.absent).Leptobrachella wumingensis sp.nov.differs from L. applebyi, L. bidoupensis, L. kalonensis, L. melica, L. minima, L. nahangensis, and L. tadungensis by the presence of shagreened and granular dorsal surface (vs smooth).
Lastly, L. wumingensis sp.nov.differs from other Leptobrachella species in terms of acoustic features such as relatively high dominant frequencies and two distinct types of callings (Fig. 3, Suppl.material 1: table S3).

Discussion
Based on morphological characters, molecular data and bioacoustics, we have identified the DMS specimens as a new species.The validation of this assignment is supported by significant genetic divergence (>7.1%), high dominant frequencies, complicated calling styles and substantial morphological characters.The DMS reserve is located in the central region of Guangxi, and previous fieldwork by many investigation teams did not discover any Leptobrachella species until we reported the first one in 2021 (Chen et al. 2021b).We have conducted annual amphibian surveys in the reserve since 2019 and have found that the population of L. wumingensis sp.nov. is rare, with only nine individuals found along with L. damingshanensis.Further field surveys are required to determine their distributions for conservation purposes.
According to previous research and our new data (AmphibiaChina 2023; Frost 2023), at least ten Leptobrachella species occur in Guangxi, including L. alpina, L. bourreti, L. damingshanensis, L. liui, L. maoershanensis, L. shangsiensis, L. shiwandashanensis, L. ventripunctata, L. wuhuangmontis and L. wumingensis sp.nov.Six of these species have been described in the past five years.The weak dispersal abilities and forest-dependent niches of Leptobrachella species may contribute to underestimating their diversity and distribution in Guangxi.Thus, more surveys are needed to understand the true species diversity and distribution of Leptobrachella in the region.

Figure 4 .
Figure 4. Morphological characters of L. wumingensis sp.nov.(NNU 01058) A dorsal view B ventral view C, D lateral view E ventral view of hand F ventral view of foot.

Figure 5 .
Figure 5.A dorsolateral view of NNU 01086 B dorsolateral view of NNU 01059 C dorsolateral view of NNU 00285 D NNU 201907009 in habitat (photo taken in situ) E NNU 00283 in habitat (photo taken in situ) F eggs (NNU 01059).

Table 1 .
DNA sequences used in this study.'*' represents type locality.

Table 2 .
Measurements of voucher specimens of L. wumingensis sp.nov.(mm).Abbreviations defined in text.