A new species of stream-living toad (Anura: Bufonidae: Bufo ) from Guangdong, China

In this work, we describe a new species of genus Bufo , Bufo exiguus sp. nov. from Mt. Nankun, Guangdong Province, China. This new species can be distinguished from all congeners by significant divergences in the mitochondrial 16S rRNA and CO1 genes and by a combination of morphological characters: small body size, tympanum absent, parotoid glands small and olive-shaped, tarsal fold absent, dorsal body with a fine vertebral line and white nuptial spinules present on dorsal and inner surfaces of fingers I and II in males. At present, Bufo exiguus sp. nov. is only known from the slow-flowing montane streams from its type locality and its conservation status should be carefully addressed.


Introduction
The genus Bufo Garsault, 1764, as a typical representative of the Old World, is widespread from temperate Eurasia and adjacent islands extending to North Africa and the Middle East, eastwards to Japan and through mainland China and southwards to Myanmar and northern Vietnam (Frost 2023).Previous systematic studies support the genus Bufo as a monophyletic group, but the phylogenetic relationships within the genus have been a controversial topic for many years and have still not been resolved (Macey et al. 1998;Liu et al. 2000;Fu et al. 2005;Igawa et al. 2006;Zhan and Fu 2011;Recuero et al. 2012;Che et al. 2020;Othman et al. 2022;Dufresnes and Litvin-chuk 2022).Despite the controversy, the monophyletic Bufo is recognised to contain 24 species at present and about three-quarters (18 species) of this genus are known to occur in China (Frost 2023).Liu and Hu (1962) described Bufo cryptotympanicus, based on a series of specimens from Huaping Nature Reserve, Longsheng, Guangxi, China.Later studies reported two more distribution sites in Guangxi (Xing'an and Ziyuan) expanding its distribution range to Guangdong (Longmen, Heyuan and Ruyuan), Yunnan (Lyuchun) and northern Vietnam (Lao Cai and Da Nang) (Yang 1991;Fei et al. 2009;Nguyen et al. 2009 and references there-in; Li et al. 2011).Yang (2008) re-identified the Lyuchun (previously transliterated as Luchun) population as a new species and transferred it to the genus Torrentophryne Yang, 1996, namely T. luchunnica Yang & Rao, 2008.Therefore, the distribution range of B. cryptotympanicus is recorded from northern Guangxi and northern Guangdong in China and northern Vietnam.
During our field surveys in Mt.Nankun, Longmen County, Guangdong Province, China (Fig. 1), we collected a series of specimens of small-sized Bufo species near mountain streams, which were previously reported as B. cryptotympanicus (Fei et al. 2009(Fei et al. , 2010(Fei et al. , 2012;;Li et al. 2011;Fei and Ye 2016).However, these specimens displayed significant morphological differences from other congeners, including small body size, tympanum absent, parotoid glands small and olive-shaped, tarsal fold absent, dorsal body with a fine vertebral line and white nuptial spinules present on dorsal and inner surfaces of fingers I and II in males.Further molecular data revealed that these specimens belong to a distinct evolutionary lineage separated from all other recognised Bufo species and, hence, it should be considered as a new species and described herein.

Specimens and morphology
Five specimens of the genus Bufo were collected from Mt. Nankun, Guangdong, China from 2015 to 2017.All specimens were fixed in 10% buffered formalin and later transferred to 70% ethanol and deposited in the Museum of Biology, Sun Yat-sen University (SYS) and Chengdu Institute of Biology, Chinese Academy of Sciences (CIB), China.
All measurements were taken with digital callipers (Neiko 01407A Stainless Steel 6-Inch Digital Caliper, USA) to the nearest 0.1 mm.Morphological characters used and their measurement methods followed Fei et al. (2009), the webbing formula followed Savage and Heyer (1997) and the computational method for sexual size dimorphism index (SSDi) followed Nali et al. (2014).
The following measurements and abbreviations are used: SVL snout-vent length (from tip of snout to vent); HDL head length (from the tip of snout to the rear of jaws); HDW head width (head width at commissure of jaws); SNT snout length (from the tip of snout to the anterior corner of eye); IND internarial distance (distance between nares); IOD interorbital distance (minimum distance between middle upper eyelids); ED eye diameter (diameter of exposed portion of eye ball); HND hand length (distance from distal end of radioulna to tip of phalanx of finger III); RAD radioulna length; FTL foot length (distance from distal end of tibia to tip of distal phalanx of toe IV); TIB tibia length (distance from knee to heel, flexed at 90°); PL, maximum parotoid gland length; PW, maximum parotoid gland width.The sex of specimens was determined by dissection or observation of secondary sexual characters (i.e., the presence of nuptial pads in males).Enlarged gonads and mature eggs were considered evidence of adulthood.
In addition, the specimens were compared with other congeners of genus Bufo on the basis of descriptions in literature (Matsui 1976;Kou 1984;Yang et al. 1996;Litvinchuk et al. 2008;Yang and Rao 2008;Zhao 2008;Kuo, 2009;Fei and Ye 2016;Rao 2022) and specimens stored in SYS.

Molecules
For phylogenetic analysis, 20 tissue samples (liver or muscle) were used.The samples were obtained from euthanised specimens and then preserved in 95% ethanol and stored at -20°C.In addition, 44 sequences of genus Bufo were obtained from GenBank for phylogenetic analyses.GenBank accession numbers of all sequences are shown in Table 1.
Genomic DNA was extracted from muscle tissue samples, using a DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.Two fragments of the mitochondrial genes that encode partial 16S ribosomal RNA gene (16S) and partial cytochrome oxidase subunit I gene (CO1) were amplified.Primers used for 16S were L3975 (5'-CGCCTGTTTACCAAAAACAT-3') and H4551 (5'-CCGGTCTGAACTCAGATCACGT-3') following Simon et al. (1994) and primers used for CO1 were dg-LCO (5'-GGTCAACAAATCATAAAGAYATYGG-3') and dgHCO (5'-AAACTTCAGGGTGACCAAARAAY-CA-3') following Meyer et al. (2005).PCR amplifications were processed with the cycling conditions of the initial denaturing step at 95°C for 5 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; and a final extending step of 72°C for 10 min.PCR products were purified with spin columns and then sequenced with both forward and reverse primers using BigDye Terminator Cycle Sequencing Kit following the guidelines, on an ABI Prism 3730 automated DNA sequencer by Wuhan Tianyi Huiyuan Bioscience & Technology Inc.
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, 987 base pairs (bp) of 16S and 635 bp of CO1, were concatenated seriatim into a 1322 bp single sequence.The dataset was partitioned according to genes and for codons for CO1 and tested respectively in jModelTest v.2.1.2with Akaike and Bayesian Information Criteria, all resulting in the best-fitting nucleotide substitution models of GTR + I + G. Sequenced data were analysed using Bayesian Inference (BI) in MrBayes 3.2.4(Ronquist et al. 2012) and Maximum Likelihood (ML) in RaxmlGUI 1.3 (Silvestro and Michalak 2012).Two independent runs were conducted in the BI analysis with 2,000,000 generations each and sampled every 1000 generations with the first 25% of samples discarded as burn-in, resulting in a potential scale reduction factor (PSRF) of < 0.005.In the ML anal-

Results
The ML and BI analyses resulted in essentially identical topologies (Fig. 2), but there were discordant topologies at some nodes within the Bufo gargarizans complex.The mean uncorrected p distances, based on 16S amongst all in-group species used in this study, are given in Table 2.The phylogenetic analyses recovered a strongly-supported monophyletic lineage containing all Bufo samples used in this study (BPP = 1.00;BS = 100), except a misidentified sequence from GenBank (KF665335).Two branches within genus Bufo are shown in our phylogenetic tree, but with low intrageneric nodal supports, so intrageneric phylogenetic relationships are not clear.Our phylogenetic analyses showed that B. bankorensis, B. gargarizans, B. tibetanus and B. tuberculatus were clustered into a species complex with low intrapopulation genetic differentiation, the systematic relationships amongst them requiring more evidence to determine.Besides, the three samples, identified as B. cryptotympanicus, were not clustered in a monophyletic group.Two samples from Guangxi, China were clustered into one lineage with low genetic differentiation (0.2%), representing the true B. cryptotympanicus.However, another B. cryptotympanicus sample from Vietnam was clustered with a topotypic sample of B. aspinia into one clade with significant genetic differentiation (2.2%).
The results recovered an unnamed independent evolutionary lineage from Mt. Nankun, Guangdong, China with strong nodal support (BPP = 1.00;BS = 100) and low intrapopulational genetic differentiation (0%), which is significantly different from other congeners in phylogeny.Therefore, this unnamed population from Mt. Nankun is considered as a separately evolving lineage and an undescribed species.
The diagnostic characters separating all species of Bufo are given in Table 4.The morphological examination on the specimens from Mt. Nankun also revealed a combination of morphological characters that distinctly differed from all other known species in the genus Bufo, including small body size, tympanum absent, parotoid glands small and olive-shaped, tarsal fold absent, dorsal body with a fine vertebral line and white nuptial spinules present on dorsal and inner surfaces of fingers I and II in males.Based on the molecular and morphological evidence, we hereby describe these specimens from Mt. Nankun as a new species, Bufo exiguus sp.nov.
Diagnosis.Bufo exiguus sp.nov. is distinguished from other species in the genus Bufo by a combination of the following morphological characters: (1) small body size (adult males with SVL 43.2-43.3mm, adult females with SVL 48.5-52.4mm); (2) tympanum absent; (3) parotoid glands small, olive-shaped; (4) tarsal fold absent; (5) dorsal body with a fine vertebral line; (6) numerous white granular nuptial spinules present on dorsal and inner surfaces of fingers I and II in males.
Etymology.The specific name exiguus means small in Latin and refers to its small body size.According to their distribution and ecological habit, we suggest the common name as "Guangdong stream toad" and Chinese formal name as "guǎng dōng xī chán" (广东溪蟾).
Radioulnar length almost equal to hand length (RAD/ HND = 1.01); hand without webbing, fingers without lateral fringes, relative finger length I = II < IV < III; tips of fingers round, not enlarged, without toe pads; subarticular tubercles prominent, rounded and not divided, present in each phalangeal joint; inner metacarpal tubercle long oval and prominent, lying at the base of finger I; outer metatarsal tubercle rounded, indistinct, boundary unclear; nuptial pad on the medial dorsal surface of the first and second fingers, bearing numerous white granular nuptial spinules.
Hind-limbs short and stout, tibio-tarsal articulation reaching middle point of parotoid gland when hind-limb stretched alongside the body; heels not meeting when flexed hind-limbs held at right angles to body axis; tibia length 0.40 of SVL and foot length 0.60 of SVL; relative toe length I < II < V < III < IV; tips of toes round, not dilated; toes with lateral fringes and with one third webbing, webbing formula I0-1 + -II0 -1 -III1-2 -IV2 --½V; subarticular tubercles rounded, not divided; inner and outer metatarsal tubercle ovoid, the former being much larger than the latter; tarsal fold absent.Dorsal surface of head and body relatively smooth, all the tubercles and granules without horny spinules; a few rounded tubercles and granules scattered on dorsum, especially in the region between parotoid glands, without large warts.Loreal region relatively smooth, only with a few flat granules; some small tubercles and granules present in temporal region, two larger tubercles at the mandibular articulation; flanks relatively smooth, only with a few granules; dorsal surfaces of limbs relatively smooth, fore-limbs only with small granules, hind-limbs with small granules, some large flat tubercles scattered on dorsal surfaces of tibia; ventral surfaces of head and body wrinkled, with scattered small granules; ventral surfaces of hands and feet with a few flat tubercles.
Colouration of holotype in preservative.Dorsal ground colour of head and body faded to greyish-brown; a dark brown "V"-shaped marking between eyes, two "inverted V"-shaped markings on the back of body; an indistinct thin vertebral line running from the snout to the upper part of the vent; dorsal surface of limbs greyish-brown, a dark brown band on forearm, some blurry blotches in upper arm, several distinct dark brown bands on femur, tibia and tarsus.Some dark brown blotches on the edge of the lips and mandibular articulation; a dark brown stripe from the nare across lower part of parotoid gland, extending to the groin.Ventral surface of throat and chest brown with a few blurry spots and worm-like markings; ventral surface of body pale yellow with dark brown worm-like markings, many markings agglomerating into a large blotch in the centre of the belly; ventral surfaces of limbs pale yellow, with dark brown spots and worm-like markings; ventral surface of hands and feet dark brown, tips of digits, subarticular tubercles and metatarsal tubercles fading to pale white.

Morphological variation.
Measurements of all individuals are given in Table 3 and the aspects of three female paratypes are given in Figure 4.
Adult females are larger than males, SSDi = 0.84.Both males have nuptial pads on the dorsal surfaces of the first and second fingers, bearing granular white nuptial spinules.All the paratypes share similar aspects with the holotype in general, apart from the following differences.The relative finger length I < II < IV < III in four paratypes (CIB 19028, SYS a005584, 5715 and 5716); tibio-tarsal articulation reaching to the posterior edge of the parotoid gland when the hind-limb is stretched alongside the body in SYS a005791.Besides, based on existing specimens, the females have rougher skin texture than males, dorsal surfaces of head and body with many rounded tubercles and granules, but all the tubercles and granules are without horny spinules; in the loreal region and temporal region, small tubercles and granules, two or three larger tubercles at the mandibular articulation are present; dorsal surfaces of limbs rough, fore-limbs with many small granules, hind-limbs with small granules and tubercles, some large tubercles scattered on dorsal surfaces of tibia; ventral surfaces of hands and feet with multiple flat tubercles.
As the general aspect of colouration of the holotype in life is not available, we describe the colouration of three female paratypes in life below.
All female paratypes share similar colouration in life.The dorsal surface of the head and body brown or yellowish-brown with a few separated dark brown and rusty red blotches; a dark brown "V"-shaped or inverted hollow triangularly-shaped marking between eyes, two "inverted V"-shaped markings on the back of body; a thin vertebral line running from the snout to the upper part of the vent.Dorsal surface of limbs have the same colour as the dorsal surface of the body, a distinct dark brown band on forearm, some indistinct blotches on the upper arm, several distinct dark brown bands on the femur, tibia and tarsus (rusty red blotches scattered on the limbs in SYS a005584).Some dark brown blotches on the edge of the lips and mandibular articulation; a dark brown stripe from the nares across the lower part of parotoid glands and flanks, extending to the groin (stripe discontinuous in the flanks in SYS a005716).Ventral surface of throat and chest light brown with a few darker spots and worm-like markings; ventral surface of body white with many dark brown worm-like markings, mostly in the centre of the belly; ventral surfaces of limbs showing the same colour as the ventral surface of the body, with dark brown spots and worm-like markings; ventral surface of hands and feet greyish-brown, tips of digits, subarticular tubercles and metatarsal tubercles faintly white.Pupil black; iris golden with black reticular markings, black blotches in four directions.

Discussion
The genus Bufo has a long history and its concept has been changed many times (Frost 2023).Similar to previous molecular phylogenetic studies, our results support the current recognition of genus Bufo as a monophyletic group with high diversity, but the intrageneric phylogenetic relationships are still not resolved.Our phylogenetic results showed that B. bankorensis, B. gargarizans, B. tibetanus and B. tuberculatus were clustered into the B. gargarizans complex with low intrapopulation genetic differentiation, but due to the lack of samples of topotype and low support at some nodes, the taxonomic placements of these four species require more evidence to determine.
In addition, we suggest the sample (AMNH 13198), reported as B. cryptotympanicus from Vietnam, is probably a misidentification of B. aspinia and, therefore, the record of this species from Vietnam requires further classification.It can be seen that the lack of available sequences and misidentified sequences for some species has affected the understanding of the systematics of the genus Bufo and the link to voucher specimens is needed to assist the identification.Besides that, frequent taxonomic changes indicate that previous knowledge on the diversity and taxonomy of genus Bufo is inadequate and more work is needed in the future.
There are generally two different ecological niches for the congeners of genus Bufo: stream-living type and terrestrial type (Fong et al. 2020).Our phylogenetic analysis shows that these two niche differentiations have occurred multiple times in the genus Bufo, but the phylogenetic relationships have not been resolved between each other.The stream-living species usually have small to medium body size, no tympanum, abdominal sucker in the tadpole stage, prefer wetter environments and even inhabit montane streams and spawn in streams rather than lentic habitats (Matsui 1976;Yang et al. 1996;Fong et al. 2020;this study).On the other hand, the terrestrial congeners usually have a medium to large body size, tympana are present and are mainly terrestrial beyond the breeding seasons.In addition, stream-living species are more vulnerable to habitat loss, human activities and other threats than terrestrial congeners (Jiang and Xie 2021a, b).The majority of these species are restricted to relatively small distribution areas and several species (Bufo ailaoanus, B. aspinius, B. luchunnicus, B. menglianus, B. exiguus and B. yongdeensis) are currently only known from their type localities, which are more vulnerable and careful attention should be paid to their conservation.

Figure 2 .
Figure 2. Bayesian inference tree inferred from 16S rRNA and CO1 genes.The Bayesian posterior probabilities (BPP) < 0.95 or the bootstrap supports (BS) < 70 are not given in the tree.

Figure 3 .
Figure 3. Morphological features of the adult male holotype SYS a005790 of Bufo exiguus sp.nov. in preservative.A dorsal; B ventral; C close-up of head; D dorsal view of right hand; E ventral view of left hand; F ventral view of left foot.Photos by Shuo Qi.

Table 1 .
Localities, voucher information and GenBank accession numbers for all samples used in this study.

3 Bufo exiguus sp. nov.
(Huelsenbeck et al. 2001;Wilcox et al. 2002)om 1000 replicates was generated.Nodes with Bayesian posterior probabilities (BPP) ≥ 0.95 and the bootstrap supports (BS) for Maximum Likelihood analysis ≥ 70 were considered as strongly supported(Huelsenbeck et al. 2001;Wilcox et al. 2002).Pairwise distances (p distances) were calculated in MEGA6 using the uncorrected model.Gaps/Missing Data Treatment used the complete-deletion option, substitutions to include the d: Transitions + Transversions option.

Table 3 .
Measurements (in mm) and ratios of the type series of Bufo exiguus sp.nov., * indicates the holotype.April and May.Both males possessed nuptial spinules, but no eggs were observed in the adult female's oviduct by dissection, suggesting their breeding season should be earlier than April.Nevertheless, the tadpoles and most of the other ecology and types of behaviour of this toad remain unknown.