One adult male and three adult females of the Brachytasophrys sp. were collected from Libo County (LB), Guizhou Province, China (see Suppl. material 1, Fig. 1). In the field, the toads were euthanised using isoflurane and the specimens were fixed in 75 % ethanol. Tissue samples were taken and preserved separately in 99 % ethanol prior to fixation. The specimens were deposited in Chengdu Institute of Biology（CIB), Chinese Academy of Sciences (CAS).

Figure 1.  

Geographical location of the type locality of Brachytarsophrys qiannanensis sp. nov., Libo County, Guizhou Province, China.

All adult specimens of the Brachytasophrys sp. were measured. The terminology and methods followed Fei and Ye (2016) and Li et al. (2020). Measurements were taken with a dial caliper to 0.1 mm. Sixteen morphometric characters of adult specimens were measured: eye diameter (ED, distance from the anterior corner to the posterior corner of the eye); foot length (FL, distance from distal end of shank to the tip of Toe IV); head length (HDL, distance from the tip of the snout to the articulation of jaw); maximum head width (HDW, greatest width between the left and right articulations of jaw); internasal distance (IND, minimum distance between the inner margins of the external nares); interorbital distance (IOD, minimum distance between the inner edges of the upper eyelids); length of lower arm and hand (LAL, distance from the elbow to the distal end of the Finger IV); lower arm width (LW, maximum width of the lower arm); snout-vent length (SVL, distance from the tip of the snout to the posterior edge of the vent); snout length (SL, distance from the tip of the snout to the anterior corner of the eye); length of foot and tarsus (TFL, distance from the tibiotarsal articulation to the end of the Toe IV); thigh length (THL, distance from vent to knee); tibia length (TL, from the outer surface of the flexed knee to the heel); maximal tibia width (TW); maximal tympanum diameter (TYD); upper eyelid width (UEW, greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis). The location of the web on the phalange articulation was designated as follows: - (distal part of phalange articulation); none (middle part of phalange articulation); + (proximal part of phalange articulation); ++ (lower part of phalange articulation) followed the protocol described by Savage (1975) and Li et al. (2020).

Sex was determined by secondary sexual characters, i.e. the presence of vocal sac and nuptial pads/spines in male (Fei and Ye 2016).

The Brachytasophrys sp. was also compared with all other Brachytarsophrys species, based on morphological characteristics. Comparative morphological data were obtained from literature for B. carinense (Boulenger 1889, Li et al. 2020), B. chuannanensis (Fei and Ye 2001, Li et al. 2020), B. feae (Boulenger 1887, Li et al. 2020), B. intermedia (Smith 1921, Li et al. 2020), B. orientalis (Li et al. 2020), B. platyparietus (Rao and Yang 1997, Li et al. 2020) and B. popei (Zhao et al. 2014, Li et al. 2020).

The advertisement calls were recorded in the field on 6 August 2021 in Libo County, Qiannan Autonomous Prefecture, Guizhou Province, China. It was recorded in the stream at ambient air temperature of 18.0°C and air humidity of 80%. SONY PCM-D50 digital sound recorder was used to record within about 50 cm of the calling individual. The sound files in wave format were resampled at 48 kHz with sampling depth 24 bits. PRAAT 6.0.27 (Boersma 2001) was used to obtain the sonograms and waveforms (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). Terminology of advertisement call analyses and description followed Köhler et al. (2017). Ambient temperature was taken by a digital hygrothermograph.

Four specimens of the Brachytasophrys sp. were included in the molecular analyses (Table 1). Total DNA was extracted using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). Three fragments of the mitochondrial 16S rRNA (16S), cytochromeoxidase subunit I (COI) and cytochrome b (Cytb) genes were amplified. For 16S, the primers P7 (5'-CGCCTGTTTACCAAAAACAT-3') and P8 (5'-CCGGTCTGAACTCAGATCACGT-3') were used following Simon et al. (1994); for COI, Chmf4 (5'-TYTCWACWAAYCAYAAAGAYATCGG-3') and Chmr4 (5’-ACYTCRGGRTGRCCRAARAATCA-3’) were used following Che et al. (2012) and for Cytb, PFGlu14140L (5'-GAAAAACCACTGTTGTHHYTCAACTA-3') and PFThr15310 (5'-CGGYTTACAAGACCGRTGCTTT-3') were used following Zhang et al. (2013). Gene fragments were amplified under the following conditions: an initial denaturing step at 95 °C for 4 min; 36 cycles of denaturing at 95 °C for 30 s, annealing at 54 °C (for 16S)/49 °C (for COI)/50 °C (for Cytb) for 40 s and extending at 72 °C for 70 s. Sequencing was conducted using an ABI3730 automated DNA sequencer in Shanghai DNA BioTechnologies Co., Ltd. (Shanghai, China). New sequences were deposited in GenBank (for GenBank accession numbers see Table 1).

For molecular analyses, the available sequence data for Brachytarsophrys were downloaded from GenBank (Table 1), primarily from previous studies (Chen et al. 2017, Li et al. 2020). For phylogenetic analyses, corresponding sequences of one Atympanophrys shapingensis Liu 1950 and one Panophrys omeimontis Liu 1950 were also downloaded (Table 1) and used as outgroups according to Chen et al. (2017). Sequences were assembled and aligned using the Clustalw module in BioEdit v.7.0.9.0 (Hall 1999) with default settings. For phylogenetic analyses of mitochondrial DNA, the dataset was concatenated with 16S, COI and Cytb gene sequences. To avoid under- or over-parameterisation (Lemmon and Moriarty 2004, McGuire et al. 2007), the best partition scheme and the best evolutionary model for each partition were chosen for the phylogenetic analyses using PARTITIONFINDER v. 1.1.1 (Robert et al. 2012). In this analysis, 16S gene and each codon position of protein-coding genes were defined and Bayesian Inference Criteria was used. As a result, the analysis suggested that the best partition scheme is 16S gene/each codon position of protein-coding genes and selected GTR + G + I model as the best model for each partition. Phylogenetic analyses were conducted using Maximum Likelihood (ML) and Bayesian Inference (BI) methods, implemented in PhyML v. 3.0 (Guindon et al. 2010) and MrBayes v. 3.12 (Ronquist and Huelsenbeck 2003), respectively. For the ML tree, branch supports were drawn from 10,000 non-parametric bootstrap replicates. In BI, two runs each with four Markov chains were simultaneously run for 50 million generations with sampling every 1,000 generations. The first 25% trees were removed as the “burn-in” stage followed by calculations of Bayesian posterior probabilities and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity, bootstrap supports (BS) and Bayesian Posterior Probabilities (BPP) are shown at the nodes. Finally, mean genetic distance between samples in this study, based on uncorrected p-distance model, was estimated using MEGA v. 6.06 (Tamura et al. 2013) with the pairwise deletion setting for the Gap/Missing Data.

All the sequences in this study were retrieved from GenBank and the accession numbers of the newly-determined sequences in this study are shown in Table 1.

Phylogenetic analyses

Aligned sequence matrix of 16S + COI + Cytb contains 2061 bp. ML and BI trees of the mitochondrial DNA dataset presented almost consistent topology (Fig. 7). Brachytarsophrys qiannanensis sp. nov. was clustered into the Brachytarsophrys clade and grouped with B. orientalis and B. popei with high supported values (node supports in ML and BI: 90 and 1.00).

Figure 7.  

Phylogenetic tree reconstructed using Bayesian Inference (BI) and Maximum Likelihood (ML) methods, based on 16S, COI and Cytb genes. Values at the nodes correspond to BPP and BS. Samples 1–36 refer to Table 1.

The mean genetic distances (p-distance) between Brachytarsophrys qiannanensis sp. nov. and its congeners was 2.1% on 16S (with B. popei), 4.9% on COI (with B. orientalis) and 6.6% on Cytb (with B. orientalis), these distances being much higher than those between some pairs of recognised congeners (Suppl. materials 3, 4, 5); for example, the COI p-distance was 4.3% between B. orientalis and B. popei, while the Cytb p-distance between B. orientalis and P. popei was 3.3%.

Molecular phylogenetic analyses showed that the population of Brachytarsophrys from Libo County, Qiannan Autonomous Prefecture, Guizhou Province, China is also distinct from its congeners.

The previous morphological studies indicated that, in the genus Brachytarsophrys, only B. carinense was recorded from Leishan and Anlong Counties of Guizhou Province (e.g. Wu et al. 1986, Fei et al. 2009, Fei et al. 2012). Lyu et al. (2014) further identified the population from Fanjing Mountain, Guizhou Province as B. chuannanensis, based on morphological comparisons. Based on molecular phyogenetic analyses and morphological comparisons, Li et al. (2020) suggested that B. platyparietus should be a valid species and populations in south-western China previously recognised as B. carinense should be re-identified as B. platyparietus, including the population from Fanjing Mountain, Guizhou Province. Moreover, Li et al. (2020) suggested that the populations from the western Hunan Province and north-western part of Guangxi Province, all adjoining Guizhou Province, belonged to B. popei and B. orientalis, respectively. Accordingly, it is inferred that the population of Brachytarsophrys from Anlong County in western Guizhou Province, near Yunnan Province, may be B. platyparietus and that from Leigong Mountain in south-eastern Guizhou Province, near the Hunan Province, may be B. popei or B. orientalis. In addition, the taxonomic assignments of the specimens from Fanjing Mountain, Guizhou Province are still doubtful because Li et al. (2020) reported that the specimen from Fanjing Mountain unexpectedly shared a common holotype on the COI gene with several specimens from the northern and central parts of Yunnan Province which is quite far from Fanjing Mountain in the eastern Guizhou Province. It is a pity that Li et al. (2020) did not provide more morphological and bioacoustics information of this specimen for comparisons with other species. Obviously, the taxonomic profiles of Brachytarsophrys in Guizhou Province are still unresolved and further investigations on this group should be conducted in the region.

Brachytarsophrys qiannanensis sp. nov. seems to be the smallest species (SVL 70.1 mm in male and SVL < 85 mm in females) in the genus Brachytarsophrys. Whether its niche characteristics promote the special morphology in this species maybe an interesting evolutionary question.

In recent years, more than 20 new amphibian species have been discovered in Guizhou Province, China (Frost 2022). However, during our frequent and extensive surveys in Guizhou Province from 2016 to 2021, we only found one adult male and three adult females of Brachytarsophrys qiannanensis sp. nov. This perhaps indicated that the population of the species in Guizhou Province is potentially small. Hence, further surveys are needed to evaluate the population status of the species.