A new species of the Music frog Nidirana (Anura, Ranidae) from Guizhou Province, China

Abstract The Music frog genus Nidirana is widely distributed in East and South Asia. Here, a new species of the genus is described from southwestern China. Phylogenetic analyses based on the mitochondrial 16S rRNA and COI gene sequences supported the new species as a clade closely related to N. leishanensis, N. hainanensis, N. chapaensis, N. daunchina, and N. yaoica. The new species could be distinguished from its congeners by a combination of the following characters: body of medium size (SVL 41.2–43.5 mm in males and 44.7 mm in female); lateroventral groove only present on toes; relative finger lengths: II < IV < I < III; three metatarsal tubercles on palm; heels overlapping when hindlimbs flexed at right angles to axis of body; tibiotarsal articulation reaching the level of eye when leg stretched forward; a pair of subgular internal vocal sacs at corners of throat in male; nuptial pad present on the inner side of base of fingers I in breeding male; tadpole labial tooth row formula with 1:1+1/1+1:2; in males, the advertisement call contains two kinds of notes and one call contains 2–6 repeated regular notes.

In all Nidirana species, N. adenopleura and N. daunchina were reported to have the widest distributional ranges in southwestern China and south-eastern China, respectively (Fei et al. 2009(Fei et al. , 2012. Recently, two species (N. yaoica and N. leishanebsis) were recognised from two populations which had been identified as N. adenopleura although they were not phylogenetically sister taxa to N. adenopleura (Lyu et al. 2019a;Li et al. 2019a). As well, it is expected that there are cryptic species in populations being recognised as N. daunchina in its wide distributional range. Wu et al. (1986) found that the population classified as N. adenopleura from Suiyang County, Guizhou Province, China (later classified as N. daunchina by Fei et al. 2009) had some morphological differences with the population of the species from its type locality (E'mei Mountain, Sichuan Province, China). Hence, deeper investigations using molecular phylogenetic approaches are necessary to evaluate the taxonomic status of these populations.
In recent years, we carried out a series of biodiversity surveys in Tongzi County, Guizhou Province, China, and collected eleven specimens of Nidirana. Molecular phylogenetic analyses, morphological comparisons, and bioacoustics comparisons indicated the specimens as an unnamed species of Nidirana. We describe it herein as a new species.

Specimens
Nine adult males, one adult female, and one tadpole of the new species were collected from Huanglian Town, Tongzi County, Guizhou Province, China from 2015 to 2019 (for voucher information see Table 1, Fig. 1, Suppl. material 1: Table S1). After taking photographs, the animals were euthanised using isoflurane, and the specimens were then fixed in 10 % buffered formalin. Tissue samples were taken and preserved separately in 95 % ethanol prior to fixation. Specimens were deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS).

Molecular data and phylogenetic analyses
Four male specimens, one female specimen, and one tadpole of the new species were included in the molecular analyses (for voucher information see Table 1). For phylogenetic comparisons, the corresponding sequences for other Nidirana species, two Babina species, and one Odorrana margaretae for which comparable sequences were available were downloaded from GenBank (Table 1). Odorrana margaretae was used as the outgroup following previous studies (Lyu et al. 2017(Lyu et al. , 2019aLi et al. 2019a).
Sequences were assembled and aligned using the Clustalw module in BioEdit v. 7.0.9.0 (Hall 1999) with default settings. Alignments were checked by eye and revised manually if necessary. For phylogenetic analyses of mitochondrial DNA, the dataset concatenated with 16S and COI 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 COI gene were defined, and Bayesian Inference Criteria was used. As a result, the analysis suggested that the best partition scheme is16S gene/each codon position of COI gene, 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 nonparametric 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. Finally, mean genetic distance between Nidirana species based on uncorrected p-distance model was estimated on the 16S gene using MEGA v. 6.06 (Tamura et al. 2011).

Morphological comparisons
All ten adults (Suppl. material 1: Table S1) and one tadpole of the new species were measured. For comparisons, three adult male specimens of N. daunchina freshly collected from its type locality (E'mei Mountain, Sichuan Province, China) were measured (Suppl. material 1: Table S1), and measurements of N. yaoica were retrieved from Lyu et al. (2019a). The terminology and methods followed Fei et al. (2005), Mahony et al. (2011), andLyu et al. (2019a). Measurements were made with a dial caliper to the nearest 0.1 mm. Twenty-four morphometric characters of adult specimens were measured: SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent); HDL head length (distance from the tip of the snout to the articulation of jaw); HDW head width (greatest width between the left and right articulations of jaw); SL snout length (distance from the tip of the snout to the anterior corner of the eye); ED eye diameter (distance from the anterior corner to the posterior corner of the eye); IOD interorbital distance (minimum distance between the inner edges of the upper eyelids); IND internasal distance (minimum distance between the inner margins of the external nares); UEW upper eyelid width (greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis); TYD maximal tympanum diameter; TED tympanum-eye distance (from anterior edge of tympanum to posterior corner of the eye); LAL length of lower arm and hand (distance from the elbow to the distal end of the finger IV); LW lower arm width (maximum width of the lower arm); HND hand length (from distal end of radioulna to tip of distal finger III); RAD radioulna length (from the flexed elbow to the base of the outer palmar tubercle); FIL first finger length (measured from the base of the second finger to the tip of the first finger); FIIL second finger length (measured from the base of the first finger to the tip of the second); FIIIL third finger length (measured from the base of the second finger to the tip of the third); FIVL fourth finger length (measured from the base of the third finger to the tip of the fourth); HLL hindlimb length (maximum length from the vent to the distal tip of the toe IV); TL tibia length (distance from knee to tarsus); TW maximal tibia width; THL thigh length (distance from vent to knee); TFL length of foot and tarsus (distance from the tibiotarsal articulation to the distal end of the toe IV); FL foot length (distance from tarsus to the tip of the fourth toe).
The stage of the tadpole was identified following Gosner (1960). Ten morphometric characters of tadpole specimen were measured: TOL total length; SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent); BH maximum body height; BW maximum body width; SL snout length (distance from the tip of the snout to the anterior corner of the eye); SS snout to spiraculum (distance from spiraculum to the tip of the snout); IOD interorbital distance (minimum distance between the inner edges of the upper eyelids); TBW maximum width of tail base; TAL tail length (distance from base of vent to the tip of tail); TAH tail height (maximum height between upper and lower edges of tail).
In order to reduce the impact of allometry, the correct value from the ratio of each character to SVL was calculated and was log-transformed for subsequent morphometric analyses. Mann-Whitney U tests were conducted to test the significance of differences on morphometric characters between the new species, N. daunchina, and N. yaoica. The significance level was set at 0.05. Due to only the measurements SVL, HDL, HDW, SL, IND, IOD, ED, TYD, TED, HND, RAD, TL, and FL of male N. yaoica being available from Lyu et al. (2019a), the Mann-Whitney U tests were conducted based on these 13 morphometric characters for the new species and N. yaoica.

Bioacoustics analyses
The advertisement calls of the new species from Huanglian Town, Tongzi County, Guizhou Province, China were recorded from the specimen CIBTZ20190608004 in the field on 8 June 2019. The advertisement calls were recorded from the ridge of a paddy field at ambient air temperature of 20 °C and air humidity of 80 %. For comparisons, the advertisement calls of N. daunchina from E'mei Mountain, Sichuan Province, China were recorded from the specimen CIB2011081603 at ambient air temperature of 20 °C and air humidity of 85 % in the ridge of paddy field on 16 August 2011; the advertisement calls of N. yaoica were retrieved from Lyu et al. (2019a). SONY PCM-D50 digital sound recorder was used to record within 20 cm of the calling individual. The sound files in wave format were resampled at 48 kHz with sampling depth 24 bits. Calls were recorded and examined as described by Wijayathilaka and Meegaskumbura (2016). Call recordings were visualised and edited with SoundRuler 0.9.6.0 (Gridi-Papp 2003-2007 and Raven Pro 1.5 software (Cornell Laboratory of Ornithology, Ithaca, NY, USA). Ambient temperature of the type and other localities was taken by a digital hygrothermograph.

Results
Aligned sequence matrix of 16S is 523 base pairs (bp) in length and 561 bp for COI. ML and BI analyses based on the 16S + COI matrix resulted in basically identical topologies (Fig. 2). All samples of the new species were clustered into one clade nested in the genus Nidirana. This new species clade was clustered into a large clade together with N. leishanensis, N. hainanensis, N. daunchina, N. yaoica, and N. chapaensis, with high supported value of 100 in ML and 1.00 in BI. On 16S gene, the mean genetic distance between the new species and the closely related species N. daunchina and N. yaoica were 1.2 % and 1.3 %, respectively, at the same level as the distance between N. adenopleura and N. okinavana (1.2 %; Table 2).
The results of Mann-Whitney U tests indicated that in males, the new species was significantly different from N. daunchina and N. yaoica on many morphometric characters (all p-values < 0.05; Table 3). The new species could also be identified from its congeners based on morphological descriptions from the literature and from our examinations of newly collected material (Table 4). More detailed descriptions of results from morphological comparisons between the new species and its congeners are presented in the following sections.
There were many differences in sonograms and waveforms of calls between the new species, N. daunchina, and N. yaoica ( Fig. 3; Table 5). Firstly, in the call duration and the note duration, the two-note call and three-note call of the new species were longer than those of both N. daunchina and N. yaoica. Secondly, the note interval of two-note call and three-note call of the new species was shorter than those of N. daunchina and N. yaoica. Thirdly, the dominant frequency of call in the new species was higher than N. yaoica.  Table 1.  Based on the molecular, morphological, and bioacoustics differences, the specimens from Tongzi County, Guizhou Province, China, represent a new species which is described as Nidirana yeae sp. nov.  Diagnosis. Nidirana yeae sp. nov. is assigned to the genus Nidirana based on molecular data and the following combination of characters: absence of thumb-like structure on finger I; disks of digits dilated, rounded; dorsolateral folds distinct; the presence of large suprabrachial gland in male.

Taxonomic account
Nidirana yeae sp. nov. could be distinguished from its congeners by a combination of the following characters: (1) body of medium size (SVL 41.2-43.5 mm in males and 44.7 mm in female); (2) lateroventral groove only present on toes; (3) relative finger lengths: II < IV < I < III; (4) three metatarsal tubercles on palm; (5) heels overlapping when hindlimbs flexed at right angles to axis of body; (6) tibiotarsal articulation reaching the level of eye when leg stretched forward; (7) a pair of subgular internal vocal sacs at corners of throat in male; (8) nuptial pad present on the inner side of base of fingers I in male in breeding season; (9) tadpole labial tooth row formula with 1:1+1/1+1:2; (10) in male, the advertisement call containing two kinds of note and the call containing 2-6 repeated regular notes.
Description of holotype. Body size medium, SVL 40.2 mm; head slightly wider than long (HDW/HDL = 1.03), flat above; snout rounded in dorsal and lateral views, slightly projecting beyond lower jaw; a maxillary gland in posterior corner of mouth from snout to tympanum, behind the gland a shoulder gland present; supratympanic fold absent; interorbital space narrower than internarial distance (IND/IOD = 1.38); eye large and convex, ED 0.76 times of SL; tympanum distinct, large and rounded, 0.76 times of ED, and close to eye; vomerine ridge present, but the outline of vomerine ridges are not sharp and almost connected to the internal nostril; tongue deeply notched posteriorly; paired subgular inner vocal sacs at corners of throat.
Forelimbs moderately robust (LW/SVL = 0.08); lower arm and hand less than a half of body length (LAL/SVL = 0.42); relative finger lengths: II < IV < I < III; tip of fingers weakly dilated, forming elongated and pointed disks; lateroventral grooves on the disks of finger absent; fingers free of webbing, with lateral fringes on fingers III and IV; subarticular tubercles prominent and rounded; week supernumerary tubercles below the base of fingers III and IV; palmar tubercles three, elliptic, distinct.
Hindlimbs relatively robust, tibia 47% of SVL; tibia longer than thigh (TL/THL = 1.04); heels overlapping when hindlimbs held at right angles to axis of body; tibiotarsal articulation reaching the level of mid-eye when hindlimb is stretched forward; toes long and thin, relative toe lengths: I < II < V < III < IV; tip of toes dilated, forming significantly elongated disks; distinct lateroventral grooves on toes; webbing weak, webbing formula: ; toes with lateral fringes; subarticular tubercles rounded, prominent; inner metatarsal tubercle elliptic, twice as long as its width; outer metatarsal tubercle indistinct, small and rounded. Dorsal skin of head and anterior part of body smooth, posterior part and flanks with several tubercles, some tubercles with black spot; a large suprabrachial gland behind base of forelimb; dorsolateral fold extending from posterior margin of upper eyelid to above groin; several granules on the dorsal surfaces of thigh, tibia, and tarsus; ventral surface of head, body, and limbs smooth, several flattened tubercles densely arranged on the rear of thigh and around vent.

Colouration of holotype in life.
In life, dorsal surface and suprabrachial gland pale brown; flank relatively smooth with dense tubercles on region nearly the dorsolateral fold; several black spots on flank, dorsum, and head; a discontinuous light yellow streak from posterior head to cloacae; dorsal forelimbs light brown and one brown stripe in front of the base of forelimb; dorsal hindlimb grey-brown with dense tubercles, three brown bands on the thigh, four on the tibia and the tarsus; tympanum and temporal region black; maxillary gland white; ventral surface smooth, throat and ventral of thigh and forelimbs incarnadine, belly and chest light yellow (Fig. 4).
Preserved holotype colouration. Dorsal surface faded to brown; black spots on dorsum and flank more distinct; limbs faded light brown and the crossbars becoming clearer; ventral surface faded to pale cream and throat fade to brownness (Fig. 5).
Variations. All adult specimens were similar in morphology but some individuals differed from the holotype in colour pattern. In some adult males, the colour of tympanum and temporal region pinkish red (Fig. 6A); in some adult males, the colour of dorsum is reddish brown (Fig. 6B); in the adult female, the colour of dorsum was brownish red and the flank was brownish under the dorsolateral fold (Fig. 6C); in some adult males, the colour of dorsum brick-red and the tubercles on flank were obvious (Fig. 6D); in some adult males, the throat was creamy and ventral surface of body was white with brown patches (Fig. 6E); in the adult female, the throat was brown and there were some patchiness on the ventral surface of the body and thigh (Fig. 6F).
Tadpole description. Measurements of specimen CIBTZ20190608019 (in mm): TOL 35.2, SVL 14.0, BW 6.1, BH 5.1, SL 3.1, SS 8.1, IOD 3.3, TAL 20.7, TAH 4.0, TBW 3.0. Body oval, body and tail yellowish brown, flattened above; several brown spots on dorsum and tail; maximum depth near posterior part of tail and more than body depth; body width longer than body height (BW/ BH = 1.53); eyes lateral, nostril near snout; spiracle on left side of body, directed dorsoposteriorly; keratodont formula: 1:1+1/1+1:2; ventral of body oval, creamy white with dense brown spots on flank of body; both upper and lower lips with labial papillae; some additional tubercles at the angles of the mouth, usually with small keratodonts; tail fusiform, approximately 1.5 times as long as snout-vent length, tail height 19.3 % of tail length; dorsal fin arising behind the origin of the tail (Fig. 7).  Table 5). Call duration was 728-2082 ms (mean 1199 ± 174 ms, N = 11). Call interval was 2000-9435 ms (mean 4586 ± 2659 ms, N = 10). The first type of note is the start note in each call and the other notes in each call are termed the second type. Amplitude modulation within strophe is apparent, beginning with moderate energy pulses, decreasing slightly to a minimum then increasing approximately to the midnote, subsequently increasing to a peak then decreasing rapidly towards the end of each note in the first type; in the second type amplitude beginning with highest pulses and decreasing towards approximately the midnote then increasing slightly then decreasing towards the end of each note. The first type of note has a longer duration than the second type (308-440 ms, N = 10 vs.135-240 ms, N = 23).The two-note call (N = 5) has a duration of 728-825 ms, and dominant frequency is 4200-5040 Hz, three-note call (N = 2) has a duration of 988-1135 ms and dominant frequency is 4620-5040 Hz, four-note call (N = 3) has a duration of 1400-1563 ms and dominant frequency is 4680-5160 Hz, six-note call (N = 1) has a duration of 2082 ms and dominant frequency is 5280 Hz (Table 5).
Secondary sexual characteristics. A pair of subgular inner vocal sacs, a pair of slitlike openings at posterior of jaw; a single light brown nuptial pad on the inner side of dorsal surface of finger I (Fig. 4C); nuptial spicules invisible; suprabrachial gland present.
Morphological comparisons. Nidirana yeae sp. nov. differs from N. leishanensis and N. lini by having smaller body size (SVL < 45 mm in the new species vs. SVL > 49 mm in males of N. leishanensis and SVL > 57 mm in females of N. lini).
Nidirana yeae sp. nov. differs from N. daunchina, N. hainanensis and N. leishanens by the presence of lateroventral groove only on toes (vs. both fingers and toes present in the latter).
Nidirana yeae sp. nov. differs from N. pleuraden by the presence of lateroventral groove only on toes (vs. both fingers and toes absent in the latter).
Nidirana yeae sp. nov. differs from N. hainanensis, N. lini, and N. nankunensis by tibiotarsal articulation reaching the level of eye when leg stretched forward (vs. reaching nostril or beyond snout in the latter).
Nidirana yeae sp. nov. differs from N. okinavana by having subgular internal vocal sacs (vs. gular vocal sacs absent in the latter).
Nidirana yeae sp. nov. differs from N. hainanensis and N. leishanensi by having nuptial pad on the inner side of base of fingers I in males in breeding season (vs. nuptial pad absent in N. hainanensis and nuptial pads on both fingers I and II in N. leishanensis).
Nidirana yeae sp. nov. is genetically closer to N. chapaensis, N. daunchina, and N. yaoica. It differs from N. chapaensis by the following characters: the relative finger lengths II < IV < I < III (vs. II < I = IV < III), tibiotarsal articulation reaching the level of eye when leg stretched forward (vs. reaching nostril), having nuptial pad on the inner side base of finger I in males in breeding season (vs. having two nuptial pads on finger I), tadpole labial tooth row formula of 1:1+1/1+1:2 (vs. 1:1+2/1+1:2); differs from N. daunchina by the presence of lateroventral groove only on toes (vs. both fingers and toes present), heels overlapping when hindlimbs flexed at right angles to axis of body (vs. heels meeting), tibiotarsal articulation reaching the level of eye when leg stretched forward (vs. reaching nostril), having significantly lower value of SVL in males and having significantly lower ratios of HDL, ED, TED, HLL, TW, TFL, and FL to SVL in males, the outline of vomerine ridges not sharp and almost connected to the internal nostril (vs. outline of vomerine ridges sharp and distinctly separated from the internal nostril; Fig. 5G, H), having longer call duration in two-note call and three-note call, having shorter note interval in the two-note call and three-note call (Table 4); differs from N. yaoica by the presence of lateroventral groove only on toes (vs. both fingers and toes present), relative finger lengths II < IV < I < III (vs. II < I < IV < III), tibiotarsal articulation reaching the level of eye when leg stretched forward (vs. reaching nostril), having significantly lower ratios of HDL, HDW, ED, TED, RAD, TL, and FL of SVL in males, having longer call duration and longer note duration in two-note call and three-note call, having shorter note interval in two-note call and three-note call, and having higher dominant frequency in call (Table 5).
Remarks. Wu et al. (1986) reported that the populations from Kuankuoshui Nature Reserve of Suiyang County, Fanjiang Mountain of Jiangkou County and Leigong Mountain of Leishan County, Guizhou Province, China belonged to N. adenopleura. Fei et al. (1990Fei et al. ( , 2009   that the population from Leigong Mountain of Leishan County should be a new species, which they named N. leishanensis. From the morphological description and morphometric data of the population from Kuankuoshui Nature Reserve of Suiyang County, some characters is very similar to Nidirana yeae sp. nov.: body of medium size (SVL 39.0-46 mm in males and 44-48 mm in females); lateroventral groove on toes present; relative finger lengths II < IV < I < III; three metatarsal tubercles on palm; a pair of subgular internal vocal sacs at corners of throat in males; nuptial pad present on the inner side of base of fingers I in males in breading season; tadpole labial tooth row formula with 1:1+1/1+1:2. This population was probably Nidirana yeae sp. nov., and detailed comparisons especially with molecular data should be conducted to establish its identity. The phylogenetic trees in our work and Lyu et al. (2019a) all supported that the population from Hejiang County, Sichuan Province, China was the closest to topotypes of N. daunchina but separated from Nidirana yeae sp. nov. and other relatives, and so this population in the south-eastern part of Sichuan Province should be N. daunchina and not Nidirana yeae sp. nov. Although the morphometric data (large body size) of the population from Fanjiang Mountain of Jiangkou County in Wu et al. (1986) indicated that it was similar to N. leishanensis, we still need detailed comparisons and molecular data to clarify its taxonomic status.
Ecology. Nidirana yeae sp. nov. is currently found from the paddy field (28.44317N, 107.02003E; ca. 1170 m a. s. l.) in Huanglian Town, Tongzi County, Guizhou Province, China. The individuals were found on the paddy field near an evergreen broadleaved forest (Fig. 8). Tadpoles of the species could be found in the water. Two sympatric amphibians, Zhangixalus omeimontis (Stejneger, 1924) and Polypedates braueri (Vogt, 1911) were also found in the type locality.
Etymology. The specific name yeae is in homage to the famous taxonomist Ye Chang-Yuan for her great contributions to Chinese amphibian research. For the common name, we suggest Ye's Music Frog (English) and Ye Shi Qin Wa (Chinese).

Discussion
Before this work, the taxonomic status for the populations of Nidirana in Tongzi County in the north part of Guizhou Province had not been reported, but populations in Suiyang County adjacent to Tongzi County were identified as N. daunchina (Fei et al. 1990(Fei et al. , 2009(Fei et al. , 2012Fei and Ye 2005). Now, the Tongzi population is revealed as a new species, Nidirana yeae sp. nov. based on integrative taxonomy using morphological comparisons, molecular phylogenetic analyses, and bioacoustics. Nidirana populations in Suiyang County, to the east of Tongzi County, are probably the new species also. Moreover, Lyu et al. (2017) recognised three specimens (included in our phylogenetic analyses) from Hejiang County, Sichuan Province as N. daunchina; of note, the straight-line geographical distance between Hejiang County and Tongzi County is ca. 110 km, much shorter than that (ca. 280 km) between Hejiang County and the type locality of N. daunchina (E'mei Mountain, Sichuan Province, China). Therefore, it could be speculated that the two closely related species, Nidirana yeae sp. nov. and N. daunchina, were probably parapatric in the region between Hejiang and Tongzi counties. Many more surveys of the surrounding areas are needed to clarify the populations of "N. daunchina" and the accurate distribution of the two species.
South-western China has long been proposed as biodiversity hotspot (Myers et al. 2000). However, Guizhou Province is an important part of south-western China, especially with the particular environments of karst rocky desertification, and knowledge of biodiversity levels and/or patterns are still seriously lacking. Recently, a series of new amphibian species were described from this province (Zhang et al. 2017;Li et al. 2018aLi et al. , b, 2019aLyu et al. 2019b;Wang et al. 2019), indicating that species diversity of amphibians in this region is highly underestimated. It is urgent for herpetologists to conduct comprehensive and in-depth surveys to discover the level of amphibian species diversity in this region under accelerating global changes.