Description of a new species of Music frogs (Anura, Ranidae, Nidirana) from Mt Dayao, southern China

Abstract A new species of Music frogs, Nidiranayaoicasp. nov. is described based on a series of adult male specimens collected from Mt Dayao, Guangxi, southern China, providing valuable new information on the phylogeny, bioacoustics, and biogeography of related species within the genus Nidirana. The new species forms the sister taxon to N.daunchina from western China and together the sister taxon to N.chapaensis from northern Vietnam. Nidiranayaoicasp. nov. can be distinguished from all known congeners by a significant genetic divergence in the mitochondrial 16S and CO1 genes, the advertisement call containing 1–3 rapidly repeated regular notes, and the combination of morphological characteristics including a medium-sized body with SVL 40.4–45.9 mm in adult males; lateroventral grooves on every digit, not meeting at the tip of disk; tibio-tarsal articulation reaching the nostril; the presence of a pair of subgular vocal sacs in males; and one single developed nuptial pad on dorsal surface of first finger in males.

During our herpetological field surveys in Mt Dayao (MDY), Guangxi, south China, we collected a series of specimens of a small-sized frog that could be assigned to the genus Nidirana by possessing large suprabrachial gland in breeding males. Further detailed comprehensive analyses of molecules, bioacoustics, and morphology indicated that this frog was distinctive from all known congeners of Nidirana. Therefore, we propose it as a new species based on this study.

Taxon sampling
Eight muscular samples of the unnamed species from MDY were used for molecular analysis. All samples were attained from euthanasia specimens and then preserved in 95% ethanol and stored at -40 °C. In addition, 36 sequences from all known Nidirana species and two sequences from the out-group Babina were obtained from GenBank and incorporated into our dataset. Detail information of these materials is shown in Table 1 and Fig. 1.

Phylogenetic analyses
DNA sequences were aligned by the Clustal W algorithm with default parameters (Thompson et al. 1997) and trimmed with the gaps partially deleted in MEGA 6 (Tamura et al. 2013). Two gene segments, 1041 base pairs (bp) of 16S and 573 bp of CO1, were concatenated seriatim into a 1614-bp sequence, and further divided into four partitions by codons. The partitions were tested in jmodeltest v2.1.2 with Akaike and Bayesian information criteria, all resulting the best-fitting nucleotide substitution models of GTR+I+G. Sequenced data was analyzed 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 a BI analysis, each of which was performed for 10,000,000 generations and sampled every 1000 generations with the first 25% samples were discarded as burn-in, resulting a potential scale reduction factor (PSRF) of < 0.005. In ML analysis, the bootstrap consensus tree inferred from 1000 replicates was used to represent the evolutionary history of the taxa analyzed.

Bioacoustic analysis
Advertisement calls of the specimen SYS a007009 from MDY were recorded in the field at the air temperature of 18 °C using a SONY PCM D100 digital sound recorder. The sound files in wave format were sampled at 44.1 kHz with 24 bits in depth. Praat 6.0.27 (Boersma 2001) was used to obtain the oscillogram, sonogram, and power spectrum (window length = 0.005 s). Raven pro 1.5 (Cornell Lab of Ornithology, 2003-2014) was used to quantify the acoustic properties (window size = 256 points, fast Fourier transform, Hanning window with no overlap). The following measurements were taken for each call: call duration (the time between onset of the first note and offset of the last note in a call) and call PF (peak frequency; the frequency at which max power occurs within the call); the following measurements were taken for each note: note duration (the time between onset and offset of a note), note rise time (the time between onset and max amplitude of a note), note interval (the time between adjacent notes in a call), note PF and note IQR-BW (inter-quartile range bandwidth; the difference between the first and third quartile frequencies within a note). Mean and standard deviation (SD) were calculated in R 3.3.2 (R Core Team 2016).

Morphology
Comparison characters of all known congeners were obtained from the literature (Boettger 1895; Boulenger 1904Boulenger , 1909Schmidt 1925;Chang and Hsu 1932;Bourret 1937;Kuramoto 1985;Chou 1999;Fei et al. 2007Fei et al. , 2009Matsui 2007;Chuaynkern et al. 2010;Lyu et al. 2017) and 55 examined museum specimens of six species which are listed in the Appendix 1. All specimens were fixed in 10% buffered formalin and later transferred to 70% ethanol, and deposited in the Museum of Biology, Sun Yatsen University (SYS), Natural History Museum of Guangxi (NHMG), and Chengdu Institute of Biology, Chinese Academy of Sciences (CIB), China.
Morphological descriptions follow the consistent definition by Fei et al. (2009), Chuaynkern et al. (2010 and Lyu et al. (2017). External measurements were made for the unnamed specimens with digital calipers (Neiko 01407A Stainless Steel 6-Inch Digital Caliper, USA) to the nearest 0.1 mm. Mean and standard deviation (SD) were calculated in R 3.3.2 (R Core Team 2016). These measurements were as follows: SVL snout-vent length (from tip of snout to posterior margin of vent); HDL head length (from tip of snout to the articulation of the jaw); HDW head width (head width at the commissure of the jaws); SNT snout length (from tip of snout to the anterior corner of the eye); IND internasal distance (distance between nares); IOD interorbital distance (minimum distance between upper eyelids); ED eye diameter (from the anterior corner of the eye to posterior corner of the eye); TD tympanum diameter (horizontal diameter of tympanum); TED tympanum-eye distance (from anterior edge of tympanum to posterior corner of the eye); HND hand length (from the proximal border of the outer palmar tubercle to the tip of digit III); RAD radio-ulna length (from the flexed elbow to the proximal border of the outer palmar tubercle); FTL foot length (from distal end of shank to the tip of digit IV); TIB tibial length (from the outer surface of the flexed knee to the heel).
Sex and age were determined by secondary sexual characters, i.e., the presence of suprabrachial glands in males. Webbing formula was written according to Savage (1975).

Results
The ML and BI analyses resulted in essentially identical topologies and were integrated in Fig. 2, in which the major nodes were sufficiently supported with the Bayesian posterior probabilities (BPP) > 0.95 and the bootstrap supports (BS) for maximum likelihood analysis > 70. This mitochondrial result is consistent with the phylogenic relationship in Lyu et al. (2017). The Nidirana specimens from MDY, southern China, grouped in a clade with strong supported values and small divergences, forming a sister taxon to N. daunchina from western China, then together forming the sister taxon to N. chapaensis from northern Vietnam.
Morphologically, the specimens from MDY significantly differ from the recognized congeners by the following characteristics: (1) medium-sized body, SVL 40.4-45.9 mm in adult males vs. 33.3-37.1 mm in N. nankunensis; (2) finger IV longer than finger I vs. equal in N. chapaensis; (3) presence of lateroventral groove on every digit vs. absent on fingers and toes in N. pleuraden; absent or barely visible on fingers in N. daunchina; absent on finger I in N. chapaensis, N. lini, N. nankunensis, N. adenopleura, and N. okinavana; (4) tibio-tarsal articulation reaches the nostril vs. beyond the snout tip in N. lini; (5) the presence of a single nuptial pad vs. absent in N. hainanensis; divided into two parts in N. chapaensis; (6) the presence of a pair of subgular vocal sacs vs. absent in N. okinavana; (7) the absence of spinules on dorsal skin vs. present in N. adenopleura, N. lini and N. pleuraden. Detail comparison between the specimens from MDY and its congeners is listed in Table 2 with the characteristics item by item.
Further, the advertisement call from the frogs from MDY is different from the congeners by: (1) containing 1-3 fast-repeated identical regular notes (vs. containing Therefore, based on the molecular, morphological, and bioacoustic differences, the specimens from MDY, southern China, represent an unnamed species which is described as a new species of genus Nidirana. Relative length of toes I < II < V < III < IV I < II < V < III < IV I < II < V < III < IV I < II < V < III < IV I < II < V < III < IV I < II < V < III < IV I < II < V < III < IV I < II < V < III < IV I < II < V < III < IV Tibio-tarsal articulation Etymology. The specific name yaoica is an adjective derived from Yao, referring to the type locality of the new species, Mt Dayao in Jinxiu Yao Autonomous County, where the settlement of the Yao people is located. We suggest its English common name to be Mt Dayao music frog and its Chinese name Yao Qin Wa (瑶琴蛙).
Description of holotype. Adult male. Body stocky, SVL 44.6 mm; head longer than wide (HDW/HDL 0.92), flat above; snout rounded in dorsal and lateral views, slightly protruding beyond lower jaw, longer than horizontal diameter of eye (SNT/ ED 1.26); canthus rostralis distinct, loreal region concave; nostril round, directed laterally, closer to the snout than to the eye; a longitudinal swollen mandibular ridge extending from below nostril through lower edges, eye and tympanum to above insertion of arm, where the ridge is intermittent, forming a maxillary gland and shoulder gland; supratympanic fold absent; interorbital space flat, narrower than internasal distance (IND/IOD 1.37); pupil elliptical, horizontal; tympanum distinct, round, TD/ED 0.72, and close to eye, TED/TD 0.38; pineal ocellus present; vomerine ridge present, bearing small teeth; tongue large, cordiform, notched behind.
Forelimbs moderately robust, lower arm 19% of SVL and hand 27% of SVL; fingers thin, relative finger lengths II < I < IV < III; tip of each finger slightly dilated and remarkable elongated, forming long pointed disks; well-developed lateroventral grooves on all fingers, not meeting at the tip of disks; fingers free of webbing; presence of weak lateral fringes on inner and outer sides of fingers II, III and IV, and on outer side of finger I; subarticular tubercles prominent and rounded; week supernumerary tubercles below the base of fingers III and IV; three elliptic, large, prominent and very distinct palmar tubercles.
Hindlimbs relatively robust, tibia 53% of SVL and foot 78% of SVL; heels overlapping when hindlimbs flexed at right angles to axis of body; tibio-tarsal articulation reaching the nostril when hindlimb is stretched along the side of the body; toes relatively long and thin, relative lengths I < II < V < III < IV; tip of each toe slightly dilated with remarkable elongated ventral callous pad, forming long and pointed disk; well-developed lateroventral grooves on toes , not meeting at the tip of disks; webbing moderate, webbing formula: I 2 -2½ II 1⅔ -3 III 2⅓ -3½ IV 3½ -2 V; presence of lateral fringes on inner and outer sides of each toes, forming distinct dermal flap on the lateral edges of toes I and V; subarticular tubercles rounded, prominent; inner metatarsal tubercle elliptic, twice as long its width; outer metatarsal tubercle indistinct, small and rounded; tarsal folds and tarsal tubercle absent.
Dorsal skin of head and anterior body smooth, posterior dorsum of body rough with dense tubercles but not bearing horny spinules; developed dorsolateral fold from posterior margin of upper eyelid to above groin but intermittent posteriorly; flank relatively smooth with dense tubercles on region nearly the dorsolateral fold; a large and smooth suprabrachial gland behind base of forelimb, slightly prominent; dorsal surface of upper arm with two longitudinal ridges and slightly extending to lower arm; the dorsal surfaces of thigh and tibia with several longitudinal ridges and tubercles bearing spinules. Ventral surface of head, body, and limbs smooth; large flattened tubercles densely arranged on the rear of thigh and around vent.
Color in life of holotype. Dorsal surface of head and body reddish brown; pineal ocellus yellowish; a longitudinal reddish brown mid-dorsal stripe edged with broad dark brown, beginning from snout, across pineal ocellus, posteriorly extending to vent; several black spots on upper eyelids and posterior dorsum of body; dorsolateral fold bicolor, upper part reddish brown and lower part black; upper flank yellowish brown with irregular black spots; lower flank yellowish white; suprabrachial gland yellowish brown. Dorsal forelimbs reddish brown; a longitudinal black stripe on the anterior surface of the forelimb; irregular black marks on dorsal surface of the forelimb; dorsal hindlimbs nonuniform dark brown, four black crossbars on the thigh, three on the tibia and three on the tarsus; irregular black marks on dorsal toes. Loreal and temporal regions black, tympanum dark brown; upper ⅓ iris bright brownish white and lower ⅔ iris reddish brown; maxillary gland and shoulder gland yellowish white. Lips and throat grey white, but two subgular vocal sacs slightly dark colored; ventral surface of body and limbs creamy white; rear thigh tinged with pink; ventral hand and foot pale white with large black patches.
Color in preservative of holotype. Dorsal surface faded, but dark brown edges of the mid-dorsal stripe more distinct; black spots on dorsum more distinct; upper flank black; limbs faded, the crossbars clearer; ventral surface faded, throat and posterior of chest with smoky gray markings.
Male secondary sexual characteristics. A pair of subgular vocal sacs, a pair of slitlike openings at posterior of jaw; a single light brown nuptial pad on the dorsal surface of first finger, nuptial spinules invisible; suprabrachial gland present.
Distribution and ecology. Currently, Nidirana yaoica sp. nov. is known only from the type locality, Mt Dayao, Jinxiu, Guangxi, in southern China. This frog inhabits in the swamps and ponds surrounded by moist subtropical secondary evergreen broadleaved forests (Fig. 4C, D). The adult male calls in the brushwood at the bank, from mid-March to late May. Nevertheless, the females, tadpoles, and much of the ecology and behavior of this species remain unknown. Vocalization. The call spectrograms are shown in Fig. 5 and the measurement parameters are listed in Table 4. The advertisement call (n = 87) of Nidirana yaoica sp. nov. contains 1-3 rapidly repeated, identical, regular notes with the PF of 516.8 Hz and note IQR-BW of 172.3 Hz or 0 generally. The one-note call (n = 25) has a duration of 43.3 ± 2.7 ms with the rise time of 10.1 ± 4.5 ms. The two-note call (n = 59)  has a duration of 355.9 ± 31.1 ms; the first note lasts 43.5 ± 2.8 ms with the rise time of 8.5 ± 4.6 ms, and the second lasts 39.6 ± 3.3 ms with the rise time of 11.6 ± 4.4 ms; the note interval last 272.8 ± 31.7 ms. basal lineage of this genus, while the monophyly of the three species groups N. adenopleura group (Fei et al. 2009;Chuaynkern et al. 2010), N. okinavana group, and N. daunchina group, was challenged. The main conflicts are: (1) N. okinavana was suggested morphologically more similar to N. daunchina and N. chapaensis (Fei et al. 2009;Chuaynkern et al. 2010) while clustered with N. adenopleura in the phylogeny; (2) N. hainanensis was suggested morphologically more similar to N. adenopleura and N. lini (Fei et al. 2009) while clustered with N. daunchina and N. chapaensis in the phylogeny; (3) N. lini was suggested morphologically more similar to N. adenopleura (Fei et al. 2009;Chuaynkern et al. 2010) while formed the basal lineage of the congeners except N. pleuraden in the phylogeny.