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Zookeys 858: 109-126 (2019)

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

Zhi-Tong Lyu', Yun-Ming Mo’, Han Wan', Yu-Long Li’,
Hong Pang’, Ying-Yong Wang'

I State Key Laboratory of Biocontrol/ The Museum of Biology, School of Life Sciences, Sun Yat-sen University,
Guangzhou 510275, China 2. Natural History Museum of Guangxi, Nanning 530012, China

Corresponding author: Ying- Yong Wang (wangyy@mail.sysu.edu.cn); Hong Pang (Isshpang@mail.sysu.edu.cn)

Academic editor: A. Ohler | Received 7 March 2019 | Accepted 28 May 2019 | Published 1 July 2019
http://zoobank. ore/8AA495F6-CCC3-4E00-B37B-BAF5A598A2ZED

Citation: Lyu Z-T, Mo Y-M, Wan H, Li Y-L, Pang H, Wang Y-Y (2019) Description of a new species of Music
frogs (Anura, Ranidae, Nidirana) from Mt Dayao, southern China. ZooKeys 858: 109-126. https://doi.org/10.3897/
zookeys.858.34363

Abstract

A new species of Music frogs, Nidirana yaoica sp. nov. is described based on a series of adult male speci-
mens 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 NV. daunchina from western China and together the sister taxon to NV. chapaensis
from northern Vietnam. Nidirana yaoica sp. nov. can be distinguished from all known congeners by a
significant genetic divergence in the mitochondrial 16S and CO1 genes, the advertisement call contain-
ing 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.

Keywords
bioacoustic, Guangxi, mitochondrial DNA, morphology, Nidirana yaoica sp. nov.

Copyright Zhi-Tong Lyu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY
4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

110 Lhi-Tong Lyu et al. | ZooKeys 858: 109-126 (2019)

Introduction

The taxonomic treatment of the Music frog genus Nidirana Dubois, 1992 was con-
troversial for a long time (Dubois 1987, 1992; Chen et al. 2005; Frost et al. 2006; Fei
et al. 2009, 2010; Chuaynkern et al. 2010). The recent contribution to the phylogeny
of genus Nidirana reconsidered it as a distinct genus, on the basis of comprehensive
evidence of morphology, molecular phylogeny, bioacoustics, and biogeography (Lyu
et al. 2017). Eight Music frog species were recognized from subtropical eastern and
southeastern Asia (Lyu et al. 2017; Frost 2019): N. okinavana (Boettger, 1895) from
Yaeyama of southern Ryukyu, and eastern Taiwan; NV. adenopleura (Boulenger, 1909)
from Taiwan and southeastern mainland China; NV. nankunensis Lyu, Zeng, Wang,
Lin, Liu & Wang, 2017 from Mt Nankun of Guangdong and UN. hainanensis (Fei, Ye,
& Jiang, 2007) from Mt Diaoluo of Hainan, both in southern China; N. daunchina
(Chang, 1933) from western China; NV. pleuraden (Boulenger, 1904) from southwest-
ern China; and NV. chapaensis (Bourret, 1937) and N. lini (Chou, 1999) from the
northeastern Indochinese peninsula.

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.

Materials and methods

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.

DNA Extraction, PCR amplification, and sequencing

Genomic DNA were extracted from muscle tissue samples, using DNA extraction kit
from Tiangen Biotech (Beijing) Co., Ltd. Two mitochondrion genes, namely partial 16S
ribosomal RNA gene (16S) and partial cytochrome C oxidase 1 gene (CO1), were am-
plified. Primers used for 16S were L3975 (5’-CGCCTGTTTACCAAAAACAT-3’) and
H4551 (S’--CCGGTCTGAACTCAGATCACGT-3’), and L2A (5’-CCAAACGAGC-

Description of a new species of Music frogs from Mt Dayao, southern China 111

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

ID Species Localities (* = type localities) Voucher 16S Col

1 Mdirana yaoica sp. nov. China: Guangxi: Mt Dayao * SYS a007009 MK882271 MK895036
2. Nidirana yaoica sp. nov. China: Guangxi: Mt Dayao * SYS a007011 MK882272 MK895037
3 Nidirana yaoica sp. nov. China: Guangxi: Mt Dayao * SYS a007012 MK882273, =MK895038
4 — Nidirana yaoica sp. nov. China: Guangxi: Mt Dayao * SYS a007013 MK882274 MK895039
5 _Nidirana yaoica sp. nov. China: Guangxi: Mt Dayao* — SYS a007014/CIB 110013. MK882275 MK895040
6 Nidirana yaoica sp. nov. China: Guangxi: Mt Dayao * SYS a007020 MK882276 MK895041
7 Nidirana yaoica sp. nov. China: Guangxi: Mt Dayao * SYS a007021 MK882277. + MK895042
8 = Nidirana yaoica sp. nov. China: Guangxi: Mt Dayao * SYS a007022 MK882278 = MK895043
9 — Nidirana adenopleura China: Zhejiang: Jingning SYS a002725 MF807827 MF807866
10 = Nidirana adenopleura China: Fujian: Nanping SYS a005911 MF807844 = MF807883
11. = Nidirana adenopleura China: Fujian: Mt Wuyi SYS 2005940 MF807851 = MF807890
12. Nidirana adenopleura China: Fujian: Mt Wuyi SYS 2005941 MF807852. MF807891
13. Nidirana adenopleura China: Fujian: Mt Wuyi XM2827 KF771281 /

14 = Nidirana adenopleura China: Taiwan: New Taipei UMMZ 189963 DQ283117 /

15. Nidirana adenopleura Not given NMNS 2384 AF458118 /

16 = Nidirana adenopleura Not given A-A-WZ001 NC018771 = NC018771
17. = Nidirana chapaensis Vietnam: Lao Cai: Sapa * ROM 28070 AF206460 /

18 = Nidirana chapaensis Vietnam: Lao Cai: Sapa * 1999.5871 KR827710 /

19 Nidirana chapaensis Vietnam: Lao Cai: Sapa * T2483/2000.4850 KR827711 KR087625
20 Nidirana chapaensis Vietnam: Gia Lai AMSR176027 KU840598 /

21 = Nidirana daunchina China: Sichuan: Mt Emei * 0609 KU840597 /

22 ~=Nidirana daunchina China: Sichuan: Mt Emei * CIB-WU37990 DQ359988 /

23. Nidirana daunchina China: Sichuan: Mt Emei * HNNU 20060103 KF185065 /

24 Nidirana daunchina China: Sichuan: Mt Emei * SYS 2004594 MF807822 MF807861
25 Nidirana daunchina China: Sichuan: Mt Emei * SYS 2004595 MF807823 MF807862
26 Nidirana daunchina China: Sichuan: Hejiang SYS 2004930 MF807824 = MF807863
27 ~Nidirana daunchina China: Sichuan: Hejiang SYS a004931 MF807825 = MF807864
28 = Nidirana daunchina China: Sichuan: Hejiang SYS 2004932 MF807826 MF807865
29 Nidirana daunchina Not given Not given / HQ395353
30 = Nidirana hainanensis China: Hainan: Mt Diaoluo * SYS a003741 MF807821 = MF807860
31 = Nidirana hainanensis China: Hainan Not given KU840596 /

32 = Nidirana lini China: Yunnan: Jiangcheng * SYS a003967 MF807818 |= MF807857
33 = Nidirana lini China: Yunnan: Jiangcheng * SYS 2003968 MF807819 =MF807858
34 = Nidirana lini China: Yunnan: Jiangcheng * SYS 2003969 MF807820 MF807859
35 Nidirana lini China: Yunnan: Lyuchun HNNULC001 KF185066 /

36 = Nidirana lini Laos: Xieng Khouang FMNH256531 KR264073 /

37 ~~ Nidirana lini Laos: Xieng Khouang FMNH256532 KR264074 /

38 = Nidirana lini Not given Not given / HQ395352
39 = Nidirana nankunensis China: Guangdong: Mt Nankun * SYS a005717 MF807838 = MF807877
40 Nidirana nankunensis China: Guangdong: Mt Nankun * SYS a005718 MF807839 =MF807878
41 Nidirana nankunensis China: Guangdong: Mt Nankun * SYS a005719 MF807840 = MF807879
42 Nidirana okinavana Japan: Okinawa: Iriomote Island * Not given NC022872 = NC022872
43 Nidirana pleuraden China: Yunnan: Mt Gaoligong SYS a003775 MF807816 = MF807855
44 Nidirana pleuraden China: Yunnan: Mt Gaoligong SYS 2003776 MF807817 = MF807856
45 Babina holsti Japan: Okinawa * Not given NC022870 = NC022870
46 Babina subaspera Japan: Kagoshima: Amami Island * Not given NC022871 = NC022871

a 2 Lhi-Tong Lyu et al. / ZooKeys 858: 109-126 (2019)

f / Yaeyama

Taiwan

e Nidirana sp. n. @ N. adenopleura
@N. daunchina @N, okinavana
Hainan @N. chapaensis © N, nankunensis
eN. hainanensis © N. lini
e Outgroups @ N. pleurade

Figure I. Localities of the samples used in this study. The numbers correspond to the ID numbers in Table 1.

CTAGTGATAGCTGGTT-3’) and H10 (5’-TGATTACGCTACCTTTGCACG-
GT-3’), and for CO1 were Chmf4 (5’-TYTCWACWAAYCAYAAAGAYATCGG-3’)
and Chmr4 (5’--ACYTCRGGRTGRCCRAARAATCA-3’), and dgLCO (5’-GGT-
CAACAAATCATAAAGAYATYGG-3’) and dgHCO (5’-AAACTTCAGGGTGAC-
CAAARAAYCA-3’), following Lyu et al. (2019). PCR amplifications were processed
with the cycling conditions that initial denaturing step at 95 °C for 4 min, 35 cycles
of denaturing at 94 °C for 40 s, annealing at 53 °C (for 16S) / 48 °C (for CO1) for
40 s and extending at 72 °C for 60 s, and a final extending step at 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 per the guidelines,
on an ABI Prism 3730 automated DNA sequencer by Shanghai Majorbio Bio-pharm
Technology Co, Ltd. All sequences were deposited in GenBank (Table 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

Description of a new species of Music frogs from Mt Dayao, southern China Tis

potential scale reduction factor (PSRF) of < 0.005. In ML analysis, the bootstrap con-
sensus 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 measure-
ments 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 1904, 1909; Schmidt 1925; Chang and Hsu 1932; Bourret
1937; Kuramoto 1985; Chou 1999; Fei et al. 2007, 2009; Matsui 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 Yat-
sen 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);

114 Lhi-Tong Lyu et al. | ZooKeys 858: 109-126 (2019)

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 IID);

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 su-
prabrachial 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 NV. 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 recog-
nized 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 NV. 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 NV. pleuraden; absent or barely visible on fingers in NV.
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 NV. ini; (5) the presence of a single nuptial pad vs. absent in NV. hainanensis; di-
vided into two parts in N. chapaensis; (6) the presence of a pair of subgular vocal sacs
vs. absent in NV. okinavana; (7) the absence of spinules on dorsal skin vs. present in NV.
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

Description of a new species of Music frogs from Mt Dayao, southern China 115

(24)
(25)
(22)
(23)
1.00/100!] “(21) | Nidirana daunchina
(26)
(28)
(29)
(27)
0.98/88 (2)
(3)
(4)
vA Nidirana sp. n.
1.00/98 (7)
1.00/100
(8)
(1)
100/100 ee
1.00/99 : 2 Nidirana chapaensis
(20)
1.00/100 ate Nidirana hainanensis
(11)
0.02 -/73 (12)
(16)
BPP/BS ES Nidirana adenopleura
aoa) ((10
0.99/94 a 4)
1.00/98 (42) | Nidirana okinavana
1.00/99 (39
1.00/1001 (40) | Nidirana nankunensis
(41)
(32)
(33)
1.00/100 (34)

(36) | Nidirana lini
1.00/100] '(37)

1.00/100; (43)
(44)

puoonog ==) | Babina subaspera

(45) | Babina holsti

Figure 2. Bayesian inference and maximum-likelihood phylogenies. Number in parenthesis corresponds
to the ID number in Table 1.

| Nidirana pleuraden

2-4 fast-repeated double-notes in N. /ainanensis; containing a significantly different
first note in NV. daunchina and N. nankunensis); (2) the call notes last 30-54 ms vs.
call notes last 115-252 ms in N. adenopleura; the first notes last 108-135 ms in N.
nankunensis; the first notes last 162-197 ms and the others last 131-150 ms in WN.
daunchina; (3) the intervals between notes last 212—372 ms vs. last 98—213 ms in WN.
adenopleura; last 12-166 ms in N. nankunensis.

Therefore, based on the molecular, morphological, and bioacoustic differences, the
specimens from MDY, southern China, represent an unnamed species which is de-
scribed as a new species of genus Nidirana.

Lhi- Tong Lyu et al. | ZooKeys 858: 109-126 (2019)

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Description of a new species of Music frogs from Mt Dayao, southern China 117

Taxonomy account

Nidirana yaoica sp. nov.

http://zoobank.org/D05423B2-1812-4AF4-890C-A0A1915BD8A6

Chresonymy. Nidirana adenopleura: Fei et al. 2009 (Mt. Dayao, Jinxiu, Guangxi); Mo
et al. 2014 (Jinxiu, Guangxi)

Holotype. SYS a007022 (Fig. 3), adult male, collected by Zhi-Tong Lyu on 1 June
2018 from Mt Dayao (24.1602N, 110.2304E; ca 1190 m a.s.l.), Jinxiu Yao Autono-
mous County, Guangxi Zhuang Autonomous Region, China.

Paratypes. SYS 2007009, 7011-7013, 7020-7021, SYS a007014/CIB 110013,
seven adult males collected by Zhi-Tong Lyu, Yu-Long Li and Cheng-Yu Yang on 30
May-—1 June 2018 from the same locality as the holotype. NHMG 1503043-47, five
adult males collected by Yun-Ming Mo and Wei-Cai Chen on 19 March 2015 from
the neighboring locality as the holotype (24.1035N,110.2294E; ca 1350 ma.s.l.).

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 (RE !).

Differential diagnosis. Nidirana yaoica sp. nov. is distinguished from its congeners
by the following combination of the morphological characteristics: (1) body medium-
size and stocky, with SVL 43.8 + 1.7 (40.4-45.9, n = 13) mm in adult males; (2) disks
of digits dilated, pointed; (3) lateroventral grooves present on every digit; (4) heels
overlapping; (5) tibio-tarsal articulation reaching at the nostril; (6) mid-dorsal stripe
present; (7) posterior of dorsal skin rough with dense tubercles but without spinules;
(8) week supernumerary tubercles below the base of fingers III and IV, palmar tuber-
cles prominent and distinct; (9) a pair of subgular vocal sacs present; (10) one single
nuptial pad present on the first finger, nuptial spinules invisible; (11) suprabrachial
gland large; (12) calling: 1-3 fast-repeated regular notes.

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 lat-
erally, closer to the snout than to the eye; a longitudinal swollen mandibular ridge ex-
tending 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 < II]; tip of each finger slightly dilated

and remarkable elongated, forming long pointed disks; well-developed lateroventral

118 Lhi-Tong Lyu et al. | ZooKeys 858: 109-126 (2019)

Figure 3. Morphological features of the adult male holotype SYS 2007022 of Nidirana yaoica sp. nov. in

life. A dorsolateral view B ventral view C left hand D poorly developed nuptial pad E left foot F surface

of posterior dorsum and hind limbs.

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 I, 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 over-
lapping when hindlimbs flexed at right angles to axis of body; tibio-tarsal articula-
tion reaching the nostril when hindlimb is stretched along the side of the body; toes
relatively long and thin, relative lengths I < H < V < III < IV; tip of each toe slightly

Description of a new species of Music frogs from Mt Dayao, southern China 119

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 - 2% II 1% - 3 III 2% - 3% IV 3% - 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 rela-
tively 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; sev-
eral 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 ir-
regular 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 non-
uniform 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, tym-
panum dark brown; upper ¥ iris bright brownish white and lower % 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.

Variations. Measurements of type series are given in Table 3. All specimens were simi-
lar in morphology. Dorsal surface light brown in SYS a007009 (Fig. 4A), 7011, 7013 and
7020; mid-dorsal stripe begins from pineal ocellus in SYS a007011, 7013, 7014, 7020
and 7021 (Fig. 4B), unclear in SYS 2007009; pineal ocellus invisible in SYS a007009.

Male secondary sexual characteristics. A pair of subgular vocal sacs, a pair of slit-
like 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

Lhi-Tong Lyu et al. | ZooKeys 858: 109-126 (2019)

120

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Description of a new species of Music frogs from Mt Dayao, southern China IPA!

| : 2 ai K te es ‘ ;
“ = : co ee eh
a * = -
4 joie 5 rt ™ os -
1 5 os . 4 :
i be ri ey a
~ aa - ay ee ce = ot ey oh eo 4 : = 2

Figure 4. A, B paratypes SYS 2007009 and SYS a007021 of Nidirana yaoica sp. nov. C habitat of
Nidirana yaoica sp. nov. in the type locality in Mt Dayao D the holotype SYS a007022 in wild.

the swamps and ponds surrounded by moist subtropical secondary evergreen broad-
leaved 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 dura-
tion of 43.3 + 2.7 ms with the rise time of 10.1 + 4.5 ms. The two-note call (n = 59)

122 Lhi-Tong Lyu et al. | ZooKeys 858: 109-126 (2019)

0 0.1s

(0) 0.58

0 1.0s

Figure 5. Advertisement call spectrograms of Nidirana yaoica sp. nov. A one-note call B two-note call;

C three-note call.

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.

Description of a new species of Music frogs from Mt Dayao, southern China 123

Table 4. Vocalization parameters of paratype SYS a007009 of Nidirana yaoica sp. nov.

one-note call (n = 25) two-note call (n = 59) three-note call (n = 3)
Call duration (ms) 37-51, 43.3 + 2.7 307-454, 355.9 + 31.1 565-678, 628.0 + 57.6
Note duration (ms) 37-51, 43.3 + 2.7 1“ note: 36-51, 43.5 + 2.8; 1“ note: 42-54, 46.7 + 6.4;
2"™ note: 30-49, 39.6 + 3.3 2™ note: 37-40, 38.7 + 1.5;
3" note: 35-52, 42.3 + 8.7
Note rise time (ms) 1.6-15.5, 10.1 + 4.5 1* note: 2.0-16.0, 8.5 + 4.6; 1* note: 3.7-13.7, 7.4 + 5.5;
2™ note: 1.7-17.9, 11.6 + 4.4 2™4 note: 13.1-15.8, 14.8 + 1.5;
34 note: 14.0-16.1, 15.3 + 1.1
Note interval (ms) / 215-372, 272.8 + 31.7 1* interval: 212—250, 234.0 + 19.7;
2"4 interval: 222—302, 266.3 + 40.7
Call PF (Hz) 516.8 516.8 516.8
Note PF (Hz) 516.8 1% note: 516.8 (98.3%) or 2584 1* note: 516.8;
(1.7%);
2™4 note: 516.8 2"™ note: 516.8;

3" note: 516.8
Note IQR-BW (Hz) 172.3 (48.0%) or 0 (52.0%) 1* note: 344.5 (8.4%), 172.3 1“ note: 172.3 (33.3%) or 0 (66.6%);
(45.8%) or 0 (45.8%);
2™ note: 172.3 (54.2%) or 0 2™ note: 172.3 (33.3%) or 0 (66.6%);
(45.8%)
3" note: 172.3 (33.3%) or 0 (66.6%)

Discussion

‘The taxonomic status for the Nidirana population in MDY was suspected and suggested
a further study by Fei et al. (2009), despite their work reported it as NV. adenopleura tenta-
tively which was followed by Mo et al. (2014). Currently this population is revealed as NV.
yaoica sp. nov. in present work. In morphology, this frog is similar to NV. hainanensis by the
presence of lateroventral groove on all digits, and further to NV. daunchina, N. chapaensis,
and JV. okinavana by the absence of spinules on dorsal skin. Bioacoustically, NV. yaoica sp.
nov. has the same calling pattern as NV. adenopleura, which contains several fast-repeated
identical regular notes, but different from the pattern in NV. daunchina and N. hainanensis.
The phylogenetic tree showed that the new species is closer to NV. daunchina with moder-
ate supports (BPP 0.98 and BS 88), and then to NV. chapaensis and N. hainanensis.

The genus \idirana was recognized as a distinct genus recently based on compre-
hensive evidence by Lyu et al. (2017). For the interspecific relationship within the genus,
Dubois (1992) constructed two morphological species groups: NV. pleuraden group for
N. pleuraden and N. adenopleura group for the other known species; Fei et al. (2009)
proposed NV. daunchina group for N. daunchina and N. psaltes Kuramoto, 1985 (= N.
okinavana), and N. adenopleura group for N. adenopleura, N. lini, and N. hainanensis, but
excluding NV. chapaensis, and placing N. pleuraden in another genus Pelophylax Fitzinger,
1843; Chuaynkern et al. (2010) suggested three species groups for Music frogs based on
the morphological characters and ecological behavior of nest construction: NV. pleuraden
group for NV. pleuraden, N. adenopleura group for N. adenopleura and N. lini, and N.
okinavana group for NV. daunchina, N. okinavana, and N. chapaensis. From the current mi-
tochondrial results (Lyu et al. 2017; this study), the NV. pleuraden consistently formed the

124 Lhi- Tong Lyu et al. | ZooKeys 858: 109-126 (2019)

basal lineage of this genus, while the monophyly of the three species groups NV. adenopleura
group (Fei et al. 2009; Chuaynkern et al. 2010), NV. okinavana group, and N. daunchina
group, was challenged. The main conflicts are: (1) N. okinavana was suggested morpho-
logically more similar to N. daunchina and N. chapaensis (Fei et al. 2009; Chuaynkern et
al. 2010) while clustered with NV. adenopleura in the phylogeny; (2) NV. hainanensis was sug-
gested morphologically more similar to NV. adenopleura and N. lini (Fei et al. 2009) while
clustered with NV. daunchina and N. chapaensis in the phylogeny; (3) NV. lini was suggested
morphologically more similar to NV. adenopleura (Fei et al. 2009; Chuaynkern et al. 2010)
while formed the basal lineage of the congeners except NV. pleuraden in the phylogeny.
Thus we propose to follow Dubois’s (1992) suggestion, regarding two species
groups within the genus Nidirana: (1) N. pleuraden group, the lateroventral groove
absent on fingers and toes: one species, NV. pleuraden; (2) N. adenopleura group, the
lateroventral groove present on toes, absent or present on fingers: eight species, NV. ad-
enopleura, N. okinavana, N. nankunensis, N. hainanensis, N. chapaensis, N. daunchina,

N. yaoica sp. nov., and N. lini.

Acknowledgements

We thank Wei-Cai Chen, Jun Wu, and Cheng-Yu Yang for their help in the fieldwork.
We are grateful to Annemarie Ohler, Yodchaiy Chuaynkern, and an anonymous re-
viewer for their valuable suggestions on the manuscript. This work was supported by
the Project of Comprehensive Scientific Survey of Luoxiao Mountains Region of Min-
istry of Science and Technology, China (No. 2013FY111500), the Specimen Platform
of Ministry of Science and Technology, China, teaching specimens sub-platform (No.
2005DKA21403-JK), and the Project of Animal Diversity Survey and Monitoring

System Construction of Guangdong Shimentai National Nature Reserve.

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Appendix |

Specimens examined

Nidirana adenopleura (29): China: Fujian: Yanping District (type locality): SYS
a005911-5916; Mt Wuyi: SYS a005939-5943; Jiangshi Nature Reserve: SYS
4004112, 4132; Mt Yashu: SYS a005890-5891, 5901-5902; Jiangxi: Tongboshan
Nature Reserve: SYS a001663-1665, 1667, 1698; Yangjifeng Nature Reserve: SYS
a0000317, 0334; Jinggangshan Nature Reserve: SYS 20040254027; Zhejiang:
Jingning County: Dongkeng Town: SYS a002725-2726.

Nidirana daunchina (5): China: Sichuan: Mt Emei (type locality): SYS a004594—
4595; Hejiang County: Zihuai Town: SYS a004930-4932.

Nidirana hainanensis (1): China: Hainan: Mt Diaoluo (type locality): SYS 2003741.

Nidirana lini (4): China: Yunnan: Jiangcheng County: Hongjiang Town (type local-
ity): SYS a003967—3970.

Nidirana nankunensis (12): China: Guangdong: Mt Nankun (type locality): SYS
4003615, 3617-3620, 4019, 4905-4907, 5717-5719 (type series).

Nidirana pleuraden (4): China: Yunnan: Mt Gaoligong: SYS a003775-3778.