ZooKeys 1059: 35-56 (202 I) A peer-reviewed open-access journal
doi: 10.3897/zookeys.1059.68140 RESEARCH ARTICLE #ZooKey S

https:/ / ZOO keys. pensoft.net Launched to accelerate biodiversity research

A new species of Music frog (Anura, Ranidae, Nidirana)
from Mt Daming, Guangxi, China

Zhi-Tong Lyu', Zhong Huang, Xiao-Wen Liao?, Li Lin?,
Yong Huang’, Ying-Yong Wang', Yun-Ming Mo?

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 3 Guangxi
Daming Mountain National Nature Reserve Administration, Nanning 530114, China 4 Guangxi University
of Chinese Medicine, Nanning 530200, China

Corresponding authors: Yun-Ming Mo (moyunming@163.com), Ying-Yong Wang (wangyy@mail.sysu.edu.cn)

Academic editor: Anthony Herrel | Received 3 May 2021 | Accepted 30 July 2021 | Published 1 September 2021
http://z00bank.org/207D BOD 1-B593-4ACD-BF5A-A386AD89399E

Citation: Lyu Z-T, Huang Z, Liao X-W, Lin L, Huang Y, Wang Y-Y, Mo Y-M (2021) A new species of Music frog
(Anura, Ranidae, Nidirana) from Mt Daming, Guangxi, China. ZooKeys 1059: 35-56. https://doi.org/10.3897/
zookeys.1059.68140

Abstract

Nidirana guangxiensis sp. nov., a new music frog species, is proposed, based on a series of specimens col-
lected from Mt Daming, Guangxi, southern China. The new species is close to NV. yeae, N. daunchina,
N. yaoica, and N. chapaensis from southwestern and south-central China and northern Indochina, while the
relationships among these species remain unresolved. Nidirana guangxiensis sp. nov. can be distinguished
from all known congeners by the genetic divergences in the mitochondrial 16S and COI genes, the be-
havior of nest construction, the advertisement call containing 6-11 rapidly repeated regular notes, and a
combination of morphological characteristics. Furthermore, the Nidirana populations recorded in Guangxi

are clarified in this work, providing valuable new information on the knowledge of the genus Nidirana.

Keywords

Bioacoustics, geography, mitochondrial DNA, morphology, nest construction

* These authors contributed equally as the first authors

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.

36 Zhi-Tong Lyu et al. / ZooKeys 1059: 35-56 (2021)

Introduction

The music frog genus Vidirana Dubois, 1992 was originally proposed as a subgenus of
Rana Linnaeus, 1758. Later, Nidirana was controversially recognized as a full genus or
a synonym of Babina Thompson, 1912 (Chen et al. 2005; Frost et al. 2006). Recently,
comprehensive morphological, molecular, bioacoustic, and biogeographical evidence has
resurrected Nidirana as a distinct genus (Lyu et al. 2017). The frogs of this genus usu-
ally inhabit the natural or artificial swamps, ponds, and paddy fields in the hilly regions
of subtropical eastern and southeastern Asia, with some species having nest construc-
tion behavior when courting (Fei et al. 2009; Lyu et al. 2017). The known diversity of
Nidirana increased dramatically from seven to 15 species since 2017 (Lyu et al. 2017,
2019, 2020a). Most of the newly described species were previously misidentified as other
congeners, due to their conservative phenotypes (Lyu et al. 2019, 2020a, 2020b). For in-
stance, Lyu et al. (2020b) revised multiple populations historically recorded as Nidirana
adenopleura (Boulenger, 1909) from China. They suggested that only the populations
from Taiwan, Jiangxi, Fujian, and southern Zhejiang are the true NV. adenopleura, and
nominated some other populations as three new species: NV. guangdongensis Lyu, Wan &
Wang, 2020, V. mangveni Lyu, Qi & Wang, 2020, and WN. xiangica Lyu & Wang, 2020.
Lyu et al.’s (2020b) work did not clarify all historic records of N. adenopleura, and the
taxonomic status for the records not involved in their study remains unresolved.

The Nidirana populations in Guangxi Zhuang Autonomous Region, southern
China, were previously recorded as N. adenopleura (Liu and Hu 1962; Zhang and
Wen 2000; Fei et al. 2009; Mo et al. 2014). Fei et al. (2009) suspected this identifica-
tion was not correct, but still tentatively followed it and suggested additional study.
Recently, the population from Mt Dayao, eastern Guangxi, has been revealed as a new
species, NV. yaoica Lyu, Mo, Wan, Li, Pang & Wang, 2019, and the population from
Mt Dupangling, northeastern Guangxi was assigned to N. xiangica (Lyu et al. 2019,
2020b). During our recent surveys in Guangxi, we collected a series of Nidirana speci-
mens from Mt Daming (MDM), central Guangxi, and Mt Jiuwan (MJW), northern
Guangxi (Fig. 1). After comprehensive analyses, the specimens from MJW are identi-
fied as NV. leishanensis Li, Wei, Xu, Cui, Fei, Jiang, Liu & Wang, 2019, while the speci-

mens from MDM are herein proposed as a new species.

Materials and methods

Phylogenetic analysis

Nine muscular samples of the unnamed species from Guangxi were used for mo-
lecular analysis, encompassing five samples from MDM and four from MJW. All
samples were obtained from euthanized specimens and then preserved in 95% etha-
nol and stored at -40 °C. In addition, 33 sequences from all known Nidirana spe-
cies and two sequences from the outgroup, Babina holsti (Boulenger, 1892) and

A new Music frogs from China 37

Sichuan %

¢

Hainan

Figure |. Map showing the collected localities for the Nidirana samples of Clade C (see Fig. 2) used
in this study.

B. subaspera (Barbour, 1908) (following Lyu et al. 2017), were obtained from Gen-
Bank and incorporated into our dataset. Detailed information on these materials is
shown in Table 1 and Figure 1.

Two mitochondrial genes, namely partial 16S ribosomal RNA gene (16S) and par-
tial cytochrome c oxidase I gene (COI), were used for phylogenetic analysis. DNA ex-
traction, PCR amplification, and sequencing conducted on the newly collected samples
followed Lyu et al. (2019). Two gene segments, 1042 base pairs (bp) of 16S and 639 bp
of COI, were concatenated seriatim into a 1681-bp matrix. The final alignment was
partitioned by gene and COI was further partitioned by codon position. ‘The partitions
were tested in jmodeltest v. 2.1.2, resulting in the best-fitting nucleotide substitution
models as GT R+1+G. Sequenced data were analyzed using maximum likelihood (ML) in
RaxmlGUI v. 1.3 (Silvestro and Michalak 2012). The bootstrap consensus tree inferred

from 1000 replicates was used to represent the evolutionary history of the taxa analyzed.

38

Zhi-Tong Lyu et al. / ZooKeys 1059: 35-56 (2021)

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

ID Species Locality Voucher number 16S COI

1 Nidirana guangxiensis sp. nov. China: Guangxi: Mt Daming* NHMG 202007001 =MZ677222 MZ678729
2 Nidirana guangxiensis sp. nov. China: Guangxi: Mt Daming* NHMG 202007002 MZ677223. MZ678730
3 Nidirana guangxiensis sp. nov. China: Guangxi: Mt Daming* NHMG 202007003. = MZ677224 MZ678731
4 Nidirana guangxiensis sp. nov. China: Guangxi: Mt Daming* NHMG 202007004. =MZ677225 MZ678732
5 Nidirana guangxiensis sp. nov. China: Guangxi: Mt Daming* NHMG 202007005 =MZ677226 MZ678733
6 Nidirana yaoica China: Guangxi: Mt Dayao* SYS a007020 MK882276 MK895041
7 Nidirana yaoica China: Guangxi: Mt Dayao* SYS a007021 MK882277. = MK895042
8 Nidirana yaoica China: Guangxi: Mt Dayao* SYS a007022 MK882278 MK895043
9 Nidirana leishanensis China: Guangxi: Mt Jiuwan NHMG 202007021 MZ677227 MZ678734
10 = Nidirana leishanensis China: Guangxi: Mt Jiuwan NHMG 202007022 MZ677228 MZ678735
11.) Nidirana leishanensis China: Guangxi: Mt Jiuwan NHMG 202007023 MZ677229 MZ678736
12. = Nidirana leishanensis China: Guangxi: Mt Jiuwan NHMG 202007025 MZ677230 MZ678737
13. Nidirana leishanensis China: Guizhou: Mt Leigong* SYS a007908 MN946453 MN945209
14 = Nidirana leishanensis China: Guizhou: Mt Fanjing SYS a007195 MN946454 MN945210
15 Nidirana xiangica China: Guangxi: Mt Dupangling SYS 2006568 MN946442, MN945198
16 = Nidirana xiangica China: Hunan: Mt Dawei* SYS 2006492 MN946434 MN945190
17. Nidirana xiangica China: Hunan: Mt Yangming SYS a007273 MN946440 MN945196
18 = Nidirana xiangica China: Jiangxi: Mt Wugong SYS a002590 MN946441 MN945197
19 = Nidirana yeae China: Guizhou: Tongzi County CIB TZ20190608004 MN295227 MN295233
20 Nidirana yeae China: Guizhou: Tongzi County CIB TZ20190608005 MN295228 MN295234
21 = Nidirana yeae China: Guizhou: Tongzi County CIB TZ20160714016 MN295231 MN295237
22. ~=Nidirana adenopleura China: Taiwan: Taichung City SYS a007358 MN946445 MN945201
23 ~—-Nidirana adenopleura China: Taiwan: Taichung City SYS a007359 MN946446 MN945202
24 Nidirana chapaensis Vietnam: Lao Cai: Sapa* MNHN 2000.4850 KR827711 KR087625
25 = Nidirana chapaensis Vietnam: Lao Cai: Sapa* MNHN 1999.5871 KR827710 /

26 Nidirana daunchina China: Sichuan: Mt Emei* SYS 004594 MF807822 MF807861
27 ~~ Nidirana daunchina China: Sichuan: Mt Emei* SYS a004595 MF807823 = MF807862
28 = Nidirana guangdongensis China: Guangdong: Yingde City* SYS a005767 MN946406 MN945162
29 = Nidirana guangdongensis China: Guangdong: Yingde City* SYS a005768 MN946407. =MN945163
30 = Nidirana hainanensis China: Hainan: Mt Diaoluo* SYS a007669 MN946451 MN945207
31 = Nidirana hainanensis China: Hainan: Mt Diaoluo* SYS 2007670 MN946452, = MN945208
32 = Nidirana lini China: Yunnan: Jiangcheng County* SYS 2003967 MF807818 |= MF807857
33 = Nidirana lini China: Yunnan: Jiangcheng County* SYS 2003968 MF807819 =MF807858
34 Nidirana mangveni China: Zhejiang: Mt Dapan* SYS 2006310 MN946424 MN945180
35 = Nidirana mangveni China: Zhejiang: Mt Dapan* SYS 2006311 MN946425 MN945181
36 = Nidirana nankunensis China: Guangdong: Mt Nankun* SYS a005718 MF807839 = MF807878
37 — Nidirana nankunensis China: Guangdong: Mt Nankun* SYS a005719 MF807840 MF807879
38 = Nidirana occidentalis China: Yunnan: Mt Gaoligong* SYS a003775 MF807816 = MF807855
39 ~—- Nidirana occidentalis China: Yunnan: Mt Gaoligong* SYS a003776 MF807817 MF807856
40 = Nidirana okinavana Japan: Okinawa: Iriomote Island* Not given NC022872 NC022872
41 = Nidirana pleuraden China: Yunnan: Kunming City* SYS a007858 MT935683. MT932858
42 = Nidirana pleuraden China: Yunnan: Wenshan City SYS a007717 MT935671 MT932850
43 Babina holsti Japan: Okinawa* Not given NC022870 = NC022870
44 = Babina subaspera Japan: Kagoshima: Amami Island* Not given NC022871 = NC022871
* Type locality.

Morphological examination

Seventeen male and two female unnamed specimens collected from MDM were ex-
amined and measured, collection information is given in the taxonomic proposal. All
specimens were fixed in 10% buffered formalin, transferred to 70% ethanol, and de-
posited in the Natural History Museum of Guangxi (NHMG) and the Museum of
Biology, Sun Yat-sen University (SYS), China.

Morphological descriptions follow the consistent definition by Lyu et al. (2017,
2019, 2020a, 2020b). External measurements of specimens were made with digital

A new Music frogs from China 39

calipers (Neiko 01407A stainless steel 6-inch digital calipers) to the nearest 0.1 mm.
These measurements are 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 cor-
ner of the eye);

HIND 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 examining the gonads. Webbing formula follows
Savage (1975).

Comparison characters of all known congeners were obtained from129 museum
specimens of 12 known congeners listed in the Appendix 1 and 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, 2019, 2020a, 2020b; Li et al. 2019; Wei et al. 2020).

Particularly, since the new Nidirana species from MDM is geographically and phy-
logenetically close to N. yaoica, and phylogenetically close to N. yeae Wei, Li, Liu,
Cheng, Xu & Wang, 2020, enhanced morphometric data of these three species were
used for statistical analyses in R v. 4.0.0. Due to the limited number of females col-
lected, only male specimens were used. Data of the MDM specimens were newly meas-
ured in this work; meanwhile data of NV. yaoica and N. yeae were obtained from the
literature (Lyu et al. 2019; Wei et al. 2020). All measurements were In-transformed to
normalize and reduce the variance. The +test was conducted with statistically similar
variances (p > 0.05 in the Levene’s test) using car R package. Boxplots were visualized
with the “ggplot2” R packages. For ¢-test and boxplots, measurements were scaled to
remove allometric effects of body size in morphological analysis, using the following
equation: X= X, —f- (SVL, -—SVL_), where X= adjusted value; x= In-transformed
measurements; § = unstandardized regression coefhcient for each species; SVL, = In-
transformed SVL; and SVL_ = overall average SVL, of all samples. Principal compo-
nent analysis (PCA) was performed to reduce the dimensionality of variation in the
data to find whether morphological variation form the basis of detectable group struc-
ture, using the “prcomp” function and “ggplot2” package.

40 Zhi-Tong Lyu et al. / ZooKeys 1059: 35-56 (2021)

Bioacoustic analysis

Advertisement calls of the Nidirana population from MDM were recorded in the field
at the air temperature of 18 °C by a Sony PCM D100 digital sound recorder on 20
April 2021. The recorded individuals were observed to ensure as the correct species
but were not captured for conservation reasons. The sound files in wave format were
sampled at 44.1 kHz with 24 bits in depth. Praat v. 6.0.27 (Boersma 2001) was used
to obtain the oscillogram, sonogram, and power spectrum (window length = 0.005 s).
Raven Pro v. 1.5 (Cornell Lab of Ornithology 2003-2014) was used to quantify the
acoustic properties (window size = 256 points, fast Fourier transform, Hanning win-
dow with no overlap). The 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) were measured for each call, and the note duration (the
time between onset and offset of a note) and note interval (the time between adjacent
notes in a call) were measured for each note.

Results

Phylogeny

The result of ML analysis was given in Figure 2, in which the supportive nodes with
the bootstrap supports (BS) > 90 were shown. This mitochondrial result is consistent
with the phylogenic relationship from previous studies (e.g. Lyu et al. 2020a), with
two species groups and four clades revealed. The Nidirana populations from MDM
(ID 1-5) and MJW (ID 9-12) are both inserted in the Clade C (clade names follow-
ing Lyu et al. 2017) of the N. adenopleura group, which are distant from the true UN.
adenopleura in Clade D in phylogeny. Within Clade C, the Nidirana population from
MJW (ID 9-12) is clustered with samples of NV. leishanensis from Mt Leigong and
Mt Fanjing, Guizhou, with strong supports (BS = 100) and small divergences, which
indicates the MJW population should be clarified as NV. leishanensis. The Nidirana
population from MDM (ID 1-5) forms an independent lineage with strong supports
(BS = 100) and almost no divergence, which is close to but diverse from the line-
ages of VV. yeae, N. daunchina (Chang, 1933), N. yaoica, and N. chapaensis (Bourret,
1937). The relationship among these five lineages remains unresolved, even though

the MDM population seems closer to NV. yeae with medium support (BS = 92).

Morphology

Detailed comparisons among all Nidirana species are listed in Table 2, which shows
the distinct differences of the Nidirana specimens from MDM (detailed comparisons
presented in the Taxonomic proposal below). The results of +test and boxplots of

morphometrics (Table 3; Fig. 3) show that the Midirana specimens from MDM are

A new Music frogs from China 4]

4
5
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. i pleuraden group
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subaspera

Figure 2. Phylogeny of Nidirana based on maximum likelihood. Number at the terminal of the branches
corresponds to the ID number in Table 1.

significantly different from NV. yeae from northern Guizhou, especially in the char-
acteristics of HDL, HDW, IND, TD, RAD, FTL, and TIB, and different from WN.
yaoica from eastern Guangxi in the characteristics of HDL, HDW, SNT, ED, TD,
and RAD. In the PCA analyses (Fig. 4), the extracted components PC1, PC2, PC3,
and PC4 eigenvectors account for 46.4%, 17.5%, 11.7%, and 8.3% of the variance,
respectively, or 83.9% cumulatively. As illustrated in the scatter plots of PC1 and
PC2, samples of each species cluster together and do not overlap with each other.

Bioacoustics

The advertisement calls of three male individuals of the Nidirana population from
MDM are recorded, and the call spectrograms are shown in Figure 5. The advertisement

35-56 (2021)

Zhi-Tong Lyu et al. / ZooKeys 1059

42

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A new Music frogs from China 43
MB cuangxiensis sp. nov. BS yeae BS yaoica

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na guangxiensis sp. nov., NV. yeae, and N. yaoica.

Table 3. Morphometric comparisons based on the #test of the morphometric measurements of males
Nidirana guangxiensis sp. nov. (N = 17), N. yeae (N= 9), and N. yaoica (N = 8). * p-values < 0.05, ** p-
values < 0.01, *** p-values < 0.001.

guangxiensis yeae yaoica guangxiensis vs yeae — guangxiensis vs yaoica
SVL 40.2-47.6(43.8 2.2) 39.2-44.5(42.44 1.8) 42.1-45.6(44.3 + 1.2) 0.1226 0.4136
HDL 17.1-19.9(18.5 + 0.7) 12.8-16.8(15.0+ 1.5) 16.3-18.6(17.3 + 0.8) 0.0002 *** 0.0011 **
HDW 15.3-18.4(16.5 0.8) 13.1-16.2(15.0+ 0.8) 15.0-16.7(16.0 + 0.6) 0.0002 *** 0.0232 *
SNT 6.4—7.8(7.2 + 0.4) 5.7—7.3(6.8 + 0.5) 6.2—7.2(6.7 £ 0.4) 0.1068 0.0031 **
IND 5.4—6.3(5.8 = 0.2) 4.7-5.9(5.4 + 0.3) 5.4—6.0(5.7 + 0.2) 0.0040 ** 0.1105
IOD 4.1-5.0(4.6 + 0.2) 3.5-5.2(4.2 + 0.6) 4.1-5.1(4.6 + 0.3) 0.1730 0.8934
ED 4,.5-4.9(4.7 + 0.1) 4,0-5.2(4.5 + 0.4) 4.6-5.4(5.1 + 0.3) 0.1964 0.0068**
TD 4,.2-4.7(4.4 + 0.1) 3.3-4.7(3.9 = 0.4) 3.2-3.9(3.7 £ 0.3) 0.0101* 0.0001***
HND 10.0-12.8(11.4+0.8) 10.1-11.9111.0+ 0.5) 10.3-12.4(11.1 + 0.8) 0.3092 0.2468
RAD 7.1-8.1(7.4 = 0.3) 7.7-9.6(8.6 + 0.7) 8.4—9.4(8.7 + 0.3) 0.0001*** 0.0000 ***
FTL 32.0-37.0(33.7 £ 1.3) 26.9-32.2(29.8£ 1.9) 33.1-35.7(34.4 + 0.8) 0.0006*** 0.1439
TIB 21.9-25.2(23.7£ 1.1) 19.6-22.8(21.5+ 1.0) 22.6—23.9(23.3 + 0.4) 0.0003*** 0.0653

calls of the Nidirana population in MDM have the duration of 1.012-1.917 s (1.461
+ 0.29, N = 20), with the PF of 1894.9 Hz generally, and consisted of 6-11 (8.4
+ 1.4, N = 20) rapidly repeated notes. All notes are identical and regular, with the
duration of 56-101 ms (77.4 + 6.7, N = 168) and the interval between them lasts
for 70-183 ms (110.4 + 21.36, N = 147). The advertisement calls of the Nidirana

44 Zhi-Tong Lyu et al. / ZooKeys 1059: 35-56 (2021)

Percentage of explained variances
0 10 20 30 40 50

1 erro] 46.4%

0.1-

sjuauodwo? |edisuiig paq9es1x9

a 10- * 1.0%

scree plot

-0.1-

-0.2- @ cuangxiensis sp. nov.
M@ yeae
A yaoica
PC1

Figure 4. Scatter plot of PC1 and PC2 of principal component analysis based on the morphometric
measurements, distinguishing Nidirana guangxiensis sp. nov., N. yeae, and N. yaoica.

0.0 0.5 1.0 1.5 2.0 Time (s)
Frequency (Hz)

0.0 0.5 1.0 1.5 2.0 Time (s)

Figure 5. Advertisement call spectrograms of Nidirana guangxiensis sp. nov. A waveform B sonogram.

population in MDM are different from the congeners by (1) all notes in a call are
identical and regular [vs containing a significantly different first note in NV. yeae, N.

daunchina, N. lini (Chou, 1999), N. nankunensis Lyu, Zeng, Wang, Lin, Liu & Wang,

A new Music frogs from China 45

2017, and N. xiangica; containing 2—4 fast-repeated double-notes in N. hainanensis
(Fei, Ye & Jiang, 2007)]; (2) containing 6-11 notes in a call [vs containing less than 6
notes in NV. leishanensis, N. chapaensis, N. yaoica, N. adenopleura, N. guangdongensis, N.
occidentalis Lyu, Yang & Wang, 2020, and N. pleuraden (Boulenger, 1904)].

Taxonomic proposal

Based on the molecular, morphological, and bioacoustic differences, the population
from MDM, Guangxi represents an unnamed species of genus Nidirana which is de-
scribed here.

Nidirana guangxiensis Mo, Lyu, Huang, Liao & Wang, sp. nov.
http://zoobank.org/4E5C27A2-D398-4758-A181-BB49D 1 DSEF42

Chresonymy.
Hylarana (Hylarana) adenopleura — Zhang and Wen 2000 (Mt. Daming, Guangxi)
Nidirana adenopleura — Mo et al. 2014 (Wuming and Shanglin, Guangxi)

Holotype. NHMG 202007003 (Figs 6, 7A, B), adult male, collected by Zhong
Huang and Xiao-Wen Liao on 7 July 2020 from Mt Daming (23.5156°N, 108.4370°E;
ca 1260 m a.s.l.), Wuming District, Nanning City, Guangxi Zhuang Autonomous
Region, China.

Paratypes. Eighteen specimens. Female NHMG 202007001 (Fig. 7C), and
males NHMG 202007002 (Fig. 7D), NHMG 202007004-005, 202007007-015,
202007019-—020, collected at the same time with the holotype. Female SYS 200881 1/
NHMG 202008003 and males SYS a008812—8813/NHMG 202008004—005, col-
lected by Yun-Ming Mo, Zhong Huang, and Xiao-Wen Liao on 18 August 2020 from
the same locality with the holotype.

Etymology. The specific name guangxiensis refers to the type locality of the new
species in Guangxi Zhuang Autonomous Region. The Zhuang language, one of the
official languages of Guangxi Zhuang Autonomous Region, is based on the dialect of
Wuming, from where the new species was collected.

Common name. “Guangxi Music Frog” in English and “) Pu4F (guang xi
qin wa)” in Chinese.

Diagnosis. Nidirana guangxiensis sp. nov. is placed in the genus Nidirana based
on the morphological characteristics of the absence of the thumb-like structure
on finger I, presence of well-developed dorsolateral folds, and the presence of su-
prabrachial glands in breeding males (Lyu et al. 2017). It is further assigned to the
N. adenopleura group by the presence of lateroventral grooves on all toes (Dubois
1992; Lyu et al. 2019).

Nidirana guangxiensis sp. nov. is distinguished from its congeners by the following
combination of the morphological characteristics: (1) body medium sized, with SVL

40.2—-47.6 mm (43.8 + 2.2, N = 17) in adult males and 49.9-51.0 mm (WN = 2) in

46 Zhi-Tong Lyu et al. / ZooKeys 1059: 35-56 (2021)

Figure 6. Morphological features of the adult male holotype NHMG 202007003 of Nidirana guangxien-
sis sp. nov. in preservative A dorsal view B ventral view C lateral view D right hand E right foot. Photos

by Shuo Qi.

adult females; (2) disks of digits dilated, pointed; (3) lateroventral grooves present on
fingers III and IV, and each toe; (4) relative finger length II < I < IV < II; (5) lateral
fringes wide on inner sides of fingers II, III and IV but absent on finger I; (6) webbing
formula on toes I 2—274 II 2—3 III 2%2~-3%4 IV 374-2 V; (7) tibio-tarsal articulation
reaching at the nostril; (8) dorsal skin rough with dense granules, several tubercles
on the posterior part, flanks, and dorsal hindlimbs, without spinules on the skin; (9)
distinct supernumerary tubercles below the base of fingers HI and IV, palmar tubercles
prominent and distinct; (10) a pair of subgular vocal sacs present; (11) a single nuptial
pad on the first finger, nuptial spinules invisible; (12) suprabrachial gland large; (13)
nest construction behavior present; (14) calling consisting of 6-11 rapidly repeated
regular notes.

Comparison. Nidirana guangxiensis sp. nov. can be significantly distinguished
from all other recognized congeners by the combination of the following characteris-
tics: (1) body medium-sized, SVL 40.2-47.6 mm (N = 17) in adult males and 49.9—
51.0 mm (N = 2) in adult females [vs SVL < 38 mm in adult male NV. nankunensis;
SVL > 50 mm in adult male NV. guangdongensis, N. mangveni, and N. xiangica; SVL
< 45 mm in adult female NV. yeae and N. nankunensis; SVL > 53 mm in adult female
N. guangdongensis, N. lini, N. mangveni, N. occidentalis, and N. xiangica]; (2) relative
fingers length IT<1<IV< II [vs <IV<I<Iin ™. yeae and N. leishanensis; Il < I

A new Music frogs from China AF

202007003 C female paratype NHMG 202007001 D male paratype NHMG 202007002 E, F uncap-
tured female and male individuals in the wild. Photos by Zhong Huang, Zhi-Tong Lyu, and Yun-Ming Mo.

=IV < [lin N. chapaensis; 1 < I< IV < Ulin N. mangvenil; (3) lateroventral grooves
present on fingers III and IV [vs absent on all fingers in NV. yeae, N. occidentalis, and
N. pleuraden; present on all fingers in NV. yaoica, N. hainanensis, N. leishanensis, and
N. xiangica; present on all fingers except finger I in NV. chapaensis, N. adenopleura,
N. guangdongensis, N. lini, N. nankunensis, and N. okinavana (Boettger, 1895)]; (4)
lateroventral grooves present on all toes [vs absent on all toes in N. occidentalis and
N. pleuraden|; (5) tibio-tarsal articulation reaches the nostril [vs beyond the snout tip
in NV. lini; at the eye in N. yeae and N. occidentalis]; (6) a single nuptial pad present on

48 Zhi-Tong Lyu et al. / ZooKeys 1059: 35-56 (2021)

finger I [vs nuptial pad absent in N. Aainanensis; nuptial pad divided into two parts
on finger I in NV. chapaensis; two nuptial pads respectively on fingers I and I in N. /eis-
hanensis]; (7) a pair of subgular vocal sacs present in males [vs absent in NV. okinavana];
(8) spinules on posterior dorsal skin absent [vs present in NV. adenopleura, N. lini, N.
mangveni, N. occidentalis, N. pleuraden, and N. xiangica).

Particularly, Nidirana guangxiensis sp. nov. is relatively close in phylogeny to
N. yeae from northern Guizhou, but it can be distinguished by: the relative fingers
length U<I<IV <I [vs I< IV<I1< Ill in \. yeae]; lateroventral grooves present on
fingers III and IV [vs absent on all fingers]; tibio-tarsal articulation reaches the nostril
[vs at the eye]; lateral fringes wide on inner sides of fingers H, IH and IV but absent
on finger I [vs present only on fingers III and IV]; webbing formula on toes I 2—2% II
2-3 III 2%4-3% IV 374-2% V [vs I 2-2 II 124-3% III 2%—-3%4 IV 374-2 V]; in males,
head larger, HDL/SVL 0.42 + 0.02 [vs 0.35 + 0.03], HDW/SVL 0.38 + 0.02 [vs
0.35 £0.01], radio-ulna length shorter, RAD/SVL 0.17 = 0.01 [vs 0.20 + 0.01], and
foot length longer, FTL/SVL 0.78 + 0.03 [vs 0.70 + 0.05].

Description of holotype. NHMG 202007003 (Figs 6, 7A, B), adult male. Body
medium-sized, SVL 43.8 mm; head relatively long and wide (HDL/SVL 0.42, HDW/
SVL 0.36), longer than wide (HDW/HDL 0.86), flat above; snout rounded in dorsal
and lateral views, slightly protruding beyond lower jaw, longer than horizontal di-
ameter of eye (SNT/ED 1.57); canthus rostralis distinct, slightly curved inwards on
the nostril; loreal region concave; nostril round, closer to the snout than to the eye;
a longitudinal swollen mandibular ridge extending from below nostril through lower
edges of 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 1.31), pineal ocellus invisible;
pupil elliptical, horizontal; tympanum distinct, round, relatively large, TD/ED 0.98;
vomerine ridge present, bearing small teeth; tongue cordiform, margin of the tongue
notched; a pair of subgular vocal sacs present.

Forelimbs moderately robust, lower arm 0.17 of SVL and hand 0.27 of SVL;
fingers thin, relative finger lengths II < I < IV < I]; tip of each finger slightly
dilated, forming rounded disks; lateroventral grooves on fingers III and IV, not
meeting at the tip of disks; fingers free of webbing; lateral fringes present and
distinct on inner and outer sides of fingers II, III and IV, but absent on finger I;
subarticular tubercles prominent and rounded; supernumerary tubercles present
below the base of fingers III and IV; palmar tubercles three, elliptic, large, promi-
nent and distinct; a single nuptial pad on the dorsal surface of finger I, nuptial
spinules invisible.

Hindlimbs robust, tibia 0.53 of SVL, and foot 0.76 of SVL; heels overlapping
when hindlimbs flexed at right angles to axis of body; tibio-tarsal articulation reach-
ing at the nostril when hindlimb is stretched along the side of the body; toes relatively
long and thin, relative lengths I < Hl < V < HI < IV; tip of each toe slightly dilated with
remarkable elongated ventral callous pad, forming long and pointed disk; lateroventral
grooves well developed on each toe, not meeting at the tip of disks; webbing moderate,

A new Music frogs from China 49

formula: I 144-2 II 144-2% III 174-3 IV 34%3-1% V; lateral fringes present on inner
and outer sides of each toe, forming distinct dermal flap on the lateral edges of toes I
and V; subarticular tubercles rounded, prominent; inner metatarsal tubercle elliptic,
length triple width; outer metatarsal tubercle indistinct, small and rounded; tarsal folds
present and tarsal tubercle absent.

Dorsal skin rough with dense granules, several tubercles on the posterior part,
flanks, and dorsal hindlimbs, not bearing spinules on the skin; developed dorsolateral
fold from posterior margin of upper eyelid to above groin but intermittent posteriorly;
a large and smooth suprabrachial gland behind base of forelimb, prominent; weak lon-
gitudinal ridges on upper arms and slightly extending to lower arm; the dorsal surfaces
of thigh and tibia relatively rough with tubercles, forming several longitudinal ridges.
Ventral surface of throat, body, and limbs smooth; large flattened tubercles densely ar-
ranged on the rear of thigh and around vent.

Color of holotype. In life (Fig. 7A, B), dorsal surface of head and body brown;
a longitudinal light brown mid-dorsal stripe faintly beginning from interorbital area,
extending posteriorly to vent and become more distinct; several black spots on pos-
terior dorsum of body; dorsolateral fold brown; upper flank brown with small black
spots; lower flank light brown; suprabrachial gland yellowish brown. Dorsal forelimbs
brown; dorsal hindlimbs brown, two olive crossbars on the thigh, three on the tibia,
and three on the tarsus; irregular olive marks on dorsal toes. Loreal and temporal
regions dark brown, tympanum pink; upper 3 iris brownish white and lower % iris
reddish brown; maxillary gland and shoulder gland creamy white. Lips, throat, ventral
surface of body and limbs creamy white; rear thigh tinged with pink and pale grey
patches; ventral hand and foot pale white.

In preservative (Fig. 6), dorsal surface becomes dark brown with the mid-dorsal
stripe and black spots more distinct; flank surface and the suprabrachial gland be-
come pale; crossbars and marks on limbs dark brown; loreal and temporal regions dark
brown; maxillary gland and shoulder gland more distinct; ventral surface pale grey;
rear thigh and ventral foot become dark grey.

Variations. Measurements of type series are given in Table 4. All specimens were
similar in morphology. Females are significantly larger than males, with relatively
smoother skin and fewer tubercles on dorsum and flanks. The colorations vary from
pale brown to reddish brown in individuals (Fig. 7C—F). The patterns of mid-dorsal
stripes are also variable but always present.

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 finger I, nuptial spinules invisible; suprabrachial gland present.

Tadpole. Body length 19.1 mm and tail length 43.1 mm in the 37" stage tadpole
SYS a008814 (Fig. 8); body oval, flattened above; snout rounded in dorsal aspect
and profile; eyes lateral; labial tooth row formula: 1:1+1/1+1:2; spiracle on left side
of body, directed dorsoposteriorly; tail depth larger than body depth; dorsal fin aris-
ing just before origin of tail, maximum depth near mid-length, tapering gradually to
narrow pointed tip.

50 Zhi-Tong Lyu et al. / ZooKeys 1059: 35-56 (2021)

Table 4. Measurements (in mm) of the type series of Nidirana guangxiensis sp. nov., * for the holotype,
M for male, and F for female.

ie) S ie) is ie) 3 ie) S ie) 5 io) 3 ie) 3 ie) = ie) = ie) =
Soe Seo Sot see Se Fee SS Sh. Sie Pak
a) zs ae) a) a) is ms zs rs xs
ZS Z& ZS ZS ZS ZS 78 ZS ZS Z8
N a N N N N N N N N
Sex M M M M M M M M M M
SVL 45.4 43.8 44.0 47.6 44.5 41.7 44.0 44.1 47.5 42.6
HDL 18.2 18.4 17.8 19.6 18.4 17.4 19.9 19.1 19.6 17.1
HDW 15.5 15.9 15.7 17.0 16.6 16.4 18.4 17.0 17.5 15.3
SNT 74 74 6.9 7.6 7.1 6.7 7.6 7.3 7.8 6.4
IND 6.0 6.0 5.7 5.9 5.6 5.7 6.0 6.1 6.3 5.4
IOD 4.6 4.6 4.8 5.0 4.7 4.1 4.5 4.6 4.8 4.1
ED 4.6 4.7 4.6 4.8 4.8 4.6 4.7 4.7 4.9 4.6
TD 4.4 4.6 4.2 4.6 44 4.3 4.4 4.3 4.7 4.3
HND 11.6 11.9 11.3 12.8 11.8 10.5 11.6 11.6 12.4 10.6
RAD ee) Fed 7.8 7.8 7.2 7.1 eo) 7.2 8.1 7.2
FTL 33.2 33.4 32.6 37.0 33.2 33.2 34.0 35.6 34.7 33.7
TIB 23.7 23.4 23.4 25.2 24.8 22.2 22.5 D522: 2522. 23.1
38 Lee io: amare 5k. eG Gk Eee Wee.
25 25 25 25 35 8828 222 35 822
mus us us mus us Sts Sts mus gis
ZS ZS ZS ZS Zs nZ223 428 ZS “29g
rx) I a I a a aq a aq aq in a
Sex M M M M M M M F F
SVL 40.2 43.2 41.5 42.5 40.3 45.1 46.5 49.9 51.0
HDL 18.0 18.3 18.0 18.5 18.6 18.5 18.5 20.3 20.4
HDW 15.8 16.2 16.2 16.7 17.0 16.1 17.1 18.7 18.8
SNT 6.7 7.3 TD) TS 7.0 7A VA) Fed: 7.8
IND 5.6 5.9 5.9 5.9 529 5.7 5.8 6.0 6.1
IOD 4.4 4.3 4.4 4.6 4.6 4.6 4.6 4.7 5.0
ED 4.5 4.7 4.6 4.7 4.7 4.8 4.6 5.1 32.
TD 4.3 4.5 4.3 4.5 4.4 4.6 4.3 4.2 4.2
HND 10.3 10.0 11.6 11.7 12:2) 11.0 11.2 12.1 12.1
RAD 7.1 Fx. 7.3 Vi 7.8 7.1 Tel 8.6 8.8
FTL 32.0 32.7 33.7 33.8 32.4 32.8 35.1 37.5 39.5
TIB 21.9 24.3 23.6 24.7 23.2 23.0 23.7 27.1 26.7

Distribution and ecology. Currently, Nidirana guangxiensis sp. nov. is known only
from the type locality, Mt Daming, which is located between Wuming District and
Shanglin County, Nanning, Guangxi (Fig. 1). This species of frog can only be found
in the alpine swamp and neighboring brushwood on the peak of Mt Daming. ‘The es-
timated extent of occurrence is less than 500 km’, and the estimated area of occupancy
is less than 50 km’. The swamp was surrounded by subtropical evergreen broadleaf
forests (Fig. 9A). Sympatric frog species observed in the swamp are Duttaphrynus mela-
nostictus (Schneider, 1799), Gracixalus jinxiuensis (Hu, 1978), Kurixalus odontotarsus
(Ye & Fei, 1993), and Polypedates mutus (Smith, 1940).

Nidirana guangxiensis sp. nov. was observed to have nest construction behavior. The
nest is in the form of a mud burrow ca 25—30 mm in diameter and near the roots of plants.
The top of the nest is open and may fill with water during the rainy season (Fig. 9B). From
April to August, males call from dusk to midnight in the nest. In late April, tadpoles at the
26°—42™ stages can be observed, with the majority at the 3337" stages.

A new Music frogs from China 51

5mm

Figure 8. Tadpole SYS 2008814 of Nidirana guangxiensis sp. nov. Photos by Shuo Qi.

Discussion

With this work, the historically recorded populations of Nidirana adenopleura from
Guangxi, are all reassigned to other recently described species, namely N. yaoica from
Mt Dayao in the east, NV. xiangica from Mt Dupangling in the northeast, NV. leishanen-
sis from Mt Jiuwan in the north, and Nidirana guangxiensis sp. nov. from Mt Daming,
central Guangxi (Fig. 1). Among them, Nidirana guangxiensis sp. nov. is phylogeneti-
cally close to NV. yaoica, while N. xiangica and N. leishanensis are sister species (Fig. 2).
The complex rivers and mountainous systems in Guangxi may play as important bar-
riers to the speciation of these species pairs.

As indicated by the etymology of the generic epithet (Dubois 1992), some spe-
cies of Nidirana were observed with the behavior of nest construction (idirana
guangxiensis sp. nov., N. okinavana, N. nankunensis, N. hainanensis, N. chapaensis,
and NV. daunchina). According to our field observations, these nest-constructing spe-
cies usually live in natural swamps and ponds with muddy bottoms (Fei et al. 2009,
2012; Lyu et al. 2017; this work). Such habitats obtain seasonal rainfall and unpre-
dictable water accumulation, which implies that constructing a nest would be help-
ful for the growth of the eggs and tadpoles. In contrast, the congeners without such
behavior (NV. adenopleura, N. guangdongensis, N. leishanensis, N. lini, N. mangveni,

52 Zhi-Tong Lyu et al. / ZooKeys 1059: 35-56 (2021)

oe ae SO ee Ce Ns ee

Figure 9. A habitat of Nidirana guanexiensis sp. nov. in the type locality in Mt Daming B a male calling
in a nest and two nests filled with half of water. Photos by Yun-Ming Mo and Shuo Qi.

N. occidentalis, N. pleuraden, and N. xiangica) usually inhabit natural or artificial
ponds and paddies with perennial water, which allows them to directly oviposit into
the water (Fei et al. 2009, 2012; Lyu et al. 2020a, 2020b). Additionally, the nest
construction behaviors of two other congeners are still unknown (NV. yaoica and
N. yeae; Table 2), but to roughly illustrate and compare the reported ecological data
which is correlated to such courtship behavior, NV. yaoica living in seasonal swamps
(Lyu et al. 2019) is likely to construct nests, and NV. yeae inhabiting paddy field with
tadpoles observed at the water surface (Wei et al. 2020) may not possess such behav-
ior. Regarded as important for breeding, this behavior was used for the species-group

A new Music frogs from China 53

divisions (Fei et al. 2009; Chuaynkern et al. 2010). Nevertheless, Lyu et al. (2019)
revised the species groups based on phylogenetic results and found that the behavior
of nest construction seems to evolve independently in different clades. As an infre-
quent habit in the family Ranidae, the evolution of nest construction behavior in
Nidirana species would be a topic worth studying and requires more ethological and
ecological work and the application of genomic data.

Based on the phylogenetic relationships, Lyu et al. (2017) partitioned the genus
Nidirana into four robustly supported clades (Fig. 2). Clade A corresponds to the
N. pleuraden group with two recognized species (Lyu et al. 2019, 2020a), while
the other clades belong to the N. adenopleura group (Lyu et al. 2019). Clade B is
monotypic and includes only N. lini, clade D is comprised of four species, and
clade C includes nine species which are more than half the members of the genus.
By bringing the phylogenetic analyses from this work and previous studies (Lyu et
al. 2019, 2020a, b), the interspecies relationships within clade C are unclear due to
the relatively lower supported values in mitochondrial genes. Species of clade C are
mostly distributed in the hilly regions throughout southwestern and south-central
China and northern Indochina (Fig. 1), at the edge of the Indo-Burma biodiversity
hotspot. In view of the extensiveness of these hilly areas and the unclear relationship
within this clade, Nidirana diversity in these areas seems still underestimated, which
suggests that further surveys are required.

Acknowledgements

We thank Wei-Liang Xie, Zhao-Chi Zeng, Shuo Qi, Jian Wang, Pi-Ning Zhou, Min
Shao, and Xi Hu for their help in the field and lab. We thank Yinpeng Zhang and
Robin Moser from Michigan State University for polishing the language. This work
was supported by National Animal Collection Resource Center, China, and Guangxi
Natural Science Foundation (grant no. 2017GXNSFDA198001).

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Appendix |

Specimens examined

Nidirana adenopleura (37): China: Taiwan: Taichung City: SYS a007358—7365; Fu-
jian: Yanping District: SYS a005911-5916; Mt Wuyi: SYS a005939-5943; Ji-
angshi Nature Reserve: SYS a004112, 4132; Mt Yashu: SYS a005890—5891,
5901-5902; Jiangxi: Tongboshan Nature Reserve: SYS a001663—1665, 1667,
1698; Yangjifeng Nature Reserve: SYS 20000317, 0334; Jinggangshan Nature Re-
serve: SYS a004025—4027; Zhejiang: Jingning County: Dongkeng Town: SYS
a002725-2726.

Nidirana daunchina (5): China: Sichuan: Mt Emei: SYS 2004594—4595 (topotypes);
Hejiang County: Zihuai Town: SYS 20049304932.

Nidirana guangdongensis (8): China: Guangdong: Shimentai Nature Reserve: SYS
a005765-5767, 5995, 5997-5998, 6879, 7688 (holotype and paratypes series).

Nidirana hainanensis (4): China: Hainan: Mt Diaoluo: SYS a003741, 7669-7671
(topotypes).

Nidirana leishanensis (13): China: Guizhou: Mt Leigong: SYS 2007908 (topotypes);
Mt Fanjing: SYS a007195—7196; Guangxi: Mt Jiuwan: NHMG 202007021-
023, 025-029, 042-043.

56 Zhi-Tong Lyu et al. / ZooKeys 1059: 35-56 (2021)

Nidirana lini (4): China: Yunnan: Jiangcheng County: Hongjiang Town: SYS
a003967—3970 (topotypes).

Nidirana mangveni (9): China: Zhejiang: Mt Dapan: SYS 20063 10-6314; Mt. Long-
men: SYS a006413-6414, 6416; Hangzhou City: SYNU 12050569 (holotype
and paratypes series).

Nidirana nankunensis (12): China: Guangdong: Mt Nankun: SYS a003615, 3617—
3620, 4019, 4905-4907, 5717-5719 (holotype and paratypes series).

Nidirana occidentalis (8): China: Yunnan: Mt. Gaoligong: SYS a003775-3778; Sh-
uangjiang County: SYS a007829-7832 (holotype and paratypes series).

Nidirana pleuraden (16): China: Yunnan: Kunming City: SYS a007585 (juvenile in-
dividual, topotype); Wenshan City: SYS a007717-7723, 7730; Xinping County:
SYS a007767, 7769-7770; Shiping County: SYS a007786-7789.

Nidirana xiangica (10): China: Hunan: Mt Dawei: SYS 2006491-6493; Mt. Yang-
ming: SYS a007269-7273; Jiangxi: Mt Wugong: SYS a002590-2591 (holotype
and paratypes series).

Nidirana yaoica (13): China: Guangxi: Mt Dayao: SYS 2007009, 7011-7014, 7020-
7022, NHMG 1503043-047 (holotype and paratypes series).