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A new species of Music frog (Anura, Ranidae, Nidirana) from Mt Daming, Guangxi, China
expand article infoZhi-Tong Lyu, Zhong Huang§, Xiao-Wen Liao§, Li Lin|, Yong Huang, Ying-Yong Wang, Yun-Ming Mo§
‡ Sun Yat-sen University, Guangzhou, China
§ Natural History Museum of Guangxi, Nanning, China
| Guangxi Daming Mountain National Nature Reserve Administration, Nanning, China
¶ Guangxi University of Chinese Medicine, Nanning, China
Open Access

Abstract

Nidirana guangxiensis sp. nov., a new music frog species, is proposed, based on a series of specimens collected from Mt Daming, Guangxi, southern China. The new species is close to N. 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 behavior 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

Introduction

The music frog genus Nidirana 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 usually 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 construction 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 instance, 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 N. adenopleura, and nominated some other populations as three new species: N. guangdongensis Lyu, Wan & Wang, 2020, N. mangveni Lyu, Qi & Wang, 2020, and N. 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 identification 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, N. 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 specimens from Mt Daming (MDM), central Guangxi, and Mt Jiuwan (MJW), northern Guangxi (Fig. 1). After comprehensive analyses, the specimens from MJW are identified as N. leishanensis Li, Wei, Xu, Cui, Fei, Jiang, Liu & Wang, 2019, while the specimens from MDM are herein proposed as a new species.

Figure 1. 

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

Materials and methods

Phylogenetic analysis

Nine muscular samples of the unnamed species from Guangxi were used for molecular analysis, encompassing five samples from MDM and four from MJW. All samples were obtained from euthanized specimens and then preserved in 95% ethanol and stored at −40 °C. In addition, 33 sequences from all known Nidirana species and two sequences from the outgroup, Babina holsti (Boulenger, 1892) and B. subaspera (Barbour, 1908) (following Lyu et al. 2017), were obtained from GenBank and incorporated into our dataset. Detailed information on these materials is shown in Table 1 and Figure 1.

Table 1.

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 a006568 MN946442 MN945198
16 Nidirana xiangica China: Hunan: Mt Dawei* SYS a006492 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 a004594 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 a007670 MN946452 MN945208
32 Nidirana lini China: Yunnan: Jiangcheng County* SYS a003967 MF807818 MF807857
33 Nidirana lini China: Yunnan: Jiangcheng County* SYS a003968 MF807819 MF807858
34 Nidirana mangveni China: Zhejiang: Mt Dapan* SYS a006310 MN946424 MN945180
35 Nidirana mangveni China: Zhejiang: Mt Dapan* SYS a006311 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

Two mitochondrial genes, namely partial 16S ribosomal RNA gene (16S) and partial cytochrome c oxidase I gene (COI), were used for phylogenetic analysis. DNA extraction, 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 GTR+I+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.

Morphological examination

Seventeen male and two female unnamed specimens collected from MDM were examined and measured, collection information is given in the taxonomic proposal. All specimens were fixed in 10% buffered formalin, transferred to 70% ethanol, and deposited 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 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 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 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 phylogenetically 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 collected, only male specimens were used. Data of the MDM specimens were newly measured in this work; meanwhile data of N. yaoica and N. yeae were obtained from the literature (Lyu et al. 2019; Wei et al. 2020). All measurements were ln-transformed to normalize and reduce the variance. The t-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 t-test and boxplots, measurements were scaled to remove allometric effects of body size in morphological analysis, using the following equation: Xa = Xln – β ∙ (SVLlnSVLm), where Xa = adjusted value; Xln = ln-transformed measurements; β = unstandardized regression coefficient for each species; SVLln = ln-transformed SVL; and SVLm = overall average SVLln of all samples. Principal component analysis (PCA) was performed to reduce the dimensionality of variation in the data to find whether morphological variation form the basis of detectable group structure, using the “prcomp” function and “ggplot2” package.

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 window 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 following Lyu et al. 2017) of the N. adenopleura group, which are distant from the true N. adenopleura in Clade D in phylogeny. Within Clade C, the Nidirana population from MJW (ID 9–12) is clustered with samples of N. 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 N. 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 lineages of N. 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 N. yeae with medium support (BS = 92).

Figure 2. 

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

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 t-test and boxplots of morphometrics (Table 3; Fig. 3) show that the Nidirana specimens from MDM are significantly different from N. yeae from northern Guizhou, especially in the characteristics of HDL, HDW, IND, TD, RAD, FTL, and TIB, and different from N. 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.

Table 2.

Diagnostic characters separating Nidirana guangxiensis sp. nov. from all congeners.

Species SVL of males (mm) SVL of females (mm) Fingers tips Lateroventral groove on fingers Relative length of fingers Toes tips Lateroventral groove on toes Tibio-tarsal articulation Subgular vocal sacs Nuptial pad Spinules on dorsal skin Nest construction Tadpole labial tooth row formula Calling References
N. guangxiensis 40.2–47.6 49.9–51.0 Dilated Present on fingers III and IV II < I < IV < III Dilated Present Nostril Present One on finger I Absent Present I: 1+1/1+1:II 6–11 rapidly repeated regular notes This study
N. yeae 41.2–43.5 44.7 Dilated Absent II < IV < I < III Dilated Present Eye Present One on finger I Absent ? (Probably absent) I: 1+1/1+1:II 2–6 notes containing a specific first note Wei et al. (2020)
N. yaoica 42.1–45.6 ? Dilated Present II < I < IV < III Dilated Present Nostril Present One on finger I Absent ? (Probably present) ? 1–3 fast-repeated regular notes Lyu et al. (2019)
N. daunchina 40.6–51.0 44.0–53.0 Dilated Absent or rarely present II < I < IV < III Dilated Present Nostril Present One on finger I Absent Present I:1+1/1+1:II or I:1+1/2+2:I 2–5 notes containing a specific first note Liu (1950); Lyu et al. (2017)
N. chapaensis 35.5–42.5 41.0–51.8 Dilated Present except finger I II < I = IV < III Dilated Present Nostril Present Two on finger I Absent or few above vent Present I:1+2/1+1:II 3 notes Chuaynkern et al. (2010)
N. adenopleura 43.1–57.6 47.6–60.7 Dilated Present except finger I II < I < IV < III Dilated Present Snout tip or eye-snout Present One on finger I Entire or posterior Absent I:1+1/1+1:II or I:0+0/1+1:I 2–5 regular notes Lyu et al. (2017, 2020b)
N. guangdongensis 50.0–58.4 55.3–59.3 Dilated Present except finger I II < I < IV < III Dilated Present Nostril Present One on finger I Entire Absent ? 2–4 regular notes Lyu et al. (2020b)
N. hainanensis 32.8–44.4 ? Dilated Present II < I < IV < III Dilated Present Nostril Present Absent Absent Present ? 2–4 fast-repeated double-notes Fei et al. (2009, 2012)
N. leishanensis 49.5–56.4 43.7–55.3 Dilated Present II < IV < I < III Dilated Present Eye-snout Present Two on fingers I and II Absent Absent I:1+2/ 1+1:II 1 single note Li et al. (2019)
N. lini 44.1–63.1 57.7–68.6 Dilated Present except finger I II < I < IV < III Dilated Present Beyond snout Present One on finger I Posterior Absent I:1+1/1+1:II 5–7 notes containing a specific first note Chou (1999); Lyu et al. (2017)
N. mangveni 53.6–59.7 59.7–65.1 Dilated Present on fingers III and IV I < II < IV < III Dilated Present Anterior corner of eye Present One on finger I Entire or posterior Absent ? 2–7 regular notes Lyu et al. (2020b)
N. nankunensis 33.3–37.1 37.8–39.5 Dilated Present except finger I II < I < IV < III Dilated Present Nostril Present One on finger I Absent or few above vent Present I:1+1/1+1:II 13–15 notes containing a specific first note Lyu et al. (2017)
N. occidentalis 44.5–53.0 55.6–61.3 Not dilated Absent II < I < IV < III Not dilated Absent Eye Present One on finger I Posterior Absent ? 3–5 regular notes Lyu et al. (2020a)
N. okinavana 35.5–42.8 44.6–48.8 Dilated Present except finger I II < I < IV < III Dilated Present Eye center-near nostril Absent Poorly one on finger I Absent Present I:1+1/1+1:II 10–25 fast-repeated notes Chuaynkern et al. (2010); Lyu et al. (2017)
N. pleuraden 46.2–52.3 46.9–61.7 Not dilated Absent II < I < IV < III Not dilated Absent Nostril Present One on finger I Posterior Absent I:1+1/1+1: II 1–4 regular notes Lyu et al. (2017, 2020a)
N. xiangica 56.3–62.3 53.5–62.6 Dilated Present II < I < IV < III Dilated Present Eye-snout Present One on finger I Entire Absent ? 2–3 notes containing a specific first note Lyu et al. (2020b)
Table 3.

Morphometric comparisons based on the t-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(42.4 ± 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) 10.1–11.9(11.0 ± 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
Figure 3. 

Boxplots of morphometrics based on the morphometric measurements, distinguishing Nidirana guangxiensis sp. nov., N. yeae, and N. yaoica.

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.

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 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 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 N. yeae, N. daunchina, N. lini (Chou, 1999), N. nankunensis Lyu, Zeng, Wang, Lin, Liu & Wang, 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 N. leishanensis, N. chapaensis, N. yaoica, N. adenopleura, N. guangdongensis, N. occidentalis Lyu, Yang & Wang, 2020, and N. pleuraden (Boulenger, 1904)].

Figure 5. 

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

Taxonomic proposal

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

Nidirana guangxiensis Mo, Lyu, Huang, Liao & Wang, sp. nov.

Chresonymy

Hylarana (Hylarana) adenopleura – Zhang and Wen 2000 (Mt. Daming, Guangxi)

Nidirana adenopleuraMo 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.

Figure 6. 

Morphological features of the adult male holotype NHMG 202007003 of Nidirana guangxiensis sp. nov. in preservative A dorsal view B ventral view C lateral view D right hand E right foot. Photos by Shuo Qi.

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 a008811/NHMG 202008003 and males SYS a008812–8813/NHMG 202008004–005, collected by Yun-Ming Mo, Zhong Huang, and Xiao-Wen Liao on 18 August 2020 from the same locality with the holotype.

Figure 7. 

Variation and colorations of Nidirana guangxiensis sp. nov. in life A, B male holotype NHMG 202007003 C female paratype NHMG 202007001 D male paratype NHMG 202007002 E, F uncaptured female and male individuals in the wild. Photos by Zhong Huang, Zhi-Tong Lyu, and Yun-Ming Mo.

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 “广西琴蛙 (guǎng xī qín wā)” 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 suprabrachial 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 (N = 2) in 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 < III; (5) lateral fringes wide on inner sides of fingers II, III and IV but absent on finger I; (6) webbing formula on toes I 2–2⅔ II 2–3 III 2½–3⅔ IV 3⅔–2½ 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 III 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 characteristics: (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 N. nankunensis; SVL > 50 mm in adult male N. guangdongensis, N. mangveni, and N. xiangica; SVL < 45 mm in adult female N. 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 II < I < IV < III [vs II < IV < I < III in N. yeae and N. leishanensis; II < I = IV < III in N. chapaensis; I < II < IV < III in N. mangveni]; (3) lateroventral grooves present on fingers III and IV [vs absent on all fingers in N. yeae, N. occidentalis, and N. pleuraden; present on all fingers in N. yaoica, N. hainanensis, N. leishanensis, and N. xiangica; present on all fingers except finger I in N. 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 N. lini; at the eye in N. yeae and N. occidentalis]; (6) a single nuptial pad present on finger I [vs nuptial pad absent in N. hainanensis; nuptial pad divided into two parts on finger I in N. chapaensis; two nuptial pads respectively on fingers I and II in N. leishanensis]; (7) a pair of subgular vocal sacs present in males [vs absent in N. okinavana]; (8) spinules on posterior dorsal skin absent [vs present in N. 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 II < I < IV < III [vs II < IV < I < III in N. 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 II, III and IV but absent on finger I [vs present only on fingers III and IV]; webbing formula on toes I 2–2⅔ II 2–3 III 2½–3⅔ IV 3⅔–2½ V [vs I 2–2 II 1⅔–3½ III 2½–3⅔ IV 3⅔–2 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 diameter 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 < III; 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, prominent 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 reaching at 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; lateroventral grooves well developed on each toe, not meeting at the tip of disks; webbing moderate, formula: I 1⅓–2 II 1⅓–2⅓ III 1⅔–3 IV 3⅓–1⅓ 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 longitudinal 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 arranged 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 posterior 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 ⅓ iris brownish white and lower ⅔ 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 become 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.

Table 4.

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

NHMG 202007002 NHMG 202007003* NHMG 202007004 NHMG 202007005 NHMG 202007007 NHMG 202007008 NHMG 202007009 NHMG 202007010 NHMG 202007011 NHMG 202007012
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 7.4 7.4 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 4.4 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 7.5 7.2 7.8 7.8 7.2 7.1 7.2 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 25.2 25.2 23.1
NHMG 202007013 NHMG 202007014 NHMG 202007015 NHMG 202007019 NHMG 202007020 SYS a008812 /NHMG 202008004 SYS a008813 /NHMG 202008005 NHMG 202007001 SYS a008811 /NHMG 202008003
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 7.2 7.3 7.0 7.4 7.5 7.9 7.8
IND 5.6 5.9 5.9 5.9 5.9 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 5.2
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 7.2 7.3 7.2 7.8 7.1 7.7 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

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 37th 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 arising just before origin of tail, maximum depth near mid-length, tapering gradually to narrow pointed tip.

Figure 8. 

Tadpole SYS a008814 of Nidirana guangxiensis sp. nov. Photos by Shuo Qi.

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 estimated extent of occurrence is less than 500 km2, and the estimated area of occupancy is less than 50 km2. The swamp was surrounded by subtropical evergreen broadleaf forests (Fig. 9A). Sympatric frog species observed in the swamp are Duttaphrynus melanostictus (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 26th–42nd stages can be observed, with the majority at the 33rd–37th stages.

Figure 9. 

A habitat of Nidirana guangxiensis 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.

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, N. xiangica from Mt Dupangling in the northeast, N. leishanensis 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 phylogenetically close to N. yaoica, while N. xiangica and N. leishanensis are sister species (Fig. 2). The complex rivers and mountainous systems in Guangxi may play as important barriers to the speciation of these species pairs.

As indicated by the etymology of the generic epithet (Dubois 1992), some species of Nidirana were observed with the behavior of nest construction (Nidirana guangxiensis sp. nov., N. okinavana, N. nankunensis, N. hainanensis, N. chapaensis, and N. daunchina). According to our field observations, these nest-constructing species 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 unpredictable water accumulation, which implies that constructing a nest would be helpful for the growth of the eggs and tadpoles. In contrast, the congeners without such behavior (N. adenopleura, N. guangdongensis, N. leishanensis, N. lini, N. mangveni, 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 (N. yaoica and N. yeae; Table 2), but to roughly illustrate and compare the reported ecological data which is correlated to such courtship behavior, N. yaoica living in seasonal swamps (Lyu et al. 2019) is likely to construct nests, and N. yeae inhabiting paddy field with tadpoles observed at the water surface (Wei et al. 2020) may not possess such behavior. Regarded as important for breeding, this behavior was used for the species-group 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 infrequent 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 1

Specimens examined

Nidirana adenopleura (37): China: Taiwan: Taichung City: SYS a007358–7365; Fujian: Yanping District: SYS a005911–5916; Mt Wuyi: SYS a005939–5943; Jiangshi 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 a0000317, 0334; Jinggangshan Nature Reserve: SYS a004025–4027; Zhejiang: Jingning County: Dongkeng Town: SYS a002725–2726.

Nidirana daunchina (5): China: Sichuan: Mt Emei: SYS a004594–4595 (topotypes); Hejiang County: Zihuai Town: SYS a004930–4932.

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 a007908 (topotypes); Mt Fanjing: SYS a007195–7196; Guangxi: Mt Jiuwan: NHMG 202007021–023, 025–029, 042–043.

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

Nidirana mangveni (9): China: Zhejiang: Mt Dapan: SYS a006310–6314; Mt. Longmen: 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; Shuangjiang County: SYS a007829–7832 (holotype and paratypes series).

Nidirana pleuraden (16): China: Yunnan: Kunming City: SYS a007585 (juvenile individual, 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 a006491–6493; Mt. Yangming: SYS a007269–7273; Jiangxi: Mt Wugong: SYS a002590–2591 (holotype and paratypes series).

Nidirana yaoica (13): China: Guangxi: Mt Dayao: SYS a007009, 7011–7014, 7020–7022, NHMG 1503043–047 (holotype and paratypes series).