Biodiversity Data Journal : Taxonomy & Inventories
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Taxonomy & Inventories
A new species of Nidirana Dubois, 1992 (Anura, Ranidae) from Chongqing Municipality, China
expand article infoQi Ma‡,§, Zhijian Wang‡
‡ State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, School of Life Sciences, Southwest University, Chongqing, China
§ Chongqing Museum of Natural History, Chongqing, China
Corresponding author: Zhijian Wang (wangzj1969@126.com)
Academic editor: Truong Nguyen
Received: 15 Feb 2023 | Accepted: 21 Apr 2023 | Published: 10 May 2023
© 2023 Qi Ma, Zhijian Wang
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.
Citation: Ma Q, Wang Z (2023) A new species of Nidirana Dubois, 1992 (Anura, Ranidae) from Chongqing Municipality, China. Biodiversity Data Journal 11: e101986. https://doi.org/10.3897/BDJ.11.e101986
ZooBank: urn:lsid:zoobank.org:pub:0B46D249-1296-4EB1-B6A5-D47E1F65FA9D
 Open Access

Abstract

Background

The Nidirana Dubois, 1992 exhibit a ubiquitous presence in East and Southeast Asia, spanning from Japan west to southern China and from northern Thailand to northern Vietnam and Laos in the south. The taxonomic categorisations pertaining to this genus continue to be a subject of debate, particularly with regard to those species that possess broad geographical distributions. In China, 18 species of Nidirana are currently recognised.

New information

We describe a new species of this genus from south-western China. Mitochondrial 16S and COⅠ gene phylogenetic analyses support the new species as an independent clade nested within the Nidirana. The new species is phylogenetically close to N. yaoica, with a genetic distance of 2.5% from its sister taxon. Morphologically, the new species can be distinguished from congeners by a combination of the following characteristics: males relatively small (SVL 41.8-43.3 mm); lateroventral grooves present on both fingers and toes; relative finger length Ⅱ < Ⅰ < Ⅳ < Ⅲ; tibio-tarsal articulation reaching to the level of the eye or nostril; a pair of external subgular vocal sacs in males; one single nuptial pad on the dorsal surface of the base of the first finger in males during the breeding season; webbing formula I 1/2 - 1 II 1/2 - 2 III 1 - 2½ IV 2 - 1V.

Keywords

Chongqing Municipality, mitochondrial DNA, morphology, Nidirana chongqingensis sp. nov.

Introduction

The genus Nidirana Dubois, 1992 is found in subtropical and tropical parts of eastern and south-eastern Asia, ranging from Japan to the south of China, to the north of Thailand, Vietnam and Laos. The members of this genus typically inhabit natural or artificial swamps, ponds and paddy fields in hilly areas and, during the breeding season, some species construct nests (Fei et al. 2009, Grosjean et al. 2015, Lyu et al. 2017). The classification of Nidirana has been a subject of debate for a long time (Chen et al. 2005, Frost et al. 2006, Chuaynkern et al. 2010, Li et al. 2019, Lyu et al. 2019). It has been proposed as a subgenus or synonym of Rana Linnaeus, 1758 and merged with Babina Thompson, 1912 (Frost et al. 2006). However, based on molecular systematics, morphology and bioacoustics, Lyu et al. (2017) suggested that Nidirana is an independent genus and sister to Babina. In the past five years, several new species of this genus have been described, namely N. leishanensis Li, Wei, Xu, Cui, Fei, Jiang, Liu and Wang, 2019, N. yaoica Lyu, Mo, Wan, Li, Pang and Wang, 2019, N. guangdongensis Lyu, Wan and Wang, 2020, N. mangveni Lyu, Qi and Wang, 2020, N. occidentalis Lyu, Yang and Wang, 2020, N. xiangica Lyu and Wang, 2020, N. yeae Wei, Li, Liu, Cheng, Xu and Wang, 2020, N. guangxiensis Mo, Lyu, Huang, Liao and Wang, 2021, N. guibeiensis Chen, Ye, Peng and Li, 2022 and N. shiwandashanensis Chen, Peng, Li and Liu, 2022. (Li et al. 2019, Lyu et al. 2019, Lyu et al. 2020a, Lyu et al. 2020b, Wei et al. 2020, Lyu et al. 2021, Chen et al. 2022a, Chen et al. 2022b).

The conservative phenotype of many newly-described species has caused them to be misidentified as other congeneric species (Lyu et al. 2019, Lyu et al. 2020a, Lyu et al. 2021). As an example, Lyu et al. (2020b) identified three new species (N. guangdongensis, N. mangveni and N. xiangica) from multiple populations that were previously believed to be N. adenopleura. In addition, Chen et al. (2022a) described another new species, N. shiwandashanensis, from Shiwandashan National Nature Reserve in Shangsi County, Guangxi Province of China.

During our field survey in Qiangjiang District of Chongqing Municipality in southern China in 2022, we collected three specimens of frogs, which were identified as a species of Nidirana, based on the comparison of their morphological features. Upon further analysis of molecular systematics and morphology, these specimens were distinct from all known species belonging to the genus Nidirana and we herein describe them as a new species.

Materials and methods

Three adult male specimens of an undescribed species of Nidirana were collected from Qianjiang District, Chongqing Municipality, China in this study (see Fig. 1). In the field, the frog was euthanised with isoflurane and the specimens were fixed in 75% ethanol. Tissue samples were taken and preserved in 100% ethanol before fixation. The specimens were subsequently deposited in the Southwest University (SWU).

Figure 1.  

Geographical location of the type locality of the new species (blue dot).

Molecular analysis

Total DNA was extracted using the Tiangen DNA Extraction Kit DP304 (Tiangen Biochemical Technology Co., Ltd.), quantified for quantity and concentration using a spectrophotometer (Thermo Scientific NanoDrop 2000), and then kept at -20°C. Two fragments of the mitochondrial 16S rRNA (16S) and the cytochrome oxidase subunit I (COI) genes were amplified. Primers used for 16S were L3975 (5’-GCCTGTTTACCAAAAACAT-3’) and H4551 (5’-CCGGTCTGAACTCAGATCACGT-3’), and L2A (5’-CCAAACGAGCCTAGTGATAGCTGGTT-3’) and H10 (5’-TGATTACGCTACCTTTGCACGGT-3’), and for COI were dgLCO (5’-GGTCAACAAATCATAAAGAYATYGG-3’) and dgHCO (5’-AAACTTCAGGGTGACCAAARAAYCA-3’), following Lyu et al. (2019). The following steps were used to carry out a standard polymerase chain reaction (PCR) amplification in a 20 μl system: de-naturing 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 COI) for 40 s and extending at 72°C for 60 s, and a final extending step at 72°C for 10 min. The PCR amplification products were detected by agarose gel electrophoresis. The products were then sent to Biotech Bioengineering (Shanghai) Co. All newly- obtained sequences have been submitted to GenBank (to be uploaded upon article acceptance). In order to investigate the current phylogenetic relationships amongst Nidirana, a dataset was constructed by concatenating 16S (~ 1102 bp) and COI (~ 562 bp) gene sequences.

From GenBank, we retrieved the homologous sequences of the related species in the genus Nidirana, as well as those of the outgroups Babina holsti and Babina subaspera (Kakehashi et al. 2013) (Table 1). Sequences that had been amplified recently were manually checked using SeqMan in the DNASTAR LASERGENE v.6 package. Sequences that had been sequenced in both directions were then combined, and the combined sequences were imported into MEGA 6 (Tamura et al. 2013) for comparison and the elimination of redundant sequences. In this study, phylogenetic trees were created using Bayesian Iinference (BI) and Mmaximum Llikelihood (ML) methods. ML was analyszed on the CIPRES Sscience Gateway with 100 rapid bootstrap replicates (Miller et al. 2010) (https://www.phylo.org/portal2). Bayesian methods were implemented in MrBayes v.3.2 (Ronquist et al. 2012) and the optimal substitution model was determined using ModelFinder (Kalyaanamoorthy et al. 2017), with the best model determined by ModelFinder in this article being GTR+I+G. In the Bayesian analysis, four Markov chains were used, operated for 10 million generations, sampled every 500 generations, and with the first 25% samples beingwere discarded as burn-in. The software Tracer v.1. 5 (Drummond and Rambaut 2007) was used to determine whether the resulting results converged (ESS > 200).

Table 1.
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Localities, voucher information and GenBank numbers for samples used in this study. An asterisk denotes type localities.

ID Species Locality (* type locality) Voucher No. 16S COI References
1 N. chongqingensis sp. nov. China: Chongqing: Qianjiang District SWU0001408 OQ846777 OQ843905 This study
2 N. chongqingensis sp. nov. China: Chongqing: Qianjiang District SWU0001439 OQ846779 OQ843907 This study
3 N. chongqingensis sp. nov. China: Chongqing: Qianjiang District SWU0001435 OQ846778 OQ843906 This study
4 N. shiwandashanensis China: Guangxi: Shangsi County NNU00238 MZ787977 MZ782098 Chen et al. (2022a)
5 N. shiwandashanensis China: Guangxi: Shangsi County NNU00239 MZ787978 MZ782099 Chen et al. (2022a)
6 N. guangxiensis China: Guangxi: Mt Daming* NHMG 202007001 MZ677222 MZ678729 Lyu et al. (2021)
7 N. guangxiensis China: Guangxi: Mt Daming* NHMG 202007002 MZ677223 MZ678730 Lyu et al. (2021)
8 N. yeae China: Guizhou: Tongzi County CIB TZ20190608005 MN295228 MN295234 Wei et al. (2020)
9 N. yeae China: Guizhou: Tongzi County CIB TZ20160714016 MN295231 MN295237 Wei et al. (2020)
10 N. daunchina China: Sichuan: Mt Emei* SYS a004594 MF807822 MF807861 Lyu et al. (2020a)
11 N. daunchina China: Sichuan: Mt Emei* SYS a004595 MF807823 MF807862 Lyu et al. (2020a)
12 N. yaoica China: Guangxi: Mt Dayao* SYS a007020 MK882276 MK895041 Lyu et al. (2019)
13 N. yaoica China: Guangxi: Mt Dayao* SYS a007021 MK882277 MK895042 Lyu et al. (2019)
14 N. chapaensis Vietnam: Lao Cai: Sapa* MNHN 2000.4850 KR827711 KR087625 Grosjean et al. (2015)
15 N. guibeiensis China: Guangxi: Xing’an: Maoershan NNU 00917 ON985180 ON968962 Chen et al. (2022a)
16 N. guibeiensis China: Guangxi: Xing’an: Maoershan NNU 00918 ON985181 ON968963 Chen et al. (2022a)
17 N. leishanensis China: Guizhou: Mt Leigong* SYS a007908 MN946453 MN945209 Lyu et al. (2021)
18 N. xiangica China: Hunan: Mt Dawei* SYS a006492 MN946434 MN945190 Lyu et al. (2020a)
19 N. guangdongensis China: Guangdong: Yingde City* SYS a005767 MN946406 MN945162 Lyu et al. (2020a)
20 N. guangdongensis China: Guangdong: Yingde City* SYS a005768 MN946407 MN945163 Lyu et al. (2020a)
21 N. hainanensis China: Hainan: Mt Diaoluo* SYS a007669 MN946451 MN945207 Lyu et al. (2020a)
22 N. hainanensis China: Hainan: Mt Diaoluo* SYS a007670 MN946452 MN945208 Lyu et al. (2020a)
23 N. adenopleura China: Taiwan: Taichung City SYS a007358 MN946445 MN945201 Lyu et al. (2020a)
24 N. adenopleura China: Taiwan: Taichung City SYS a007359 MN946446 MN945202 Lyu et al. (2020a)
25 N. mangveni China: Zhejiang: Mt Dapan* SYS a006310 MN946424 MN945180 Lyu et al. (2020a)
26 N. mangveni China: Zhejiang: Mt Dapan* SYS a006311 MN946425 MN945181 Lyu et al. (2020a)
27 N. okinavana Japan: Okinawa: Iriomote Island* Not given NC022872 NC022872 Kakehashi et al. (2013)
28 N. nankunensis China: Guangdong: Mt Nankun* SYS a005718 MF807839 MF807878 Lyu et al. (2017)
29 N. nankunensis China: Guangdong: Mt Nankun* SYS a005719 MF807840 MF807879 Lyu et al. (2017)
30 N. lini China: Yunnan: Jiangcheng County* SYS a003967 MF807818 MF807857 Lyu et al. (2017)
31 N. lini China: Yunnan: Jiangcheng County* SYS a003968 MF807819 MF807858 Lyu et al. (2017)
32 N. occidentalis China: Yunnan: Mt Gaoligong* SYS a003775 MF807816 MF807855 Lyu et al. (2020a)
33 N. occidentalis China: Yunnan: Mt Gaoligong* SYS a003776 MF807817 MF807856 Lyu et al. (2020a)
34 N. pleuraden China: Yunnan: Kunming City* SYS a007858 MT935683 MT932858 Lyu et al. (2020b)
35 N. pleuraden China: Yunnan: Wenshan City SYS a007717 MT935671 MT932850 Lyu et al. (2020b)
36 Babina subaspera Japan: Kagoshima: Amami Island* Unknown NC022871 NC022871 Kakehashi et al. (2013)
37 Babina holsti Japan: Okinawa* Unknown NC022870 NC022870 Kakehashi et al. (2013)

Morphological characteristics

Measurements were taken with an electronic digital caliper (SHAHE brand, range 0-200 mm) with a measurement accuracy of 0.1 mm. The methods for measuring adult external morphological traits were according to Fei et al. (2009). The morphological characteristics are as follows: SVL, snout-vent length (the length from the snout to the posterior margin of the vent); HL, head length (the length from the snout to the articulation of the jaw); HW, head width (the widest distance between the two sides of the head); SL, snout length (the distance from the snout to the anterior corner of the eye); IND, internasal distance (minimum distance between the inner margins of the external nares); IOD, interorbital distance (the shortest distance between the inner edges of the upper eyelids); NSL, nostril-snout length (the length from the centre of the nostril to the tip of the snout); NEL, nostril-eye length (the length from the centre of the nostril to the anterior corner of the eye); UEW, upper eyelid width (margins measured perpendicular to the anterior-posterior axis); ED, eye diameter (distance from the anterior corner to the posterior corner of the eye); TD, tympanum diameter (horizontal diameter of tympanum); FAHL, forearm and hand length (the length from the elbow joint to the tip of finger IV); HAL, hand length (the length from proximal edge of inner palmar tubercle to the tip of the third finger); FAW, maximum width of forearm (the widest distance between the two sides of the forearm); HLL, hind limb length (the length from tip of fourth toe to vent); THL, thigh length (distance from vent to knee); TBL, tibia length (distance from knee to tarsus); TFL, length of tarsus and foot (the length from the tibiotarsal articulation to the tip of the toe IV); FTL, foot length (distance from distal end of shank to the tip of Toe IV).

Sex can be determined by examining the nuptial pad, vocal sac and suprabrachial gland, the webbing formula following Savage and Heyer (1997).

The unnamed species is also separated from other species of Nidirana based on morphological features. Morphological data for comparison were obtained from literature (Boettger 1895, Boulenger 1904, Boulenger 1909, Schmidt 1925, Chang and Hsü 1932, Bourret 1937, Kuramoto 1985, Chou 1999, Fei et al. 2007, Matsui 2007, Fei et al. 2009, Chuaynkern et al. 2010, Lyu et al. 2017, Li et al. 2019, Lyu et al. 2020a, Lyu et al. 2020b, Wei et al. 2020, Chen et al. 2022a, Chen et al. 2022b).

Data resources

The accession numbers of the newly-discovered sequences in this analysis are displayed in Table 1 and all of the sequences in this study were obtained from GenBank.

Taxon treatment

Nidirana chongqingensis Ma & Wang sp. nov.

Materials   Download as CSV 
Holotype:
  1. scientificName:
    Nidirana chongqingensis
    ; kingdom:
    Animalia
    ; phylum:
    Chordata
    ; class:
    Amphibia
    ; order:
    Anura
    ; family:
    Ranidae
    ; genus:
    Nidirana
    ; higherGeography:
    South-western China
    ; country:
    China
    ; municipality:
    Chongqing
    ; locality:
    Gaolu Village, Mala Town, Qianjiang District
    ; verbatimElevation:
    1419 m
    ; verbatimCoordinates:
    29°14'31.32"N 108°54'7.93"E
    ; georeferenceSources:
    Google Earth
    ; eventDate:
    03/07/2022
    ; individualCount:
    1
    ; sex:
    male
    ; lifeStage:
    adult
    ; catalogNumber:
    SWU0001408
    ; recordedBy:
    Qi Ma
    ; identifiedBy:
    Qi Ma
    ; basisOfRecord:
    PreservedSpecimen
    ; occurrenceID:
    C3B1F1AD-5D4C-5F35-8F0C-7020A48D4D15
Paratype:
  1. scientificName:
    Nidirana chongqingensis
    ; kingdom:
    Animalia
    ; phylum:
    Chordata
    ; class:
    Amphibia
    ; order:
    Anura
    ; family:
    Ranidae
    ; genus:
    Nidirana
    ; higherGeography:
    South-western China
    ; country:
    China
    ; municipality:
    Chongqing
    ; locality:
    Gaolu Village, Mala Town, Qianjiang District
    ; verbatimElevation:
    1419 m
    ; verbatimCoordinates:
    29°14'31.32"N 108°54'7.93"E
    ; georeferenceSources:
    Google Earth
    ; eventDate:
    03/07/2022
    ; individualCount:
    2
    ; sex:
    male
    ; lifeStage:
    adult
    ; catalogNumber:
    SWU0001439
    ; recordedBy:
    Qi Ma
    ; identifiedBy:
    Qi Ma
    ; basisOfRecord:
    PreservedSpecimen
    ; occurrenceID:
    A44D36F5-8AD4-5B9E-A834-324FA55C768E

Diagnosis

The new species was placed in the genus Nidirana based on molecular data (16S and COI genes).

The new species could be distinguished from its congeners by a combination of the following characters: (1) body size small (SVL 41. 8-43. 3 mm in males); (2) indistinct canthus rostralis; (3) no longitudinal ridges on the upper arm; (4) lateroventral grooves on the ventral side of all fingers and toes; (5) supernumerary tubercles below the base of fingers III and IV, inconspicuous; (6) metacarpal tubercles distinct and prominent; (7) well-developed dorsolateral folds, but intermittent posteriorly; (8) supratympanic fold absent; (9) males with large, smooth and protruding suprabrachial glands in breeding period; (10) dorsal skin relatively smooth, without horny spines on the back; (11) mid-dorsal stripe present; (12) ventral surface of body milky white, but the throat, chest and ventral side of the limbs are densely covered with brownish-red or brown-black spots; (13) tibio-tarsal articulation reaching the angle of the eye or the nostril when adpressed along body, heels not meeting or just meeting when hind limbs flexed at a straight angle to the body's axis; (14) a pair of external subgular vocal sacs in males; (15) males with one single nuptial pad on the dorsal surface first finger in the breeding period; (16) finger tips not dilated, tip of each toe slightly dilated (Table 2).

Table 2.
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Measurements of the new species (in mm). Abbreviations defined in Materials and Methods.

Measurement

SWU0001408 (Male, holotype)

SWU0001435 (Male, paratype)

SWU0001439 (Male, paratype)

SVL

43. 3

42. 0

41. 8

HL

17. 4

17. 3

16. 2

HW

14. 7

13. 4

13. 3

SL

7. 2

6. 5

6. 8

IND

5. 8

5. 5

5. 0

IOD

4. 9

4. 6

3. 2

NSL

3. 8

3. 4

3. 7

NEL

3. 7

3. 2

3. 0

UEW

4. 1

3. 9

3. 3

ED

5. 5

5. 3

5. 2

TD

4. 0

3. 1

3. 4

FAHL

18. 0

18. 1

17. 9

HAL

10. 7

11. 1

11. 4

FAW

3. 8

3. 5

2. 8

HLL

70. 1

67. 8

66. 5

THL

22. 6

20. 2

20. 4

TBL

23. 8

22. 5

22. 8

TFL

32. 5

31. 9

31. 2

FTL

22. 6

22. 2

22. 2
Etymology: 

The specific name "Chongqing" refers to the type locality of the new species in Chongqing Municipality, where the new species was collected.

Common name: 

“Chongqing Music Frog” in English and “重庆琴蛙 (Chóng qìng qín wā)” in Chinese.

Description of holotype: 

An adult male, SVL 43.3 mm, head longer than width (HL: HW = 1.18), flat above; snout rounded in dorsal view, slightly protruding beyond lower jaw; loreal region slightly inclined outwards, slightly concave in the middle; canthus rostralis indistinct; pupil elliptical, horizontal; nostril rounded, directed laterally, closer to the eye than to the snout; internasal distance larger than interorbital distance (IND:IOD = 1.18); from below anterior corner of the eye and tympanum to axial region, forming a maxillary gland in posterior corner of mouth, with a soybean size gland behind; tympanum distinct, tympanum diameter smaller than eye diameter (TD/ED=0.73); supratympanic fold absent; the presence of vomerine ridge, small teeth on the surface; a pair of external subgular vocal sacs present at the corners of the throat; tongue cordiform, deeply notched posteriorly.

Forelimbs moderately robust, with forearm and hand length being half of the body length (FAHL:SVL = 0.42); a large and smooth suprabrachial gland present behind the base of the forelimb, prominent; absent longitudinal ridges on the upper arm; tips of fingers rounded, not dilated; lateroventral grooves of fingers meeting at the disc's tip; fingers thin, free of webbing, with narrow lateral fringes; subarticular tubercles clearly visible, rounded and with a distinct protuberance; supernumerary tubercles below the base of finger present on fingers III and IV; relative length of fingers: II < I < IV < III; palmar tubercles three, which are long and elliptic.

Hind limbs relatively robust, tibia length 55% of SVL and foot length 52% of SVL; heels not meeting or just meeting when hind limbs flexed at right angles to axis of body; tibio-tarsal articulation reaching the eye angle or nostril when hind limb is stretched along the side of the body; several longitudinal ridges on the back of the thigh and tibia; thigh length shorter than tibia length (THL:TBL = 0.89), length of tarsus and foot longer than tibia length (TFL:TBL = 1.37); toes relatively long and thin, relative lengths I < II < V < III < IV; webbing moderate, webbing formula: I 1/2 - 1 II 1/2 - 2 III 1 - 2½ IV 2 - 1V; toes with lateral fringes; prominent and rounded subarticular tubercles; inner metatarsal tubercles oval, outer metatarsal tubercles small and rounded.

Dorsal anterior region of skin relatively smooth; several large warts on the middle and posterior back; posterior dorsum of body rough with small protrusions, but no horny spinules on them; dorsolateral fold relatively thin and distinct, extending from posterior corner of the eye to above of groin, but discontinuous in the posterior; a noticeable, large, smooth suprabrachial gland behind the base of the forelimb; ventral skin smooth, small tubercles around the region close to dorsolateral fold; body side and limbs relatively smooth; dorsum of tibia relatively rough, forming several longitudinal ridges; ventral skin of the body is smooth, flattened tubercles densely distributed on the rear of thigh and around vent.

Colouration of holotype in life: 

The colouration of the dorsal skin varies, mostly being brown, several tubercles on the flanks and posterior region, some of which have a black spot. A light brown mid-dorsal stripe starts from the mid-dorsal region and begins at the eye angle area, extending backwards to vent, becoming more distinct posteriorly, without dark brown edges; dorsolateral folds brown on the upper part and dark brown on the lower part; flanks cream-yellow on the upper part and light grey on the lower part; limbs brown with black-brown transverse bars; loreal and temporal regions dark brown; tympanum red-brown; upper 1/3 iris bright yellow and lower 2/3 brown-red; ventral surface of body milky white; ventral surfaces of throat, anterior chest, upper limbs and flank of trunk densely covered with brown-red or brown-black spots, while spots on lower limbs relatively sparse; maxillary gland yellow-white; ventral side of the forelimbs milky white; upper part of the thigh ventral side light yellow, lower part flesh red; ventral side of the tibia light yellow (Fig. 2).

Figure 2.  

Holotype of Nidirana chongqingensis sp. nov. (SWU0001408) in life. A lateral view; B dorsal view; C ventral view; D ventral view of hand; E ventral view of foot; F dorsal view of foot.

Colouration of holotype in preservation: 

Colouration of dorsal surface fading to blackish-brown; black spots on dorsum and flank more distinct; upper limbs fading to grey-white and lower limbs fading to brownish; transverse bars on the limbs brownish-black; ventral surface of body fading to grey-white; ventral surface of thigh buff-coloured (Fig. 3).

Figure 3.  

Holotype of Nidirana chongqingensis sp. nov. (SWU0001408) in preservative. A dorsal view; B ventral view; C view of oral cavity; D lateral view; E ventral view of hand; F ventral view of foot.

Variation: 

Measurements of the type specimens are listed in Table 2. Paratypes are similar to the holotype in morphology and colouration. The dorsal side colouration is light brown or dark brown; a light brown mid-dorsal stripe starts from the area between the eyes and extends backwards to vent and becomes more obvious; ventral side colouration milky white or grey-white; transverse stripes on the limbs light brown or dark brown; black spots on the back sparser and darker (Fig. 4).

Figure 4.  

Colouration variation of the male paratype of Nidirana chongqingensis sp. nov. (SWU0001435) in life. A lateral view; B dorsal view.

Secondary sexual characters: 

A pair of external subgular vocal sacs present in males; nuptial pad on the inner side of base of fingers Ⅰ; nuptial spinules invisible; the presence of suprabrachial gland.

Distribution and habitats: 

Nidirana chongqingensis sp. nov. is known from the type locality in Qianjiang District, Chongqing Municipality, China, at elevations between 1400 and 1500 metres above sea level.

Ecology

Nidirana chongqingensis sp. nov. inhabits mountain swamps, ponds, aquatic grassland and nearby weed thickets (Fig. 5). Due to the dense weeds, the frog is hard to find while it is hiding in them. The males were seen to call at night.

Figure 5.  

Habitat of N. chongqingensis sp. nov. in the type locality, Qianjiang District, Chongqing Municipality, China.

Analysis

Phylogenetic

The aligned sequence matrix of 16S + COI comprised 1664 bp. Our ML and BI analyses produced nearly identical phylogenetic topologies, which was consistent with the result of Chen et al. (2022b). The phylogenetic results showed that the Nidirana genus included two species groups that could be further divided into four highly-supported clades, denoted as A, B, C and D (following Lyu et al. (2017)) (Fig. 6). The Nidirana populations from Qianjiang, Chongqing (ID 1-3) comprising of three specimens were assigned to Clade C of the N. adenopleura group and were distant from true N. adenopleura in Clade D in the phylogeny. Uncorrected p-distances amongst all Nidirana species used in this study are presented in Table 3.

Table 3.
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Uncorrected p-distances based on COI genes amongst all Nidirana species (in 0.1%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1 N. chongqingensis sp. nov.
2 N. shiwandashanensis 3.9%
3 N. guangxiensis 4.1% 2.7%
4 N. yeae 3.4% 2.7% 1.8%
5 N. daunchina 4.8% 4.8% 4.3% 3.9%
6 N. yaoica 2.5% 3.9% 3.0% 2.3% 3.7%
7 N. chapaensis 3.2% 4.3% 3.4% 3.0% 4.8% 2.8%
8 N. guibeiensis 5.9% 6.4% 5.9% 5.5% 6.2% 5.5% 5.5%
9 N. leishanensis 5.5% 6.0% 5.5% 5.9% 6.2% 5.5% 5.5% 2.7%
10 N. xiangica 6.0% 7.3% 6.0% 5.7% 6.4% 5.7% 5.3% 2.8% 3.0%
11 N. guangdongensis 6.4% 6.8% 6.6% 5.2% 5.9% 5.3% 6.0% 5.7% 6.2% 6.4%
12 N. hainanensis 4.3% 5.3% 5.2% 4.4% 5.3% 4.3% 4.3% 6.0% 5.5% 5.7% 5.7%
13 N. adenopleura 8.9% 9.1% 8.5% 8.2% 8.9% 7.5% 8.2% 9.1% 9.6% 10.5% 8.9% 8.4%
14 N. mangveni 9.8% 10.3% 10.0% 9.4% 9.6% 9.1% 9.3% 10.1% 10.7% 10.9% 9.8% 9.4% 4.8%
15 N. nankunensis 10.0% 11.2% 10.3% 9.6% 10.9% 10.0% 9.6% 9.8% 9.8% 10.1% 9.1% 10.5% 7.1% 6.9%
16 N. lini 9.4% 9.6% 9.6% 8.5% 9.3% 9.6% 8.5% 10.1% 11.0% 10.9% 9.6% 9.4% 9.6% 9.8% 9.3%
17 N. occidentalis 12.3% 11.4% 12.5% 12.1% 11.2% 11.7% 11.0% 12.8% 13.2% 12.8% 12.6% 11.7% 11.2% 11.2% 12.5% 11.4%
18 N. pleuraden 12.6% 11.7% 12.6% 11.6% 11.9% 12.1% 12.3% 14.2% 14.2% 14.1% 11.7% 11.7% 11.0% 12.8% 11.7% 10.9% 8.4%
19 N. okinavana 10.1% 9.6% 8.9% 8.9% 9.4% 8.9% 8.5% 10.1% 10.7% 11.0% 9.3% 9.4% 4.6% 4.8% 7.1% 10.3% 11.4% 11.2%
Figure 6.  

Phylogenetic tree reconstructed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods, based on mitochondrial 16S + COI genes. Numbers denote the Maximum Likelihood bootstrap values (left) and Bayesian posterior probabilities (right). Samples 1–37 refer to Table 1. The symbol “–” represents value below 50/0.6.

The new species is embeded in the same clade with N. shiwandashanensis, N. guangxiensis, N. yeae, N. daunchina, N. yaoica and N. chapaensis. The relationships amongst these seven lineages remain unresolved, although the Qianjiang, Chongqing population appears to be more closely related to N. yaoica.

For the COI gene, the genetic distance between the new species and the closely-related species N. yaoica was 2.5%, respectively, approximately the same level as the distance between N. guangxiensis and N. yeae (1.8%), N. yaoica and N. yeae (2.3%) (see Table 3).

Molecular phylogenetic analyses revealed that the Nidirana population from Qianjiang District, Chongqing Municipality, China is distinct from its congeners.

Morphology

A summary of morphological characters is provided in Suppl. material 1. Nidirana chongqingensis sp. nov. differs from its congeners in the following characteristics: (1) small-sized body, SVL 41.8-43.3 mm in adult males [vs. SVL < 38 mm in adult male N. nankunensis; SVL > 46.2 mm in adult male N. shiwandashanensis, N. guangdongensis, N. guibeiensis; N. leishanensis, N. mangveni, N. pleuraden and N. xiangica]; (2) relative length of fingers: II < I < IV < III [vs. Ⅰ < Ⅱ < Ⅳ < Ⅲ in N. mangveni; Ⅱ < Ⅰ = Ⅳ < Ⅲ in N. chapaensis; Ⅱ < Ⅳ < Ⅰ < Ⅲ in N. yeae, N. shiwandashanensis and N. leishanensis; II < IV < I < III in N. guibeiensis]; (3) lateroventral grooves present on all fingers [vs. absent on all fingers in N. yeae, N. occidentalis and N. pleuraden; absent or rarely present in N. daunchina; present on all fingers, except finger I in N. nankunensis, N. chapaensis, N. okinavana, N. adenopleura, N. lini, N. guangdongensis and N. guibeiensis; present on fingers Ⅲ and IV in N. guangxiensis and N. mangveni]; (4) lateroventral grooves present on all toes [vs. absent on all toes in N. occidentalis and N. pleuraden]; (5) tibio-tarsal articulation reaches anterior corner of the eye or nostril [vs. beyond the snout in N. lini; at the eye in N. yeae and N. occidentalis]; (6) dorsal skin relatively smooth, without horny spines on the back [vs. white horny spinules on the entirely dorsum, dorsolateral folds, flanks and dorsal hind limbs in N. guangdongensis; white horny spinules on the entirely dorsum, dorsolateral folds, flanks, dorsal limbs, loreal region and temporal region including tympanum in males in N. xiangica, white horny spinules on the posterior or entire dorsum in males in N. mangveni; present in N. adenopleura, N. lini and N. pleuraden]; (7) during the breeding season, nuptial pads present on the first finger [vs. no nuptial pad is present in N. hainanensis; the nuptial pad of the first finger is divided into two parts in N. chapaensis; nuptial pads present on the first and second finger respectively in N. leishanensis]; (8) males possess a pair of external subgular vocal sacs in males [vs. absent in N. okinavana]; (9) finger tips not dilated [vs. dilated in N. shiwandashanensis, N. adenopleura, N. chapaensis, N. daunchina, N. guangdongensis, N. guangxiensis, N. guibeiensis, N. hainanensis, N. leishanensis, N. lini, N. mangveni, N. nankunensis, N. okinavana, N. xiangica, N. yaoica and N. yeae]; (10) tip of each toe dilated [vs. not dilated in N. occidentalis and N. pleuraden]; (11) supratympanic fold absent [vs. weak supratympanic fold present in N. mangveni]; (12) heels not meeting or just meeting when hind limbs flexed at right angles to axis of body [vs. heels overlapping in N. adenopleura, N. guangdongensis, N. guangxiensis, N. leishanensis, N. lini, N. mangveni, N. nankunensis, N. okinavana and N. yaoica]; (13) indistinct canthus rostralis [vs. distinct in N. adenopleura, N. daunchina, N. guangdongensis, N. guangxiensis, N. hainanensis, N. lini, N. mangveni, N. shiwandashanensis and N. xiangica]; (14) no longitudinal ridges on upper arms [vs. weak longitudinal ridges on upper arms and slightly extending to lower arm in N. guangxiensis]; (15) supernumerary tubercles below the base of finger III and IV, inconspicuous [vs. distinct in N. guangxiensis, N. mangveni and N. xiangica; absent in N. okinavana]; (16) dorsal skin relatively smooth [vs. rough with dense granules in N. guangxiensis].

Phylogenetically, N. chongqingensis sp. nov. is closest to N. yaoica. However, N. chongqingensis sp. nov. differs from N. yaoica in the following characteristics: (1) indistinct canthus rostralis [vs. distinct in N. yaoica]; (2) maxillary gland from below anterior corner of the eye [vs. from below nostril in N. yaoica]; (3) the absence of longitudinal ridges on the upper arm [vs. present in N. yaoica]; (4) finger tips not dilated, rounded [vs. discs of digits dilated, pointed]; (5) lateroventral grooves meeting at the disc's tip [vs. not meeting in N. yaoica]; (6) heels not meeting or just meeting when hind limbs flexed at right angles to axis of body [vs. heels overlapping in N. yaoica]; (7) absent wide dark brown edges of the mid-dorsal strip [vs. dark brown edges of the mid-dorsal stripe more distinct in N. yaoica].

Discussion

To date, within the confines of Chongqing Municipality, a duo of acknowledged organisms of the genus Nidirana have been identified, namely N. adenopleura and N. daunchina. Owing to their cautious phenotypic traits, the Nidirana species located in the southern region of China typically fall under the classification of N. adenopleura (Lyu et al. 2017, Lyu et al. 2020a, Lyu et al. 2020b). The distribution range of N. adenopleura is deemed to be the most extensive, encompassing Iriomote and Ishigaki Islands, as well as the Yaeyama group of Ryukyu Island in Japan, Taiwan and several Provinces in China, such as Guizhou, Anhui, Zhejiang, Jiangxi, Hunan, Fujian, Guangdong and Guangxi (Fei et al. 2009, Fei et al. 2012, Li et al. 2019, Frost 2023). As stated by Fei et al. (2009), the survival of this particular species is heavily reliant on stillwater habitats. Given its extensive range of distribution and specific habitat demands, it is presumed that population divergence and potentially even species divergence, could be facilitated. Our newly-discovered species N. chongqingensis sp. nov. indicate that previous studies, which only regarded the Nidirana population from Chongqing Municipality as N. adenopleura, need further verification.

Acknowledgements

We would like to express our gratitude to the editor for their work on this manuscript. We also extend our appreciation to Truong Nguyen and the anonymous reviewers for their valuable feedback on our paper. Constructive suggestions provided by Jianping Jiang, Bin Wang and Shengchao Shi from the Chengdu Institute of Biology, Chinese Academy of Sciences, during the writing of this article are also acknowledged. We are also grateful for the assistance of Ming Zhong during fieldwork. This research was supported by the Supplementary Investigation Project of National Key Protected Terrestrial Wildlife in Chongqing (No. sxxycq-2021-086).

Author contributions

Qi Ma conceived and designed the experiments, performed the experiments, analysed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Zhijian Wang conceived and designed the experiments, analysed the data, approved the final draft.

References

Supplementary material

Suppl. material 1: Diagnostic characters separating Nidirana chongqingensis sp. nov. from all congeners 
Authors:  Qi Ma, Zhijian Wang
Data type:  morphological
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