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Research Article
Discovery of a new limestone karst-restricted odorous frog from northern Guangdong, China (Anura, Ranidae, Odorrana)
expand article infoShi-Shi Lin‡, Yuan-Hang Li‡, Hong-Lin Su‡, Hui Yi§, Zhong Pan|, Yan-Jun Sun, Zhao-Chi Zeng‡, Jian Wang‡
‡ Guangdong Polytechnic of Environmental Protection Engineering, Foshan, China
§ Department of Ecology and Environment of Guangdong Province, Guangzhou, China
| Guangzhou Shengheng Forestry Co., LTD, Guangzhou, China
¶ Shenzhen Landscape Co., LTD, Shenzhen, China

Corresponding author: Zhao-Chi Zeng ( zengzhch5@mail2.sysu.edu.cn )

Corresponding author: Jian Wang ( wangj-1994@outlook.com )

Academic editor: Angelica Crottini
© 2022 Shi-Shi Lin, Yuan-Hang Li, Hong-Lin Su, Hui Yi, Zhong Pan, Yan-Jun Sun, Zhao-Chi Zeng, Jian 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: Lin S-S, Li Y-H, Su H-L, Yi H, Pan Z, Sun Y-J, Zeng Z-C, Wang J (2022) Discovery of a new limestone karst-restricted odorous frog from northern Guangdong, China (Anura, Ranidae, Odorrana). ZooKeys 1120: 47-66. https://doi.org/10.3897/zookeys.1120.87067
ZooBank: urn:lsid:zoobank.org:pub:398862C6-FCA8-4B0F-B084-F34D8B86EA1B
Open Access

Abstract

Karstic landscapes play an important role in biodiversity formation and often contain high levels of endemism. However, site-endemic taxa in karstic landscapes are being threatened by exploitation and weak legal protection. In this study, we describe Odorrana concelata Wang, Zeng, & Lin, sp. nov., a limestone karst-restricted odorous frog from northern Guangdong, China. This new species shows distinctive genetic divergence and morphological differences from its congeners. Phylogenetic results suggest that the new species represents an independent lineage that is grouped with O. lipuensis and O. liboensis based on the mitochondrial 16S and 12S ribosomal RNA genes. We recommend the new species be listed as Vulnerable (VU) in the IUCN categorization as it is only known from the type locality with limited microhabitats and is threatened by habitat degradation.

Keywords

Conservation, endemism, karstic landscapes, phylogeny, taxonomy

Introduction

Karstic landscapes in Asia, ranging from China to western Melanesia, play an important role in biodiversity formation and often contain high levels of endemism (Clements et al. 2006; Grismer et al. 2021). The genus Odorrana Fei, Ye & Huang, 1990 currently contains 62 species that are widely distributed in the subtropical and tropical regions of East and Southeast Asia (AmpibiaWeb 2022; Frost 2022). Almost all of the congeners are montane stream dwellers except for Odorrana wuchuanensis (Xu, 1983), O. lipuensis Mo, Chen, Wu, Zhang, & Zhou, 2015, O. liboensis Luo, Wang, Xiao, Wang, & Zhou, 2021, and O. mutschmanni Pham, Nguyen, Le, Bonkowski, & Ziegler, 2016, which occur in and are endemic to karstic landscapes (Fei et al. 2009, 2012; Liu and Wang 2014; Mo et al. 2015; Pham et al. 2016a, b; Luo et al. 2021).

The unique evolutionary lineage composed of two of the karst-dwellers (Odorrana lipuensis and O. liboensis) appears to have diverged from the rest of their congeners early and form an ancestral evolutionary branch of the genus. During herpetological surveys in karstic landscapes in northern Guangdong (Fig. 1, solid circle), China, several specimens of the genus Odorrana were collected. The specimens possess the common characteristics of this lineage (i.e., dorsum with mixed irregular moss-green speckles and brown mottling, males lacking vocal sacs, karst-endemic dwelling habit, etc.). However, subsequent morphological and phylogenetic studies support the newly collected specimens as a distinct taxon that can be distinguished reliably from all known congeners, especially from the closely related Odorrana lipuensis (Fig. 1, solid squares) and O. liboensis (Fig. 1, solid triangle). Therefore, we describe the first known karst-dwelling Odorrana population of Guangdong as a new species below.

Figure 1. 

Distribution of Odorrana concelata sp. nov. (solid circle), O. lipuensis (solid squares), and O. liboensis (solid triangle).

Materials and methods

Sampling

In total, 71 samples including 11 outgroup samples were used in this study, encompassing six newly sequenced individuals and others downloaded from GenBank. Detailed information for all samples is given in Table 1.

Table 1.
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Localities, voucher information, and GenBank accession numbers for all samples used in this study.

Species Locality Voucher 12S 16S
Odorrana concelata sp. nov. Longlinchang Village, Qingyuan, Guangdong,China GEP a050 OP137167 OP137161
Odorrana concelata sp. nov. Longlinchang Village, Qingyuan, Guangdong,China GEP a051 OP137168 OP137162
Odorrana concelata sp. nov. Longlinchang Village, Qingyuan, Guangdong,China GEP a052 OP137169 OP137163
Odorrana concelata sp. nov. Longlinchang Village, Qingyuan, Guangdong,China GEP a053 OP137170 OP137164
Odorrana concelata sp. nov. Longlinchang Village, Qingyuan, Guangdong,China GEP a054 OP137171 OP137165
Odorrana concelata sp. nov. Longlinchang Village, Qingyuan, Guangdong,China GEP a055 OP137172 OP137166
O. absita Xe Kong, Laos FMNH 258107 – EU861542
O. amamiensis Tokunoshima, Ryukyu, Japan KUHE:24635 AB200923 AB200947
O. anlungensis Anlong, Guizhou, China HNNU1008I109 KF185013 KF185049
O. aureola Phu Rua, Loei, Thailand FMNH 265919 – DQ650564
O. bacboensis Khe Moi, Nghe An, Vietnam ROM 13044 AF206099 AF206480
O. banaorum Tram Lap, Vietnam ROM 7472 AF206106 AF206487
O. chapaensis Lai Chau, Vietnam AMNH A161439 DQ283372 DQ283372
O. chloronota Ha Giang, Vietnam AMNH A163935 DQ283394 DQ283394
O. daorum Sa Pa, Vietnam ROM 19053 AF206101 AF206482
O. dulongensis Dulongjiang, Yunnan, China KIZ035027 MW128102 MW128102
O. exiliversabilis Mt. Wuyi, Fujian, China HNNU0607032 KF185020 KF185056
O. fengkaiensis Heishiding Nature Reserve, Fengkai, Guangdong, China SYS a002262 KT315354 KT315375
O. geminata Ha Giang, Vietnam AMNH 163782 – EU861546
O. grahami Kunming, Yunnan, China HNNU1008II016 KF185015 KF185051
O. graminea Wuzhishan, Hainan, China HNNU0606123 KF185002 KF185038
O. hainanensis Wuzhishan, Hainan, China HNNU0606105 KF184996 KF185032
O. hejiangensis Hejiang, Sichuan, China HNNU1007I202 KF185016 KF185052
O. hmongorum Lao Cai, Vietnam ROM 38605 – EU861556
O. hosii Kuala Lumpur, Malaysia IABHU 21004 AB511284 AB511284
O. huanggangensis Mt. Wuyi, Fujian, China HNNU0607001 KF185023 KF185059
O. ishikawae Amami Island, Japan IABHU 5275 AB511282 AB511282
O. jingdongensis Jingdong, Yunnan, China 20070711017 KF185014 KF185050
O. junlianensis Junlian, Sichuan, China HNNU002JL KF185022 KF185058
O. kuangwuensis Nanjiang, Sichuan, China HNNU0908II185 KF184998 KF185034
O. kweichowensis Lengshuihe Nature Reserve, Jinsha, Guizhou, China CIBjs20171014001 MH193539 MH193551
O. leporipes Shaoguan, Guangdong, China HNNU1008I099 KF185000 KF185036
O. liboensis Maolan National Nature Reserve, Libo, Guizhou, China GZNU20180608007 MW481339 MW481350
O. liboensis Maolan National Nature Reserve, Libo, Guizhou, China GZNU20180608009 MW481340 MW481351
O. liboensis Maolan National Nature Reserve, Libo, Guizhou, China GZNU20180608003 MW481341 MW481352
O. lipuensis Lipu, Guangxi, China NHMG1303018 MH665670 MH665676
O. lipuensis Lipu, Guangxi, China NHMG1303019 – KM388701
O. lipuensis Lung Tung Village, Ha Lang, Cao Bang, Vietnam IEBR: A2015_63 – LC155910
O. lipuensis Coong Village, Ha Lang, Cao Bang, Vietnam IEBR: A2015_65 – LC155911
O. livida Prachuap Kirikhan, Thailand FMNH 263415 KF771294 DQ650613
O. lungshengensis Longsheng, Guangxi, China HNNU70028 KF185018 KF185054
O. margaretae Mt. Emei, Sichuan, China HNNU20050032 KF184999 KF185035
O. morafkai TramLap, Vietnam ROM 7446 AF206103 AF206484
O. mutschmanni Cao Bang , Vietnam IEBR 3725 KU356762 KU356766
O. narina Okinawa Island, Japan AB511287 AB511287
O. nasica HaTinh, Vietnam AMNH A161169 DQ283345 DQ283345
O. nasuta Mt. Wuzhishan, Hainan, China HNNU051119 KF185017 KF185053
O. sangzhiensis Sangzhi, Hunan, China CSUFT 4308220046 MW465705 MW464864
O. schmackeri Yichang, Hubei, China HNNU0908II349 KF185011 KF185047
O. supranarina Iriomotejima, Ryukyu KUHE:12898 AB200926 AB200950
O. swinhoana Nantou, Taiwan, China HNNUTW9 KF185010 KF185046
O. tianmuii Lin’an, Zhejiang, China HNNU707071 KF185004 KF185040
O. tiannanensis Hekou, Yunnan, China HNNUHK001 KF185008 KF185044
O. tormota Huangshan, Anhui, China No. AM04005 DQ835616 DQ835616
O. utsunomiyaorum Iriomotejima, Ryukyu KUHE:12896 AB200928 AB200952
O. versabilis Leigongshan Nature Reserve, Leishan, Guizhou, China HNNU003 KF185019 KF185055
O. wuchuanensis Wuchuan, Guizhou, China HNNU019L KF185007 KF185043
O. yentuensis Guangxi, China NHMG1401035 MH665669 MH665675
O. yizhangensis Nanling Nature Reserve, Ruyuan, Guangdong, China HNNU1008I075 KF185012 KF185048
O. yunnanensis Longchuan, Yunnan, China HNNU001YN KF185021 KF185057
Amolops loloensis Shimian, Sichuan, China SM-ZDTW-01 NC029250 NC029250
A. mantzorum Xiling Snow Mountain, Dayi, Sichuan, China NC024180 NC024180
A. granulosus Mt. Wawu, Sichuan, China 20130258 NC044901 NC044901
A. ricketti Mt. Wugong, Jiangxi, China AM13988 NC023949 NC023949
A. hongkongensis Mt. Wuyi, Fujian, China DYTW-WYS-001 KX233864 KX233864
Sylvirana guentheri Fuzhou, Fujian, China SCUM-H002CJ KX269219 KX269219
S. spinulosa Wuzhishan, Hainan, China HNNU051117 KF185031 KF185067
Glandirana tientaiensis Huangshan, Anhui, China SCUM0405192CJ KX269222 KX269222
Pelophylax nigromaculata Hongya, Sichuan, China SCUM045199CJ KX269216 KX269216
Nidirana daunchina Mt. Emei, Sichuan, China HNNU20060103 KF185029 KF185065
Rana weiningensis Weining, Guizhou, China SCUM0405171 KX269217 KX269217

All specimens were fixed in 10% buffered formalin, later transferred to 70% ethanol for preservation, and deposited at the Guangdong Polytechnic of Environmental Protection Engineering (GEP), Foshan City, Guangdong, China; tissue samples were preserved in 95% ethanol for molecular studies.

DNA Extraction, PCR and sequencing

For the newly collected samples, genomic DNA were extracted from muscle tissue, using DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. Two mitochondrial genes namely 16S ribosomal RNA gene (16S) and 12S ribosomal RNA gene (12S) were amplified. Primers used for 16S were L3975 (5’-CGCCTGTTTACCAAAAACAT-3’) and H4551 (5’-CCGGTCTGAACTCAGATCACGT-3’), for 12S were L33 (5’-CTCAACTTACAMATGCAAG-3’) and H56 (5’-CGATTATAGAACAGGCTCCT-3’). PCR sequencing methods followed Lyu et al. (2017).

Phylogenetic analyses

DNA sequences were aligned in MEGA 11 (Tamura et al. 2021) by the Clustal W package with default parameters (Thompson et al. 1997). The two gene segments, which are 733 bp for 12S and 1081 bp for 16S, were concatenated into an 1814 bp length sequence. PartitionFinder (Lanfear et al. 2012) was used to select partitioning schemes and their corresponding best-fitting nucleotide substitution models. This resulted in two partitions for the alignment (one partition for 16S and one partition for 12S), with GTR+I+G being found as the best-fitting model for both. Phylogenetic trees were constructed using maximum likelihood (ML) implemented in RaxmlGUI 1.3 (Silvestro and Michalak 2012), and Bayesian inference (BI) using MrBayes 3.2.4 (Ronquist et al. 2012). For ML analysis, the maximum likelihood tree inferred from 1000 replicates was used to represent the evolutionary history of the analyzed taxa. Branches corresponding to partitions reproduced in less than 60% of bootstrap replicates were collapsed. For BI analysis, two independent runs with four Markov Chain Monte Carlo simulations were performed for ten million iterations and sampled every 1000 iterations. The first 25% of samples were discarded as burn-in. Pairwise distances (p-distance) for the 16S rRNA gene were calculated in MEGA 11 using the uncorrected p-distance model.

Morphometrics

Measurements followed Fei et al. (2009) and were taken with a digital caliper to the nearest 0.1 mm. These measurements are as follows:

SVL snout-vent length (from tip of snout to vent);

HDL head length (from tip of snout to rear of jaws);

HDW head width (head width at commissure of jaws);

SNT snout length (from tip of snout to anterior corner of eye);

ED eye diameter (from anterior corner to posterior corner of the eye);

IOD interorbital distance (minimum distance between upper eyelids);

IND internasal distance (distance between nares);

TD tympanum diameter (horizontal diameter of tympanum);

HND hand length (from tip of third digit to proximal edge of inner palmar tubercle);

RAD radioulnar length (from the flexed elbow to the proximal border of the outer palmar tubercle);

TIB tibia length (from knee to heel);

FTL foot length (from the distal end of the shank to the tip of digit IV).

Sex was determined by direct observation of the presence of nuptial pads in males, and the presence of eggs in the abdomen seen via external inspection in females. Comparative morphological data of Odorrana species were obtained from the references listed in Table 2.

Table 2.
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Data sources of the currently known species of the genus Odorrana.

ID Odorrana species Literature
1 O. absita (Stuart & Chan-ard, 2005) Stuart and Chan-ard (2005)
2 O. amamiensis (Matsui, 1994) Matsui (1994)
3 O. anlungensis (Liu & Hu, 1973) Hu et al. (1973)
4 O. aureola Stuart, Chuaynkern, Chan-ard, & Inger, 2006 Stuart et al. (2006)
5 O. bacboensis (Bain, Lathrop, Murphy, Orlov, & Ho, 2003) Bain et al. (2003); Wang et al. (2015)
6 O. banaorum (Bain, Lathrop, Murphy, Orlov, & Ho, 2003) Bain et al. (2003)
7 O. bolavensis (Stuart & Bain, 2005) Stuart and Bain (2005)
8 O. cangyuanensis (Yang, 2008) Yang (2008)
9 O. chapaensis (Bourret, 1937) Bourret (1937)
10 O. chloronota (Günther, 1876) Günther (1876); Che et al. (2020)
11 O. dulongensis Liu, Che, & Yuan, 2021 Liu et al. (2021)
12 O. exiliversabilis Li, Ye, & Fei, 2001 Fei et al. (2001b)
13 O. fengkaiensis Wang, Lau, Yang, Chen, Liu, Pang, & Liu, 2015 Wang et al. (2015)
14 O. geminata Bain, Stuart, Nguyen, Che, & Rao, 2009 Bain et al. (2009)
15 O. gigatympana (Orlov, Ananjeva, & Ho, 2006) Orlov et al. (2006)
16 O. grahami (Boulenger, 1917) Boulenger (1917)
17 O. graminea (Boulenger, 1900) Boulenger (1900)
18 O. hainanensis Fei, Ye, & Li, 2001 Fei et al. (2001a)
19 O. hosii (Boulenger, 1891) Boulenger (1891)
20 O. hejiangensis (Deng & Yu, 1992) Deng and Yu. 1992
21 O. huanggangensis Chen, Zhou, & Zheng, 2010 Chen et al. (2010a)
22 O. ichangensis Chen, 2020 Shen et al. (2020)
23 O. ishikawae (Stejneger, 1901) Stejneger (1901)
24 O. indeprensa (Bain & Stuart, 2006) Bain and Stuart (2006 “2005”)
25 O. jingdongensis Fei, Ye, & Li, 2001 Fei et al. (2001a)
26 O. junlianensis Huang, Fei, & Ye, 2001 Ye and Fei (2001)
27 O. khalam (Stuart, Orlov, & Chan-ard, 2005) Stuart and Chan-ard (2005)
28 O. kuangwuensis (Liu & Hu, 1966) Hu et al. (1966)
29 O. kweichowensis Li, Xu, Lv, Jiang, Wei, & Wang, 2018 Li et al. 2018
30 O. livida (Blyth, 1856) Blyth (1856)
31 O. liboensis Luo, Wang, Xiao, Wang, & Zhou, 2021 Luo et al. 2021
32 O. lipuensis Mo, Chen, Wu, Zhang, & Zhou, 2015 Mo et al. 2015; Pham et al. 2016a
33 O. leporipes (Werner, 1930) Werner (1930)
34 O. lungshengensis (Liu & Hu, 1962) Liu and Hu (1962)
35 O. macrotympana (Yang, 2008) Yang (2008)
36 O. margaretae (Liu, 1950) Liu (1950)
37 O. mawphlangensis (Pillai & Chanda, 1977) Pillai and Chanda (1977); Mahony 2008
38 O. mutschmanni Pham, Nguyen, Le, Bonkowski, & Ziegler, 2016 Pham et al. (2016a)
39 O. monjerai (Matsui & Jaafar, 2006) Matsui and Jaafar (2006)
40 O. morafkai (Bain, Lathrop, Murphy, Orlov, & Ho, 2003) Bain et al. (2003)
41 O. nasica (Boulenger, 1903) Boulenger (1903)
42 O. nasuta Li, Ye, & Fei, 2001 Fei et al. (2001b)
43 O. narina (Stejneger, 1901) Stejneger (1901)
44 O. nanjiangensis Fei, Ye, Xie, & Jiang, 2007 Fei et al. (2007a)
45 O. orba (Stuart & Bain, 2005) Stuart and Bain (2005)
46 O. rotodora (Yang & Rao, 2008) Yang (2008)
47 O. sangzhiensis Zhang, Li, Hu, & Yang, 2021 Zhang et al. (2021)
48 O. schmackeri (Boettger, 1892) Boettger (1892); Shen et al. (2020)
49 O. sinica (Ahl, 1927) Ahl (1927 “1925”); Bain et al. (2003)
50 O. swinhoana (Boulenger, 1903) Boulenger (1903)
51 O. supranarina (Matsui, 1994) Matsui (1994)
52 O. splendida Kuramoto, Satou, Oumi, Kurabayashi, & Sumida, 2011 Kuramoto et al. (2011)
53 O. tianmuii Chen, Zhou, & Zheng, 2010 Chen et al. (2010b)
54 O. tiannanensis (Yang & Li, 1980) Yang and Li 1980
55 O. tormota (Wu, 1977) Wu (1977)
56 O. trankieni (Orlov, Le, & Ho, 2003) Orlov et al. (2003)
57 O. utsunomiyaorum (Matsui, 1994) Matsui (1994)
58 O. versabilis (Liu & Hu, 1962) Liu and Hu (1962)
59 O. wuchuanensis (Xu, 1983) Wu et al. (1983)
60 O. yentuensis Tran, Orlov, & Nguyen, 2008 Tran et al. (2008); Lu et al. 2016
61 O. yizhangensis Fei, Ye, & Jiang, 2007 Fei et al. (2007b)
62 O. yunnanensis (Anderson, 1879 “1878”) Anderson (1879 “1878”); Fei et al. 1990

Results

Molecular results

The ML and BI analyses resulted in identical topologies (Fig. 2). Most of the nodes have sufficient support with bootstrap support (BS) ≥ 70 and the Bayesian posterior (BPP) ≥ 0.90, and the relationship among the Odorrana species in our result corresponds to those in previous studies (Chen et al. 2013; Liu et al. 2021; Luo et al. 2021). The Odorrana specimens from northern Guangdong form a monophyletic clade and is grouped with O. lipuensis and O. liboensis, and (Clade A) with strong node supports (BS = 100; BPP = 1.00). In addition, the clade formed by the northern Guangdong specimens shows strong divergence to the other two species in Clade A. Although we do not use genetic distances to diagnose the new species, we note that the mean p-distance between the new collected Odorrana specimens and its most closely-related congeners is 4.6% (between the new lineage and O. liboensis) and 4.7% (between the new lineage and O. lipuensis) in 16S rRNA gene (Suppl. material 1: Table S1). The smallest divergence between the new Odorrana specimen and other Odorrana species is 2.9% in 16S rRNA gene (between the new lineage and O. geminata), which approximates the level of genetic divergence observed in uncontroversial species within Odorrana. Moreover, detailed morphological examination (see Taxonomic account below) has revealed discrete, diagnostic (non-overlapping ranges in traditional characters) differences between the specimens from this independent lineage and all other congeners. Therefore, both phylogenetic result and morphological comparison support the Odorrana specimen from northern Guangdong as an undescribed new species, and we herein describe as below.

Figure 2. 

Maximum-likelihood and Bayesian inference phylogenies. Numbers at nodes indicate the bootstrap support (BS)/Bayesian posterior (BPP) for the topology. ‘-’ means BS < 70 or BPP < 0.90.

Taxonomic account

Odorrana concelata Wang, Zeng, & Lin, sp. nov.

https://zoobank.org/63E81BE8-F60F-47F4-B49A-3403C4AB82D3
Moss-speckled Odorous Frog (in English) / Tai Ban Chou Wa (苔斑臭蛙 in Chinese) Figs 3, 4, 5

Holotype

GEP a055, adult male, collected by Shi-Shi Lin, Hong-Lin Su and Yuan-Hang Li on 20 April 2022 from Longlinchang Village (24°04'47"N, 112°40'37"E; ca. 280 m a.s.l.), Jintan Town, Qingyuan City, Guangdong, China.

Paratypes

Three adult males, GEP a052–054, and two adult females, GEP a050–051, the same collection data as the holotype.

Etymology

The specific epithet, concelata, is a feminine adjective that means disguised, in reference to the highly concealed coloration of the new species in its mossy habitat.

Diagnosis

(1) Small body size, SVL 34.0–36.8 mm in males (n = 4), SVL 41.4–46.0 mm in females (n = 2); (2) dorsolateral folds absent; (3) relative finger lengths II < I < IV < III; (4) pectoral spines absent; (5) vocal sacs absent; (6) nuptial pads present on base of finger I, medially along inner side of fingers II and III in males; (7) eggs of females uniformed beige; (8) dorsum with mixed irregular grass green speckles and brown mottling, ventral skin of body greyish white with light brown mottling.

Comparisons

Odorrana concelata sp. nov. is phylogenetically closest to the clade composed of O. lipuensis and O. liboensis (Fig. 2). However, the new taxon can be distinguished by possessing a smaller body size, SVL 34.0–36.8 mm in males and 41.4–46.0 mm in females (vs. SVL 40.7–49.8 mm in males and 51.1–60.1 mm in females of O. lipuensis; SVL 47.1–49.9 mm in males and 55.8–58.2 mm in females of O. liboensis); presence of pineal body (vs. absent in O. lipuensis and O. liboensis); presence of nuptial pads on base of finger I, medially along inner side of fingers II and III (vs. presence of nuptial pad on finger I in males of both O. lipuensis and O. liboensis); relative finger lengths II < I < IV < III (vs. I = II < IV < III in O. lipuensis); absence of conical spines on upper lip except skin of commissure of jaw (vs. presence of conical spines on entire upper lip in O. lipuensis); tibiotarsal articulation reaches to nostril (vs. reaches to anterior of eye in O. lipuensis); presence of tiny conical spines on temporal region except tympanum, skin of commissure of jaw, upper edge of eyelid, and along dorsolateral sides of body (vs. absent in O. liboensis). Odorrana concelata sp. nov. further differs from another karst-dweller O. wuchuanensis by the smaller body size (vs. 71.1–76.5 mm in males and 75.8–99.6 mm in females), and absence pectoral spines (vs. present).

Odorrana concelata sp. nov. can be easily distinguished from O. absita, O. amamiensis, O. anlungensis, O. aureola, O. bacboensis, O. banaorum, O. bolavensis, O. cangyuanensis, O. chapaensis, O. chloronota, O. dulongensis, O. exiliversabilis, O. fengkaiensis, O. geminata, O. gigatympana, O. grahami, O. graminea, O. hainanensis, O. hejiangensis, O. huanggangensis, O. indeprensa, O. ichangensis, O. ishikawae, O. jingdongensis, O. junlianensis, O. khalam, O. kweichowensis, O. lungshengensis, O. macrotympana, O. morafkai, O. nanjiangensis, O. nasica, O. nasuta, O. orba, O. sangzhiensis, O. schmackeri, O. swinhoana, O. tianmuii, O. tiannanensis, O. tormota, O. trankieni, O. utsunomiyaorum, O. versabilis, O. yentuensis, O. yizhangensis and O. yunnanensis, by the absence of vocal sacs (vs. present; internal vocal sacs present in O. grahami, O. hainanensis, O. jingdongensis, O. junlianensis, O. yunnanensis); and from O. absita, O. amamiensis, O. banaorum, O. bolavensis, O. exiliversabilis, O. gigatympana, O. graminea, O. indeprensa, O. hosii, O. khalam, O. livida, O. leporipes, O. monjerai, O. narina, O. nasica, O. nasuta, O. orba, O. supranarina, O. tormota, O. trankieni, O. utsunomiyaorum, O. versabilis, and O. yentuensis, by the absence of dorsolateral folds (vs. present).

Odorrana concelata sp. nov. differs from the remaining seven congeners by the marked differences in dorsal and ventral coloration; the smaller body size, SVL 34.0–36.8 mm in males and 41.4–46.0 mm in females (vs. 57.2 mm in male and 66.0–71.4 mm in females in O. kuangwuensis, 78.0–88.0 mm in males and 93.0–113.0 mm in females in O. margaretae, 85.8–91.6 mm in males and 108.7–110.1 mm in females in O. mutschmanni, 80.0 mm in males and 84.3–106.0 mm in females in O. mawphlangensis, 44.0–55.0 mm in males and 86.0–97.0 mm in females in O. rotodora, 66.6 mm in male in O. sinica, and 74.4–124.4 mm in males and 94.6–137.4 mm in females in O. splendida).

Description of holotype

Adult male. Body slender and small, SVL 36.8 mm. Head length larger than head width, HDW/HDL ratio 0.88; snout short, rounded in dorsal view, projecting beyond lower jaw, snout length larger than eye diameter, SNT/ED ratio 1.35; canthus rostralis distinct; nostril rounded, located laterally, closer to tip of snout than eye; internasal distance larger than interorbital distance, IND/IOD ratio 1.09; loreal region slightly concave and oblique; eye large and prominent; tympanum rounded, large, TD/ED ratio 0.86, edge of tympanum slightly elevated relative to tympanum; strong vomerine ridges bearing vomerine teeth; tongue deeply notched distally; pupil horizontally oval; pineal body present, small; vocal sac absent.

Forelimbs slender, HND/SVL ratio 0.28, RAD/SVL ratio 0.22; fingers slender, relative finger lengths II < I < IV < III; tips of fingers expanded into disc, all with circummarginal grooves, horizontal grooves present, without webbing and lateral fringes; subarticular tubercles prominent: 1, 1, 2, 2; inner metacarpal tubercle oval, elongate; medium and outer metacarpal tubercles oval; nuptial pads present on base of finger I, medially along inner side of fingers II and III.

Hindlimbs slender, FTL/SVL ratio 0.70, TIB/SVL ratio 0.50; heels overlapping when thighs are appressed at right angles with respect to body; tibiotarsal articulation reaches to nostril when leg stretched forward; relative toe lengths I < II < III < V < IV; toes entirely webbed; tips of toes expanded into disc with circummarginal grooves; subarticular tubercles prominent: 1, 1, 2, 3, 2; inner metatarsal tubercle oval, elongate, almost equal length to first toe; outer metatarsal tubercle absent.

Dorsal skin relatively smooth, granular; skin of loreal region smooth; weak supratympanic fold from posterior corner of eye to posterior edge of tympanum; dorsolateral folds absent; tiny conical spines present on temporal region except tympanum, skin of commissure of jaw, upper edge of eyelid, and along dorsolateral sides of body. Ventral skin smooth.

Coloration of holotype in life

Skin of dorsal body, dorsal limbs and flanks with irregular moss-green speckles and brown mottling; dorsal skin of limbs with distinct brown transverse bands; ventral skin of body greyish white with light brown mottling; ventral skin of forelimb greyish white, ventral skin of hindlimb purplish brown. Iris black, with irregular gold-green reticulated mottles; pineal body light green; tympanum dark brown; nuptial pad creamy white.

Figure 3. 

Morphological features of the male holotype GEP a055 in life: A dorsolateral view B ventral view C ventral view of hand, showing nuptial pads on fingers I, II and III D ventral view of foot.

Coloration of holotype in preservative

Skin of dorsal body, dorsal limbs and flanks greyish brown, with brown mottling and dark brown transverse bands, moss-green speckles absent; ventral skin of body greyish white with brown mottling; ventral skin of thighs greyish white, ventral skin of shank and foot dark grey with dark brown mottling.

Variations

Mensural data of the type series are listed in Table 3. Most of the paratypes are similar to the holotype in morphology and color pattern, except for the following: (1) skin of dorsal trunk lacking tiny spines (vs. present in the male paratype GEP a052); (2) sparse spines on temporal region except tympanum, skin of commissure of jaw, upper edge of eyelid, and along dorsolateral sides of body; nuptial pads absent; and larger body size in female paratypes (Fig. 4).

Table 3.
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Measurements (minimum–maximum (mean ± SD); in mm) of Odorrana concelata sp. nov.

Voucher GEP a052 GEP a053 GEP a054 GEP a055 Range Voucher GEP a050 GEP a051
Sex Male Male Male Male Males (n = 4) Sex Female Female
SVL 34.0 35.7 35.2 36.8 34.0–36.8 (35.4 ± 1.2) SVL 46.0 41.4
HDL 11.8 12.4 12.5 12.8 11.8–12.8 (12.4 ± 0.4) HDL 15.3 13.1
HDW 10.9 11.2 11.1 11.3 10.9–11.3 (11.1 ± 0.2) HDW 14.6 12.2
SNT 5.1 5.1 5.2 5.3 5.1–5.3 (5.2 ± 0.1) SNT 6.7 5.6
IND 3.3 3.6 3.2 3.2 3.2–3.6 (3.3 ± 0.2) IND 4.2 3.5
IOD 3.0 3.1 3.0 3.0 3.0–3.1 (3.0 ± 0.1) IOD 3.3 3.3
ED 4.1 3.8 4.1 4.0 3.8–4.1 (4.0 ± 0.2) ED 4.5 4.2
TD 3.2 3.3 3.4 3.4 3.2–3.4 (3.3 ± 0.1) TD 3.4 3.2
HND 9.8 9.6 9.6 10.2 9.6–10.2 (9.8 ± 0.3) HND 12.9 12.3
RAD 7.1 7.8 7.7 8.0 7.1–8.0 (7.6 ± 0.4) RAD 9.8 9.1
FTL 24.0 24.8 24.7 25.6 24.0–25.6 (24.8 ± 0.7) FTL 32.3 29.6
TIB 17.3 17.6 18.0 18.5 17.3–18.5 (7.8 ± 0.6) TIB 23.3 20.8
Figure 4. 

Morphological features of the female paratype GEP a050 in life: A dorsolateral view B ventral view C ventral view of hand D ventral view of foot.

Distribution and habits

Currently, Odorrana concelata sp. nov. is known only from its type locality (Fig. 1, solid circle). The nocturnal karst-dweller inhabits mossy rocks and damp forest floors in subtropical evergreen broad-leaved forests and secondary forests at elevations between 200–300 m (Fig. 5A, B). They are completely hidden in their habitat by their coloration (Fig. 5D, E). During breeding season (March to June), they congregate in and around the small and steep moss-covered waterfalls which flows out of karst caves (ca. 1–2 m width). Juveniles were observed in June (Fig. 5C). No individuals were found during surveys in mid-July.

Figure 5. 

Microhabitat of Odorrana concelata sp. nov. (A, B) and the uncaptured individuals of juvenile (C), female (D), and male (E) in situ.

Discussion

The history of the formation and the ecological niches afforded by complex terrains of the karstic landscape contribute to a unique biological pattern (Culver et al. 2000; Engel 2007). In the phylogenetic tree (Fig. 2, Clade A), the unique evolutionary lineage composed of three karst-dwellers, i.e., Odorrana concelata, O. lipuensis, and O. liboensis, appears to have diverged from the rest of their congeners early on and form an ancestral evolutionary branch of the genus. Moreover, the phylogenetic placement of Odorrana concelata and the range extension of O. lipuensis (Pham et al. 2016a) also provides new insights into the ancestral distribution of the genus in the karstic landscape straddling Guangdong, Guangxi, and Guizhou of China and northern Vietnam.

The exploitation and weak legal protection of karstic landscapes has caused site-endemic taxa to be under threat (Clements et al. 2006; Grismer et al. 2021). Odorrana concelata is the fourth known karst-endemic species within the genus in China and is a site-endemic species only known from its type locality despite our frequent surveys in northern Guangdong. They are only found in wet mossy habitats, which limit the distribution of the species. Habitat degradation due to tourism development and local religious activities are major threats. The influx of tourists brings much waste such as plastic products. Also, local worship activities cause the destruction of microhabitats. Therefore, we recommend Odorrana concelata to be listed as Vulnerable (VU) [IUCN Red List criteria A1cd+B1b(iii)+D2].

Acknowledgements

We thank Angelica Crottini, Cuong Pham, and an anonymous reviewer for their constructive comments on the manuscript. This work was supported by the Project of Background survey of biosafety in Guangdong Province (STST-2021-10) by the Department of Ecology and Environment of Guangdong Province.

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Supplementary material

Supplementary material 1 

Table S1

Shi-Shi Lin, Yuan-Hang Li, Hong-Lin Su, Hui Yi, Zhong Pan, Yan-Jun Sun, Zhao-Chi Zeng, Jian Wang

Data type: excel file

Explanation note: Pairwise distances based on 16S gene among all species used in this study.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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