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Research Article
A new cryptic species in the Theloderma rhododiscus complex (Anura, Rhacophoridae) from China–Vietnam border regions
expand article infoLingyun Du‡, Jian Wang§|, Shuo Liu, Guohua Yu‡
‡ Guangxi Normal University, Guilin, China
§ Hainan Normal University, Haikou, China
| Honghe University, Mengzi, China
¶ Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China

Corresponding author: Shuo Liu ( liushuo@mail.kiz.ac.cn )

Corresponding author: Guohua Yu ( yugh2018@126.com )

Academic editor: Angelica Crottini
© 2022 Lingyun Du, Jian Wang, Shuo Liu, Guohua Yu.
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: Du L, Wang J, Liu S, Yu G (2022) A new cryptic species in the Theloderma rhododiscus complex (Anura, Rhacophoridae) from China–Vietnam border regions. ZooKeys 1099: 123-138. https://doi.org/10.3897/zookeys.1099.80390
ZooBank: urn:lsid:zoobank.org:pub:5C7E35BD-A831-4403-9831-7FFAC17DA0A7
Open Access

Abstract

We describe a new species of Theloderma from southern Yunnan, China and northern Vietnam based on morphological and molecular evidence. Theloderma hekouense sp. nov., which had been recorded as T. rhododiscus, is the sister to T. rhododiscus. The new species differs genetically from T. rhododiscus by 4.2% and 10.7% in 16S rRNA and COI genes, respectively, and it can be morphologically distinguished from T. rhododiscus by having more densely spaced white warts on the dorsal surface, red subarticular tubercles, red metacarpal tubercles, a red metatarsal tubercle, and black dorsal and ventral surfaces in preservative. Currently the new species is only known from the China–Vietnam border regions of Yunnan and Ha Giang, while T. rhododiscus has a wide distributional range in China including Guangxi, Guangdong, Hunan, Fujian, Jiangxi, and presumably Guizhou and eastern Yunnan. Including the new species, there are currently 10 Theloderma species in China and seven Theloderma species in Yunnan, where more species will probably be found.

Keywords

16S rRNA, COI, southern Yunnan, Theloderma hekouense sp. nov.

Introduction

Theloderma Tschudi, a genus of the family Rhacophoridae, occurs in southern and eastern areas of Asia and currently contains 26 species (Frost 2021), of which nine are recognized from China and seven are known from Yunnan including T. albopunctatum (Liu & Hu), T. baibungense (Jiang, Fei & Huang), T. bicolor (Bourret), T. gordoni Taylor, T. moloch (Annandale), T. pyaukkya Dever, and T. rhododiscus (Liu & Hu) (Du et al. 2020).

Theloderma rhododiscus was originally described from Mt Dayao, Guangxi, China in 1962 (Liu and Hu 1962; Fig. 1) and now is widely recorded from Fujian, Jiangxi, Hunan, Guangdong, Yunnan (Fei et al. 2012; Hou et al. 2017a, 2017b; Zeng et al. 2017), and northern Vietnam (Bain and Nguyen 2004). It was characterized by fingers and toes with orange-red disks, dorsal surface tea-brown, and a dorsum covered with white tubercles interweaved as a network (Liu and Hu 1962).

Figure 1. 

Map showing the collection sites of T. hekouense sp. nov. (circle) and T. rhododiscus (triangle) in this study 1 Hekou (type locality of the new species) 2 Ha Giang 3 Longlin 4 Huanjiang 5 Jinxiu (type locality of T. rhododiscus)

Numerous studies have shown that widely recorded amphibian species might actually be composed of multiple cryptic species (e.g., Lyu et al. 2019; Yu et al. 2019a, 2019b). Although Zeng et al. (2017) confirmed that the records of T. rhododiscus from Guangdong (Mt Nankun) and Jiangxi (Mts Jiulian and Sanbai) are conspecific with T. rhododiscus from the type locality based on morphological and molecular evidence, records of T. rhododiscus from other places need further confirmation from both morphological and molecular perspectives. Our earlier phylogenetic analysis of Theloderma (Hou et al. 2017b) showed that the clade consisting of populations from Yunnan and northern Vietnam is separated from the clade consisting of the topotypes with a relative large genetic divergence, which indicates that more studies are needed to test whether the records of T. rhododiscus from Yunnan and Vietnam belong to T. rhododiscus or not.

In this study, we compared the T. rhododiscus specimens from Yunnan with the topotypes of this species from both morphological and molecular perspectives. Our results supported that the records of T. rhododiscus from Yunnan and northern Vietnam warrant distinct taxonomic recognition. Additionally, we confirmed two new distribution sites of T. rhododiscus in northwestern (Longlin County) and northern Guangxi (Huanjiang County).

Materials and methods

Sampling

Specimens were collected by Guohua Yu during fieldwork in Jinxiu and Longlin counties, Guangxi, China in April and June of 2020, by Jian Wang during fieldwork in Hekou County, Yunnan, China in May and September 2020 and 2021, and by Shuo Liu during field surveys in Huanjiang County, Guangxi in September 2019. Specimens were fixed and then stored in 75% ethanol. Liver tissues were preserved in 99% ethanol. All specimens were deposited at Guangxi Normal University (GXNU).

Morphology

Morphometric data were taken using digital calipers to the nearest 0.1 mm. Morphological terminology follows Yu et al. (2019a). Measurements include: snout–vent length (SVL, from tip of snout to vent); head length (HL, from tip of snout to rear of jaws); head width (HW, width of head at its widest point); snout length (SL, from tip of snout to anterior border of eye); internarial distance (IND, distance between nares); interorbital distance (IOD, minimum distance between upper eyelids); upper eyelid width (UEW, maximum width of upper eyelid); eye diameter (ED, diameter of exposed portion of eyeball); tympanum diameter (TD, the greater of tympanum vertical and horizontal diameters); forearm and hand length (FHL, from elbow to tip of third finger); tibia length (TL, distance from knee to heel); foot length (FL, from proximal end of inner metatarsal tubercle to tip of fourth toe); length of foot and tarsus (TFL, from tibiotarsal joint to tip of fourth toe). Comparative morphological data of other Theloderma species were taken from their original descriptions or redescriptions (Taylor 1962; Stuart and Heatwole 2004; Orlov and Ho 2005; Orlov et al. 2006; McLeod and Ahmad 2007; Rowley et al. 2011; Poyarkov et al. 2015, 2018; Nguyen et al. 2016; Sivongxay et al. 2016; Dever 2017; Du et al. 2020).

Molecular phylogenetic analyses

Total genomic DNA was extracted from liver tissues. Tissue samples were digested using proteinase K, and subsequently purified following a standard phenol/chloroform isolation and ethanol precipitation. Sequences of 16S rRNA (16S) and cytochrome oxidase subunit I (COI) genes were amplified using the primers and experimental protocols of Du et al. (2020). Sequencing was conducted directly using the corresponding PCR primers. All new sequences were deposited in GenBank under accession numbers OL843957–OL843967 and OL843972–OL843982 (Table 1). Available homologous sequences of members of Theloderma were obtained from GenBank (Table 1). Buergeria oxycephala, Liuixalus hainanus, Gracixalus jinxiuensis, and Nyxtixalus pictus were selected as hierarchical outgroups according to Yu et al. (2009) and Du et al. (2020).

Table 1.
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Samples used in molecular analyses of this study.

Species Voucher number Locality 16s COI
Buergeria oxycephala MVZ 230425 Hainan, China KU244359 KU244459
Liuixalus hainanus LJT V15 Hainan, China KC465826 –
Gracixalus jinxiuensis KIZ 061210YP Guangxi, China EU215525 –
Nyctixalus pictus KUHE 53517 Malaysia LC012863 –
Theloderma albopunctatum VNMN JR2887 Vinh Phuc, Vietnam KU244375 KU244431
Theloderma laeve NAP01644 Lam Dong, Vietnam KT461907 –
Theloderma leporosum LJT W46 Malaysia KC465841 –
Theloderma palliatum NAP02516 Lam Dong, Vietnam KT461903 –
Theloderma vietnamense AMS R174047 Mondol Kiri, Cambodia JN688171 KU244460
Theloderma stellatum Stel1 Chanthaburi, Thailand KT461918 –
Theloderma truongsonense VNMN 4402 Khanh Hoa, Vietnam LC012847 –
Theloderma ryabovi VNMN 3924 Kon Tum, Vietnam LC012860 –
Theloderma phrynoderma CAS247910 Myanmar KJ128283 KU244449
Theloderma nebulosum ROM 39588 Kon Tum, Vietnam KT461887 –
Theloderma licin MVZ 9458 Indonesia KU244368 KU244447
Theloderma lateriticum VNMN 1216 Bac Giang, Vietnam LC012851 –
Theloderma lacustrinum NCSM 84683 Vientiane, Laos KX095246 –
Theloderma horridum KUHE 52582 Negeri Sembilan, Malaysia LC012861 –
Theloderma gordoni MVZ 226469 Vinh Phuc, Vietnam KU244363 KU244451
Theloderma corticale MVZ 223905 Vinh Phuc, Vietnam KU244364 KU244452
Theloderma auratum ZMMU A5828 Gia Lai, Vietnam MG917767 –
Theloderma annae NAP05558 Hoa Binh, Vientam MG917766 –
Theloderma asperum ZRC1.1.9321 Malaysia GQ204725 –
Theloderma baibungense YPX31940 Motuo, Tibet, China KU981089 –
Theloderma bicolor LC1 Lvchun, Yunnan, China KY495632 –
Theloderma moloch GXNU YU000115 Yingjiang, Yunnan, China MT509809 –
Theloderma pyaukkya GXNU YU000116 Yingjiang, Yunnan, China MT509810 MT522176
Theloderma petilum HNUE MNA2012.0001 Dien Bien, Vietnam KJ802925 –
Theloderma rhododiscus CIB GX200807017 Jinxiu, Guangxi, China LC012842 –
Theloderma rhododiscus KIZ060821063 Jinxiu, Guangxi, China EF564533 –
Theloderma rhododiscus KIZ060821170 Jinxiu, Guangxi, China EF564534 –
Theloderma rhododiscus SCUM 061102L Jinxiu, Guangxi, China EU215530 –
Theloderma rhododiscus CIB GX200807048 Jinxiu, Guangxi, China KJ802921 –
Theloderma rhododiscus GXNU YU000069 Jinxiu, Guangxi, China OL843957 OL843972
Theloderma rhododiscus GXNU YU000070 Jinxiu, Guangxi, China OL843958 OL843973
Theloderma rhododiscus GXNU YU000309 Huanjiang, Guangxi, China OL843959 OL843974
Theloderma rhododiscus GXNU YU000318 Longlin, Guangxi, China OL843960 OL843975
Theloderma rhododiscus GXNU YU000319 Longlin, Guangxi, China OL843961 OL843976
Theloderma rhododiscus C051 Jinxiu, Guangxi, China – KP996753
Theloderma rhododiscus C089 Jinxiu, Guangxi, China – KP996786
Theloderma rhododiscus C090 Jinxiu, Guangxi, China – KP996787
Theloderma hekouense sp. nov. GXNU YU000397 Hekou, Yunnan, China OL843962 OL843977
Theloderma hekouense sp. nov. GXNU YU000398 Hekou, Yunnan, China OL843963 OL843978
Theloderma hekouense sp. nov. GXNU YU000412 Hekou, Yunnan, China OL843964 OL843979
Theloderma hekouense sp. nov. GXNU YU000413 Hekou, Yunnan, China OL843965 OL843980
Theloderma hekouense sp. nov. GXNU YU000495 Hekou, Yunnan, China OL843966 OL843981
Theloderma hekouense sp. nov. GXNU YU000496 Hekou, Yunnan, China OL843967 OL843982
Theloderma hekouense sp. nov. AMNH A163893 Vi Xuyen, Ha Giang, Vietnam DQ283393 –
Theloderma hekouense sp. nov. HHU-WJHK01 Hekou, Yunnan, China KY495639 –
Theloderma hekouense sp. nov. HHU-WJHK02 Hekou, Yunnan, China KY495640 –

Sequences were aligned using MUSCLE with the default parameters in MEGA v. 7 (Kumar et al. 2016). Uncorrected pairwise distances between species were calculated in MEGA v. 7. The best substitution model was selected using the Akaike Information Criterion (AIC) in jMODELTEST v. 2.1.10 (Darriba et al. 2012). Bayesian inferences were performed in MRBAYES v. 3.2.6 (Ronquist et al. 2012) under the selected substitution model (GTR + I + G). Two runs were performed simultaneously with four Markov chains starting from random tree. The chains were run for 3,000,000 generations and sampled every 100 generations. The first 25% of the sampled trees were discarded as burn-in after the standard deviation of split frequencies of the two runs was less than a value of 0.01, and then the remaining trees were used to create a consensus tree and to estimate Bayesian posterior probabilities (BPPs).

Results

The obtained sequence alignments of the 16S and COI genes were 784 bp and 561 bp, respectively. Our phylogenetic analysis strongly supported that specimens from Yunnan and Vietnam form a clade (clade A), which is the sister to the clade consisting of topotypes and other specimens from Guangxi (clade B; Figs 2, 3). The genetic divergence between these two clades is 4.2% and 10.7% in 16S and COI genes, respectively.

Figure 2. 

Bayesian phylogram of Theloderma inferred from 784 bp of 16S rRNA gene. The values above the branches are Bayesian posterior probabilities.

Figure 3. 

Bayesian phylogram of Theloderma inferred from 561 bp of COI gene. The values above the branches are Bayesian posterior probabilities.

The specimens from Hekou, Yunnan, China can be morphologically distinguished from topotypes of T. rhododiscus by a series of characters: i.e., red subarticular tubercles, red metacarpal tubercles, a red metatarsal tubercle, and denser white warts on dorsal surface. Therefore, based on the molecular and morphological evidence, we consider the Hekou specimens to represent a cryptic species and describe this species below.

Theloderma hekouense sp. nov.

http://zoobank.org/65A68280-DECC-4559-BB78-1FBA7F82474B
Figs 4, 5A–C

Holotype

GXNU YU000496, adult male, collected on 9 September 2021 by Jian Wang from Hekou, Yunnan, China (22°54'N, 103°42'E, 2109 m a.s.l.; Fig. 1).

Paratypes

GXNU YU000397 and GXNU YU000398, two adult males, collected from the type locality by Jian Wang on 1 May 2021; GXNU YU000413 and GXNU YU000495, two adult males collected from the type locality by Jian Wang on 28 May 2020 and 9 September 2021, respectively; GXNU YU000412, one adult female, collected from the type locality by Jian Wang on 28 May 2020.

Etymology

The specific epithet is named after the type locality, Hekou County, Yunnan, China. We suggested “Hekou Bug-eyed frog” for the common English name and 河口棱皮树蛙 (Hé Kǒu Léng Pí Shù Wā) for the common Chinese name.

Diagnosis

The new species was assigned to genus Theloderma by its phylogenetic position and the following morphological characters: distinct tympanum, terminal phalanx with Y-shaped distal end, intercalary cartilage between terminal and penultimate phalanges of digits, tips of digits expanded into large discs bearing circummarginal grooves, head skin not co-ossified to skull (Poyarkov et al. 2018). Theloderma hekouense sp. nov. can be distinguished from T. rhododiscus and other congeners by having a combination of the following characters: 1) small body size; 2) dorsal surface coarsely rough with large ridges and tubercles; 3) dense warts on dorsal surface; 4) absence of white markings on dorsal surface; 5) iris uniformly reddish brown; 6) discs, metacarpal tubercles, metatarsal tubercles, and subarticular tubercles red; 7) webbing between fingers, vocal sac, and vomerine teeth absent.

Description of holotype

Adult male (SVL 25.7 mm; Table 2); head width (HW 8.5 mm) nearly equal to head length (HL 8.9 mm); snout slopes upward towards the tip, slightly protruding beyond lower jaw in ventral view; canthus rostralis distinct; loreal region sloping; nostrils oval, lateral, nearer tip of snout; interorbital distance (IOD 3.0 mm) greater than internarial distance (IND 2.4 mm) and upper eyelid width (UEW 2.6 mm); pineal spot absent; pupil oval, horizontal; tympanum distinct (TD 2.2 mm), rounded, greater than half eye diameter (ED 3.1 mm); supratympanic fold indistinct; vomerine teeth absent; choanae oval; tongue cordiform, wide deeply notched posteriorly; no vocal sac.

Table 2.
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Measurements (in mm) of Theloderma hekouense sp. nov. from the type locality (holotype is marked with asterisk).

Character GXNU YU000397 GXNU YU000398 GXNU YU000412 GXNU YU000413 GXNU YU000495 GXNU YU000496*
Sex M M F M M M
SVL 25.9 27.2 26.8 25.9 26.2 25.7
HL 8.9 9.0 8.9 8.8 8.9 8.9
HW 8.6 9.0 9.1 8.7 8.5 8.5
SL 3.7 3.8 3.8 3.6 3.6 3.5
IND 2.4 2.5 2.4 2.4 2.3 2.4
IOD 2.9 2.9 3.0 3.0 2.8 3.0
UEW 2.3 2.5 2.7 2.4 2.4 2.6
ED 3.2 3.1 3.3 3.2 3.2 3.1
TD 2.2 2.2 2.3 2.1 2.1 2.2
DNE 2.4 2.5 2.5 2.3 2.2 2.2
FHL 13.7 14.2 14.5 13.5 14.1 13.3
TL 13.9 14.2 14.9 14.0 14.9 13.9
TFL 19.8 20.3 21.2 19.4 20.3 18.9
FL 12.8 13.2 13.9 12.8 13.7 12.3

Forelimbs moderately robust; relative length of fingers I<II<IV<III; all fingertips expanded into discs with circummarginal grooves, relative width of finger disks I<II<IV<III; nuptial pad present on base of finger I; webbing between fingers absent; subarticular tubercles prominent and rounded, formula 1, 1, 2, 2; supernumerary tubercle prominent; two metacarpal tubercles, the outer divided into two.

Hindlimbs long; tibiotarsal articulation reaching tip of snout when hindlimb stretched alongside of body; heels overlapping when legs positioned at right angles to body; tarsal glands absent; relative length of toes I<II<III=V<IV; toe I with preaxial dermal fringe and toe V with postaxial dermal fringe; all toe tips expanded into discs with circummarginal grooves; toes webbed, webbing formula I2-2II1.5–3III2-3IV3-1.75V; subarticular tubercles prominent and rounded, formula 1,1,2,3,2; inner metatarsal tubercle prominent, light red; outer metatarsal tubercle absent.

Dorsolateral fold absent; dorsal surface very rough with prominent irregular ridges, conical tubercles, and dense white small warts on dorsum, top of head, upper eyelids, and dorsal of limbs; head side and body flank rough, scattered with warts; no warts on tympanum; dorsal skin of digits relatively smooth, scattered with white warts; white tubercles and warts around vent; chest, belly, body flank, and ventral surface of forearm and thigh coarsely granular, more so on venter; white tubercles and warts scattered on venter of tarsus and feet.

Coloration in life

Dorsal surface tea-brown with black spots between the nostrils and eyes, between eyes, and on dorsum and dorsal surface of limbs; head side almost uniformly tea-brown, with few white dots on tympanum region; body flank tea-brown, scattered with black spots enclosed by white stripes; a large black spot on sacral area extended to dorsum and connected with the black band on thigh when thigh adhered to body; ventral surface brownish black with white spots on chin and white marbled network on belly and limbs; dorsal and ventral surfaces of discs orange-red; subarticular tubercles, metacarpal tubercles, and metatarsal tubercle semitransparent with light red; nuptial pad greyish white; toe webbing orange-red mottled with dark; iris red-brown.

Coloration in preservative

Dorsal surface faded to brownish black with black spots, pattern as in life; tubercles and warts white; ventral surface brownish black with white spots and white marbled network; discs, subarticular tubercles, metacarpal tubercles, and metatarsal tubercles faded to white (Fig. 5A, B).

Morphological variation

The new species is sexually dimorphic in that the female has no nuptial pad. Black spots on dorsal surface varied among individuals in that 1) GXNU YU000398 and YU000495 have no distinct black spots between snout and eyes, 2) GXNU YU000398 and YU000413 have only one large black spot on dorsum whereas other types have two or more, and 3) GXNU YU000397 has two large black spots between eyes whereas other types have only one.

Distribution

In addition to the type locality, Hekou, Yunnan, China, the new species also occurs in Ha Giang, northern Vietnam (Bain and Nguyen 2004) because our molecular analyses revealed that the samples from Ha Giang also belong to the clade of the new species. The new species inhabits shrubs and prefers to breed in water-filled tree hollows. All specimens from Yunnan were found in an artificial breeding trap constructed using water bottles for surveillance of amphibian diversity (Fig. 6).

Comparisons

Orlov et al. (2006) identified three groups in Theloderma based on SVL, including small (28–35 mm), medium-sized (40–45 mm), and large (48–75 mm). Here the new species (adult SVL 25.7–27.2 mm) is referred to the small group, and therefore can be easily distinguished from members of the other two groups including: T. bicolor, T. corticale (male SVL 61 mm, n = 1), T. gordoni (male SVL 36.4–46.7 mm), T. horridum (SVL 37.1–48.7 mm, n = 4), T. leporosum (SVL 62.6 mm, n = 1), T. moloch (SVL 39.6–46.3 mm in two females and SVL 40.2 mm in one male), T. nagalandense (male SVL 52.8 mm, n = 1), T. phrynoderma (SVL 41.4–44.6 mm), and T. ryabovi (male SVL 43.8 mm, n = 1).

A morphological comparison between small-bodied Theloderma species is summarized in Table 3. The new species can be distinguished from its sister-species T. rhododiscus, with which it was previously confused, by the denser white warts on dorsal surface (vs relatively sparse), red subarticular tubercles (vs white), red metacarpal tubercles (vs white), a red metatarsal tubercle (vs white), and dorsal and ventral surfaces blackish in preservative (vs tea-brown) (Fig. 4).

Table 3.
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Morphological comparison of members of Theloderma with small size (SVL < 35 mm). “?” means unknown.

Species Iris color Finger webbing Color of discs Dorsal colour Ventral colour Vomerine teeth Vocal sac Dorsal skin Metacarpal, metatarsal, and subarticular tubercles
T. hekouense sp. nov. red brown absent both dorsal and ventral surfaces orange red tea-brown with no white markings brownish black with white marbled network absent absent coarsely rough with large asperities red
T. annae greyish green absent both dorsal and ventral surfaces greyish white greyish green greyish white absent absent smooth gray
T. albopunctatum red brown present both dorsal and ventral surfaces brown brown with white markings dark olive with white stripes absent present smooth with small asperities greyish white
T. asperum reddish brown absent both dorsal and ventral surfaces brown dark grey-brown with white markings marbled black and bluish grey/white absent present rough with large asperities ?
T. auratum golden above and black below absent dorsal surface dark brown and ventral surface grey golden yellow greyish blue with brown blotches absent absent smooth gray
T. baibungense red brown absent dorsal surface black brown and ventral surface grey brown with white markings black with white stripes absent present smooth with small asperities white
T. lacustrinum uniformly bronze absent dorsal and ventral surfaces bronze light brown uniformly gray absent ? smooth with small asperities gray
T. lateriticum deep brick-red absent both dorsal and ventral surfaces grey brick-red grey-brown with white spots absent absent granular with small bumps gray-brown
T. laeve grey above and dark brown below absent both dorsal and ventral surfaces grey beige with thin light middorsal stripe uniformly violet-grey absent absent smooth grey
T. licin red present dorsal surface black-brown pale whitish brown to light brown white with brown reticulation absent present nearly smooth with fine asperities ?
T. nebulosum pale gold above and reddish brown below absent both dorsal and ventral surfaces brown brown with dark patterning dark brownish black with pale blue/white marbling absent ? nearly smooth with very sparsely distributed minute asperities brown
T. palliatum pale gold above and dark red below absent both dorsal and ventral surface brown to greyish brown pale to medium brown with dark brown blotches dark warm brown with pale bluish white marbling absent absent weakly rugose with sparsely scattered minute asperities faint white
T. petilum reddish brown above and grey below absent dorsal surface lavender and ventral surface creamy-white light brown with dark brown reticulations creamy white present ? nearly smooth with small, white asperities creamy white
T. pyaukkya uniformly red absent dorsally red and ventrally brown brown with white markings brown with cream marbling absent present rough with fine asperities grayish white
T. rhododiscus uniformly red-brown absent both dorsal and ventral surface red tea-brown with black blotches brownish black with gray-white network absent absent rough with large asperities white
T. stellatum dark gold with black present dorsal surface reddish and ventral surface grey brown with white markings cream with purplish-brown flecks or spots absent absent rough with small or large asperities flesh-white
T. truongsonense golden yellow above and black below absent dorsal surface beige to black brown and ventral surface yellow-goldish with dark brown dark gray with black speckles absent ? smooth with small asperities gray
T. vietnamense golden-brownish present dorsally reddish and ventrally grey brown with white markings dark brown to blackish with slight whitish to bluish reticulations absent present rough with large ridges and warts whitish to bluish
Figure 4. 

Views of holotype of Theloderma hekouense sp. nov. (GXNU YU000496) in life.

Figure 5. 

Theloderma hekouense sp. nov. and T. rhododiscus A–C dorsal and ventral views of Theloderma hekouense sp. nov. A, B holotype (GXNU YU000496) in preservative C paratype (GXNU YU000495) in life D–F T. rhododiscus D, E topotype (GXNU YU000069) in preservative F topotype (GXNU YU000417) in life.

Figure 6. 

Habitat of Theloderma hekouense sp. nov. A habitat at the type locality B an individual found in a water bucket that was set up in the field as potential breeding site of treefrog preferred breeding in water-filled tree holes by the authors for amphibian monitoring.

Theloderma hekouense sp. nov. is distinguishable from T. annae, T. auratum, T. laeve, T. lacustrinum, T. lateriticum, T. licin, T. nebulosm, T. palliatum, T. petilum, and T. truongsonense by having the dorsal surface coarsely roughened with large ridges and tubercles (vs smooth or weakly rugose with small asperities), and from T. albopunctatum, T. asperum, T. baibungense, T. pyaukkya, T. stellatum, and T. vietnamense by absence of white markings on the dorsal surface (vs present).

The new species further differs from T. annae, T. auratum, T. lacustrinum, T. laeve, T. nebulosm, T. palliatum, T. petilum, T. stellatum, T. truongsonense, and T. vietnamense by the uniformly reddish-brown iris (vs lacking red colouration or bicoloured); from T. albopunctatum, T. licin, T. stellatum, and T. vietnamense by lacking webbing between the fingers (vs present); from T. albopunctatum, T. asperum, T. baibungense, T. licin, T. pyaukkya, and T. vietnamense by lacking a vocal sac (vs present); from T. petilum by lacking vomerine teeth (vs present); from T. annae, T. albopunctatum, T. asperum, T. auratum, T. baibungense, T. lacustrinum, T. lateriticum, T. laeve, T. licin, T. nebulosum, T. palliatum, T. petilum, T. pyaukkya, T. stellatum, T. truongsonense, and T. vietnamense by having both dorsal and ventral surfaces of the discs reddish brown (vs lacking red colouration or red only on the dorsal surface); and from all small-bodied congeners in having red metacarpal, metatarsal, and subarticular tubercles (vs lacking red colouration).

Discussion

Theloderma rhododiscus was thought to have a broad distribution ranging from eastern China to southwestern China and northern Vietnam (Zeng et al. 2017). Although previous molecular studies have revealed relatively large genetic divergence between samples from the type locality and limited samples from Yunnan and Vietnam (e.g., Poyarkov et al. 2015; Hou et al. 2017b), the taxonomic status of T. rhododiscus from the western part of its distribution (Yunnan and Vietnam) has never been doubted in previous publications. In this study, our molecular data and morphological comparison supports that the taxon known as T. rhododiscus from Yunnan, China and adjacent northern Vietnam should be considered representing a sibling species of T. rhododiscus, from which the new species differs morphologically by denser white warts on the dorsal surface and red subarticular, metacarpal, and metatarsal tubercles, and genetically by 4.2% and 10.7% divergence in 16S rRNA and COI genes, respectively.

With the exclusion of Yunnan and northern Vietnam from the geographic range of T. rhododiscus, the range of T. rhododiscus should be revised to include Guangxi, Guangdong, Hunan, Fujian, and Jiangxi. In Guangxi, T. rhododiscus was previously known from three areas including Jinxiu (Dayao Mt National Natural Reserve), Longsheng (Huaping National Natural Reserve), and Nanning (Daming Mt National Natural Reserve) (Zeng et al. 2017). In this study, we found two new occurrences of T. rhododiscus in northern and northwestern Guangxi, including Longlin and Huanjiang counties. The former is adjacent to southwestern Guizhou and eastern Yunnan and the latter is adjacent to southern Guizhou. Therefore, it can be expected that T. rhododiscus will be found from Guizhou and eastern Yunnan in the future.

Yunnan is the region richest in species of bug-eyed frogs in China. With the addition of T. hekouense sp. nov., there are now 10 Theloderma species in China and seven of them are distributed in Yunnan including T. albopunctatum, T. baibungense, T. bicolor, T. gordoni, T. moloch, T. pyaukkya, and T. hekouense sp. nov. Most of these species were recorded from there recently (e.g., Hou et al. 2017b; Qi et al. 2018; Du et al. 2020), indicating that species diversity of Theloderma in Yunnan was obviously underestimated probably owing to that Theloderma species are not easy to be found because of their preference of breeding in water-filled tree hollows. Taxonomic progress of amphibians from Yunnan in recent years (e.g., Yuan et al. 2018; Yu et al. 2019a, 2019b; Du et al. 2020; Jiang et al. 2020) reflects that amphibian diversity in Yunnan remains to be poorly known. Beside T. rhododiscus mentioned above, we expect that more Theloderma species known from adjacent regions will be found from southern Yunnan, China (e.g., T. corticale, T. lateriticum, and T. petilum).

Acknowledgements

We thank our colleagues from Daweishan National Nature Reserve for their assistance during the fieldwork. This work was supported by grants from the National Natural Science Foundation of China (32060114), Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education (ERESEP2020Z22), and Guangxi Key Laboratory of Rare and Endangered Animal Ecology, Guangxi Normal University (19-A-01-06).

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