﻿A new cryptic species in the Thelodermarhododiscus complex (Anura, Rhacophoridae) from China–Vietnam border regions

﻿Abstract We describe a new species of Theloderma from southern Yunnan, China and northern Vietnam based on morphological and molecular evidence. Thelodermahekouensesp. 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.

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. 2017aHou et al. , 2017bZeng 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).
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. 2019aYu et al. , 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).

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: snoutvent 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. 2015Poyarkov et al. , 2018Nguyen 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).
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. 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.  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.
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.
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 waterfilled 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  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.
(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 , 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).

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. 2019aYu et al. , 2019bDu 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).