A new species of the genus Zhangixalus (Amphibia: Rhacophoridae) from Vietnam

ABSTRACT A new rhacophorid species is described from Vietnam on the basis of nine specimens collected in Quan Ba District, Ha Giang Province, northeastern Vietnam. The new species is morphologically similar to Zhangixalus pinglongensis, Z. dorsoviridis, and Z. nigropunctatus, however, it differs from the latter by having the flank cream with large black blotches on axilla and groin. The genetic distance between the new species and Zhangixalus pinglongensis, Z. dorsoviridis and Z. nigropunctatus is >3.57% (16S mtDNA gene fragment). Zhangixalus jodiae sp. nov. can be distinguished from all other species of Zhangixalus and other small rhacophorid species from Southeast Asia by a combination of the following characters: size small (SVL 36.1–39.8 mm in males); head as long as wide; vomerine teeth present; dorsal surface of head and body green without spots; axilla cream with large black blotches, groin and front-rear parts of the thigh, ventral surface of tibia black with orange blotches; lower jaw region greyish, chest and belly cream. http://www.zoobank.org/urn:lsid:zoobank.org:pub:89597718-024F-4FFC-B0AE-2005F12CF66C


Introduction
The genus Zhangixalus was recently erected by Jiang et al. (2019) and Polypedates dugritei David, 1872 was designated as the type species. The genus currently contains 37 species, found in northeastern India, Nepal, Bhutan, southern China, Myanmar, northern Thailand, Laos, northern Vietnam, Taiwan, and Japan, southwards to Indonesia, Brunei, and Malaysia. (Jiang et al. 2019;Frost 2020). In Vietnam, eight species have been recorded so far, mainly occurring in the northern part of the country (Frost 2020).
During our recent fieldwork in northeastern Vietnam, a small tree frog species was collected from Ha Giang Province. Based on molecular and morphological data it proved to be a representative of the genus Zhangixalus (Jiang et al. 2019). The specimens from Ha Giang Province can be assigned to the genus Zhangixalus on the basis of the following morphological characters: snout round; the presence of intercalary cartilage between the terminal and penultimate phalanges of digits, distal end of the terminal phalanx in Y-shape; tips of digits expanded into large disks, bearing circum-marginal grooves; fingers webbed; the presence of a supra-cloacal dermal ridge; and pupil horizontal. Morphologically, the specimens from Ha Giang resemble Zhangixalus pinglongensis (Mo et al. 2016), a recently described species from Guangxi, China, in size and colour pattern. However, closer morphological examination revealed distinct morphological differences between them. The phylogenetic analyses showed that the new species is nested in the same clade with Zhangixalus nigropunctatus and Z. pinglongensis with strong support value; however, the tree frogs from Ha Giang represent a distinct species with genetic distances of 3.57-11.27% from aforementioned Zhangixalus species. Based on these results, we describe the newly discovered tree frog population from Ha Giang Province as a new species.

Sampling
Field surveys were conducted from 14 to 27 March 2018 by T. T. Nguyen in Tung Vai Commune, Quan Ba District, Ha Giang Province, northern Vietnam. Specimens were collected between 19:00 and 24:00 h. After taking photograph in life, specimens were euthanised in a closed vessel with a piece of cotton wool containing ethyl acetate (Simmons 2002), fixed in 80% ethanol for 5 h and subsequently stored in 70% ethanol. Specimens were deposited in the collections of the Vietnam National Museum of Nature (VNMN) and the Institute of Ecology and Biological Resources (IEBR), Hanoi, Vietnam.

Molecular data and phylogenetic analyses
We used the protocols of Kuraishi et al. (2013), modified by Nguyen et al. (2015), for DNA extraction, amplification, and sequencing. Fragments of mitochondrial DNA gene 16S rRNA were amplified using the primers following Kuraishi et al. (2013). For the phylogenetic analyses, sequences of 25 species of Rhacophorus and Zhangixalus genera were used in combination with a sequence of Buergeria buergeri (Temminck and Schlegel) as outgroup according to Nguyen et al. (2014) (Table 1).
Chromas Pro software (Technelysium Pty Ltd., Tewantin, Australia) was used to edit the sequences, which were aligned using MAFFT version 7 (Katoh and Standley 2013) with default settings. We then checked the initial alignments by eye and adjusted slightly. Phylogenetic trees were constructed by using maximum likelihood (ML) and Bayesian inference (BI). Prior to ML and Bayesian analyses, we chose the optimum substitution models for entire sequences using Kakusan 4 (Tanabe 2011) based on the Akaike information criterion (AIC). The best model selected for ML was the general time-reversible model (GTR: Tavaré 1986) with a gamma shape parameter (G: 0.306 in ML and 0.359 in BI). The BI summarised two independent runs of four Markov Chains for 10,000,000 generations. A tree was sampled every 100 generations and a consensus topology was calculated for 70,000 trees after discarding the first 30,001 trees (burn-in 1,000,000). We checked parameter estimates and convergence using Tracer version 1.6 (Rambaut and Drummond 2013). The strength of nodal support in the ML tree was analysed using non-parametric bootstrapping (MLBS) with 1,000 replicates. We regarded tree nodes in the ML tree with bootstrap values of 75% or greater as sufficiently resolved (Hillis and Bull 1993), and nodes with a BPP of 95% or greater as significant in the BI analysis (Leaché and Reeder 2002). Pairwise comparisons of uncorrected sequence divergences (p-distance) were calculated for 16S rRNA fragments only between species of the genus Zhangixalus.

Morphological characters
Measurements were taken with a dial calliper to the nearest 0.1 mm . Terminology for describing webbing formula followed that of Glaw and Vences (1997). Sex was determined by the presence of nuptial pads and gonadal inspection. We compared morphological characters of the new species with four congeners from Vietnam and China, which showed similar characters.

Bioacoustics
Advertisement calls of a single individual were recorded with a handphone (Iphone 7, as an Mp4 file with a sampling rate of 44.1 kHz). Calls were recorded in the natural habitats at a distance of approximately 0.5 m to the calling tree frogs, and environmental parameters including temperature and humidity were also recorded. Calls were analysed with Raven Pro© v.1.5 software (http://www.birds.cornell.edu/raven). Audiospectrograms provided in figures were calculated with fast-Fourier transform (FFT) of 512 points, 50% overlap using Hanning windows. The units of a call group and pulse were defined according to Duellman (1970), and we define a single call as a vocalisation produced during a single expiration (Brown and Richards 2008). Temporal and spectral parameters of calls were measured using the definitions of Cocroft and Ryan (1995). For each call recording, we measured the call duration (ms), intercall interval (ms), number of calls per call group, call repetition rate (calls/s), number of pulses per call, and dominant frequency (kHz).

Phylogenetic analyses
Aligned, combined sequences yielded a total of 1.084 characters. Of 1.084 nucleotide sites, 362 were variable and 315 were parsimony informative within the in-group. The ML and Bayesian analyses produced topologies with -lnL = 5762.932 and 6763,088, respectively.
Phylogenetic analyses employing ML and BI methods yielded slightly different topologies only among referenced species, and only the ML tree is presented in Figure 1.
The interspecific uncorrected genetic p-distances (16S rRNA gene) examined between the unnamed Zhangixalus species from Ha Giang and other congeners varied from 3.57% (compared with Z. nigropunctatus) to 18.56% (compared with R. orlovi) ( Table 2). Thus, we describe the species of Zhangixalus from Ha Giang Province, Vietnam, as a new species in the following: Zhangixalus jodiae sp. nov. (Figure 2 Paratypes. VNMN 07120, VNMN 07122-07128, eight adult males, the same collection data as the holotype, collected between 24 and 26 March 2018 at elevations between 1180 and 1320 m a.s.l. For measurements of the type series see Table 3. Diagnosis. The new species is distinguished from its congeners and other small rhacophorid species by a combination of the following characters: (1) Size small (SVL 36.1-39.8 mm in males); (2) head as long as wide; (3) vomerine teeth present; (4) snout round; (5) dorsal skin smooth; (6) dermal appendage at vent absent; (7) hand-webbing formula I1-1II1-1III2-1IV and toe webbing formula I1-1II½-1III0-1½IV1-½V; (8) dorsal surface of head and body green without spots; (9) axilla cream with large black blotches, groin, anterior and posterior thighs and ventral surface of tibia black with orange blotches; and (10) lower jaw region greyish, chest and belly cream.
Description of holotype. Size small, body robust (SVL 36.1 mm); head moderately compressed, as long as wide (HL 14.8 mm, HW 14.7 mm), slightly convex above; snout round anteriorly, slightly protruding beyond lower jaw in lateral view, longer than horizontal diameter of eye (SNL 6.5 mm, ED 4.8 mm); canthus rostralis round, loreal region oblique, concave; interorbital distance wider than internarial distance and upper eyelid (IOD 5.7 mm, IN 4.3 mm, UEW 3.8 mm); distance between anterior corners of eyes about 67% of distance between posterior corners of eyes; nostril round, without lateral flap of skin, closer to the tip of snout than to eye; pupil oval, horizontal; tympanum distinct, round, about half of eye diameter, about twice greater than the distance between tympanum and eye; pineal ocellus absent; spinules on upper eyelid absent; vomerine teeth well developed, in two oblique ridges; choanae rounded; tongue deeply notched posteriorly; supratympanic fold distinct, extending from behind eye to beyond level of axilla.
Forelimbs. Upper arm short, about one-third of forearm length (UAL 6.2 mm, FAL 18.7 mm), dermal fringe along the outer edge of forearm present, not well developed; relative lengths of fingers I< II<V< III; tips of fingers with enlarged discs with distinct  (1) Zhangixalus jodiae sp. nov.
Hindlimbs. Heels overlapping when held at right angles to the body; tibia length about three times greater than tibia width (TbL 17.3 mm, TbW 5.1 mm), slightly longer than thigh length (FeL 17.1 mm), shorter than foot length (FoL 24.9 mm); relative lengths of toes I< II<III<V< IV; tips of toes with enlarged discs with distinct circum-marginal grooves, discs slightly smaller than those of fingers; webbing formula I1-1II½-1III0-1½IV1-½V; subarticular tubercles distinct, blunt, round, formula 1, 1, 2, 3, 2; inner metatarsal tubercle small (IMT 2.07 mm); dermal ridge along the outer edge of tibia and tarsus present; dermal projection at tibiotarsal articulation present. Skin texture. Dorsal surface of head and body smooth; dorsolateral folds absent; throat and chest smooth, belly granular; ventral surface of fore and hind limbs granular; dermal appendage above vent absent.
Colouration in life. Iris silver, pupil horizontal, black; dorsal surface of the head and body green without spots; tympanum and supratympanic region green; dorsal surface of forelimbs and hindlimbs green; flanks cream, axilla and groin with large black blotches; anterior and posterior parts of thigh, and ventral surface of tibia black with orange blotches; ventral surface of arm and thigh cream; lower jaw and throat region greyish, chest and belly cream; supracloacal area white; dermal fringes on outer edges of fore and hind limbs white; ventral side of webbing orange in hindlimbs and cream in forelimbs, nuptial pads yellow.
Colouration in preservative. As in life, but with green fading to grey and pale orange fading to white. Male secondary sexual characters. Nuptial pad present; external single subgular vocal sac, throat greyish.
Advertisement call. Call descriptions are based on two advertisement calls of one paratype (VNMN 07126). Advertisement calls were recorded at 17.3°C ambient temperature. Each call was 1.1 s in duration and consisted of six notes, each~6 ms in duration and uniformly spaced~15 ms. The two notes were 8.8 s apart (Figure 3). The dominant frequency was uniformly 2.0 kHz, with harmonics at 3.7 and 5.5 kHz. Calls were amplitude modulated, with the last note of each call of lower amplitude than the others. Three calls of a more distant frog were also detected in the recording, each with six notes, and consistent with those described above.

Comparison
Based on our molecular data, Zhangixalus jodiae sp. nov. is closely related to Z. nigropunctatus, Z. yaoshanensis, Z. pinglongensis, and Z. chenfui. The genetic distance between Zhangixalus jodiae sp. nov. and the latter is approximately 3.6%, which is indicative of species-level divergence in anurans (Vences et al. 2005). Morphologically, these species are similar to each other in the following characters: small size in males SVL 32.0-37.0 mm in Z. nigropunctatus , 32-38.5 mm in Z. pinglongensis (Mo et al. 2016), and 31.6-36.4 mm in Z. yaoshanensis (Chen et al. 2018); dorsum green, smooth, without spots; webbing moderately developed; dermal flap on heel and supracloacal dermal ridge absent. However, Zhangixalus jodiae sp. nov. differs from Z. nigropunctatus ) by colouration on flank and thigh (cream with irregular black and orange blotches vs. flank and thigh cream with black blotches in Z. nigropunctatus); from Z. pinglongensis by colouration on flank and thigh (large black blotches in the axilla, black and orange blotches in the groin, thigh and ventral side of the tibia vs. irregular white blotches interposed by black from axilla to flank and thigh), ventral aspect of feet and webbing (cream vs. tangerine); from Z. yaoshanensis (Chen et al. 2018) by having different flank colour pattern (irregular black and orange blotches vs. cream with small greyish spots), the colouration on the thigh (irregular black blotches interposed by orange vs orange-red without spots); from Z. chenfui by the absence of a white line isolating the dorsal part from the ventral part (Liu 1945).
The new species differs from other green rhacophorid tree frogs recorded from Vietnam and the neighbouring countries as follows: Zhangixalus jodiae sp. nov. can be distinguished from Z. duboisi, Z. omeimontis, Z. burmanus, Z. dennysi, Z. pachyproctus, and Z. feae by having a smaller size (SVL<60 mm vs. SVL>60 mm). The new species differs from the other tree frogs in the same size by colour pattern: dorsal surface of head and body green without spots and axilla cream with large black blotches, groin, anterior and posterior part of thighs and ventral surface of tibia black with orange blotches vs. axilla and groin yellowish with small grey spots in Z. zhoukaiyae (Pan et al. 2017); axilla and groin yellow with black dots in Z. dorsorviridis (Bourret 1937); flanks as well as anterior and posterior part of thigh cream with scattered mottling in Z. minimus (Rao et al. 2006); varies from dark yellowish brown to light green on dorsal and lateral surfaces, with numerous light-brown spots that have dark yellowish brown edges in Z. wui and Z. hongchibaensis (Li et al. 2012); flank and front-rear of thigh cream without spot in Z. hungfuensis (Liu and Hu, 1961); back green with round brown spot in Z. dugritei (David, 1872); front and rear of thigh red-orange with dark spot with dorsal surface of foot green with dark spots in Z. moltrechti (Fei et al. 2012); dorsum green with numerous dark spots in Z. arboreus (Okada and Kawano, 1924); flank and groin cream without spot in Z. schlegelii (Günther, 1858).
The male advertisement calls of the most closely related species Z. chenfui, Z. nigropunctautus and Z. yaoshanensis are unknown. The male advertisement call of Zhangixalus jodiae sp. nov. differs from that of Z. pinglongensis by having six, singlepulsed notes of 1.1 s duration (versus two notes with six pulses, each~0.4 s duration) per call (Mo et al. 2016).
Distribution. Zhangixalus jodiae sp. nov. is currently known only from the type locality in Ha Giang Province, northeastern Vietnam ( Figure 4).
Natural history. The specimens were found between 19:00 and 24:00 h on the ground, near streams. The surrounding habitat was undisturbed forest of large hardwoods, shrub and liane ( Figure 5). The air temperature ranged from 17.0-21.4°C at night and the relative humidity was 88-92% at the site. The breeding biology, females and tadpoles of Zhangixalus jodiae sp. nov. are unknown.
Conservation status. Currently known only from the type locality in Cao Ma and Ta Van communes, Quan Ba District, Ha Giang Province, northeastern Vietnam. However, the actual distribution range of this species should be confirmed in further studies. Given the available information, we suggest this species be considered as Data Deficient following IUCN's Red List categories (IUCN 2016).  Etymology. The specific epithet is in honour of Dr. Jodi Rowley from the Australian Museum for her great contribution to amphibian taxonomy in Asia. We propose the following colloquial names: Jodi's Treefrog (English) and Ếch cây jô-đi (Vietnamese).

Discussion
The family Rhacophoridae is the most diverse group of amphibians in Vietnam and the species number has remarkably increased from 70 (Nguyen et al. 2009) to 79 at present (Frost 2020).
Within Rhacophoridae, Zhangixalus was considered as the sister taxon of Leptomantis and Rhacophorus. The distribution range of this genus is restricted to South Asia, from India and southern China, eastward to Japan, throughout the Indochina region and southwards to Malaysia (Frost 2020). Hence, the discovery of Zhangixalus jodiae from Ha Giang Province, northeastern Vietnam is not surprising. The mountainous border of southern China and northern Vietnam is an extraordinary region of high amphibian diversity within the Northern Subtropical Indochina Forests Ecoregion (Buckley and Jetz 2007). This high amphibian diversity has been attributed to this region being located at the intersection of tropical and subtropical zones and biotic influences of three biogeographic units, viz. southern China, Indochina, and coastal Indochina (Sterling et al. 2006). The surveys conducted in this area indicated that the amphibian fauna of the tropical rain forests at high elevation and adjacent regions is still imperfectly studied.
Furthermore, our molecular results demonstrated that Zhangixalus jodiae is embedded in the same subclade with Z. nigropunctatus, Z. yaoshanensis, Z. pinglongensis and Z. chenfui. All these species inhabit limestone karst forests (Orlov et al. 2001;Mo et al. 2016;Chen et al. 2018). Z. nigropunctatus was discovered from Long chu, Weining, Guizhou, China at an elevation of about 2,134 m, while Z. yaoshanensis was discovered from Yaoshan, Guangxi, China at an elevation of 1100 m asl. Zhangixalus pinglongensis has been known from the Pinglong mountain, Shangsi, Guangxi China, at the elevation of approximately 500 m asl., and Z. chenfui was found in Omei mountain, Sichuan, China at an elevation of 1100 m. The new species from Vietnam was found in the undisturbed limestone karst forest at elevations from 1180 to 1320 m asl. in Quan Ba District, Ha Giang Province. The type locality of Zhangixalus jodiae is sharing the national border with Malipo District of Guangxi Province, China. Our new discovery brings the species number of Zhangixalus to nine in Vietnam.