INTRODUCTION

The Vietnam Flying Frog, Rhacophorus calcaneus Smith, 1924, is endemic to the Annamite Mountains of southern Vietnam (type locality: Langbian peaks, see Smith, 1924). The taxonomic history of the R. calcaneus species group is complicated (Orlov et al., 2012). R. calcaneus was previously extensively confused with the recently described R. robertingeri (Inger et al., 1999), and another recently described species, R. chuyangsinensis (Orlov et al., 2008), which was subsequently synonymized with R. calcaneus (Orlov et al., 2012). According to Frost (2017), the latter species is known with certainty only from Chu Yang Sin National Park, Krong Bong and Lak districts, Dak Lak Province, and from Bidoup - Nui Ba National Park, Lam Dong Province. Because of the complicated taxonomic history, not much is known about the natural history of R. calcaneus sensu stricto (Van Dijk & Nguyen, 2009). We herein report the species for the first time from Khanh Hoa Province. Based on tadpoles collected in Hon Ba Nature Reserve, which were genetically matched with topotypic metamorphosed frogs and R. calcaneus from Dak Lak Province (Nguyen et al., 2014) and subsequently identified as R. calcaneus we herein further describe the larval morphology of the species for the first time.

MATERIAL AND METHODS

Material examined: Collected specimens were deposited in the herpetological collection of the Vietnam National Museum of Nature (VNMN), Vietnam Academy of Science and Technology in Hanoi. One adult male (VNMN 06317), one adult female (VNMN 0965), one juvenile (VNMN 0969) and six Rhacophorus tadpoles (VNMN 06318-06323) were collected in Hon Ba Nature Reserve, Khanh Hoa Province, Vietnam. Two adult males of R. calcaneus (VNMN 0608, 0610) from Chu Yang Sin National Park, Dak Lak Province, Vietnam (Nguyen et al., 2014) served for morphological and molecular comparisons (Table 1).

Molecular analysis: For the molecular analysis we used the protocols of Kuraishi et al. (2013), modified by Nguyen et al. (2014), for DNA extraction, amplification, and sequencing. Fragments of the mitochondrial DNA gene 16S rRNA was amplified using the primers following Kuraishi et al. (2013). Chromas Pro software (Technelysium Pty Ltd., Tewantin, Australia) was used to edit the sequences, which were aligned using MAFFT version 7 (Katoh & Standley, 2013) with default setting (FFT-NS-2 algorithm). We then checked the initial alignments by eye. Phylogenetic trees were constructed by using maximum likelihood (ML) and Bayesian inference (BI). Prior to ML and Bayesian analyses, the optimum substitution models for the 16S rRNA partition was selected by Kakusan 4 (Tanabe, 2011), based on the Akaike information criterion (AIC). We performed ML analyses with Treefinder version March 2011 (Jobb, 2011), while we estimated BI and Bayesian posterior probabilities (BPP) with MrBayes v.3.2.1 (Ronquist & Huelsenbeck, 2003). The best model selected for ML was the general time reversible model (GTR: Tavaré, 1986) with a gamma shape parameter (G: 0.255 in ML and 0.259 in BI). The BI summarized 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 = 3,000,000). We checked parameter estimates and convergence using Tracer version 1.5 (Rambaut & Drummond, 2009). The strength of nodal support in the ML tree was analyzed 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 supported (Hillis & Bull, 1993), and nodes with a BPP of 95% or greater as significant in the BI analysis (Leaché & Reeder, 2002). Pairwise comparisons of uncorrected sequence divergences (p-distance) were calculated for 16S rRNA fragments. Resulting DNA sequences from this analysis were submitted to GenBank (Accession Numbers: LC375231 to LC375239; Table 1). For tadpole matching and specific identification, sequences of the 16S rRNA of the tadpoles from Hon Ba Nature Reserve, Khanh Hoa Province, were compared with these from topotypic adults and a topotypic juvenile together with one male and one female of R. calcaneus from Chu Yang Sin National Park, Dak Lak Province (Nguyen et al., 2014). Morphological identification of adult frogs followed Smith (1924), Orlov et al. (2008, 2012) and Tran et al. (2011).

Table 1.

Samples of Vietnamese Rhacophorus and other rhacophorid species used for DNA analysis in this study together with information on voucher, collection locality and GenBank accession numbers. Voucher abbreviations: KIZ = Kunming Institute of Zoology; KUHE = Graduate School of Human and Environmental Studies, Kyoto University; VNMN = Vietnam National Museum of Nature.

Morphological analysis: Terminology for morphometric data and abbreviations followed Altig & McDiarmid (1999) and Grosjean (2005). Tadpoles were staged according to Gosner (1960). The labial tooth row formula (LTRF) was determined according to Altig & McDiarmid (1999) and for general larval types see Orton (1953). Tadpoles were photographed alive in a cuvette, subsequently euthanized with ethyl acetate and preserved in 70% ethanol. Prior to preservation, a piece of the lower tail fin and tail musculature was taken from each tadpole and preserved in a 98% ethanol solution for further genetic analysis.

The measurements were taken with a dial calliper to the nearest 0.1 mm. Abbreviations are as follow: BH: maximum body height; BL: body length; BW: maximum body width; HT: maximum tail height; LF: maximum height of lower tail fin; IND: internarial distance (measured between centers of narial apertures); PP: interpupilar distance (measured between centers of pupils); RND: rostro-narial distance (measured between the tip of the snout and the center of the nostril); SS: distance from tip of snout to opening of spiracle; SU: distance from the tip of snout to insertion of upper tail fin; TL: total length; TAL: tail length; UF: maximum height of upper tail fin; VT: distance from vent to tip of tail; TMH: height of the tail musculature at base; TMW: width of tail musculature at base; FL: forelimb length; HLL: hindlimb length; SVL: Snout-vent length; ODW: oral disc width; ED: maximum diameter of eye (horizontal); LTRF: Labial Tooth Row Formula with A (number of rows on anterior labium) and P (number of rows on posterior labium); NPD: naro-pupilar distance (measured between the center of the nostril and the center of the pupilla).

RESULTS

Phylogenetic analyses: Aligned, combined sequences of 16S rRNA yielded a total of 457 characters. Of 457 nucleotide sites, 2 were variable within the in-group. The ML and Bayesian analyses produced topologies with -lnL = 1029.838 and 1057.656, respectively. Phylogenetic analyses employing ML and BI methods yielded identical topologies, and only the ML tree is presented (Fig. 1).

The comparisons of the resulting 457 bp long fragment of the 16S rRNA between the tadpoles and the topotypic frogs from Hon Ba Nature Reserve, Khanh Hoa Province, Vietnam, and adults from Chu Yang Sin National Park, Dak Lak Province, Vietnam and, showed only a negligible single base pair difference (corresponding to 0.4%), and thus an unambiguous specific assignment of the tadpoles both to the topotypic frogs and adult R. calcaneus from Dak Lak Province is guaranteed. Furthermore, the adults from Hon Ba Nature Reserve morphologically well agreed with the original description of R. calcaneus provided by Smith (1924), and with subsequent reviews (Orlov et al., 2008; Tran et al., 2011; Orlov et al., 2012), as well as with adults from Chu Yang Sin National Park, Dak Lak Province, from where R. calcaneus already was proven to occur with certainty. Thus the identity of the tadpoles from Hon Ba Nature Reserve to represent R. calcaneus is sufficiently proven.

Morphological analysis: The collected tadpoles from Hon Ba Nature Reserve, Khanh Hoa Province, Vietnam were in the developmental stages 32, 36, 37, 41, and 42 according to Gosner (1960) (Tabs 2–3, Figs 2–3). Larvae are exotrophic, lentic: benthic; larva type IV after Orton (1953); for comparisons with larvae of other Rhacophorus species occurring in Vietnam (Tab. 4).

In the following we provide a detailed description of a larva of Rhacophorus calcaneus at stage 32 (VNMN 06318).

Dorsal view: Body somewhat elliptically protracted, with a slightly pointed snout; widest portion being at midbody; maximum body width 0.68 times of body length (BW 11.48 mm; BL 16.86 mm). Nares small, rounded, positioned dorsolaterally in anterolaterally direction; naris closer to the pupil than to the tip of snout, rostro-narial distance 1.11 times of naro-pupilar distance (RND 3.2 mm; NPD 2.88 mm); internarial distance about 0.44 of interpupilar distance (IND 2.83 mm; PP 6.34 mm). Eyes of moderate size (ED 1.36 mm); eye diameter 0.12 times of maximum body width and 0.08 times of body length, positioned dorsolaterally, directed more laterally than anteriorly, slightly bulging. The tail musculature is of moderate size, tail muscle width 0.28 times of body width (TMW 3.19 mm; BW 11.48 mm).

Lateral view: Body slightly depressed, body height 0.5 times of body length (BH 8.51 mm; BL 16.86 mm); spiracle single, sinistral, ventrolaterally positioned at midbody, oriented in posterodorsal direction and entirely attached to the body; opening of the spiracle oval. Distance from tip of snout to opening of spiracle 0.6 times of body length (SS 10.05 mm; BL 16.86 mm).

Tail tapered and long; body length 0.64 times of tail length (BL 16.86 mm; TAL 26.26 mm); tail musculature moderately developed; maximum height of tail musculature 0.67 times body height and 0.46 times maximum tail height (TMH 5.7 mm; BH 8.51 mm; HT 12.43 mm). Tail musculature from the proximal to its distal half in parallel, then gradually tapering, reaching the tip of the tail. Upper and lower tail fins almost equal in size (LF 4.15 mm; UF 4.16 mm), enlarged, maximum at the end of tail; tip of tail rounded; distance from tip of snout to insertion of upper tail fin 0.83 times body length (SU 13.99 mm; BL 16.86 mm); maximum height of upper and lower tail fin 0.33 times of maximum tail height (UF 4.16 mm; LF 4.15 mm; HT 12.43 mm). Vent tube dextral, located directly at end of body between limbs; posterior part of vent tube coadunate with lower tail fin, moderate size, margin thick.

Lateral line organ present and well developed on body and along the apex of the caudal musculature.

Oral disc: Anteroventrally positioned, of nearly triangular shape in expanded state (see Fig. 2), and laterally emarginated; two short rows of papillae situated laterally of the lower labium and margin of the mouth present, which is restricted by two side corners of the mouth; absence of papillae on margin of the upper labium; lower lip with medial gap; in preservative, tip of the papillae rounded, white; base of the papillae brown. Jaw sheaths black and convex; both upper and lower jaw sheaths serrated; upper jaw sheath semicircular and narrow, and stretched into a wide arch; lower jaw sheath V-shaped. Mouth part medium-sized (ODW 3.8 mm); oral disc width 0.33 times of maximum body width and 0.23 times of body length (BW 11.48 mm; BL 16.86 mm). Keratodont row formula (LTRF): 7(2–7)/3(1). Upper and lower labium with black keratodont rows; keratodonts positioned at margin; lateral keratodont rows absent; keratodont rows with numerous small black keratodonts. The upper labium with seven keratodont rows, A1 positioned at margin, continuous and in curved shape, A2 – A7 divided and separated by upper jaw sheath. The lower labium with three parallel keratodont rows, P1 divided, P2–P3 continuous.

Fig. 1.

Maximum-likelihood (ML) and Bayesian inference (BI) tree based on the partial 16S rRNA mitochondrial gene. Numbers above and under branches are ML bootstrap values and Bayesian posterior probabilities.

Table 2.

Developmental stages, total length in mm and morphological characters of the six collected larvae of Rhacophorus calcaneus from Hon Ba Nature Reserve, Khanh Hoa Province; stage diagnostic characteristics according to Gosner (1960) are italicized.

Table 3.

Measurements (in mm) of the six collected larvae of Rhacophorus calcaneus from Hon Ba Nature Reserve, Khanh Hoa Province; for abbreviations see Material & Methods.

Table 4.

Comparison of the larvae of Rhacophorus calcaneus with these of other Rhacophorus species occurring in Vietnam (after Hendrix et al., 2007; Wildenhues et al., 2010, 2011; Rowley et al., 2010; Vassilieva et al., 2013, 2016; Grosjean & Inthara, 2016).

Fig. 2.

Drawings of the preserved tadpole (VNMN 6318) of Rhacophorus calcaneus from Hon Ba Nature Reserve in Gosner Stage 32: lateral view (A), dorsal view (B) (scale bar = 1 cm); oral apparatus (C) (scale bar = 0.5 mm).

Fig. 3.

Drawings of the preserved tadpoles of Rhacophorus calcaneus from Hon Ba Nature Reserve in advanced Gosner stages. (A) Stage 36. (B) Stage 37. (C) Stage 41 and (D) Stage 42 (scale bar = 1 cm).

Coloration: Dorsal surface of head and body brown, with several small dark dots; the tail brown, with numerous small dark dots, edge and end of the tail light yellowish brown. Ventral surface whitish; vent tube region opaquewhite; abdominal fins slightly lighter than tail; limbs white.

In general, the collected tadpoles of R. calcaneus in Gosner stages 32–37 can be diagnosed as follows: medium-sized; oral disk anteroventrally positioned; LTRF: 7(2–7)/3(1); upper jaw sheath semicircular and narrow; spiracle sinistral, single, ventrolaterally positioned at midbody; vent tube open, round, dextral and located directly at end of body between limbs; tail tip round; body and tail brown with numerous small dark dots.