A new species of the genus Kurixalus from Yunnan, China (Anura, Rhacophoridae)

Abstract A new species of the genus Kurixalus (Anura: Rhacophoridae) is described from Yunnan, China based on morphological and molecular evidence. The new species, Kurixalus lenquanensis sp. n., is distinguished from other congeneric species by a combination of (1) smaller body size (SVL in males ranges from 25.0 to 28.9 mm), (2) obtusely pointed snout with no prominence on tip, (3) rough and brown dorsum with darker mark, (4) absence of large dark spots on ventral surface, (5) brownish clouded chin, (6) granular throat, chest, and belly, (7) presence of vomerine teeth, (8) serrated dermal fringes along outer edge of limbs, (9) slight nuptial pad, (10) golden brown iris, and (11) single internal vocal sac. The new species is known only from montane scrub vegetation at the type locality (Lenquan Village, Mengzi City, Yunnan Province) and Yangjiatian Village, Gejiu City, Yunnan Province. Genetically, the new species is nested within a clade of Taiwanese Kurixalus and recovered as the sister taxon to Kurixalus idiootocus with strong support values, indicating that the ancestor of this new species might have come from Taiwan Island or the ancestor of this new species may have been widespread in southern China and the descendent species in between Taiwan and Yunnan has become extinct.


Introduction
est 0.1 mm. Morphological terminology follows Fei (1999). 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); distance from nostril to eye (DNE, from nostril to anterior border of eye); forelimb length (FLL, 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); and thigh length (THL, from vent to knee).
A multivariate principal component analysis (PCA) was conducted using SPSS 17.0 (SPSS Inc.) based on a correlation matrix of size-standardized measurements (all measurements divided by SVL). Scatter plots of the scores of the first two factors of PCA were used to examine the differentiation among the new species, K. idiootocus, K. bisacculus, and K. odontotarsus.

Molecular analyses.
In order to support the generic placement of the new species, the phylogenetic position of the new species was investigated based on molecular data. Total genomic DNA was extracted from liver tissue stored in 99% ethanol. Tissue samples were digested using proteinase K, and subsequently purified following a standard phenol/chloroform isolation and ethanol precipitation. A fragment of the mitochondrial 16S rRNA gene was amplified and sequenced. The primers and experiment protocols used in this study are the same as with Yu et al. (2010). Homologous sequences of other Kurixalus species were obtained from GenBank and all new sequences have been deposited in GenBank under Accession Nos. KY768931-KY768944 (Table 1). Philautus abditus Inger, Orlov, & Darevsky, 1999 and Raorchestes menglaensis (Kou, 1990) were selected as outgrups based on Yu et al. (2013).
Sequences were aligned using CLUSTAL X v1.83 (Thompson et al. 1997) with the default parameters and the alignment was revised by eye. Pairwise distances between species were calculated in MEGA 5 (Tamura et al. 2011). The best substitution model was selected using the Akaike Information Criterion (AIC) in MODELTEST v3.7 (Posada and Crandall 1998). Bayesian phylogenetic inference and Maximum likelihood analysis were performed in MRBAYES 3.1.2 (Huelsenbeck and Ronquist 2001) and RAXML-GUI 1.5b1 (Silvestro and Michalak 2012), respectively, based on the selected substitution model. For the Bayesian analysis, two runs were performed simultaneously with four Markov chains starting from random tree. The chains were run for 4,000,000 generations and sampled every 100 generations. The first 25% of the sampled trees was 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 50% majority-rule consensus tree and to estimate Bayesian posterior probabilities (BPPs). For the maximum likelihood analysis, node support was estimated by 1,000 rapid bootstrap replicates.
Morphological data. We retained the first two principal components which accounted for 43.7% of the total variance and had eigenvalues above 1.0. Loadings for PC 1 were all positive except for TD and DNE and were most heavily loaded on HW, IND, and ED (Table 3). No difference was found along the PC 1 axis between the four species. The second principal component (PC 2) accounted for 20.96% of the total variance and loaded heavily and positively on SL and DNE and negatively on FL. Differentiation was found along the PC 2 axis between the new species and K. idiootocus, K. odontotarsus, and K. bisacculus (Fig. 3). The result indicates that the new species differs from K. idiootocus, K. odontotarsus, and K. bisacculus by smaller ratio of SL divided by SVL, smaller ratio of DNE divided by SVL, and higher ratio of FL divided by SVL.   Etymology. The name lenquanensis refers to Lenquan Village, the locality where the new species was found.
Diagnosis. The new tree frog species is assigned to the genus Kurixalus based on a combination of the following characters: tips of digits enlarged to discs, bearing circum-marginal grooves; small body size (adult males SVL range of 25.0-28.9 mm; Table 4); finger webbing poorly developed and toe webbing moderately developed; serrated dermal fringes along outer edge of forearm and tarsus; an inverted triangle-shaped dark brown mark between eyes; dorsal brown ") (" saddle-shaped or X-shaped marking maybe present; and coarse dorsal and lateral surfaces with small, and irregular tubercles. Kurixalus lenquanensis sp. n. can be distinguished from its congeners by a combination of the following characters: smaller body size (mean SVL 27 mm in males); obtusely pointed snout with no prominence on tip; curved canthus rostralis; slight nuptial pad; brown dorsal color; rough dorsum; chin clouded with brown; absence of large dark spots on ventral surface; presence of vomerine teeth; gold brown iris; single internal vocal sac; dermal fringes along outer edge of limbs; rough flanks; and granular throat and chest.
Description of holotype. A small rhacophorid (SVL 27.2 mm); HL 89.4% of HW; snout obtusely pointed, no dermal prominence on tip, projecting slightly beyond margin of lower jaw in ventral view; SL (3.7 mm) shorter than ED (4.1 mm); canthus rostralis blunt and curved; lore region oblique, slightly concave; nostril oval, slightly protuberant, closer to tip of snout than eye; IND (2.5 mm) narrower than IOD (2.8 mm) and slightly wider than UEW (2.3 mm); pineal spot absent; pupil oval, horizontal; tympanum distinct (TD 1.6 mm), rounded, less than half ED; supratympanic fold distinct, curves from posterior edge of eye to insertion of arm; vomerine teeth in two oblique patches touching inner front edges of oval choanae; tongue notched posteriorly; single internal vocal sac.
Limbs slender; relative length of fingers is I < II < IV < III. Tips of all four fingers expanded into discs with circum-marginal and transverse ventral grooves; disc on    finger I small, slightly wider than phalanx width; disc width shorter than tympanum width; relative width of discs is I < II < III < IV. Nuptial pad slight; fingers webbed at base, webbing formula is I2-2.5II2-3.5III3-2.5IV following Myers and Duellman (1982). Fringe of skin on edge of all fingers; subarticular tubercles prominent and rounded, formula 1, 1, 2, 2; supranumerary tubercles present; two metacarpal tubercles, outer slightly narrower than inner; row of white warts forming serrated fringe along outer edge of forearm.
Heels overlapping when legs at right angle to body; relative length of toes is I < II < V < III < IV. Tips of toes expanded into discs with circum-marginal and transverse ventral grooves; toe discs smaller than finger discs; disc on toe I same with width as phalanx width; relative size of discs is I < II < III < IV < V. Webbing moderate on all toes, webbing formula is I2-2.5II1.5-3III1.5-3IV2.75-1.5V. Subarticular tubercles prominent and rounded, formula 1, 1, 2, 3, 2; supernumerary tubercles absent; inner metatarsal tubercle distinct, oval; outer metatarsal tubercle absent; series of tubercles forming serrated dermal fringe along outer edge of tarsus and fifth toe.
Numerous small or large tubercles scattered on top of head, upper eyelids, dorsum, and flanks; patch of white tubercles below vent; white conical tubercle on tibiotarsal articulation; throat and chest finely granulated and abdomen coarsely granulated; dorsal surface of limbs tuberculate and ventral surface of thighs finely granulated.
Color of holotype in life. Iris golden brown; dorsal surface grayish brown with dark brown saddle-shaped mark on dorsum, beginning behind eye; dark brown inverted triangle-shaped mark between eyes; lateral head and tympanic region brown with dark brown spot below canthus; broad dark brown bar along canthus rostralis; limbs dorsally brown with clear dark brown barring; rear, anterior, and venter of thigh light yellow with scattered brown spots, more spots on rear; rear of flank faint yellow with brown spots; chest and abdomen white, nearly immaculate; chin clouded with black.
Color of holotype in preservative. In preservative (Fig. 4), dorsal ground color brown, pattern same as in life. Chest and abdomen white; chin clouded with dark brown; flank dirty white with dark brown spots; rear, anterior, and venter of thigh dirty white with scattered brown spots, more so on rear.
Variations. Morphometric data are summarized in Table 4. Because the holotype and paratypes of the new species are all male, sexual dimorphism could not be determined. Differing from the nearly immaculate abdominal surfaces of the holotype and paratypes from the type locality, abdominal surfaces of three paratypes (KIZ 170183Y, 170184Y, 170186Y) from Yangjiatian, Gejiu are scattered with fine brown spots (Fig.  7). TL is longer than FL in the holotype and most paratypes, but TL is shorter than FL in paratype KIZ 170182Y (Table 4). Additionally, pattern of dark brown mark on dorsum varies among specimens. The holotype and most paratypes have a saddleshaped dorsal mark, but the two paratypes KISD 1506203 and KISD 1506204 have an X-shaped dorsal mark and the paratype KIZ 170183Y has no obvious dark brown mark on dorsum.
Ecology. At present, the new species is known only from the type locality, Lenquan Village, Mengzi City, Yunnan Province and Yangjiatian Village, Gejiu City, Yunnan Prov-  (Fig. 1). The holotype was found calling on a tree branch approximately 0.5 m above near a dry puddle in a fruit garden in Lenquan Village (Fig. 8). All other specimens were found on vegetation near the dry puddle in Lenquan Village or vegetation near a reservoir in Yangjiatian Village. Males began to call at about 19:30 when sky was getting dark and all specimens were encountered at night (20:00-22:45). Males called loudly, but no females or eggs were found. Hyla annectans (Jerdon, 1870), Kaloula verrucosa Boulenger, 1904, andMicrohyla heymonsi Vogt, 1911 were also encountered at the type locality.
Comparisons. The new species, Kurixalus lenquanensis sp. n., is morphologically similar to K. idiootocus in that it has a small body size (mean male SVL of 27 mm in new species versus mean male SVL of 27.5 mm in K. idiootocus; Table 5). However, the new species can be distinguished from K. idiootocus by its obtusely pointed snout with no prominence on tip, absence of a pair of symmetrical large dark patches on chest, single internal vocal sac, and absence of supernumerary plantar tubercles (versus pointed snout with a small prominence on tip, presence of a pair of symmetrical large dark patches on chest, single external vocal sac, and small supernumerary plantar tubercles; Kuramoto and Wang 1987;Figs 9-10). In addition, besides that snout of the new species is shorter than that of K. idiootocus, the PCA analysis showed that the new species also differs from K. idiootocus by greater ratio of FL divided by SVL (Table 3 and Fig. 3).
Kurixalus lenquanensis sp. n. is distinguished from K. berylliniris by gold brown iris, obtusely pointed snout with no prominence on tip, smaller body size, tubercles on upper eyelid, slight nuptial pad, and coarsely granular abdomen (versus emerald to light green iris, pointed snout with a small prominence on tip, larger body size [mean SVL in males = 35 mm], lack of palpebral tubercles, greatly expanded nuptial pad, and smooth abdomen; Wu et al. 2016; Fig. 10).
In addition, the new species can be distinguished from K. wangi by a lack of prominence on snout tip, smaller body size, presence of tubercles on dorsum, coarse skin on flanks, and slight nuptial pad (versus pointed snout with small prominence on tip, larger body size [mean SVL in males = 30 mm], absence of tubercles on dorsum, smooth skin on flanks, and greatly expanded nuptial pad; Wu et al. 2016;Fig. 10) and from K. eiffingeri by smaller body size, slight nuptial pad, oblique loreal region, and curved canthus rostralis (versus larger body size [mean SVL of 31.1 mm in males], greatly expanded nuptial pad, vertical loreal region, and straight canthus rostralis; Wu et al. 2016 ).
Kurixalus lenquanensis sp. n. further differs from Kurixalus appendiculatus (Günther, 1858) by smaller body size, absence of dermal prominence on snout tip, and tympanum less than half of eye diameter (versus larger body size [male SVL = 30-37 mm], presence of prominence on snout tip, and tympanum half eye diameter; Günther 1858, Inger et al. 1999; from K. baliogaster by smaller body size (SVL in males = 25.0-27.4 mm), absence of prominence on obtusely pointed snout tip, absence of large dark spots on ventral surface, tuberculated dorsal and lateral skin, presence of tubercles on eyelids, granular throat, and presence of dermal fringes on limbs (versus larger body size [male SVL = 33.0-33.3 mm], pointed snout with prominence on tip, large dark spots on ventral surface, smooth dorsal and lateral skin, absence of tubercles on eyelids, smooth throat, and absence of dermal fringes on limbs; Inger et al. 1999); and from Kurixalus banaensis (Bourret, 1939) by smaller body size, obtusely pointed snout being shorter than eye, presence of vomerine teeth, and tuberculate flanks (versus larger body size [mean SVL in males = 29.7 mm], markedly pointed snout being longer than eye, absence of vomerine teeth, and smooth flanks in K. banaensis; Nguyen et al. 2014a, Nguyen et al. 2014b, Bossuyt and Dubois 2001. The new species differs from Kurixalus viridescens Nguyen, Matsui, & Duc, 2014 by tuberculate dorsum, brown dorsal color, dark bands on dorsum and limbs, brownish clouded pattern on chin, and presence of vomerine teeth (versus nearly smooth dorsum, uniformly greenish dorsal color, no dark markings on dorsum and limbs, pinkish cream without marking on chin, and absence of vomerine teeth in K. viridescens; Nguyen et al. 2014b); from Kurixalus ananjevae (Matsui & Orlov, 2004) by smaller body size, presence of vomerine teeth, presence of dermal fringes on limbs, and finely granular throat surface (versus larger body size [32 mm in one male], absence of vomerine teeth, absence of dermal fringes on limbs, and smooth throat surface; Matsui and Orlov 2004); and from Kurixalus motokawai Nguyen, Matsui, & Eto, 2014 by obtusely pointed snout tip, presence of vomerine teeth, and clouded chin with brown (versus pointed snout tip, absence of vomerine teeth, and small dark brown spots scattered on chin; Nguyen et al. 2014a) Currently, two species of Kurixalus (K. bisacculus and K. odontotarsus) are recognized in Yunnan, China (Yu et al. 2010). The new species can be distinguished from K. bisacculus and K. odontotarsus by smaller body size, absence of large black spots on belly, and obtusely pointed snout with no prominence on tip (versus larger body size [mean SVL in males at more than 33 mm], presence of large black spots on belly, and markedly pointed snout with a prominence on tip extending beyond lower jaw in K. bisacculus and K. odontotarsus; Table 5; Fig. 9). Moreover, the PCA analysis revealed that the new species further differs from K. odontotarsus and K. bisacculus by smaller ratio of SL/SVL, smaller ratio of DNE/SVL, and bigger ratio of FL/SVL (Table 3 and Fig. 3).
Additionally, the new species differs from K. verrucosus found in Myanmar by smaller body size, snout shorter than diameter of eye, interorbital distance wider than upper eyelid, tympanum less than half of eye diameter, moderate toe webbing, granular throat and chest, and absence of large brown spots on belly and throat (versus larger body size [mean SVL in males = 29.9 mm], snout as long as diameter of eye, interorbital space as broad as upper eyelid, tympanum half eye diameter, nearly entirely developed toe webbing, smooth throat and chest, and presence of large brown spots on belly and throat; Boulenger 1893); and from Kurixalus naso (Annandale, 1912) by smaller body size, obtusely pointed snout with no dermal prominence on tip, moderately developed toe webbing, and absence of large dark spots on chest and belly (versus larger body size [male SVL at more than 30 mm], pointed snout with a dermal prominence on tip, almost completely developed toe webbing, and presence of large dark spots on chest and belly in K. naso; Annandale 1912, Mathew andSen 2008).

Discussion
Although morphological synapomorphies of the genus Kurixalus are still not very clear (Yu et al. 2013, Nguyen et al. 2014b, intuitively K. lenquanensis sp. n. can be placed in Kurixalus because of its morphological similarity to other members of the genus (e.g. small body size, inner and outer fingers not opposable, poorly developed finger webbing, moderately developed toe webbing, and serrated dermal fringes on forearm and tarsus). Other small rhacophorid species in the genera Feihyla, Gracixalus, Chiromantis, or Philautus generally lack serrated fringes on forearm and tarsus and lack vomerine teeth (Fei 1999, Fei et al. 2010. Additionally, inner (first and second) and outer (third and fourth) fingers are opposable in all species of Feihyla and Chiromantis (Fei 1999, Fei et al. 2010. This assignment is supported by the molecular data, which indicates that K. lenquanensis sp. n. is nested in the genus Kurixalus with strong support values. It is very interesting biogeographically that K. lenquanensis sp. n. is nested within a clade consisting of Taiwanese Kurixalus with strong support, indicating that the ancestor of the new species may have been from Taiwan Island. Another plausible scenario is that the ancestor of this new species may have been widespread in southern China and the descendent species in between Taiwan and Yunnan has become extinct. Although K. ananjevae and K. naso are not included in the present study, absence of them would have no impact on the phylogenetic position of the new species because K. ananjevae likely does not belong to the genus (Yu et al. 2013), and morphologically, K. naso is more similar to members of the K. odontotarsus species group than to K. lenquanensis sp. n. in body size and ventral color pattern.
Reproductive behavior among Taiwanese relatives of the new species varies; Kurixalus idiootocus lays pigmented eggs on land near the edge of water or in depressions where rainfall accumulates (Kuramoto and Wang 1987), whereas K. eiffingeri, K. berylliniris, and K. wangi lay eggs inside tree hollows or cut bamboos with water (Fei et al. 2010, Wu et al. 2016. Although no eggs of K. lenquanensis sp. n. were found, reproductive behavior of this new species probably is closer to that of K. idiootocus than to that of the other three Taiwanese species because 1) it is the sister taxon to K. idiootocus and 2) no tree hollows or cut bamboos were found at the type locality.
Species boundaries among members of the genus Kurixalus were previously confusing and our earlier work, based on molecular data (Yu et al. 2010), supported that there are three valid members of Kurixalus in mainland China. Therefore, with the new species described here, there are currently four Kurixalus species in mainland China: K. bisacculus, K. lenquanensis sp. n., K. odontotarsus, and K. verrucosus. However, considering the obvious geographical discontinuity in distribution between K. lenquanensis sp. n. and its congeneric relatives from Taiwan Island, additional undiscovered species of Kurixalus may exist in south China.