A New Species of the Genus Pachytriton (Caudata: Salamandridae) from Hunan and Guangxi, Southeastern China

Zootaxa 4085 (2): 219–232 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2016 Magnolia Press ISSN 1175-5334 (online edition) http://doi.org/10.11646/zootaxa.4085.2.3 http://zoobank.org/urn:lsid:zoobank.org:pub:E153BEED-8C53-482F-B70A-25554AEC2D17 A new species of the genus Pachytriton (Caudata: Salamandridae) from Hunan and Guangxi, southeastern China

ZHI-YONG YUAN1,2,4, BAO-LIN ZHANG1,2,4 & JING CHE1,3 1State Key Laboratory of Genetic Resources and Evolution State, Kunming Institute of Zoology, Chinese Academy of Sciences, Kun- ming 650223, Yunnan, China 2Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650204, Yunnan, China 3Corresponding author. E-mail: [email protected] 4These authors contributed equally

Abstract

Despite recent descriptions of multiple new species of the genus Pachytriton (Salamandridae), species richness in this Chi- na-endemic newts genus likely remains underestimated. In this study, we describe a new species of Pachytriton from northeastern Guangxi and southern Hunan, southeastern China. Both molecular analyses and morphological characters reveal that the new species can be distinguished from its congeners. The mitochondrial gene tree identified the new lineage highly divergent (uncorrected p-distance > 5.8 % by mitochondrial gene) from currently recognized species and placed it as the sister species of P. xanthospilos and P. changi. Furthermore, a nuclear gene haplotype network revealed a unique haplotype in the new populations. Statistical species delimitation using Bayes factor strongly supported the evolutionary independence of the new species from the closely-related P. xanthospilos. Morphologically, the new species is characterized by a uniformly dark brown dorsum without bright orange dots or black spots; irregular orange blotches on the venter; tips of fingers and toes orange on the dorsal side; moderately developed webs on the side of digits; absence of costal grooves between the axilla and groin; and widely open vomerine tooth series.

Key words: Amphibians, cryptic diversity, Bayes factor species delimitation, Pachytriton wuguanfui sp. nov.

Introduction

Asian newts (Salamandridae) have a high species diversity, comprising 57 described species, most of which occur in China (AmphibiaWeb 2015; Frost 2015). There are six genera of Asian salamandrids comprising two major lineages: the “modern Asian newts”, which includes Cynops, Laotriton, Paramesotriton and Pachytriton, and the “primitive newts” which includes Echinotriton and Tylototriton (Zhang et al. 2008; AmphibiaWeb 2015). Multiple new taxa described from China every year since 2010 (e.g., Nishikawa et al. 2011a,b; Wu et al. 2009, 2010a,b; Yuan et al. 2013, 2014; Hou et al. 2014) suggest that the true species richness of Asian newts is probably much higher than currently recognized. The Chinese stout newts (Pachytriton) are endemic to small montane streams in broadleaf forests of southeastern China. According to Amphibian Species of the World 6.0 (Frost 2015) and AmphibiaWeb (2015), this genus currently consists of eight species: P. archospotus Shen, Shen & Mo; P. brevi pes Sauvage; P. changi Nishikawa, Matsui & Jiang; P. feii Nishikawa, Jiang & Matsui; P. granulosus Chang; P. inexpectatus Nishikawa, Jiang, Matsui & Mo; P. moi Nishikawa, Jiang & Matsui; and P. xanthospilos Wu, Wang & Hanken. Five of these species were described after 2010, although there is some controversy regarding the status of P. changi and P. xanthospilos (Nishikawa et al. 2013; Wu & Murphy 2015). The recent increase in known species diversity within Pachytriton is due to recent field surveys, comparative morphological reviews, and large-scale molecular phylogenetic studies (Nishikawa et al. 2011a; Wu et al. 2012 a,b). The number of species in the genus is likely remain underestimated in southeastern China, particularly given the preservation of a few isolated primeval forests. Our recent fieldwork in southern China found two populations of Pachytriton from Mt. Gupo, northeastern

Accepted by J. Rowley: 18 Jan. 2016; published: 2 Mar. 2016 219 Guangxi, and Mt. Jiuwei, southern Hunan with distinct morphological characters, indicating that they may belong to an undescribed species. Both localities (Figure 1) are far from the town and only accessible to local villagers. Herein, we compare the external morphology between the two recently-discovered populations (referred to as the “Gupo-Jiuwei populations”) and their congeners, analyze genetic divergence within the genus using mitochondrial and nuclear DNA markers, and describe the above-mentioned two populations as a new species of Pachytriton.

Material and methods

Specimen collection. We collected seven specimens from Mt. Gupo, Guangxi (locality 5, Fig. 1) and twelve specimens from Mt. Jiuwei, Hunan (locality 6, Fig. 1) during two fieldtrips in 2010 and 2012, respectively. Newts were collected at the bottom of the stream at night by searching with torchlights and headlamps. Specimens were euthanized by immersion in a solution of chlorobutanol, fixed in 10% buffered formalin, and then stored in 70% ethanol. Livers or muscles were taken from freshly euthanized specimens in the field and preserved in 10% RNAlater. All these specimens were deposited in the Museum of the Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences.

FIGURE 1. Sampling localities used in this study of Pachytriton. Locality numbers refer to Table 1 and Figure 2. Different species are represented by different symbols (shown in Figure 2).

Morphological examination. A total of 8 well-preserved adults, including two males (KIZ08756, KIZ08759) and three females from Mt. Gupo (KIZ08755, KIZ08757–58) and three females from Mt. Jiuwei (KIZ021705–06, KIZ021708), were used for morphological measurements, which were obtained using digital calipers that were calibrated to the nearest 0.1 mm. All measurements followed the descriptions in Wu et al. (2012): SVL, snout-vent length, length from the tip of the snout to the posterior vent; HL, head length, length from the tip of the snout to the posterior angle of the parotoid gland; HW, head width, maximum width of head; HD, head depth, measured at the posterior angle of the jaw; SL, snout length, measured from the tip of the snout to the posterior angle of the jaw; IC, intercanthal distance, distance between the anterior corner of two eyes; IN, internostril distance, distance between nostrils; SF, snout-to-forelimb length, measured from the tip of the snout to the base of forelimb; SHW, shoulder width, distance between the bases of two forelimbs; TAL, tail length, measured from the posterior angle of the vent

220 · Zootaxa 4085 (2) © 2016 Magnolia Press YUAN ET AL. to the tail tip; TAD, tail depth, measured at the base of tail; TAW, tail width, measured at the base of tail; AL, measured from base of forelimb to tip of longest finger; PL, measured from base of hindlimbs to tip of longest toe. Toe 1–5 and finger 1–4 represent the length of each toe and finger, respectively. The maturity and sex of the specimens were checked by dissections. Molecular analyses. Phylogenetic analyses were based on two mitochondrial genes and a fragment of nuclear DNA for 34 specimens of Pachytriton. The two mitochondrial genes were NADH dehydrogenase subunit 2 (ND2) and the cytochrome b (cytb). Both genes have been frequently used in phylogenetic studies of Pachytriton (Wu et al. 2010c, 2013, 2015). The nuclear fragment was a partial coding sequence of proopiomelanocortin gene (POMC) based on the study of Vieites et al. (2007). PCR and sequencing conditions of ND2 and cytb were performed as in Wu et al. (2010c); and POMC were performed as in Vieites et al. (2007). All currently recognized species of Pachytriton were included in our phylogenetic analysis (see Table 1). However, P. changi, which was described from two captive animals obtained in a pet-shop without exact locality data (indicated as “possibly, China”), and now deposited at Kyoto University (Nishikawa et al. 2012), lacks ND2 and POMC data because the collecting locality and tissue samples were not available to us. Laotriton laoensis, Paramesotritron chinensis and Paramesotritron guangxiensis were chosen as outgroup taxa based on phylogenetic relationships from previous studies (Weisrock et al. 2006; Zhang et al. 2008). Because mitochondrial DNA is linked as a single locus and the two mitochondrial genes give congruent phylogenetic signals in Pachytriton (Wu et al. 2010c), we concatenated ND2 and cytb into a single alignment for further analysis. We used MEGA 5 (Tamura et al. 2011) to align sequences and calculate interspecific mean uncorrected p-distance among species after trimmed the sequences to same length. Bayesian inference (BI) and maximum likelihood (ML) methods were used to reconstruct phylogenetic relationships. We used jModelTest 2 (Darriba et al. 2012) to select the best fitting model of nucleotide evolution for codon position and tRNA of each gene under the Akaike Information Criterion. The best fitting model for each partition was GTR+I+G. Bayesian inference was performed in MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003) with two runs of 4 million generations, each of which had four incrementally heated Markov chains using default heating values. The Markov chains were sampled at intervals of 100 generations. The first 25% of the trees were discarded as burn-in after chain convergence was assessed in Tracer v1.5 (Rambaut & Drummond 2007). ML analysis was performed using the program RAxML v7.0.4 (Stamatakis et al. 2008). Heterozygous nucleotide sites were detected in the POMC dataset. Therefore, we used the software Phase v2.1.1 (Stephens et al. 2001) to infer haplotypes. Haplotype network was reconstructed with the phased sequences of the POMC fragment using the software NETWORK v4.5 (Bandelt et al. 1999). Following Wu et al. (2015), we advocate the incorporation of statistical species delimitation into species discovery. To test whether the Gupo-Jiuwei populations represent an evolutionary lineage that is independent from congeners, we used Bayes factors to evaluate competing delimitation hypotheses based on both mitochondrial and POMC data set. Pachytriton changi was excluded due to its missing data in two of the three genes used in this study. The analysis was performed using the *BEAST option in BEAST v1.8.2 (Drummond & Rambaut 2007; Heled & Drummond 2010). Marginal model likelihood (i.e., marginal likelihood for each delimitation hypothesis) was calculated via both path-sampling (PS; Baele et al. 2012) and stepping-stone sampling (SS; Xie et al. 2011) methods. Substitution model HKY was selected for the POMC data set based on the result of jModelTest 2. Population size model was set to piecewise linear with a constant root. We used an inverse gamma prior (α = 4, β = 0.003) to model the population sizes (θ) with a mean of 0.001. The Markov chains were run for 20 million generations sampled every 2000 generations. Marginal model likelihood estimation was run for one million generations of 100 path-steps (total 100 million generations). The Bayes factor (2lnBf) for competing delimitation hypotheses were evaluated following the recommendations of Kass & Raftery (1995): 2lnBf >2 is considered positive support and 2lnBf > 6 means strong support for the better model.

Results and discussion

Morphological measurements and variations were summarized in Table 2. We generated an alignment of 2253 bp mitochondrial DNA sequence, which consisted of 1124 bp of ND2 and 1129 bp of cytb sequences. The concatenated data set consisted of 634 variable sites and 571 parsimony-informative sites among sampled

A NEW PACHYTRITON FROM CHINA Zootaxa 4085 (2) © 2016 Magnolia Press · 221 specimens of Pachytriton. Interspecific mean uncorrected pairwise p-distances in the concatenated mitochondrial gene data set ranged 5.8–11.0% between the Gupo-Jiuwei populations and congeners (Table 3). Particularly, the Gupo-Jiuwei populations differentiated from their geographic neighbors, P. inexpectatus in the west and P. xanthospilos in the east, by 11.0% and 6.3%, respectively. These distances were not only higher than or comparable to distances between currently recognized species (Table 3), but also higher than interspecific divergence in other Asian salamander genera such as Tylototriton (2.3–3.4% among T. podichthys, T. shanjing, and T. verrucosus, Phimmachak et al. 2015).

FIGURE 2. Bayesian inference tree based on concatenated mitochondria ND2 and cytb. Asterisks (*) indicate highly support in ML bootstrap values (BS) and Bayesian posterior probabilities (BPP) (> 70% BS and > 95% BPP). Numbers following the species’ name refer to localities shown in Figure 1 and Table 1.

Tree topologies recovered by both BI and ML are nearly identical, thus only the BI tree is shown here (Fig. 2). Interspecific relationships within Pachytriton were well resolved and highly congruent with those reported by Wu et al. (2013). The Gupo-Jiuwei populations from locality 5 and locality 6 constituted a monophyletic lineage, which formed the sister group to the clade including P. xanthospilos and P. changi with strong nodal support. The POMC data set represented a subset of the mitochondrial data with an alignment of 479 bp from 13 individuals that had ten haplotypes. The Gupo-Jiuwei populations were characterized by a unique haplotype that could be clearly separated from congeners in the haplotype network (Fig. 3).

222 · Zootaxa 4085 (2) © 2016 Magnolia Press YUAN ET AL. ! ……continued on the next page page next the on ……continued " #$%& ND2 cytb ! 1 ()0 & - /, "0 '#)*+&#), '-.* ! " #$%23456#$%23453 * (2 * (2 #)674227 #)673879 #)674229 N #)673878 #)674265 #$%25653#$%25656#$%289#$%2896 D 0A% A(4 D 0A% A(4 ? A(7 ? A(7 #)67424 #)673885 #)67425 #)674236 #)673887 #)67427 N #)673889 #)67429 N #)673888 N ($ 8485#$%2985#$%2989#)*+6896#)*+68756 > " ." >=(#$%239 ; A(6#$%238 ; A(6 ) #$%2362 @62655 ) #$%2874 @626556 N * 0 (3 * 0 (3 * 0 (3 #)6742 BA; C % A(5 #)67389 #)67422 N #)67389 #)674267 N #)6742 #)67423 N #)67388 #)6742 #)673884 #)674268 #)673886 #)67426 #)67423 0 5697 N #)673883 N 0 569728 N N $ &( $ " archospotus archospotus ($ 8783 ; <=(>= >?827969 >?827864 N " # Pachytriton # $ "% '( brevipes ($ 8784 #$%279 ; <=(>= (8 >?827968 >?827865 #)674228 N #)673892 #)674269 changi changi feii granulosus

A NEW PACHYTRITON FROM CHINA Zootaxa 4085 (2) © 2016 Magnolia Press · 223 ! cytb ND2 #$%297 >* (3 #)67426 #)67422 #)674233 #$%2426#$%2423 / =( / =( #)67428 #)674222 #)674262 #)674234 #)67422 N ($ 995($ 9939#$%27757 D D C ( D D C ( - =(6 @626489 @626564 @626522 N @626567 N #)67426 #)674226 N #$%27759 - =(6 #)674266 #)674223 #)674235 #$%25742 - * (7 #)67427 #)673899 #)674239 #$%2574 - * (7 #)67429 #)673898 N #$%2574 - * (7 #)67428 #)673882 #)674237 #$%29 /! (9 #)67422 #)67388 N #$%2975#$%2724 =(4 > * (5 #)67426 #)673893 #)67423 #)674232 #)673894 N #$%25664 <> % A( #)674263 #)674224 N -.*4434 D +)99269 +)99269 N #$%2725 > * (5 #)67424 #)673895 #)67423 #$%894 . =( #)674264 #)674225 N #$%2727 > * (5 #)67425 #)673897 N #$%29745 =(4 #)6742 #)673896 N sp. nov. sp. chinensis laoensis laoensis ( inexpectatus moi xanthospilos xanthospilos wuguanfui wuguanfui guangxiensis guangxiensis Paramesotriton Paramesotriton Laotriton Laotriton #

Measurements Holotype Females (N=6, include Holotype) Males (N=2) KIZ08758 Range Mean±SE Range Mean±SE SVL 83.5 79.6–97.9 82.8±6.1 85.6–86.7 86.1±0.8 HL 20.2 20.0–23.8 21.6±1.6 22.3–22.4 22.4±0.1 HW 16.3 14.5–18.2 16.1±1.4 16.0–18.5 17.3±1.7 HD 9.5 7.6–9.7 8.5±1.0 7.7–9.7 8.7±1.4 SF 23.7 21.9–27.4 24.6±1.8 22.4–22.8 22.6±0.2 SL 7.7 6.6–7.6 7.4±0.5 7.6–7.9 7.8±0.2 IC 7.7 7.4–8.0 7.8±0.3 8.0–8.3 8.1±0.2 IN 3.8 3.9–4.8 4.2±0.3 3.9–4.2 4.1±0.2 SHW 13.1 11.7–17.0 14.3±2.1 14.0–14.6 14.3±0.5 TAL 72.2 65.7–84.5 75.0±7.0 69.9–77.1 73.5±5.1 TAD 7.0 7.0–9.5 8.2±0.9 7.2 –8.3 7.8±0.8 TAW 10.1 8.7–11.3 10.3±0.9 8.7–11.1 9.9±1.7 AL 14.7 11.9–16.8 14.9±1.9 14.7–15.1 14.9±0.3 PL 17.3 17.3–20.8 18.6±1.5 18.0–18.8 18.4±0.6 Toe 1 0.9 1.1–1.6 1.4±0.2 1.1–1.3 1.2±0.2 Toe 2 3.0 2.2–3.4 3.0±0.4 2.3–2.7 2.5±0.2 Toe 3 3.6 3.29–4.9 4.2±0.6 3.8–3.9 3.8±0.1 Toe 4 3.1 3.2–4.3 3.6±0.5 2.9–3.1 3.0±0.2 Toe 5 1.0 1.0–2.0 1.6±0.4 1.0–1.1 1.1±0.1 Finger 1 1.1 0.9–1.3 1.1±0.1 1.0–1.1 1.1±0.1 Finger 2 2.4 1.6–3.0 2.3±0.5 2.3–2.4 2.3±0.1 Finger 3 2.9 3.1–3.6 3.3±0.2 2.7–3.4 3.0±0.5 Finger 4 1.6 1.6–2.6 1.9±0.4 1.3–2.1 1.7±0.5

FIGURE 3. Haplotype network for Pachytriton constructed from the nuclear gene POMC. Colors correspond to each species in Figure 2.

A NEW PACHYTRITON FROM CHINA Zootaxa 4085 (2) © 2016 Magnolia Press · 225 TABLE 3. Uncorrected p-distance between the Gupo-Jiuwei populations of Pachytriton and congeners based on concatenated mitochondrial data set gene (ND2 and cytb). “-” indicates genetic distance less than 0.1%. P. sp. (Gupo-Jiuwei) P. changi P. xanthospilos P. archospotus P. sp. (Gupo-Jiuwei) 0.6% P. changi 5.8% - P. xanthospilos 6.3% 3.0% 0.4% P. archospotus 8.1% 8.0% 7.8% 1.8% P. brevipes 7.1% 6.9% 6.5% 8.1% P. feii 7.0% 6.3% 6.3% 7.1% P. granulosus 7.4% 6.2% 7.0% 7.9% P. inexpectatus 11.0% 8.5% 10.6% 10.2% P. moi 10.3% 8.5% 9.3% 8.8% continued. P. brevipes P. feii P. granulosus P. inexpectatus P. moi P. sp. (Gupo-Jiuwei) P. changi P. xanthospilos P. archospotus P. brevipes 1.2% P. feii 5.9% - P. granulosus 7.3% 6.7% 2.6% P. inexpectatus 11.1% 10.1% 10.5% 3.3% P. moi 9.4% 9.3% 9.2% 9.0% -

Given the sister relationship between P. xanthospilos and the populations in question, we used Bayes factor to evaluate two competing delimitation hypotheses: 1) the Gupo-Jiuwei populations belong to one species with P. xanthospilos; 2) the two populations represent a separate species from P. xanthospilos (Table 4). PS and SS methods yielded very similar marginal likelihoods for the two hypotheses. Both methods estimated the 2lnBf > 7, suggesting strong support for the second hypothesis. However, the available genetic data is insufficient to test conspecificity between the Guopo-Jiuwei populations and P. changi or between P. xanthospilos and P. changi. But the Guopo-Jiuwei populations differ from P. changi in dorsal coloration (see below comparisons). Moreover, the Gupo-Jiuwei populations were unlikely conspecific with P. changi based on mitochondrial divergence. Therefore, the two populations from Mt. Gupo and Mt. Jiuwei should be considered an independently evolving lineage in the genus Pachytriton and thus warrant description as a new species.

TABLE 4. Marginal likelihood estimates and Bayes factor species delimitation results for the Gupo-Jiuwei populations of Pachytriton. According to Kass and Raftery (1995), 2lnBf > 6 means strong support for the better model, namely the “independent” model. Competing delimitations Path sampling Stepping-stone sampling P. sp. (Gupo-Jiuwei) conspecific to P. xanthospilos -7919.41 -7919.67 P. sp. (Gupo-Jiuwei) independent from P. xanthospilos -7915.48 -7915.85 2lnBf 7.86 7.64

Holotype. KIZ08758 (Fig. 5), an adult female from Mt. Gupo, Hezhou city, Guangxi Zhuang Autonomous Region, China; 24.64º N, 111.53º E, elevation 1202 m, collected by Zhiyong Yuan on August 17, 2010. Paratypes. KIZ08755–08757 (three females), 08759 (male), collected at the same time and locality as the holotype. KIZ021705–06 (two females), 021708 (female), 021711 (female), 021712 (male), KIZ021832 (female), 021833–35 (three males) from Chahuaping, Dao country, Hunan province, 25.22º N, 111.79º E, elevation 649 m, collected by Zhiyong Yuan and Limin Ding on June 18, 2012. Diagnosis. Pachytriton wuguanfui sp. nov. is assigned to the genus Pachytriton by its molecular phylogenetic position and the following morphological characters: head oval and flat; skin very smooth; labial folds obvious on upper jaw; limbs short; tips of fore- and hindlimbs widely separated when limbs adpressed against body flank; tail long, base broad; posterior half of tail gradually laterally compressed; dorsal caudal fin evident. This species can be differentiated from congeners by the following combination of morphological characters: dorsum uniformly dark brown without bright orange dots and black spots over the body; tips of fingers and toes orange on dorsal side; costal grooves between axilla and groin not discernable; Ʌ-shaped vomerine tooth series widely open.

FIGURE 4. Dorsal (A) and ventral (B) views of living female Pachytriton wuguanfui sp. nov. (KIZ08755).

A NEW PACHYTRITON FROM CHINA Zootaxa 4085 (2) © 2016 Magnolia Press · 227 FIGURE 5. Dorsal (A) and ventral (B) views of the holotype of Pachytriton wuguanfui sp. nov. (KIZ08758) in preservation. The scale shows 20 mm.

FIGURE 6. Ventral views of (A) forelimb and (B) hindlimb of the holotype of Pachytriton wuguanfui sp. nov. (KIZ08758) in preservation.

228 · Zootaxa 4085 (2) © 2016 Magnolia Press YUAN ET AL. Description of the holotype. This specimen is a moderately sized newt (TTL = 155.7 mm). Head oval and flat, much longer than wide (HL/HW = 1.24). Snout truncate, projects slightly beyond lower jaw. Nostril small, nearly to the tip of snout. Eyes small and do not protuberate in dorsal view. Labial fold well developed on upper jaw. Gular folds weak. Parotoid gland prominent and protuberant. Tongue pad elliptical, adheres to mouth floor. Vomerine tooth series Ʌ-shaped. Dorsal vertebral groove conspicuous along the dorsal midline. Skin smooth. Costal grooves between axilla and groin lacking. Limbs very short (AL/SVL = 0.18, PL/SVL = 0.21). The forelimb is slightly shorter than the hindlimb (AL/PL = 0.85); tips of forelimbs and hindlimbs are well separated when adpressed to body flank. Fingers and toes moderately webbed on the sides (so digits appear flat) and without palmar tubercles. The relative finger lengths are 1 < 4 < 2 < 3 and relative toe lengths are 1 < 5 < 2 < 4 < 3, the fifth toes are very short. Dorsal caudal fin evident extends from tail-base to tail-tip. Ventral caudal fin prominent on posterior half of tail. Tail-tip rounded. Cloaca short slit, small and not protuberant. Color in life. In life, dorsal color uniform dark brown; venter and chin brown and scattered with large, irregular, orange blotches; cloaca, underside of limbs and tail also orange. Tips of digits appear orange red when viewed from above. Color in preservative. In preservation, dark brown coloration turns to grey black; orange coloration fades to milky white. Variation. The cloaca in males is larger and more swollen than that in females, with papillae on the cloacal wall. Sexual dimorphism in body size could not be determined because only two well-preserved adult males. Ventral coloration varies greatly from a large amount of orange, irregular blotches (KIZ08756–08759, KIZ021705– 06, 021708, 021712, 021832, 021833, 021835) to only a few small orange spots (KIZ08755, 021711, 021834). Etymology. The specific epithet wuguanfui is a patronym honouring Guanfu Wu (Chengdu Institute of Biology, Chinese Academy of Sciences), a prominent Chinese herpetologist and educator, for his great contribution to the taxonomy, karyology, and osteology of amphibians and reptiles in China.

FIGURE 7. Habitat at the type locality of Pachytriton wuguanfui sp. nov., Mt. Gupo, Hezhou, Guangxi, China.

A NEW PACHYTRITON FROM CHINA Zootaxa 4085 (2) © 2016 Magnolia Press · 229 FIGURE 8. Vomerine tooth morphology in adult (A) Pachytriton wuguanfui sp. nov. (KIZ021834) and (B) Pachytriton inexpectatus (KIZ014508, Mt. Maoer, Guangxi). The scale shows 1.7 mm.

Habitat and distribution. This species inhabits small montane streams in broadleaf forests. Streams inhabited by these newts are ca. 2 meters wide and 0.5–1 meters in depth; slope is steep, with fast flowing clean and cold water. Many large boulders are scattered in or around the streams. Stream substrates include gravels, scattered small rocks, leaves and sands. Frogs in the genera Megophrys, Amolops and Quasipaa were also found in these habitats. Newts were found along the stream at night. We did not encounter the species during the day. Pachytriton wuguanfui sp. nov. were found at Mt. Jiuwei in Hunan and Mt. Gupo in Guangxi and it likely occurs in nearby mountains between the distribution of P. inexpectatus in the west and P. xanthospilos in the east. Comparisons. Pachytriton wuguanfui sp. nov. described here distinctly differs from P. archospotus, P. brevipes, and a few populations of P. granulosus by the lack of black spots on the whole body (vs. presence of black spots in these species). It differs from P. changi and P. xanthospilos by lacking orange dots on the dorsolateral side (vs. presence of orange spots or blotches that extend ribbon-like along the dorsolateral sides of the body in both species) and from P. moi by having orange blotches on venter (vs. lack of orange blotches on venter in P. moi). It differs from P. inexpectatus, P. granulosus and P. feii by lacking costal grooves between axilla and groin (vs. all these three species present costal grooves). The anterior end of the vomerine tooth series is also more widely open in the new species than P. inexpectatus (Fig. 8). Finger and toe tips in life appear orange on the dorsal side in P. wuguanfui sp. nov., which distinguishes the new species from P. feii (digit tips appear black in life).

Acknowledgements

We thank Limin Ding and local conservation departments for their help in the field. We greatly thank Yunke Wu, Jodi J. L. Rowley, Theodore J. Papenfuss and Nikolay A. Poyarkov for the invaluable comments on the manuscript. This work was supported by grants of the Ministry of Science and Technology of China (MOST 2011FY120200), and the Animal Branch of the Germplasm Bank of Wild Species of Chinese Academy of Sciences (the Large Research Infrastructure Funding).

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