A new species of the genus Takydromus (Squamata, Lacertidae) from southwestern Guangdong, China

Abstract A new species, Takydromus yunkaiensis J. Wang, Lyu, & Y.Y. Wang, sp. nov. is described based on a series of specimens collected from the Yunkaishan Nature Reserve located in the southern Yunkai Mountains, western Guangdong Province, China. The new species is a sister taxon to T. intermedius with a genetic divergence of 8.0–8.5% in the mitochondrial cytochrome b gene, and differs from all known congeners by a combination of the following morphological characters: (1) body size moderate, SVL 37.8–56.0 mm in males, 42.6–60.8 mm in females; (2) dorsal ground color brown; ventral surface green to yellow-green, but light blue-green on chin and throat, posteriorly green in adult males; (3) dorsolateral lines paired, strikingly yellowish-white bordered by black above and below, invisible or indistinct in juveniles and adult females; (4) flanks of body blackish brown with light brown marks in adult males; (5) presence of four pairs of chin-shields; (6) four supraoculars on each side; (7) presence of a row of supracilary granules that separate supracilaries from supraoculars; (8) two postnasals; (9) enlarged dorsal scales in six longitudinal rows on trunk of body, with strong keel; (10) enlarged ventral scales in six longitudinal rows, strongly keeled in males, smooth but outermost rows weakly keeled in females; (11) enlarged and keeled lateral scales in a row above ventrals; (12) femoral pores 2–3 on each side; (13) subdigital lamellae 20–23 under the fourth finger, 23–30 under the fourth toe; and (14) the first 2–3 subdigital lamellae under the fourth toe divided. The discovery of Takydromus yunkaiensissp. nov. brings the total number of species of this genus to 24, of which nine occur in mainland China.

Previous studies have revealed the very high biodiversity level of the genus Takydromus in southern China, for which the species diversity is just below that of Taiwan Island (Lue and Lin 2008;Wang et al. 2017). During repeated field surveys in the Yunkai Mountains, located in southwestern Guangdong Province (Fig.  1), a number of lacertid specimens were collected that could be assigned to the genus Takydromus by a combination of diagnostic characters defined by Arnold et al. (2007) and Zhao et al. (1999): (1) body slender with an extra-long tail, tail length usually more than two times larger than snout-vent length, (2) dorsal scales enlarged and keeled, ventral scales enlarged, keeled or smooth, (3) scales on flanks small and granular, (4) lateral teeth tricuspid, (5) temporal scales usually keeled, (6) 0-5 femoral pores on each side. Close examination of the external morphology and subsequent molecular analyses revealed that these specimens from Yunkaishan Nature Reserve, Guangdong Province, represented a distinct taxon. They are described below as a new species.

Sampling
Samples sequenced for molecular analyses were obtained from two specimens of the undescribed Takydromus species from Yunkaishan Nature Reserve, Guangdong Province; the paratype specimen (SYS r001292) of T. albomaculosus; two specimens of T. amurensis; four specimens of T. intermedius including a topotypic specimen (SYS r001602) from Mt. Emei, Sichuan; four specimens of T. kuehnei; three specimens of T. septentrionalis; two specimens of T. sexlineatus; one specimen of T. sylvaticus; and two specimens of T. wolteri, all freshly collected from China. Additional 14 sequences of T. dorsalis, T. formosanus, T. hsuehshanensis, T. sauteri, T. smaragdinus, T. stejnegeri, T. tachydromoides, and T. toyamai were obtained from GenBank and three sequences of Eremias persica Blanford, 1875, E. strauchi Kessler, 1878, and E. velox (Pallas, 1771 also from GenBank were used as the out-groups. Details of samples sequenced for mitochondrial cytochrome b gene and their associated GenBank accession numbers are listed in Table 1. All specimens were fixed in 10 % buffered formalin and later transferred to 70% ethanol for preservation, and deposited at the Museum of Biology, Sun Yat-sen University (SYS); liver tissue samples were separately preserved in 95% ethanol for molecular studies.

DNA Extraction, PCR and sequencing
DNA was extracted from liver tissue using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). The fragment of mitochondrial cytochrome b gene was PCR amplified and sequenced using the primers L14919 5'-AACCACCGTT-GTTATTCAACT-3' and H16064 5'-CTTTGGTTTACAAGAACAATGCTTTA-3' (Burbrink et al. 2000). PCR amplifications were performed in a 20 µL reaction volume with the following cycling conditions: an initial denaturing step at 95 °C for five min.; 35 cycles of denaturing at 95 °C for 40 s, annealing at 53 °C for 40 s, and extending at 72 °C for one min., and a final extending step of 72 °C for 10 min. PCR products were purified with spin columns. The purified products were sequenced with both forward and reverse primers using a BigDye Terminator Cycle Sequencing Kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's guidelines. The products were sequenced on an ABI Prism 3730 automated DNA sequencer (Shanghai Majorbio Bio-pharm Technology Co., Ltd).

Phylogenetic analyses
Sequence alignments were first conducted using Clustal X 2.0 (Thompson et al. 1997), with default parameters and the alignment being checked and manually revised, if necessary. The data were tested in jmodeltest v2.1.2 with Akaike and Bayesian information criteria, resulting in the best-fitting nucleotide substitution models of GTR + I + G. Phylogenetic relationships were reconstructed using Maximum Likelihood (ML) as implemented in RaxmlGUI 1.3 (Silvestro and Michalak 2012), and Bayesian Inference (BI) using MrBayes 3.12 (Ronquist et al. 2012). For ML analysis, we used the rapid-bootstrapping algorithm (1000 replicates) with the thorough ML search option. Bootstrap values less than 60 were collapsed. For BI analysis, two independent runs with four Markov Chain Monte Carlo simulations were performed for ten million iterations and sampled every 1000 th iteration. The first 25 % of samples were discarded as burn-in. Convergence of the Markov Chain Monte Carlo simulations was assessed using Tracer v.1.4 (http://tree. bio.ed.ac.uk/software/tracer/). We also calculated pairwise sequence divergence based on uncorrected p-distance implemented in MEGA 6 (Tamura et al. 2013).

Morphometrics
Measurements of all specimens were taken with a digital caliper to the nearest 0.1 mm. Abbreviations of measurements followed the convention of Lue and Lin (2008): ALL arm-leg length (from insertion of the forelimb to insertion of hindlimb); HH head height (measured at the highest point); HL head length (from tip of snout to anterior margin of ear opening with claw); HLL hindlimb length (from groin to tip of fourth toe); HW head width (measured at the broadest point); LTL length of fourth toe excluding claw; RUL radius-ulna length; SAL snout-arm length (from tip of snout to anterior insertion margin of forelimb); SEL snout-eye length (from tip of snout to anterior margin of eye); SKL skull length (from tip of snout to posterior margin of occipital); SVL snout-vent length (from tip of snout to anterior margin of cloaca); TaL tail length (from cloaca to tip of tail); TFL tibia-fibula length.
Moreover, 20 external morphological characters were examined from the specimens listed in Appendix 1. Modified abbreviations of these characters followed Arnold (1997), Lue and Lin (2008) and Wang et al. (2017) as follows:

Results
BI and ML phylogenetic trees were constructed based on DNA sequences of the mitochondrial cytochrome b gene with a total length of 1074 -bp. The two analyses resulted in essentially identical topologies and are integrated in Figure 2, in which the Bayesian posterior probabilities (BPP) > 0.75 and the bootstrap supports (BS) for ML analysis > 60 were retained. The specimens from Yunkaishan Nature Reserve grouped in a strongly supported clade (BPP 1.00 and BS 98) with small divergence (p-distance 0.2 %), forming the sister taxon to Takydromus intermedius with strong support (BPP 1.00 and BS 92) and significant divergences (p-distance 8.0-8.5 %), and then to T. dorsalis, T. sylvaticus, and T. albomaculosus (BPP 1.00 and BS 95), indicating that the population from Yunkaishan Nature Reserve represents a separate evolutionary lineage.
Morphologically, the unnamed specimens can be clearly distinguished from its congeners by the following characters: (1) body size moderate, SVL 37.8-56.0 mm in males, 42.6-60.8 mm in females; (2) dorsal ground color brown; ventral surface green to yellow-green, but light blue-green on chin and throat, posteriorly green in adult males; (3) dorsolateral lines paired, strikingly yellowish-white bordered by black above and below, invisible or indistinct in juveniles and adult females; (4) flanks of body blackish brown with light brown marks in adult males; (5) the presence of four pairs of chin-shields; (6) four supraoculars on each side; (7) presence of a row of supracilary granules that separate supracilaries from supraoculars; (8) two postnasals; (9) enlarged dorsal scales in six longitudinal rows on trunk of body, with strong keel; (10) enlarged ventral scales in six longitudinal rows, strongly keeled in males, smooth but outermost rows weakly keeled in females; (11) enlarged and keeled lateral scales in a row above ventrals; (12) femoral pores 2-3 on each side; (13) subdigital lamellae 20-23 under the fourth finger, 23-30 under the fourth toe; and (14) the first 2-3 subdigital lamellae under the fourth toe divided.
Based on the comprehensive evidence of molecular and morphological analyses, we hereby describe these specimens from Yunkaishan Nature Reserve as a new species, Takydromus yunkaiensis sp. nov. Now, the genus Takydromus contains 24 species, nine of which are recorded from mainland China. Etymology. The specific epithet, yunkaiensis, is in reference to the type locality of the new species. We propose the standard name "Yunkai grass lizard" and the Chinese name "Yun Kai Cao Xi (云开草蜥)".
(1) body size moderate, SVL 37.8-56.0 mm in males, 42.6-60.8 mm in females; (2) dorsal ground color brown; ventral surface green to yellow-green, but light blue-green on ventral head and neck, posteriorly green in adult males; (3) dorsolateral lines paired, strikingly yellowish-white bordered by black above and below, invisible or indistinct i n juveniles and adult females; (4) flanks of body blackish brown with light brown marks in adult males; (5) the presence of four pairs of chin-shields; (6) four supraoculars on each side; (7) presence of a row of supracilary granules that separate supracilaries from supraoculars; (8) two postnasals; (9) enlarged dorsal scales with strong keel in six longitudinal rows on trunk of body; (10) enlarged ventral scales in six longitudinal rows, strongly keeled in males, smooth but outermost rows weakly keeled in fe-   Comparisons. In this study we only compare the new species with the other 22 recognized species, excluding Takydromus haughtonianus, which is currently an uncertain species and poorly known (Jerdan 1870; Arnold 1997). Measurements, body proportions, and scale counts of the new species are listed in Tables 3 and 4; comparative data of the new species and nine other recognized members of the genus Takydromus occurring on the Chinese mainland are listed in Tables 5 and 6.
In our phylogenetic tree, Takydromus yunkaiensis sp. nov. is a sister taxon to T. intermedius, from which it differs by having two postnasals (only one in T. intermedius), having a pair of strikingly yellowish-white dorsolateral lines in adult males (vs. always absent or indistinct in T. intermedius), flanks of body blackish brown with light brown spots in adult males (vs. pure brown without spots in T. intermedius), ADSR 9-10, PDSR 7 (vs. ADSR 6-8, PDSR 6 in T. intermedius).
Description of holotype. Adult male. Body size slightly small, SVL 43.0 mm; trunk of body short, ALL 19.7 mm, 46 % of SVL; head slightly long, HL 11.1, HW 6.7 mm, HH 5.2 mm, HL 26 % of SVL; skull length larger than head length, SKL 11.9 mm; snout moderately long, SEL in 5.0 mm, SEL 45 % of HL. Rostral large, pentagonal, visible in dorsal view, in contact with the first supralabials posteriorly on both sides, and supranasals dorsolaterally; nostril surrounded by a supranasal, two postnasals and the first supralabial on each side; one supranasal on each side, large, in contact with each other dorso-medially, separating rostral from frontonasal, and in contact with the upper postnasal posteriorly, not in contact with the anterior loreal; postnasals two, both in contact with the anterior loreal posteriorly, the upper one in contact with supranasal dorsolaterally, with frontonasal dorsally, the lower one in contact with the first supralabial ventrally; supralabials six on each side, the fifth one largest, under the eye; two loreals on each side, anterior one smaller than posterior one; posterior loreal in contact with anteriormost supraocular and anteriormost supraciliary scale posteriorly; four supraoculars on each side, the posteriormost one much smaller than others; supraciliaries four on left side, the second one longest; supraciliaries two on right side, the first one longest; supracilary granules arranged in a row, separated supracilaries from supraoculars; frontonasal large, smooth, hexagonal, separated from frontal by a pair of prefrontals; prefrontals two, weakly keeled, in contact with each other medially, with frontal and anterior two supraoculars posteriorly, with loreals laterally, respectively; a single frontal hexagonal, weakly keeled, in contact with second and third supraoculars laterally, with frontoparietals posteriorly; frontoparietals two, pentagonal, in contact with each other medially, with parietal and interparietal posteriorly, respectively; interparietal diamond, surrounded by two frontoparietals, two parietals and the single occipital; parietal pit located in the central of interparietal, distinctly visible; parietals two, large, weakly keeled, slightly in contact with each other medially; a single occipital between two parietals; temporal scales granular, slightly keeled; supratemporals three on each side, keeled, anteriormost one largest, longer than total length of posterior two; mental large, semielliptical; infralabials six on each side; four pairs of chin-shields, anterior two pairs in contact with each other medially, posterior two pairs separated from each other by gular scales; following gular scales gradually increasing in size, keeled, and become imbricated; enlarged, strongly keeled median gular scales extending anteriorly to the line joined posterior edges of ears; collars clear, composed of scales in ten rows pointed backwards, and forming a free serration; enlarged, imbricated dorsal scales on body with strong keel oriented posteriorly that form continuous ridges, extending anteriorly beyond forelimbs on to the nape, in nine rows in position of forelimbs, seven rows in position of hindlimbs; seven rows at mid-body, including a much smaller and discontinuous central row; longitudinal dorsal scales (LDSN) 47; ventrals in six rows, imbricate, strongly keeled and pointed posteriorly; enlarged and keeled lateral scales in a row above ventrals; longitudinal ventral scales (VN) 24; small flat and granular scales in a transverse row on flank at mid-body (SSRF) 14 on left side and 15 on right side, including a row of scales (enlarged and keeled, shorter than ventrals) adjoining the ventrals; four rows of scales on lower flanks reduced, flattened, keeled; nine rows of small granular scales on upper flanks on left side and ten on right side; a discontinuous row of scales adjoining outermost dorsal scale row reduced, flattened, keeled; a total of 42 scales (MBSR) in a transverse row in mid-body region; a single precloacal entire, enlarged, surrounded by eight continuous moderately sized scales anteriorly and laterally; three femoral pores on each side.
Tail original, TaL 111.3 mm, TaL/SVL ratio 259%, SVL/TaL ratio 39 %, with strongly keeled scales in 15 rows at base (fifth subcaudal scale), in 13 rows in position of the 13 th to 15 th subcaudal scales (CSR); paired vertebral series of large scales on tail extending on to hind body.
Coloration of holotype in life. Dorsal surface of head, body, limbs, and tail bright brown, with a pair of strikingly yellowish-white dorsolateral lines bordered by black above and below, each beginning from the posterior margin of the most last supratemporal, running along outermost dorsal scale row, posteriorly extending to the forepart of the tail; flanks of body blackish brown with light brown marks; a pair of orange ventrolateral lines beginning from axilla, running along lower part of flanks, posteriorly extending to the groin; labial series, mental, chin-shields, granular scales on throat, collars light blue-green, posteriorly yellowish green from chest, venter, until to subcaudal region; ventral surface of limbs brown, tinged with green.
Coloration of holotype in preservative. Dorsal surface of head, body, limbs and tail brown; labial series, mental, chin-shields, granular scales on throat, ventral surface of body and tail pale blue; mottles on flanks blurry, color of mottles on flanks faded; ventral surface of limbs beige; dorsolateral stripes greyish white with black-brown edges at the inner sides; color of ventrolateral stripes faded, greyish white.
Variations and sexual dimorphism. Measurements, body proportions, and scale counts of the type series of Takydromus yunkaiensis sp. nov. are listed in Tables 2 and 4.
In the holotype SYS r001580, there are four supraciliaries on left side and two on right side, the first one longest on right side, the second supraciliary longest on left side (vs. four supraciliaries on each side, and the second one longest in the paratypes SYS r001439, 1440,1442,1513,1514,1581,1684,1901; three supraciliaries on each sides, and the second one longest in the paratype SYS r001507); prefrontal in contact with the anterior two supraoculars posteriorly in the holotype (vs. prefrontal only in contact with the first supraocular posteriorly on the right side of the paratype SYS r001439); three pairs of femoral pores in the holotype (vs. only two pairs present in the paratypes SYS r001513, 1514); tail relatively longer in two of the female paratypes, TaL/SVL 2.97 in SYS r001581 and 3.02 in SYS r001901 (vs. TaL/SVL 2.59 in the holotype).
Takydromus yunkaiensis sp. nov. exhibits noticeable sexual dimorphism: (1) enlarged ventral scales strongly keeled in males (vs. smooth but outermost rows weakly keeled in females); (2) dorsolateral lines strikingly yellowish-white bordered by black above and below (vs. invisible or indistinct in adult females, also in juveniles); (3) a pair of orange ventrolateral lines present on lower flanks (vs. invisible in females, also in juveniles); (4) flanks of body blackish brown with light brown marks in adult males (vs. absent in females).

Distribution and habits.
Currently, Takydromus yunkaiensis sp. nov. is known only from its type locality of Dawuling Forestry Station, adjacent Xianrendong Scenic Area located in the southern Yunkai Mountains in western Guangdong Province, China (Fig. 1).
The diurnal species was found to be very active in daytime and rapidly escapes when disturbed, and is usually observed resting on fern leaves at night. The surrounding environment was covered by well-preserved montane evergreen broad-leaved forest or mixed forest (Fig. 5) at altitudes of 900-1600 m.

Discussion
The description of Takydromus yunkaiensis sp. nov. brings the total number of species of this genus to 24, nine of which occur in mainland China. As noted, six species were recorded from Guangdong Province: T. albomaculosus, T. kuehnei, T. septentrionalis, T. sexlineatus, T. sylvaticus, and Takydromus yunkaiensis sp. nov., which further support the very high biodiversity level of the genus in southern China (Zhao et al. 1999;Lue and Lin 2008;Yang and Wang 2010;Wang et al. 2017).
Most of the early descriptions of Takydromus species only listed relatively limited diagnostic characteristics, resulting in considerable challenges in field identification of the species, and causing ambiguities in taxonomy. Moreover, in recent years, a number of new or cryptic species were discovered and described from southern mainland China and Taiwan Island (Lin et al. 2002;Lue and Lin 2008;Wang et al. 2017). These discoveries confirm the substantially underestimated species diversity within the tropical genus Takydromus, and more field research is required to increase the knowledge of the diversity.
Located in the western Guangdong Province, the Yunkai Mountains have gradually been recognized for its unique biodiversity. During herpetological surveys during the last several years, we have discovered a number of new species including some cryptic species, as well as providing new regional records of amphibians and reptiles (Yang et al. 2011;Lyu et al. 2018;Wang et al. 2018a;Wang et al. 2018b;Lyu et al. 2019), suggesting that future herpetological exploration will likely continue to yield new discoveries from the region.