﻿A new species of Hemiphyllodactylus (Squamata, Gekkonidae) from southwest Yunnan, China

﻿Abstract A new species of gekkonid, Hemiphyllodactylusgengmaensissp. nov., is described based on six specimens from Gengma Dai and Wa Autonomous County, Yunnan, China. The new species can be distinguished from its congeners by a significant genetic divergence of greater than 9.7% in the mitochondrial ND2 gene and a combination of the following characters: a maximum SVL of 43.24mm; 8 or 9 chin scales; six circumnasal scales; 2 or 3 internasal scales; 9–11 supralabial scales; 8 or 9 infralabial scales; 11–18 dorsal scales; 8–10 ventral scales; a manual lamellar formula of 5–5–5–4 or 5–6–5–4 and a pedal lamellar formula of 5–5–6–5; 20–25 precloacal and femoral pore-bearing scales contiguous in males; dark postorbital stripes or striping on body; dark dorsal transverse blotches present; and a brown postsacral mark bearing anteriorly projecting arms. The discovery of this new species brings the number of Hemiphyllodactylus species in China to 15.

During our herpetological survey in Banxing Village, Gengma Dai and Wa Autonomous County, Yunnan, China, we collected six specimens belonging to the genus Hemiphyllodactylus.These specimens are distinguished from known species of Hemiphyllodactylus based on molecular and morphological data.Therefore, we describe them as a new species below.

Sampling
Six specimens were collected from in Banxing Village, Gengma Dai and Wa Autonomous County of Yunnan Province in China on 15 May 2014 (Fig. 1).All specimens were preserved in 80% ethanol, and their muscle and liver tissues were preserved in 95% ethanol.Specimens were deposited in Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS).

Molecular data and phylogenetic analyses
We used Trelief Hi-Pure Animal Genomic DNA Kit for genomic DNA extraction following the manufacturer's protocol (https://www.tsingke.com.cn).We amplified and sequenced the complete mitochondrial NADH dehydrogenase subunit 2 gene (ND2), totaling 1,038 bp useing the primers L4437b and H5934 (Macey et al. 1997).The protocol for polymerase chain reaction (PCR) amplifications followed Agung et al. (2021).Genomic DNA extraction, PCR processes, and sequencing were executed at Beijing Tsingke Biotechnology Co., Ltd.All specimen sequences have been deposited in GenBank, with accession numbers from PP540021 to PP540025 .A total of 47 ND2 sequences from GenBank, containing 2 ND2 sequences of outgroup taxa (Hemiphyllodactylus harterti Werner, 1900) and 45 sequences of extant Hemiphyllodactylus species, was downloaded; these with our five new sequences are listed in Table 1.Sequences were assembled and manual proofread in SeqMan (DNASTAR, Inc., Madison, WI, USA), then aligned using Clustal W (Thompson et al. 1994) implemented in MEGA 7 (Kumar et al. 2016).For phylogenetic relationships analysis, we considered maximum likelihood (ML) and Bayesian inference (BI) using IQ-TREE v. 2.2.0 (Nguyen et al. 2015) and MrBayes v. 3.2.7a(Ronquist et al. 2012) in the Phylosuite application (Zhang D et al. 2020;Xiang et al. 2023), respectively.After alignment, we used Gblock 0.91b (Talavera and Castresana 2007) to remove misaligned positions.Mod-elFinder v. 2.2.0 (Kalyaanamoorthy et al. 2017) was used to select the best-fitting model of evolution based on the Bayesian Information Criterion (BIC).A maximum-likelihood (ML) analysis was conducted using TPM+F+G4 as the best-fit substitution model for codon position one, TPM+F+G4 for position two, and TIM+F+G4 for position three.We applied 1,000 bootstrap pseudoreplicates with the ultrafast bootstrap approximation algorithm (UFBoot) (Agung et al. 2021), where nodes having values 95 and above were considered highly supported (Minh et al. 2013).A Bayesian-inference (BI) analysis was conducted using GTR+I+G+F model following the methods by Agung et al. (2021), except that instead of discarding 10% of the trees, we discarded the first 25% of the sampled as burn-in.Nodes with Bayesian posterior probabilities (BPP) of 0.95 and above were considered highly supported (Huelsenbeck et al. 2001;Wilcox et al. 2002).Uncorrected pairwise divergences were calculated using MEGA 7 (Kumar et al. 2016).

Morphological data
Mensural data were taken with a digital calipers to the nearest 0.01 mm under a dissecting microscope (Jiangnan XTB-01) following Zug (2010), Grismer et al. (2013), and Agung et al. (2021): snout-vent length (SVL), taken from the tip of the snout to the vent; tail length (TL), taken from the vent to the tip of the tail; trunk length (TrunkL), taken from the posterior margin of the forelimb at its insertion point on the body to the anterior margin of the hind limb at its insertion point on the body; head length (HL), measured from the posterior margin of the retroarticular process of the lower jaw to the tip of the snout; head width (HW), measured at the angle of the jaws; eye diameter (ED), the greatest horizontal diameter of the eyeball; snout-eye length (SnEye), measured from anterior-most margin of the eyeball to the tip of snout; nares-eye length (NarEye), measured from the anterior margin of the eyeball to the posterior margin of the external nares; and snout width (SnW), measured between the external nares.
For meristic characters and color pattern, we measured and evaluated them according to the methods of Agung et al. (2021).

Results
Our results of ML and BI analyses were similar to those obtained by Agung et al. (2021): the specimens from Gengma County were recovered as members  2).Furthermore, the new species also could be distinguished from its congeners by body proportions, CN, VS, Lamellar formulae hands and feet II-V, SL1T and total number of femoroprecloacal pores.Therefore, we describe them here as a new species.Diagnosis.Hemiphyllodactylus gengmaensis sp.nov.can be distinguished from its congeners by the combination of the following characters: maximum SVL of 43.24 mm; 8-9 chin scales; enlarged postmentals; 6 circumnasal scales; 2-3 internasal scales; 9-11 supralabial scales; 8-9 infralabial scales; 11-18 dorsal scales; 8-10 ventral scales; a manual lamellar formula of 5-5-5-4 or 5-6-5-4 and a pedal lamellar formula of 5-5-6-5; 20-25 precloacal and femoral pore-bearing scales contiguous in males.dark postorbital stripes or striping on body; dark dorsal transverse blotches; and a brown postsacral mark bearing anteriorly projecting arms.
Description of holotype.Adult female, SVL 38.52 mm; head triangular in dorsal profile, depressed, distinct from neck (HL 10.80 mm; HW 7.36 mm); lores flat; snout short (SnEye 3.94 mm; SnEye/HL 36%), narrow (SnW 1.78 mm; SnW/ HW 24%); eye large (ED 2.12 mm; ED/HL 20%); rostral scale wider than high, bordered posteriorly by two large supranasals and three internasals (IS); nares bordered anteriorly by rostral scale, ventrally by first and second supralabial scale, dorsally by supranasal scale, posteriorly by three postnasals; supralabials square, 10/9 (left/right), tapering from rostral to a point in line with posterior margin of orbit (SL); infralabials square, 9/9 (left/right), tapering from mental to a point in line with posterior margin of orbit (IL); scales on head small, rounded, largest on rostrum; mental triangular, eight chin scales touching internal edges of infralabials from juncture of the second and the third on left and right and mental scale (Chin); scales in gular region rounded, non-overlapping, becoming larger and more ovoid on venter.Robust body type and small, (TrunkL/SVL 45%), dorsoventrally compressed; dorsal body scales small, granular, 18 dorsal scales at midbody contained within one eye diameter; ventral body scales smooth and flat, much larger than dorsal scales, subimbricate, 10 ventral scales at midbody contained within one eye diameter; granular scales on the limbs; finger I is vestigial, clawless, and with rectangular subdigital lamellae, while fingers II-V are well developed; the proximal subdigital lamellae are undivided and rectangular, while the distal subdigital lamellae are divided, angular, U-shaped, except for the terminal lamellae, which are rounded and undivided; the forefoot and hindfoot digital formulae are unidentifiable; white cloacal spur present, one on each side.Tail length (TL/SVL = 73%), with dorsal scales on the tail larger than those on the body and head, but smaller than the subcaudals.The ventral scales are large and flat.
Coloration in ethanol.The dorsal surface of head and body is light brown; dark brown stripes extend from the posterior corner of the eye socket to the neck; the back is covered with irregular, dark-brown stripes that interconnect to form a net-like pattern; the dorsal surfaces of the limbs are brown, with irregular, dark-brown stripes; the tail is brown, with several dark-brown, transverse stripes; the regenerated tail is gray and without transverse stripes; the ventral surfaces of the head and limbs are cream-grey.

A B C
Variation.Variation of mensural and meristic data are presented in Table 3. Dark dorsal transverse blotches on the body of this species are relatively small, with those of two specimens (2014002302, 2014002300) being indistinct and fragmented.Furthermore, females are slightly larger than males.The postsacral mark, bearing anteriorly projecting arms, of one individual ( 2014002300) is indistinct, possibly due to prolonged preservation.
Distribution.This species is currently known to be distributed at the type locality Banxing Village, Gengma Dai and Wa Autonomous County of Yunnan Province in China (Fig. 1).
Natural History.Hemiphyllodactylus gengmaensis sp.nov.was found at an elevation of 664 m a.s.l., around 21:00.The specimens were found on a restaurant's wall, which was rough and with crevices.When illuminated with a flashlight, the animals quickly crawled into the crevices.
Etymology.The scientific name "gengmaensis" is derived from its type locality Gengma Dai and Wa Autonomous County in Yunnan province.We suggest Gengma Slender Gecko in English and "耿马半叶趾虎 (Gěng Mǎ Bàn Yè Zhǐ Hǔ)" in Chinese.
Our study increases the number of recognized species in the Hemiphyllodactylus in China to 15. Apart from H. typus, H. dupanglingensis, and H. hongkongensis, all other species in the genus Hemiphyllodactylus are montane species.Hemiphyllodactylus gengmaensis sp.nov.was found at an elevation of 664 m a.s.l.The discovery of the new species may represent an intermediate elevation type given by the known ranges, suggesting that species in the Hemiphyllodactylus may have a wide distribution range in southern China, spanning elevations from 120 m (Sung et al. 2018) to 2,169 m (unpublished data).Additionally, Grismer et al. (2018b) discovered 12 new gecko species within two weeks in a single study of karsts in Myanmar, with similar climatic and habitat conditions likely to exist in southern China.Moreover, some populations previously considered as H. yunnanensis in China have also been described as new species (Deng et al. 1998;Shi et al. 2011;Che et al. 2020;Zhang B et al. 2020).Therefore, it is possible that there are still numerous undiscovered cryptic species in southern China.

Figure 1 .
Figure 1.Distribution map of the genus Hemiphyllodactylus in Yunnan Province, China.

Figure 2 .
Figure 2. Maximum-likelihood consensus tree based on 1038 bp mitochondrial ND2 gene.Numbers by the nodes indicate ML bootstrap support values.

Figure 3 .
Figure 3. Phylogenetic tree by Bayesian inference based on 1038 bp mitochondrial ND2 gene.Numbers by the nodes indicate posterior probability values of the BI.

Figure 5 .
Figure 5. Paratype (2014002301) of Hemiphyllodactylus gengmaensis sp.nov.A, B lateral views of head, red lines indicate IL and SL, respectively C ventral view, red lines indicate chin scales D ventral view, red lines indicate femoroprecloacal pores.

Table 1 .
List of specimens used for phylogenetic analyses in this study.

Table 2 .
The mean percentage of the uncorrected p-distance among the Hemiphyllodactylus species studied based on mitochondrial ND2 gene fragments.
Hemiphyllodactylus gengmaensis sp.nov.from Yunnan Discussion Our research supports the recognition of the Hemiphyllodactylus gengmaensis sp.nov.as a new species, belonging to clade 3 of Agung et al. (2021).It is sister taxa to H. longlingensis, H. zalonicus, H. changningensis, and H. zhutangxiangensis.Except for H. zalonicus, all species of clade 3 occur in China.Considering the 292 km of unexplored area between H. zalonicus and its closest distance species (H.longlingensis), there may be numerous undescribed species in northern Myanmar and Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, China.The 56 km distance between H. gengmaensis sp.nov.and side ZooKeys 1197: 197-213 (2024), DOI: 10.3897/zookeys.1197.117359Hongxin Zhou et al.: