A New Species of Hemiphyllodactylus Bleeker,1860 (Reptilia:Squamata) from Hunan Province,China

Bing ZHANG ,Tianyu QIAN ,Xiangjin JIANG ,Bo CAI ,Xuejian DENG and Daode YANG

1 Institute of Wildlife Conservation,Central South University of Forestry and Technology,Changsha 410004,Hunan,China

2 Yueyan Branch of Hunan Dupangling National Nature Reserve Bureau,Yongzhou 425300,Hunan,China

3 Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,Sichuan,China

4 Hunan Normal University,Changsha 410081,Hunan,China

Abstract A new species of gekkonid,Hemiphyllodactylus dupanglingensis sp.nov.,is described based on six specimens from Hunan Province,China.The new species is phylogenetically close to H.zugi,H.hongkongensis,H.dushanensis,and H.huishuiensis,and can be distinguished from its congeners by the following combination of characters:9-11 chin scales; postmental scales enlarged; 9-12 infralabials,11-14 supralabials; 14-16 dorsal scales and 10-11 ventral scales longitudinally arranged at the midbody contained with one eye diameter; a manual lamellar formula of 4-4-5-4 or 4-5-5-4 and a pedal lamellar formula of 4-5-5-5 or 5-6-6-4; 21-25 continuous precloacal and femoral pores in males.

Keywords Hemiphyllodactylus dupanglingensis sp.nov.,molecular phylogeny,morphology,slender gecko,taxonomy

1.Introduction

The genusHemiphyllodactylusBleeker,1860 consists of 35 species of small geckos distributed in South and Southeast Asia and the Indo-Pacific region (Zug,2010;Agarwalet al.,2019; Eliadeset al.,2019).Morphological similarity makes species identification difficult.Before 2013,only nine species and three subspecies had been recognized mainly based on morphological characters:H.typusBleeker,1860,H.aurantiacusBeddome,1870,H.larutensisBoulenger,1900,H.harteriWerner,1900,H.yunnanensisBoulenger,1903,H.insularisTaylor,1918,H.margarethaeBrongersma,1931,H.ganoklonisZug,2010,andH.titiwangsaensisZug,2010,and three subspeciesH.yunnanensis dushanensisZhou and Liu,1981,H.y.jinpingensisZhou and Liu,1981,andH.y.longlingensisZhou and Liu,1981.Integrative taxonomy,using both molecular phylogenetic and morphological analyses (Grismeret al.,2013),has revealed a high level of species diversity.The subspeciesH.y.dushanensis,H.y.jinpingensis,andH.y.longlingensishave been raised to full species status,and occurrences of at least nine possibly undescribed species in Indochina and Southeast Asian Islands were suggested.Agarwalet al.(2019) reported three additional undescribed clades in this genus from peninsular India in South Asia.

Most species ofHemiphyllodactylusare small and nocturnal with cryptic body colora tion a nd low population density.As such,researchers seldom encounter them in the field (Zug,2010; Grismeret al.,2013; Agarwalet al.,2019; Eliadeset al.,2019).During the wild terrestrial vertebrate surveys in Dupangling,Dao County,Hunan Province,China,in 1998,three specimens ofHemiphyllodactylusfrom Kongshuyan in Dupangling were accidentally collected.After a prelimina ry morphological description,these specimens were tentatively assigned to the speciesH.yunnanensis(Denget al.,1998).H.yunnanensisis widely distributed ranging through China,Myanmar,Thailand,Laos,and Vietnam(Zug,2010).Some species ofHemiphyllodactylus,once considered asH.yunnanensis,have now been described as new species by using an integrative taxonomic approach.This genus has high species diversity,localized population distribution,and little movement between populations.Due to its isolation of other collection localities,the taxonomic status of the Dupangling population from Hunan Province was reconsidered.However,we could not obtain access to the preserved material of the three specimens and were uncertain of their taxonomic status.During herpetological surveys in Dupangling,Hunan Province,from 2018 to 2019,we made six collections ofHemiphyllodactylusin the same locality reported by Denget al.(1998).Based on close examination,comparison with its congeners,and a phylogenetic analysis,we determined that the geckos from Dupangling are an undescribed species.The phylogeny results show that the population ofHemiphyllodactylusfrom Dupangling is a sister group ofH.zugi,H.hongkongensis,H.dushanensis,andH.huishuiensis.

2.Materials and Methods

2.1.Phylogenetic AnalysisGenomic DNA was extracted from 95% ethanol-preserved liver tissues,by using a TSINGKE (https://www.tsingke.net)DNA extraction kit.The mitochondrial ND2 (NADH dehydrogenase subunit 2) gene from three samples was amplified by PCR using primer pairs ND2f101A(5’-CAACAGAAGCCACAACAAAAT-3’) (modified from ND2f101; Nguyenet al.2013) a nd HemiR(5’-GAAGAAGAGGCTTGGKAGGCT-3’) (Nguyenet al.,2013).The amplifications program followed the sequence of 98°C for 2 min; 35 cycles of 98°C for 10 s,55°C for 10 s,72°C for 10 s; and a final extending step at 72°C for 2 min.PCR purification and sequencing were performed by Beijing Tsingke Biological Technology Co.Ltd.We downloaded sequences of the orthologous gene ofHemiphyllodactylusand an outgroup available from GenBank,following the recent phylogenetic and taxonomic studies ofHemiphyllodactylus(Grismeret al.,2017; Sukprasertet al.,2018; Sunget al.,2018; Agarwalet al.,2019; Eliadeset al.,2019) (Table S1).DNA sequences were aligned by the Clustal W algorithm with default parameters (Thompsonet al.,1997) and trimmed with gaps partially deleted in MEGA 6 (Tamuraet al.,2013).The partitions were tested in jmodeltest v2.1.2 with Akaike and Bayesian information criteria,all resulting in the best-fitting nucleotide substitution models of GTR+I+G.Sequenced data were analyzed using Bayesian inference (BI) in MrBayes 3.2.4 (Ronquistet al.,2012),and maximum likelihood (ML) in RaxmlGUI 1.3 (Silvestro and Michalak,2012).Two independent runs were conducted in a BI analysis,each of which was performed for 10 000 000 generations and sampled every 1000 generations with the first 25% samples discarded as burn-in,resulting in a potential scale reduction factor (PSRF) of ＜ 0.005.In ML analysis,the bootstrap consensus tree inferred from 1000 replicates was utilized to represent the evolutionary history of the taxa analyzed.Genetic distances between and within species were calculated in MEGA 6 using the uncorrectedp-distance model base on ND2 gene.

2.2.Morphological AnalysisMeasurements were taken for the specimens from Dupangling by a digital caliper (0.01 mm,nearest to 0.1 mm),and meristic traits were counted under a binocular scope (Leica Z16 APO/DFC550) (Table 1).High-quality images of the holotype were taken with a Leica Z16 APO/DFC550.The mensural and meristic characters and abbreviations followed Zug(2010),Grismeret al.(2013) and Grismeret al.(2018).These were eye diameter (EyeD),the maximum horizontal diameter of the exposed eyeball; head length (HeadL),the distance from the posterior margin of the retroarticular process of the lower jaw to the tip of the snout; head width(HeadW),measured at the angle of the jaws; nares-eye length (NarEye),measured from the anterior margin of the eyeball to the posterior margin of the external nares;snout-eye length (SnEye),measured from the anteriormost margin of the eyeball to the tip of snout; internarial width (SnW),measured between the nares across the rostrum; snout-vent length (SVL),taken from the tip of snout to the vent; tail length (TailL),taken from the vent to the tip of the tai,original or regenerated; and trunk length (TrunkL),taken from the posterior margin of the forelimb at its insertion point on the body to the anterior margin of the hindlimb at its insertion point on the body.The meristic characters included chin scales (Chin),the number of scales touching internal edge of infralabials and mental from juncture of 2nd and 3rd infralabials on left and right; circumnasal scales (CircNa),the number of scales contacting the nares; scales between nasals (SnS),the number of scales touching rostral scale between left and right supranasals; dorsal scales (Dorsal),the number of scales longitudinally at midbody on dorsum containedwithin one EyeD; ventral scales (Ventral),the number of scales longitudinally at midbody on ventral contained with one EyeD; infralabial scales (Inflab) and supralabial scales (Suplab),the number of enlarged scales from rostral to top of mouth curve,usually equivalent to end of orbit; femoral and precloacal pores series (Pore),the total number of precloacal and femoral pores in males; the number of subdigital lamellae wider than long on the first finger (1FingLm) and toe (1ToeLm); lamellar formula,determined as the number of U-shaped subdigital lamellae on the digital pads on digits 2-5 of the hands (2-5FingLm)and feet (2-5ToeLm); and the number of cloacal spurs(CloacS).The coloration characters include the presence/absence of dark pigmentation in the gonadal tracts and caecum; a dark postorbital stripe extending to at least the neck; and a linear series of white postorbital or trunk spots,light-colored anteriorly projecting arms of the postsacral marking.

Table 1 Measurements and scale counts of Hemiphyllodactylus dupanglingensis sp.nov.

We examined morphological characters of the specimens from Dupangling and compared them with those of the known species ofHemiphyllodactylusbased on relevant literature includingH.arakuensis(Agarwalet al.,2019),H.aurantiacus(Zug,2010; Agarwalet al.,2019),H.banaensis(Ngoet al.,2014),H.bintik(Grismeret al.,2015),H.changningensis(Guoet al.,2015),H.chiangmaiensis(Grismeret al.,2014b),H.cicak(Coboset al.,2016),H.dushanensis(Zhouet al.,1981; Grismeret al.,2013),H.engganoensis(Grismeret al.,2014a),H.flaviventris(Sukprasertet al.,2018),H.ganoklonis(Zug,2010),H.harterti(Zug,2010; Grismeret al.,2013),H.hongkongensis(Sunget al.,2018),H.huishuiensis(Yanet al.,2016),H.indosobrinus(Eliadeset al.,2019),H.insularis(Zug,2010;Grismeret al.,2013),H.jinpingensis(Zhouet al.,1981;Grismeret al.,2013),H.jnana(Agarwalet al.,2019),H.khlonglanensis(Sukprasertet al.,2018),H.kiziriani(Nguyenet al.,2014),H.kolliensis(Agarwalet al.,2019),H.larutensis(Zug,2010; Grismeret al.,2013),H.linnwayensis(Grismeret al.,2017),H.longlingensis(Zhouet al.,1981; Grismeret al.,2013),H.margarethae(Zug,2010; Grismeret al.,2013),H.montawaensis(Grismeret al.,2017),H.serpispecus(Eliadeset al.,2019),H.tehtarik(Zug,2010; Grismeret al.,2013),H.titiwangsaensis(Zug,2010; Grismeret al.,2013),H.tonywhitteni(Grismeret al.,2017),H.typus(Zug,2010; Grismeret al.,2013),H.uga(Grismeret al.,2018),H.yunnanensis(Zhouet al.,1981; Grismeret al.,2013),H.ywanganensis(Grismeret al.,2018),andH.zugi(Nguyenet al.,2013).

3.Results

Phylogenetic analyses.The ML and BI phylogenetic trees were constructed based on DNA sequences of the mitochondrial ND2 gene with a total length of 670 bp.The two analyses resulted in identical topologies,suggesting that the population ofHemiphyllodactylusfrom Dupangling is sister to a clade comprisingH.zugi,H.hongkongensis,H.dushanensis,andH.huishuiensis(Figure 1).Furthermore,the three specimens from Dupangling were grouped together with a small genetic variation to each other (＜ 0.001) and reciprocal monophyly toH.zugiwith a moderate mtDNA sequence divergence (pairwise genetic distance ranged from 5.2%-5.8%,which were larger than the divergence level betweenH.linnwayensisandH.ywanganensis(3.6%)) (Table S2).Combined with the morphological characteristics of these specimens from Dupangling(see below),our molecular genetic results show that they deserve recognition as a new species ofHemiphyllodactylus.

Taxonomic account.

Hemiphyllodactylus dupanglingensissp.nov.Zhang,Qian and Yang

Holotype.CSUFT 00402,adult male collected by Benjun WANG on 10 August 2018 in Hunan Dupangling National Nature Reserve (25°34′23.23" N,111°23′21" E; 437 m a.s.l.)(Figure 2).

Paratypes.CSUFT 00403,00404,00405,three adult males,were collected by Benjun WANG from August to September 2018 in Hunan Dupangling National Nature Reserve (25°34′23.23" N,111°23′21" E; 437 m a.s.l.).CSUFT 00401,one adult female,and CSUFT 00406,one adult male,were collected by Bing ZHANG and Jifu ZHOU on August 21,2019,in Hunan Dupangling National Nature Reserve (25°34′23.23" N,111°23′21" E; 437 m a.s.l.) (Figure 3).

Diagnosis.Hemiphylodactylus dupanglingensissp.nov.can be distinguished from all its congeners by a combination of following characters:(1) bisexual,presence of both males and females; (2) medium body size (SVL 39.4-44.7 mm in adult males,40.8 mm in one adult female); (3)9-11 chin scales; (4) postmental scales enlarged; (5) 3-4 circumnasal scales; (6) 2-3 scales between internasals; (7)9-12 infralabials,11-14 supralabials; (8) 14-16 dorsal scales and 10-11 ventral scales longitudinally arranged at midbody contained with one eye diameter; (9) a manual lamellar formula of 4-5-5-4 (n=1) or 4-4-5-4 (n=4),a pedal lamellar formula of 4-5-5-5 (n=4) or 5-6-6-4 (n=1); (10) four or five subdigital lamellae on first finger and first toe; (11) 21-25 continuous precloacal and femoral pores in males; (12) cloacal spurs 1-2; (13) no enlarged subcaudal scales; (14) dark transverse blotches on dorsal;(15) dark postorbital stripe present; (16) a linear series of white spots present on flank; (17) postsacral marking lack anterior projecting arms; and (18) pigmented gonadal tracts and caecum.

Description of holotype.Adult male,body slender,SVL 41.0 mm; tail regenerated,29.3 mm in length; trunk length 19.4 mm (TrunkL/SVL 0.47); head longer than wide(HeadL 11.2 mm,HeadW 7.5,HeadW/HeadL 0.67),head 4.5 mm in depth (HeadD); eye diameter 2.6 mm (EyeD/HeadL 0.23),snout-eye length (SnEye) 4.7 mm; internarial distance (SnW) 1.5 mm.

Snout robust; rostral with a suture on upper edge,visible from above; 12/11 supralabials,enlarged from rostral to below eye; 9/9 infralabials; 3/3 circumnasal scales; two scales between supranasals; dorsal scales in one EyeD 15; ventral scales in one EyeD11; a manual lamellar formula of 4-4-5-4,a pedal lamellar formula of 4-5-5-5; four lamellar on the first finger and four on the first toe;precloacal a femoral pore series continuous,24 in number.

Coloration in life.We describe the coloration in life using a series of photos of paratype (CSUFT 401,adult female:Figure 4).Lateral view of head,presence of a white line beginning at loreal region through orbital to the back of the head,which gets thinner at neck and extends to dorsolateral.Two lines separated by some black-centered yellow pigments in the middle of the head front.Two dark postorbital lines present at edge of the white line.Iris copper red with brown reticulations,and pupil vertical with four black spots linked by black lines.

Dorsal color light yellow with dark reticulation transverse stripes starting behind the head and ending at postsacral marking; postsacral marking pale buff,chevron shaped; a series of light spots present on both sides on dorsalateral; fingers and toes are light yellow with black pigmentation on dorsal.

Ventral view of body and limbs pinkish white with light yellow edge ventral scales,and dense black dots present on each scale; gonadal tracts and caecum pigmented.Ventral view of tail orange.

Coloration in alcohol.Dorsal surface of head,body,and limbs greyish-brown,light orange postsacal mark faded to white.Ventral surface greyish-white,pigmented gonadal tracts,and caecum,light yellow edge on scales faded.

Variation.Measurements are shown in Table 1.Specimens vary in scale counts,subdigital lamellar,number of precloacal and femoral pores and body size.All recognized specimens share the same 2-5FingLm 4-4-5-4 except CSUFT 00401 (4-5-5-4).All recognized specimens share the same 2-5ToeLm 4-5-5-5 except CSUFT 00404 (5-6-6-4).Specimen CSUFT 00403 shows a different dorsal color pattern.Instead of dark transverse bars,light paler irregular veins distributed on the dorsal of the head and body.Darker coloration on flanks extends from under-eye area to anterior portion of forefoot,with small white dots on flanks.

Figure 1 Bayesian inference tree of Hemiphylodactylus species based on partial DNA sequences of the mitochondrial ND2 gene.Outgroups are not shown.Numbers before slashes indicate Bayesian posterior probabilities (＞ 0.6 retained) and numbers after slashes are bootstrap support for maximum likelihood(1000 replicates) analyses (＞ 60 retained).The symbol “-” represents the bootstrap value below 0.60/60%.

Figure 2 Holotype of Hemiphyllodactylus dupanglingensis sp.nov.in alcohol.A:dorsal view of head; B:lateral view of head; C:ventral view of head; D:ventral view of pubis and hind limbs showing pore series; E:ventral view of tail in middle part.Scale bars equal 0.5 mm.

Comparisons.We show the diagnostic characters separatingH.dupanglingensissp.nov.with all named species ofHemiphyllodactylus(Table S3).We compared this species with eight closely related species (H.banaensis,H.dushanensis,H.hongkongensis,H.huishuiensis,H.kiziriani,H.serpispecus,H.yunnanensis,andH.zugi) (Table 2).Hemiphyllodactylus dupanglingensissp.nov.can be differentiated from its eight closely related species by a combination of the following characters:maximum SVL 44.7 mm (vs.＞ 50 mm inH.dushanensis,H.huishuiensis,andH.banaensis); chin scales 9-11 (vs.＜ 9 inH.hongkongensis);internasal scales two or three (vs.4-11 inH.banaensis);dorsal scales 14-16 (vs.＞ 16 inH.banaensis,H.kiziriani,H.serpispecus,andH.zugi); ventral scales 10 or 11 (vs.＞ 11 inH.zugiand ＜ 10 inH.dushanensis,andH.huishuiensi); total lamellae on hand 17 or 18 (vs.＜17 inH.hongkongensis,H.kiziriani,H.serpispecus,H.yunnanensis,andH.zugi);total lamellae on foot 19-21 (vs.＜19 inH.hongkongensis,H.kiziriani,H.serpispecus,andH.yunnanensis); subdigital lamellae on the first finger four or five (vs.＜4 inH.huishuiensis); subdigital lamellae on the first toe four or five (vs.＜4 inH.huishuiensis); and continuous precloacal and femoral pores 21-25 (vs.＜21 inH.huishuiensis).

Etymology.The scientific name “dupanglingensis” is derived from its type locality Dupangling in Hunan province.As a common name we suggest Dupangling Slender Gecko in English and “Du Pang Ling Ban Ye Zhi Hu (都龐嶺半葉趾虎)” in Chinese.

Distribution and natural history.The new species is currently known only from its type locality,Kongshuyan from Dupangling Nature Reserve,Hunan Province,China(Figure 5).This new species is nocturnal.In the daytime,individuals hide under wall seams,tile eaves,cupboards,and other concealed places,and vacate these at night to forage.All the individuals were found on the walls of human houses-that were surrounded by evergreen broad-leaf forests.

4.Discussion

The recognition ofH.dupanglingensissp.nov.increases the number of described species ofHemiphyllodactylusin China to eight,along withH.changningensis,H.dushanensis,H.hongkongensis,H.huishuiensis,H.jinpingensis,H.longlingensis,andH.yunnanensis(Grismeret al.,2013;Guoet al.,2015; Yanet al.,2016; Sunget al.,2018).These highlight the diversification ofHemiphyllodactylusspecies within China.H.dushanensis,H.hongkongensis,andH.huishuiensisin ChinaHemiphyllodactylusare close toHemiphyllodactylus dupanglingensissp.nov.in geographical range.The type locality ofH.dushanensisis about 380km west of the collection locality ofH.dupanglingensissp.nov.(Zhouet al.,1981).The average mitochondrial ND2 sequence difference betweenH.dupanglingensissp.nov.andH.dushanensisis 5.3%.The type locality ofH.huishuiensisis about 470 km west of the collection locality ofH.dupanglingensissp.nov.(Yanet al.,2016).The average mitochondrial ND2 sequence difference betweenH.dupanglingensissp.nov.andH.huishuiensisis 7.8%.The type localities ofH.dushanensisandH.huishuiensisare all located in the Miaoling mountains.The type locality ofH.hongkongensisis Aberdeen Country Park,Hong Kong,about 470 km southeast of the collection locality ofH.dupanglingensissp.nov.(Sunget al.,2018).The average mitochondrial ND2 sequence difference betweenH.dupanglingensissp.nov.andH.hongkongensisis 9.8%.Hemiphyllodactylusspecimens with an uncorrected mitochondrial ND2 sequence divergence value of ≥ 5%were suspected to be different species (Grismeret al.,2013).Consistent with Grismeret al.(2013),the ND2 sequence differences betweenH.dupanglingensissp.nov.andH.dushanensis,H.hongkongensis,orH.huishuiensisall exceed 5%.The collection locality ofH.dupanglingensissp.nov.and the type localities of the three species are separated by much warmer and drier low elevation habitats or other mountains.The discovery ofH.dupanglingensissp.nov.expands the distribution area of ChinaHemiphyllodactyluseastward to the Nanling mountains.The orogeny of this region and the common habits of the restricted,montane distribution of mostHemiphyllodactylusspecies may be the major driver for the formation ofH.dupanglingensissp.nov.

Table 2 Diagnostic characters (bold) separating Hemiphyllodactylus dupanglingensis sp.nov.from closely related species of Hemiphyllodactylus.

Figure 3 Type series of Hemiphyllodactylus dupanglingensis sp.nov.from specimens preserved in alcohol.

Figure 4 Living specimen (CSUFT 00401) of Hemiphyllodactylus dupanglingensis sp.nov.A:dorsal view; B:plantar view of left foot; C:volar view of right hand; D:ventral view; E:dorsal view of tail base; F:lateral view of head; G:dorsal view of head.

Most species of this genus are geographically restricted to highlands and island habitats (Grismeret al.,2013).In China,onlyH.hongkongensislives in a lower island habitat (Sunget al.,2018).OtherHemiphyllodactylusspecies are all restricted to high elevation habitats (＞ 900 m) (Zhouet al.,1981; Guoet al.,2015; Yanet al.,2016),which represent mid-to high-elevation taxa.However,the type locality ofH.dupanglingensissp.nov.is about 430 m.Dupangling is located in the middle part of Nanling mountains.This unique geographical location contains large expanses of limestone karst forest.It is the most typical and representative transition zone from the middle subtropical zone to the south subtropical area in China,which contains high reptile diversity.The karst landforms of Dupangling produce rock substrates with a variety of microhabitats under the forest canopy.These provide effective thermoregulation,a stable microclimate,low illumination,and foraging opportunities for geckos and other reptiles (Grismeret al.,2017).Many geckos are restricted to karst forest areas (Zug,2010; Grismeret al.,2013; Grismeret al.,2017; Grismeret al.,2018; Agarwaletal.,2019; Eliadeset al.,2019).We have only collected this species of gecko in Kongshuyan of Dupangling,and we urge additional searches in the Nanling mountains for other “cryptic species.”

AcknowledgementsWe thank Benjun WANG,Jifu ZHOU,Xiang LIU,and Keyuan LI from Yueyan Branch of Hunan Dupangling National Nature Reserve Bureau,and Dan PAN for their assistance in field work.We thank Shuo QI and Chaohui ZENG for helping with references and manuscript preparation,Simin WU for making a map of the collection locations,and Dr.Hannan WANG for photographing the holotype.This study was supported by the National Natural Science Foundation of China (Grant No.31472021) and the Project for Endangered Wildlife Investigation,Supervision,and Industry Regulation of the National Forestry and Grassland Bureau of China (No.2019072-HN-001).

Appendix

Table S1 GenBank accession numbers,localities,and voucher information for all specimens used in this study.

(Continued Table S1)