Description of a new karst-adapted species of the subgenus Japonigekko (Squamata: Gekkonidae: Gekko ) from Guangxi, southern China

A new species of the genus Gekko Laurenti, 1768 , Gekko paucituberculatus sp. nov. , is described here, based on two specimens from Tianyang District, Baise City, Guangxi Zhuang Autonomous Region, China. It was placed in subgenus Japonigekko on the basis of morphological and molecular phylogenetic analysis and can be distinguished from all congeners in this subgenus by significant divergences in the mitochondrial 16S and ND2 genes and by a combination of the following morphological characters: Moderate body size, SVL 77.2 mm in the adult male and SVL 85.9 mm in the adult female; tubercles only present along dorsolateral trunk and absent on other regions; fingers and toes with weak webbing; continuous precloacal pores 12 in the male, absent in the female; a single postcloacal tubercle on each side; a light-coloured vertebral line from nape to tip of tail; dorsum greyish-brown, with 7–8 dirty-white bands between nape and sacrum. Meanwhile, the distribution of G. palmatus in China has been confirmed as occurring in Guangxi and Guangdong Provinces. This study brings the total species of the subgenus Japonigekko in China to 19.


Introduction
The genus Gekko Laurenti, 1768, currently containing 88 recognised species assigned to seven subgenera, is a widely distributed group of nocturnal lizards (Wood et al. 2020;Uetz et al. 2023;Zhang et al. 2023).Amongst these subgenera, G. (Japonigekko) is the most diverse group with many karst-dwellers.At present, there are three described G. (Japonigekko) species-adapted karst ecosystems in China, namely G. adleri Nguyen et al., 2013 andG. kwangsiensis Yang, 2015 from Guangxi and G. liboensis Zhou & Li, 1982 from Guizhou and Guangxi (Nguyen et al. 2013;Jono et al. 2015;Yang 2015).During the field survey in the karst forest of western Guangxi, southern China (Fig. 1), two gekkonid individuals were collected.Morphologically, the specimens possess the common characteristics of subgenus G. (Japonigekko), but are apparently distinct from all known congeners by the following characters: Moderate body size, tubercles only present along dorsolateral trunk, continuous precloacal pores 12 in the male, a single postcloacal tubercle on each side and a light-coloured vertebral line from nape to tip of tail.Phylogenetic studies reveal that these specimens belong to a separated taxon most closely related to G. kwangsiensis.Hence, we consider it as a new species and describe it below.

Specimens and morphology
Two specimens were collected from Tianyang District, Baise City, Guangxi Zhuang Autonomous Region, China on 3 August 2023.The specimens were euthanised and then fixed in 10% buffered formalin, later being transferred to 75% ethanol and deposited in the Museum of Biology, Sun Yat-sen University (SYS), Guangzhou, China.Liver tissue samples were preserved in 95% ethanol for molecular analysis.
Measurements were taken with digital callipers (Deli DL91200 Digital Vernier Caliper) to the nearest 0.1 mm on the right side of the body and scalation features were counted under a binocular scope (Leica EZ4 HD).Bilateral scale counts are given as left/right.External measurements, meristic traits and their abbreviations follow Lyu et al. (2021) and Grismer et al. (2022).They are snoutvent length (SVL, from tip of snout to anterior margin of cloaca); tail length (TaL, from posterior margin of cloaca to tip of tail); axillia-groin length (AG, distance between axilla and groin); head length (HL, maximum head length from tip of snout to posterior margin of ear opening); head width (HW, measured at the angle of the jaws); head height (HH, from the top of the head posterior to the eyes to the bottom of the lower jaw); snout length (SNT, from snout tip to anterior corner of eye); maximum eye diameter (ED); maximum ear opening diameter (EOD); maximum rostral width (RW); maximum rostral height (RH); maximum mental width (MW); maximum mental length (ML); nasals (N, nasorostrals, supranasals and postnasals); intersupranasals (I, scales between supranasals, in contact with rostral); supralabials and infralabials (SPL and IFL, number of scales from commissure of jaw to the rostral /mental scale); interorbitals (IO, number of scales in a line between anterior corners of eyes); preorbitals (PO, number of scales in a line from nostril to anterior corner of the eye); postmentals (PM, scales bordering the mental); gulars bordering the postmentals (GP); dorsal tubercle rows at mid-body (DTR); granules surrounding dorsal tubercles (GSDT); scales in a line from mental to the front of cloacal slit (SMC); ventral scale rows at midbody (V); scale rows at mid-body (SR, including ventral scales); subdigital lamellae under entire first finger (LF1); subdigital lamellae under entire fourth finger (LF4); subdigital lamellae under entire first toe (LT1); subdigital lamellae under entire fourth toe (LT4); precloacal pores (PP); postcloacal tubercles (PAT); dorsal scale rows in the middle of the third caudal whorl (S3W).In addition, the specimens were compared with other Gekko (Japonigekko) congeners on the basis of descriptions in literature (Zhou et al. 1982;Song 1985;Ota et al. 1995;Toda et al. 2008;Phung and Ziegler 2011;Rösler et al. 2011;Nguyen et al. 2013;Luu et al. 2014Luu et al. , 2015Luu et al. , 2017;;Jono et al. 2015;Ngo et al. 2015;Yang 2015;Lin and Yao 2016;Hou et al. 2021;Lyu et al. 2021;Sitthivong et al. 2021;Zhang et al. 2023).

Phylogenetic sampling and analyses
A total of 24 samples of subgenus Gekko (Japonigekko) were used for molecular analysis in this study.All samples were attained from euthanasia specimens and then preserved in 95% ethanol and stored at -40°C.Furthermore, 22 sequences were obtained from GenBank and incorporated into our dataset for phylogenetic analysis.G. gecko and G. reevesii, belonging to subgenus G. (Gekko), were used to root the tree, based on Rösler et al. (2011) and Lyu et al. (2021).Detailed information of these samples is given in Table 1.
Genomic DNA was extracted from liver tissue using a DNA extraction kit (Tiangen Biotech Co., Ltd, Beijing).Two fragments of the mitochondrial genes that encode the partial 16S ribosomal RNA gene (16S) and partial NADH dehydrogenase subunit 2 gene (ND2) were amplified.The primers used for two genes are listed in Table 2. PCR amplifications were processed with the following cycling conditions: Initial denaturing step at 95°C for  (Edgar 2004).Parti-tionFinder2 was used to determine the best partitioning scheme (Lanfear et al. 2017) and jModelTest v.2.1.2was used to determine the best fitting nucleotide substitution models (Darriba et al. 2012), resulting in the partitions by gene.ND2 was further partitioned by codon position and the best fit models for all partitions was GTR+I+G.Sequenced data were analysed using Bayesian Inference (BI) in MrBayes 3.2.4 (Ronquist et al. 2012) and Maximum Likelihood (ML) in raxmlGUI 1.3 (Silvestro and Michalak 2012).Two independent runs were conducted in the BI analysis with 2,000,000 generations each and sampled every 1000 generations with the first 25% of samples discarded as burn-in, resulting in a potential scale reduction factor (PSRF) of < 0.005.In the ML analysis, a bootstrap consensus tree inferred from 1000 replicates was generated.MEGA11 was used to calculate uncorrected pairwise sequence divergence for 16S and ND2 amongst and with-in species using the complete deletion option which removes missing data and gaps (Tamura et al. 2021).

Results
The aligned dataset contained a total of 1578 nucleotide base pairs (bp), with 566 bp for 16S and 1012 bp for ND2.The BI and ML analyses resulted in essentially identical topologies (Fig. 2).The mean uncorrected p distances, based on 16S and ND2 used in this study, are given in Tables 3 and 4.
All Gekko (Japonigekko) samples used in this study formed a strongly-supported monophyletic lineage (BPP = 1.00;BS = 100).However, intrageneric relationships within this lineage remained unclear due to low intrageneric nodal supports.Two samples from Baise City, Guangxi were placed in subgenus G. (Japonigekko), which clustered together with strong support (BPP = 1.00;BS = 100) and exhibited low intra-lineage genetic differentiation (0%).This unnamed lineage is the sister taxon to G. kwangsiensis from Wuming County, Guangxi and these together formed the sister group to G. hokouensis Pope, 1928.The lowest genetic distance between the unnamed lineage and a known Gekko species were, on average, 8.96% in 16S and 15.54% in ND2, as compared to G. kwangsiensis, although we did not use genetic divergences to make species delimi-  tation, as it served as a reference only.The newly-collected specimens showed recognisable morphological differences from all known congeners (see Taxonomic account below).Hence, the unnamed population from Baise City is considered as an undefined species.
In addition, two samples from Mt. Dinghu, Guangdong grouped together with other G. palmatus Boulenger, 1907 samples from Vietnam and Guangxi, China and exhibit a robust monophyletic lineage with strong support (BPP = 1.00;BS = 100) and low intrapopulational genetic differentiation (0.34% in 16S and 0.82% in ND2).Morphologically, the characters of the specimens from Guangdong mostly agree with the original description and re-description of G. palmatus Boulenger, 1907(Ota et al. 1995).

Gekko
Original tail (broken when capturing), longer than body (TaL 89.3 mm, TaL/SVL 1.16); distinctly swollen at base, oval in section; dorsal scales small, flat, smooth; caudal whorl distinct, 10 dorsal scale rows in the middle of the third one; subcaudals enlarged, arranged in a longitudinal row.

Colouration of holotype.
In life, the dorsal regions of head and body are greyish-brown in colour, with scattered white spots on the anterior of head.An inverted U-shaped marking is present on the occipital region and there are seven regularly arranged, dirty-white bands between the nape and sacrum.The first band, located at the nape, extends forwards and backwards to the posterior corners of eye and the second band, respectively.A light-coloured vertebral line is present from the nape to the tail terminal.Some light spots are visible on the lateral sides.Limbs are light brown with many indistinct pale marks.Dorsal tail is black with seven bands, from dirty white to pure white in colour towards terminal.The ventral surface of the holotype is light flesh-coloured.The body colour becomes darker after capture.
In preservative, the dorsal ground colour of head, body and limbs is greyish-black; ventral surface fading to greyish-white.
Morphological variation.Measurements and scale counts of two individuals are shown in Tables 5 and 6 and the female paratype is shown in Figure 5.
Precloacal pores are absent in the female.In the male, the postcloacal tubercle is significantly larger than in the female.The paratype specimen exhibits eight light bands between the nape and sacrum.
Furthermore, the new species differs from G. liboensis by tubercles only present along the dorsolateral surface not on other regions (vs.present from occipital region to tail base) and having regular and broad bands between nape and sacrum (vs.irregular and thin bands with many scattered round-shaped spots).
Distribution and ecology.Currently, Gekko paucituberculatus sp.nov. is limited to Tianyang District, Baise City, in Guangxi Zhuang Autonomous Region of China.The new gecko species is a rock-dwelling specialist.Both of the two individuals were discovered on rocks near the entrance to a limestone karst cave at night.

Discussion
Gekko palmatus was initially described by G. A. Boulenger in 1907 (as Gecko palmatus), based on a single female specimen from the mountains of Man Son, Vietnam (Boulenger 1907).Poyarkov et al. (2023) cited Nguyen et al. (2009) who argued that G. palmatus occurs in China, but it was not recognised due to lack of evidence (Wang et al. 2020;Cai et al. 2022).Both this study and Lyu et al. (2021) confirmed distributions of G. palmatus in Napo County and Nonggang Natural Reserve in Guangxi and Mt.Dinghu in Guangdong of China.Regarding morphology, the specimens from Guangdong share the diagnoses with G. palmatus: Nostril in contact with rostral; internasals 0-2, much smaller than supranasal when present; tubercles present on dorsum of body, lacking on forelimb and thigh; webs well developed; male preanal pores 24-27; cloacal spur single; one pair of dark, roundish or somewhat elongated spots in occipital region; smaller, but more distinct dark spot in nuchal region; light broken mid-dorsal stripe evident on body (Ota et al. 1995).
The discoveries of Gekko paucituberculatus sp.nov.and G. palmatus bring the number of known species in the subgenus G. (Japonigekko) in China to 19, with seven in Guangxi, the provincial district with the highest diversity.Four of them are karst dwellers: Gekko adleri, G. kwangsiensis, G. liboensis and G. paucituberculatus.
The multiple landforms (e.g.peak cluster, depression, cave and peak forest) in the karst region have led to the formation of fragmented and unique microhabitats, resulting in a comparatively significant biodiversity and the presence of numerous endemic species (Clements et al. 2006;Grismer et al. 2021).The globally-concentrated karst is primarily found in south-central Europe, eastern North America and southern China (Li and Xiong 2021).Karstic landscapes from southern China are considered one of the most representative regions of tropical-subtropical karst formation (Xiong et al. 2008).In the last decade, researchers have discovered several narrow-ranged  species of amphibian and reptile that are specifically adapted to the karst ecosystems in this region (Mo et al. 2015;Qi et al. 2020;Luo et al. 2021;Agung et al. 2022;Lin et al. 2022).However, these studies have been limited to only very recent and unconnected survey areas, leaving much of the region unexplored.Additionally, the slow soil formation rate of limestone in karst creates a thin soil layer that is prone to soil erosion and rocky desertification (Yang 1990).These features render the karst ecosystem highly vulnerable and challenging to restore after damage.Consequently, conducting a thorough and comprehensive survey of amphibians and reptiles in this expansive karst region is imperative to assess their diversity and the hazards they encounter.This will enable the development of effective conservation strategies to prevent further loss of unidentified species.

Figure 4 .
Figure 4. Morphological features of the adult male holotype SYS r002806 of Gekko paucituberculatus sp.nov.A Dorsal view of body; B ventral view of body; C dorsal head; D ventral head; E precloacal pores; F left hand; G left foot; H dorsal scalation at midbody; I ventral tail.Photos by Hao-Tian Wang and Han-Ming Song.

Figure 5 .
Figure 5. Morphological features of the adult female paratype SYS r002807 of Gekko paucituberculatus sp.nov.A Dorsal view of body; B ventral view of body; C dorsal head; D ventral head; E right hand; F right foot; G dorsal scalation at mid-body.Photos by Hao-Tian Wang and Han-Ming Song.

Table 1 .
Localities, voucher information and GenBank accession numbers for all samples used in this study.
4 min and 5 min (16S and ND2, respectively), 35 cycles of denaturing at 95°C for 40 s, annealing at 53°C for 34 s (16S) and 55°C for 40 s (ND2), extending at 72°C for 60 s and a final extending step at 72°C for 10 min.PCR products were purified with spin columns and then sequenced with a forward primer using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA).Sequencing was performed on an ABI Prism 3730 automated DNA sequencer by Wuhan Tianyi Huiyuan Bioscience and Technology Inc.DNA sequences were aligned by the MUSCLE algorithm with default parameters

Table 2 .
Primers used in this study.

Table 3 .
Uncorrected p distances (%) of the 16S gene amongst species of Gekko (Japonigekko) used in this study.

Table 4 .
Uncorrected p distances (%) of the ND2 gene amongst species of Gekko (Japonigekko) used in this study.Body slender, elongate (AG/SVL 0.41); dorsals smooth, round or oval, granular and juxtaposed; tubercles flattened, only present on dorsal lateral surface, two rows on each side, surrounded by eight dorsal scales, absent on other regions; lateral fold present, without tubercles; ventrals distinctly larger than dorsal scales, smooth, imbricate and largest in middle of belly; ventral scale rows at mid-body 42; scale rows around mid-body 140; ventral scales in a row between mental and cloacal slit 189; precloacal scales enlarged, but no enlarged scales on thighs; precloacal pores 12, in a continuous row; postcloacal tubercle 1/1, large.

Table 5 .
Measurements (in mm) and body proportions of the type series of Gekko paucituberculatus sp.nov.See Materials and Methods section for abbreviations."*" regenerated tail.

Table 6 .
Scalation features of the type series of Gekko paucituberculatus sp.nov.Bilateral scale counts are given as left/right.