Taxonomic reassessment and phylogenetic placement of Cyrtodactylus phuketensis (Reptilia, Gekkonidae) based on morphological and molecular evidence

Abstract The taxonomy and phylogeny of the Cyrtodactylus pulchellus complex along the Thai-Malay Peninsular region has been the focus of many recent studies and has resulted in the recognition of 17 species. However, the majority of these studies were focused on Peninsular and insular Malaysia where there were specimens and genetic vouchers. The taxonomic status and phylogenetic relationships of the Thai species in this complex remain unresolved, due to the lack of genetic material of some species, especially C. phuketensis and C. macrotuberculatus from Thai populations. In this study, we investigated the phylogenetic relationship between C. phuketensis and its closely related species C. macrotuberculatus, using both morphometric and molecular data. Phylogenetic analyses of mitochondrial NADH dehydrogenase subunit 2 (ND2) gene revealed that C. phuketensis is embedded within a C. macrotuberculatus clade with 1.45–4.20% (mean 2.63%) uncorrected pairwise sequence divergences. Morphological comparisons showed nearly identical measurements of C. phuketensis and C. macrotuberculatus and overlapping ranges in meristic characters. Based on these data, C. phuketensis is considered to be a variant of C. macrotuberculatus, thus rendering C. phuketensis a junior synonym of C. macrotuberculatus.


Introduction
Cyrtodactylus is a genus of the bent-toed geckos which is widely distributed across South Asia to Melanesia (Wood et al. 2012;Grismer et al. 2020Grismer et al. , 2021a. This genus is the most speciose group of gekkotans, with 306 species currently recognized (Uetz et al. 2020). Due to discoveries of hidden taxa within species complexes over decades, species diversity of Cyrtodactylus has remarkably increased, especially in Southeast Asia (e.g., Grismer et al. 2012Grismer et al. , 2018Riyanto et al. 2017;Pauwels et al. 2018;Murdoch et al. 2019;Quah et al. 2019). In the last decade, the integrative approach of molecular and morphological data has been applied to study species boundaries, evaluate taxonomic status, and the adaptive evolution in habitat preference in Cyrtodactylus (Grismer et al. 2015(Grismer et al. , 2021aNielsen and Oliver 2017).
Cyrtodactylus phuketensis was described as a new species from Ban Bangrong, Thalang District, Phuket Province by Sumontha et al. (2012). It was similar to C. macrotuberculatus in having tuberculation on ventral surface of the forelimbs, gular region and ventrolateral folds, and relatively larger ventral scales (compared to other species in C. pulchellus complex). In the original description, C. phuketensis was separated from C. macrotuberculatus by having three dark bands between limb insertions, 19 subdigital lamellae on the 4 th toe, the presence of a precloacal groove in both sexes, and eight dark caudal bands on an original tail.
During our field surveys, nine specimens of C. phuketensis were collected from the type locality and nearby areas and we found variation in the number of body bands and overlap in the ranges of putatively diagnostic meristic characters when compared to C. macrotuberculatus. Therefore, this study aims to reassess the taxonomic status of C. macrotuberculatus and C. phuketensis using morphological and genetic data from the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene and flanking tRNAs. The analyses were performed on newly collected specimens from southern Thailand and from the type specimens of both species.

Specimen sampling
During October 2017 and June 2019, field surveys were conducted at five localities in southern Thailand, including the type locality of C. phuketensis ( Fig. 1; Table 1). Specimens were investigated and captured by hand during the night . Liver or muscle tissues were individually preserved in 95% ethyl alcohol and stored at -20 °C for molecular analysis. Specimens were fixed in 10% formalin and later transferred to 70% ethyl alcohol. Preserved specimens were deposited in the herpetological collections of the Zoological Museum, Kasetsart University, Thailand (ZMKU). Additional specimens were also examined from the Princess Maha Chakri Sirindhorn Natural History Museum (PSU), Prince of Songkhla University, Thailand; Thailand Natural History Museum (THNHM), Thailand; La Sierra University Herpetological Collection (LSUHC), La Sierra University, Riverside, California, USA; and the Zoological Reference Collection (ZRC) of Lee Kong Chian Natural History Museum at National University of Singapore, Singapore.

Molecular analyses
Total genomic DNA was extracted from 95% ethanol-preserved muscle or liver tissue using a NucleoSpin Tissue Kit (Macherey-Nagel GmbH & Co. KG, Germany). Mitochondrial NADH dehydrogenase subunit 2 (ND2) gene and flanking tRNAs were amplified via double-stand Polymerase Chain Reaction (PCR) using primers L4437a (tRNAmet: 5' AAGCTTTCGGGCCCATACC 3') and H5934 (COI: 5' AGRGT-GCCAATGTCTTTGTGRTT 3') (Macey et al. 1997). PCR amplification occurred with an initial denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 sec, annealing at 48-52 °C for 30 sec, extension at 72 °C for 1 min 30 sec, and final extension at 72 °C for 7 min. Amplification products were purified using NucleoSpin Gel and PCR Clean-Up kit (Macherey-Nagel GmbH & Co. KG, Germany) and visualized on 1.0% agarose gel electrophoresis. Purified PCR products were sequenced in both directions using amplifying primers on an ABI 3730XL DNA Sequencer (Applied Biosystems, CA, USA). Sequences were manually edited and aligned in Geneious R11 (Biomatters, Ltd, Auckland, New Zealand). The ND2 nucleotide sequences were translated to amino acid for the protein-coding region and to ensure the lack of stop codons. All sequences were deposited in GenBank under the accession numbers MW809294 to MW809309 (Table 1).

Figure 1.
Map illustrating the known geographic distribution of Cyrtodactylus macrotuberculatus and C. phuketensis. Yellow star: the type locality of C. macrotuberculatus at Gunung Raya, Pulau Langkawi, Kedah, Malaysia. Green star: the type locality of C. phuketensis at Thalang District, Phuket Island, Phuket Province. Yellow circles: C. macrotuberculatus samples used in this study. Green circle: C. phuketensis samples used in this study. Yellow squares: the distribution of C. macrotuberculatus taken from Grismer et al. (2012), andQuah et al. (2019). The samples used correspond to those in Table 1. Phylogenetic relationships were inferred using two model based approaches, Bayesian Inference (BI) and Maximum Likelihood (ML). Outgroup species used to root the tree were Hemidactylus frenatus, Agamura persica, Tropiocolotes steudneri, C. elok, C. intermedius, C. interdigitalis, and Cyrtodactylus sp. based on Wood et al. (2012). The best-fit nucleotide substitution model for each of the three codon partitions and tRNAs was selected under the Bayesian Information Criterion (BIC) in PartitionFinder2 on XSEDE (Lanfear et al. 2016) using CIPRES (Cyberinfrastructure for Phylogenetic Research; Miller et al. 2010). BI analysis was executed in MrBayes 3.2.6 on XSEDE (Ronquist et al. 2012) using CIPRES with the TRN+I+G for the 1 st and 2 nd codon position, and TIM+I+G for the 3 rd codon position and tRNAs. Four chains (three hot and one cold) were run for 10,000,000 generations and sampled every 1,000 generations using Markov chain Monte Carlo (MCMC). To build a consensus tree, we discarded the first 25% of each run as burn-in and assessed stationarity by plotting log-likelihood score in Tracer ver. 1.7.1. (Rambaut et al. 2018). The ML analysis was performed on the web server W-IQ-TREE (Trifinopoulos et al. 2016) with 1,000 bootstrap replicates using ultrafast bootstrap approximation (Minh et al. 2013). Nodes having Bayesian posterior probabilities (BPP) of ≥ 0.95 and ultrafast bootstrap support (UFB) of ≥ 95 were considered to be strongly supported (Huelsenbeck and Ronquist 2001;Wilcox et al. 2002;Minh et al. 2013). Uncorrected pairwise sequence divergence was calculated to assess within and among species differences using the default settings in MEGA X 10.0.5 (Kumar et al. 2018).

Morphological measurements
Morphological and meristic characters were modified from the previous studies of Grismer and Ahmad (2008) and Grismer et al. (2012). Measurements were taken with digital calipers to the nearest 0.1 mm for the following sixteen characters: SVL snout-vent length, taken from the tip of snout to the vent; TW tail width, taken at the base of the tail immediately posterior to the postcloacal swelling; TL tail length, taken from vent to the tip of the tail, original or regenerated; FL forearm length, taken from the posterior margin of the elbow while flexed 90° to the inflection of the flexed wrist; TBL tibia length, taken from the posterior surface of the knee while flexed 90° to the base of the heel; AG axilla to groin length, 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; HL head length, the distance from the posterior margin of the retroarticular process of the lower jaw to the tip of the snout; HW head width, measured at the angle of the jaws; HD head depth, the maximum height of head from the occiput to the throat; ED eye diameter, the greatest horizontal diameter of the eye-ball; EE eye to ear distance, measured from the anterior edge of the ear opening to the posterior edge of the eye-ball; ES eye to snout distance, measured from anterior most margin of the eye-ball to the tip of snout; EN eye to nostril distance, measured from the anterior margin of the eye-ball to the posterior margin of the external nares; IO inter orbital distance, measured between the anterior edges of the orbit; EL ear length, the greatest vertical distance of the ear opening; IN internarial distance, measured between the nares across the rostrum.
Additional scale counts and non-meristic characters evaluated were the number of supralabial and infralabial scales counted from the largest scale immediately posterior to the dorsal inflection of the posterior portion of the upper jaw to the rostral and mental scales, respectively; the number of paravertebral tubercles between limb insertions counted in a straight line immediately left of the vertebral column; the number of longitudinal rows of body tubercles counted transversely across the center of the dorsum from one ventrolateral fold to the other; the number of longitudinal rows of ventral scales counted transversely across the center of the abdomen from one ventrolateral fold to the other; the presence or absence of tubercles on the ventral surface of the forearm; the presence or absence of tubercles in the gular region, throat, and lateral margins of the abdomen; the number of subdigital lamellae beneath the fourth toe counted from the base of the first phalanx to the claw; the total number of precloacal and femoral pores (i.e., the contiguous rows of femoral and precloacal scales bearing pores combined as a single meristic referred to as the femoroprecloacal pores); the presence or absence of a precloacal depression or groove; the degree of body tuberculation, weak tuberculation referring to dorsal body tubercles that are low and rounded whereas prominent tuberculation refers to tubercles that are raised and keeled; the width of the dark body bands relative to the width of the interspace between the bands; number of dark caudal bands on the original tail; the white caudal bands of adults immaculate or infused with dark pigment; and whether or not the posterior portion of the original tail in hatchlings and juveniles less than 50 mm SVL was white or whitish and faintly banded or boldly banded.

Morphological analyses
All statistical analyses were performed using the base statistical software in RStudio v. 1.2.1335 (RStudio Team 2018). To remove potential effects of allometry, mensural characters were scaled to SVL using the following allometric equation: X adj = X-β(SVL-SVL mean ), where X adj = adjusted value; X = measured value; β = unstandardized regression coefficient for each OTU; SVL = measured snout-vent length; SVL mean = overall average SVL of each OTU (Thorpe 1975(Thorpe , 1983Turan 1999;Lleonart et al. 2000). Male and female measurements were analyzed separately to remove potential effects of sexual dimorphism. For morphological analyses, TL (tail length) was excluded due to their different conditions (e.g., original, broken, and regenerated). Importantly, the following type material and topotypic specimens were included in the analysis: C. macrotuberculatus (holotype and three paratypes) and C. phuketensis (holotype, paratype and three topotypes). Prior to the morphological analyses, individuals were assigned on the basis of molecular data except C. phuketensis based on its distribution into three groups (= species): C. macrotuberculatus, C. phuketensis, and C. pulchellus.
Principal component analysis (PCA) was implemented in the R package Facto-MineR (Lê et al. 2008) to discover or reduce dimensionality of the original character variables in order to recover characters bearing the highest degree of variation among groups. Fifteen scaled morphometric and seven meristic characters (scalations) were concatenated and used for the PCA analyses separately by sex. For females, femoroprecloacal pore counts were excluded from the PCA due to their presence in only males.
For univariate analyses, all transformed mensural characters were tested for normality using the Shapiro-Wilk Test. Equality of variances was tested using F-tests. Morphological differences of both males and females between C. macrotuberculatus and C. phuketensis were examined using a t-test (for normally distributed and equal variance data), Welch's t-test (for unequal variance data) and Mann-Whitney U test (for non-normally distributed data) at a significant level of 95%.

Phylogenetic relationships
The aligned matrix contained 1,453 bp of ND2 gene and its flanking tRNAs for 101 samples of the C. pulchellus complex including outgroups ( Table 1). The standard deviation of split frequencies among the two Bayesian runs was 0.003263, and the Estimated Sample Size (ESS) of all parameters were ≥ 200. The BI and ML analyses generated similar topologies and strong nodal support for most clades, and only the ML tree is shown (Fig. 2). According to phylogenetic analyses, C. phuketensis is nested within C. macrotuberculatus with strong support (1.00 BPP, 100 UFB), thus rendering C. macrotuberculatus paraphyletic. Cyrtodactylus macrotuberculatus (including C. phuketensis) was recovered as sister lineage to a clade containing C. pulchellus and C. evanquahi. Uncorrected pairwise sequence divergence (p-distance) between C. phuketensis and this sister lineage was higher than 8.45% and within the C. phuketensis and C. macrotuberculatus clade, it ranged from 1.45-4.20% (mean 2.63%; Table 2). The p-distance within species ranged from 0.00-0.36% (mean 0.14%) for C. phuketensis and 0.00-4.38% (mean 2.48%) for C. macrotuberculatus.

Morphology
A total of 45 preserved specimens from three species groups (C. macrotuberculatus = 29, C. phuketensis = 10, and C. pulchellus = 6) were used for principal component analysis (Table 3). The PCA of males showed complete overlap between C. macrotuberculatus and C. phuketensis, and they were completely separated from C. pulchellus along the first two principal components (Fig. 3A). The first three principal components of males accounted for 53.17% of the variation. The first principal component (PC1) accounted for 25.78% of the variation and was most heavily loaded on HL adj , ES adj , EN adj and ventral scales; the PC2 accounted for 17.56% of the variation and was most heavily loaded on TBL adj , IO adj , supralabial and infralabial scales; and the PC3 accounted for 9.83% of the variation and was loaded most heavily on longitudinal tubercles (Table 3).
Along the first two PC plots, the PCA of females revealed complete overlap between C. macrotuberculatus and C. phuketensis, which were distinctly separated from C. pulchellus (Fig. 3B). The first three principal components of females accounted for 52.99% of the variation. The first principal component (PC1) accounted for 23.14% of the variation and was most heavily loaded on TW adj , AG adj , ES adj , EN adj , ventral scales and number of the 4 th toe lamellae; the PC2 accounted for 16.59% of the variation and was most heavily loaded on HL adj , ED adj , supralabial and infralabial scales; the PC3 accounted for 13.26% of the variation and was loaded most heavily on TBL adj and EL adj (Table 3).

Systematics
The phylogenetic analyses recovered C. phuketensis as being nested within the C. macrotuberculatus and bearing a low genetic divergence (mean 2.63%) which was similar to that within C. macrotuberculatus populations (mean 2.48%). In concordance, the statistical analyses of meristic and mensural characters of C. phuketensis widely overlap with those of C. macrotuberculatus. Based on these data, we propose that C. phuketensis from Phuket Island, Phuket Province is a junior synonym of C. macrotuberculatus which can be recognized as follows. Figure 4 Cyrtodactylus macrotuberculatus Grismer & Ahmad, 2008: 55;Grismer 2011: 406;Grismer et al. 2012 (Grismer and Ahmad 2008;Sumontha et al. 2012) and this study based on type materials and newly additional specimens. / = data unavailable.  (Table 1).
Body relatively short (AG/SVL 0.43-0.51) with well-defined, tuberculate, ventrolateral folds; dorsal scales small, granular, interspersed with large, trihedral, regularly arranged, keeled tubercles separated by no more than three granules at their base; tubercles extend from top of head onto approximately one-half of tail but not onto regenerated tail; tubercles on occiput and nape relatively small, those on body largest; approximately 19-27 longitudinal rows of dorsal tubercles at the mid body; approximately 37-49 paravertebral tubercles; 17-28 flat, imbricate, ventral scales and much larger than dorsal scales; precloacal scales large, smooth; deep precloacal groove (= depression).
Forelimbs moderate in stature, relatively short (FL/SVL 0.15-0.17); virtually no granular scales on dorsal surface of forelimbs, only large, trihedral, keeled tubercles; palmar scales slightly rounded; digits well-developed, inflected at basal, interphalangeal joints; subdigital lamellae nearly square proximal to joint inflection, only slightly expanded distal to inflection; digits more narrow distal to joints; claws well-developed, sheathed by dorsal and ventral scale; hind limbs more robust than forelimbs, moderate in length (TBL/SVL 0.18-0.21), virtually no granular scales on dorsal surfaces of hind limbs, only large, trihedral, keeled tubercles; ventral scales of thigh flat, smooth, imbricate; ventral, tibial scales flat, imbricate, slightly keeled; two rows of enlarged, flat, imbricate, femoroprecloacal scales extend from knee to knee through precloacal region where they are continuous with enlarged, pore-bearing precloacal scales; 28-42 contiguous, pore-bearing femoroprecloacal scales forming an inverted T bearing a deep, precloacal groove; eight to eleven pores bordering groove; postfemoral scales immediately posterior to the pore-bearing scale row conical, forming an abrupt union on posteroventral margin of thigh; plantar scales low, slightly rounded; digits welldeveloped, inflected at basal, interphalangeal joints; subdigital lamellae proximal to joint inflection nearly square, only slightly expanded distal to inflection; digits more narrow distal to joints; claws well-developed, sheathed by a dorsal and ventral scale; 19-23 subdigital lamellae on the 4 th toe.
Coloration of adult male ZMKU R 00871 in life (Fig. 5). Ground color of head, body, limbs, and dorsum light-brown to yellowish brown; wide, dark-brown nuchal band edged anteriorly and posteriorly by thin, creamy-white lines bearing tubercles extends from posterior margin of one eye to posterior margin of other eye; four similar body bands between nuchal loop and hind limb insertions edged anteriorly and posteriorly by thin, creamy-white lines bearing tubercles, first band terminates at shoulders, second and third bands terminate just dorsal to ventrolateral folds, the fourth band terminates at femurs; dark body bands slightly larger than light-colored interspaces; one additional dark-brown band posterior to hind limbs; original portion of tail bearing eight ringed, dark-colored bands separated by seven, narrower, off-white bands infused with dark pigmentation; ventral surfaces of head smudged with brown; abdomen and limbs beige, slightly darker, lateral regions.
Coloration in preservative (Fig. 6). Color pattern of head, body, limbs, and tail similar to that in life with some fading. Ground color of head, body, limbs, and dorsum tan; dark body and dark caudal bands lighter than in life.
Variation. Cyrtodactylus macrotuberculatus usually varies in coloration and banding pattern (Figs 7-8). In females, a precloacal groove and pores are absent (Fig. 9). PSUZC-RT 2010.58 and THNHM 15378 have a shallow precloacal groove. Three dark body bands occur in PSU 2010.58, THNHM 15378, ZMKU R 00889-00894 and ZMKU R 00897. In ZMKU R 00887, the second dorsal band bifurcates just dorsal to the ventrolateral fold. ZMKU R 00895 has four bands and the third band is incomplete. The third body band in ZMKU R 00896 is broken on the left of the midline and contacts the fourth body band bilaterally. Nuchal loop and body bands of ZMKU R 00883, ZMKU R 00895, and ZMKU R 00898 edged anteriorly and posteriorly by thin, light-yellow lines and tubercles; and dorsal superciliaries are light-yellow (Fig. 8). Variation in morphometric and meristic data are shown in Table 6.
Natural history. Based on specimens in Thailand, all individuals were found in similar habitat type, lowland forest habitat along granitic rock streams and surrounding areas (elevation 7-186 m asl) during a night survey (1900-2200; Fig. 10). The geckos were found mostly on rock boulders, vegetation (trunk of tree, buttress root, rotting wood and vines), and sometimes on the ground with leaf litter and high humidity (26.3-30.8 °C in temperature, 73.8-100% in relative humidity). Gravid female (ZMKU R 00876) contained four eggs during December. One juvenile (ZMKU R 00898, 56.50 mm in SVL) was found on a tree trunk in January. The varied microhabitats within which this species occurs, are consistent with its characterization as a habitat generalist (Grismer et al. , 2021b and may account for its wide peninsular and insular distribution relative to other species of the pulchellus group whose distributions are much less extensive or site-specific (Grismer et al. , 2014(Grismer et al. , 2016Quah et al. 2019;Wood et al. 2020).
In Thailand, C. macrotuberculatus were found sympatric with other gecko species, Cnemaspis adangrawi Ampai et al., 2019 on Adang and Rawi Islands, Satun Province   19-23 subdigital lamellae on the fourth toe; 28-42 femorprecloacal pores in males; deep precloacal groove in males; no scattered white spots on dorsum; 7-10 dark-ringed caudal bands on original tail; white caudal bands on original tail infused with dark pigmentation in adults. Additional comparisons between C. macrotuberculatus and other species in C. pulchellus complex are in Table 7.  Based on molecular data, C. macrotuberculatus is the sister lineage to a clade composed of C. pulchellus and C. evanquahi. It can be separated from those two species by having tubercles on ventral surface of forelimbs, gular region, and in ventrolateral body folds (vs. absent in C. evanquahi and C. pulchellus); 17-28 ventral scales (vs. 29-33 in C. evanquahi and 29-34 in C. pulchellus); deep precloacal groove in males (vs. a shallow in C. evanquahi); three or four dark dorsal bands (vs. six or seven bands in C. evanquahi and only four bands in C. pulchellus); white posterior caudal region absent (vs. present in C. evanquahi); hatchlings and juveniles without white tail tip (vs. present in C. evanquahi).

Discussion
Cyrtodactylus macrotuberculatus and C. phuketensis are considered to be conspecific with the latter restricted to Phuket Island whereas C. macrotuberculatus is found on the Thai-Malay Peninsula and adjacent islands. The distinct characteristics between these two species were based solely on morphological comparisons by Sumontha et al. (2012). Our study provided additional morphology and molecular evidence to reassess the taxonomic status of C. macrotuberculatus and C. phuketensis from Thai populations and determine that these closely related populations are conspecific. Phylogenetic Table 7. Diagnostic characters of Cyrtodactylus macrotuberculatus and related species within C. pulchellus complex. W = weak; P = prominent; / = data unavailable.
Some information was collected from the following literature (Grismer et al. , 2014(Grismer et al. , 2016Quah et al. 2019;Wood et al. 2020). analyses from this study are concordant with the phylogenetic studies of Grismer et al. ( , 2014Grismer et al. ( , 2016, Quah et al. (2019), andWood et al. (2020). Based on the dataset of ND2 gene and its flanking tRNA, the phylogenetic analyses recovered a clade of C. macrotuberculatus -including C. phuketensis -as a strongly supported monophyletic group consisting of multiple insular populations. Some substructuring occurs within the C. macrotuberculatus which could be the result of limited gene flow among isolated populations (Hurston et al. 2009;Jang et al. 2011) or local adaptation to different selection pressures in widely distributed habitat generalist. Sumontha et al. (2012) diagnosed C. phuketensis by the number of bands between the limb insertions and the presence of a precloacal groove in the female paratype. We re-examined the type series of both species (except female paratype of C. phuketensis, QSMI 1170) and newly collected specimens. Variation in the number of bands was found in both species, similar to several species of the C. pulchellus group such as C. bintangrendah, C. australotitiwangsaensis and C. lenggongensis (Grismer et al. , 2016. Within the C. pulchellus group, a continuous series of enlarged femoroprecloacal scales forming an inverted T in the precloacal region is present in both sexes; however, the precloacal groove was found only in males. In the present study, the newly collected female specimens from the type locality of C. phuketensis had a continuous series of enlarged femoroprecloacal scales but lacked a precloacal groove (or depression) (Fig. 9). Therefore, this character is the same as in C. macrotuberculatus and all other species in the C. pulchellus group. The presence of a precloacal groove in the female specimen of C. phuketensis examined in Sumontha et al. (2012) was an erroneous observation ( fig. 4 in Sumontha et al. 2012). The absence of a precloacal depression was used as a diagnostic character separating C. macrotuberculatus from C. phuketensis (see Grismer and Ahmad 2008;Sumontha et al. 2012). Based on the terminology of the precloacal depression in Mecke et al. (2016), the described specimens were re-examined and the presence of a precloacal depression (as precloacal groove) was observed in both C. macrotuberculatus (deep depression) and C. phuketensis (shallow depression). The PSUZC-RT 2010.58 and THNHM 15378 specimens are two males of C. phuketensis, in which the precloacal grooves are shallow (all others are deep) and could result from their poor state of preservation; thus, the character of this specimen was not included in the present diagnostic characters of C. macrotuberculatus.
Evidence from both overlapping ranges of morphology and relatively low sequence divergence indicate that C. phuketensis is an inconsistent pattern variation of C. macrotuberculatus. We concluded that C. phuketensis should be treated as a junior synonym of C. macrotuberculatus based on the priority of names designated by International Code of Zoological Nomenclature (ICZN). Additional surveys should be conducted to determine their geographic distribution and the degree of variation and patterns of gene flow within this species.