A new species of rock-dwelling Hemidactylus Goldfuss, 1820 (Squamata: Gekkonidae) from the southern Eastern Ghats, India

A new large-bodied (SVL 101–109 mm) gecko of the genus Hemidactylus is described from the Gingee Hills in the Eastern Ghats of India. The new species is closely related to H. graniticolus and the recently described H. easai , from which it can be distinguished by its lower femoral pores count. The new species described here was previously identified as H. cf. graniticolus based only on the molecular data, pending its formal description. Our findings were consistent with the results from the molecular DNA analyses, showing that this population is morphologically distinct from other closely related species. As a result, we formally describe this lineage as a new species, providing a comprehensive description of its morphological characteristics based on a type series of five specimens and compare it with its congenerics.


Introduction
The Eastern Ghats of India comprises a discontinuous series of mountains and hills, along the eastern coast of peninsular India extending from the state of Odisha in the north to Tamil Nadu in the south. The forested regions of peninsular India, which were once ancestral, became more arid during the early Oligocene epoch, followed by accelerated aridification in Miocene (Dettman et al. 2001). This trend continued into the Pleistocene and Holocene, because of the weakening of the Indian monsoon (Riedel et al. 2021). This resulted in the fragmentation of the forests, now primarily confined to small hill ranges and isolated hillocks, with a few remaining isolated forest blocks on the plains. These isolated hillocks have been previously claimed to act as microclimatic refugia  and are known to harbor several micro-endemic geckonids (Agarwal et al. 2019a(Agarwal et al. , 2022. The speciose gekkonid genus Hemidactylus Goldfuss, 1820 comprises 188 species of which 54 are known from India (Pal and Mirza 2022;Kumar et al. 2022; also see Uetz et al. 2023). Among the South Asian Hemidactylus, the well-supported prashadi group comprise twenty-two species of large-bodied geckos with a snout vent length (SVL) > 100 mm distributed throughout peninsular India and Sri Lanka (Agarwal et al. 2019b;Pal and Mirza 2022). Members of this group primarily inhabit granitic boulders, except for H. scabriceps (Annandale, 1906), which is terrestrial ). Among the Indian members of this group, nine species are restricted to the Western Ghats, while the rest are distributed throughout peninsular India (Agarwal et al. 2019b(Agarwal et al. , 2011Das et al. 2022;Pal and Mirza, 2022) with two species more widely reported from the Western Ghats, the Eastern Ghats and the Mysore plateau (Mirza et al. 2018). Within the prashadi group, the graniticolus clade (sensu Agarwal et al. 2019b) currently comprises three described species Hemidactylus graniticolus Agarwal et al., 2011 andH. kolliensis Agarwal et al., 2019 from the eastern Ghats and Mysore plateau and the recently described H. easai Das et al., 2022 from the Western Ghats. Both Agarwal et al. (2019b) and Das et al. (2022) recognized three undescribed lineages from the Eastern Ghats in their phylogenetic analysis and tentatively identified them as H. cf. graniticolus.
In this study, we analyzed the morphological features of one of these three undescribed lineages, utilizing fresh materials from the Gingee Hills in the southern Eastern Ghats. Additionally, we obtained mitochondrial ND2 sequences from fresh specimens. Based on our results from both morphological and molecular analysis, we herein describe the monophyletic lineage from the Gingee Hills as a new species.

Specimen collection
Five specimens (BNHS 2907-BNHS 2911) of Hemidactylus sp. were collected from Pakkamalai Hill, Tamil Nadu, India (12.164819°N, 79.250909°E) (Fig. 1). Collected individuals were euthanized using isoflurane after taking live photographs. Specimens were then fixed in 4% formalin after collecting liver/tail tissues that are stored in absolute ethanol (99.9%) at -20°C for molecu-lar analysis. Fixed specimens were then washed and kept in water overnight and subsequently transferred to 70% ethanol.

Molecular analysis
We extracted genomic DNA from liver tissue samples stored in absolute ethanol at -20°C, using the DNeasy (Qiagen TM ) blood and tissue kit. We amplified the partial sequence (1041 base pairs) of the mitochondrial ND2 gene, for two specimens (BNHS 2907 & BNHS 2911) using the following primers: MetF1 and H5934 (Macey et al. 1997). PCR conditions were as described in Das et al. (2022).
Bidirectional sequences were manually checked using the CHROMAS v.2.6.6 software (http://technelysium. com.au/wp/chromas) and aligned using ClustalW (Higgins et al. 1994) with default prior settings implemented in MEGA v.7 (Kumar et al. 2016). We checked for unexpected stop codons by translating the sequence to amino acids in MEGA v.7 (Kumar et al. 2016). The new sequences generated in this study was aligned with 39 sequences of Hemidactylus from the prashadi group deposited in Genbank and two other species, Dravidogecko septentrionalis Chaitanya et al., 2019 andD. douglasadamsi Chaitanya et al., 2019 that are used as outgroups for the phylogenetic analysis (see Supplementary Material 1: Table S1).
Maximum Likelihood (ML) analysis for the final dataset was carried out using IQTREE (http://iqtree.cibiv.univie.ac.at) (Trifinopoulos et al. 2016). The best-fit models for partition scheme suggested by the Partitionfinder v1.1.1 (Lanfear et al. 2017) were determined using the in-built Modelfinder (Kalyaanamoorthy et al. 2017) for the ML analysis. The best-fit models for the ML analysis are as follows, ND2 position1: TVM+F+G4, ND2 posi-tion2: TPM3u+F+G4, ND2 position3: TIM+F+I+G4. A Bayesian Inference (BI) analysis was carried out using MrBayes v.3.2 (Ronquist et al. 2012), with default prior settings. For this analysis, we used the best-fit models suggested by Partitionfinder for each partition as follows, ND2 position1: TVM+I+G, ND2 position2: TVM+G, ND2 position3: TrN+I+G. Four separate runs were set up with eight Metropolis-Coupled Markov Chain (MC3) Monte Carlo each initiated from random trees and allowed to run for 10 × 10 6 million generations, sampling every 100 generations. Analyses were terminated when the standard deviation of split frequencies was less than 0.001, the first 25% of trees were discarded as "burn-in", and trees were constructed under 50% majority consensus rule. We obtained ESS values using the Tracer software and confirmed the convergence for all the priors (ESS > 200). Support for internal branches in ML and BI trees was quantified using 1000 pseudoreplicates (ultrafast bootstrap UFB) and posterior probability PP, respectively. We considered PP above 0.95 and UFB above 95 as strong support. The uncorrected pairwise genetic distance (p-distance) was calculated in MEGA7 using default settings (pairwise deletion).

Morphological analysis
All the morphological characters (morphometric and meristic) examined in this work were implemented following Agarwal et al. (2019). Measurements and meristic data from the collected specimens for this study were taken under a Nikon SMZ1270 stereo microscope and using a Mitutoyo digital vernier caliper (accuracy 0.1mm). Measurements and meristic characters were as follows: snout vent length (SVL, from tip of snout to the cloacal opening; axilla to groin length (AGL, from posterior margin of forelimb insertion to anterior margin of hind-limb insertion); forearm length (FL, from posterior margin of elbow while flexed 90º to distal end of wrist); crus length (CL, from the posterior surface of the knee while flexed 90º to the base of the heel); tail length (TL, from the cloacal opening to tip of tail); head length (HL, distance from the posterior margin of the retroarticular process to the tip of the snout); head width (HW, maximum width of head); head depth (HD, maximum head depth at occiput); eye diameter (ED, greatest horizontal diameter of eye); eye to naris distance (EN, distance between anterior margin of eye and posterior edge of nostril); eye to snout distance (ES, distance between anterior margin of eye and tip of snout); eye to ear distance (EE, distance from anterior edge of ear opening to posterior margin of eye); ear length (EL, maximum length of ear opening); internarial distance (IN, distance between nares); interorbital distance (IO, shortest distance between left and right supraciliary scale rows). Additional meristic characters include: longitudinal rows of enlarged dorsal tubercles at midbody (DTR); number of paravertebral tubercles between limb insertions (PVT); the number of ventral scale rows at midbody between the lowest rows of dorsal scales (MVSR); femoral pores in the femoral region in males; the number of pore-less scales between the series of femoral pores and the number of undivided lamellae on all the digits in manus and pes.
Comparative data and other morphological data for the large-bodied prashadi group were obtained from literature and the original descriptions (Chaitanya et al. 2018;Giri, 2008;Khandekar et al. 2021;Mirza et al. 2018;Mirza and Sanap 2014;Srikanthan et al. 2018;Amarasinghe et al. 2021;Das et al. 2022). The museum abbreviation are as follows: BNHS: Bombay Natural History Society, Mumbai, India; CES: Centre for Ecological Sciences, Indian Institute of Science, Bengaluru, India.

Molecular analysis
Both ML and BI analyses recovered similar topology for the prashadi group. The graniticolus clade is recovered as a strongly supported (UFB: 100, PP: 1.0) monophyletic clade with two subclades, one that includes H. easai + H. graniticolus and the other clade that includes the new species here described with two other undescribed lineages (Fig. 2). The new species described here is nested together with a sample reported in Lajmi and Karanth (2020) collected from a hillock within the Gingee Hills cluster, with strong support (UFB: 100, PP: 1.0). These three samples are together sister to two other samples identified as H. cf. graniticolus 1 from the adjacent Shevaroy landscape with low support in ML and BI (UFB: 59, PP: 0.86). The uncorrected pairwise genetic distance between the new species and other lineages (including the two undescribed lineages) within the graniticolus clade range between 5.6% and 10.3%, in the mitochondrial ND2 gene (Table 1).

Morphological analysis
Morphological measurements and counts are summarised in Table 2 and in the comparison section below. The overall morphology and colour pattern for the members of the graniticolus clade and the new species described here are similar. We found significant differences in the femoral pores between these species and dorsal pholidosis. The new species described here is characterized by a combination of non-overlapping characteristics like the number of femoral pores, dorsal tubercle rows and ventral scales along the midbody. Etymology. The specific epithet is a toponym named after its type locality Pakkamalai Hill. We suggest an English name Pakkamalai rock gecko and a Tamil name பாக் கமைல பாைறப் பல் லி.

Diagnosis.
A large-sized gecko of the genus Hemidactylus, snout-vent length up to a maximum SVL of 109 mm (n = 5). Dorsal pholidosis heterogeneous, composed of roughly circular, granular scales intermixed with much enlarged, fairly regularly arranged longitudinal rows of 19-22 striated subtrihedral tubercles at midbody. Enlarged tubercles on the two most medial parasagittal rows are small, subconical, strongly keeled and rounded, gradually increasing in size and becoming conical towards flanks, last two to three rows on flanks smaller, conical. Two well-developed pairs of postmentals, the inner pair longer than the outer pair and mental, and in broad contact behind the mental. Paravertebral rows with 21-24 tubercles; 34-38 transverse ventral scale rows at mid-body. Digits with enlarged scansors, lamellae in straight transverse series; two or three undivided basal lamellae beneath first finger and two or three beneath first toe; one or two undivided basal lamellae beneath fourth toe; ten or 11 lamellae (including undivided and divided) beneath first finger and nine or ten beneath first toe; 12 or 13 lamellae (including undivided and divided) beneath fourth finger and 12-14 beneath fourth toe; males with series of 19-21 femoral pores on each side separated by four or five poreless scales. Original tail depressed, oval in transverse section with a median dorsal furrow; scales on the tail slightly larger than dorsals, striated, with longitudinal series of 4-8 large, keeled, striated, posteriorly pointed tubercles.
Dorsal colouration dull-brown with a series of four or five transverse pale saddles from occiput to sacrum, tail with distinct alternating light and dark bands.    Description of holotype (BNHS 2907). The holotype, an adult male, (SVL 104 mm) is well preserved. Head short (HL/SVL 0.29), slightly elongated (HW/HL 0.66), not strongly depressed (HH/HL 0.29), distinct from neck. Loreal region slightly inflated, and canthus rostralis indistinct (Fig. 4C). Snout short (SE/HL 0.40); slightly longer than twice eye diameter (OD/SE 0.45); scales on the snout, canthus rostralis, forehead and inter-orbital region heterogeneous, mostly granular and conical; scales on the snout and canthus rostralis much larger than those on occiput, forehead and inter-orbital regions; skin in the frontal slightly damaged. Eye small (OD/HL 0.18); pupil vertical with crenulated margins; supraciliaris small, mucronate, gradually increasing in size towards the front of the orbit, largest about one-third the way from the anterior edge of orbit; a few supraciliaris in the suprocular region in the left eye damaged and missing. Ear opening elliptical (2.8 mm); a row of small keeled tubercles above the ear.
Rostral scale wider than deep (RL/RW 0.68), partially divided dorsally by a weakly developed rostral groove; nasals, enlarged and separated by three small scales; one supranasal on each side, smaller than internasals; two diminutive postnasals on each side; rostral in contact with nasal scale, supralabial I, internasals and the anterior small scale separating the internasals; nostrils small (0.8 mm), oval; nasal scale surrounded by supranasal, internasal, rostral, supralabial I and three postnasals on either side; five (on the anterior) and three (on the posterior) rows of scales separate orbit from supralabials.
Mental triangular; two well-developed pair of postmentals, the inner pair smaller (3.4 mm) than the mental (4.6 mm), and in strong contact with each other (1.2 mm) behind mental, outer pair shorter (2.2 mm) than the inner pair and separated from each other by inner pair (Fig. 4B). Inner postmentals bordered by mental, infralabial I and II on left and right, outer postmental and 10 small gular scales on each side; outer postmentals bordered by infralabial II on the right and left, inner postmental, and five gular scales on the right and six gular scales in the left along with two other large scales between the gular scales and the infralabials; one to three additional rows of scales below infralabials III to VIII are enlarged and weakly imbricate. Supralabials (on both sides) to mid orbital position 10, to angle of jaw 12 on right and left; infralabials 10 and nine on right and left side, respectively. Body relatively stout (BW/SVL 0.22), ventrolateral folds not very distinct.
Dorsal pholidosis heterogeneous, composed of subcircular granular scales intermixed with enlarged, fairly regularly arranged strongly keeled. Pointed tubercles in 21 longitudinal rows, extending from occiput to tail, that are heterogeneous in shape and size; enlarged tubercles on the two most medial parasagittal rows slightly smaller than rest on dorsum and the rows most broadly spaced from one another, gradually increasing in size and becoming conical towards flanks, last two rows on flanks slightly smaller than medial parasagittal rows and strongly conical; each enlarged tubercle surrounded by a rosette of 13-16 small granules with 2-5 granules between two longitudinally adjacent enlarged tubercles (5-11 between parasagittal rows at midbody); enlarged tubercles on nape and shoulder smaller and conical, those on occiput and the temporal region still smaller, conical.
Ventral scales larger than granular scales on the dorsum, smooth, imbricate, slightly larger on precloacal and femoral region than on chest and abdominal region; midbody scale rows across belly 34; gular region with small, granular scales, becoming slightly larger and imbricate on anterior and lateral aspect. Scales on palm and sole smooth, imbricate, subcircular; scales on the dorsal aspect of upper arm slightly smaller than the last row of enlarged tubercles on dorsum, flat, weakly pointed, imbricate, keeled; dorsal aspect of forearm with smaller, granular scales, intermixed with a few enlarged, conical tubercles, those on anterior aspect are smooth, flat, imbricate; scales on dorsal part of thigh and shank granular, imbricate, intermixed with enlarged, conical tubercles, which are larger on thigh compared to shank that are subequal to those tubercles in the dorsal aspect of the body; anterior aspect of thigh with flatter scales, posterior aspect with granular scales.
Tail complete regenerated (about 30% of the tail original); depressed, flat beneath, verticillate, with well-defined median furrow; scales on the dorsal aspect of tail subimbricate, larger than granules on dorsum, with a series of 4-6 much enlarged, strongly pointed, moderately keeled tubercles; ventral scales enlarged, imbricate, median row (subcaudal plates) covering almost entire base of the tail, bordered laterally by two or three rows of larger pointed, smooth, imbricate scales; those close to vent small, smooth, flat and imbricate. Two indistinct postcloacal spurs on each side, much smaller than dorsal tubercles at midbody.

Colour in life and preservative (based on holotype).
Dorsal aspect of the body, uniformly brownish with three indistinct transverse bands from the neck to the hind limb insertion and one on the tail, bordered by discontinuous brownish patches extending towards the lateral side of the body (Fig. 6A). Dorsal aspect of the head is brownish with inconspicuous white markings on the parietal and neck region. Ventral aspect of the head is mostly cream-coloured with light brown mottling on the gular region and mental, infralabials, post mentals and a few other adjacent scales mottled with dark brown. Fore and hindlimbs are brownish speckled with irregular black patches and a few scattered brownish-yellow spots on the dorsal side. Ventral aspect of the body, forelimbs and hindlimbs predominantly cream coloured with sprinkled and or mottled with brown colour. In preservative, the overall colouration is the same as in life except in dark transverse bands on the dorsum that became less prominent.
Distribution and natural history. Hemidactylus pakkamalaiensis sp. nov. is currently known from the Gingee Hills cluster. This species is nocturnal and appears to be locally abundant. In addition to the collected specimens, we encountered 56 live individuals in and around the collection site during our four-day fieldwork. All the specimens in the type series were gathered from rock boulders within a limited area at the type locality, mostly after 19:00 hrs. These hills are predominantly formed of granitic rock boulders and feature native vegetation, including thorny scrub jungle, moist deciduous forest, and tropical dry evergreen forest (Arulappan et al. 2015).

Discussion
The description of Hemidactylus pakkamalaiensis sp. nov. expands the known diversity of Hemidactylus in India to 55 and the prashadi group to 23 Uetz et al. 2023). Although this lineage was previously identified through phylogenetic data, it had not been formally described until now (Lajmi and Karanth 2019;Agarwal et al. 2019;Das et al. 2022). The Gingee Hills are part of the larger Southern Granulitic Terrain (SGT), which consists mostly of granitic gneiss with an origin in the mid-Archean to the Neoproterozoic era (Fermor 1936). The Gingee Hills are characterized by a significant presence of granitic rock boulders that provide a suitable micro-habitat for rock-dwelling geckos. These hills are unique in this regard, and despite being situated in the drier part of peninsular India, Pakkamalai Hill is home to a diverse range of vegetation types, including the distinctive Tropical Dry Evergreen Forest (Arullappan et al. 2009). This combination of vegetation and the microhabitats availability likely provided an ambient microclimatic condition that facilitated the diversification of this species. However, our survey found that H. pakkamalaiensis sp. nov. was present in all habitat types. This would suggest that the substrate (rock boulders) is the primary requirement of this species rather than the type of vegetation. Previous studies have demonstrated a high diversity in other gekkonid genera that are also restricted to small hill ranges within this region (Agarwal et al. 2019a(Agarwal et al. , 2022. Agarwal et al. (2020) proposed that microclimatic conditions within the granitic hill ranges in the region were a potential reason for the diversifica-tion in the genus Cnemaspis. The current study concurs with the previous recognition of this lineage as a distinct species based on morphological evidence. We follow the evolutionary species concept to delimit the monophyletic lineage from Gingee Hills as a distinct species (Simpson 1951;Wiley 1978;de Queiroz 2007).
Although Hemidactylus pakkamalaiensis sp. nov. is genetically related to H. easai and H. graniticolus, it differs significantly from these closely related species by having fewer femoral pores, with a count of 19-21 as compared to 24-28 in the latter two species. One of the samples (CES 12565) used here in the molecular analyses, collected from the adjacent hill to the Pakkamalai (Lajmi and Karanth 2020), shows a shallow divergence (2.8%) with the samples of H. pakkamalaiensis sp. nov. collected in the present study. While this specimen could not be located and examined, we recover it as H. pakkamalaiensis sp. nov. solely based on the low level of divergence, which is consistent with the intraspecific divergence observed within the H. graniticolus sensu stricto.
In our phylogeny, we recovered two sister lineages to H. pakkamalaiensis sp. nov. that could potentially represent distinct lineages. Previously, Agarwal et al. 2019b recognized these two as distinct lineages based on their species delimitation analyses and the genetic divergence cut-off. Hemidactylus pakkamalaiensis sp. nov. is geographically separated from the sister lineage H. cf. graniticolus 1 from the Shervaroys landscape by the intervening plains which are an unsuitable habitat for these large rock-dwelling geckos. We concur with the aforementioned hypothesis as these lizards have disjunct distributions and are isolated by geographical separation with no obvious contact zones. However, as previously stated in Figure 7. Habitat at the type locality (Pakkamalai Hill, Tamil Nadu, India) of Hemidactylus pakkamalaiensis sp. nov. Das et al. (2022), we failed to locate these specimens in the collections and their morphological affinity with H. pakkamalaiensis sp. nov. remains unclear.
Globally nine new species of Hemidactylus were described in the year 2022 out of which seven were from peninsular India (Adhikari et al. 2022;Kumar et al. 2022;Pal and Mirza 2022; also see Uetz et al. 2023). Additional surveys are required to uncover the true species diversity of Hemidactylus in peninsular India. Furthermore, finescale sampling is required to understand genetic structuring within species occurring in isolated hillocks across this landscape.