A new species of large-bodied Hemidactylus Goldfuss, 1820 (Squamata: Gekkonidae) from the Western Ghats of India

A new large (94–107mm SVL) species of gecko of the genus Hemidactylus is described from the drier parts of the Western Ghats of India. The new species is closely related to H. graniticolus, from which it can be distinguished based on dorsal pholidosis at midbody, the structure of tubercles on the dorsum, dorsal pholidosis on the tail. The new species is also 6.6–7.2% divergent from H. graniticolus in the ND2 mitochondrial gene.


Introduction
The gekkonid genus Hemidactylus Goldfuss, 1820 is one of the most specious groups of geckos with 180 known species distributed across the globe (Uetz et al. 2021). Nearly one-third of this diversity, 48 species, is found in India (Uetz et al. 2021). A recent study showed the role of the Eocene-Oligocene cooling that facilitated the early diversification of Hemidactylus spp. in peninsular India (Lajmi and Karanth 2020). Furthermore, it is shown that most of the diversification in the endemic Indian radiation of Hemidactylus spp. occurred in the drier zones with Vertebrate Zoology 72, 2022, 81-94 | DOI 10.3897/vz.72.e76046 Copyright  multiple dispersals into Sri Lanka (Lajmi et al. 2019). Among the south Asian Hemidactylus, prashadi clade is well-supported (Agarwal et al. 2019a andLajmi et al. 2019) and comprises 20 described species that are largely restricted to peninsular India and Sri Lanka (Khandekar et al. 2021). This group can be differentiated from the other members of the genus by their having mostly medium to large adult body size .

Molecular analysis
We collected four specimens of Hemidactylus sp. and generated DNA sequence for two specimens of Hemidactylus sp. (ZSI/WGRC/IR.V/3471 & BNHS 3103) from Pattimalam, Attapadi, Palakkad district, Kerala, India (11.146397°N, 76.65939°E, 530m asl) (Fig. 1). Specimens were euthanized and fixed in 4% formalin and later washed and transferred to 70% ethanol for preservation. We extracted genomic DNA from liver tissue sample stored in absolute ethanol at -20°C, using the DNeasy (QiagenTM) blood and tissue kit. We amplified the complete sequence (1041 base pairs) of the mitochondrial ND2 gene using the following primers: MetF1 (Forward) and H5934 (Reverse) (Macey et al. 1997). PCR conditions were as follows: initial denaturation at 95°C for 3 min, followed by 39 cycles of denaturation at 95°C for 35 sec, annealing at 55°C for 40 sec and extension at 72°C for 1 min 30 sec. The final extension was at 72°C for 5 min.
Bidirectional sequences were manually checked using the CHROMAS 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 7 (Kumar et al. 2016). We checked for unexpected stop codons by translating the sequence to amino acids in MEGA7 (Kumar et al. 2016 A Maximum Likelihood (ML) analysis was carried out with RAxML GUI version 2.0 (Edler et al. 2021) implementing the GTRGAMMAI for the dataset partitioned by codon position. A Bayesian Inference (BI) analysis was carried out using the program MrBayes 3.2 (Ronquist et al. 2012), with default prior settings. The dataset was partitioned by codon positions using PartitionFinder v1.1.1 (Lanfear et al. 2017) with default settings to find the best-fit model of sequence evolution. Partition finder suggested three partitions as follows, position1: TVM+I+G, position2: TVM+G, position3: TrN+I+G. Four separate runs were set up with eight Markov chains 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 replicates (thorough bootstrap) and posterior probability, respectively. We considered BI>0.95, ML>85 as strong support. The uncorrected pairwise genetic distance was calculated in MEGA7 using default settings (pairwise deletion).

Morphological analysis
All the morphological characters examined (mensural and meristic) follow Agarwal et al. (2019a). Measurements and meristic data from the collected specimens for this study were taken under a Nikon microscope and were measured using a Mitutoyo digital Vernier calliper to the nearest 0.1mm. Photographs were taken using Canon EOS 7D mounted with a 100mm macro lens and two external flashes. Measurements and meristic characters include: snout vent length (SVL, from tip of snout to vent); axilla to groin length (AGL, from posterior margin of forelimb insertion to anterior margin of hindlimb insertion); body width (BW, maximum body width); 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 vent to tip of tail); tail width (TW, measured at widest point 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 DTR: longitudinal rows of enlarged dorsal tubercles at midbody; PVT: number of paravertebral tubercles between limb insertions; MVSR: the number of ventral scale rows at mid-body between the lowest rows of dorsal scales; 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.

Molecular analysis
In both ML and BI analyses, the new species is recovered as a sister to the Hemidactylus graniticolus sensu stricto clade with strong support (ML 88, BI 1.0) and are together forming a strongly supported (ML 100, BI 1.0) monophyletic group with three other putative species identified as H. cf. graniticolus (see, Agarwal et al. 2019a) (Fig.  2). The uncorrected pairwise genetic distance is 6.6-7.2% between the new species and other samples of H. graniticolus (Table 1).
Description of holotype. The holotype, an adult male, (SVL 105.8 mm) well preserved and is in good condition. Head short (HL/SVL 0.30), slightly elongate (HW/ HL 0.76), not strongly depressed (HH/HL 0.41), distinct from neck. Loreal region slightly inflated, canthus rostralis indistinct (Fig. 4C). Snout short (SE/HL 0.46); slightly longer than twice eye diameter (OD/SE 0.45); scales on snout, canthus rostralis, forehead and inter-orbital region heterogenous, mostly granular and conical; scales on the snout and canthus rostralis much larger than those on occiput, forehead and inter-orbital regions. Eye small (OD/ HL 0.19); pupil vertical with crenulated margins; supraciliaries small, mucronate, gradually increasing in size towards the front of the orbit, largest about one-third the way from anterior edge of orbit. Ear opening elliptical (greatest diameter 2.2 mm); a row of small pointed tubercles above the ear; eye to ear distance slightly greater than the diameter of eye (EE/OD 0.71). Rostral wider than deep (RL/RW 0.65), partially divided dorsally by a weakly developed rostral groove; two internasals, 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 two postnasals on either side; 4-6 rows of scales separate orbit from supralabials. Mental triangular; two well-developed postmentals, the inner pair smaller (3.3 mm) than the mental (4.4 mm), and in strong contact with each other (1.1 mm) behind mental, outer pair shorter (1.7 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 six small gular scales on each side; outer postmentals bordered by infralabial II on the right and separated by a large scale on the left, inner postmental, and five gular scales on both side along with one large scale on the right side between the third infralabial and the outer postmental; four to five additional rows of scales below infralabials III to VIII are enlarged and weakly imbricate. Supralabials (on both sides) to mid orbital position nine, to angle of jaw 11 on right and left; infralabials 9 on each side. Body relatively stout (BW/SVL 0.20), ventrolateral folds distinct. Dorsal pholidosis heterogeneous, composed of subcircular granular scales intermixed with enlarged, fairly regularly arranged strongly keeled, pointed tubercles in 18 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 12-15 small granules with 2-5 granules between two longitudinally adjacent enlarged tubercles (3-6 between parasagittal rows at midbody); enlarged tubercles on nape and shoulder smaller and conical, those on occiput and the temporal region still smaller, conical.
Tail regenerated; 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 4-10 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 (Fig. 3D). Two subequal rounded postcloacal spurs on each side are much smaller than dorsal tubercles at midbody (Fig. 3D).
Variations in the paratypes. Variations in the paratypes in some of the meristic and mensural characters are provided in the Table 2. Two paratypes are females. BNHS 3104 has an incomplete tail and partially broken in BNHS 3103 at the base, but intact (Fig. 6B). Internasals in BNHS 3103 are separated by two small scales. In ZSI/WGRC/ IR.V/3472 & BNHS 3104 outer post mental on the left side is in contact with the third supralabial and a distinct small scale is present between the mental and the inner post mental on the right side in ZSI/WGRC/IR.V/3472. Skin in the frontal region of the head in BNHS 3104 is slightly damaged but intact (Fig. 6C). In life, all the paratypes were comparatively lighter in dorsal colouration and this, however, is not significant in preservative.
Color in life (based on holotype). Dorsal aspect of the body, uniformly brownish with four indistinct transverse bands from the neck to the hind limb insertion and two on the tail, bordered by discontinuous black patches (Fig.  7A). Dorsal aspect of the head without any dark markings in the parietal and frontal region. A preorbital yellow streak extends from the posterior of the naris to the anterior of the eye on both sides. Ventral aspect of the head mostly cream coloured with light brown mottling on the gular region and mental, infralabials, post mentals and few other adjacent scales mottled with dark brown. Fore and hindlimbs brownish speckled with irregular black patches on the dorsal side. Ventral aspect of the body, forelimbs and hindlimbs predominantly cream coloured with sprinkled and or mottled with brown colour.
From the closely related Hemidactylus graniticolus, H. easai sp. nov. can be differentiated by the presence of moderate to strongly keeled tubercles on the dorsum (vs. weakly keeled or smooth tubercles on the dorsal aspect) (Fig. 5); enlarged tubercles on the two most medial parasagittal rows subconical, strongly keeled (vs. tubercles on the two most medial parasagittal rows flattened, smooth to weakly keeled); dorsal pholidosis of tail with slightly larger granular, striated scales and longitudinal series of 6-8 large, keeled, striated, posteriorly pointed tubercles (vs. dorsal pholidosis of tail with small, imbricate, striated scales and a series of four enlarged, keeled weakly striated and flattened tubercles).
Correct spelling of Hemidactylus paaragowli Srikan than, Swamy, Mohan & Pal 2018. Srikanthan et al. (2018), in the original description, included two different spellings, "Hemidactylus paaragowlipaaragowli"(abstract) or "Hemidactylus paaragowli" (keywords, description). However, the original authors or the subsequent authors working on the systematics of Hemidactylus from India did not correct this nomenclatural error but used the name "Hemidactylus paaragowli" (Mirza et al. 2018;Agarwal et al. 2019a;Lajmi and Karanth 2020;Khandekar et al. 2020 1999), we act as the first reviser and choose Hemidactylus paaragowli as the correct spelling since this spelling is used in the formal description part and higher usage (20 times) over the incorrect "Hemidactylus paaragowlipaaragowli" which is cited only once in the abstract of the original description (Srikanthan et al. 2018).

Discussion
The rate at which Hemidactylus are being described from India is high in the last decade, with 16 new descriptions of the total 48 known species (Uetz et al. 2021;Khandekar et al. 2021 5F) collected from "Malabar", is morphologically similar to H. graniticolus than to the new species but a precise location for this specimen is lacking. We also examined the specimen (CES08026) reported as H. graniticolus by Lajmi and Karanth (2020) from the Nilgiri Hills in the Western Ghats and we tentatively refer to this specimen as H. easai sp. nov., because of the presence of prominent and moderately keeled tubercles along the paravertebral rows but, we were unable to check its phylogenetic posi- tion since there is no ND2 sequence data. Furthermore, in our phylogeny, five samples representing three distinct lineages, previously identified as "H. cf. graniticolus" are closely related to H. easai sp. nov. We were unable to trace these specimens at the CES collections and hence the identity of these specimens remains unknown. However, all these three lineages, phylogenetically appear to be independent lineages and the genetic pairwise distance between any of these pairs including H. easai sp. nov. is 5.7%-10% and thus do not challenge our hypothesis of H. easai sp. nov. as a new species.
The dry zones of peninsular India are known for its high endemism in many squamate reptiles (Hemidactylus: Lajmi et al. 2019Lajmi et al. , 2020Hemiphyllodactylus: Agarwal et al. 2019b;Sitana: Deepak & Karanth 2018;Cnemaspis: Agarwal et al. 2020), largely due to the landscape heterogeneity. The isolated hills in peninsular India have played an important role in speciation of lizards (Agarwal et al. 2019b. Furthermore, in some cases the rivers in peninsular India have also played a role in reproductive isolation and speciation (e.g Sitana: Deepak & Karanth 2018;Platyceps: Deepak et al. 2021). Hemidactylus easai sp. nov. and Hemidactylus graniticolus sensu stricto (including samples identified as H. cf. graniticolus) are also geographically separated by the river Cauvery.
With the current description and the available distributional data, it is likely that H. graniticolus may be restricted to the Mysore plateau and parts of the southern Eastern Ghats. Hemidactylus easai sp. nov. is currently known from the north of the Palghat gap and is nested within the clade containing species from the Mysore plateau and the southern Eastern Ghats, in our phylogenetic analyses (Fig. 2). Although H. easai sp. nov. is currently known only from the type locality, it is likely that this species is distributed in the adjacent regions with similar habitat. However, we have not sampled extensively in the Palghat gap nor immediately south of the gap for Hemidactylus species. Given the patchy sampling and incomplete data, we refrain from commenting on the relationships of these species and their distribution in the south of Palghat gap. Additional sampling in this region and the eastern slopes of southern Western Ghats may unveil actual distribution of H. easai sp. nov. and the true diversity of Hemidactylus in peninsular India and their systematic relationships.