Cnemaspis flaviventralis , a new species of gecko ( Squamata : Gekkonidae ) from the Western Ghats of Maharashtra , India

Cnemaspis flaviventralis, a new species of day gecko, is described from the forests of Amboli, Sindhudurg District, Maharashtra State, northern Western Ghats, India. The new species was previously confused with the sympatric species Cnemaspis girii, C. indraneildasii, C. kolhapurensis and C. goaensis. It is distinguished from C. giri by having spine-like tubercles on flanks, granular dorsal scales intermixed with large, depressed, slightly keeled scales (vs. lack of spine-like tubercles on flanks, granular dorsal scales, intermixed with large smooth scales); from C. indraneildasii by having dorsal scales heterogeneous (vs. homogenous), lacking a series of enlarged median sub-caudal scales, and 28–29 (vs. 20) ventral scales across mid-body; from C. kolhapurensis by having heterogeneous (vs. homogenous) dorsal scalation, lacking spine-like tubercles on flanks and lacking pre-cloacal pores (vs. 24–28 pre-cloacal-femoral pores); and from C. goaensis by lacking pre-cloacal pores and lacking a series of enlarged median sub-caudal scales. We further provide partial mitochondrial 16S rRNA gene sequences for the new species and for the sympatric species C. girii, C. kolhapurensis and C. goaensis, and show that the new species is genetically distinct.


INTRODUCTION
The species-rich lizard genus Cnemaspis Strauch, 1887 in the family Gekkonidae includes at least 120 Old World species (Uetz & Hošek 2015) distributed from Africa to South-east Asia.As presently understood, the widespread genus Cnemaspis appears to be polyphyletic (Gamble et al. 2012;Pyron et al. 2013;Grismer et al. 2014).Further, cryptic and underestimated diversity is also very high in Cnemaspis (e.g., Manamendra-Arachchi et al. 2007;Grismer et al. 2014;Amarasinghe et al. 2015).The Western Ghats-Sri Lanka biodiversity hotspot is becoming a hotspot for Cnemaspis with several new species described in recent years (e.g., Manamendra-Arachchi et al. 2007;Giri et al. 2009;Cyriac & Umesh 2014;Mirza et al. 2014;Vidanapathirana et al. 2014;Srinivasulu et al. 2015).Given the extreme morphological similarity among species within the group, their small size and the generally overlooked nature of the South Asian herpetofauna, discovering new species is not surprising.
Based on a new collection from the northern Western Ghats of Maharashtra, India, we describe here a new species of Cnemaspis to make a name available and facilitate their conservation.We also provide a molecular analysis based on 16S rRNA genomic sequences that may facilitate future studies in the region.

Specimen collection
Specimens of the type series were collected from Amboli (15.960 0 N & 73.999 0 E, elevation 735m), Sindhudurg District, Maharashtra, India.Four males and four female specimens were collected during night.Two specimens of Cnemaspis girii Mirza, Pal, Bhosale & Sanap, 2014 were collected from Kaas plateau (17.724 0 N & 73.819 0 E), the type locality of the species; two specimens of C. kolhapurensis Giri, Bauer & Gaikwad, 2009 were collected from Amboli (15.971 0 N & 73.979 0 E); and two specimens of C. goaensis Sharma, 1976, were collected from Goa (15.290 0 N & 74.006 0 E).They were anaesthetized, fixed in formalin, and preserved in 70% ethyl alcohol.Geographic coordinates and atmospheric temperature readings were taken at the collection localities with the aid of a Kestrel 4500 weather meter.

Museum details
Specimens from the current study are deposited in the collections of the Bombay Natural History Society, Mumbai (BNHS) and the Zoological Survey of India, Western Regional Center (ZSI-WRC), Pune, Maharashtra.
Comparative materials were examined at the Zoological Survey of India, Kolkata (ZSI-K).

Morphometric and meristic data collection
Following measurements were taken using a Yamayo digimatic caliper to the nearest 0.1mm following Giri et al. (2009) and Mirza et al. (2014), and include snout vent length (SVL, from tip of snout to vent), trunk length (TRL, distance from axilla to groin measured from posterior edge of forelimb insertion to anterior edge of hind limb insertion), trunk width (TrW, maximum width of body), tail length (TL, from vent to tip of tail), tail width (TaW, measured at widest point of tail), head length (HL, distance between retroarticular process of jaw and snout-tip), head width (HW, maximum width of head), head depth (HD, maximum depth of head, from occiput to underside of jaws), forearm length (FL, from base of palm to elbow), tibia length (TBL, knee to tarsal), eye to nares distance (E-N, distance between anteriormost point of eye and nostril), eye to snout distance (E-S, distance between anteriormost point of eye and tip of snout), eye to ear distance (E-E, distance from anterior edge of ear opening to posterior corner of eye), Ear length (EL, greatest diameter of ear opening), internarial width (IN, distance between nares), orbital diameter (OD, greatest diameter of orbit), inter orbital distance (IO; greatest distance between left and right supraciliary scale rows).Following meristic data were taken for all specimens under a Leica stereo microscope: supralabial scales (SupraL), infralabial scales (InfraL), femoral pores (FPores), lamellae under digits of manus (MLam) and pes (PLam) for both left (L) and right (R) sides (lamellae counts taken from the scale just behind claw to first interphalangeal joint excluding large scansors), longitudinal scale rows across the belly (in between the lowest rows of lateral granular scales (MVS).

Genetic analysis
Muscle tissue harvested from two fresh specimens of the new species (ZSI-WRC 1042, 1043) collected from same locality of holotype, two specimens of C. girii (BNHS 2445(BNHS , 2446)), two specimens of C. goaensis (ZSI R/1044 and 1045) and two specimens of C. kolhapurensis (BNHS 2447(BNHS , 2448)).Tissue samples were digested at 55 0 C using STE buffer (50 mM Tris-HCl, 20 mM EDTA and 50μl of 10% SDS) with 10 μL of 20 mg/ ml Proteinase K. RNase treatment was given for two hours at 37 0 C. Final extraction process was done using Additional sequences for phylogenetic analysis were downloaded from NCBI database.Eublepharis macularius (Blyth, 1854), a member of family Eublepharidae, was used as an outgroup.Gene sequences were aligned using MUSCLE (Edgar 2004).Best fit nucleotide substitution model was selected from 56 models available in PhyML (Guindon et al. 2010) using TOPALi v2 (Milne et al. 2008) based minimum Bayesian Information Criterion (BIC) value (Schwarz 1978;Nei & Kumar 2000).Best nucleotide substitution matrix was used to perform maximum likelihood analysis using PhyML (Guindon et al. 2010).Reliability of the phylogenetic tree was estimated using bootstrap values run for 1000 iterations.Phylogenetic tree was edited in FigTree v1.4.2 (Rambaut 2009). Raw (p) distances between pairs of sequences were calculated in MEGA 6 (Tamura et al. 2013).
Diagnosis: A medium-sized Cnemaspis, maximum known SVL less than 37.0mm (30.4-36.4,n = 8), granular dorsal scales on trunk heterogeneous, intermixed with large depressed slightly keeled scales, large keeled conical tubercles present on flanks, postcloacal spurs in both sexes, ventral scales larger than dorsal scales, imbricate, smooth, without a series of enlarged median sub-caudal scales, 28-29 scales across the belly, 8-9 lamellae under first digit and 10-11 lamellae under fourth digit of manus, 8-9 lamellae under first digit and 10-12 lamellae under fourth digit of pes, males with three femoral pores on each side and no pre-cloacal pores.

Variation
Variation in mensural and meristic data as in Table 1.Some paratypes are duller than the holotype in overall appearance.

Etymology
The species epithet is a combination name derived from the Latin words 'flavus' meaning yellow and 'ventralis' meaning ventral or pertaining to the belly, referring to the distinct yellow colouration of the ventral part of the body of the new species.
Suggested common name: Yellow-bellied Day Gecko.

Distribution
Cnemaspis flaviventralis sp.nov. is known only from its type locality (Image 5).

Natural history
Cnemaspis flaviventralis sp.nov. is a semi-arboreal species.Four specimens were collected from a tree trunk about two metres above ground level, another two found in leaf litter, and two specimens were collected from rock surfaces.All specimens were collected in October 2012 and in November 2013 between 2200 and 0200 hr (atmospheric temperature was 22-27 0 C).Individuals were found to be more active between 22 0 C and 25 0 C at dusk.The specimens were observed in a dense forest (Image 6) in Amboli.Field observations suggest that September to November is the breeding season for the species and egg laying occurs in October and November.After mating females deposit two eggs under rocks, on boulders, or in tree holes.Co-occurring herpetofauna include: Bungarus caeruleus (Elapidae); Trimeresurus malabaricus (Viperidae); Lycodon travancoricus and Macropisthodon plumbicolor (Colubridae); Uropeltis sp.(Uropeltidae); Hemidactylus prashadi, H. cf.brookii, Geckoella albofasciatus and Cnemaspis kolhapurensis (Gekkonidae); Raorchestes ghatei, Pseudophilautus amboli and Rhacophorus malabaricus (Rhacophoridae); Indirana chiravasi (Ranixalidae); and Xanthophryne tigerina (Bufonidae).

Molecular analysis
Model selection suggested general time reversible with gamma distribution (GTR+G, lnL = -2672.85, BIC = 5548.48, G = 0.283, df = 32, n = 565) as the best fit model for nucleotide substitution.Maximum likelihood analysis (Image 7) suggested that Cnemaspis flaviventralis sp.nov.forms a monophyletic group with other species of Cnemaspis found in sympatry, with C. girii as its sister taxon.Uncorrected raw genetic distance between the two specimens of C. flaviventralis was 0.6%, whereas the distances between C. flaviventralis and other northern Western Ghats congeners was 7.5-8.1% with C. girii, 9.1-10.1% with C. goaensis and 15.4-15.7%with C. kolhapurensis.

Comparisons
Cnemaspis flaviventralis sp.nov.can be distinguished from its congeners from the northern Western Ghats on the basis of the following characters: presence of spinelike tubercles on flanks, granular dorsal scales intermixed with large, depressed, slightly keeled scales (vs.lack of

DISCUSSION
It is already established that the Cnemaspis species in the Western Ghats and Sri Lanka are distinct (Manamendra-Arachchi et al. 2007).Nevertheless, it is essential to note that Giri & Vithoba (2001) reported Cnemaspis kandiana (Kelaart, 1852), a species restricted      and the generic level to resolve their taxonomy.In our genetic analysis (Image 7), Cnemaspis species from northern Western Ghats formed a monophyletic clade that was well separated from Southeast Asian C. limi Das & Grismer, 2003, with a species of Hemidactylus in between.Although the current placement could be an artifact of limited genetic data for 16S rRNA, previous molecular studies (Pyron et al. 2013) with other genetic markers have also shown similar trends.More extensive molecular work and comprehensive taxonomic revisions are therefore essential to understand and to resolve the taxonomy of this group.Moreover, the species diversity within the Western Ghats/Sri Lanka biodiversity hotspot is obviously high.
Given that several species of Western Ghats Cnemaspis are globally threatened (Srinivasulu et al. 2014), understanding both the unknown diversity and assessment of distribution of previously recognized species is essential to design and implement future conservation.

to
Sri Lanka (Manamendra-Arachchi et al. 2007), from Amboli, the type locality of the new species.Manamendra-Arachchi et al. (2007) have already suggested that records of C. kandiana outside Sri Lanka are based on misidentifications.Further, Mirza et al. (2014) suggested that reports of C. kandiana from southern Maharashtra should be attributed either to C. indraneildasii or are likely to belong to an undescribed species.Cnemaspis flaviventralis sp.nov.cannot be confused with either C. kandiana or C. indraneildasii.Cnemaspis flaviventralis differs from C. kandiana in having 28-29 ventral scales across mid-body (vs.19); conical (vs.spine-like) tubercles present on flanks; pre-cloacal pores absent (vs.present); and sub-digital lamellae on digit IV of pes 10-12 (vs.18-20).Cnemaspis flaviventralis differs from C. indraneildasii based on characters provided in the comparison section above.As presently understood, 28 species of Cnemaspis are known from India (Uetz & Hošek 2015).The description of a new species has elevated the number of Indian species to 29.Within the genus Cnemaspis, more work remains to be done at both the species © Amit Sayyed Image 6. Habitat of Cnemaspis flaviventralis sp.nov. at Amboli, (a) view of the moss covered boulders and rocky outcrops in the dense evergreen forest, and (b) view of dense evergreen forest.

Cnemaspis flaviventralis sp. nov. Sayyed et al.
were analyzed byBLAST tool (Altschul et al. 1990) for similar sequences in the NCBI (www.ncbi.gov)database.These sequences have been deposited in GenBank under the accession numbers KX269819-KX269826.