Descriptions of two new endemic and cryptic species

Zootaxa 4434 (2): 327–365 http://www.mapress.com/j/zt/ Copyright © 2018 Magnolia Press

Article

ISSN 1175-5326 (print edition)

ZOOTAXA

ISSN 1175-5334 (online edition)

https://doi.org/10.11646/zootaxa.4434.2.5 http://zoobank.org/urn:lsid:zoobank.org:pub:D1264EF3-FBC1-4F5D-8A3A-545BE4BE924D

Descriptions of two new endemic and cryptic species of Sitana Cuvier, 1829 from peninsular India V. DEEPAK1,2,4, AKSHAY KHANDEKAR1, R. CHAITANYA3 & PRAVEEN KARANTH1 1

Centre for Ecological Sciences, Indian Institute of Science, Bengaluru, 560012, India. Department of Life Sciences, The Natural History Museum, London SW7 5BD, United Kingdom. 3 508, 8 B Cross, Asha Township, Doddagubbi village, Bengaluru, 560077, India 4 Corresponding author. E-mail: [email protected] 2

Abstract Two new cryptic species of the agamid genus Sitana Cuvier, 1829 from Peninsular India are described herein. Sitana gokakensis sp. nov. from Gokak, Karnataka closely resembles Sitana thondalu sp. nov. from Nagarjuna Sagar, Andhra Pradesh. The two species can be distinguished based on their subtle morphological differences, genetic difference and geographic distribution. Sitana gokakensis sp. nov. have a relatively depressed head compared to Sitana thondalu sp. nov. Additionally, the vertebral scale counts differ in females of the two new species (Sitana gokakensis sp. nov. 45–47 vs Sitana thondalu sp. nov. 49–53). Genetic divergence between them is comparable to those between previously described Sitana species. Furthermore, the two new species are distributed ca. 500 km apart and are endemic to their respective landscapes that lie in similar latitudes of peninsular India. We urge the use of large sample size in new species descriptions especially those dealing with cryptic species like Sitana. The discovery of the two new cryptic species from these rocky terrains in peninsular India highlights need for more herpetological exploration in this region. Key words: cryptic species, DNA, fan-throated lizards, peninsular India, Sitana, taxonomy

Introduction The South Asian lizard genus Sitana Cuvier, 1829 currently comprises of ten species. Five of these species occur in Peninsular India and they belong to two distinct clades namely the Sitana ponticeriana clade and the Sitana spinaecephalus clade (Deepak et al. 2016a). The three species occuring in the Terai of Nepal Sitana fusca Schleich & Kästle, 1998, Sitana sivalensis Schleich, Kästle & Shah, 1998 and Sitana schleichi Anders & Kästle, 2002 belong to the S. sivalensis complex (Deepak et al. 2016a). Most of the species of the genus Sitana were described in the last two decades (Schleich & Kästle, 1998; Schleich, Kästle & Shah, 1998; Anders & Kästle, 2002; Amarasinghe et al. 2015; Deepak et al. 2016a, Deepak et al. 2016b). This surge in new species descriptions of Sitana in recent years highlights two lacunae: 1. a lack of systematic studies on Sitana and 2. poor sampling on lizards in general from peninsular India. Extensive, focused surveys targetting species of this genus led by the first author over the past five years have resulted in discovery of multiple species (Deepak et al. 2016a,b). Deepak and Karanth (2018) used molecular species delimitation methods and morphology to identify five undescribed Sitana spp. from the Indian subcontinent. In this study, we describe two out of these five species from peninsular India. We augmented a subset of multilocus data from Deepak and Karanth (2018) to show the genetic difference between these two species. We did a thorough morphological comparison of the two new species. We sampled extensively in the areas around and intervening the two new species to find out if they are geographically isolated.

Materials and methods Sampling and study area. The locality where we found Sitana gokakensis sp. nov. is in Gokak taluk, which is the Accepted by L. Avila: 4 Apr. 2018; published: 15 Jun. 2018

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headquaters of Belagavi district in Karnataka State. It is located at the confluence of two rivers, the Ghataprabha and Markandeya. These two rivers are mostly rocky (rocky substrate and river bank) isolating the plateau from the surrounding landscape (Fig. 1, Table 1). Sitana thondalu sp. nov. was found in two adjacent localities near Nagarjuna Sagar reservoir in Guntur district at the foothills, north of the Nallamala hill range (Fig. 1, Table 1). The first site is located on the east bank of Nagarjuna Sagar reservoir. Macherala the second site is located 17 km east of the dam site. Molecular phylogenetics. Tissue samples were extracted from atleast one individual per location for the twelve sampled localities (Fig. 1, Table. 1). Specimens were fixed in 4% formaldehyde for 24 hours, washed in water and subsequently preserved in 70% ethanol for long-term storage. Genomic DNA was extracted from tissue samples collected from the liver or tail tip of individuals. These samples were stored in 99.9% ethanol and refrigerated at -20oC. DNeasy (Qiagen ®) blood and tissue kits were used to extract DNA. Partial sequences of the NADH dehydrogenase subunit 2 (ND2) mitochondrial gene (~1026 base pairs, bp) were amplified and sequenced using the primers L4437b (forward) and H5540 (reverse) from Macey et al. (1997, 2000). A 665 bp fragment of partial R35 gene was amplified and sequenced using the primers R-35 (forward) and R-35 (reverse) (Leaché, 2009). Sequences were obtained using Sanger sequencing method and deposited in GenBank (Table 1).

FIGURE 1. Map showing sampling localities where we found different Sitana spp. during this study. Rivers and hills referred in this study are labelled. Locality where the two new species were collected are marked in red (S. gokakensis sp. nov.) and blue (S. thondalu sp. nov.). Localities referred in table 1.

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TABLE 1. Sampled localities and sequence numbers for the samples used in this study. * Samples not shown on the map. Locality Species No.

Location

Lat.

Long.

Voucher

GenBank accession no. ND2

1

S. laticeps

Kagal, Maharashtra Tumarguddi, Karnataka

16.611 74.344 CES 13517

R35

MG641387 MG641298

2

S. laticeps

15.918 74.651 CES 141134

MG641342 MG641268

3

S. gokakensis sp. nov. Gokak, Karnataka

16.186 74.76

MG641434 MG641308

4

S. laticeps

Bagalkot, Karnataka

16.173 75.642 CES 13588

MG641388 MG641299

5

S. sp 1

Devadurga, Karnataka

16.420 76.906 CES 141114

MG641389 _

6

S. spinaecephalus

Guddegudda, Telangana

16.681 77.826 CESG 518

MG641425 _

7

S. sp 1

Srisailem, Andhra Pradesh

16.057 78.881 CESG 245

MG641390 MG641301

8

S. thondalu sp. nov.

Nagarjuna Sagar east bank, Andhra Pradesh

16.556 79.301 CES 141173

MG641401 MG641309

BNHS 2490

9

S. thondalu sp. nov.

Macherla, Andhra Pradesh

16.450 79.429 CES 141200

MG641400 _

10

S. ponticeriana

Venikonda, Andhra Pradesh

15.621 79.373 CES 14521

MG641368 _

11

S. ponticeriana

Kothapatnam, Andhra Pradesh

15.437 80.178 CES 14516

MG641369 _

12

S. ponticeriana

Machilipattnam, Andhra Pradesh

16.245 81.240 CES 14548

MG641367 _

13*

S. spinaecephalus

Halol, Gujarat

22.432 73.630 NCBSAQ055 KT831332

_

14*

S. laticeps

Saswad, Maharashtra

18.347 73.898 BNHS 2323

KT831323

KT831284 KT831292

15*

S. visiri

Tuticorin, Tamil Nadu

8.785

78.156 CES 13594

KT831338

16*

S. marudhamneydhal

Kanyakumari, Tamil Nadu

8.079

77.532 VDE 241

KX371915 KX371918

17*

Sarada deccanensis

Jalna, Maharashtra

19.828 75.879 CES 14610

KT831315

KT831279

In the phylogenetic study undertaken by Deepak and Karanth (2018) the two new species were in “clade C” thus in our phylogenetic analysis all the samples from clade C and representative samples from sister clade (Clade B) were included. Sarada deccanensis (Jerdon, 1870) was used as the outgroup (see Deepak et al. 2016a; Deepak & Karanth, 2018). Sequences were aligned using ClustalW using default settings and uncorrected genetic distances were calculated using MEGA 5 (Tamura et al. 2011). The nuclear and mitochondrial markers were concatenated and phylogenetic analysis were undertaken on the combined dataset. PartitionFinder v1.1.1 (Lanfear et al. 2012) was used (default MrBayes settings) to find the best-fit partition scheme for the concatenated dataset and model of sequence evolution for each partition. The best-fit scheme comprised five partitions (Table 2). We estimated phylogenetic relationships using maximum likelihood (ML; Felsenstein 1981) and Bayesian inference (BI; Huelsenbeck & Ronquist 2001) as implemented in RaxML 1.3.1 (Stamatakis et al. 2005) and MrBayes 3.2 (Ronquist et al. 2012), respectively. ML analysis in RaxML takes only one model of sequence evolution in the analysis, therefore we used GTR+ Γ for all five partitions. We used GTR+ Γ model in RAxML which is recommended over the GTR + Γ + I because the 25 rate categories account for potentially invariant sites (Stamatakis, 2006). The GUI version of RAxML (Silvestro & Michalak 2012) was used, employing the ML+ rapid bootstrap method to search for best trees, with branch support quantified via 1000 non-parametric bootstrap replicates. The BI analysis used two Markov chains initiated from random trees and run for 1,000,000 generations, sampling every 100 generations. The BI analysis was terminated when the standard deviation of split frequencies was less than 0.005, and the first 25% trees were discarded as “burn-in”. Support for clades in the BI tree was quantified using posterior probabilities. Morphological comparisons. Materials examined directly are listed in Appendix 1. Voucher specimens have the following museum prefixes/field register number: BNHS (Bombay Natural History Society), CES (Centre for Ecological Sciences. Bangalore), ZSI (Zoological Survey of India, Kolkatta), ZSIP (Zoological Survey of India, Pune), VDE (V. Deepak field register number specimens at CES). We obtained measurements and meristic characters in accordance with Deepak et al.(2016a), modified from Zug et al (2006). All measurements were taken NEW SITANA CUVIER FROM PENINSULAR INDIA

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to the nearest 0.01 mm with digital calipers (Mitutoyu TM). SVL (snout-vent length): from the tip of the snout to the anterior border of the cloaca, HL (head length): from the snout tip to posterior border of the tympanum, HW (head width): distance from the left to right outer edge of the head at its widest point, HH (head height): Dorso-ventral distance from top of the head to underside of the jaw at a transverse plane intersecting angle of jaws, SE (snout-eye length): from snout tip to the anterior border of the orbit, ET (eye-tympanum length): from posterior border of the orbit to the anterior border of tympanum, JL (jaw length): from rostrum to the corner of the jaw, IO (interorbital width): transverse distance between antero-dorsal corners of left and right orbits, NE (naris to eye): distance from the anterior ridge of the orbit to posterior edge of naris, IN (internasal distance/snout width): transverse distance between left and right nares, TD (tympanum diameter): greatest diameter of tympanum, OD (orbit diameter): distance between anterior and posterior margins of the orbit, LAL (lower arm length): distance from elbow to proximal end of the wrist or just the underside of the forefoot, UAL (upper arm length): distance from anterior insertion of forelimb to elbow, F4 (finger length): distance from juncture of 3rd and 4th digits to distalmost extent of 4th finger, FEL (femur length): length of femur from groin to knee, CL (crus length): length of crus/tibia from knee to heel, HFL (hind foot length): distance from proximal end (heel) of hind foot to distal most point of the fourth toe, T4 (toe length): distance from juncture of 3rd and 4th digits to distal end of 4th digit of hind foot, TrL (trunk length): distance from forelimb insertion to hind limb insertions, TL (tail length): from posterior border of cloacal opening to the tip of the tail, TH (tail height): vertical length of the tail, TW (tail width): width of tail at the base, DWL (dewlap length): distance between the posterior end of dewlap and tip of lower jaw, DWLT (extent of dewlap in trunk): measured from the axilla till the end of the dewlap. In order to extract the dewlap area, we took photographs with extended dewlap with a metric scale. The outline of the throat-fan was traced from the photographs in the program imageJ (Rasband, 2004) which was used to measure the dewlap area. Nine meristic characters were collected from each specimen. 1) MBS (mid body scales rows): number of scale rows around the trunk at midbody, 2) VEN (ventral scales): number of scales from below mental to the anterior border of cloaca, 3) VEND (ventral scales on dewlap): number of scales from below mental to until the last dewlap scale, 4) VENB (ventral scales on the belly): number of scales below dewlap to the anterior border of cloaca, 5) VS (vertebral scales): number of scales on the vertebral counted at mid-dorsal first nuchal spine to the level directly above cloacal opening, 6) LAM4 (lamellae from the 4th toe): from first lamellae at the digit’s cleft to the most distal lamellae, 7) ESD (enlarged scales on dewlap): number of enlarged scales on the dewlap, 8) SL (supralabials): posterior end defined by the last enlarged scale that touches infralabial at rear corner of mouth and 9) IL (infralabials): posterior end defined by the posterior most enlarged scales that contact the supralabial at the rear corner of the mouth. TABLE 2. Partitions and models of sequence evolution used in the Maximum Likelihood (ML) and Bayesian Inference (BI) phylogenetic analyses. Partitions P1

Sites st

ND2 1

ML

BI

GTR+ Γ

HKY+ Γ

P2

ND2 2 , R35 3

GTR+ Γ

HKY+ Γ

P3

ND2 3rd

GTR+ Γ

GTR+ Γ

P4

R35 1st

GTR+ Γ

K80+I

P5

R35 2nd

GTR+ Γ

JC

nd

rd

We everted and prepared the hemipenes during specimen preparation by injecting 4% formaldehyde under the tail base in order to evert the hemipenes. One hemipenis was removed for each species, stained using Alzarin red in 70% ethanol. The stained hemipenes were photographed with Canon 750D with 100mm macro lens. Hemipenis morphology was documented using standard hemipenial nomenclature (Dowling & Savage, 1960). Morphological data were analysed in software R (R Core Team, 2013), the package ggplot2 was used to prepare plots (Wickham, 2009). Due to low sample size of females, only males were used in the PCA and DFA analysis. We did morphometric comparisons between Sitana gokakensis sp. nov., Sitana thondalu sp. nov., S. spinaecephalus Deepak, Vyas, Giri, 2016 and S. laticeps Deepak & Giri, 2016 for the PCA analysis. Apart from the two new species we used DEWL and TrL measurements of S. laticeps (33 males, 29 females) and S. spinaecephalus (16 males, 12 females) (Appendix 1). One of the undescribed species of Sitana (Sitana sp1)


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delimited in Deepak and Karanth (2018) occurs in and around the areas where we found the two new species, we refer to this species in our diagnosis section. In total 25 males and 5 females of S. sp1 were used for morphological comparison (Appendix 1). All individuals used for morphological comparisons are adult males identified based on the hemipenal bulge at the tail base. Since we collected during breeding season males were also identified based on the fully developed breeding colouration on dewlap whereas females could be distinguished by the presence of eggs. We used 19 morphometric variables and three 3 meristic variables in two different Principal Component Analysis (PCA) with varimax rotation (Table 4). The dataset used for PCA and DFA had 69 individuals (10 S. gokakensis sp. nov., 15 S. thondalu sp. nov., 13 S. spinaecephalus and 31 S. laticeps) because we did not have some measurements for the already described species. All variables were log transformed before PCA analysis. R package MASS (Venables & Ripley, 2002) was used to carry out Linear Discriminant Function Analysis (DFA). Each of the 19 measurement variables were tested for normality using Shapiro-Wilk normality test (Appendix 2). Overall ten out of the 19 measurements were non-normal. Therefore, we did our first DFA analysis with all the four species and 9 variables (Appendix 2). Since our interest was to identify differences for the two-new species we did a second DFA analysis in which we dropped S. laticeps which had two additional non-normal variables (Appendix 2). In the second DFA we had three species and 11 variables. In both the DFA analyses the dataset was divided into two subsets: (1) training subset which included 60% of randomly selected from the data and (2) testing subset which included remaining 40% of the data. We used a confusion matrix to predict and test the model accuracy using this two subsets of the data.

Results Phylogenetic analysis. The maximum likelihood (ML) and Bayesian trees (BI) had similar tree topology (Fig. 2). Sitana gokakensis sp. nov. and Sitana thondalu sp. nov. are deeply divergent sister lineages with well supported phylogenetic relationship (Fig. 2). The support for relationship of this lineage with other Sitana spp. is poor in both ML and BI analysis (Fig. 2). Sitana gokakensis sp. nov. has an uncorrected p-distance of 11% in the ND2 gene, from its sister Sitana thondalu sp. nov. (Appendix 3). The clade containing the two new species has an uncorrected p-distance in mtDNA (ND2) of 14%–17% with the S. spinaecephalus clade and 15%–18% with the S. ponticeriana clade (Appendix 3). The samples found in nine other localities were nested with already described species (S. laticeps, S. ponticeriana, S. spinaecephalus) and two of them (locations 5,7 in Fig. 1) were genetically 6–8 % divergent from S. laticeps which were delimited as a different species (Sitana sp1 in Deepak & Karanth, 2018).

FIGURE 2. Maximum Likelihood tree with highest likelihood score based on the concatenated dataset. ML bootstrap support and Bayesian posterior probability are shown at each node. Numbers in parentheses refer to location ID in Table 1.

NEW SITANA CUVIER FROM PENINSULAR INDIA


331

FIGURE 3. PCA plots showing overlap in morphometric characters of S. gokakensis sp. nov. (red circles) with S. thondalu sp. nov. (blue circles), S. laticeps (green circles) and S. spinaecephalus (purple circles). A. Screeplot showing variance explained by each axis B. PC1 plotted against PC2. and C. PC3 plotted against PC4. The arrows are pointing in the direction of the variables, as projected into a 2-dimension plane of the biplot.


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FIGURE 4. DFA plots showing overlap in morphospace of S. gokakensis sp. nov. (red circles) with S. thondalu sp. nov. (blue circles), S. laticeps (green circles) and S. spinaecephalus (purple circles). A–B. Results of DFA analysis 1 and C. Results of DFA analysis 2. The arrows are pointing in the direction of the variables with respect to their coefficient values.



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FIGURE 5. Boxplot showing the spread of body measurements for the top two variables with highest factor loadings on PC1 (SVL, LAL) and PC2 (EE, NE); VENB and VEND are the two out of the three count variables used in the second PCA with higher factor loadings. Gray points in and around the boxplots are values of individual measurements/counts.

Morphological comparisons. The two new species are more or less similar in external morphology but have diagnostic characters that differentiate them from the other known species in the genus Sitana (see diagnosis). However, we compared morphology of Sitana gokakensis sp. nov. and Sitana thondalu sp. nov. with other sister species with plain dewlap. In the PCA analysis for measurements, axis 1 explained 59.4 % of the variance, axis 2 explained 11.7 % of the variance (Fig. 3 a,b; Appendix 4) and 5.0% variance was explained by axes 3 and 4.6% of the variance was explained by axis 4 (Fig. 3b; Appendix 4). PCA for counts, axis 1, 2 and 3 explained 49.2%, 32.6% and 18.3% variance respectively (Appendix 5). Sitana gokakensis sp. nov. and Sitana thondalu sp. nov. have extensive overlap in external morphology (Fig. 3–6). LAL, SVL, CL, HEL, and HL in PC1 and EE, NE, DEWLT and SW in PC2 had higher factor loadings (Fig. 3c; Appendix 4). In the PCA using count variables VEND and VENB had higher factor loadings on PC1 and PC2 respectively (Appendix 5). However, even the top two variables with high factor loading in both the PCA’s have extensive overlaps between these two species (Fig. 5). The training subset data in both DFA analysis had high model prediction probability of 1 (DFA analysis 1) and 0.85


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(DFA analysis 2) (Appendix 6), which means the species were correctly classified all the time. However, in the testing data subset the prediction probability was moderate 0.77 (DFA analysis 1) and 0.68 (DFA analysis 2) (Appendix 6). In the first DFA analysis axis 1, 2 and 3 explained 60.9%, 25.5% and 4.2% of variance in the dataset (Fig 4a). There is extensive overlap of morpho space between all the four species (Fig. 4a,b). In the second DFA analysis there was overlap in morphospace between S. gokakensis sp. nov. and S. thondalu sp. nov. S. gokakensis sp. nov. and S. spinaecephalus on axis 1(Fig. 4c). There is a clear separation between S. gokakensis sp. nov. and S. spinaecephalus on axis 1(Fig. 4c). All three species have some amount of overlap in axis 2, S. gokakensis sp. nov. and S. thondalu sp. nov. have a marginal overlap in this axis (Fig. 4c). The ranges for VS, which was different for females of the two new species, overlapped in males (Table 3). Dewlap area also had complete overlap between the two species (Fig. 6). Most of the meristic characters were also overlapping in the two new species (Table 3) except, that females of S. gokakensis sp. nov. had lower vertebral scale counts (45–47) compared to females of S. thondalu sp. nov. (49–53) (Table 3). Given that most morphological characters are highly conserved in these two species we used a combination of genetic difference and geography to delineate these two species.

FIGURE 6. Boxplot showing dewlap area of the two new species, points in and around the boxplots are values of individual measurements. Representative dewlap photographs for the two new species are shown on top. Scale bar 10 mm.



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Taxonomy Sitana gokakensis sp. nov. Fig. 7–9 & 11; Table 3 & 4; Appendix 7 & 8 Holotype. BNHS 2490, adult male (Fig. 7 & 8A) from Gokak plateau, Belagavi district, Karnataka, India (16.18618°N, 74.75952°E), 255 m elevation, collected on 08.08.2013 by V. Deepak and Aparna Lajmi. Paratypes. BNHS 2491, adult female (Fig. 9b; Appendix 7b) collected by V. Deepak and Aparna Lajmi; CES 141136, an adult male (Appendix 7a) collected by V. Deepak and Kunal Arekar on 12.5.15; both same collection data as holotype. Diagnosis. A large sized Sitana with a maximum SVL of 53.1 mm, distinguished from its congeners by the following combination of characters: 1) dewlap feebly serrated without bright orange patches in breeding males ( vs well serrated with bright orange patches in breeding males in S. ponticeriana, S. visiri, S. marudhamneydhal and S. devakai); 2) dewlap extending beyond forearm insertion (vs not extending in S. sivalensis, S. schleichi and S. fusca); 3) four prominent enlarged non spine like scales bordering occipital region (vs enlarged spine like scales in S. spinaecephalus); 4) dewlap large sized extending up to 55% of trunk (vs up to 44 % of trunk in S. laticeps in southern most population which appears to be an outlier for the species; up to 29% of trunk in remaining population (Fig. 10) and vs up to 42% of trunk in Sitana sp1 (Fig. 10); 5) body size (male & females averaged) relatively large (SVL: mean 48.8 +/- 3.6 SE; range 42.4–53.1; n=14) vs (body size relatively small in S. laticeps SVL: mean 47.8 +/- 3.2 SE; range 40.7– 54.6; n=63 and in S. sp1 SVL: mean 46.2 +/- 3.2 SE; range 38.9–54.1; n=31 and S. spinaecephalus SVL: mean 47.3 +/- 3.7 SE; range 39.5– 56.6; n=25). It has to be noted here that the southernmost population of S. laticeps appears to have some relatively large bodied individuals (SVL: mean 50.1 +/- 4.2 SE; range 45.2–54.6; n=6) but need a larger sample size to further confirm.

FIGURE 7. Holotype of Sitana gokakensis sp. nov. (BNHS 2490) from Gokak, Karnataka. (A) full body dorsal (B) flank region (C) dorsal view of head (D) lateral view of head. Scale bar:10 mm.


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FIGURE 8. Sitana gokakensis sp. nov. colouration in life. A. Holotype (BNHS 2490) in life, B. another adult male collected during 2016 from the type locality.



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FIGURE 9. Sitana gokakensis sp. nov. colouration in life. A. An adult male from type locality, B. paratype BNHS 2491, C & D. two different uncollected adult females from the type locality.


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Description of holotype. The holotype is in good condition; hemipenis partially everted, exposed and seen on both sides when viewed dorsally (Fig. 7a). Tail entire, curved slightly towards the right; loose folds of skin on the dorsum and a small incision of 4mm to extricate tissue, are artefacts of preservation. An adult male, SVL 51.2 mm. Head relatively long (HL/SVL ratio 0.29), wide (HW/HL ratio 0.70), not depressed (HH/HL ratio 0.51), distinct from neck. Snout short (SE/HL ratio 0.39), longer than diameter of orbit (OD/SE ratio 0.73), obtusely pointed in profile when viewed dorsally (Fig. 7c); rostral much wider than high, contacted laterally on either side by first supralabial, a prenasal and dorsally by two smaller, keeled scales (Fig. 7e). Canthus rostralis and supraciliary edge, sharp. Nostril roughly circular, laterally positioned and placed in the centre of a large, undivided nasal. Nasal scale bordered by eight scales on either side: one supranasal, three postnasals, one prenasal, the last two scales form a series of enlarged scales bordering the supralabials and a small scale separates them from the first supralabial. Ten supralabials on the right side (11 on left side), first slightly higher than others, broader than long, roughly rectangular, rest more elongate, weakly keeled, bordered above by a row of slightly smaller, rectangular, weakly keeled scales, which start at posterior margin of first supralabial, decreasing in size posteriorly and terminating after the posterior margin of orbit. Twelve infralabials on the right side (13 on left), first slightly smaller, the rest elongate, keeled increasing in size posteriorly. Loreal region concave, with scales of heterogeneous shape and size. Canthals enlarged, overlapping, slightly protruding on supraorbital ridge laterally. Eye large (ED/HL ratio 0.27); pupil rounded, covered under the eyelids; eyelids covered with scales that are heterogeneous in shape and size; larger scales on the upper eyelids, keeled, elongate and bluntly pointed, rest smooth; supraciliaries longer than broad. Orbital scales small but not granular. Scales on postorbital and temporal region, heterogeneous, subimbricate, strongly keeled, directed backward and upwards. Tympanum naked. Scales on dorsal surface of snout, forehead, interorbital and occipital region highly heterogeneous in size, shape, mostly elongate, strongly keeled longitudinally (Fig. 7c); scales on snout large in size, those on forehead slightly larger, interorbital region largest; occipital region with much smaller imbricate scales relative to other dorsal head scales; four relatively enlarged scales on a transverse row in the occiput region; nine scales anterior and 14 scales posterior of eyelids in the interorbital region; supraorbital scales along the supraciliary edge elongate, keeled, decreasing in size posteriorly, following curvature of orbit. Parietals larger than surrounding scales, longer than broad, tricarinate, separated from each other by two inter-parietal scales; anterior large, roughly triangular, tricarinate; posterior roughly pentagonal, bicarinate, with distinctly visible pineal eye. Parietals and the posterior inter-parietal part of a series of nine large, strongly keeled scales traversing the forehead. Mental shield narrower than rostral, roughly pentagonal, pointed posteriorly, a pair of roughly hexagonal postmentals, slightly shorter than mental, completely divided by a smaller gular scale (Fig. 7d); scales on the gular region homogenous in shape, those behind mental smooth, increasing in size and carination posteriorly. Dewlap large (DEWL/SVL ratio 0.69), extends posteriorly over 55% of trunk, extending much beyond axilla (Fig. 7b); about nine rows of anterior dewlap scales smaller, elongate, pointed, keeled; remainder of scales much enlarged, keeled, pointed, gradually increasing in size towards margin; single marginal row largest with lanceolate scales. Enlarged scales on dewlap in 23 rows. Nuchal and dorsal crest absent (Fig. 7a). Scales on nuchal region smaller, less than half the size of those on interorbital region, imbricate, strongly keeled. Body slender, 60 rows of scales around midbody; vertebral scale row with 46 scales, vertebral scale row partially paired and alternating. Five enlarged dorsal scale rows, on either side of the vertebral scale row (except in 3 places the five rows on either side are in contact with each other), the 5 dorsal scale rows starts from back of neck until groin, sub equal in size and shape, imbricate, pointed, keeled, directed backwards forming regularly arranged longitudinal rows; scales on flanks heterogeneous in size, shape, smaller than those on back, pointed, keeled, upper rows directed backwards and upwards, lower rows backwards and downwards; ventral scales subimbricate, keeled, homogenous in size and shape, arranged in 101 rows. Fore and hindlimbs relatively slender, tibia short (CL/SVL ratio 0.33); digits moderately long, ending in strong, elongate, slightly recurved claw; inter-digital webbing absent; subdigital lamellae entire, bi-mucronate, 22 subdigital lamellae on toe IV including claw sheath; relative length of right fingers 3 > 4 > 2 > 5 > 1, right toes 4 > 3 > 2 > 1. Fore and hindlimbs covered above and below with regularly arranged, enlarged, pointed, strongly keeled, scales. Tail long (TL/SVL ratio 2.35), entire, base swollen, uniformly covered with similar sized, keeled, pointed, regularly arranged, backwardly directed imbricate scales; subcaudal scales keeled, weakly pointed near base, becoming pointed posteriorly. Colour of holotype in life. Head darker than body, dorsum of the torso dark brown, darker compared to flank and tail (Fig. 7a). Neck region with blue and dark pink colouration. Iris yellow in colour, tympanum cream coloured. Dorsum with five black blotches the one on neck darker. Flank region brick red colour with mottled dark



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brown patches. Enlarged scales on the flank and thigh cream in colour. Belly off white in colour with brown speckles on most scales. Forelimbs and hindlimbs dark brown suffused with red on dorsal side, cream patch on the back of thigh extending to the saccaral region and pale on the ventral side. Lower jaws cream mostly, dark blue colouration starts from the mentum (on 7–8 horizontal scale rows (Fig. 7d) and extends along midline of dewlap. Dewlap white, each scale on dewlap with clustered brown speckles. Colour of holotype in preservative. Head dark brown, darker than the body—with scattered blue patches on the neck. Two broken black stripes start below the eye approximately in the middle directed downwards that broaden and end after the forearm insertion. Tympanum cream coloured, slightly lighter than the surrounding scales with small light brown speckles. Dorsum dark brown with two black blotches on the neck; 3 broad, dark brown bars at the tail base and multiple smaller bars found throughout the tail. The five enlarged rows of scales on the dorsum flanking the vertebral scale rows dark brown coloured, a pale brown broken white stripe starting from neck bordering enlarged scale row on the dorsum ends near the tail base, upper 3/4th of the flanks dark brown with mixed light brown scales, the lower end of flanks paler. Venter mottled with brown on off white scales. Brown bars present on the dorsal side of limbs. Tail with dark brown bands throughout. Enlarged scales on dewlap with dark brown/black spots, mottled, with some scales having dark brown/black spots with a white spot in the center. Throat region on either side of the enlarged scale on dewlap off white with bluish dark gray /black speckles and the dark blue colouration on the throat is prominent 5 scales after the mentum and its surrounding scales, the rest are faint bluish gray.

FIGURE 10. Box plot showing proportion of dewlap in trunk. Note red circle highlighing the southern most population (Bagalkot & Tummargudi) of S. laticeps which have relatively larger dewlaps compared to all other populations.

Variation in paratypes. The two paratypes agree with the holotype in overall scalation with some exceptions. CES 141136 has 101 ventral scales and 32 belly ventral scales, 58 mid-body scales, 49 dorsal scales, one less infralabial on each side and one additional supralabial on the left. The female BNHS 2491 agrees with the holotype in overall scalation except that it lacks a dewlap, and has 67 ventral scales, one less infralabial on each side. CES 141136 differs in colouration from the holotype in having a light brown dorsum (an artefact of preservation), four distinct dark brown patches on the dorsum the first one darker. No blue patches on the neck. The stripe below the


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eye, indistinguishable compared to the surrounding scale, only a short light brown stripe ends near the forearm insertion. Blue line on the dewlap/throat darker than the holotype. Broad bands are present throughout the tail. BNHS 2491 differs in colouration from the holotype in having head colouration similar to body, three rhomboidal/ oval patterns on the dorsum, pale white stripe on the dorsum absent. Head brown with patches of brick red and grey (likely due to shedding scales), no blue patches on the neck. Throat region pale white in colour with dark black spots and speckles. One rhomboidal marking on the dorsum of tail base.

FIGURE 11. Hemipenis of Sitana gokakensis sp. nov. (CES 141136): (A) sulcal view; (B) asulcal view; C) lateral view; (D) apical view. Scale bar:10 mm.

Variation in live colouration. Most of the male specimens collected during this study matched with the holotype in live colouration. There were some specimens which were different from the rest which we describe NEW SITANA CUVIER FROM PENINSULAR INDIA


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herein. Three out of the ten breeding males were found with blue colouration on the upper eyelids. Seven out of the ten males observed in-situ had mostly dark pink and tinge of blue on the nuchal region. The fifth scale on the enlarged scale rows flanking vertebral scales on the dorsum was bordered with continuous cream coloured/reddish stripe starting from neck till the vent (e.g, see Fig. 9 a,b,d), in some individuals these stripes were broken. The rhomboidal patch on the neck was single in many individuals but the 5 rhomboid patches on the dorsum were separated by a cream line on the vertebral region (e.g, see Fig. 9c). One of the adult males had blue colouration on the tail which became prominent immediately after euthanization (Appendix 8). Hemipenial morphology. Four samples were examined (CES 141136–141139). Hemipenis bilobed, relatively small, longer than wide and shallowly forked. Sulcus spermaticus bifurcated and the fork continues onto the apical lobes (Fig. 11a). Sulcal lips raised and papillate, sulcus smooth originating from the side of the base. Apex with small serrated row of calyces and the sulcal region of apex nude (Fig. 11d), medial projection absent. Ornamentation is differentiated and combination of flounces and calyces observed. Papillae present between the apical lobes. Apical regions on the lobes of sulcal side calyces are serrated and continuous; calyces are relatively larger and non-serrated at the base of the lobes (Fig. 11a,b). Calyces are deep regular pits on the asulcal side and become shallow at basal region (Fig. 11b). Ridges between the calyces are thin and show micro-ornamentation which are scalloped. Flounces present, eight to ten in number, all prominent on the asulcal side (Fig. 11B,C). Etymology. The specific epithet is an adjectival toponym and refers to the Gokak plateau of Belagavi district in Karnataka, to which this species is endemic. Suggested common name. Gokak fan-throated lizard. Distribution. Sitana gokakensis sp. nov. is endemic to Gokak plateau in Belagavi district, Karnataka (Fig. 1). The samples collected from north and south outside this plateau (Nipani, Ramdurga, Bagalkot) were of S. laticeps. The Ghataprabha is a rocky river and a potential geographical barrier for this species.

FIGURE 12. Habitat of Sitana gokakensis sp. nov. at the type locality, Gokak, Karnataka.

Habitat and natural history. Sitana gokakensis sp. nov. is only known from the open rocky habitat in Gokak plateau (Fig. 12). Gokak hills receives 820 mm of average annual rainfall. The habitat is xeric and is dominated by thorny and dwarf succulent species (Malpure et al. 2016). The recently described Euphorbia gokakensis Yadav,


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Malpure, Chandore, 2016 is endemic to this plateau which is found alongside other succulents such as E. antiquorum L., E. caducifolia Haines and Opuntia elatior Mill. and non-succulents like Mundulea sericea (Willd.) A.Chev. (Malpure et al. 2016). Ophisops sp and Hemidactylus murrayi Gleadow, 1887 are the two sympatric lizards found in this plateau. Breeding males were recorded during May and August months.

Sitana thondalu sp. nov. Fig. 13–14 & 16; Table 3 & 4; Appendix 9 & 10 Holotype. BNHS 2492, adult male (Fig.13, 14a) from East Bank of Nagarjuna Sagar reservoir, Guntur district, Andhra Pradesh, India (16.55557°N, 79.30134°E), 200 m asl, collected on 08.08.2015 by V. Deepak, Praveen Karanth, Aniruddha Datta-Roy, Rama Harvey and Aparna Lajmi. Paratypes. CES 141175 adult male (Appendix 9b) and CES 141178 adult female (Appendix 9a) collected on 08.08.2015 by V. Deepak, Praveen Karanth, Aniruddha Datta-Roy, Rama Harvey and Aparna Lajmi both same collection data as holotype. Diagnosis. A large sized Sitana with a maximum SVL of 56.3 mm, distinguished from its congeners by the combination of characters: 1) dewlap feebly serrated without bright orange patches (vs well serrated in S. ponticeriana, S. visiri, S. marudhamneydhal, S. devakai with bright orange patches in breeding males), 2) dewlap extending beyond forearm insertion (vs not extending in S. sivalensis and S. fusca), 3) four prominent enlarged non spine like scales bordering occipital region (vs enlarged spine like scales in S. spinaecephalus); 4) relatively higher head HH/HL: mean 0.53 +/- 0.05 SD and HH/HW: mean 0.79 +/-0.06 (vs relatively depressed head HH/HL: mean 0.50 +/- 0.02 SD and HH/HW: mean 0.73 +/- 0.03 SD in S. gokakensis sp. nov.) (Fig. 15). Although these ratios have overlaps S. gokakensis overall have a depressed head compared S. thondalu sp. nov. (Fig. 15). 5) higher number of vertebral scales in females 49–53 (vs lower 45–47 in S. gokakensis sp. nov. however, this needs further verification with a larger sample size). 6) Geographic location confined to Northern Andhra, Guntur District (vs known only from Gokak plateau S. gokakensis sp. nov.). 7) Sitana thondalu sp. nov. has an uncorrected p-distance of 11% in the ND2 gene and 1.5 % in R35 gene, from its sister species Sitana gokakensis sp. nov. (Appendix 3 & 11). Description of holotype: The holotype is in good condition; hemipenis everted, exposed and seen on both sides when viewed dorsally (Fig. 13a). Tail entire, curved towards the right; loose folds of skin on the dorsum are artefacts of preservation. An adult male, SVL 54.25 mm. Head relatively long (HL/SVL ratio 0.26), wide (HW/HL ratio 0.67), not depressed (HH/HL ratio 0.56), distinct from neck. Snout short (SE/HL ratio 0.38), longer than eye diameter (OD/SE ratio 0.88), obtusely pointed in profile when viewed dorsally (Fig. 13c); rostral wider than high (RH/RW ratio 0.55), contacted laterally on either side by first supralabial, a prenasal and dorsally by two large scales. Canthus rostralis and supraciliary edge sharp (Fig. 13e). Nostril roughly circular, laterally positioned and placed roughly in the centre of a large, undivided nasal scale (Fig. 13e). Nasal scale bordered by seven scales on the right and six on the left, one supranasal, two postnasals, one prenasal, the last two of a series of enlarged scales bordering the supralabials, first supralabial only on the right. Ten supralabials on the right side (11 on left side), first higher than others, broader than high, roughly rectangular, rest more elongate, weakly keeled, bordered above by a row of slightly smaller, rectangular, weakly keeled scales, which start at posterior margin of first supralabial, decreasing in size posteriorly and terminating above the eigth (right side). Twelve infralabials on the right side (11 on left), first slightly smaller, the rest elongate, weakly keeled increasing in size posteriorly. Loreal region concave, with scales of heterogeneous shape and size. Canthals enlarged, overlapping, slightly protruding on supraorbital ridge laterally. Loreal region with few weekly keeled scales. Eye large (ED/HL ratio 0.34); pupil rounded, covered under the eyelids; eyelids covered with scales that are heterogeneous in shape and size, predominantly smooth; supraciliaries longer than broad. Orbital scales small but not granular. Scales on postorbital and temporal region heterogeneous (size and shape), sub-imbricate, strongly keeled, directed backward and upwards. Tympanum naked. Canthal scales, and orbit bordered below by a row of fourteen scales that are heterogeneous (size and shape) anteriorly, roughly rectangular under the eye, weekly keeled, starting at the posterior margin of nasal scale and terminating after the posterior margin of the orbit. Scales on dorsal surface of snout, forehead, interorbital and occipital region highly heterogeneous (size and shape), mostly elongate, imbricate, strongly keeled longitudinally (Fig. 13c); scales on snout large, those on forehead slightly larger, interorbital region largest; occipital region with much smaller scales; 11 scales anterior and 13 scales posterior to eyelids in the interorbital region; supraorbital NEW SITANA CUVIER FROM PENINSULAR INDIA


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scales along the supraciliary edge elongate, keeled, decreasing in size posteriorly, following curvature of orbit. Parietals larger than surrounding scales, longer than broad, strongly keeled, separated from each other by a series of three smaller scales; single inter-parietal, roughly pentagonal, with no visible pineal eye. Mental shield narrower than rostral, roughly pentagonal, pointed posteriorly, a pair of elongate, curved postmentals, slightly longer than mental, in strong contact with each other (Fig. 13d); scales on the gular region homogenous in shape, those behind mental smooth, increasing in size and carination posteriorly. Dewlap large (DEW/SVL ratio 0.62), extends posteriorly over 41% of trunk; about seven to eight rows of anterior dewlap scales smaller, elongate, pointed, keeled; remainder of scales much enlarged, keeled, ending obtusely, gradually increasing in size towards margin; single marginal row largest. Enlarged scales on dewlap in 21 rows. Nuchal and dorsal crest absent (Fig. 13a). Scales on nuchal region smaller, less than half the size of those on interorbital region, imbricate, strongly keeled. Body slender (TORW/SVL ratio 0.23), 60 rows of scales around midbody; vertebral scale row with 49 scales, four or five dorsal scale rows on either side of the slightly smaller alternating pair of vertebral scale row, the 4 or 5 dorsal scale rows are larger than those on the neck and the largest enlarged scale on the flank, these scales starts from back of neck until groin, sub equal in size, shape, imbricate, pointed, keeled, directed backwards forming regularly arranged longitudinal rows; those on flanks heterogeneous in size and shape, smaller than those on back, pointed, keeled, upper rows directed backwards and upwards, lower rows backwards and downwards; ventral scales imbricate around the dewlap, subimbricate posteriorly, keeled, homogenous in size and shape, arranged in 94 rows. Fore and hindlimbs relatively slender, tibia short (CL/SVL ratio 0.32); digits moderately long, ending in strong, elongate, slightly recurved claw; inter-digital webbing absent; subdigital lamellae entire, bi-mucronate, 23 subdigital lamellae on toe IV including claw sheath; relative length of fingers (right) 3 > 4 > 2 > 5 > 1, toes (right) 4 > 3 > 2 > 1. Fore and hindlimbs covered above and below with regularly arranged, enlarged, pointed, strongly keeled scales. Tail long (TL/SVL ratio 2.72), entire, base swollen, uniformly covered with similar sized, keeled, pointed, regularly arranged, backwardly directed imbricate scales; subcaudal scales keeled, weakly pointed near base, becoming pointed posteriorly.

FIGURE 13. Holotype of Sitana thondalu sp. nov. (BNHS 2492) from Nagarjun Sagar, Andhra Pradesh. (A) full body dorsal (B) flank region (C) dorsal view of head (D) lateral view of head. Scale bar: 10 mm.


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FIGURE 14. Sitana thondalu sp. nov. colouration in life. A. Holotype (BNHS 2492) in life, B. another adult male (CES 141174) collected during 2016 from the type locality. Note the blue colouration on the eyelids, labials, nuchal and nasal region and two cream stripes on the neck that are characteristic of males in this species.



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FIGURE 15. Box plot showing head ratios of the two new species of Sitana. Note this data includes both males and females, Sitana gokakensis sp. nov. have an overall depressed head compared to Sitana thondalu sp. nov. which is variable.

Colour of holotype in life. Head visibly darker than body (Fig. 14a). Dorsum of the torso dark brown, darker than flank region and tail. Blue tinge on the upper labials, neck and upper eyelids. Iris yellow, lower jaws pale


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cream, tympanum cream coloured. Forebody with two prominent cream yellow/orange stripes. First stripe starts from the nasal scale and extends till above the forearm insertion, the second shorter and thinner stripe starts above the tympanum and extends to the dorsum. Confluent with the second thinner stripe are the two prominent dark brown stripes on the dorsum which are discontinous and broken by lighter patches starting from the neck and end before 1/4th of the tail. Enlarged scale rows on the dorsum grayish brown in contrast to flank region which is brick red in colour (Fig. 14a). Some enlarged scales on the flank and thigh cream in colour. Most belly scales were cream yellow, scales adjoining the dewlap with brown speckles. Most scales on belly without speckles. Forelimbs and hindlimbs dark brown on dorsal side and pale on the ventral side; both limbs with dark brown bands on the dorsal side. Dewlap white with most scales having clustered brown speckles and some plain. Tail with broad dark brown bands, darker on the dorsal than the ventral side (Fig. 14a).

FIGURE 16. Sitana thondalu sp. nov. colouration in life. A, B. An uncollected male photographed in situ, C. an uncollected female from the type locality, D. one of the females collected from the type locality.

Colour of holotype in preservative. Head dark brown, darker than body, with a faded Carolina blue patch on the neck and a pale white stripe with brown speckles that starts below the eye approximately in the middle directed downwards which broadens and ends at the anterior edge of the forearm insertion. A dark brown stripe below the off-white stripe on the head starting from the 10th supralabials on the left and 9th supralabial on the right, extends into the neck and ends near the forearm insertion. Tympanum off white with small light brown speckles, slightly lighter than the surrounding scales. A prominent dark brown band between the supraciliaries on top of the head. Dorsum light brown with 5 rhomboidal markings on the back that are variable in size and almost indistinguishable from the dorsal colouration; behind the hind limb insertion is a small rhomboidal marking on the dorsal side of tail base with small dark brown bars found throughout the tail. The 4–5 enlarged rows of scales on the dorsum markings, dark brown coloured. A off white stripe starting from neck bordering the 4th and the 5th lower enlarged scale rows on the dorsum ends near the tail base. Flanks light brown with the ventro-lateral scales paler with dark brown speckles. Venter off white (Fig 13b). Faint brown bars on the dorsal side of limbs. Enlarged scales on dewlap mottled with dark brown/black spots with a pale white spot in the center that is interspersed with dark brown/black spots. Anterior of each dewlap scale pale white without any spots/speckles, the throat region on either side of the enlarged scale on dewlap pale white with the dark blue colouration on the throat visible. NEW SITANA CUVIER FROM PENINSULAR INDIA


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FIGURE 17. Hemipenis Sitana thondalu sp. nov. (CES 141177): (A) sulcal view; (B) asulcal view; C) lateral view; (D) apical view. Scale bar:10 mm.

Variation in paratypes. The two paratypes agree with the holotype in overall scalation with some exceptions. CES 141175 has 105 ventral scales, 32 belly ventral scales (VENB) and 46 dorsal scales. The female CES 141178 agrees with the holotype in overall scalation except that it lacks a dewlap, and has 68 ventral scales, one additional supralabial on the right and one less supralabial on the left, 53 vertebral scales. CES 141175 differs in colouration from the holotype in having a light brown stripe staring below the eye and ending near the forearm insertion. The thin black stripe near the labium starts from the angle of the jaw instead of the supralabials. The throat region on either side of the enlarged scale on dewlap off white with blue speckles. The first three bands on the tail broader than the rest. CES 141178 differs in colouration from the holotype in having head colouration similar to body, rhomboidal patterns prominent, pale white stripe on the dorsum, absent. Instead, the edges of the enlarged row on the dorsum that is dark brown starting from neck to tail base, forms two stripes on the dorsum. Light brown stripe starting below the eye ends anterior to the tympanum. Stripe near labium faint and brown in colour. Venter mottled with pale brown colouration. Throat region pale white in colour. Bands on the tail broader than the holotype.


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FIGURE 18. Habitat of Sitana thondalu sp. nov. in Guntur district, Andhra Pradesh A. Type locality Nagarjuna Sagar, East bank, B. Macherla.



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TABLE 3. Range of select scale counts of the two new Sitana species including the type series, sample size in brackets. Abbreviations, “_” = NA. 0 = absent. Lam4 = lamellae on fourth toe; SL = supralabials; IL = infralabial; MBS = mid body scales; ENL = enlarged scales on the flank region; ESD = enlarged scale rows in dewlap; VEN = number of ventral scales, VEND= number of ventral scales on dewlap; VENB= number of ventral scales on belly; VS = vertebral scales Sitana gokakensis sp. nov.

Sitana thondalu sp. nov.

♂

♀

♂

♀

Lam4

19–23(10)

20–21(4)

20–23(9)

20–23(4)

SL

9–12(10)

10–11(4)

9–11(9)

10–11(4)

IL

9–13(10)

11–12(4)

10–14(9)

11–12(4)

MBS

57–61(10)

58–60(4)

54–64(15)

59–63(4)

ESD

19–23(10)

0

19–25(9)

0

VEN

94–102(10)

67–68(4)

93–122(15)

68–71(4)

VEND

63–73(10)

0

61–89(15)

0

VENB

29–34(10)

0

30–35(15)

0

VS

45–49(10)

45–47(4)

43–51(15)

49–53(4)

ENL

14–18(10)

13–19(4)

10–15(9)

11–16(4)

Variation in live colouration. Some of the male specimens collected during this study matched with the holotype in live colouration but there were some specimens which were different from the holotype. All of the breeding males had blue colouration on the eyelids, upper labials, nasal and the nuchal region (e.g. 14A & B; 16A & B). In two individuals, the blue colouration on the nuchal region extended most of the dorsum most prominent immediately after euthanization (Appendix 10). The gray band marking between the supraciliaries on top of the head, were darker in some males. The last row on the enlarged scale rows on the dorsum were bordered with continous/broken cream coloured or dark brown stripes till the vent. The rhomboidal patch on the neck was relatively darker than the other 4–5 rhomboidal markings which were faint and broken (Fig. 16 a,b). Hemipenial morphology. Hemipenis of seven specimens including the type and the paratype were examined (BNHS 2492, CES 141175, CES 141198, CES 141200, CES 141201, CES 141202–141203). Hemipenis bilobed, relatively small, longer than wide and shallowly forked (Fig. 17 a,b). Sulcus spermaticus bifurcated (Fig. 17a). Sulcal lips raised and papillate. Sulcus smooth, originating from the side of the base. Apex with close irregular row of small calyces and the sulcal region of apex nude, medial projection absent (Fig. 17a,d). Ornamentation is differentiated and combination of flounces, calyces observed (Fig. 17a–d). Papillae present between the apical lobes. Calyces are relatively larger at the base of the lobes. Calyces are deep regular pits on the asulcal side and become shallow at basal region (Fig. 17b). Ridges between the calyces are smooth. Eight to eleven flounces present, all of them, prominent on the asulcal side. Etymology. The specific epithet is a noun in apposition, derived from the Telugu word thondalu for an agamid lizard in Andhra Pradesh and Telangana states. Suggested English name. Nagarjuna Sagar fan-throated lizard Distribution. Sitana thondalu sp. nov. is found in Macherala and Nagarjuna Sagar, Guntur District, in Andhra Pradesh state. This species was only recorded from these two localities during this study (Fig. 1). The altitudinal distribution is between 180 and 200 m a.s.l. Habitat and Natural history. Sitana thondalu sp. nov. is found in dry rocky outcrops with boulders (Fig. 18a) and with sparse vegetation predominantly shrubs and few stunted trees (dominated by Mundulea sericea. Macherla receives annual rainfall between 380 to 508 mm (Talukder & Pal, 2007). In the second site Macherla, the habitat is dominated by the invasive Prosopis juliflora (Sw.) DC. (Fig. 18b). Calotes versicolor (Daudin, 1802) and Eutropis nagarjunensis (Sharma, 1969) are the two sympatric diurnal lizards found in this region. Breeding males were recorded during June and August months.


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69/

7/ B B B B B B B B B B B B B B B B B

Discussion The lack of morphological characters to delimit species is one of the persistent problems while describing new cryptic species (Marin et al. 2013). The advent of DNA sequencing opened a new window and rapid method in identifying cryptic species (Bickford et al. 2007). However, systematists and taxonomists have recommended the use of more than one line of evidence (Wiens & Penkrot, 2002; Wiens, 2007; De Queiroz, 2007; Padial et al. 2010; Karanth, 2017). DNA, morphology, geography, ecology and reproduction could be used as broad criteria to delimit species (Padial et al. 2010). The fan-throated lizards have complex communication systems and species with similar dewlap morphology are reported to have similar display patterns (Kästle, 1998; Kamath, 2015). However, we did not systematically document any behaviour of the two new species and this can be investigated in future. Previous studies have highlighted the compulsion to use geography as one criterion to delimit species (De Queiroz, 2007; Vijayakumar et al. 2014) and peninsular India is known for cryptic lizard species with subtle morphological differences (e.g. Cyrtodactylus srilekha Agarwal, 2016 and Cyrtodactylus rishivalleyensis, Agarwal, 2016). In the present study we have used a relatively large sample size for each species and have done thorough morphological comparisons. The divergence between Sitana gokakensis sp. nov. and Sitana thondalu sp. nov. is comparable to those between other described species (Appendix 3 & 11). In addition, the samples collected in the surrounding and inbetween areas (Fig. 1) of the two type localities, we found other species namely: Sitana laticeps, Sitana sp1, Sitana spinaecephalus and Sitana ponticeriana. This further reinforces the fact that the two species are local endemics. Eventhough we did not investigate why these species are endemic to these regions, both the study areas are previously known for their unique and endemic flora and fauna (Sharma, 1969; Malpure et al. 2016). In the case of Sitana gokakensis sp. nov., we speculate that the rocky river surrounding the flat rocky top plateau in Gokak might have acted as a barrier for its dispersal. Sitana in general have a physical disadvantage because of the lack of 5th toe which is associated with climbing (Russell & Rewcastle, 1979). Therefore, rivers which are primarily rocky, often barren with peaks and troughs are barriers for Sitana compared to sandy rivers often with vegetation (See Deepak & Karanth, 2018 for other examples). Similarly, Sitana thondalu sp. nov. is most likely restricted in the plains north of Nallamala hills which are surrounded by the Krishna River in the North and the higher Nallamala hills in the south. However, further fine scale sampling is required to identify the exact extent of distribution of these two species. This highlights the importance of the two study areas (Gokak, Nagarjuna Sagar) and systematic explorations for other groups of reptilian fauna in this region may yield new endemic species. Krishna river in peninsular India was identified as a significant barrier for the widely distributed Hanuman langur (Semnopithecus spp.) (Nag et al. 2011; Nag et al. 2014; Ashalakshmi et al. 2015) and could potentially be a biogeographic barrier for small terrestrial lizards like Sitana (See Deepak & Karanth, 2018). Breeding colouration in Sitana is subjective as some species are known to change colour upon handling (pers. Obs. V. Deepak). Furthermore, the colouration also varies with the time of the breeding season (i.e. pre/peak/post breeding) (pers. Obs. V. Deepak). Sitana thondalu sp. nov. have more blue colouration on the head compared to S. gokakensis sp. nov. However, we did not use this as a diagnostic character because it is relative and we did not systematically quantify colour. Deepak et al (2016b) provided detailed morphological comparisons of Sitana ponticeriana complex in which they showed extensive overlap between S. ponticeriana and S. bahiri Amarasinghe, Ineich & Karunarathna, 2015. Deepak and Karanth (2018) used multiple molecular species delimitation methods to show that S. bahiri is indeed S. ponticeriana. Based on these lines of evidence we consider S. bahiri as a junior synonym of S. ponticeriana. In this study, we found out that the two new species have extensive overlap in morphology which was possible to investigate only due to our large sample size. We also found out that there is potential population level variation in dewlap size in the case of southern population of S. laticeps (Fig. 10). Therefore, it is imperative to compare large sample sizes in conjugation with thorough molecular comparison before describing a new taxon in the genus Sitana.

Acknowledgement We thank the Karnataka and Andhra Pradesh forest Department for their support. Kunal Arekar, Eleni Foui, Aniruddha Datta-Roy, Prudhvi Raj G and Aparna Lajmi for support in field work. VD thanks Department of



353

Biotechnology for DBT-RA fellowship. VD thanks Director Zoological Survey of India. P.S. Bhatnagar at ZSI Pune for his support. Staff at ZSI Pune and Kolkata for facilitating this work. Staff at Bombay Natural History Society museum, namely Rahul Khot, Curator-Reptile collections and Saunak Pal for their support. VD thanks Aparna Lajmi’s family for logistic support during field work. VD thanks Rufford Small Grants for funding the field work and Asian Nature Conservation Foundation (ANCF) for their support. VD thanks Rohini Balakrishnan for allowing us to use the Camera-Lucida facility in her lab. VD thanks Natalie Cooper for teaching and introducing to various packages in R. VD’s contribution was support, in part, by Marie Skłodowska-Curie Fellowship. We thank Varad Giri for his support. Navendu Page for identification of some plant species from the type localities. We thank Ishan Agarwal and Abhijit Das for their comments which improved this manuscript.

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APPENDIX 1. Comparative material used in this study Sitana gokakensis sp. nov. CES 141131–141133; CES 141137–141140 (7 males), CES 141141–141143 (3 females) collected by V. Deepak and Kunal Arekar on 12.5.15. VDE 168—adult male collected by V. Deepak and Aparna Lajmi on 08.08.13 all specimens collected from Gokak plateau, Belagavi district, Karnataka (N16.186, E74.760), 255 m elevation. Note. VDE 168 was cleared and stained and stored at CES. Sitana thondalu sp. nov. CES 141170–141174 (5 males), CES 141176– 141177 (2 males), 141179 (female) from East Bank of Nagarjun Sagar reservoir, Guntur district, Andhra Pradesh, India (N 16.556, E79.301), 200 m elevation, collected by V. Deepak, Praveen Karanth, Aniruddha Datta-Roy, Rama Harvey and Aparna Lajmi. CES 141197–141198; 141200–1411203 (6 males) and CES 141199, Ces 141204 (2 females) from Macherla, Guntur district, Andhra Pradesh, India (N 16.556, E79.301), 200 m elevation, collected by V. Deepak, R. Chaitanya, Aniruddha Datta-Roy Sitana spinaecephalus CES 141164, CES 13521–13522, CES 13525, CES 13527, CES 13535–13536, CES 13539–13542, CES 13541– 13542 (18 males); ZSIK 6333, 6336, CES 13523–13524, 13526, 13528, 13530–13531, 13534, 13537–13538, BNHS 2320 (12 females) from Gujarat. Refer to Deepak et al (2016a) for additional information on locality. Sitana laticeps Southern population CES 13586–13588 (3 males) from Bagalkot, Karnataka; CES 141134 (male) and CES 141135 (female) Tummarguddi, Karnataka and CES 141106 (male) from Koppal, Karnataka. Populations from throughout the species range in Maharashtra: VDE 94, VDE97–99 from Kagal, Maharashtra (4 males), ZSIP R/613c–d, CES G354, ZSIP R/779, ZSIP R/548, ZSIP R/564, ZSIP R/958, ZSIP R/549, ZSIP R/616, ZSIP R/900, ZSI 21571 a–f, ZSI 21573 a–d, ZSI 21581, ZSI 21578, ZSI 21585, CES 141165 (28 males); ZSI21571a–b, ZSI21571e, ZSI21571i-j, ZSI21573a–f, ZSI21578a, ZSI21584, ZSIK/R549b, ZSIK/R900b, ZSIK/R548b, ZSIK R701, ZSIK/R958b, ZSIK 21579a–c, ZSIK/R776, ZSIK/R613c, ZSIK/R613d, VDE 202, VDE 223, VDE95–96 (28 Females) from Maharashtra. Refer to Deepak et al (2016a) for additional information on locality. Sitana sp1 Ces14534–14535, Ces14538–14541 (6 males) Ahobillum, Andhra Pradesh; CES 141168, CESG245–G246 (3 males) Srisalem, Andhra Pradesh; CES 13501 (male) Kanakpura, Karnataka; CES 14650 & CES 13509 (2 males) Ullodu, Karnataka; CES 13502 (male) Bailur, Karnataka; CES 13550–13551, VDE 138 (3 females) & VDE 139 & 142, CES 13552–13554 (5 males) Hampi, Karnataka; CESG 212–213, CES 141146–141147, CES 141149–141152 (8 males) & CES 214, CES 141148 (2 females) Challekere, Karnataka. Refer to Deepak & Karanth (2018) supplementary material for additional information on locality.


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APPENDIX 2. Shapiro-Wilk normality test for the 19 variables used in this study. significant values indicated with *. S.no

Variable

Shapiro-wilk test

S. gokakensis

S. thondalu

S. spinaecephalus

S. laticeps

1

HL

W

0.79

0.96

0.88

0.97

p-value

0.012*

0.62

0.08

0.58

2

HW

W

0.87

0.98

0.95

0.98

p-value

0.11

0.98

0.55

0.79

3

HH

W

0.88

0.94

0.92

0.95

p-value

0.13

0.48

0.25

0.2

W

0.88

0.90

0.98

0.99

p-value

0.13

0.13

0.95

0.99

W

0.78

0.95

0.97

0.99

p-value

0.01*

0.58

0.9

0.96

W

0.93

0.87

0.96

0.92

p-value

0.47

0.04*

0.76

0.03*

W

0.93

0.91

0.94

0.95

p-value

0.42

0.12

0.48

0.17

W

0.85

0.96

0.88

0.98

p-value

0.052*

0.62

0.07

0.7

W

0.9

0.93

0.94

0.95

p-value

0.22

0.29

0.49

0.14

4 5

EE IO

6

JL

7

NE

8 9

SE SW

10

DEWL

W

0.86

0.95

0.94

0.87

p-value

0.08

0.51

0.51

0.001*

11

DEWLT

W

0.98

0.97

0.93

0.94

p-value

0.98

0.86

0.42

0.15

W

0.97

0.94

0.97

0.91

p-value

0.93

0.4

0.83

0.4

W

0.89

0.96

0.96

0.83

p-value

0.18

0.72

0.69

0.001*

12 13

4thT FL

14

CL

W

0.78

0.95

0.87

0.87

p-value

0.001*

0.46

0.06

0.001*

15

HEL

W

0.91

0.88

0.94

0.88

p-value

0.26

0.04*

0.426

0.001*

W

0.83

0.92

0.97

0.91

p-value

0.03*

0.18

0.86

0.01*

W

0.86

0.92

0.97

0.97

p-value

0.09

0.21

0.86

0.64

16 17

LAL UAL

18

TRL

W

0.9

0.97

0.99

0.97

p-value

0.21

0.83

0.99

0.63

19

SVL

W

0.81

0.87

0.9

0.87

p-value

0.01*

0.03*

0.15

0.001*



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Sitana laticeps

Sitana laticeps

Sitana VS

Sitana VS

Sitana spinaecephalus

Sitana spinaecephalus

Sitana thondalu VSQRY

Sitana thondalu VSQRY

6LWDQDgokakensisVSQRY

Sitana ponticeriana

Sitana ponticeriana

Sitana ponticeriana

Sitana visiri

Sitana marudhamneydhal

Sarada deccanensis

B

Sitana laticeps

6SHFLHV

Sitana laticeps

B

B

B

B

B

B

B

B

B

B

B

B

B

B

B

$33(1',;8QFRUUHFWHGSVHTXHQFHGLYHUJHQFH1'GDWDVHW IRUJHQHWLFVDPSOHVLQFOXGHGLQWKLVVWXG\1XPEHUVLQEUDFNHWVFRUUHVSRQGVWRORFDOLW\QXPEHUVLQ7DEOH

B

APPENDIX 4. Factor loadings on the principal components (PC) for each morphometric variable derived from PCA Variables

PC1

PC2

PC3

PC4

CL

0.27

0.14

0.02

-0.08

DEWL

0.26

0.23

-0.03

-0.07

DEWLT

0.22

0.37

0.02

-0.04

EE

0.11

-0.51

0.00

-0.24

FL

0.26

0.19

-0.11

-0.04

HEL

0.27

0.00

0.05

0.32

HH

0.23

-0.07

-0.17

-0.26

HL

0.27

-0.11

0.02

-0.07

HW

0.19

-0.16

-0.02

-0.33

IO

0.19

-0.32

-0.24

-0.23

JL

0.26

-0.06

0.07

0.17

LAL

0.28

0.04

-0.06

0.12

NE

0.14

-0.38

0.47

0.16

SE

0.20

-0.23

0.34

-0.07

SVL

0.28

0.09

-0.03

-0.03

SW

0.08

-0.31

-0.70

0.45

TRL

0.24

0.13

-0.06

-0.22

UAL

0.25

0.16

-0.03

0.06

X4thT

0.21

-0.03

0.23

0.52



359

APPENDIX 5. PCA plot showing overlap in merestic characters of S. gokakensis sp. nov. (red circles) with S. thondalu sp. nov. (blue circles). The arrows are pointing in the direction of the variables, as projected into a 2-dimension plane of the biplot.


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APPENDIX 6. Confusion matrix for the DFA model predictions (in rows) and actual (in columns) for the training and testing dataset. Probability values indicate model accuracy for correct classification of species in a given dataset. DFA analysis1

Training dataset

Probability 0.85

S. gokakensis

S. thondalu

S. spinaecephalus

S. laticeps

S. gokakensis

5

0

0

1

S. thondalu

1

7

0

0

S. spinaecephalus

1

0

9

1

S. laticeps

0

1

2

19

Testing dataset

Probability 0.77

S. gokakensis

2

1

1

0

S. thondalu

1

5

0

0

S. spinaecephalus

0

0

1

1

S. laticeps

0

1

0

9

DFA analysis 2

Training dataset

Probability 1.00

S. gokakensis

S. thondalu

S. spinaecephalus

S. gokakensis

7

0

0

S. thondalu

0

8

0

S. spinaecephalus

0

0

10

Testing dataset

Probability 0.67

S. gokakensis

S. thondalu

S. spinaecephalus

S. gokakensis

1

1

1

S. thondalu

2

6

0

S. spinaecephalus

0

0

1



361

APPENDIX 7. Dorsal profile of the two paratypes of S. gokakensis sp. nov. A. CES 141136 (Male) B. VDE 169/BNHS (female). Scale bar 10 mm.


DEEPAK ET AL.

APPENDIX 8. Image showing blue coloration on the scales in the tail region of one of the male S. gokakensis sp. nov. immediately after euthanization of the specimen.



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APPENDIX 9. Dorsal profile of the two paratypes of S. thondalu sp. nov. A. CES 141175(male) B. CES 141178 (female). Scale bar 10 mm.


DEEPAK ET AL.

APPENDIX 10. Image showing blue coloration on the scales in the dorsum of S. thondalu sp. nov. males immediately after euthinisation of the specimen.

APPENDIX 11. Uncorrected p sequence divergence (R35 data set) for genetic samples included in this study. Numbers in brackets corresponds to locality numbers in Table 1. Species

1

2

3

1

Sitana laticeps (4)

_

2

Sitana laticeps (14)

0.013

_

3

Sitana laticeps (2)

0.010

0.010

_

4

5

6

7

8

9

4

Sitana sp1 (7)

0.011

0.018

0.015

_

5

Sitana sp1 (5)

0.013

0.000

0.010

0.018

_

6

Sitana thondalu sp. nov. (8)

0.021

0.018

0.018

0.020

0.018

_

7

Sitana gokakensis sp. nov. (3)

0.020

0.018

0.016

0.018

0.018

0.015

_

8

Sitana visiri (15)

0.016

0.015

0.016

0.015

0.015

0.018

0.013

_

9

Sitana marudhamneydhal (16)

0.016

0.011

0.013

0.015

0.011

0.015

0.013

0.010

_

10

Sarada deccanensis (17)

0.050

0.053

0.050

0.050

0.053

0.060

0.055

0.052

0.052


10

_


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