Indirana salelkari , a new species of leaping frog ( Anura : Ranixalidae ) from Western Ghats of Goa , India

1,4 Department of Biodiversity, MES Abasahab Garware College, Karve Road, Pune, Maharashtra 411004, India 2 Indian Institute of Science Education and Research (IISER), G1 Block, Dr. Homi Bhabha Road, Pashan, Pune, Maharashtra 411008, India 2 Systematics, Ecology and Conservation Laboratory, Zoo Outreach Organization (ZOO), 96 Kumudham Nagar, Vilankurichi Road, Coimbatore, Tamil Nadu 641035, India 3 Department of Zoology, Willingdon College, Sangli, Maharashtra 416416, India 4 Department of Zoology, MES Abasahab Garware College, Karve Road, Pune, Maharashtra 411004, India 1 nikhilsmodak@gmail.com, 2 n.dahanukar@iiserpune.ac.in, 3 ninad.gosavi4@gmail.com, 4 anand.padhye@mesagc.org (corresponding author)

During the field surveys in Goa region of the Western Ghats, we came across a population of Indirana which was found to be morphologically and genetically different from other known species of the genus.The new species is described here.

Study site and specimen collection
Specimens of the new species were collected from the Tanshikar Spice Farm at Netravali (Neturlim) in Sanguem Taluk of South Goa, India (15.095 0 N & 74.211 0 E; elevation 78m).Four male and four female specimens were collected and preserved in absolute alcohol for further analysis.Ten tadpoles of different stages were collected from the lateritic rocks near the same locality.

Museum details
Specimens studied in this paper are deposited in the museum of the Bombay Natural History Society (BNHS), Mumbai; the Wildlife information Liaison Development (WILD) Society, Coimbatore; the Zoological Survey of India, Western Regional Center (ZSI-WRC), Pune and Abasaheb Garware College, Zoology Research Laboratory (AGCZRL), Pune, India.Type specimens from the Natural History Museum (BMNH), London and the Muséum National d'histoire Naturelle (MNHN), Paris, were studied for comparison by the first author.

Morphometry
Morphometric measurements were carried out with the help of a digital caliper (Ocean Premium measuring instruments) to the nearest 0.1mm.A total of 27 characters were measured following Padhye et al. (2014), viz.: SUL (Length of specimen from snout to the visible tip of urostyle); HL (Head length: from the posterior border of tympanum to the tip of snout); HW (head width: width of head between to posterior borders of tympanum); SL (Snout length: from the anterior orbital border to the tip of snout); EL (Eye Length: Horizontal length of eye between orbital borders); TYL (maximum tympanum length); UEW (upper eyelid width); SNL (snout to nostril distance); ENL (eye to nostril distance); INL (internarial distance); IOL (inter-orbital distance: minimum distance between two eyelids); UAL (Upper arm length); FoAL (Fore-arm Length); F1 to F4 (Finger 1 to Finger 4 length from the base of the sub-articular tubercle); THL (thigh length from hip joint to joint between thigh and shank); TL (Tibia/shank length from joint between thigh and shank to joint between shank and tibiotarsal articulation); ACL (Astragalo-calcaneal length from joint between shank and tibiotarsal articulation to the base of the inner metatarsal tubercle); FOL (Foot length: from the base of the inner metatarsal tubercle to the tip of the fourth toe); TFOL (Total foot length: from the tibiotarsal articulation to the tip of fourth toe) and T1 to T5 (Toe1 to Toe5 length from the base of the respective subarticular tubercle).Webbing formula was determined following the method provided by Savage & Heyer (1967) with modifications by Myers & Duellman (1982).We also measured the characters related to the roof of buccal cavity (Fig. 1a) using stage and ocular micrometer scale (least count 0.01mm) in Leica 58AP0 dissecting microscope.The depth of the buccal cavity (Fig. 1b) was determined by measuring the difference in the focal planes of upper lip and vomerine region of the buccal roof using scale (least count 0.002mm) on the fine focus knob of Zeiss Primostar compound microscope.

Statistical analysis
Statistical analysis of the morphometric data was performed on size adjusted measurements by taking all measurements as percent of SUL to remove the bias due to body size variation.Multivariate normality of the data was checked using Doornik & Hansen (2008) omnibus.Discriminant Analysis (DA) was performed to understand whether related species form significantly different clusters (Huberty & Olejnik 2006) in the genus Indirana.Pillai's trace statistic was used to test the null hypothesis that the mean vectors of different clusters are equal (Harris 2001).Mahalanobis distances (Harris 2001) between pair of individuals were calculated and were used for computing Fisher's distances (distance between the centroids of the clusters, divided by the sum of their standard deviations) between two clusters to check if the clusters were significantly different.Statistical analysis was performed in PAST 3.0 (Hammer et al. 2001).

Molecular analysis
Thigh muscles of the three specimens (BNHS 5931, WILD-15-AMP-551 and AGCZRL-Amphibia-210) were used for extracting DNA and conducting molecular analyses.Genomic DNA extraction, Polymerase Chain Reaction (PCR) for two mitochondrial (12S and 16S) and one nuclear (rho) genes, PCR product purification and sequencing was performed following the protocols detailed in Padhye et al. (2014).Sequences were checked by BLAST tool (Altschul et al. 1990) to identify the nearest congeners.These sequences have been deposited in GenBank (KP826821 to KP826829).Additional sequences of related species were retrieved from NCBI GenBank database (http://www.ncbi.nlm.nih.gov/).GenBank accession numbers of the sequences used for the analysis are provided in Appendix A. Gene sequences were aligned separately using MUSCLE (Edgar 2004) implemented in MEGA 6 (Tamura et al. 2013) and were concatenated to make a combined matrix of 921 nucleotides.Best fit model for nucleotide substitution was selected from 24 models using MEGA 6 (Tamura et al. 2013) based on minimum Bayesian Information Criterion (BIC) value (Schwarz 1978;Nei & Kumar 2000).This best fit model was also used for constructing the phylogenetic trees using maximum likelihood in MEGA 6 (Tamura et al. 2013).Reliability of the phylogenetic tree was estimated using bootstrap values run for 1000 iterations.Phylogenetic tree was edited in FigTree v1.4.2 (Morariu et al. 2009).Pairwise raw genetic distances using 16S rRNA gene and combined matrix of 12S, 16S and rho genes were calculated using p distances method in MEGA 6 (Tamura et al. 2013).

Molecular clock analysis
A subset of concatenated sequences, with monophyletic clades, were used for molecular clock analysis.Separation of Nasikabatrachidae (149.5 mya); separation of Nyctibatrachidae (91.6 mya) and separation of Micrixalidae (89.7 mya) were used as calibration points obtained from time tree (Hedges et al. 2006).Aligned sequences were used for finding the best fit model for nucleotide substitution using J Model test (Darriba et al. 2012).The best fit models for the three partitions (12S: TIM2ef + G; 16S: TIM2 + G; Rho: K80 + Inv) were used for molecular clock analysis using BEAST v.1.8.0 (Drummond et al. 2012).Phylogenetic tree was edited in FigTree v1.4.2 (Morariu et al. 2009).Time of taxa split are expressed as mean ± 95% Highest Posterior Density (HPD).
Diagnosis: Indirana salelkari sp.nov.differs from all other congeners based on the following combination of characters: medium-sized frog (20.9-30.9mm SUL), head longer than wide, distinct canthus rostralis, first finger longer than or equal to second, presence of double outer palmar tubercle, elongated inner metatarsal tubercle, webbing moderate (I1-2II1-2½III1¼-3IV3-1¼V), discs of fingers and toes with crescentic deep marginal grooves restricted only to the anterior side of the discs, buccal cavity narrow and deep, vomerine teeth large and acutely placed closer to each other with a distance less than the length of vomerine teeth series, oval choanae, dorsally skin with glandular folds but without warts, ventrally skin granular with some mottling on throat and palms and soles dark brown.

Description
General appearance of holotype as in Image 1 and of female paratype as in Image 2. Morphometric details as in Table 1.
Dorsal and ventral skin smooth; few longitudinal folds on dorsal side; lateral and ventral side granular.

Coloration in life (Image 4)
Dorsum Pale to dark brown, some specimens were also observed with pinkish dorsum; dark band between the eyes which continues on the upper eyelid; interrupted W shaped mark on the back of the head may or may not be present; upper and lower mandible barred with brown stripes which are sometimes interrupted or absent on upper mandible; a dark brown stripe running from the tip of the snout to shoulder through the eye and tympanum; forelimbs and hind limbs bearing transverse bands which are also present on fingers and toes which may not be quite distinct in darker specimens (usually these bands are paler in females); lateral margin of forelimbs and hind limbs densely spotted with dark brown or black (fewer in females) which may continue on ventral side in forelimbs; palm dark brown in color; foot and soles dark brown; ventrally white, throat in some specimens mottled with brown.

Coloration in preservation (Image 1 and 2)
Dorsal pale to dark brown, dark band between the eyes which continues on the upper eyelid; interrupted W shaped mark on back of the head which may or may not be present; upper and lower mandible barred with brown strips sometimes interrupted or absent on upper mandible; dark brown strip running from tip of snout to shoulder through eye and tympanum visible; few dark spots on the lateral side of abdomen; forelimbs and hindlimbs barred with dark brown strips which may not be quite distinct in darker specimens; lateral margin of forelimbs and hind limbs densely spotted with dark brown or black which may continue on ventral side in forelimbs; region near outer palmer tubercle darker; sole and foot dark brown; ventrally creamish to white; brown mottling on the throat of some specimens.

Sexual Dimorphism
In the breeding season males bear nuptial pad on the outer side of first finger and femoral glands on thighs.

Etymology
The species is named after Prakash Salelkar, Range Forest Officer, Netravali, Goa, to honor his dedicated work on the conservation of wildlife in Goa State and for his continual help since 2003 during field work in Goa.

Distribution
The species is currently known only from its type locality in Sanguem Taluk of South Goa, India (15.095 0 N & 74.211 0 E; 78m) (Image 5).

Habitat
General habitat at type locality is shown in Image 6.The specimens were collected from Tanshikar Spice Farm.The species was seen to occupy nearby riparian habitats.Some sub adults were seen under leaf litter.The tadpoles were collected from the exposed laterite in the vicinity of the type locality.

Natural history and description of tadpoles
Tadpoles of various stages in prometamorphic (Stage 36, stage 39 and stage 41) and metamorphic (stage  (Dubois 1994) as 4[A 1 -A 4 ]/4[P 1 -P 2 ] (Image 8).The oral apparatus is divided into two lateral parts by large horny beak.The first anterior keratodont ridge A 1 is divided while three succeeding anterior keratodont ridges A 2 -A 4 are placed lateral to the horny beak.On the posterior labia keratodont ridge P 1 is marginal and keratodont ridge P 2 is placed lateral to the horny beak and P 3 and P 4 are continuous.

Common name
Netravali Leaping Frog.

Genetic analysis
Best fit model for 16S rRNA barcoding gene was GTR+G (BIC = 6137.51,lnL = -2496.68,G = 0.37).The best fit model for the nucleotide substitution for cconcatenated genetic sequences (921 bases) of mitochondrial 12S and 16S rRNA genes and nuclear rho gene was GTR+G+I (BIC = 10429.89,lnL = -4596.32,I = 0.32, G = 0.58).Maximum likelihood analysis of the genetic data (Figs. 1 and 2) suggested that Indirana salelkari sp.nov. is a monophyletic group genetically distinct from the other Indirana species for which genetic data are available.The sister taxa for I. salelkari is I. chiravasi from which it differs with the raw distance of 3.8% in 16S rRNA gene and 3.1-3.2% in concatenated sequences.Molecular clock analysis (Fig. 4) suggested that Indirana salelkari separated from I. chiravasi about 10.9 myr ago (95% HPD 14.5-7.4).

Statistical analysis
Size corrected morphometric data was not significantly different from multivariate normal (Doornik and Hansen omnibus, within group Ep = 74.91,P = 0.0512).MANOVA suggested that there were significantly distinct clusters among the species (Pillai's trace = 5.13, F 234,333 = 1.886,P < 0.0001).Discriminant Analysis extracted nine factors out of which first three canonical axes explained 86.77% of the total variation in the data where the first axis explained 40.13%, second axis explained 26.26% and third axis explained 20.38% of the total variation (Fig. 3c).First two canonical axes readily separated Indirana salelkari sp.nov.from I. diplosticta, I. leptodactyla and I. phrynoderma (Fig. 3a).Indirana salelkari sp.nov.was separated from I. leithii, I. beddomii, I. brachytarsus, I. chiravasi, I. gundia and I. semipalmata on the third canonical axis (Fig. 3b).DA loadings of morphometric characters on the first three canonical axes are shown in  (vs.first finger shorter than second).Indirana salelkari sp.nov.differs from I. tenuilingua in having head longer than broad (vs.head slightly wider than long), inter-orbital distance equal to or wider than inter-narial distance (vs.interorbital width more than twice the distance between the nostrils) and toes and fingers with crescentic deep marginal grooves restricted only to the anterior side of the discs (vs.semicircular groove in front of the toes and fingers absent, faint or indistinct).
Indirana salelkari sp.nov.can be distinguished from I. brachytarsus in having moderate webbing I1-2II1-2½III1¼-3IV3-1¼V (vs.extensive webbing, webbing formula, I1-2II1-2½III1-3IV3-1V), longer upper arm (17.1-I. chiravasi, however it differs from I. chiravasi in having thin elongated metatarsal tubercle in males and females (vs.thin shovel shaped in males and thin elongated in females) and deep and narrow buccal cavity (vs.shallow and broad, Image 3, Table 3).Both the species also differ in their placement and structure of vomerine teeth in relation to choanae (Image 3, Table 3) where I. salelkari has a much longer and acutely placed vomerine teeth series, which are placed closer to each other with a distance less than the length of vomerine teeth series (vs.shorter vomerine teeth series placed at a distance larger than the length of vomerine teeth series) (Table 3).Further, I. salelkari has oval choanae that are more laterally placed in the buccal cavity as compared to I. chiravasi which has circular and dorsally placed choanae (Image 3, Table 3).Indirana salelkari is genetically different from I. chiravasi with a genetic distance 3.8% in 16S rRNA gene and 3.1-3.2% in concatenated sequences.

DISCUSSION
Indirana salelkari sp.nov. is the twelfth species of the monogeneric family Ranixalidae, endemic to the Western Ghats of India.Indirana chiravasi, the sister species of I. salelkari, is a widely distributed species in northern Western Ghats from 15.4 -18.5 0 N latitudes (Modak et al. in prep.).Indirana salelkari is known from just south of this range (15.1 0 N), it is morphologically distinct species and forms a monophyletic group in genetic analysis.Although, the raw genetic distance between the two species is only 3.8%, Vences et al. (2005) have observed differentiation among species ranging from 1-16.5 %.Further, we are delineating the species based on morphology, by studying the available types of known species, and genetic evidence is just used as a support to bolster our claims.The other genetically closely related individuals are identified as I. beddomii by Nair et al. (2012) with voucher numbers IND/AA/ 231,200,227 and 230.However, our study of the type material of I. beddomii clearly suggests that I. salelkari is distinct from I. beddomii.
Oral apparatus structure in the tadpoles of I. chiravasi, I. leithii and I. semipalmata (Sekar 1992;Gopalan et al. 2012;Padhye et al. 2014) are similar to that of I. salelkari.Bonacci et al. (2008) have suggested that the oral apparatus structure can be related to feeding habits and dietary specializations.We have observed similar feeding habits in I. leithii, I. chiravasi and I. salelkari, where the semiterrestrial tadpoles occupy wet rocks and boulders to feed on the algal matter.Owing to similar food and feeding habits, the members of this genus might have developed similar oral apparatus.Unfortunately, very little information is available on the ecology of Indirana and more studies in this respect are essential.
In the IUCN redlist of threatened taxa (IUCN 2014), six species of Indirana are currently listed under threatened categories and include I. gundia and I. phrynoderma under Critically Endangered; I. brachytarsus, I. diplosticta and I. leptodactyla under Endangered; and I. leithii under Vulnerable.Two species, I. gundia and I. phrynoderma, are also Alliance for Zero Extinction (AZE) species (Parr et al. 2009).Further, there are reports of chytrid infection in Indirana brachytarsus and I. leithii (Nair et al. 2012, Dahanukar et al. 2013;Molur et al. 2015).The fact that half of the known species in this endemic family are threatened calls for immediate attention towards their conservation.Given that the taxonomy and distribution of several of these species is still ambiguous and there is also possibility of discovering cryptic species within the genus (Nair et al. 2011;Modak et al. 2014), further explorations and molecular studies are essential to reveal conservation status of this taxon.

Figure 1 .
Figure 1.Diagrammatic illustration of the roof of buccal cavity indicating the measured distances.(a) ventral view of the roof and (b) sagittal section.
at the type locality at Tanshikar Spice Farm.

Figure 5 .
Figure 5. Discriminant analysis of size corrected morphometric data in first two axis for all the species (a) and first three axis for the taxa closely related to Indirana salelkari sp.nov.(b).Scree plot is given in (c).

Table 2 .
Relatively higher values of characters such as TFOL, FOL, TL, THL, T1 and ACL and lower values of TYL separated Indirana salelkari sp.nov.from other related species.

with other species of Indirana Indirana
salelkari sp.nov.differs from I. diplosticta, I. leithii, I. leptodactyla, I. longicrus and I. phrynoderma in having first finger equal to or longer than second finger

Indirana salelkari -new leaping frog Modak et al.
750524.9%SVL vs. 16.1% SVL), deep and narrow buccal cavity (vs.shallower and broader), large choanae (vs.small) and thick vomerines (vs.thin) (Image 3).Furthermore, genetic distance between I. salelkari and I. brachytarsus is 8.3% for 16S gene and 7.0% for concatenated sequences.Indirana salelkari sp.nov.differs from I. gundia in having tympanum flushing with the lateral side of the head (vs.tympanum protruding out of the lateral side of the head) and discs have marginal groove (vs.discs of males and females have sub-marginal groove).Genetic distance between I. salelkari and I. gundia is 4.3-4.5 % for 16S gene and 3.2-3.3%for concatenated sequences.Indirana salelkari sp.nov. is morphologically similar to