Raorchestes ghatei , a new species of shrub frog ( Anura : Rhacophoridae ) from the Western Ghats of Maharashtra , India

1,3 Department of Zoology, MES’s Abasaheb Garware College, Pune, Maharashtra 411004, India 2 Wildlife Protection and Research Society, 40 Rajaspura Peth, Satara, Maharashtra 415001, India 4 Indian institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune, Maharashtra 411008, India 4 Zoo Outreach Organization, 96 Kumudham Nagar, Villankurittchi Road, Coimbatore, Tamil Nadu 641035, India 1 anand.padhye@mesagc.org (correspondence author), 2 amitsayyedsatara@gmail.com, 3 anushreejadhav@gmail.com, 4 n.dahanukar@iiserpune.ac.in


INTRODUCTION
The Western Ghats of India harbors a rich diversity of amphibians with high levels of endemism (Dinesh & Radhakrishnan 2011).With recent descriptions of several new species and genera of amphibians from the Western Ghats (Biju et al. 2011;Zachariah et al. 2011;Seshadri et al. 2012;Abraham et al. 2013), it is clear that the amphibian diversity within this region is subject to Linnean shortfall, where several species are not yet formally described (Bini et al. 2006), and detailed surveys and studies are essential to overcome it.
In the Western Ghats, tree frogs of the family Rhacophoridae are grouped under seven genera, namely Beddomixalus Abraham et al., 2013, Ghatixalus Biju, Roelants & Bossuyt, 2008, Mercurana Abraham et al., 2013, Polypedates Tschudi, 1838, Pseudophilautus Laurent, 1943, Raorchestes Biju et al., 2010and Rhacophorus Kuhl & Van Hasselt, 1822.The genus Raorchestes was recently erected to accommodate a monophyletic clade of shrub frogs characterized by adult snout-vent length between 15 and 45 mm, vomerine teeth absent, large gular pouch transparent while calling, nocturnal habit and direct development without free-swimming tadpoles (Biju et al. 2010).Currently, 49 species are recognized under Raorchestes by Frost (2013) and the genus is distributed in Western Ghats, southern China, Laos and Vietnam (Biju et al. 2010).Annandale (1919) described Ixalus bombayensis (= Raorchestes bombayensis) from Castle Rock and mentioned that the species is also present in Khas (= Kaas) in Satara and Khandalla (= Khandala) in Poona (= Pune).In this communication, based on morphological and molecular analysis, we describe a new species of shrub frog and show that the populations of the Raorchestes from Satara District and Pune District, earlier reported as R. bombayensis are those of the new species.We further show that the character of tubercle on the humeral bone, which was previously thought as a specific character of R. tuberohumerus (Kuramoto & Joshy, 2003), is a sexually dimorphic character possessed by males but not by females.

Study area
The Western Ghats of Maharashtra state extend from south of Amboli to north of Surgana covering around 600km of mountain ranges, parallel to the Arabian Sea coast, extending east as well as west, at some places even up to the seashore.A formation of a series of 200-300 m high cliffs, which extends almost throughout the length of this part of the Western Ghats, forms a hurdle in the way of southwestern monsoon clouds.This results in a very high rainfall (average 6,000mm), with some places like Tamhini, Mahabaleshwar and Bhimashankar receiving rainfall up to 10,000mm.The major habitat in this part of Western Ghats is scrub and grasslands, both at the foothills and on the mountaintops.The hilly regions show some primary evergreen forest patches and comparatively more secondary evergreen and moist deciduous forests.The Western Ghats of Maharashtra is rich in biodiversity especially in that of amphibians (Padhye & Ghate 2002).

Morphometry
Measurements were taken to the nearest 0.1mm using a digital caliper and using a binocular microscope.The following measurements, as defined by Biju & Bossuyt (2009), were taken: snout-vent length (SVL); head length (HL); head width (HW); rear of the mandible to the nostril (MN); rear of the mandible to the anterior orbital border of the eye (MFE); rear of the mandible to the posterior orbital border of the eye (MBE); snout length (SL); eye length (EL); inter upper eyelid width (IUE); maximum upper eyelid width (UEW); internal front of eyes (IFE); internal back of eyes (IBE); forelimb length (FLL); hand length (HAL); third finger length (TFL); disc width on finger III (FDIII); width of finger III (FWIII); shank length (ShL); thigh length (TL); foot length (FOL); distance from the heel to the tip of the fourth toe (TFOL).Tympanum diameter was measured both vertically (TYDV) and horizontally (TYDH).

Genetic analysis
Muscle tissue was harvested from nine fresh specimens of the new species (ZSI-WRC A/1484; 100,104,105;127,128,130) collected from different localities and three specimens of R. bombayensis (WILD-13-AMP-230, 231; AGCZRL-Amphibia-172) collected from the type locality of the species and was preserved in absolute ethanol.The tissue was digested at 55°C for two hours using the STE buffer (0.1M NaCl, 0.05 M Tris-HCl, 0.01M EDTA, 1%SDS) with 15µl proteinase K (20mg/ml) per 500ml of STE buffer.DNA was extracted using conventional phenol-chloroform method and re-suspended in nuclease free water.Polymerase chain reaction was performed to amplify partial 16S rRNA gene using primer pair 16SF (5'-CGC CTG TTT ATC AAA AAC AT-3') and 16SR (5'-CCG GTC TGA ACT CAG ATC ACG T-3') (Palumbi et al. 2002).PCR reaction was performed in a 25µl reaction volume containing 5µl of template DNA (~200ng), 5µl of 5X reaction buffer (100 mM Tris pH 9.0, 500 mM KCl, 15 mM MgCl 2 , 0.1% Gelatin), 3µl of 25mM MgCl 2 , 1µl of 10mM dNTPs, 1µl of each primer, 0.5µl Taq polymerase (Promega) and 8.5µl nuclease free water.The thermal profile was 10min at 95 0 C, and 35 cycles of 1min at 94 0 C, 1min at 50 0 C and 2min at 72 0 C, followed by extension of 10min at 72 0 C. Amplified DNA fragments were purified using the 'Promega Wizard Gel and PCR clean up' system and sequenced.The purified PCR products were sequenced using ABI prism 3730 sequencer (Applied Biosystems, USA) and Big dye terminator sequencing kit (ABI Prism, USA).Sequences were analyzed by BLAST tool (Altschul et al. 1990).These sequences have been deposited in GenBank (accession numbers are provided in Appendix A).
We retrieved additional sequences on other related species from NCBI GenBank database (http://www.ncbi.nlm.nih.gov/)details of which are provided in Appendix A. Gene sequences were aligned using MUSCLE (Edgar 2004).Best fit model for nucleotide substitution was selected from 24 models available in MEGA 5 (Takamura et al. 2011) based on minimum Akaike Information Criterion (AIC) value (Posada & Crandall 2001).Maximum Parsimony analysis was performed in MEGA 5. General time reversal nucleotide substitution model with gamma distribution (GTR+G), obtained as a best fit model in the model test (AIC = 4653.9,lnL = -2204.2),was used for constructing phylogenetic tree based on maximum likelihood method in MEGA 5 and Bayesian analysis using MrBayes (Huelsenbeck & Ronquist 2001) integrated in TOPALi v2.5 (Milne et al. 2004).Reliability of the phylogenetic tree using maximum likelihood method was estimated using bootstrap values run for 1000 iterations.For Bayesian analysis two runs were performed for 1,000,000 generations with sample frequency of 10 and burn percentage of 25.

Statistical analysis
Statistical analysis of the morphometric data was performed on size adjusted measurements by taking all measurements as percent of SVL.For analysis, we used the morphometry of related species Raorchestes bombayensis and R. tuberohumerus given by Biju & Bossuyt (2009) and data collected in the present study.Multivariate normality of the data was checked using Doornik & Hansen (2008) omnibus.Multivariate Analysis of Variance/Canonical Variates Analysis (MANOVA/CVA) was performed to understand whether related species of Raorchestes form significantly different clusters (Huberty & Olejnik 2006).We performed Pillay's trace statistic to find the significant difference between the clusters (Harris 2001).Statistical analysis was performed in PAST (Hammer et al. 2001).A.D. Padhye, N. Dahanukar and A. Sayyed;BNHS 5581, 23.viii.2012, 1 male, SVL 25.5mm, Jaichiwadi (17.42

Diagnosis
Raorchestes ghatei sp.nov.can be distinguished from all related taxa by following combination of characters:

Description
Morphometric data are listed in Table 1.General body shape as in Image 1. Dorsal, ventral and lateral view of head as in Image 2. Maximum size 25.5mmSVL in male and 29.8mm SVL in female.
Snout with granular dorsal skin, inter-orbital space with smooth skin; skin on the back with minute sparsely located horny spinules; less in number as well as less conspicuous (Image 2d) as compared to R. bombayensis (Image 2e).Some variants show tubercles on the back.
Outer margins of dorso-lateral bands with inconspicuous dorso-lateral glandular folds, more evident in live specimens.Skin coarsely granular laterally.Gular skin rather smooth.Ventral skin in trunk region coarsely granular from chest to groin.Granulation extends up to thigh.
Colouration in life (Image 6): Dorsum greyish-brown with dark brown marbling.Black dorso-lateral band extending from tympanum converging in and then diverging to groin is present.Fore limbs dark without crossbars while thigh and shank with single cross bar.Creamish-yellow spots on dark background near the groin.Ventral skin in the trunk region is creamish in colour.A few dark spots present near fore limbs.Medium sized frog (SVL 25.5), with robust body; head length (HL 8.9) shorter than head width (HW 9.7; MN 8.0; MFE 6.2; MBE 3.8); outline of snout in dorsal view mucronate; snout length (SL 3.2) slightly longer than horizontal diameter of eye (EL 3.0); canthus rostralis angular, loreal region obtuse, concave; ratio of distance between anterior margins of the eyes (IFE .4.3) to distance between posterior margins of eyes (IBE 6.0) 1:1.40; tympanum (TYD 1.3) indistinct, rounded, almost one-third of the eye diameter; supratympanic fold distinct, from posterior corner of upper eyelid to shoulder; tongue bifid, without papilla but with a lingual pit; infratympanic fold distinct, from posterior margin of lower jaw joining to the supratympanic fold; interorbital distance (IUE 3.4) 1.3 times greater than width of upper eyelid (UEW 2.6).Fore limbs: hand length (HAL 6.9) > humeral length (6.6) > forelimb (FLL 5.5); fingers with lateral (inner as well as outer) dermal fringes, webbing absent; subarticular tubercles prominent, rounded, single; single palmer tubercles present; supernumerary tubercles indistinct; finger length 3 (TFL 3.6) > finger length 2 (2.5) > finger length 4 (2.2) > finger length 1 (1.9); bony tubercle on humerus at the end of deltoid ridge absent.
Hind limbs: moderately long, shank (ShL 11.4) shorter than thigh (TL 11.9), longer than the distance from the base of the inner metatarsal tubercle to the tip of the fourth toe that is foot (FOL 9.8); distance from the heel to tip of the fourth toe (TFOL 15.9) > thigh length (TL 11.9) > shank length (ShL 11.4) > distance from the base of the inner metatarsal tubercle to the tip of the fourth toe (FOL 9.8); webbing reduced; reaching up to penultimate subarticular tubercle on either side of IV toe; webbing formula (I2-2II2-2½III2-3IV2½-2V); dermal fringe absent; subarticular tubercles single, round; tarsal fold and tarsal fringe absent; toe length 4 (5.0)> toe length 3 (3.7)= toe length 5 (3.7) > toe length 2 (2.3) > toe length 1 (1.8); toe discs prominent with circummarginal groove; inner metatarsal tubercle simple, oval; outer metatarsal tubercle absent (Image 4b); heels Colouration in life (Image 7): Dorsum blackish with creamish-white marbling.Black dorso-lateral bands extending from tympanum converging in and then diverging to groin are present.Fore limbs and hind limbs dark with crossbars.Creamish-yellow spots on dark background near the groin.Ventral skin in the trunk region is creamish in colour.A few dark spots present near fore limbs.Yellowish-orange colour towards the posterior end of trunk.Gular skin creamish in colour marbled with dark spots.
In preservation: Colour pattern in alcohol preserved specimen more or less similar as in live condition except for the creamish marbling on the back, which is lost in preservation.

Colour variation
Variation in colour pattern is shown in Image 8. Colour variation on the flanks and thigh region is shown in Image 9. A faint white stripe between anterior margins of upper eye lids (absent in holotype) may or may not be present.

Sexual dimorphism
Males of the species have a bony tubercle on the humerus at the end of deltoid ridge as its extension, which is absent in females (Image 5); males also posses single, sub-gular vocal sac, however, nuptial pads are absent.

Etymology
The species is named after Dr. H.V. Ghate for his contributions to the herpetology of Western Ghats of Maharashtra.

Natural history
Distribution: The species is widely distributed in the Western Ghats of Maharashtra (Fig. 1) and is currently known from Jaichiwadi (17.42 0 N & 73.85 0 E, 1005m) in the south and Taleghar near Bhimashankar (19.08 0 N & 73.64 0 E, 1025m) in the north.
Habitat: Usually inhabit semi-evergreen forests and scrub patches (Image 10).Females usually found underneath loose stones while males are found calling on the branches of shrubs or even on trunks of trees, up to 5m above ground.
Biology: The species has a direct development mode without free-swimming tadpoles.The eggs are laid in soil under loose stones on the forest floor (Image 11a).Development occurs within the egg (inside the vitelline membrane) (Image 11b) and fully metamorphosed juvenile emerges from the egg (Image 11c).

DISCUSSION
Phylogenetically, Raorchestes ghatei sp.nov. is nested within a monophyletic group consisting of R. bombayensis and R. tuberohumerus from the Western Ghats and R. gryllus, R. longchuanensis and R. menglaensis from Viet Nam and China.Yu et al. (2009Yu et al. ( , 2010) ) have already suggested that some species of Raorchestes  from China and Viet Nam are nested within the Western Ghats radiation of Raorchestes.Raorchestes ghatei sp.nov.has a distribution in between the southern most member R. tuberohumerus in this clade and the species in China and Viet Nam.Biogeographical significance of this finding, however, needs further investigations.Raorchestes ghatei sp.nov.can be differentiated from the closely related species from the phylogenetic tree as follows.Raorchestes ghatei sp.nov.differs from R. bombayensis in absence of tongue papilla (vs.presence), absence of nuptial pad (vs.presence), dorsal skin finely granulated (vs.coarsely granulated) and lower SL/EL ratio (vs.higher).Raorchestes ghatei sp.nov.can be differentiated from R. tuberohumerus in having robust body (vs.slender body), longer SVL (vs.smaller), ventrally snout slightly projecting beyond mouth (vs.projecting beyond mouth), wider IUE (vs.narrow).Raorchestes ghatei sp.nov.differs from R. gryllus in having round pupil (vs.horizontal), small and indistinct tympanum (vs.big and distinct) relative to eye diameter, ventral surface coarsely granular (vs.slightly granular) and absence of dermal fringe (vs.well developed dermal fringe).Raorchestes ghatei sp.nov.differs from R. longchuanensis in ventrally snout slightly projecting beyond mouth (vs.projecting beyond mouth) and head wider than long (vs.longer or equal to wide).Raorchestes ghatei sp.nov.differs from R. menglaensis in having dorsal skin smooth or finely granulated (vs.coarsely granulated) and snout mucronate (vs.acutely pointed).
Osteological and genetic study of Raorchestes ghatei sp.nov.has revealed that the tubercle on the humeral bone, a character which was previously considered specific for R. tuberohumerus, is in fact a sexually dimorphic character present only in males.In the description of R. tuberohumerus, Kuramoto & Joshy (2003) mentioned that they failed to collect females of the species.However, we believe that even in R. tuberohumerus, the females may be devoid of the tubercle on the humeral bone.Ecological significance of this sexually dimorphic character is yet to be determined but our initial observations suggests that it might be used by males for clasping the females during amplexus.Another possibility is that the tubercle might be useful in clinging to the small shrubs in windy habitats in Western Ghats.This is especially true for males who are found on the trunks giving the advertisement calls, while the females are mostly found on the ground.
Presence of a lingual pit in the case of Raorchestes ghatei sp.nov.(Image 3a), apposed to a papilla in the case of R. bombayensis is an interesting finding of our study.In an extensive review on median lingual processes in frogs, Grant et al. (1997) have suggested that the presence of a median lingual pit does not necessarily mean the presence of a median lingual process.While taking an example from the museum collection of American Museum of Natural History (AMNH), Grant et al. (1997) argued that in R. bombayensis there is presence of a pit rather than a retracted process.However, the specimen (AMNH 40044) photographed by Grant et al. (1997) is from Satara, which is within the distributional range of R. ghatei sp.nov.Therefore, the arguments raised by Grant et al. (1997) are for R. ghatei sp.nov.and not for R. bombayensis.Raorchestes bombayensis has a welldefined papilla which can be seen in live specimens (Image 3b) as well as the holotype of R. bombayensis (Appendix D).In R. tuberohumerus an ill-defined pit is present even though a papilla is absent (Image 3c).
Raorchestes ghatei sp.nov. is widely distributed in the Western Ghats of Maharashtra and is common in most of the forest patches.Even though no specific threats could be identified for the species, continuous deforestation in these areas leading to habitat fragmentation could be a threat to the species.Several localities from which the species is currently known are also subject to tourism and recreational activities.There is a large wind farm near the type locality at Chalkewadi and the other localities are also potential wind farm sites.Pande et al (2013) have discussed threat of wind farms to avifauna wherein they discuss the windmill erection activity to be a measured threat to general diversity as well.Recently, Dahanukar et al. (2013) reported the presence of chytrid fungal infection in morphologically identified R. bombayensis from Taleghar, which should be attributed to the R. ghatei sp.nov.based on the current study.Therefore, it can be suggested that the species is prone to chytrid infection.Even though the effect of chytrid on this species is not available, further studies are essential on this topic.Furthermore, a detailed study on the ecology, distribution, population status and threats to the populations is essential to evaluate the conservation status of this species.
of tongue in Raorchestes ghatei sp.nov.(a), R. bombayensis (b) and R. tuberohumerus (c).In R. ghatei sp nov.and R. tuberohumerus tongue devoid of papilla but with a lingual pit (pointed with red arrow), while in R. bombayensis a well-defined papilla is present.colour towards the posterior end of trunk.Gular skin marbled with dark spots, sometimes with reddish tinge.In preservation (Image 1): Colour pattern in alcohol preserved specimens as in live conditions albeit faded Allotype (BNHS 5582, female) (all measurements in mm):

Figure 2 .
Figure 2. Maximum likelihood analysis of partial 16S rRNA gene.Tree for all Raorchestes species for which 16S rRNA gene data is available where the new species is depicted in red.Values on the node are percent bootstrap values for 1000 iterations.Pseudophilautus species from Western Ghats are used as outgroup.

Image 9 .
Colour and pattern variation on the flank and thigh region of Raorchestes ghatei sp.nov. in life.All specimens are from the single population at Thoseghar.Specimens not collected.Image 10. Habitat at the type locality of Raorchestes ghatei sp.nov.stages of Raorchestes ghatei sp.nov.(a) Eggs clutches under the stone on forest floor in Dongarwadi, (b) developing embryo in the egg and (c) newly hatched juvenile.

Figure 3 .
Figure 3. MANOVA/CVA of size adjusted morphometric data as percentage of SVL for adult males.Raorchestes ghatei sp.nov.forms a separate cluster distinct from R. bombayensis and R. tuberohumerus on the first canonical axis.
Figure 5. Distribution of three species based on current study and data provided in Biju & Bossuyt (2009).