Hydrophylax bahuvistara, a new species of fungoid frog (Amphibia: Ranidae) from peninsular India

1,2 Department of Zoology, Abasaheb Garware College, Pune, Maharashtra 411030, India 2 Current Address: Department of Zoology, Savitribai Phule Pune University, Ganeshkhind, Pune, Maharashtra 411007, India 1,3 Department of Biodiversity, Abasaheb Garware College, Pune, Maharashtra 411030, India 4 Centre for Wildlife Studies, College of Forestry, Kerala Agricultural University, Thrissur, Kerala 680656, India 5 Indian Institute of Science Education and Research (IISER), G1 Block, Dr. Homi Bhabha Road, Pashan, Pune, Maharashtra 411008, India 5 Systematics, Ecology and Conservation Laboratory, Zoo Outreach Organization (ZOO), 96 Kumudham Nagar, Vilankurichi Road, Coimbatore, Tamil Nadu 641035, India 1 anand.padhye@mesagc.org, 2 anushreejadhav@gmail.com, 3 nikhilsmodak@gmail.com, 4 nameer.po@kau.in,, 5 n.dahanukar@iiserpune.ac.in (corresponding author)

The genus Hydrophylax is diagnosed based on a combination of characters including presence of a postocular mask, robust body, rear of thighs with strong vermiculations, large rictal gland, prominent humeral gland, and ventrolateral grooves sometimes absent on finger 1 (Oliver et al. 2015). Currently recognized species in the genus are widely distributed in Asia and are known from Sri Lanka, India, Bangladesh, southern Myanmar and western Thailand (Frost 2015;Oliver et al. 2015).
In a recent review of golden-backed frogs from the Western Ghats of India and Sri Lanka, Biju et al. (2014) considered Rana malabarica as a member of the genus Hylarana. They suggested that this widespread species, which is spread across peninsular India, harbors two genetically distinct haplogroups, one restricted to the Western Ghats of Kerala and Tamil Nadu and the other distributed in the Western Ghats of Karnataka, Goa and Maharashtra and extending as far as Madhya Pradesh in central India. While the specimens from Western Ghats of Kerala and Tamil Nadu could be attributed to Hydrophylax malabaricus sensu stricto, Biju et al. (2014) refrained from describing the haplogroup north of Kerala as a distinct species owing to limited data and absence of diagnostic characters for the separation of the haplogroups, and considered it as 'Hylarana malabarica haplogroup 1'.
Here we report the description of a new species of fungoid frog Hydrophylax bahuvistara, considered as 'Hylarana malabarica haplogroup 1' by Biju et al. (2014), based on the study of type and topotypic material of Hydrophylax malabaricus and specimens collected from a wide range within peninsular India from Karnataka, Goa, Maharashtra and Madhya Pradesh states. We show that the new species can be delineated from typical H. malabaricus based on both genetic and multivariate morphometric analysis, and is diagnosable based on morphological combination of characters.

Specimen collection
Specimens of the new species were collected in India from Goa State and Pune, Raigad, Sindhudurg, Ratnagiri, Thane, Chandrapur and Satara districts of Maharashtra State. The specimens were collected from a variety of habitats, including roadside paddy fields and pools, grasslands, temporary rainwater pools on plateaus, lake shores and stream banks in semi-evergreen forests. A total of 20 specimens (Table 2) were collected and not more than two specimens were collected from each locality except for the type locality at Tamhini, where four specimens were collected. Four specimens of H. malabaricus were collected from the main campus of the Kerala Agricultural University, Thrissur, Kerala, India. These specimens were considered as topotypes as the area is within the type locality in Malabar mentioned in the original description, and specimens agree in morphology with the lectotype of H. malabaricus. Specimens were preserved in 70% ethanol with 5% glycerol.

Statistical analysis
All the measurements showed a positive linear relationship with SVL. Thus to remove the effect of size, morphometric data were normalized by expressing measurements as a percentage of SVL. Multivariate normality of the data was checked using the Doornik & Hansen (2008) omnibus. Discriminant Analysis (DA) was performed to understand whether related species of Hydrophylax form significantly different clusters (Huberty & Olejnik 2006). 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 used for computing Fisher's distances (distance between the centroids of the clusters, divided by the sum of their standard deviations) between clusters to determine if the clusters were significantly different. Statistical analysis was performed in PAST 3.0 (Hammer et al. 2001).

Molecular analysis
Thigh muscle tissue was harvested from twelve specimens of the new species (11 marked by asterisk in Table 1 and WILD-13-AMP-011) and four specimens of topotypic H. malabaricus (KAUNHM201501, KAUNHM201502, KAUNHM201503 and KAUNHM201504). Tissues were preserved in absolute ethanol. DNA extraction, PCR amplification of 16S rRNA gene and sequencing protocols followed Padhye et al. (2014). Sequences were analyzed by the BLAST tool (Altschul et al. 1990). These sequences have been deposited in GenBank under the accession numbers KP826810 to KP826820 and KT334413. Additional 16S gene sequences were retrieved from the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/). GenBank accession numbers for the sequences used in the study are provided in Appendix A. Gene sequences were aligned separately using MUSCLE (Edgar 2004). Molecular phylogenetic analysis was performed using MEGA 6 (Tamura et al. 2013). Pairwise raw phylogenetic distances were calculated in MEGA 6 (Tamura et al. 2013). The best fit model for nucleotide substitution was selected from 24 models using MEGA 6 (Tamura et al. 2013) based on the minimum Bayesian Information Criterion (BIC) value (Schwarz 1978;Nei & Kumar 2000). The best fit nucleotide substitution model was used for testing the phylogenetic hypothesis using maximum likelihood method. This analysis was not carried out to thoroughly resolve the deep phylogeny of the genus but to assign individuals to genetically homogenous clusters. Reliability of the phylogenetic tree was estimated using bootstrap values from 1000 replicates. Phylogenetic tree was edited in FigTree v1.4.2 (Rambaut 2009 Boulenger (1920), Lalremsanga et al. (2007) and Biju et al. (2014).

Additional material
Details of additional material studied is provided in Appendix B.

Diagnosis
Hydrophylax bahuvistara sp. nov. can be separated from its congeners based on a combination of characters including HW/HL ratio in the range of 0.83-0.98, IUE/HL ratio in the range of 0.27-0.36, outline of snout in dorsal view truncated, finger and toe tips without lateroventral groove, foot moderately webbed with webbing formula I1¾-2II1-3III2-3½IV3½-2V, metatarsals of 4 th and 5 th toes closely set, outer metatarsal tubercle small, foot length 39.6-49.4 % of SVL, dorsal parts of shank without glandular folds and sparse horny spinules, and heels touch each other when the legs are folded at right angles to the body.

Description
Morphometric measurements are listed in Table 2. General body topology as in Image 1 and 2. Palm and foot structures as in Image 3. Coloration in life as per Image 4.
Hind limbs: moderately long, tibio-tarsal articulation reaches middle of eye; thigh (TL 32.6), shank (SHL 32.8) and foot (FOL 33.1) subequal in length; toes long, thin, with flattened tips with rounded edges, without groove; webbing moderate, reaching between the 3 rd and 4 th subarticular tubercles on either side of toe IV; subarticular tubercles single, oval; supernumerary tubercles absent; dermal ridge on the outer side of the toe V present; tarsal fold absent; toe length formula 1 < 2 < 3 ≈ 5 < 4; inner metatarsal tubercle short, oval and distinct; outer metatarsal tubercle small, prominent (Image 3b). Heels touch each other when the legs are folded at right angles to the body.
Skin texture: Head smooth, finely granular on the dorsal side of the body, with moderately developed conspicuous dorsolateral glandular folds. Mandibular margin having thick granulations. Rictal gland present. Gular skin smooth. Skin on venter smooth, with coarse granulations on the posterior-ventral surface of the thighs. Ventral skin in the trunk region smooth, coarsely granular on the posterio-ventral surface of the thighs.
Hind limbs: moderately long, tibio-tarsal articulation   (FOL 30.5) almost equal in length; toes long, thin, with flattened tips with rounded edges, without groove; webbing moderate, reaching between the 3 rd and 4 th subarticular tubercles on either side of toe IV; subarticular tubercles single, oval; supernumerary tubercles absent; dermal ridge on the outer side of the toe V present; tarsal fold absent; toe length formula 1 < 2 < 3 ≈ 5 < 4; inner metatarsal tubercle short, oval and distinct; outer metatarsal tubercle small, prominent; heels touch each other when the legs are folded at right angles to the body. Skin texture: Head smooth, finely granular on the dorsal side of the body, with moderately developed conspicuous dorsolateral glandular folds. Mandibular margin having thick granulations. Rictal gland present. Gular skin smooth. Skin on venter smooth, with coarse granulations on the posterior-ventral surface of the thighs. Ventral skin in the trunk region smooth, coarsely granular on the posterio-ventral surface of the thighs.

Coloration
In life: Dorsum with a wide band of pale orange color extending from tip of snout to vent, interspersed occasionally with black spots. Golden yellow dorsolateral fold, extending from tympanum to groins, separates bright orange dorsum from a dark brown dorsolateral region. Dorsolateral region of body bears creamy to golden yellow spots on dark brown background. Belly creamy white with light brown marbling. Fore limbs and hind limbs dark, with creamy white to yellow marbling. Lateral region of head dark brown; mandibular margin and rictal gland golden yellow in color. Throat yellowish, marbled with brown.
In alcohol preservation (Image 1): Dorsum color changes from pale orange to yellowish-brown. The color of other body regions remains same as in live specimens.

Variations
In life, dorsal coloration varies from pale orange to reddish-orange (Image 4), which changes from pale orange to yellowish-brown and reddish-orange to crimson in alcohol preservation. Ventral coloration varies from plain creamy white to yellow to dark brown or black. Creamy white or yellow marbling usually present on the dark belly, while pale brown marbling may or may not be present on creamy white or yellow belly. Male individuals usually attain more yellowish color during the breeding season. Morphometric variations for male and female types are provided in Table 1 with mean and standard deviation for all characters.

Sexual dimorphism
Males of the species have single internal vocal sac visible as loose skin on the throat in live specimens; dark, swollen patch on upper arm -humeral gland and a prominent, thick nuptial pad, which are absent in females.

Distribution
Type material of the species comes from a wide distribution in the Western Ghats of Maharashtra and eastern Maharashtra, however, based on genetic data available in Kurabayashi et al. (2005), Biju et al. (2014) and Hasan et al. (2014) and localities for additional material from this study and distributional data for Hydrophylax malabarica Haplogroup 1 from Biju et al. (2014), the species is widespread in peninsular India distributed in Maharashra, Karnataka, Goa and Madhya Pradesh (Table 3; Fig. 4).

Habitat, ecology and natural history
Hydrophylax bahuvistara sp. nov. is usually found near human habitation and in agricultural fields. It is also found on the forest floor and near ephemeral or permanent water bodies, but mainly during breeding season. The eggs are laid in shallow water in the paddy fields or on the banks of small ponds or lakes. Calling behavior of an adult male is shown in movie clip (Appendix C). Adults usually gather in large numbers at potential breeding habitats. A loud chorus of calling males is heard at such places (Appendix D). The loud chorus is audible form a distance of up to a kilometer on quiet nights. Occasionally, smaller groups of adult males are also seen calling from the periphery of temporary rain water pools.

Comparison
Diagnosis of the genus and a key to the species of Hydrophylax are provided in Box 1.
Hydrophylax bahuvistara sp. nov. differs from H. leptoglossa by the absence of lateroventral groove on finger and toe tips (vs. present) and eyes separated from each other with a greater distance with IUE/HL ratio of 0.27-0.36 (vs. eyes placed closer to each other with IUE/ HL ratio of 0.16-0.26).   Biju et al. (2014) considered H. bahuvistara as "Hylarana malabarica Haplogroup 1" on the basis of genetic studies. They highlighted the close morphological resemblance of the haplogroup from H. malabaricus sensu stricto. However, our field observations, study of H. malabaricus type series and topotypes, and study of H. bahuvistara specimens from a wide range of its distribution suggests that the two species are morphologically distinct and can be delineated based on discrete characters.
All the six populations of H. malabaricus studied by Padhye et al. (2012) should now be attributed to the new species H. bahuvistara based on the genetic analysis presented in the current study. Padhye et al. (2012) showed that within the six populations of H. bahuvistara there are morphological as well as genetic variations in Randomly Amplified Polymorphic DNA markers. In the current study, however, we show that there is little genetic distance in the 16S rRNA gene among the populations of H. bahuvistara. Nevertheless, it is also true that the populations of H. bahuvistara are highly fragmented because of habitat fragmentation and therefore, the population variation reported by Padhye et al. (2012) could be due to recent fragmentation of populations of H. bahuvistara.
There is confusion regarding the type series of Hydrophylax malabaricus. Original description by Tschudi (1838) does not mention the number of specimens in the type series. However, Guibé (1950) mentioned six specimens as syntypes of H. malabaricus originating from Malabar, India and collected by Roux and Dussumier. While examining the type series of H. malabaricus we observed that four specimens MNHN  MNHN 1989.3451, MNHN 0771 andMNHN 1989.3452 (Image 5), collected by Dussumier, originated from Malabar and two of the syntypes MNHN 4439 and MNHN 1989.3448 (Image 6) are labeled as originating from Bangale, Inde (= Bengal, India) on the specimen bottle. As there is a contradiction in the information provided in Guibé (1950) and the information available on the specimen bottle for the later two specimens, we have not considered them as syntypes of H. malabaricus and they are not included in the morphometric analysis. Biju et al. (2014)  Six specimens MNHN 1893.458-463 from Burma (Myanmar) in the Muséum national d'Histoire naturelle, France, have two labels: "Rana malabarica" and "Rana granulosa". These specimens have a close similarity with the syntypes of Hydrophylax leptoglossa (MCZ A-1588, A-125024 and A-125025) and therefore they should be identified as H. leptoglossa.
Currently, H. malabaricus is listed as a Least Concern species in IUCN Red List of Threatened Species, owing to its wide distribution in the peninsular India (Biju et al. 2004). However, based on the current study, the populations north of Kerala belong to H. bahuvistara, which is a widely distributed species in the peninsular India, while H. malabaricus appears to be restricted to the Western Ghats of Kerala and Tamil Nadu. It is therefore essential to reassess the conservation status of H. malabaricus.