Monograph Leaping frogs (Anura: Ranixalidae) of the Western Ghats of India: An integrated taxonomic review

Leaping frogs of the family Ranixalidae are endemic to the Western Ghats of India and are currently placed in a single genus, Indirana. Based on specimens collected from their entire range and a comprehensive study of type material defining all known species, we propose a revised taxonomy for the leaping frogs using an integrative approach including an analysis of the mitochondrial 16S rRNA and nuclear rhodopsin genes, as well as multivariate morphometrics. Both genetic and morphological analyses suggest that the genus Indirana is paraphyletic and a distinct monophyletic group, Walkerana gen. nov., is described herein. The new genus is separated from Indirana sensu stricto by an apomorphic character state of reduced webbing, with one phalange free on the first and second toe (vs. no free phalanges), two phalanges free on the third and fifth toe (vs. one free phalange), and three phalanges free on the fourth toe (vs. 2–21⁄2 phalanges free). This review includes (i) identification of lectotypes and redescription of three species of the genus Walkerana; (ii) identification of lectotypes for Indirana beddomii and I. semipalmata and their redescription; (iii) redescription of I. brachytarsus and I. gundia; and (iv) descriptions of four new species, namely, I. duboisi and I. tysoni from north of the Palghat gap, and I. yadera and I. sarojamma from south of the Palghat gap; and (iv) a key to the genera and species in the family Ranixalidae.


INTRODUCTION
The taxonomic history of anurans currently grouped into the genus Indirana, in the monotypic family Ranixalidae, dates back to Günther (1876), who described four species, Polypedates beddomii, P. brachytarsus, P. brevipalmatus and Ixalus diplostictus from the Malabar Coast of southwestern India. Boulenger (1882) transferred these species to the genus Rana; however, as the name brevipalmata was preoccupied by R. brevipalmata Peters, 1871, Boulenger (1882) provided a replacement name R. leptodactyla for P. brevipalmatus. Boulenger (1882) also synonymized P. brachytarsus with R. beddomii and described two other related species, R. phrynoderma and R. semipalmata. Subsequently, Boulenger (1888) described another member of the same group, R. leithii from Matheran in the northern Western Ghats. The combination of these species into a single group was a result of Boulenger's (1920) monograph on Rana, in which he defined the subgenus Discodeles by using characters including "toes, often also fingers, dilated at the end, the dilation, or disc, bearing a crescentic or horseshoe-shaped horizontal groove, outer metatarsals united or separated only in the distal third; omosternum forked at the base". Although Boulenger (1920) included three other species from the Solomon Islands, Oceania, in the same subgenus, he specifically considered a distinct 'Ranae beddomianae' group to consolidate the six species, namely, R.  Rao (1937) later added R. (D.) tenuilingua from Kemphole Forest, Karnataka, India to the same group, elevating the number of species to seven. Laurent (1986) erected the genus Indirana and transferred the seven species to it, but he was apparently unaware that Inger et al. (1984) had already resurrected R. brachytarsus from the synonymy with R. beddomii. During the same year, Dubois (1986) erected a new genus, Ranixalus, in his description of Ranixalus gundia. Dubois (1987a) subsequently transferred the six 'Ranae beddomianae' group members, R. tenuilingua, as well as Rana brachytarsus, to Ranixalus, making the total number of species under Ranixalus nine. Dubois (1987b), based on the priority in publication date, synonymized Ranixalus with Indirana. In their review of the frog genus Philautus, Bossuyt & Dubois (2001) transferred P. longicrus Rao, 1937 to genus Indirana, elevating the number of species under Indirana to 10. With recent description of two species Modak et al. 2015), the genus Indirana currently comprises 12 species, including: I. beddomii (Günther, 1876), I. brachytarsus (Günther, 1876), I. chiravasi Padhye et al., 2014, I. diplosticta (Günther, 1876), I. gundia (Dubois, 1986), I. leithii (Boulenger, 1888), I. leptodactyla (Boulenger, 1882), I. longicrus (Rao, 1937), I. phrynoderma (Boulenger, 1882), I. salelkari Modak et al., 2015, I. semipalmata (Boulenger, 1882), and I. tenuilingua (Rao, 1937). Dubois (1987a) proposed the tribe Ranixalini, later treated as the subfamily Ranixalinae by Dubois (1992), with the type genus Ranixalus (now a synonym of Indirana) and included two other genera, Nannophrys and Nyctibatrachus, based on the presence of femoral glands in males. On the basis of priority, the subfamily Indiraninae Blommers-Schlösser, 1993, is a junior synonym of Ranixalinae Dubois, 1987. Van Bocxlaer et al. (2006 in their taxonomic analysis using three nuclear and one mitochondrial genetic markers elevated the taxon to the family Ranixalidae with the genus Indirana as its sole member. More recent molecular studies (Nair et al. 2012a;) have revealed that several species in the genus Indirana are undescribed. Although some studies have been published that attempt to resolve these issues by the study of topotypic material to assist in delineating geographic distribution boundaries of species (e.g., , and by defining new species using an integrated taxonomic approach Modak et al. 2015), a detailed taxonomic review of leaping frogs is still pending.
In the current study, using type material of known species, and with freshly collected material from throughout the Western Ghats, emphasizing type localities of known species, we present genetic barcodes for species of Indirana and provide species delimitation based on genetic gap analysis. We describe a new genus, redescribe the known species in the family, and describe four new species of Indirana.

Study site and specimen collection
Specimens of the family Ranixalidae were studied from the Western Ghats mountain ranges in the Indian states of Kerala,Tamil Nadu,Karnataka,Goa,Maharashtra,and Gujarat (Fig. 1

Molecular analysis
Thigh muscles of 70 specimens (Table 1) were used for extracting DNA and conducting molecular analyses. Genomic DNA extraction, polymerase chain reaction (PCR) for the mitochondrial 16S rRNA and nuclear rhodopsin partial genes, PCR product purification, and sequencing were performed following the protocols detailed in Padhye et al. (2014). Sequences were checked using the BLAST tool (Altschul et al. 1990) to identify the nearest congeners. Sequences are deposited in GenBank under the accession numbers KX641759-KX641828 for 16S rRNA and KX641829-KX641886 for rhodopsin genes. Additional sequences of related species from the study by Modak et al. ( , 2015 and  and five outgroups, namely Nyctibatrachus aliciae, N. major, Micrixalus fuscus, M. kottigeharensis and Nasikabatrachus sahyadrensis, were retrieved from the NCBI GenBank (http://www.ncbi.nlm.nih.gov/). GenBank accession numbers of the sequences used for the analysis are provided in Table 1. Gene sequences were aligned separately for 16S rRNA and rhodopsin using MUSCLE (Edgar 2004) implemented in MEGA 6 (Tamura et al. 2013). Genes were concatenated to make a super gene alignment with 922 bases using DAMBE (Xia 2013). Alignment was partitioned into four charsets corresponding to 16s rRNA gene and three codon positions of rhodopsin gene to create a full partition. We used a greedy strategy (Lanfear et al. 2012) implemented in IQ-TREE (Nguyen et al. 2015) to find the best partition scheme for the data using minimum Bayesian information criterion (BIC) value (Schwarz 1978;Nei & Kumar 2000). The best fit partition scheme was used to perform maximum likelihood analysis using IQ-TREE (Nguyen et al. 2015). Reliability of the phylogenetic tree was estimated using ultrafast bootstrap values run for 1000 iterations (Minh et al. 2013). The phylogenetic tree was edited in FigTree v1.4.2 (Rambaut 2009). This tree was also used to understand the phylogeographic distribution of Indirana in the Western Ghats using GenGIS 2.5 (Parks et al. 2013). Digital elevation map for phylogeography analysis was downloaded from Spatial Data Access Tool (http://webmap.ornl.gov/wcsdown/).
Since 16S rRNA gene has been suggested as a better barcoding region (Vences et al. 2005), we used this marker for analysis. Raw (p) distances between pairs of 16S rRNA sequences were calculated in MEGA 6 (Tamura et al. 2013). The frequency distributions of genetic distances was plotted to understand whether the distribution of the distances followed a bimodal distribution with a natural gap separating the two peaks. Such a gap is an indication of separation of intra-and interspecies genetic distances, which can be used to identify a gap in genetic distances that can be reliably used to separate two closely related species (Meyer & Paulay 2005;Meier et al. 2008). Intra-and interspecific distances were plotted as the mean, with minimum and maximum value indicated as error bars, to identify genetic gaps for delineating species. To substantiate our genetic gap analysis we also analyzed the 16S rRNA sequences using Automatic Barcode Gap Discovery (ABGD) software with simple distances (Puillandre et al. 2012) so as to understand species delimitation.
Further, we identified taxonomically important sites (defined below) within the 16S rRNA gene sequence for delineating and diagnosing eight species of Indirana that are morphologically similar. All available sequences for the given species were aligned with the complete 16S rRNA gene of Fejervarya cancrivora (Gravenhorst) extracted from whole mitochondrial genome sequence EU652694.
Character ( were identical within a species but showed variation between the species, alone or in combination, were considered taxonomically informative sites and used as diagnostic for the given species.

RESULTS
Identification of topotypes, putative topotypes, and their barcodes for known species Günther (1876) did not provide an exact type locality for Indirana beddomii, and the syntypes of this species came from Malabar, Travancore, Anamallays (=Anamalai), and Sevagherry (=Sivagiri). In fact, the syntypes of I. beddomii represent a species complex, which necessitates the designation of a lectotype from among the syntypes (see Taxonomy section below). The original illustration provided by Günther (1876: Plate LXIII B) depicts specimen BMNH 1947.2.27.72, originating in Malabar, and we hereby designate this specimen as the lectotype of I. beddomii. This specimen closely resembles the population we studied at Peruvannamuzhi, Malabar Wildlife Sanctuary (11.599 0 N & 75.819 0 E, elevation 38m), located north of the Palghat gap. Because Malabar is not a precise locality (see Biju 2001), being the first revisers we designate Peruvannamuzhi, Malabar Wildlife Sanctuary, Kerala, India as the putative type locality of I. beddomii. This is pertinent, given that our specimen (WILD-14-AMP-414) from this locality closely resembles the lectotype, based on the following key characters: (i) one phalange free of webbing on the inner side of the third toe; (ii) heels just overlap when thighs are held at right angles to body axis, loreal region more oblique; and (iii) structure and placement of vomerine teeth. Further, Peruvannamuzhi also falls into the larger Malabar region of the British era. Therefore, the 16S rRNA gene sequence KX641760 is topotypic and can be considered as a barcode to allow genetic identification of I. beddomii.
In the original description of Indirana brachytarsus, Günther (1876) suggested that the species hailed from Anamalai and Sivagiri, both south of the Palghat gap. From among the syntypes, Inger et al. (1984) designated the specimen originating from Anamalai (BMNH 1947.2.27.92) as lectotype of the species. Specimen WILD-15-AMP-609 from our collection, originating from Topslip of Anamalai in Tamil Nadu (10.471 0 N & 76.842 0 E, elevation 748m), is conspecific with the lectotype according to the following key characters: (i) 1¼ phalange free of webbing on inner side of third toe; (ii) buccal cavity shallow 8.4-19.2% HL; (iii) heels strongly overlap when thighs are held at right angles to body axis; and (iv) structure and placement of vomerine teeth and choanae in buccal cavity. We therefore consider this specimen as a putative topotype and its 16S rRNA gene sequence KX641773 as a genetic barcode to identify I. brachytarsus. Padhye et al. (2014) provided genetic data for paratypes of Indirana chiravasi. As these paratypes were from the same locality as that of the holotype, they are also isotypes and therefore the 16S rRNA gene sequences KM386530 and KM386531 represent topotypic material of I. chiravasi and are therefore genetic barcodes for identifying I. chiravasi.
Günther (1876) did not provide exact type locality for Indirana diplosticta, and the syntypes came from Malabar.
Because three syntypes are available, designation of a lectotype is essential to stabilize taxonomy. We could not decipher which of the three specimens was used in the original illustration (Günther 1876: Plate LXIII C), however, BMNH 1947.2.2.21 is most similar to the illustration. We therefore designate BMNH 1947.2.2.21 as lectotype for the species (see Taxonomy section below). This specimen closely resembles the population we studied at Bonacaud (8.686 0 N & 77.183 0 E, elevation 877m), Peppara Wildlife Sanctuary. Since Malabar is not a precise locality, as first revisers we designate Bonacaud, Peppara Wildlife Sanctuary, Kerala, India, as the putative type locality of I. diplosticta. This is pertinent, given that our specimen (WILD-15-AMP-640) from this locality closely resembles the lectotype. Therefore, the 16S rRNA gene sequence KX641828 is topotypic and can be considered as a genetic barcode to allow genetic identification of I. diplosticta. Padhye et al. (2014) provided genetic information of Indirana gundia from the type locality Gundia, Karnataka, India. We can therefore consider sequences KM386532 and KM386533 as 16S rRNA gene barcodes for identifying I. gundia.  provided the 16S rRNA gene sequence KF590637 for Indirana leithii from its type locality in Matheran, Maharashtra, India, and considered this as topotypic sequence. We follow these authors and consider this sequence as a genetic barcode for I. leithii.
In the discussion of his replacement name Rana leptodactyla for Günther's (1876) Polypedatus brevipalmatus, Boulenger (1882) suggested that several types of Günther's species came from Anamalai and Malabar. We could not locate any syntypes originating from Anamalai in the collection of BMNH. However, three syntypes, from Malabar (BMNH 1947.2.29.39, BMNH 1947.2.29.40 and BMNH 1947.41) were studied. Although we also examined several other specimens collected by Col. R.C. Beddome (BMNH 1874.4.28.503-509) and Jerdon (BMNH 1874.4.28.503-509), the type status of these specimens are uncertain (see Taxonomic section below). Because three syntypes are available, designation of a lectotype is essential to stabilize taxonomy. We therefore designate BMNH 1947.2.29.39 as lectotype for the species (see Taxonomy section below), based on the priority in voucher number as no illustration or specific comments on any one of the syntypes is available in the original description. This specimen closely resembles the population we studied at Eravikulam National Park (10.145 0 N & 77.038 0 E, elevation 1940m), and because Malabar is not a precise locality, as first revisers we designate Eravikulam National Park, Kerala, India, as the putative type locality of I. leptodactyla. This is pertinent, given that our specimen (WILD-13-AMP-184) from this locality closely resembles lectotype. Therefore, the 16S rRNA gene sequence KX641827 is topotypic and can be considered as a genetic barcode to allow genetic identification of I. leptodactyla.
Boulenger (1882) described Rana phrynoderma from Anamalai based on two syntypes (BMNH 1947.2.3.8 and1947.2.3.9). To stabilize taxonomy, we designate BMNH 1947.2.3.8 as the lectotype for I. phrynoderma (see Taxonomy section below), based on the priority in voucher number as no illustration or specific comments on any one of the syntypes is available in the original description. Our specimen WILD-14-AMP-509 from Parambikulam Tiger Reserve (10.354 0 N & 76.815 0 E, elevation 928m) in Anamalai of Kerala is conspecific with the syntypes of I. phrynoderma and therefore we consider the sequence KX641824 as a genetic barcode that can serve in identifying the species. Modak et al. (2015) provided the 16S rRNA gene sequence KP826824 of the holotype while describing Indirana salelkari, which is the genetic barcode for identifying the species.
In his description of Indirana semipalmata, Boulenger (1882) mentioned Malabar as the type locality. Because two syntypes (BMNH 1947.2.29.50 and1947.2.29.51) are available, designation of a lectotype is essential to stabilize taxonomy. We could not decipher which of the two specimens was used in the original illustration (Boulenger 1882 precise locality, as first revisers we designate Painavu, Idukki Wildlife Sanctuary, Kerala, India, as the putative type locality of I. semipalmata. This is pertinent, given that our specimen (WILD-14-AMP-354) from this locality closely resembles the lectotype, based on the following key characters: (i) reduced webbing with at least ½ phalange free on 3 rd and 5 th toe and 2¼ phalanges free on 4 th toe; and (ii) tympanum diameter more than 50% of eye diameter. Therefore, the 16S rRNA gene sequence KX641813 is topotypic and can be considered as a genetic barcode to allow genetic identification of I. semipalmata.
Genetic barcodes for I. longicrus and I. tenuilingua cannot be assigned because of taxonomic ambiguity as explained under the Taxonomy section.

Phylogenetic analysis
Analysis of best partition scheme for mitochondrial 16S rRNA gene and three codon positions of nuclear rhodopsin gene implied transition model with rate heterogeinity (TIM2+R3, BIC = 11498.60, lnL=-5186.11, df=165) as the best nucleotide substitution model for all partitions. In the maximum likelihood tree (Fig. 2), a deep branching clade separates species with highly reduced webbing (I. phrynoderma, I. leptodactyla, I. diplosticta) from other species of Indirana. We identify this clade as a distinct genus and erect Walkerana gen. nov. (see Taxonomy section below) within the family Ranixalidae. Intra-and interspecific genetic raw uncorrected p distances in 16S rRNA gene for species of Indirana are provided in Table 2, for Walkerana gen. nov. in Table 3, and between species of Indirana and Walkerana gen. nov. in Table 4. In the genus Indirana, maximum intraspecific genetic distance in 16S rRNA gene is 1.9% in I. semipalmata, while the minimum interspecific distance is 2.4% between I. duboisi sp. nov. and I. salelkari. The gap between the two is marked as a genetic gap for species delimitation in Fig. 3. This gap is also evident in a frequency distribution of pairwise distances for all samples of Indirana (Fig. 3 inset). This genetic gap revealed presence of 11 species in genus Indirana, which included three proposed new species. Analysis using Automatic Barcode Gap Discovery (ABGD) software also revealed same grouping into 11 species with partition having prior maximal distance P = 0.00596. We had limited samples of species in Walkerana gen. nov., as most of the species occur within protected areas with limited access and permission to collect only a few individuals. Nevertheless, we find that the maximum intraspecific genetic distance is 0.4% in W. leptodactyla comb. nov., and the minimum interspecific

Morphometric analysis
Fisher's distances between clusters were significantly different after sequential Bonferroni correction at twotailed level, except in two cases, Indirana beddomii vs. I. brachytarsus and I. semipalmata and I. chiravasi, which were only marginally significant at one-tailed level (Table 5). Nevertheless, both these pairs of species had different extent of webbing (see Taxonomy section below). Discriminant analysis of all species of Ranixalidae extracted 13 factors (Fig. 5, inset). Species of Walkerana gen. nov. are morphometrically distinct from species of Indirana along the first two; and fourth discriminant axes (Fig. 5). This separation occurs largely because of smaller TYL and ENL and longer THL, FOL, TL and TFOL in species of Walkerana gen. nov., when all the lengths are corrected for size (Table 6). Species under Walkerana gen. nov. form distinct clusters in first two dimensions of PCA (Fig. 6). Walkerana diplosticta comb. nov. separated based on higher EL, IOL and T4W; W. leptodactyla comb. nov. separated based on higher TYL, F3D, FOL and T5; and W. phrynoderma comb. nov. separated based on higher SL, UEW, SNL and INL (Table  6). Discriminant analysis of Indirana species extracted 10 factors, of which the first three factors explain 74.45% of the total variation in the data (Fig. 7, inset). Indirana leithii forms a distinct cluster in the first two dimensions (Fig. 7a, b), while I. tysoni sp. nov. forms a distinct cluster in the third dimension (Fig. 7c). Indirana leithii forms a distinct cluster from all other species under Indirana (Fig. 7a, b) based on comparatively higher values of ENL, F2 and F3D (Table 6); while I. tysoni sp. nov. forms a distinct cluster from other species of Indirana based on relatively higher values of characters such as SNL, INL and F3D (Table 6). The remaining species of Indirana (Fig. 8) and members of 'beddomii group' (Fig. 9) (see Taxonomy section for details) form distinct clusters in DA only in higher dimensions. Factor loading for the first three dimensions in Figs. 8 and 9 are provided in Table 6.

Species
(1)     Rao, 1937(= P. crnri Dutta, 1985, a replacement name to avoid homonymy with Ixalus longicrus Boulenger, 1894, a species now part of the genus Philautus) was transfered to Indirana based on the arguments put forth by Bossuyt & Dubois (2001), who suggested that Rao's (1937) mention of absence of vomerine teeth and a lingual papilla in P. crnri were 'defects of observations'. We disagree with Bossuyt & Dubois (2001) that Rao (1937) made a mistake listing these characters as absent in P. crnri, because his descriptions of other species based on these characters are correct. Since we have been unable to identify any species of Indirana from the type locality of P. crnri that matches the original description, and since the holotype is now lost and the species description and illustration are not adequate to reliably determine whether the species belong to the families Rhacophoridae, Micrixalidae or Ranixalidae (members of the first two families lack vomerine teeth), we treat Philautus longicrus Rao, 1937 Table 5. Fisher's distances (above diagonal blue cells) and associated P values (below diagonal red cells) between clusters of species. P values in bold are significant only at one tailed level. Dutta, 1985) as incertae sedis within the Order Anura until further information becomes available.

(= Philautus crnri
Family: Ranixalidae Dubois, 1987 Type genus: Indirana Laurent, 1986 Diagnosis of the family: Y shaped terminal phalanges, digital discs, femoral glands, and semi-terrestrial tadpoles with 3-5/3-4 rows of labial teeth, elongated bodies and low tail fins, with an ability to make long jumps on the ground to escape predators (Dubois 2003 Type species: Ixalus diplostictus Günther, 1876 Diagnosis: Walkerana gen. nov. represents a genetically distant clade within the family Ranixalidae and differs from its sister taxon Indirana in having extremely reduced webbing with one phalange free on first and second toes (vs. nil), and three phalanges free on the fourth toe (vs. 2-2½). Further, Walkerana differs from Indirana in consistently having the first finger shorter than second (vs. equal to or longer than second, except in I. leithii).
Etymology: The genus is named after Ms. Sally Walker in recognition of her selfless service to the improvement of zoos in South Asia, as well as her contributions to in situ conservation of neglected and non-charismatic wild fauna and flora through the work of Zoo Outreach            Organization. An unsung hero, this is also in recognition of her voluntary service to conservation in India and South Asia for well over 35 years. The generic name is a combination of Sally Walker's last name and the Latin name for frogs, Rana, and is used as a noun. Included species: Walkerana diplosticta (Günther, 1876), W. leptodactyla (Boulenger, 1882), W. phrynoderma (Boulenger, 1882).
Distribution: The new genus Walkerana is endemic to the Western Ghats of India and is currently known from south of the Palghat gap ( Fig. 10). All records of the species under Walkerana gen. nov. from north of the Palghat gap need genetic confirmation.
Dorsal and ventral skin smooth; very few longitudinal folds on dorsal side; lateral side granular.
Coloration: In alcohol preservation, dorsal reddishbrown, dark band between the two upper eyelids; lower mandible barred with brown stripes inconspicuous on upper mandible; dark brown stripe running from tip of snout to shoulder through eye and tympanum visible; symmetrical black spots on the flanks -one just posterior to the axil, another on the middle of the flank, a third anterior to the loin; forelimbs and hindlimbs barred with dark brown stripes; lateral margin of forelimbs and hind limbs densely spotted with dark brown or black; sole and foot pale brown dorsally, dark brown ventrally.
Variation: Morphometric variation provided in Table  7. Color in life as in Image 2. Some of the symmetrical black spots on the flanks (except those on the loin) may not be present or could be very small in size, or may be lost in preservation.
Distribution: In the current study, the species was collected only from Bonacaud (8.686 0 N & 77.183 0 E, elevation 877m), Kerala Western Ghats, south of the Palghat gap (Fig. 10). In our surveys we could record W. diplosticta only from south of Shencottah. Except Athiramala (Biju et al. 2004h), Ponmudi (Inger et al. 1984;Nair et al. 2012b), Kalakkad-Mundanthurai Tiger Reserve (Johnsingh 2001;Vasudevan et al. 2001Vasudevan et al. , 2008Kumar et al. 2002), all other previous records of this species, including Ranipuram (Andrews et al. 2005), Anamalai Hills (Boulenger 1920), Indira Gandhi National Park (Biju et al. 2004h Remarks: During the study of types of Walkerana diplosticta, two specimens of the species collected by T.C. Jerdon from Malabar were also photographed (Image 3) but as they were not types they were not considered for morphometric study.
Dorsal and ventral skin smooth; longitudinal folds on dorsal side; lateral side granular.
Coloration: In alcohol preservation, dorsal dark brown; white band between the two upper eyelids followed by a dark band posterior; upper and lower mandible barred with brown stripes; narrow dark brown stripe running from tip of snout to shoulder through eye Variation: Morphometric variation is provided in Table 7. Color in life as in Image 5. Dorsal side sometimes with a white middorsal line; ventral side creamy white mottled with brown or brown with white dots; anterior and posterior sides of the thigh and tibia mottled with brown.
Dorsal skin warty; few longitudinal folds on dorsal side; W shaped skin fold on the posterior side of head; lateral side granular; ventral side smooth.
Coloration: In alcohol preservation, dorsal brown with few scattered dark brown spots; dark band between the two upper eyelids; upper and lower mandible barred with brown stripes; indistinct narrow dark brown stripe running from tip of snout to shoulder through eye and tympanum; forelimbs and hindlimbs barred with dark brown stripes; sole and foot dark brown; ventrally cream.
Variation: Morphometric variation is provided in Table  7. Live coloration as in Image 9. Ventrally sometimes dotted with whitish W-shaped fold sometimes dark brown.
Distribution: We recorded the species from Anamalai  During the current study, we collected specimens from same locality (i.e. Mulshi) and we confirm that they were misidentifications of Indirana leithii.
Diagnosis: Indirana forms a genetically distinct clade within the family Ranixalidae and differs from its sister taxon Walkerana in having more extensive webbing with no phalange free on first and second toe (vs. one) and 2-2½ phalanges free on fourth toe (vs. three). Further, Indirana differs from Walkerana in consistently having first finger equal to or longer than second, except I. leithii (vs. shorter than second).
Incertae sedis: 'Indirana' tenuilingua (Rao, 1937) There are no known surviving types of I. tenuilingua as they have been considered lost by Dubois (1984). The original description of I. tenuilingua is not adequate to diagnose it from other known species in Indirana, further; there are several discrepancies in the description, morphometry and the illustration. For instance, in the original description, Rao (1937) mentions head wider than long; however, the figure provided by Rao (1937) suggests that head should be longer than wide. Further, Rao (1937) mentions inter-orbital distance more than twice the internarial distance; however the measurement data provided by the author does not reflect this. From the type locality mentioned by Rao (1937) as Kempholey ghats, we could only collect I. gundia despite extensive and repeated surveys for three consecutive years (2013)(2014)(2015)(2016). Due to lack of comparative type material for phylogenetic analysis and unavailability of similar specimens from the type locality and discrepancies between the description, morphometry table and figure in the original publication, we consider I. tenuilingua incertae sedis under the genus Indirana as the correct generic status cannot be ascertained.
Remarks: Although we provide a key to separate the species of the beddomii group, it is essential to note that some of these characters are subjective. Nevertheless, the species of the beddomii group are well separated in both genetic and multivariate morphometric analyses. A more reliable method to diagnose the species within the beddomii group is the use of an integrated approach with separation based on morphological, genetic and geographic distribution information. Western Ghats mountain ranges have three geographical gaps (see Robin et al. 2010;Van Bocxlaer et al. 2012): the Goa gap, the Palghat gap and the Shencottah gap (see Fig.  1). From the beddomii group, Indirana chiravasi and I. salelkari are distributed north of the Goa gap (Fig.  11a); I. beddomii, I. gundia and I. duboisi sp. nov. are distributed between the Goa and Palghat gaps ( Figure  11b); and I. brachytarsus, I. sarojamma sp. nov. and I. yadera sp. nov. are distributed south of the Palghat gap (Fig. 11c). Taxonomically informative sites that can be used in combination for identification of species based on 16S rRNA gene sequence are provided in Table 8. Based on these data, we provide an alternate key for the separation of species in the beddomii group.        (1888). We could only examine photograph of the specimen (see Remarks below) and therefore detailed description is not possible. Since the original description is not in detail for comparison of the species with its congeners, we provide description of topotypic female (BNHS 5590, Image 11), which is of comparable size to the holotype.
Dorsal skin smooth with dense glandular longitudinal folds arranged in irregular rows; lateral side granular with sparse granulation below the tympanum; ventral side smooth; glandular W-shaped mark at the posterior margin of head.
Coloration: In life (Image 12a), dorsal uniform pinkish-brown; dark band between upper eyelids; upper and lower mandible barred with brown stripes; distinct dark brown stripe running from tip of snout to shoulder through eye and tympanum; forelimbs and hindlimbs barred with brown stripes; sole and foot dark brown; ventrally cream to light brown. In preservation, color as above but faded.
Variation: Morphometric variation is provided in Table 9 and 10. Coloration in life varies as in Image 12. Dorsum coloration ranges from golden brown to dark brown. Darker brown spots and W-shaped mark on dorsum sometimes absent. Throat sometimes mottled with brown.
Distribution: The species is distributed in the northern Western Ghats between 16 0 N to 21 0 N latitudes (Fig. 11a, and Fig. 6 in . Remarks: We could not examine the type of the species during a visit to BMNH as the specimen was misplaced; however, the specimen was traced later and photographs of the same have been examined.  diagnosed from all other members of the genus Indirana based on the following combination of characters: (i) first finger longer than or equal to second, (ii) double outer palmar tubercle, and (iii) reduced webbing with two phalange free on 3 rd and 5 th toe and 3½ phalanges free on 4 th toe. From its morphological closest congener I. tysoni, I. semipalmata differs in the webbing formula I1¼-2¼II1¼-3III2-3½IV3½-2V (vs. I1-2II1-2½III1½-3¼IV3¼-1½V) and TYL more than 50% of EL (vs. less than 50% of EL).
Dorsal skin smooth with few glandular longitudinal folds arranged in irregular rows; lateral side smooth; ventral side smooth. Coloration: In alcohol preservation, dorsal brown with irregular dark brown spots; white band followed posteriorly by dark band between upper eyelids; upper and lower mandible barred with brown stripes; distinct dark brown stripe running from tip of snout to shoulder through eye and tympanum; forelimbs and hindlimbs barred with dark brown stripes; sole and foot dark brown; ventrally cream with light brown throat.
Dorsal skin smooth with dense glandular longitudinal folds arranged in irregular rows; lateral side granular; ventral side smooth.
Coloration: In alcohol preservation, dorsal greyishbrown with irregular dark brown spots; W-shaped mark at the posterior border of head; two dorso-lateral brown streaks posterior to W-shaped mark; white band followed posteriorly by dark band between upper eyelids; upper and lower mandible barred with brown stripes; distinct dark brown stripe running from tip of snout to shoulder through eye and tympanum; forelimbs and hindlimbs barred with dark brown stripes; sole and foot dark brown; ventrally cream; thighs and tibia orange.
Dorsal skin smooth with dense glandular longitudinal folds arranged in irregular rows; lateral side granular with dense granulation below the tympanum; ventral side smooth; posteoventral side of femur dense granular.
Coloration: In alcohol preservation, dorsal uniformly pale brown; white band followed posteriorly by dark band between the two upper eyelids; upper and lower mandible barred with brown stripes; distinct narrow dark brown stripe running from tip of snout to shoulder through eye and tympanum; forelimbs and hindlimbs barred with dark brown stripes; sole and foot dark brown; ventrally brown.
Variation: Morphometric variation is provided in Table  9 (Fig. 11b). Because the species is a part of a complex, we do not consider any previous distribution records as valid until further genetic analyses of specimens from those localities are done.
Remarks: Fourteen syntypes of I. beddomii originate from Malabar, Travancore, Anamalais and Sivagiri. Of these syntypes, Inger et al. (1984) designated BMNH 1947.92 as lectotype of I. brachytarsus. Other seven specimens from Anamalais and Sivagiri are conspecific to the lectotype of I. brachytarsus and therefore we consider them as paralectotypes of I. brachytarsus. Of the remaining six specimens, four from Malabar are not conspecific with the two from Travancore. To stabilize taxonomy, we designate BMNH 1947.2.27.72, a specimen originating from Malabar pictured in the original publication as I. beddomii, as lectotype and other three specimens from Malabar as paralectotypes of I. beddomii. Malabar is not a specific locality; however, since the lectotype closely resembles with the population we studied from Peruvannamuzhi, Malabar Wildlife Sanctuary (11.599 0 N & 75.819 0 E, elevation 38m) north of the Palghat gap, we consider this locality as putative type locality of the species. Further, Peruvannamuzhi also falls into the larger Malabar region of the British era. The remaining two syntypes BMNH 1947.2.27.87 and1947.2.27.88 originating from Travancore are conspecific to I. sarojamma described below.
Dorsal skin smooth with dense glandular longitudinal folds arranged in irregular rows; lateral side granular with dense granulation below the tympanum; ventral side smooth; postero-ventral side of femur dense granular.
Coloration: In alcohol preservation, dorsal brown with a thick mid-dorsal stripe interrupted by dark band between upper eyelids; upper and lower mandible barred with brown stripes; distinct dark brown stripe running from tip of snout to shoulder through eye and tympanum; ventrally cream with brown throat; forelimbs and hindlimbs barred with dark brown stripes; sole and foot dark brown; ventrally cream with brown throat. Variation: Morpholmetric variation is provided in Table 9 (Fig. 11c). Because the species of the beddomii group are morphologically similar, we do not consider any previous distribution records as valid until further genetic analyses of specimens from those localities are done.
For differences from I. beddomii and I. brachytarsus see comparison section for the respective species.
Coloration: See Padhye et al. (2014). Variation: Morphometric variation is provided in Table 9 (Fig. 11a and figure 1 in Padhye et al. 2014). can be diagnosed based on following combination of characters: (i) first finger equal to or longer than second, (ii) double outer palmar tubercle, (iii) extensive webbing with a webbing formula I1-2II1-2III1-3IV3-1V, (iv) tibio-tarsal articulation reaching beyond snout, (v) vomerine teeth in slightly oblique rows its length about 9.5-10.5% of HL, (vi) choanae round to slightly oblong its maximum to minimum ratio 1.0-1.2, (vii) buccal cavity slightly deep 10.0-17.0% of HL, (viii) FOL less than 55% of SUL, (ix) TL more than 55% of SUL, (x) PAL more than 25% of SUL, and (xi) UAL 19 -21% of SUL. Genetically, the species can be diagnosed from other members of beddomii complex with 16S rRNA gene using unique characters at position 1057: T, 1161: C (see Table 8). Geographically, the species is distributed in Western Ghats between the Palghat and Goa gaps.
For differences between I. beddomii, I. brachytarsus and I. chiravasi see comparison section for the respective species.
Dorsal skin smooth with dense glandular longitudinal folds arranged in irregular rows; lateral side granular; ventral side smooth; postero-ventral side of thighs densely granular; thigh bearing large femoral glands.
Coloration: In alcohol preservation, dorsal brown with irregular dark brown spots and W shaped mark at the posterior border of head; white band followed posteriorly by dark band between upper eyelids; upper and lower mandible barred with brown stripes; distinct dark brown stripe running from tip of snout to shoulder through eye and tympanum; forelimbs and hindlimbs barred with dark brown stripes; sole and foot dark brown; ventrally cream to light brown.
Variation: Morphometric variation is provided in Table 9 and 10. Variation in coloration in life as per Image 24. Mid-dorsal broad white stripe may be present. Dorsum coloration ranges from pale to dark brown. Darker brown spots and W-shaped mark on dorsum sometimes absent. W-shaped mark sometimes has a glandular fold. Ventral surface sometimes granular.
Coloration: In alcohol preservation (Image 27), dorsal pinkish-brown; white band followed posteriorly with a dark brown band between the two upper eyelids; upper and lower mandible barred with brown stripes; distinct dark brown stripe running from tip of snout to shoulder through eye and tympanum; ventrally cream; forelimbs and hindlimbs barred with dark brown stripes; sole and foot dark brown; ventrally cream. In life (Image 28), coloration similar to that in alcohol, but slightly darker.
Etymology: The species is named after Mrs. S. Saroja, who has contributed immensely to the functioning of the Zoo Outreach Organization for three decades and helping Sally Walker to volunteer at Mysore Zoo for five years before that. The name is in recognition of her selfless service committing all her time and forsaking her family life for the organization. Sarojamma is the name she is known by and she is the 'office mom'. It is a noun used in apposition.
Variation: Morphological variation is provided in Table 9 and 10. Mid-dorsal broad white stripe may be present, interrupted by dark band between the upper eyelids. Dorsum coloration ranges from pale to dark brown with dark brown spots and W-shaped mark on dorsum sometimes present.
Dorsal skin smooth with dense glandular longitudinal folds arranged in irregular rows; lateral side granular; ventral side smooth; posteoventral side of femur dense granular.
Coloration: In alcohol preservation, dorsal pinkishbrown with irregular dark brown spots and W-shaped mark at the posterior border of head; white band followed posteriorly by dark band between upper eyelids; upper and lower mandible barred with brown stripes; distinct dark brown stripe running from tip of snout to shoulder through eye and tympanum; forelimbs and hindlimbs barred with dark brown stripes; sole and foot dark brown; ventrally cream; throat mottled with brown; thigh and tibia orangish-yellow.
Etymology: The species is a combination name of Yamini, Deepa and Ravisankaran, a family of good friends who met with untimely demise. The species is named after the three as a dedication to Ravisankaran's service to conservation, Deepa's support and Yamini's spirit. The combination name is used as a noun in apposition.
Variation: Morphological variation is provided in Table 9 and 10. Live coloration as in Image 31. Dorsum coloration ranges from grey to dark brown. Mottling on throat could be absent. Thigh and tibia could be cream. W-shaped mark sometimes absent.

Molecular phylogeny of species under family
Ranixalidae revelead the presence of two distinct clades distinguished from each other by the extent of webbing. It is therefore clear that the clade previously recognized as Indirana was polyphyletic with respect to the extent of webbing. We therefore recognize Walkerana gen. nov. to resolve the polyphyly and obtain two monophyletic clades. Although the taxonomic sampling in Modak et al. (2015) is not extensive, it can be inferred from their molecular clock analysis based on two mitochondrial and one nuclear gene, that Indirana and Walkerana split from each other around 58.4 mya (95% HPD interval 70.0-48.5 mya).
Molecular phylogeny and dating has revealed that Ranixalidae probably had a delayed diversification in the early cenozoic (Roelants et al. 2004;Van Boxclaer et al. 2012). Ranixalidae probably split from its sister taxon Micrixalidae around 89.7 mya (95% HPD interval 90.5-86.9 mya) and the members of Ranixalidae did not diverge much until about 58.4 mya (95% HPD interval 70.0-48.5 mya) before splitting into Indirana and Walkerana (Modak et al. 2015). Genus Indirana further showed a lag till 33.4 mya (95% HPD interval 41.5-26.0 mya) after which it started diverging rapidly (Modak et al. 2015). It is therefore likely that since the divergence of members of the beddomii group is relatively recent they are morphologically quite similar; although the species form monophyletic clades, are separated by distinct geographical areas and are morphologically distinct in multivariate morphometric space.
Morphological simlarities among the members of beddomii group are also evident from following  Inger et al. (1984) used characters such as larger size and amount of webbing. It is interesting to note that while describing the members of the beddomii group, I. gundia, the author did not provide any comparison for separating the species from closely allied species (see Dubois 1986). Dubois (1987b) thought that I. gundia could be a synonym of I. brachytarsus based on their morphological similarity. Although Padhye et al. (2014) and Modak et al. (2015) provided some characters to separate the newly described species, I. chiravasi and I. salelkari respectively, from the beddomii group, their analysis was based on the study of only the type material which consisted of limited number of specimens. Our study, based on a much larger sampling clearly revealed that there are only few discrete morphological characters to separate the members of the beddomii group. These characters include extent of webbing on the inner side of the third toe, extent of the overlap of the heels when thighs are held at right angle to the body axis, depth of buccal cavity and length and placement of vomerine teeth series as mentioned in the key to the species of Indirana. A more reliable way to separate the species is by using integarted taxonomy. It is also essential to note that the keys by Boulenger (1920) and Daniel & Sekar (1989) separate I. semipalmata, I. leithii and members of Walkerana from I. beddomii; however, none provide a key to separate members of the beddomii group. Because members of the beddomii group are morphologically similar, Nair et al. (2012a) misidentified several of the known species from this group, which we could rectify (Table 11) based mainly on genetic analysis of topotypic material of the known species. Several sequences available on NCBI for members of the genus Indirana and Walkerana are based on misidentifications. For these sequences we provide the correct species identification (Table 11) based on analysis of 16S rRNA gene. Some of the sequences identified as I. diplosticta (JQ596653 to JQ596657) formed a monophyletic clade separated from I. semipalmata and therefore are considered here as I. cf. semipalmata. Sequences of I. gundia submitted to GenBank prior to the publication of Padhye et al. (2014) have been largely misidentified as I. beddomii. Sequence JQ596677 deposited as I. cf. beddomii by Nair et al. (2012a) belongs to I. sarojamma sp. nov. Sequences JQ596674, 75 and 76 submitted as I. cf. beddomii by Nair et al. (2012a) as well as KT282219 identified as I. beddomii by Jesmina & George (2015) and other sequences KJ585610-13 submitted as Indirana sp. are conspecific with I. yadera sp. nov. Sequence JQ596673 of I. leithii identified by Nair et al. (2012a) has already been suggested as not conspecific with topotypic I. leithii and was therefore considered as I. cf. leithii by . Sequences AF215392 (from Ooty, approx. 11.406 0 N & 76.693 0 E), JQ596658-62, KF991280, KJ585595-99 identified as Indirana sp. can be assigned to I. beddomii based on current study. Sequences (GU136104, JQ596642-44, AF215391 and KF991281) that are currently assigned to I. brachytarsus have been identified previously as I. beddomii, I. cf. beddomii, and Indirana sp. Further, sequences KT282223, JQ596681-85 identified as I. leptodactyla are not conspecific with topotypic Walkerana leptodactyla comb. nov. and are considered as Walkerana cf. leptodactyla comb. nov.
We have provided the first comprehensive review of species in the family Ranixalidae and clarify the identity as well as distribution of several species. We caution the readers from relying on the review of Indirana provided by Nair et al. (2012b) for the following reasons: (i) they do not provide detailed taxonomic comments on the species; (ii) they are heavily dependent on the earlier literature for records of the species without critically validating the authenticity of the reports; (iii) although they mention observation of the types and other voucher specimens in the collections of several museums, they provide no details of the material examined; (iv) they acknowledge their own study (Nair et al. 2012a) for suggesting that some species, such as I. beddomii, are a complex, but do very little to resolve the taxonomic issue; and (v) some of the records are likely based on misidentification. For instance,  showed that the species considered as I. leithii by Nair et al. (2012a), and subsequently used prominently in Nair et al. (2012b), is not conspecific with the topotypic I. leithii, especially in genetic analysis; hence the information provided regarding the distribution of this species is erroneous.
In the IUCN Red List of Threatened Species, Indirana gundia and Walkerana phrynoderma are listed as Critically Endangered (Biju et al. 2004c; I. brachytarsus, W. diplosticta and W. leptodactyla as Endangered (Biju et al. 2004b); I. leithii as Vulnerable (Biju et al. 2004g); I. beddomii and I. semipalmata as Least Concern (Biju et al. 2004a; and I. tenuilingua as Data Deficient (Biju et al. 2004f). The IUCN status of most of these species need