Molecular and Morphological Study of Leaping Frogs (Anura, Ranixalidae) with Description of Two New Species

<div><p>The monotypic anuran family Ranixalidae is endemic to India, with a predominant distribution in the Western Ghats, a region that is home to several unique amphibian lineages. It is also one of the three ancient anuran families that diversified on the Indian landmass long before several larger radiations of extant frogs in this region. In recent years, ranixalids have been subjected to DNA barcoding and systematic studies. Nearly half of the presently recognized species in this family have been described over the last three years, along with recognition of a new genus to accommodate three previously known members. Our surveys in the Western Ghats further suggest the presence of undescribed diversity in this group, thereby increasing former diversity estimates. Based on rapid genetic identification using a mitochondrial gene, followed by phylogenetic analyses with an additional nuclear gene and detailed morphological studies including examination of museum specimens, new collections, and available literature, here we describe two new species belonging to the genus <i>Indirana</i> from the Western Ghats states of Karnataka and Kerala. We also provide new genetic and morphological data along with confirmed distribution records for all the species known prior to this study. This updated systematic revision of family Ranixalidae will facilitate future studies and provide vital information for conservation assessment of these relic frogs.</p></div

Ventral view of hands showing the relative length of first and second finger in ranixalid species.

<p>(A–E) Species with first finger shorter than second finger. (A) <i>Indirana leithii</i>, SDBDU 2014.2515. (B, C) <i>Sallywalkerana diplosticta</i>, SDBDU 2015.2957. (B) Ventral surface. (C) Spinular projections on dorsal surface of first finger. (D) <i>S</i>. <i>leptodactyla</i>, SDBDU 2011.1058A. (E) <i>S</i>. <i>phrynoderma</i>, SDBDU 2002.1181. (F–I) Species with first finger longer than second finger. (F) <i>Indirana beddomii</i>, SDBDU 2010.225. (G) <i>I</i>. <i>sarojamma</i>, SDBDU 2002.334. (H) <i>I</i>. <i>tysoni</i>, SDBDU 2012.74. (I) <i>I</i>. <i>yadera</i>, SDBDU 2012.2744. (J–Q) Species with first finger equal or nearly to second finger. (J) <i>I</i>. <i>brachytarsus</i>, SDBDU 2015.2931. (K) <i>Indirana bhadrai</i> sp. nov., ZSI/WGRC/V/A887. (L) <i>I</i>. <i>Chiravasi</i>, SDBDU 2014.2483. (M) <i>I</i>. <i>Duboisi</i>, SDBDU 2003.1086. (N) <i>I</i>. <i>Gundia</i>, MNHN 1985.0633. (O) <i>Indirana paramakri</i> sp. nov., SDBDU 2005.3741. (P) <i>I</i>. <i>Salelkari</i>, SDBDU 2011.1330. (Q) <i>I</i>. <i>Semipalmata</i>, SDBDU 2015.3035.</p

DNA barcoding and status of IUCN assessment of ranixalid species of the Western Ghats.

<p>(A) Neighbor-Joining (NJ) tree based on Kimura-2-parameter model for 16S mitochondrial gene sequences from 269 ranixalid samples, representing all previously known and newly recognized species from the Western Ghats. Terminal node labels indicate voucher numbers that are cross-referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166326#pone.0166326.s012" target="_blank">S2 Table</a>; clade numbers are indicated towards the internal nodes of the tree; mean and range of interspecific genetic distances (in percent) are shown between subsequent clades. (B) Frequency of intra (grey) and interspecific (black) uncorrected pairwise distances for 16S. (C) Number of ranixalid species in the different IUCN categories [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166326#pone.0166326.ref066" target="_blank">66</a>].</p

Distribution localities of Leaping frog species reported from the Western Ghats of India in the present study.

<p>(A) Sampling localities of all species. Species in genus <i>Indirana</i> are categorized into two morphological groups recognized in the study. (B) Sampling localities of seven species in the <i>Indirana beddomii</i> group. (C) Sampling localities of six species in the <i>Indirana semipalmata</i> group. (D) Sampling localities of three species in genus <i>Sallywalkerana</i>. Colors of sampling localities correspond with group/species labels on the right panel of each map. The Western Ghats biodiversity hotspot region is shaded orange.</p

Genus <i>Sallywalkerana</i> in life.

<p>(A–D) <i>S</i>. <i>diplosticta</i>. (A, B) An adult male (SDBDU 2015.2956), from Pandipath. (A) Dorsolateral view. (B) Dorsal view. (C) An adult male (SDBDU 2003.40103), from Athirimala. (D) An adult male (SDBDU 2011.283), from Pandimotta. (E–G) <i>S</i>. <i>leptodactyla</i>. (E) An adult female (SDBDU 2013.911), from Mattupetti, dorsolateral view. (F) An adult female (SDBDU 2002.917), from Kodaikanal. (G) An adult female (SDBDU 2004.40336), from Valparai. (H, I) <i>S</i>. <i>phrynoderma</i>, an adult male (SDBDU 2002.1181), from Grass Hills. (H) Dorsolateral view. (I) Ventral view.</p

<i>Indirana semipalmata</i> group in life.

<p>(A–C) <i>I</i>. <i>chiravasi</i>. (A) An adult male (not preserved), from Amboli. (B) An adult female (SDBDU 2012.2124), from Amboli. (C) An adult male (SDBDU 2012.2125), from Amboli. (D–F) <i>I</i>. <i>duboisi</i>. (D, E) An adult male (SDBDU 2014.2517), from Agumbe. (D) Dorsolateral view. (E) Frontal view. (F) An adult male (SDBDU 2011.1399), from Charmadi Ghats. (G–H) <i>I</i>. <i>gundia</i>. (G) An adult male (SDBDU 2008.433), from Kempholey. (H) An adult male (SDBDU 2002.1115), from Kempholey. (I) <i>Indirana paramakri</i> sp. nov., holotype, an adult female (ZSI/WGRC/V/A888), from Suganthagiri. (J) <i>I</i>. <i>salelkari</i>, an adult female (SDBDU 2011.1330), from Dandeli, dorsolateral view. (K–L) <i>I</i>. <i>semipalmata</i>. (K) An adult female (SDBDU 2015.3033), from Parambikulam. (L) An adult male (SDBDU 2015.3014), from Singappara.</p

<i>Indirana beddomii</i> group in life.

<p>(A–B) <i>I</i>. <i>beddomii</i>. (A) An adult male (SDBDU 2010.225), from Kakkayam. (B) An adult female (SDBDU 2011.960), from Silent Valley. (C) <i>Indirana bhadrai</i>, holotype, an adult female (ZSI/WGRC/V/A887), from Muthodi. (D–H) <i>I</i>. <i>brachytarsus</i>. (D) An adult female (not preserved), from Ponmudi. (E) An adult male (SDBDU 2011.541), from Parambikulam. (F) An adult male (SDBDU 2006.4800), from Anchuruli. (G) An adult female (SDBDU 2011.280), from Pandimotta. (H) An adult male (SDBDU 2015.2995), from Upper Manalar. (I, J) <i>I</i>. <i>leithii</i>. (I) An adult female (SDBDU 2014.2514), from Matheran. (J) An adult male, (SDBDU 2011.1095), from Bhimashankar. (K) <i>I</i>. <i>sarojamma</i>, an adult female (SDBDU 2002.516) from Chathankod–Bonnacaud. (L–N) <i>I</i>. <i>tysoni</i>. (L, M) An adult female (SDBDU 2012.73), from Coorg. (L) Dorsolateral view. (M) Frontolateral view. (N) An adult male (SDBDU 2012.2228), from Coorg. (O–R) <i>I</i>. <i>yadera</i>. (O) An adult male (SDBDU 2012.2744), from Methooty. (P, Q) An adult male (SDBDU 2015.2984), from Kozhikana. (P) Frontal view. (Q) Dorsolateral view. (R) An adult female (SDBDU 2015.3155), from Vazhachal.</p

Two new <i>Indirana</i> species from the Western Ghats.

<p>(A–E) Holotype of <i>Indirana bhadrai</i> sp. nov., ZSI/WGRC/V/A887, female, in preservation. (F–J) Holotype of <i>Indirana paramakri</i> sp. nov., ZSI/WGRC/V/A888, female, in preservation. From left to right: Dorsal view, ventral view, lateral view of head, ventral view of hand, ventral view of foot.</p

Maximum likelihood tree for a combined mitochondrial (16S rRNA) and nuclear (Rag1) DNA data set of 1063 bp showing phylogenetic relationships among 16 recognized ranixalid species and one outgroup species.

<p>Bayesian Posterior Probabilities and RaxML Bootstrap values >50% are indicated above and below the branches, respectively. Morphological groups are referenced in the text.</p

Schematic illustration of foot webbing in ranixalid species.

<p>(A–G) <i>Indirana beddomii</i> group. (A) <i>I</i>. <i>beddomii</i>, lectotype of <i>Polypedates beddomii</i>, NHM 74.4.29.208 (ex BMNH 1947.2.27.72), female. (B) <i>Indirana bhadrai</i> sp. nov., holotype, ZSI/WGRC/V/A887, female. (C) <i>I</i>. <i>brachytarsus</i>, lectotype of <i>Polypedates brachytarsus</i>, NHM 74.4.29.1307 (ex BMNH 1947.2.27.92), female. (D) <i>Indirana leithii</i>, holotype of <i>Rana leithii</i>, NHM 69.8.28.50 (ex BMNH 1947.2.28.17), female. (E) <i>Indirana sarojamma</i>, SDBDU 2002.516, female. (F) <i>I</i>. <i>tysoni</i>, SDBDU 2012.73, female. (G) <i>I</i>. <i>yadera</i>, SDBDU 2012.2744, male (left), SDBDU 2015.2984, female (right). (H–M) <i>Indirana semipalmata</i> group. (H) <i>I</i>. <i>chiravasi</i>, SDBDU 2014.2423, male (left), SDBDU 2015.3087, female (right). (I) <i>I</i>. <i>duboisi</i>, SDBDU 2003.1086, male. (J) <i>I</i>. <i>gundia</i>, holotype of <i>Ranixalus gundia</i>, MNHN 1985.0633, male. (K) <i>Indirana paramakri</i> sp. nov., SDBDU 2015.3741, male (left), ZSI/WGRC/V/A888, female (right). (L) <i>I</i>. <i>salelkari</i>, SDBDU 2011.1330, female. (M) <i>I</i>. <i>semipalmata</i>, lectotype of <i>Rana semipalmata</i>, NHM 74.4.29.605 (ex BMNH 1947.2.29.50), female. (N–P) Genus <i>Sallywalkerana</i>. (N) <i>S</i>. <i>diplosticta</i>, lectotype of <i>Ixalus diplostictus</i>, NHM 74.4.29.1412 (ex BMNH 1947.2.2.21), female. (O) <i>Sallywalkerana leptodactyla</i>, lectotype of <i>Rana leptodactyla</i>, NHM 74.4.29.593 (ex BMNH 1947.2.29.39), female. (P) <i>Sallywalkerana phrynoderma</i>, lectotype of <i>Rana phrynoderma</i>, NHM 82.2.10.21 (ex BMNH 1947.2.3.8), female.</p