Description of four new species of Burrowing Frogs in ...

Zootaxa 4277 (4): 451–490 http://www.mapress.com/j/zt/ Copyright © 2017 Magnolia Press

Article

ISSN 1175-5326 (print edition)

ZOOTAXA

ISSN 1175-5334 (online edition)

https://doi.org/10.11646/zootaxa.4277.4.1 http://zoobank.org/urn:lsid:zoobank.org:pub:71A18DA8-2AFF-4998-AE87-00D713EC531D

Description of four new species of Burrowing Frogs in the Fejervarya rufescens complex (Dicroglossidae) with notes on morphological affinities of Fejervarya species in the Western Ghats SONALI GARG1 & S.D. BIJU1,2 1 2

Systematics Lab, Department of Environmental Studies, University of Delhi, Delhi, India Corresponding author. E-mail: [email protected]

Abstract The Rufescent Burrowing Frog, Fejervarya rufescens, is thought to have a wide distribution across the Western Ghats in Peninsular India. This locally abundant but secretive species has a short breeding period, making it a challenging subject for field studies. We sampled 16 populations of frogs morphologically similar to F. rufescens in order to understand the variation among populations found across the Western Ghats. Our study shows significant morphological and genetic differences among the sampled populations, suggesting that F. ‘rufescens’ is a complex of several undescribed species. Using evidence from morphology and genetics, we confirm the presence of five distinct species in this group and formally describe four as new. The new species were delineated using a phylogeny based on three mitochondrial genes (16S, COI and Cytb) and a haplotype network of a nuclear gene (Rag1). Hereafter, the distribution of F. rufescens is restricted to the state of Karnataka and adjoining regions of northern Kerala. Three new species (Fejervarya kadar sp. nov., Fejervarya manoharani sp. nov. and Fejervarya neilcoxi sp. nov.) are from regions south of Palghat gap in the state of Kerala, and one (Fejervarya cepfi sp. nov.) from the northern Western Ghats state of Maharashtra. These findings indicate that Fejervarya frogs of the Western Ghats are more diverse than currently known. Our results will also have implications on the conservation status of F. rufescens, which was previously categorized as Least Concern based on its presumed wide geographical distribution. Furthermore, in order to facilitate a better taxonomic understanding of this region’s fejervaryan frogs, we divide all the known Fejarvarya species of the Western Ghats into four major groups—Fejervarya nilagirica group, Fejervarya rufescens group, Fejervarya sahyadris group and Fejervarya syhadrensis group, based on their morphological affinities. Key words: Amphibians, bioacoustics, multi-gene DNA barcoding, India, integrative taxonomy, molecular phylogeny, new species, species diversity, Western Ghats

Introduction The anuran genus Fejervarya Bolkay (Dicroglossidae: Dicroglossinae) is one of the most widely distributed genera in Asia. The distribution of fejervaryan frogs extends from South and Southeast Asia (largely the Indian subcontinent, mainland Indochina and islands of the Malay archipelago) up to East Asia (China and Japan). Currently represented by 40 species (Frost 2016), this genus is noted for various taxonomic problems mainly due to high degree of morphological similarity between species and unavailability or doubtful status of several type specimens (e.g., Jerdon 1870; Dubois 1984; Dubois & Ohler 2000; Biju 2001; Dubois et al. 2001; Kuramoto et al. 2008 “2007”). As a consequence, Fejervarya frogs have often been misidentified throughout their ranges (e.g., Dutta 1997; Kuramoto & Joshy 2001; Chanda 2002; Kadadevaru et al. 2002; Daniels 2005) thereby propagating misconceptions regarding the identity of various poorly known and cryptic taxa (Biju 2001; Ohler et al. 2009). More recently molecular studies have addressed the systematics of fejervaryan frogs but the phylogenetic relationships of the genus and its various members still remain unresolved (e.g., Kurabayashi et al. 2005; Frost et al. 2006; Kuramoto et al. 2008 “2007”; Wiens et al. 2009; Kotaki et al. 2010; Pyron & Wiens 2011; Ohler et al. 2014; Dinesh et al. 2015). However, molecular systematics has facilitated the discovery of several cryptic species

Accepted by M. Vences: 27 Apr. 2017; published: 20 Jun. 2017

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from populations previously identified as single wide-ranging species (e.g., Veith et al. 2001; Kuramoto et al. 2008 “2007”; Matsui et al. 2008 “2007”; Djong et al. 2011). Despite the existing taxonomic uncertainties, a total of eleven new Fejervarya species have been described over the past decade (Kuramoto et al. 2008 “2007”; Matsui et al. 2008 “2007”; Ohler et al. 2009; Howlader 2011; Djong et al. 2011; Purkayastha & Matsui 2012; Dinesh et al. 2015; Howlader et al. 2016). The possible occurrence of several more undescribed species within the genus has also been suggested, particularly from poorly explored regions of Asia or from areas known to have high anuran diversity (e.g., Kurabayashi et al. 2005; Kuramoto et al. 2008 “2007”; Sumida et al. 2007; Islam et al. 2008; Kotaki et al. 2008, 2010; Hasan et al. 2012). India is a center for anuran diversity, with 27 species presently recognized in the genus Fejervarya (AmphibiaWeb 2016; Frost 2016). Of these, 15 species were described from the Western Ghats of Peninsular India, a region that is regarded as a global biodiversity hotspot (Myers et al. 2000). However, in contrast to the dramatic increase in species descriptions in other anuran groups of the Western Ghats (Biju et al. 2014a) understanding of species diversity within the genus Fejervarya has remained relatively poor. The major impediment being the unavailability of type specimens of species described by Rao (1920, 1922, 1937), absence of new collections or insights regarding the status of several poorly studied taxon such as F. modesta, F. murthii, F. mysorensis, F. nilagirica, F. parambikulamana and F. sauriceps, and range assumptions and misidentifications of species such as F. syhadrensis (e.g., Kuramoto & Joshy 2001; Kadadevaru et al. 2002; Daniels 2005). At the same time, none of the studies so far have attempted a comprehensive taxonomic revision including all the known Fejervarya members in this region. Among members of the Western Ghats, Fejervarya rufescens is the oldest known species, originally described as Pyxicephalus rufescens Jerdon from “Malabar Coast”, South India. Subsequently, this species was considered as a member of various genera such as Rana (Boulenger 1882), Rana (Tomopterna) (Boulenger 1920), Rana (Fejervarya) (Dubois 1984), Limnonectes (Fejervarya) (Dubois 1987 “1986”), Tomopterna (Dutta 1997; Das & Dutta 1998; Chanda 2002; Daniels 2005; Matsui et al. 2008 “2007”), Zakerana (Howlader 2011) and the present designation Fejervarya (e.g., Iskandar 1998; Dubois 1999; Dubois & Ohler 2000; Fei et al. 2002; Kuramoto et al. 2008 “2007”; Kotaki et al. 2008, 2010; Dinesh et al. 2015). The taxonomic placement of this species in genus Tomopterna was largely because of the overall morphological similarity with its South Asian members, which are now in the genus Sphaerotheca (see the ‘results’ section for detailed discussion). Apart from this, Fejervarya rufescens can be considered as one of the morphologically most distinct species of the genus and has never been confused with other members. It is also the only recognized Fejervarya species that is known to exhibit burrowing behavior, from which its common name Rufescent Burrowing Frog (Das & Dutta 1998; Daniels 2005) is aptly derived. Because of its secretive lifestyle, F. rufescens is usually difficult to encounter despite its local abundance. This species is considered as widely distributed, with its range presumed to extend from southern Western Ghats in Kerala up to Maharashtra in the north (e.g., Dutta 1997; Chanda 2002; Daniels 2005; Frost 2016). So far, the available information for this species includes male advertisement calls along with ecological and behavioral observations (Kadadevaru et al. 2000; Kuramoto & Dubois 2009; Grosjean & Dubois 2011), and karyotypes and cbanding patterns (Joshy & Kuramoto 2008). Kuramoto et al. (2008 “2007”) also indicated that F. rufescens is likely to be a species complex. We sampled various populations of Fejervarya ‘rufescens’ from its known distribution range, in order to understand the intra and interspecific morphological and genetic variations. A detailed study of our collections suggests the presence of several undescribed cryptic species morphologically related to F. rufescens. More specifically, the populations from southern Kerala and Maharashtra are found to be distinct from the typical populations. These findings are confirmed using an integrated taxonomic approach and four new species are formally described.

Materials and methods Field surveys and specimen collection. Frogs were sampled during routine amphibian surveys carried out between the years 2007–2015 in three Indian states encompassing the Western Ghats region—Kerala, Karnataka and Maharashtra. Specimens were found either during opportunistic surveys conducted in the day and night time, or by locating calling males after sunset during the breeding season. Geographical coordinates and altitude of the

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surveyed localities were recorded with a Garmin 76CSx GPS using the WGS84 datum system, and the locality information was used to prepare maps in QGIS (http://www.qgis.org). Live animals were photographed immediately after collection and later euthanised in MS-222 solution (Tricaine methane sulphonate). Prior to fixation, tissue samples were taken from the thigh muscle of selected representatives, preserved in absolute ethanol, and stored at -20°C in the Systematics Lab, University of Delhi for molecular studies. Specimens were fixed in 4% formalin for several days and transferred to 70% ethanol after being thoroughly washed under running water. Type specimens are deposited in the Zoological Survey of India–Western Ghats Regional Centre (ZSI-WGRC), Calicut, and referred specimens are available at the Systematics Lab, University of Delhi (SDBDU). Molecular study. Total genomic DNA was extracted from 16 tissue samples with the Qiagen DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA), following manufacturer’s protocol. Fragments of three mitochondrial (mt) genes—16S rRNA (16S, ≈540 bp), Cytochrome oxidase subunit I (COI, ≈650 bp) and Cytochrome b (Cytb, ≈580 bp), and one nuclear (nu) gene Rag1 (≈550 bp), were PCR-amplified using primers published by Simon et al. (1994), Bossuyt & Milinkovitch (2000), Che et al. (2012) and Biju & Bossuyt (2003), respectively. Cyclesequencing was performed in both directions using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) on ABI 3730 automated DNA sequencer (Applied Biosystems). Sequence data was checked and assembled in ChromasPro v1.34 (Technelysium Pty Ltd.) and deposited in the GenBank under accession numbers KY447308–KY447323 and KY820721–KY820766. Details of DNA sequences used for molecular analyses are provided in Table 1. Sequences were aligned using the ClustalW tool in MEGA 6.0 (Tamura et al. 2013) and alignments were manually optimised. First, a mitochondrial dataset of 16S gene (545 characters) was compiled using DNA sequences generated from 16 new samples of Fejervarya frogs and GenBank retrieved sequences (total 28) representing previously known Fejervarya species and two Sphaerotheca species used as outgroups. The data matrix was executed in PAUP* 4.0b10 (Swofford 2002) to determine an appropriate model of DNA evolution by implementing the Akaike Information Criterion in ModelTest 3.4 (Posada & Crandall 1998). Using the best-fit model (GTR+I+G) and obtained parameters, a phylogenetic analysis was performed using the Maximum likelihood (ML) criterion. Heuristic ML searches were executed in PAUP* 4.0b10 and clade support was assessed with 10,000 rapid bootstrap replicates executed using RAxML 7.3.0 (Stamatakis 2006; Stamatakis et al. 2008) in raxmlGUI 1.1 (Silvestro & Michalak 2012). Bayesian Posterier Probabilities (BPP) for the clades were estimated in MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003). Bayesian analyses were performed using the GTR+I+G model, with two parallel runs of four MCMC chains executed for 10 million generations. The sampling frequency was set at ten and the results were summarised by discarding the first 25% of trees as burn-in. Based on the phylogenetic position of new samples, uncorrected pairwise distances among members of the Fejervarya rufescens group were computed for the 16S gene in PAUP* 4.0b10 to determine the levels of genetic divergence. Uncorrected pairwise distances were also computed for two other mitochondrial genes (COI and Cytb). Based on the mitochondrial analyses, another molecular dataset was assembled combining 2314 basepairs of mitochondrial (16S, COI and Cytb) and nuclear (Rag1) DNA for 14 selected taxa representing members of the Fejervarya rufescens group. Fejervarya granosa was included as outgroup taxon for the phylogenetic analyses. Heuristic ML searches were performed in PAUP* 4.0b10 using the GTR+I+G model with all parameters estimated. Clade support was assessed with 10,000 rapid bootstrap replicates using RaxML as well as Bayesian analyses executed for 20 million generations with sampling frequency of 100 and 25% burn-in. Furthermore, datasets of the mitochondrial and nuclear genes were also analysed independently to understand species-level distinctness within the Fejervarya rufescens group. To separate Rag1 sequences into haplotypes, the PHASE algorithm (Stephens et al. 2001) as implemented in DnaSP Ver 5 (Librado & Rozas 2009) was applied and the phased data was used to construct Median Joining (MJ) networks in the software Network 4.6.1.0 (www.fluxus-engineering.com). Morphological study. Our collections were morphologically compared with the available type specimens and other referred specimens of closely related members within the genus. Sex and maturity were determined either by the presence of secondary sexual characters or by examining the gonads through a small lateral or ventral incision. Only adult specimens were used for morphometric studies. Measurements and associated terminologies follow Biju et al. (2014b) and Garg & Biju (2016). The following measurements were taken to the nearest 0.1 mm using a digital slide caliper or a binocular microscope with a micrometer ocular: SVL (snout–vent length), HW (head width, at the angle of the jaws), HL (head length, from rear of mandible to tip of snout), SL (snout length, from tip of snout to anterior orbital border), EL (eye length, horizontal distance between bony orbital borders), IUE (inter

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upper eyelid width, the shortest distance between the upper eyelids), UEW (maximum upper eyelid width), IN (internarial distance), NS (distance from the nostril to the tip of the snout), EN (distance from the front of the eye to the nostril), TYD (greatest tympanum diameter), TYE (distance from the tympanum to the back of the eye), FAL (forearm length, from flexed elbow to base of outer palmar tubercle), HAL (hand length, from base of outer palmar tubercle to tip of third finger), FLI–IV (finger length), TL (thigh length), SHL (shank length), FOL (foot length, from base of inner metatarsal tubercle to tip of fourth toe), TFOL (total foot length, from heel to tip of fourth toe), IMT (length of inner metatarsal tubercle), OMT (length of outer metatarsal tubercle) and ITL (inner toe length). All measurements provided in the taxonomy section are in millimetres. The webbing formulae follow Savage & Heyer (1967) as modified by Myers & Duellman (1982), and the degree of webbing relative to subarticular tubercles is described by numbering the tubercles 1–3, starting from the toe discs. All measurements and photographs were taken for the right side of the specimen, except when a character was damaged, in which case the measurement was taken on the left side. For the convenience of discussion, Fejervarya species of the Western Ghats are grouped as small (male SVL 17.0–25.0 mm), medium (male SVL 25.1–45.0 mm) and large (male SVL 45.1–65.0 mm); webbing is defined as basal (slightly above or beyond the basal subarticular tubercles on all toes), small (webbing on toe IV beyond the third subarticular tubercle but below the second subarticular tubercle on either side), medium (webbing on toe IV beyond the second subarticular tubercle but below the first subarticular tubercle on either side), and large (webbing on toe IV extending beyond the first subarticular tubercle on either side). Principal Component Analysis (PCA) and Discriminant Function Analysis (DFA) were performed on the morphometric data from adult male specimens using Statistica v7.1 (StatSoftInc). PCA was performed on 17 morphometric variables that were size-corrected to nullify the influence of body size. Size correction was obtained by expressing the measurements of head characters (HW, SL, EL, TYD, TYE, EN, NS, IUE, UEW and IN) as percent of head length (HL) and other body measurements (HL, FAL, HAL, TL, SHL, FOL and TFOL) as percent of snout to vent length (SVL). Factor scores of the first two Principal Components (PC) were observed on a scatterplot to assess the degree of morphological differentiation among members of the Fejervarya rufescens group. Discriminant Function Analysis (DFA) was performed using the highest weighted variables. For this, PCA factors with eigenvalues >1.0 were used as input variables to determine the classification success of our samples. Bioacoustics. Advertisement calls of single males were recorded for two species using a Marantz PMD620 solid-state digital recorder (44.1 kHz sampling rate, 16-bit resolution) and a Sennheiser ME 66 unidirectional microphone. Recorded individuals were collected for identification using both genetic and morphological tools. A total of five temporal properties (call duration, call rise time, call fall time, number of pulses per call and pulse rate) and one spectral property (dominant frequency) were measured using Raven Pro 1.4 (Charif et al. 2010). Dominant frequency was measured after averaging spectrum over the entire call. Spectrogram figures were prepared by selecting the call on one-second time frame. Terminologies and graphical representation of the analysed call properties follow Bee et al. (2013a, 2013b). Air temperature (dry bulb and wet bulb) at the calling site was recorded to the nearest 0.1◦C using a Jennson Delux thermometer. Abbreviations. Museum acronyms and frequently used terms are as follows: Systematics Lab, University of Delhi (SDBDU), ZSI/WGRC (Zoological Survey of India, Western Ghats Regional Centre, formerly Zoological Survey of India, Western Ghats Field Research Station, WGFRS, Calicut, India), MNHNP (Museum National d'Histoire Naturelle, Paris, France), ZSIC (Zoological Survey of India, Kolkata, India), WLS (Wildlife Sanctuary), SDB (S.D. Biju) and SG (Sonali Garg).

Results Our integrative approach provided evidence for concordant differentiation of five lineages in the Fejervarya rufescens group, four of which are described as new species in the present work. Anticipating this taxonomic conclusion, we refer to these under their new names in the following sections, whereas formal taxonomic descriptions are provided subsequently.


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FIGURE 1. Maximum Likelihood phylogram based on the 16S mitochondrial DNA dataset of 545 basepairs. Bayesian Posterior Probabilities (BPP) and RaxML bootstrap values of >50% are indicated above and below the branches, respectively. The uncorrected pairwise distances between species in the Fejervarya rufescens group are shown near the clade labels. Voucher number and locality details of the samples are referenced in Table 1.



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FIGURE 2. Phylogenetic analyses. A. Maximum Likelihood phylogram based on mitochondrial (16S, COI and Cytb) and nuclear (Rag1) DNA of 2314 basepairs, showing phylogenetic relationships among members of the Fejervarya rufescens group. Bayesian Posterior Probabilities (BPP) and RaxML bootstrap values of >50% are indicated above and below the branches, respectively. Fejervarya granosa (2) was used as the outgroup taxon. Voucher number and locality details of the samples are referenced in Table 1; B. Median Joining haplotype network inferred from 554 basepair fragment of the nuclear Rag1 gene. Circle sizes are proportional to the number of haplotypes, branch lengths are proportional to the number of mutational steps, and black bars represent mutational steps. Number of individuals used for each species: Fejervarya cepfi (3), Fejervarya kadar (1), Fejervarya manoharani (3), Fejervarya neilcoxi (2), and Fejervarya rufescens (5).


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FIGURE 3. Geographical distribution of five species in Fejervarya rufescens group of the Western Ghats, Peninsular India. The Western Ghats biodiversity hotspot region is shown in orange colour. Locality details are referenced in Table 3.

Genetic diversity. All the newly sampled populations formed a well-supported clade along with Fejervarya rufescens in the 16S Maximum Likelihood (ML) tree (Fig. 1). This clade, identified as the Fejervarya rufescens group, may be defined as the most inclusive clade containing F. rufescens and the four new species, but none of the other previously known Fejervarya members. Furthermore, all the Fejervarya species of the Western Ghats formed a larger clade along with some species from neighbouring regions such as Bangladesh, Nepal and Sri Lanka, in FOUR NEW FEJERVARYA SPECIES FROM THE WESTERN GHATS


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South Asia, but none of the other Fejervarya species from Southeast and East Asia, including the type species of the genus F. limnocharis. The relationships among known species were largely in agreement with the previously published phylogenies (e.g., Kurabayashi et al. 2005; Wiens et al. 2009; Kotaki et al. 2010; Pyron & Wiens 2011; Dinesh et al. 2015). Phylogenetic relationships within the Fejervarya rufescens group were inferred based on the multi-gene (mt + nu) ML tree (Fig. 2A). The new species Fejervarya kadar sp. nov. is sister to F. rufescens, whereas F. manoharani sp. nov. is closely related to F. neilcoxi sp. nov. Phylogenetically, F. cepfi sp. nov. is the most divergent member of the group and occupies a basal position to the clades containing (F. kadar + F. rufescens) and (F. manoharani + F. neilcoxi). The average uncorrected pairwise distances for all five species (F. rufescens and four new species) with their closest relatives were >3.2% for 16S (range 2.8–6.3%), >8.9% for COI (range 8.9–12.3%) and >8.2% for Cytb (range 8.0–14.6%) (Table 2). Based on previously observed molecular divergences within the genus Fejervarya (e.g., Kuramoto et al. 2008 “2007”; Dinesh et al. 2015; Howlader et al. 2016), closely related dicroglossid genera such as Euphlyctis and Hoplobatrachus (e.g., Hasan et al. 2012) and other anuran groups in the Western Ghats (e.g., Biju et al. 2011, 2014a, 2014b; Garg & Biju 2016; Garg et al. 2017), we considered these genetic distances as thresholds for delineating candidate species in the present study. The maximum intraspecific pairwise distances were observed for Fejervarya rufescens—2.1% for 16S, 4.8% for COI and 4.6% for Cytb, largely due to high genetic divergence between the populations from Karnataka and Kerala (Tables 1–2; Figs. 1–2). Detailed intra and interspecific pairwise comparisons for the three mitochondrial genes analyzed in our study are provided in Table 2. The haplotype network for the nuclear Rag1 gene showed sharing of haplotypes among some of the species in the F. rufescens group. However, a clear differentiation was observed for some species such as F. manoharani sp. nov. that did not share haplotypes despite inclusion of three individuals, and F. cepfi sp. nov. and F. neilcoxi sp. nov., both of which also had at least one haplotype distinctly different from all other species (Fig. 2B). Importantly, F. kadar sp. nov. did not share haplotypes with F. neilcoxi sp. nov., despite very close geographical proximity between the collection localities of these two species (Table 3; Fig. 3).

Morphological grouping of species in the genus Fejervarya Based on morphology, species belonging to the genus Fejervarya sensu lato in the Western Ghats can be assigned to four distinguishable groups: (1) Fejervarya nilagirica group (nine species), (2) Fejervarya rufescens group (four species), (3) Fejervarya sahyadris group (two species), and (4) Fejervarya syhadrensis group (five species). Fejervarya nilagirica group. This group can be distinguished from other Fejervarya groups of the Western Ghats by following suite of characters: medium to large adult size (male SVL 32.0–50.0 mm, female SVL 36.0–65.0 mm); elongate or robust body; head longer than wide or sub-equal; large webbing between toes (beyond the first subarticular tubercle on either side of toe IV); long and cylindrical inner metatarsal tubercles; dorsal skin with prominent folds, either continuous or discontinuous, with glandular warts or prominent granulations; dorsal chevron present; groin and thigh with prominent reticulations (Figs. 4A–B). Members included. Fejervarya brevipalmata, F. keralensis, F. kudremukhensis, F. mudduraja, F. murthii, F. mysorensis and F. nilagirica. Based on original descriptions, F. parambikulamana (Rao 1937) and F. sauriceps (Rao 1937) are provisionally considered as members of this group. Fejervarya rufescens group. This group can be distinguished from other Fejervarya groups of the Western Ghats by following suite of characters: medium adult size (male SVL 28.0–35.0 mm, female SVL 32.0–36.0 mm); stout body; head wider than long or sub-equal; presence of rictal glands at labial commissures of the mouth; small webbing between toes (beyond the third subarticular tubercle but not beyond the second subarticular tubercle on either side of toe IV); shovel-shaped inner metatarsal tubercles; dorsal skin with glandular projections, spines or warts, but without longitudinal skin folds; dorsal chevron present; groin without reticulations; thigh with or without faint reticulations (Fig. 4C). Members included. Fejervarya cepfi sp. nov., F. kadar sp. nov., F. manoharani sp. nov., F. neilcoxi sp. nov. and F. rufescens. Fejervarya sahyadris group. This group can be distinguished from other Fejervarya groups of the Western Ghats by following suite of characters: small adult size (male SVL 17.0–22.0 mm, female SVL 21.0–24.0 mm); slender body; head longer than wide; presence of rictal glands at labial commissures of the mouth and a white horizontal band along the upper lip; basal webbing between toes (slightly above the basal subarticular tubercles on all toes); long and cylindrical inner metatarsal tubercles; dorsal skin with weakly developed longitudinal skin folds


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FIGURE 4. Four morphologically recognized Fejervarya groups in the Western Ghats. A–B. Fejervarya nilagirica group: A. Fejervarya nilagirica in life (male, SDBDU 2014.2772); B. Fejervarya keralensis in life (male, SDBDU 2015.3139); C. Fejervarya rufescens group, Fejervarya rufescens in life (male, SDBDU 2015.3070); D. Fejervarya sahyadris group, Fejervarya sahyadris in life (male, SDBDU 2015.3046); E–F. Fejervarya syhadrensis group: E. Fejervarya syhadrensis in life (male, SDBDU 2015.2934); F. Fejervarya granosa in life (male, SDBDU 2014.2541). Numbers on the panels represents: 1. dorsal view, 2. ventral view of foot showing the inner metatarsal tubercle, 3. schematic illustration of foot webbing, 4. thighs showing the marking and colour pattern, 5. lateral view showing the groin markings.



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FIGURE 5. Morphological comparison between members of the genus Sphaerotheca and Fejervarya rufescens group of the Western Ghats. A–H. Genus Sphaerotheca: A–E. Sphaerotheca cf. breviceps, in life (SDBDU 2014.2822): A. dorsolateral view, B. ventral view, C. ventral skin texture, D. lateral view showing the groin markings, E. thighs showing the marking and colour pattern; F–H. Inner metatarsal tubercle: F. Sphaerotheca breviceps (syntype ZMB 3351), G. S. dobsonii (Holotype BMNH 1947.2.28.45), H. S. rolandae (Holotype BMNH 1973.3024). I–R. Fejervarya rufescens group: I–K. F. rufescens in life (SDBDU 2015.3070): I. dorsolateral view, J. ventral view, K. ventral skin texture; L–M. F. kadar sp. nov. (Holotype ZSI/ WGRC/V/A/940): L. lateral view showing the groin markings, M. thighs showing the marking and colour pattern; N–R. Inner metatarsal tubercle: N. F. cepfi sp. nov. (Holotype ZSI/WGRC/V/A/937), O. F. kadar sp. nov. (Holotype ZSI/WGRC/V/A/ 940), P. F. manoharani sp. nov. (Holotype ZSI/WGRC/V/A/945), Q. F. neilcoxi sp. nov. (Holotype ZSI/WGRC/V/A/951), R. F. rufescens (SDBDU 2015.3070).

or granulations; dorsal chevron absent; groin without reticulations; thigh with faint reticulations (Fig. 4D). Members included. Fejervarya gomantaki and F. sahyadris. Fejervarya syhadrensis group. This group can be distinguished from other Fejervarya groups of the Western Ghats by following suite of characters: medium adult size (male SVL 25.0–31.0 mm, female SVL 27.0–42.0 mm); elongate body; head longer than wide; medium webbing between toes (beyond the second subarticular tubercle but


GARG & BIJU

not beyond the first subarticular tubercle on either side of toe IV); long and cylindrical inner metatarsal tubercles; dorsal skin with prominent discontinuous skin folds, with or without scattered granular projections; dorsal chevron present; groin without reticulations; thigh with faint reticulations (Figs. 4E–F). Members included. Fejervarya caperata, F. granosa and F. syhadrensis. Based on the original description, F. modesta (Rao 1920) is provisionally considered to be a member of this group. Morphological comparison of groups. The Fejervarya rufescens group differs from all other members of the genus Fejerarya by its stout body shape (vs. slender or robust), skin with glandular projections, spines or warts, but without longitudinal skin folds (vs. presence of dorsal skin folds), and shovel-shaped inner metatarsal tubercles (vs. long and cylindrical) (Fig. 4). Morphologically Fejervarya rufescens and the four new species could be confused with Sphaerotheca members in the Western Ghats due to similarities such as stout body shape, dorsal skin texture with relatively less prominent granulations or folds, head wider than long or nearly equal, finger and toe tips blunt and rounded, reduced webbing between toes, and prominent shovel-shaped inner metatarsal tubercles. However, members of the Fejervarya rufescens group differ from Sphaerotheca species found in the Western Ghats by the presence of small and circular outer metatarsal tubercles on foot (vs. absent), distance from the tip of inner metatarsal tubercle to the tip of first toe larger than the length of inner metatarsal tubercle (vs. shorter), smooth belly (vs. prominently granular), and presence of fejervaryan line on both sides of the belly (vs. absent) (Fig. 5). Detailed morphological comparisons between members of the Fejervarya rufescens group are provided in the respective species descriptions.

FIGURE 6. PCA plot for the first two principal component axes showing morphometric differentiation among adult males of five species in the Fejervarya rufescens groups of the Western Ghats. Factor loadings for each component are provided in Table 4.



461


GARG & BIJU

Parambikulam, Kerala Parambikulam, Kerala Kattalapara, Shendurney WLS, Kerala Chathankod-Bonnacad, Kerala Chathankod-Bonnacad, Kerala Chathankod-Bonnacad, Kerala Guddekere, Agumbe RF, Karnataka Pozhuthana, Kerala Peruvannamuzhi, Kerala Kollur, Mookambika WLS, Karnataka Manipal, Karnataka Madikeri, Karnataka Bajipe, Mangalore, Karnataka Thavalakuzhipara, Kerala Phansad WLS, Maharashtra Koyna, Maharashtra Koyna, Maharashtra Amboli, Maharashtra Madikeri, Karnataka BR Hills, Karnataka Bentota, Sri Lanka Chitwan, Nepal India Bangladesh Bangladesh Aralam, Kerala Codal village, Goa Karnoor, Karnataka Kudremukh, Karnataka Nuwara Eliya, Sri Lanka

Laggalla, Sri Lanka

Talapu, Madikeri, Karnataka Western Ghats, India Orissa, India Java, Indonesia

Fejervarya neilcoxi (1) Fejervarya neilcoxi (2) Fejervarya manoharani (1)

Fejervarya rufescens (5) Fejervarya rufescens (6) Fejervarya rufescens (7) Fejervarya kadar (1) Fejervarya cepfi (1) Fejervarya cepfi (2) Fejervarya cepfi (3) Fejervarya cepfi (4) Fejervarya granosa (1) Fejervarya granosa (2) Fejervarya cf. syhadrensis Fejervarya ‘pierrei’ Fejervarya ‘syhadrensis’ Fejervarya asmati Fejervarya dhaka Fejervarya sahyadris Fejervarya gomantaki Fejervarya caperata Fejervarya kudremukhensis Fejervarya greenei

Fejervarya kirtisinghei

Fejervarya mudduraja Fejervarya keralensis Fejervarya orissaensis Fejervarya iskandari

Fejervarya manoharani (2) Fejervarya manoharani (3) Fejervarya manoharani (4) Fejervarya rufescens (1) Fejervarya rufescens (2) Fejervarya rufescens (3) Fejervarya rufescens (4)

Collection locality

Species

BNHS 4645 – 16S-ori 16S-isk

MNHN 2000.620

SDBDU 2015.3070 – 030526-03 ZSI/WGRC/V/A/940 SDBDU 2012.1429 SDBDU 2007.1561 SDBDU 2007.1569 ZSI/WGRC/V/A/938 BNHS 4649 SDBDU 2014.2541 – – IN023 FaCSE_17 F1DSE_23 RBRL 050714-01 CESF 2294 BNHS 4657 BNHS 4653 MNHN 2000.617

SDBDU 2011.275 ZSI/WGRC/V/A/950 ZSI/WGRC/V/A/945 SDBDU 2013.2669 SDBDU 2013.2505 SDBDU 2015.2882 SDBDU 2008.407 SDBDU 2009.4712

ZSI/WGRC/V/A/955 ZSI/WGRC/V/A/951

Voucher number

AB355833 GQ478322 AB277304 AB277303

AY014380

KY447321 AB488897 AB167945 KY447312 KY447311 KY447309 KY447310 KY447308 AB355836 KY820766 AB488892 AB488888 AY882955 KP849815 KP849819 AB530604 KR781085 AB355842 AB167949 AY014378

KY447315 KY447313 KY447314 KY447319 KY447323 KY447322 KY447320

16S KY447317 KY447318 KY447316

KY820747 – – KY820739 KY820736 KY820737 – KY820738 – KY820750 – – – – – – – – – – – – – – –

– – – – –

KY820741 KY820742 – KY820745 KY820748 KY820749 KY820746

KY820732 – – KY820724 KY820721 KY820722 – KY820723 – KY820735 – – – – – – – – – –

KY820726 KY820727 – KY820730 KY820733 KY820734 KY820731

Accession number COI Cytb KY820728 KY820743 KY820729 KY820744 KY820725 KY820740

– – – –

–

KY820762 – – KY820754 KY820751 KY820752 – KY820753 – KY820765 – – – – – – – – – –

KY820756 KY820757 – KY820760 KY820763 KY820764 KY820761

Rag1 KY820758 KY820759 KY820755

Kuramoto et al. 2008 “2007” Meenakshi et al. 2010 Kotaki et al. 2008 Kotaki et al. 2008 ...continued on the next page

Kosuch et al. 2001

This study Kotaki et al. 2010 Kurabayashi et al. 2005 This study This study This study This study This study Kuramoto et al. 2008 “2007” This study Kotaki et al. 2010 Kotaki et al. 2010 Unpublished Howlader et al. 2011 Howlader et al. 2016 Hasan et al. 2014 Dinesh et al. 2015 Kuramoto et al. 2008 “2007” Kurabayashi et al. 2005 Kosuch et al. 2001

This study This study This study This study This study This study This study

This study This study This study

Reference

TABLE 1. List of DNA sequences used in the study. Taxa are arranged in the order of their phylogenetic position in Figure 1 (Top to bottom).



463

Collection locality

Iriomote Island, Japan Hiroshima, Japan Green Island, Taiwan Java, Indonesia Husa, China Ubon Ratchatani, Thailand Selangor, Malaysia Luzon Island, Philippines India/Sri Lanka Bajipe, Karnataka

Table 1. continued Species

Fejervarya sakishimensis Fejervarya ‘limnocharis’ Fejervarya ‘limnocharis’ Fejervarya limnocharis Fejervarya multistriata Fejervarya triora Fejervarya cancrivora Fejervarya vittigera Sphaerotheca breviceps Sphaerotheca dobsonii

– – – 16S-limno – – – PNM7826 – 16S-dob

Voucher number 16S AB488886 AB488887 AB488885 AB277302 AB488884 AB488883 AB488882 AY313683 AF249042 AB277305

COI – – – – – – – – – –

Accession number Cytb – – – – – – – – – –

Rag1 – – – – – – – – – –

Kotaki et al. 2010 Kotaki et al. 2010 Kotaki et al. 2010 Kotaki et al. 2008 Kotaki et al. 2010 Kotaki et al. 2010 Kotaki et al. 2010 Evans et al. 2003 Bossuyt & Milinkovitch 2000 Kotaki et al. 2008

Reference


GARG & BIJU

Fejervarya kadar Fejervarya manoharani Fejervarya neilcoxi Fejervarya rufescens Fejervarya manoharani Fejervarya neilcoxi Fejervarya rufescens Fejervarya neilcoxi Fejervarya rufescens Fejervarya rufescens

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