Nyctibatrachus mewasinghi, a new species of night frog (Amphibia: Nyctibatrachidae) from Western Ghats of Kerala, India

Communication

 

 

 

INTRODUCTION

 

 

 

Genus Nyctibatrachus is diagnosed based on a combination of characters including small to large body sizes (10.0–77.0 mm snout to vent length); oval or diamond shaped pupil; shagreened, glandular or wrinkled dorsal skin; tympanum not visible externally, or with borders obscured by rugose skin; vomerine teeth present; tongue notched or emarginated posteriorly; tips of fingers with or without discs, discs with or without distinct dorso-terminal grooves; webbing absent on fingers; tips of toes with or without discs, discs with or without dorso-terminal grooves; and subocular gland present on all species (Biju et al. 2011). Although originally considered as a member of the family Ranidae by Boulenger (1882), based on molecular phylogenetic studies by Roelants et al. (2004), Bossuyt et al. (2006) and Frost et al. (2006), this genus is now placed under the family Nyctibatrachidae. The Western Ghats endemic genus Nyctibatrachus has 35 known species (Garg et al. 2017).

While studying the distribution of species of Nyctibatrachus along the Western Ghats mountain ranges using molecular analysis of mitochondrial NADH dehydrogenase subunit 1 (ND1) and 16S rRNA genes, we discovered a population that is sister taxa to, but genetically divergent from N. kempholeyensis and N. athirappillyensis. We describe this population as a new species, Nyctibatrachus mewasinghi, based on morphology, multivariate morphometric analysis and molecular analysis.

 

 

MATERIALS AND METHODS

 

Specimen collection and museum deposition

Specimens of Nyctibatrachus were studied from the Western Ghats of Kerala, Tamil Nadu, Karnataka and Goa, India. Specimen collection was kept to a minimum with collection of mainly road kills, injured or potentially infected individuals. Specimens from protected areas were collected with permissions from the Kerala Forest Department (permit no. WL WL10-3548/2013) and Karnataka Forest Department (permit no. PS/PCCF/WL/CR/22/2013-14). Specimens were preserved in either absolute ethanol or 4% formaldehyde. Studied specimens are in the museum collections of the Bombay Natural History Society (BNHS), Mumbai, and the Wildlife Information Liaison Development (WILD), Coimbatore (Appendix 1).

 

Morphometry and morphological description

Measurements were taken to the nearest 0.1mm using a digital caliper (Mitutoyo) as defined in Biju et al. (2011), and include: SVL (snout-vent length), HW (head width), HL (head length), MN (distance from rear of mandible to nostril), MFE (distance from rear of mandible to anterior orbital border), MBE (distance from rear of mandible to posterior orbital border), SL (snout length), EL (eye length), IUE (inter upper eyelid width), UEW (maximum upper eyelid width), IFE (internal front of eyes), IBE (internal back of eyes), FAL (forearm length), HAL (hand length), FDIII (disc width of finger III), FWIII (width of finger III), ShL (shank length), TL (thigh length), FOL (foot length), TFOL (distance from heel to tip of fourth toe), TD (disc width of toe IV), TW (width of toe IV). Methods for morphological descriptions follow Biju et al. (2011) for ease of comparison. Webbing formula was determined following the method provided by Savage & Heyer (1967) with modifications by Myers & Duellman (1982).

 

Statistical analysis

All the measurements showed a positive linear relationship with SVL. Thus to remove the effect of size, morphometric data were normalized by expressing measurements as a ratio of SVL. Apart from the N. kempholeyensis and N. athirappillyensis comparative material (see Appendix 1) we also used data from Biju et al. (2011) and Garg et al. (2017). Discriminant analysis (DA) was performed to understand whether related species of Nyctibatrachus form significantly different clusters (Huberty & Olejnik 2006). Statistical analysis was performed with PAST 3.09 (Hammer et al. 2001).

 

Molecular analysis

Thigh muscle tissue was harvested from 10 specimens (WILD-13-AMP-144, 341, WILD-14-AMP-400, 403, 431, WILD-15-AMP-561, 623, 624, 625 and BNHS 5983). DNA was extracted using QIAamp DNA Mini Kit (QIAGEN, USA) following manufacturer’s protocol. Mitochondrial ND1 gene sequence was amplified using the primer pair NDH-L (5’-AAA CTA TTT AYY AAA GAR CC-3’) and NDH-M (5’-GGG TAT GAN GCT CGN ACT CA-3’) (Roelants & Bossuyt 2005), and 16S rRNA gene was amplified using primer pair 16SF (5’-CGC CTG TTT ATC AAA AAC AT-3’) and 16SR (5’-CCG GTC TGA ACT CAG ATC ACG T-3’) (Palumbi et al. 1991). PCR reaction was performed in a 25μl reaction volume containing 5μl of template DNA (~200ng), 2.5μl of 10X reaction buffer (100 mM Tris pH 9.0, 500 mM KCl, 15 mM MgCl2, 0.1% Gelatin), 2μl of 25 mM MgCl2, 1μl of 10 mM dNTPs, 1μl of each primer, 1μl Taq polymerase and 16.5μl nuclease free water. The thermal profile for both the genes was 10 minutes at 950C, and 35 cycles of 1 minute at 940C, 1 minute at 500C and 2 minutes at 720C, followed by extension of 10 minutes at 720C. Amplified DNA fragments were purified using the Promega Wizard Gel and PCR clean up system and sequenced. The purified PCR products were sequenced using ABI prism 3730 sequencer (Applied Biosystems, USA) and Big dye terminator sequencing kit (ABI Prism, USA). These sequences have been deposited in GenBank (accession numbers are provided in Appendix 2).

Sequences were analyzed for similar sequences in NCBI GenBank database (http://www.ncbi.nlm.nih.gov/) using BLAST (Altschul et al. 1990). Additional ND1 and 16S gene sequences for the genus Nyctibatrachus and the outgroup Lankanectes corrugatus were retrieved from the NCBI GenBank database. GenBank accession numbers for the sequences used in the study are provided in Appendix 2. Gene sequences were aligned separately using MUSCLE (Edgar 2004) and genes were concatenated. Data was partitioned in four parts, one for 16S rRNA gene and three for respective codon positions of ND1 gene to create a full partition, and then the greedy strategy (Lanfear et al. 2012) implemented in IQ Tree (Nguyen et al. 2015) was used to find the right partitioning scheme based on minimum Bayesian Information Criterion (BIC) (Schwarz 1978; Nei & Kumar 2000). Maximum likelihood analysis was performed in IQ Tree (Nguyen et al. 2015) with ultrafast bootstrap support (Minh et al. 2013) for 1000 iterations. The phylogenetic tree was edited in FigTree v1.4.2 (Rambaut 2009). Kimura two parameter (K2P) genetic distances between sequences were determined in MEGA 7 (Kumar et al. 2016).

 

 

 

RESULTS

 

Molecular analysis

Best partition scheme suggested nucleotide substitution model TPM2+I+G4 for 16S rRNA gene and first two codon positions of ND1 gene (BIC = 23697.804) and TIM2+I+G4 for the third codon position of ND1 gene (BIC = 10121.790). Maximum likelihood analysis (Fig. 1) indicates that Nyctibatrachus mewasinghi is genetically distinct from known congeners. Nyctibatrachus mewasinghi formed a monophyletic group with its sister taxa N. athirappillyensis and N. kempholeyensis.

For 16S rRNA gene, K2P distance between N. mewasinghi and N. athirappillyensis was 2.1–2.5 %, while between N. mewasinghi and N. kempholeyensis was 4.0–4.7 %. These distances were more than for other known species, including 2.1% distance between N. shiradi and N. vrijeuni, 1.5% between N. jog and N. petraeus, 1.4% between N. gavi and N. acanthodermis, and 1.2% between N. periya and N. deveni.

For ND1 gene, K2P distance between N. mewasinghi and N. athirappillyensis was 6.8%, while between N. mewasinghi and N. kempholeyensis was 12.2–12.9 %. These distances were more than for other known species, including 4.6% distance between N. shiradi and N. vrijeuni, 4.8% between N. jog and N. petraeus, 8.1% between N. gavi and N. acanthodermis, and 4.0% between N. periya and N. deveni.

The phylogeographic map of the subtree containing only N. mewasinghi, N. athirappillyensis and N. kempholeyensis (Image 1) shows that N. mewasinghi and its sister taxon N. athirappillyensis are separated by the Palghat gap. Although, the latitudinal distribution of N. mewasinghi and N. kempholeyensis overlap, we did not record N. kempholeyensis in the type locality of N. mewasinghi.

 

Morphometric analysis

Nyctibatrachus mewasinghi, N. athirappillyensis and N. kempholeyensis formed distinct clusters in multivariate morphometric space (Fig. 2). Nyctibatrachus mewasinghi differs from N. athirappillyensis and N. kempholeyensis in having relatively larger EL and smaller HL, HW, ShL, FOL, TFOL and SL (Table 1).

 

 

 

 

 

 

 

Taxonomy

Nyctibatrachus mewasinghi sp. nov.

(Images 2, 3a,b)

urn:lsid:zoobank.org:act:CF14F2CB-2107-4DC8-86BD-6C6866D8C334

 

 

Holotype: BNHS 5984, female, 22.1mm SVL, 26.xii.2013, Peruvannamuzhi (11.599 N & 75.819 E, 38m elevation), Malabar Wildlife Sanctuary, Kozhikode District, Kerala, India, coll. K. Krutha & B. Kumar.

Paratypes: 3 ex., WILD-15-AMP-623 (female, 21.4mm SVL), WILD-15-AMP-624 (male, 22.3mm SVL) and WILD-15-AMP-625 (male, 22.1 SVL), 26.xii.2013, collected along with the holotype; 2 ex., WILD-13-AMP-341 (female, 23.0mm SVL) and WILD-14-AMP-403 (male, 21.9mm SVL), 26.xii.2013, same location as holotype, coll. K. Krutha & B. Kumar.

Diagnosis: Nyctibatrachus mewasinghi sp. nov. can be distinguished from all its known congeners by a combination of characters including: small adult size (21.4–23.0 mm SVL); head equal to or slightly wider than long (HW/HL ratio 1.0–1.3); internal back of eyes to eye length ratio 2.0–2.4; less wrinkled dorsal skin with prominent granular projections; absence of dorso-lateral glandular folds separated by an ‘X’ pattern on anterior half of back; a ridge extending from the lip over the tip of the snout to between the nostrils bifurcate posteriorly producing an inverted ‘Y’; finger and toe discs well developed; third finger disc slightly wider than finger width (FDIII/FWIII ratio 1.3–2.5); fourth toe disc almost equal to or slightly wider than toe width (TD/TW ratio 1.0–1.8); third finger and fourth toe discs with dorso-terminal groove, cover rounded distally; presence of two palmar tubercles; webbing small, reaching above the second subarticular tubercle on either side of toe IV; thigh nearly equal to shank (TL/SHL ratio 0.9–1.1); and shank nearly equal to foot length (SHL/FOL ratio 0.9–1.0).

Comparisons: Nyctibatrachus mewasinghi sp. nov. differs from N. anamallaiensis, N. beddomii, N. manalari, N. minimus, N. minor, N. pulivijayani, N. robinmoorei and N. sabarimalai in presence of webbing between toes (vs. absent). Nyctibatrachus mewasinghi differs from congeners N. acanthodermis, N. dattatreyaensis, N. gavi, N. humayuni, N. major, N. petraeus, N. pillaii, N. poocha, N. radcliffei, N. sylvaticus and N. vrijeuni in having dorsal skin less prominently wrinkled (vs. prominently wrinkled). Further, Nyctibatrachus mewasinghi differs in having the third finger with a dorso-terminal groove (vs. without groove in N. acanthodermis, N. dattatreyaensis, N. deccanensis, N. gavi, N. grandis, N. indraneili, N. major, N. radcliffei, N. sanctipalustris, N. vrijeuni and N. webilla), fourth toe disc with a dorso-terminal groove, cover rounded distally (vs. bifurcate distally in N. aliciae, N. anamallaiensis, N. beddomii, N. deveni, N. kumbara, N. manalari, N. minimus, N. minor, N. periyar, N. pillaii, N. poocha, N. pulivijayani, N. robinmoorei, N. sabarimalai and N. vasanthi; or notched distally in N. athirappillyensis and N. shiradi). From species that have finger and toe discs, covered rounded distally, Nyctibatrachus mewasinghi differs in having reduced webbing with webbing formula I1–2-II1–2½III1–2¼IV2¼–1V (vs. extensive webbing with webbing formula I1–1II1–2+III1–2+IV2+–1V in N. danieli; I1–1II1–1¾III1–2+IV2+–1V in N. humayuni; I1–1¼II1–1½III1–2¼IV2¼–1V in N. jog; I1–1II1–1III1–1½IV1½–1V in N. karnatakaensis; I1–1½II1–2III1–2+IV2+–1V in N. petraeus; I1–1¾II1–2+III1–2+IV2+–1V in N. sylvaticus). Nyctibatrachus mewasinghi also has flesh or off-white ventral coloration in life (vs. red, reddish-orange or reddish-brown in N. deccanensis and N. webilla). Nyctibatrachus mewasinghi differs with a genetic distance between 7.8–13.7 % in 16S rRNA gene and 25.0–32.4 % in ND1 gene from all its congeners except N. athirappillyensis and N. kempholeyensis.

Compared to its sister taxa, Nyctibatrachus mewasinghi sp. nov. differs from N. athirappillyensis in having larger eye length to snout length ratio (EL/SL ratio 0.8–0.9 vs. 0.6–0.7), smaller internal back of eyes to eye length ratio (IBE/EL ratio 2.0–2.4 vs. 2.5–2.7), fourth toe with a dorso-terminal groove and cover rounded distally (vs. fourth toe with a dorso-terminal groove and cover notched distally) and webbing formula I1–2-II1–2½III1–2¼IV2¼–1V (vs. extensive webbing with webbing formula I1–1¾II1–2+III1–2+IV2+–1V). The genetic distance between N. mewasinghi and N. athirappillyensis is 2.1–2.5% for 16S rRNA gene and 6.8% for ND1 gene.

Nyctibatrachus mewasinghi sp. nov. differs from N. kempholeyensis in having two palmar tubercles (vs. single), a significantly shorter head (HL/SVL = 31.0 ± 3.2 vs. 37.0 ± 4.5, t = 2.999, df = 21, P = 0.007), significantly narrower head (HW/SVL = 36.0 ± 2.1 vs. 39.5 ± 2.9, t = 2.669, df = 21, P = 0.014) and webbing formula I1–2-II1–2½III1–2¼IV2¼–1V (vs. I1–2−II1–2¼III1–2½IV2½–1V). The genetic distance between N. mewasinghi and N. kempholeyensis is 4.0–4.7% for 16S rRNA gene and 12.2–12.9% for ND1 gene.

Description of the female Holotype BNHS 5984 (all measurements are in mm): Small sized frog (SVL 22.1); head slightly wider than long (HL 7.2; HW 6.4); outline of snout in dorsal and ventral view oval (Image 2a,b), snout (SL 3.2) longer than horizontal diameter of eye (EL 2.6); loreal region obtuse, canthus rostralis indistinct; interorbital area (IUE 3.1) wider than upper eyelid (UEW 1.0); distance between posterior margins of eyes (IBE 6.2) about 2.1 times the distance between anterior margins of eyes (IFE 2.9); supratympanic fold distinct, from posterior corner of upper eyelid to near the shoulder (Image 2c); choanae small, its largest diameter less than half of the largest diameter of vomerine teeth patch (Image 2d).

Forearm (FAL 4.2), smaller than hand (HAL 4.8); fingers with dermal fringes, third finger discs with a dorso-terminal groove, cover rounded distally, third finger disc two times wider than finger (FDIII 0.4, FWIII 0.2); all subarticular tubercles present, distinct, oval; pre-pollex distinct, oval; two palmar tubercles, oval, distinct.

Hind limbs moderately long, shank (ShL 9.7) slightly smaller than thigh (TL 9.9) and larger than foot length (FOL 9.3); distance from heel to tip of toe IV (TFOL 8.6); webbing small, webbing formula I1–2-II1–2½III1–2¼IV2¼–1V; reaching beyond the second subarticular tubercle on either side of toe IV (Image 2f, g); fourth toe disc with dorso-terminal groove, cover rounded distally, fourth toe disc 1.3 times wider than toe (TD 0.5, TW 0.4); subarticular tubercles prominent, oval, single, all present.

Skin of snout and between eyes shagreened, upper eyelids sparsely tuberculated; anterior and posterior parts of back wrinkled (Images 2a, 3a); two large longitudinal folds on the back extending from just behind upper eyelid towards dorsum; a ridge extending from the lip over the tip of the snout to between the nostrils bifurcate posteriorly producing an inverted ‘Y’; a glandular fold between the eyes; dorsal parts of forelimb, thigh, and shank have longitudinal folds; ventral side smooth; glandular projections on margin of lower jaws and anterior margin of thighs.

Coloration of holotype: In life (Image 3a,b), dorsum light grayish-brown, lateral side lighter than dorsum; a dark stripe between the eyes connecting upper eyelids mid-dorsally, demarcating a triangular light brown patch on dorsal side of the snout; a pair of light reddish-brown longitudinal dorsal bands extend from the back of the eyelids to the middle of dorsum; glandular folds brown; limbs dorsally light brown; fore limbs, hind limbs, fingers and toes with dark brown cross-bands; ventral side off-white with very few and scattered grayish-brown spots on throat and ventral side of fore limb and hind limb; belly plain; upper and lower jaw margin grayish with white glandular projections on lower jaw margin; palm and feet grayish-brown; fingers, toes and webbing dark gray. In preservation (Image 2), coloration as per life just more faded, longitudinal dorsal bands cream to light brown color.

Variation: Morphometric variation in five paratypes is provided in Table 2. Males have a prominent femoral gland (Image 3e).

Distribution and habitat: Nyctibatrachus mewasinghi sp. nov. is known only from its type locality Peruvannamuzhi in Malabar Wildlife Sanctuary, Western Ghats of Kerala, north of Palghat Gap (Image 4). The species was seen in a stream with riparian cover (Image 5) running alongside a wall after the toe drain of the Peruvannamuzhi Dam. Males were observed calling on top of rocks and leaves around 19:00hr.

Etymology: The species is named after Dr. Mewa Singh, Distinguished Professor (for Life), University of Mysore; J.C. Bose Fellow, Institution of Excellence, and Honorary Professor of Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru. This species is in honour of his extraordinary contributions in behavioral ecology and primatology, and his immense contribution to the conservation of Indian primates.

Suggested common name: Mewa Singh’s Night Frog.

 

 

DISCUSSION

 

 

Nyctibatrachus mewasinghi sp. nov. and N. athirappillyensis are sister taxa within a monophyletic clade that includes N. kempholeyensis. Genetic divergence of N. mewasinghi from N. athirappillyensis can be attributed to their separation across the Palghat gap, as observed for Nyctibatrachus species earlier (see Van Bocxlaer et al. 2012) and other related taxa such as Indirana (see Dahanukar et al. 2016). The genetic divergence of N. mewasinghi from N. kempholeyensis, however, raises new questions on the biogeography. While N. kempholeyensis is widely distributed from 11.5–14.30N (Image 4) with little genetic divergence (Fig. 1; Image 1), the type locality of N. mewasinghi, which falls within this latitudinal range has only a single species with high genetic divergence from N. kempholeyensis. The genetic divergence between N. mewasinghi and N. athirappillyensis and between N. mewasinghi and N. kempholeyensis is much above the expected barcode gap as evidenced from the fact that for both the genes there are several other species pairs with much lower genetic divergence (see molecular analysis above).

Nyctibatrachus athirappillyensis has been recorded from a new location in Idukki District, while N. kempholeyensis is recorded from four additional locations (see Image 2 and Appendix 1).

Family Nyctibatrachidae with its two extant genera (Frost et al. 2006), namely the monotypic genus Lankanectes endemic to Sri Lanka (Dubois & Ohler 2001) and the genus Nyctibatrachus endemic to the Western Ghats of India (Biju et al. 2011), is an ancient frog lineage that emerged about 85–61mya (Roelants et al. 2004). In an important contribution, Biju et al. (2011) revised the genus, clarified taxonomy of several known species, described 12 new species based on new genetic and morphological information and provided novel behavioral observations. This was followed by discovery of a new mud packing behavior and description of a new species by Gururaja et al. (2014) and seven new species of small sized night frogs by Garg et al. (2017). All these papers elucidated our limitations in the knowledge of amphibian diversity in Western Ghats biodiversity hotspot. Description of a new species in the current communication further bolsters this assertion and raises possibilities of discovering more species from this region.

 

 

 

 

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