Nyctibatrachus kumbara sp. nov. (Table 2S, Figures 5, 6, 8–9, 10 c.) Holotype. ZSI/ WGFRS /V/A/860, adult male collected on 09-06-2006 by KVG and SA along a flowing rivulet of Sharavathi river basin (14.27323°N; 74.74756°E, 590m amsl) at Kathalekan, Uttara Kannada District, Karnataka, India. Paratypes. ZSI/ WGFRS /V/A/861 and ZSI/ WGFRS /V/A/863, adult females collected on 03-10-2006 by KVG and SA along a flowing rivulet of Sharavathi river basin (14.27499°N; 74.73695°E, 583m amsl) at Malemane, Uttara Kannada District, Karnataka, India; ZSI/ WGFRS /V/A/862 and ZSI/ WGFRS /V/A/864, adult males collected on 09-06-2011 by KVG and KPD along a flowing rivulet of Sharavathi river basin (14.27323°N; 74.74756°E, 590m amsl) at Kathalekan, Uttara Kannada District, Karnataka, India. Diagnosis. This species is assignable to the genus Nyctibatrachus because of its semi aquatic to aquatic habitat, medium to large size, pupil rhomboidal, glandular wrinkled skin, presence of vomerine teeth, notched tongue, finger and toe discs with disc, absence of webbing on fingers and presence of webbing on toes and presence of sub ocular gland (Biju et al. 2011). Nyctibatrachus kumbara sp. nov. can be easily distinguished from all other species in the genus by the following combination of characters: (1) adult male size medium to large (SVL 46.5± 0.74 mm); (2) head wider than long (HW 18.7–20.9 mm; HL 14.8–15.7 mm); (3) dorsum glandular without any spiny projections in the anterior half, glandular corrugations irregular without specific pattern; in males, throat and chest finely dotted with glandular folds rest of the region smooth, belly white; (4) webbing on toes medium (I ½- 1 II ½- 1 III 0- 2 IV 2-0 V); (5) nuptial pad and femoral glands prominent in adult males; (6) dorsal body color dark brown, ventrally buff colored except belly; (7) finger disc weakly developed (fd3 0.9± 0.24 mm; fw3 0.8± 0.19 mm); (8) toe disc moderately developed (td4 1.7± 0.32 mm; tw4 0.8± 0.05 mm); (9) third finger disc with dorso-terminal groove, cover notched distally, fourth toe disc with dorso-terminal groove cover bifurcate distally; (10) hand-stand oviposition, fertilized egg clutch covered with mud pack; (11) genetically belongs to N. sanctipalustris clade including N. dattatreyaensis (3.41% divergent on mitochondrial 16S rRNA), N. vrijeuni (4.02%), N. shiradi (4.02%), N. karnatakaensis (4.62%) and N. sanctipalustris (4.62%). Description of the holotype. ZSI/WGRC/V/A/860, adult male (Figure 9), terminology follows Biju et al. (2011) and Dinesh et al. (2008a). Morphometric data are given in Table 2S. A medium-sized species of Nyctibatrachus (SVL 46.2 mm); habitus compact and squat; head wider (HW 18.7 mm) than long (HL 15 mm; MN 11.9 mm; MFE 9.6 mm; MBE 3.4 mm), rounded in anterior view; snout rounded (SL 7.1 mm), marginally protruding, its length longer than horizontal diameter of eye (EL 5.7 mm); canthus rostralis rounded; loreal region concave; interorbital space flat (IUE 6.3 mm) and double the upper eye lid width (UEW 3.1 mm); internarial distance (IN 4.3 mm) less than interorbital distance (IUE 6.3 mm); distance between back of eyes (IBE 14.5 mm) is greater than double the distance between front of eyes (IFE 6.9 mm); nostrils oval, without a flap of skin, and is closer to eye (EN 2.9 mm) than snout tip (NS 3.5 mm); eyes large (EL/HL = 0.38%) protruding on the sides of head, its diameter more than eye to nostril distance (EL/EN=1.96); pupil rhomboidal; tympanum indistinct; subocular gland distinct (Figure 9 C); pineal ocellus absent; vomerine ridge present posterior to choanae, oval bearing three on left and six sharp spinose teeth on right ridge; symphysial knob ‘W’ shaped, moderately developed; tongue moderate, oval slightly emarginated, median lingual process absent; parotoid glands, cephalic ridges and co-ossified skin absent. Forelimbs (FLL 10.9 mm) strong and sub-equal to hand length (HAL 10.1 mm); third finger thin, long, rounded (TFL 5.3mm); thin dermal fringe on finger III; webbing absent (Figure 9 D); relative length of fingers, shortest to longest: I10 and 30cm from water=3) and position of male and female at the time of oviposition (upright=1, handstand=2) among the overlapping and non-overlapping groups (stress value = 0.11). There is a clear clustering of allopatric species of Nyctibatrachus to sympatric species (Figure 12). For the co-occurring congeneric (COCG) species call characteristic features were subjected to statistical analysis. Overall call characteristic features (duration and peak frequency) between all COCG species were significantly different (ANCOVA, F=669.1, P0.5), remaining COCG species exhibited significant difference (Table 6). Amplexus in N. jog is loose amplexus, while in N. kempholeyensis (KVG personal observation) and in N. kumbara it is axillary. Oviposition site for N. jog is wet rock surfaces, overhanging leaves and tree bark above 15 cm to ~ 200 cm from the bottom of stream, but it was only 4−10 cm for N. kumbara and within 30 cm for N. kempholeyensis. Egg clutch size significantly differed among COCG species (Table 5). Most significant reproductive character displacement is in terms of parental care. Nyctibatrachus kumbara covers eggs with mud, while N. jog and N. kempholeyensis do not cover the eggs, but attend the egg clutches. In terms of breeding period, again there is an overlap between N. kumbara and N. kempholeyensis, but they differ from N. jog which breeds only during monsoon (KVG personal observation). Anurans exhibit diverse reproductive strategies to overcome the limitation posed by external or internal fertilisation (Beck 1998), selective pressure of predators on aquatic eggs and tadpoles (Prado et al. 2005), prevailing environmental conditions both in space and time; and as a part of evolutionary process (Jameson 1955; Duellman & Trueb 1994; Wells 2007; Pfenning & Stewart 2010). Also r- and K-selection (MacArthur & Wilson 1967) influence the reproductive strategies in anurans (Nichols et al. 1976). However, according to Stearns (1992) the concept of r- and K-selection is less meaningful due to varied range in life-history strategies seen in nature and does not make precise predictions. In the present study, we compared three co-occurring species of Nyctibatrachus, namely N. jog, N. kempholeyensis and N. kumbara and three allopatric species, namely N. humayuni, N. petraeus and N. jog (Figure 10). Nyctibatrachus jog is alloptric with the N. humayuni clade, however it is sympatric with N. kempholeyensis and N. kumbara. Significance of courtship. Mate identification through tactile cues is an important non-vocal signal in anurans (Duellman & Trueb 1994). Male individuals of N. kumbara called from a fixed site and amplected with an approaching female, which is similar to the species of Bombina, Discoglossus, Pelobates, Scaphiopus, Bufo and Rana (Wells 2007). In N. kumbara the courtship behaviour exhibited by males and females by touching each other is perhaps to recognise the potential mate and to gauge the size of both individuals. However, all this remain very speculative and calls for further studies to ascertain the behaviour. Significance of mud pack on egg clutches. Parental care in amphibians is the energy investment into offspring after fertilisation (for details refer to Wells 2007). Egg attendance by male individuals has been attributed to protection of eggs from being damaged by intruding males, and deterring egg predation and preventing desiccation (in Hyalinobatrachium valerioi (Vockenhuber et al. 2009)). Parental care involving both male and female, only females and only males are well known (Wells 2007). So far, there is no report of any other anuran species covering egg clutch with mud. Mud packing on egg clutches by N. kumbara might be attributed to, 1. minimising clutch dehydration as the outer capsule of terrestrial amphibian eggs are susceptible to dehydration by losing moisture to drier air (Duellman & Trueb 1994), 2. camouflage eggs as the eggs are very prominently pigmented to avoid predators (Crump 1995; Wells 2007). Reproductive Character Displacement. Observed reproductive characters in N. jog, N. humayuni and N. petraeus are similar with loose to abbreviated amplexus (Kunte 2004; Narahari et al. 2011; Biju et al. 2011, detailed in Table 4). They belong to the N. humayuni clade from the Northern Western Ghats (Bocxlaer et al. 2012 and Figure 7). The minimal aerial distance between the known populations of this clade are N. jog-N. petraeus is 97 km; N. jog-N. humayuni is 331 km and N. petraeus-N. humayuni is 164 km (distance calculated from the nearest verified population of the respective species using Google Earth). However, information on reproductive characters from other clades of Nyctibatrachus from the Western Ghats is lacking. Despite such limitation, on comparing reproductive characters between spatially non-overlapping allopatric to overlapping sympatric clades, we found differences in reproductive characters among the spatially overlapping taxa and reproductive character similarities in spatially non-overlapping taxa in a suite of character, which could be an indication for reproductive character displacement. Significance of new species. Taxonomy for the genus Nyctibatrachus has been stabilised in the recent past (Das & Kunte 2005; Dinesh et al. 2007; Dinesh et al. 2008b) and a complete revision with the description of twelve new species has been recently made by Biju et al. (2011) providing baseline for comparisons across taxa. The discovery of N. kumbara reiterates that there is need for systematic sampling across the Western Ghats for providing a comprehensive listing of amphibian species in the region. For delimiting species ranges and to appreciate mountain associated clade endemism in Nyctibatrachus, sampling has to be very systematic covering altitudinal, latitudinal and longitudinal gradients. Although N. kumbara sp. nov. is distinct from known species of Nyctibatrachus in morphology, acoustics, breeding behavior and single gene genetic study, we still warrant multiple gene phylogenetic studies on N. kumbara to substantiate its specific phylogenetic relationship with the other members of Nyctibatrachus. Apart from taxonomy and phylogeny, we stress the importance of studies on breeding behaviour, ecology and parental care in amphibians of the Western Ghats, which would provide a holistic and inclusive approach to understand the evolutionary significance of anurans in the Western Ghats.

Published as part of Gururaja, Kotambylu Vasudeva, Dinesh, K. P., Priti, H. & Ravikanth, G., 2014, Mud-packing frog: A novel breeding behaviour and parental care in a stream dwelling new species of Nyctibatrachus (Amphibia, Anura, Nyctibatrachidae), pp. 33-61 in Zootaxa 3796 (1) on pages 41-52, DOI: 10.11646/zootaxa.3796.1.2, http://zenodo.org/record/286069