Range extension of Ferguson’s Toad Duttaphrynus scaber (Schneider) (Amphibia: Anura: Bufonidae) up to the northern most limit of Western Ghats, with its advertisement call analysis

1 Department of Zoology, Abasahab Garware College, Karve Road, Pune, Maharashtra 411004, India 2 Department of Biodiversity, Abasahab Garware College, Karve Road, Pune, Maharashtra 411004, India 3 Ela Foundation, C-9, Bhosale Park, Sahakarnagar-2, Pune, Maharashtra 411009, India 4,6 National Center for Cell Sciences (NCCS), Ganeshkhind, Pune, Maharashtra 411007, India 5 Indian Institute of Science Education and Research (IISER), Sai Trinity, Sus Road, Pashan, Pune, Maharahtra 411021, India and Zoo Outreach Organization, 96 Kumudham Nagar, Vilankurichi Road, Coimbatore, Tamil Nadu 641035, India 1 adpadhye@gmail.com (corresponding author), 2 rohanpandit87@gmail.com, 3 rajnpatil@gmail.com, 4 swapy28@gmail.com, 5 n.dahanukar@iiserpune.ac.in, 6 yogesh@nccs.res.in


Western Ghats Special Series
Duttaphrynus scaber (type locality 'Orientali India') was described by Schneider (1799). Boulenger (1892) described Bufo fergusonii (type locality 'on the Cavalry Parade ground' Thiruvananthapuram, Kerala) as a different species. However, after examining the lectotypes of D. scaber and comparing them with the holotype of B. fergusonii, Dubois & Ohler (1999) found these two species to be conspecific and hence they considered B. fergusonii as a junior subjective synonym of D. scaber.
To date, Duttaphrynus scaber has not been reported from Maharashtra and Gujarat states, despite a number of amphibian surveys having been carried out (Padhye & Ghate 2002 and references therein;Naik & Vinod 1992, 1993aBhatta et al. 1999;Vyas 2000aVyas ,b, 2002Vyas , 2004aVyas ,b, 2007Vyas , 2008Dinesh et al. 2009). There is a record of Bufo fergusonii (now Duttaphrynus scaber) in the list of amphibians from Shoolpaneshwar Wildlife Sanctuary, Gujarat (Sabnis & Amin 1992) but Vyas (2008; has raised the question about its validity. According to Vyas (2008), Naik & Vinod (1992, 1993a never claimed any records of the species from Shoolpaneshwar Wildlife Sanctuary, which shows that the record of this toad species has been mistaken.
Therefore, we report this species from both the states with molecular evidence for the first time and describe its advertisement call.

Materials and Methods
We carried out amphibian surveys of Surgana Taluka in Nasik District, Maharashtra State and Ahwa Taluka, in Dang District, Gujarat State to assess the amphibian diversity of the northern most regions of the Western Ghats. Surveys were conducted from 1830-0230 hr on the 17 and 18 of July 2010. Specimens were identified in the field down to the species level and most were released. The presence of species was also reported on the basis of calls. Two specimens of Duttaphrynus scaber were collected from a road side puddle and brought back to the laboratory for confirmation. One of the two specimens was preserved in absolute ethanol and the other was preserved in 10% formalin and then transferred to 70% ethanol. These specimens were deposited at the museum of Zoology Research Laboratory, Abasaheb Garware College, Pune, under the accession numbers AGCZRL Amphibia 41 and AGCZRL Amphibia 42, respectively. Morphometry was performed using digital Vernier caliper (Areospace® China, least count 0.01mm).
Advertisement calls of two frogs were recorded with a mobile phone in AMR (Adaptive Multi-Rate) format at 8kHz sampling rate. Because the calls recorded on the mobile phone have limited sampling frequency we provide a very preliminary account of the advertisement call. Nevertheless, our analysis is important since there is no available information on the calls of this species. Calls were recorded at a distance of approximately 0.5m from the calling frogs. The toads were sitting on the sides of a temporary rain-water pool. The atmospheric temperature was 21.2 0 C. Calls were analysed with Raven Pro 1.4 software (Bioacoustics Research Program 2003) and Praat sound analysis program version 5.2 (Boersma & Weenink 2009). Audio spectrograms were calculated with fast-Fourier transform (FFT) of 256 points, 50% overlap and 31.3 Hz grid-spacing, using Hanning windows. We tested the hypothesis that all different call parameters were same for both the individuals by performing unpaired t test.

Results and Discussion
During our field surveys we observed a total of three D. scaber. A single male D. scaber (not collected) was observed on 17 July 2010 on the banks of Ahwa Lake (20 0 45'9"N & 73 0 40'39"E, elevation 455m), Ahwa, District Dang, Gujarat at 2340hr. This male was calling in the open on the edge of the reservoir (Image 1). The two collected males were found calling in roadside puddles, which were flanked by paddy fields (20 0 37'18"N & 73 0 38'56"E, elevation 356m). These males were collected on 18 July 2010 at Supdahad, Ahwa Taluka, Dang District, Gujarat, at 0050hr. There was sporadic rain on the same day. Further, calling individuals of this species were observed near the Village Satkhamb, Taluka Surgana, District Nasik, Maharashtra (20 0 35'31"N & 73 0 34'47"E, elevation 385m). Judging from the number of calling males heard, the species appears to be locally abundant. The overall habitat was road surrounded by paddy fields and fragmented patches of deciduous forest (Image 2). Our new records of D. scaber represent a substantial northwestern range extension to the northernmost limits of the Western Ghats of India (Fig. 1).
Duttaphrynus scaber individuals were identified by the presence of distinct parietal ridges on head, numerous spiny warts on the entire body, a fawn coloured underside, the 1 st and 2 nd fingers equal in length, toes scarcely webbed, parotid glands rounded and tympanum less than half the diameter of eye (Daniel 1963;Dubois & Ohler 1999;Daniels 2005; Mathews & Sen 2010) (Image 3a-c). The specimens were also compared with a previously collected museum specimen from Thrissur, Kerala (Voucher No. AGCZRL Amphibia 98), which was identified by Dr. Annemarie Ohler (Museum National d'Histoire Naturelle, Paris). Morphometrics of the two specimens collected during the current survey and the specimen from Thrissur are given in Table 1.
Further identification of the specimens was confirmed by comparing the 12S and 16S mitochondrial rDNA partial sequences with the available Gene Bank sequence of Duttaphrynus scaber and other allied species. Percent divergence in partial sequences of 12S and 16S genes between the freshly collected specimens of D. scaber and GenBank sequences was 0.4%. As compared to other Duttaphrynus species considered in the study, D. scaber in our collection showed a minimum divergence of 5.3% and maximum of 8%. Modeltest performed in MEGA 5 revealed that the nucleotide substitution rates could be best described by General Time Reversal model with Gamma Distribution (AIC = 5247.40, lnL = -2595.61). Phylogenetic analysis based on maximum likelihood  method suggests further conspecificity with a known Duttaphrynus scaber specimen from Western Ghats for which the sequences are available in GenBank (Fig. 2).
The advertisement call of Duttaphrynus scaber is a sustained call that is mildly harsh and scratchy in tone (Audio 1). It has a regular beat pattern giving the feel of a small saw quickly going back and forth with a brief interlude at each end. The call file is provided as supplementary data. The frequency of the call is spread over a band mostly in the region of 2.5-4.5 kHz (Fig. 3a). The dominant frequency was 3.6 kHz (Table 2, Fig. 3c). Though the sampling freq was 8 kHz the software used for analysis could render the upper bound unto 4 kHz that is evident from the fig. 6a. Due to the limitations of the sampling frequency the energy levels were cut off at 4 kHz however it can be predicted that the frequency range could reach 4.5 kHz which is evident from the Fig.  3a. Each note thus had an average duration of 155.8 (sd = 24.50) ms, the note interval was about 85.6 (sd = 10.60) ms and number of notes per minute was 251 (Table 2). The waveform (Fig. 3b) shows the beat pattern of the call and the rapid amplitude modulation within each note highlights the scratchy sound each note makes. Comparison of the two calls from two different males revealed that there was no significant difference in higher frequency (t = -0.562, df = 40, P = 0.577), dominant frequency (t = -1.959, df = 40, P = 0.057) and call duration (t = 1.295, df = 40, P = 0.203) of the two calls but there was significant difference in the lower frequency (t = -3.683, df = 40, P = 0.001) and inter-note duration (t = 2.495, df = 38, P = 0.017). This indicates that  except for the lower frequency and inter-note duration the call pattern for two individuals was not significantly different. Even though the recorded calls had limitations in terms of sampling frequency, the preliminary analysis provided here is important as, to our knowledge, there is no other information about the calls of this species. Our report of Duttaophrynus scaber based on both morphological and genetic data extends the geographical distribution of the species up to the northern most limit of the Western Ghats of India. From the previous reports, this species was known from southern Western Ghats up to Karnataka, Eastern Ghats up to Odisha and Manipur from northeastern India. However its presence in northern Western Ghats indicates that the species may be distributed throughout most of peninsular India. Shoolpaneshwar Wildlife Sanctuary, the place from where Sabnis & Amin (1992) reported the species, is far away from the location indicated by the current locality records. Further, the specimens collected by them (if any) should be confirmed by DNA analysis. This emphasizes the need for detailed surveys for the presence of this species in Satpura ranges as well as plains of Maharashtra State.