Journal of Threatened Taxa | www.threatenedtaxa.org | 26 July 2013 | 5(11): 4579–4585 Western Ghats Special Series ISSN Range extension of Ferguson’s Toad Duttaphrynus scaber Online 0974–7907 Communication Short (Schneider) (Amphibia: Anura: Bufonidae) up to the northern Print 0974–7893 most limit of Western Ghats, with its advertisement call OPEN ACCESS analysis Anand Padhye 1, Rohan Pandit 2, Rajgopal Patil 3, Swapnil Gaikwad 4, Neelesh Dahanukar 5 & Yogesh Shouche 6 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 [email protected] (corresponding author), 2 [email protected], 3 [email protected], 4 [email protected], 5 [email protected], 6 [email protected] Abstract: Duttaphrynus scaber (Schneider) is known to occur in the Duttaphrynus scaber (type locality ‘Orientali southern Western Ghats, Eastern Ghats and northeastern India. However, there is no report of its occurrence from the northern India’) was described by Schneider (1799). Boulenger Western Ghats from the states of Maharashtra and Gujarat. Here we (1892) described Bufo fergusonii (type locality ‘on the report the occurrence of this species from the northernmost limit of Cavalry Parade ground’ Thiruvananthapuram, Kerala) the Western Ghats, a substantial range extension (approx. 550km) from the nearest known locality. We confirm our identification as a different species. However, after examining the on the basis of morphology by comparing our specimens with a lectotypes of D. scaber and comparing them with the previously collected Duttaphrynus scaber specimen from Thrissur holotype of B. fergusonii, Dubois & Ohler (1999) found in Kerala State as well as on the molecular basis. Analysis based on the advertisement call of Duttaphrynus scaber, albeit preliminary, is these two species to be conspecific and hence they provided for the first time for this species. considered B. fergusonii as a junior subjective synonym of D. scaber. Keywords: Advertisement call analysis, Bufo fergusonii, Duttaphrynus scaber, range extension. Duttaphrynus scaber has been reported in India from DOi: http://dx.doi.org/10.11609/JoTT.o3345.4579-85 | ZooBank: urn:lsid:zoobank.org:pub:14242B29-CB85-461F-A8E4-81C59C225BE8 Editor: Anonymity requested. Date of publication: 26 July 2013 (online & print) Manuscript details: Ms # o3345 | Received 13 September 2012 | Final received 24 June 2013 | Finally accepted 29 June 2013 Citation: Padhye, A., R. Pandit, R. Patil, S. Gaikwad, N. Dahanukar & Y. Shouche (2013). 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.Journal of Threatened Taxa 5(11): 4579–4585; http://dx.doi.org/10.11609/JoTT.o3345.4579-85 Copyright: © Padhye et al. 2013. Creative Commons Attribution 3.0 Unported License. JoTT allows unrestricted use of this article in any medium, reproduction and distribution by providing adequate credit to the authors and the source of publication. Funding: None. Competing interest: None. Acknowledgements: We thank authorities of NCCS for providing the facility for molecular work. We also thank Principal Abasaheb Garware College as well as head of the Zoology Department for the infrastructural facilities. Thanks to Ankur Padhye, Makarand Bhagwat, Vinayak Khade, Vitthal Bhoye and driver Anil Pujari who helped us in the field surveys. Special thanks to Dr. H.V. Ghate for his valuable suggestions during the work and preparation of manuscript. The publication of this article is supported by the Critical Ecosystem Partnership Fund (CEPF), a joint initiative of l’Agence Française de Développement, Conservation International, the European Commission, the Global Environment Facility, the Government of Japan, the MacArthur Foundation and the World Bank. 4579 Range extension of Bufo scaber Padhye et al. Thriuvananthapuram in Kerala (Daniels 2005); Mysore count 0.01mm). (Daniel 1963), Lakkavalli State Forest (Krishnamurthy Thigh muscles of AGCZRL Amphibia 41 were used 1999) and Dharwad (Dutta 1997) in Karnataka; Chennai for the molecular analysis. The tissue was digested at in Tamil Nadu (Rao 1915; Ravichandran 1996; Daniels 500C for two hours using the extraction buffer (0.1M 2005); Banjara Hills, Hyderabad (Donahue & Daniel NaCl, 0.05M Tris-HCl, 0.01M EDTA, 1%SDS) with 15µl 1966) in Andhra Pradesh and Sambalpur District (Dutta Proteinase K (20mg/ml). DNA was then extracted 1988) in Odisha. Recently the species has been recorded using the conventional phenol:chloroform method from the northeastern state of Manipur by Mathew & (Sambrook et al. 1989). The partial fragment of each Sen (2009, 2010). Outside of India, the species has also of the mitochondrial ribosomal RNA genes 12S (~400 been reported from Sri Lanka (Dutta & Manamendra- bp) and 16S (~520 bp) were amplified using PCR. The Arachchi 1996). Largely because of its wide distribution, 12S segment of mitochondrial DNA was amplified using IUCN Red List status of this species is Least Concern universal primers L1091 (5’-AAACTGGGATTAGATACCCCA (Dutta & Manamendra-Arachchi 2004). CTA-3’) and H1478 (5’-GAGGGTGACGGGCGGTGTGT-3’) as To date, Duttaphrynus scaber has not been reported described by Kocher et al. (1989). While for 16S rDNA we from Maharashtra and Gujarat states, despite a number used primers 16SA (5’-CGCCTGTTTATCAAAAACAT-3’) and of amphibian surveys having been carried out (Padhye & 16SB (5’-CCGGTCTGAACTCAGATCACGT -3’) as described Ghate 2002 and references therein; Naik & Vinod 1992, by Simon et al. (1994). PCR products were purified 1993a,b; Bhatta et al. 1999; Vyas 2000a,b, 2002, 2004a,b, by using PEG-NaCl protocol (Sambrook et al., 1989). 2007, 2008, 2012; Dinesh et al. 2009). There is a record Products were sequenced on ABI 3730 DNA analyzer of Bufo fergusonii (now Duttaphrynus scaber) in the list (Applied Biosystems, Hitachi), as per the manufacturer’s of amphibians from Shoolpaneshwar Wildlife Sanctuary, instructions. Gujarat (Sabnis & Amin 1992) but Vyas (2008; 2012) has Sequences were manually checked for any ambiguity raised the question about its validity. According to Vyas using BioEdit (Hall 1999) and were deposited in GenBank (2008), Naik & Vinod (1992, 1993a,b) never claimed any (http://www.ncbi.nlm.nih.gov/) under the accession records of the species from Shoolpaneshwar Wildlife number JQ898085 (12S rDNA) and JQ898086 (16S Sanctuary, which shows that the record of this toad rDNA). Additional sequences of 12S and 16S rDNA for species has been mistaken. comparison were downloaded from GenBank. We Therefore, we report this species from both the states retrieved 12S and 16S rDNA sequence for Duttaphrynus with molecular evidence for the first time and describe scaber (FJ882785), Duttaphrynus melanostictus its advertisement call. (DQ283333), Duttaphrynus stomaticus (FJ882787), Duttaphrynus parietalis (FJ882784), Duttaphrynus Materials and Methods hololius (FJ882781), Duttaphrynus brevirostris We carried out amphibian surveys of Surgana Taluka (FJ882786), Pedostibes tuberculosus (FJ882793), in Nasik District, Maharashtra State and Ahwa Taluka, Xanthophryne koynayensis (FJ882782) and Ansonia in Dang District, Gujarat State to assess the amphibian ornata (FJ882797) for comparision. Microhyla ornata diversity of the northern most regions of the Western from family Microhylidae was used as an out-group and Ghats. Surveys were conducted from 1830-0230 hr on 12S (AB201176) and 16S (AY948728) sequence of the the 17 and 18 of July 2010. Specimens were identified same were retrieved from GenBank. Sequences were in the field down to the species level and most were aligned independently for each gene using MUSCLE released. The presence of species was also reported (Edgar 2004). The individual gene sequences were on the basis of calls. Two specimens of Duttaphrynus trimmed using freeware DAMBE 5.2.77 (Xia & Xie 2001). scaber were collected from a road side puddle and Both the gene sequences were concatenated before brought back to the laboratory for confirmation. One performing the phylogenetic analysis. Phylogenetic of the two specimens was preserved in absolute and molecular evolutionary analyses were conducted ethanol and the other was preserved in 10% formalin using MEGA5 (Tamura et al. 2011). Best fit model for and then transferred to 70% ethanol. These specimens nucleotide substitution was selected based on minimum were deposited at the museum of Zoology Research Akaike Information Criterion (AIC) value (Posada & Laboratory, Abasaheb Garware College, Pune, under the Crandall 2001). We constructed phylogenetic trees accession numbers AGCZRL Amphibia 41 and AGCZRL based on maximum parsimony and maximum likelihood. Amphibia 42, respectively. Morphometry was performed Reliability of the phylogenetic tree was estimated using using digital Vernier caliper (Areospace® China, least bootstrap values run for 1000 iterations. 4580 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 July 2013 | 5(11): 4579–4585