Short Communication |
Corresponding author: Madhu Chetri ( manaslu.conservation@gmail.com ) Academic editor: Kristofer M. Helgen
© 2016 Madhu Chetri, Yadvendradev Jhala, Shant Raj Jnawali, Naresh Subedi, Maheshwar Dhakal, Bibek Yumnam.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Chetri M, Jhala YV, Jnawali SR, Subedi N, Dhakal M, Yumnam B (2016) Ancient Himalayan wolf (Canis lupus chanco) lineage in Upper Mustang of the Annapurna Conservation Area, Nepal. ZooKeys 582: 143-156. https://doi.org/10.3897/zookeys.582.5966
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The taxonomic status of the wolf (Canis lupus) in Nepal’s Trans-Himalaya is poorly understood. Recent genetic studies have revealed the existence of three lineages of wolves in the Indian sub-continent. Of these, the Himalayan wolf, Canis lupus chanco, has been reported to be the most ancient lineage historically distributed within the Nepal Himalaya. These wolves residing in the Trans-Himalayan region have been suggested to be smaller and very different from the European wolf. During October 2011, six fecal samples suspected to have originated from wolves were collected from Upper Mustang in the Annapurna Conservation Area of Nepal. DNA extraction and amplification of the mitochondrial (mt) control region (CR) locus yielded sequences from five out of six samples. One sample matched domestic dog sequences in GenBank, while the remaining four samples were aligned within the monophyletic and ancient Himalayan wolf clade. These four sequences which matched each other, were new and represented a novel Himalayan wolf haplotype. This result confirms that the endangered ancient Himalayan wolf is extant in Nepal. Detailed genomic study covering Nepal’s entire Himalayan landscape is recommended in order to understand their distribution, taxonomy and, genetic relatedness with other wolves potentially sharing the same landscape.
Himalayan wolf, wolf-dog clade, Canis lupus chanco , Trans-Himalaya, Annapurna Conservation Area, Nepal
The presence of wolf (Canis lupus) in the Trans-Himalayan regions of Nepal has been reported for centuries (
During a recent survey in the Trans-Himalayan region of Upper Mustang, Annapurna Conservation Area, Nepal, wolves were encountered several times and their physical features were observed carefully (Figure
In the present paper, we describe for the first time the extant mitochondrial lineage of wolves that inhabit the Trans-Himalayan region in Upper Mustang of Annapurna Conservation Area, Nepal, based on DNA extracted from fecal samples collected in the wild. We identified a novel mtDNA CR haplotype that clustered within the monophyletic Himalayan wolf clade of C. l. chanco.
During October 2011, six fecal samples suspected to have originated from wolves were collected from Upper Mustang in the Annapurna Conservation Area of Nepal at an elevation ranging from 4,750 to 5,050 m asl (Figure
MtDNA CR sequences were aligned and edited using Bioedit 5.0 (
We successfully obtained mtDNA control region sequences (~ 220 bp) from five (Table
Phylogenetic trees constructed using 229 bp of aligned CR sequence data. The values at nodes correspond to A bootstrap support > 50% in maximum likelihood (ML) and maximum parsimony (MP) analyses; and B Bayesian posterior probability > 0.50. Scat samples sequenced in this study are highlighted in bold. Four samples (D2137, D2138, D2139 and D2143) represented a novel haplotype HWF within the Himalayan wolf clade, while a fifth sample (D2140) matched with existing domestic dog haplotypes.
Specimen ID, location and area name with GenBank account number of the sequenced samples.
Specimen ID | Species | Clade | Locality (datum WGS84); Altitude (m) | Area name | Haplotype | GenBank Acc # | Date | Collector |
---|---|---|---|---|---|---|---|---|
D2137 | Canis lupus chanco | Himalayan wolf | N29°12'14.3", E084°8'24.8"; 5020 | Yarsa | HW-F | KT321360 | 11.10.2011 | Madhu Chetri |
D2138 | Canis lupus chanco | Himalayan wolf | N29°12'58.4", E084°6'32.1"; 4740 | Yarsa | HW-F | KT321360 | 11.10.2011 | Madhu Chetri |
D2139 | Canis lupus chanco | Himalayan wolf | N29°10'24.2", E084°8'3.5"; 5050 | Dharkeko pass | HW-F | KT321360 | 12.10.2011 | Madhu Chetri |
D2143 | Canis lupus chanco | Himalayan wolf | N29°10'24.2", E084°8'3.5"; 5050 | Dharkeko pass | HW-F | KT321360 | 12.10.2011 | Madhu Chetri |
D2140 | Canis familiaris | Domestic dog | N29°13'25.5", E084°6'10.6"; 4740 | Dhalung | ID-H | KT321361 | 14.10.2011 | Madhu Chetri |
The sequences of these four scat samples which clustered within the monophyletic Himalayan wolf clade are new and not identical to haplotypes identified previously in GenBank. We therefore designated this novel haplotype HWF, in line with the five existing HW (A to E) haplotypes identified previously by
The sequence of a fifth scat sample fell within the domestic dog clade (C. familiaris). BLAST analyses in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) indicated 100% sequence match to many well represented domestic dog breeds from different regions of the world (see the Blast report pdf file in Suppl. material
The results of molecular analysis support our initial assumption, based on morphological observations, that the wolves found in Upper Mustang region of the Annapurna Conservation Area, Nepal, include individuals that belong to the genetically distinct and ancient Himalayan wolf clade (Figures
As part of the ongoing long term ecological research on wolves, both formal and informal interviews with herders, livestock owners, nomads and village elite were conducted in order to understand the status of human-wolf conflict, local attitudes and perceptions. Formal interview involves semi-structured questionnaire survey (n=354) which covers all the potential areas of wolf distribution in Mustang and Manang Districts of Annapurna Conservation Area. Informal interview (n=61) was mainly through discussion when herders were encountered while herding their livestock or while visiting their herding camps/corrals. Our preliminary assessment revealed that local communities persecuted wolves mainly in retaliation for livestock depredation. In some parts of the conservation area, livestock depredation from wolves was found to be a cause of concern for local livelihoods. These genetically distinct Himalayan wolves deserve special conservation attention, at the same time that the conservation of this species in a context of human-wildlife conflict is challenging. A species action plan needs be formulated that develops mechanisms to minimize conflict, and strategies for motivating local communities towards wolf conservation.
We are grateful to the National Trust for Nature Conservation’s Annapurna Conservation Area Project for providing us the research permission to carry out field work. Long term ecological study of the wolf in Nepal has been funded by USAID/Hariyo Ban Nepal Ko Dhan Program and Hedmark University of Applied Sciences , Norway. We are also thankful to the Wildlife Institute of India for collaborating on this research and providing an access to database of sequences. We are grateful to Broughton Coburn (Wyoming, USA) for assistance with editing. We would like to thank all the field staff of National Trust for Nature Conservation’s Annapurna Conservation Area Project and local communities who were directly and indirectly involved in the field work. We also wish to thank both reviewers, especially Rob Fleischer at the Center for Conservation and Evolutionary Genetics (Smithsonian Institution, USA), whose insightful and constructive comments immensely improved the manuscript.
Best model selection for Bayesian analysis
Data type: DOCX file
Explanation note: Estimating the best model of molecular substitution inferred using Log Bayes Factors (LBF) from Bayesian posterior distributions in Mr Bayes. Rate variation for tested models - gamma distributed rate variation across sites (gamma); gamma distributed with proportion of invariable sites (invgamma); rate variation with proportion of invariable sites (propinv); equal rate variation across sites (equal).
Genebank sequences analysed in this study
Data type: DOCX file
Explanation note: GenBank accession numbers for sequences analyzed in this study.
Aligned CR sequences of selected samples of wolves and dogs from GenBank, with the obtained scat samples
Data type: DOCX file
Explanation note: Aligned CR sequences of selected samples of wolves, dogs from GenBank, with scat samples obtained in this study. Numbers refer to mtDNA nucleotide positions referenced with respect to the complete mtDNA genome of gray wolf (GenBank Accession No. KF857179). Scat sequences D2137, D2138, D2139 and D2143 match each other differs from known Himalayan wolf haplotypes by at least two substitutions, while D2140 completely matches domestic dog and a wolf sequence in GenBank.
Median-joining networks of Himalayan wolf and related wolf and dog clades
Data type: TIF file
Explanation note: Median-joining networks of Himalayan wolf and related wolf and dog clades. Golden jackal and Ethiopian wolf haplotypes are shown for comparison. Circle size and branches are proportional to sampled haplotype frequency and number of nucleotide mutation steps among haplotypes, respectively. Branch numbers refer to mutation steps separating individual haplotypes. Scat samples sequenced in this study are represented by arrows falling within the Himalayan wolf (HWF) and Indian feral dog (IDH) haplotypes. Nomenclature for the African wolf follows
Blast report of sample D2140
Data type: PDF file
Explanation note: Blast search result for D2140 scat sequence.