Wolves in Bhutan Are Part of the Ancient Himalayan Wolf (Canis lupus chanco) Lineage

Three wolf lineages are recognised in South Asia, which include the Himalayan wolf (Canis lupus chanco), inhabiting the high Himalayas and the Tibetan Plateau; the Indian wolf (Canis lupus pallipes), found on the Indian subcontinent; and the Holarctic grey wolf, represented by multiple Canis lupus subspecies, found in adjacent regions of Central Asia. The Himalayan wolf represents an evolutionarily distinct high-elevation wolf lineage, yet genetic information remains scarce in the eastern Himalaya, including Bhutan, constituting a critical knowledge gap. During Bhutan's nationwide snow leopard survey (2022-2023), putative snow leopard scats were collected and a subset was genetically confirmed as C. lupus. We extracted DNA and amplified the mitochondrial control region (D-loop) and cytochrome b sequences from 13 samples (Jigme Dorji National Park: n = 1; Wangchuck Centennial National Park: n = 12). Phylogenetic analyses placed all Bhutanese sequences within the monophyletic Himalayan wolf clade, and median-joining haplotype networks provided concordant support for this placement. All detected haplotypes matched those previously reported from northwestern Nepal, indicating that Bhutanese wolves fall within the known Himalayan wolf mtDNA clade. These results provide the first genetic evidence that the Himalayan wolf lineage is present in Bhutan and provide an initial assessment of mtDNA diversity. Expanded non-invasive sampling across Bhutan, including areas with wolf records but lacking genetic representation (e.g., Jigme Khesar Strict Nature Reserve and Paro Forest Division), coupled with individual identification and nuclear markers, will be necessary to quantify genetic diversity and population connectivity, and provide first population size estimates. Such sampling will be critical for assessing fine-scale population structure and informing national conservation planning for this threatened wolf lineage endemic to High Asia.