Small felids play a critical role in the ecosystems they inhabit by regulating populations of small prey, facilitating nutrient cycling and maintaining habitat health (Litvaitis & Villafuerte, Reference Litvaitis and Villafuerte1996; Crooks & Soulé, Reference Crooks and Soulé1999; Bandyopadhyay et al., Reference Bandyopadhyay, Banerjee, Mazzamutto, Koley, Koprowski, Qureshi and Jhala2024). However, despite their ecological importance and the threats they face, these species often remain underrepresented in research and conservation, especially compared to charismatic large carnivores (Nowell & Jackson, Reference Nowell and Jackson1996; Mugerwa et al., Reference Mugerwa, Adhya, Ratnayaka, Thudugala, Napolitano and Sanderson2020; Bandyopadhyay et al., Reference Bandyopadhyay, Banerjee, Mazzamutto, Koley, Koprowski, Qureshi and Jhala2024). One such species is Pallas’s cat Otocolobus manul, a small, elusive felid with a wide but fragmented distribution across Central Asia from Iran to Mongolia and southern Siberia (Greenspan & Giordano, Reference Greenspan and Giordano2021; Lorestani et al., Reference Lorestani, Hemami, Rezvani and Ahmadi2022). Studies of the species’ potential habitat have enhanced knowledge of its distribution in Afghanistan, Tibet and parts of India, Nepal, China, Bhutan and Pakistan (Hameed et al., Reference Hameed, Ud din, Shah and Kabir2014; Shrestha et al., Reference Shrestha, Ale, Jackson, Thapa, Gurung and Adhikari2014; Dendup et al., Reference Dendup, Shrestha, Mahar, Kolipaka, Regmi and Jackson2019; Ross et al., Reference Ross, Barashkova, Dhendup, Munkhtsog, Smelansky, Barclay and Moqanaki2020). The species typically inhabits rocky areas, montane grasslands, shrublands and cold montane deserts with extreme continental climate, low rainfall, low humidity and large temperature variations (Nowell & Jackson, Reference Nowell and Jackson1996; Munkhtsog et al., Reference Munkhtsog, Ross and Brown2004; Ross et al., Reference Ross, Barashkova, Dhendup, Munkhtsog, Smelansky, Barclay and Moqanaki2020). Pallas’s cat plays an important role in cold dry montane ecosystems by regulating small mammal populations, particularly pikas Ochotona spp. (Greenspan & Giordano, Reference Greenspan and Giordano2021), the availability of which has a strong influence on the distribution of this felid (Seimon et al., Reference Seimon, Lim, Nightingale, Elmore and Seimon2022). Pallas’s cat is categorized as Least Concern on the IUCN Red List, but data on its occurrence comprises mostly individual records and thus there have been no estimates of population size or trend (Ross et al., Reference Ross, Barashkova, Dhendup, Munkhtsog, Smelansky, Barclay and Moqanaki2020).

The c. 15,000 km² Nubra Valley lies in the northernmost region of the district of Leh within the Union Territory of Ladakh, in the Trans-Himalayan biogeographical zone of India. The Ladakh mountain range lies to its south and the Karakoram Range to the north, with some permanently glaciated areas. The valley was designated as a protected area in 1987, as the Karakoram (Nubra-Shayok) Wildlife Sanctuary (Fig. 1), spanning an elevation range of 2,690–8,572 m. This region also harbours the snow leopard Panthera uncia, Central Asian lynx Lynx lynx isabellinus, Tibetan wolf Canis lupus chanco, Siberian ibex Capra sibirica, blue sheep Pseudois nayaur and Ladakh urial Ovis vignei vignei. However, as a result of the remoteness of the area and the extreme climate, there has been no comprehensive documentation of the fauna of the region.

Fig. 1 Nubra Valley, northern Ladakh, India, showing the location (Hunder Dok) in which we collected a scat of Pallas’s cat Otocolobus manul.

During our study on the dietary patterns of sympatric carnivores in the Karakoram Wildlife Sanctuary from February 2021 to December 2022, we encountered an ambiguous scat sample in the Hunder Dok area, at an altitude of 4,424 m, which we could not assign to any of the known carnivore species in the area based on its morphological characteristics. We collected the scat in a sterile falcon tube, sprayed it with absolute ethanol, and stored it at the Wildlife Biology Laboratory at the University of Kashmir, at −20 °C, for later genetic analysis.

We extracted genomic DNA from the scat using the QIAamp® Fast DNA Stool Mini Kit (QIAGEN, Germany) following the manufacturer’s protocol. The extracted DNA was PCR amplified using the carnivore-specific primer targeting the 148-bp mitochondrial Cytochrome-b gene with the forward sequence 5’ -TATTCTTTATCTGCCTATACATRCACG-3’ and reverse sequence 5’ AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3’ (Farrell et al., Reference Farrell, Roman and Sunquist2000). We conducted a PCR reaction in a total volume of 15 μl, containing 7.5 μl of 2X Premix EmeraldAmp GT PCR Master Mix (Takara, Japan), 5.9 μl of ddH₂O, 0.3 μl of each forward and reverse primer, and 1.0 μl of template DNA. The PCR assays entailed an initial denaturation at 94 °C for 5 minutes, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, elongation at 72 °C for 1 minute, and a final extension at 72 °C for 10 minutes. We used electrophoresis with 1.2% agarose gel to confirm the success of the PCR amplification, and sequenced the successful PCR amplicon using a BigDye Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, USA) on an ABI 3730XL sequencer (Applied Biosystems) in both the forward and reverse directions, with the same primer pair used for PCR amplification. We manually edited sequences to remove low-quality bases and primer sequences at the ends, corrected any base-calling errors, and also generated contig and consensus sequences using CodonCode Aligner (CodonCode Corporation, USA). To identify the species, we did an NCBI nucleotide blast of the consensus DNA sequence. The cytochrome b sequence that we generated matched the reference sequences of Pallas’s cat in GenBank (top three hits: MH978908.1, KR132585.1, KF990330.1, identity > 98.70%). We submitted the DNA sequence to NCBI to generate an accession number (PQ066009.1).

We then used standard micro-histology of mammalian hair remains in the scat (Vogel et al., Reference Vogel, Somers and Venter2019) to identify any prey, using the modified point-frame method (Ciucci et al., Reference Ciucci, Tosoni and Boitani2004). Oven-dried scat samples were evenly spread in a gridded tray, and 40–50 hairs located nearest to the grid intersections were randomly selected for microscopic examination. Under a microscope, the cuticular and medullary patterns of these hairs were compared with reference slides of potential prey species in the region (Bahuguna et al., Reference Bahuguna, Sahajpal, Goyal, Mukherjee and Thakur2010). Using this method, we found undigested hair remains of pikas and the Himalayan marmot Marmota himalayana in the scat.

To our knowledge, this is the first confirmed occurrence of Pallas’s cat in the Karakoram Wildlife Sanctuary in the Trans-Himalayan region of northern Ladakh. In India, the species was previously reported only from the Trans-Himalayan areas of Ladakh (Mahar et al., Reference Mahar, Shrotriya, Habib, Singh, Takpa and Hussain2017), Himachal Pradesh (Sharma et al., Reference Sharma, Thuktan, Tobge, Angrup, Chhering and Sherab2024), Uttarakhand (Pal et al., Reference Pal, Bhattacharya and Sathyakumar2019) and Sikkim (Chanchani, Reference Chanchani2008; Menon, Reference Menon2023). Within Ladakh, previous records have confirmed its presence in the Hanle, Staklung and Lal Pahari areas of the Changthang Wildlife Sanctuary and in the Rupshu region (Mallon, Reference Mallon1991; Pfister, Reference Pfister2004; Mahar et al., Reference Mahar, Shrotriya, Habib, Singh, Takpa and Hussain2017; Maeshwari et al., Reference Maheshwari, Chapman and Bhadula2023). Nubra Valley shares its eastern boundary with the Changthang plateau, where there have been frequent sightings of the species (Mahar et al., Reference Mahar, Shrotriya, Habib, Singh, Takpa and Hussain2017; Maeshwari et al., Reference Maheshwari, Chapman and Bhadula2023), suggesting that Pallas’s cat in this area may be ecologically connected to the population on the adjoining Tibetan Plateau. Our finding expands the known distribution of Pallas’s cat into northern Ladakh and suggests biogeographical connectivity with eastern Ladakh. The new record also provides evidence to support connectivity of this region with other parts of the Indian Trans-Himalaya where the species has already been documented, and with neighbouring countries, including China (Mallon, Reference Mallon2002; Fox & Dorji, Reference Fox and Dorji2007) and Nepal (Shrestha et al., Reference Shrestha, Ale, Jackson, Thapa, Gurung and Adhikari2014; Werhahn et al., Reference Werhahn, Kusi, Karmacharya and Sherchan2018, Reference Werhahn, Gurung, Lama, Tamang, Tamang and Pathak2024), and Gilgit-Baltistan in Pakistan (Hameed et al., Reference Hameed, Ud din, Shah and Kabir2014).

Similar biogeographical extensions have also been observed in other high-altitude carnivores, such as the Tibetan sand fox Vulpes ferrilata, which was first reported from the Indian Trans-Himalaya in Changthang, Ladakh (Namgail et al., Reference Namgail, Bagchi, Bhatnagar and Wangehuk2005), expanding its known range beyond the Tibetan Plateau. The finding of pika and marmot hairs in the scat of Pallas’s cat reaffirms the species’ dependence on small-sized prey and its potential role in regulating the population of small mammals in this high altitude ecosystem (Ross et al., Reference Ross, Munkhtsog and Harris2010, Reference Ross, Barashkova, Dhendup, Munkhtsog, Smelansky, Barclay and Moqanaki2020). Given the limitations of a single sample, however, we recommend a detailed study to provide a baseline of the species’ distribution, status and diet. Our finding nevertheless suggests this region may provide suitable habitat for Pallas’s cat (Plate 1), and provides scope for monitoring to inform species conservation and habitat management in the region.

Plate 1 Habitat of Pallas’s cat Otocolobus manul in the Karakoram Wildlife Sanctuary, Nubra Valley, Ladakh, India. Photo: Chamba Wangmo.