2. Materials and Methods

2.1 Study area

The cats were discovered in the Gyatong Grassland (Chindu County, Yushu Prefecture, Qinghai Province of China). Geographically, the grassland is located in a central plain surrounded by gentle steppe mountains at elevations of 4200-4700 m, which also serve as the boundaries between Qinghai and Sichuan provinces (Fig. 1b). The central plain is typical alpine meadow with the Ya-lung River flowing eastwards, covered with herbs, while shrubs are scarcely distributed in the shady sides of surrounding mountains (Fig. 1c). Vegetation as such make the place a perfect habitat for the plateau pika (Ochotona curzoniae ), thus providing abundant food resources for meso-carnivores such as the Tibetan fox (Vulpes ferrilata ), red fox (Vulpes vulpes ), Pallas’s cat (Otocolobus manul ), leopard cat (Prionailurus bengalensis ) and the Chinese mountain cat.
Other than wildlife, the meadow also hosts eleven villages of Tibetan herdsmen. Their settlements and winter pastures are generally located in the central plains, as well as in the bottom of surrounding mountains, while summer pastures are located in surrounding mountain valleys and steppe slopes. The dens of the Chinese mountain cats were all found in a valley and were only several hundred meters away from human settlements (Fig. 1b).

2.2 Collection and identification of carcasses

The discovery and monitoring of the breeding family of Chinese mountain cats were described in Han et al. (2020). The family was closely monitored by camera-traps until both kittens left the mother on 24 Dec 2018. Shortly after their independence, the female kitten (CMC-1) was first found dead on 30 Jan 2019. About one week later, on 7 Feb, the dead body of the adult female (CMC-2) was also found by a local field ranger (Fig. 2). The identification of both dead cats was based on the distinctive black stripes on their tails (compared with the information provided in Han et al. , 2020).
Both bodies were carefully recovered and stored under -20 °C for following examination and genetic analysis. Moreover, we also interviewed the locals who live close to where the bodies were found, to acquire more information on the cats’ demise.

2.3 Free-ranging dogs survey

Tibetan dogs, typically Tibetan mastiff (Canis familiaris ), are usually raised to protect herds and family property, with large body size, aggressive nature, and a strong territorial defense (Wang et al. , 2004). In our study area, dogs are commonly free-roaming (i.e., not permanently chained or under human control) and thus there is no clear boundary between domestic dogs (loose after sunset and during summer when herdsmen moved away from their village to summer pasture), stray dogs and feral dogs (Messerschmidt, 1983). Hereinafter, we used ‘free-ranging dogs’ as a general designation for dogs in our study area, while specifically we used ‘domestic dogs’ to refer to dogs belonging to certain households and ‘stray dogs’ to those that are not. These numerous free-ranging dogs pose great threats to local wildlife (Hughes & Macdonald, 2013), including the Chinese mountain cat. Consequently, in spring 2019, we investigated the population of domestic and stray dogs in the area where the cats occur. Later on, in May 2019, we interviewed a total of 15 herdsmen about their dogs (e.g., number, rearing patterns, abandonment), stray dogs around their village (e.g., number, population trend, potential origin, interactions with their dogs, livestock, and wildlife), and their attitudes to stray dogs (e.g., feeding behaviors, management measures) (see Table S1 for full interview questions). The participation of herdsmen in the interview was voluntary and anonymous.

2.4 Genetic analysis of carcasses

For the genetic analysis, tissue samples (CMC1, 2) were collected from the carcasses of the two Chinese mountain cats and stored in 95% alcohol at -20 °C. DNA was extracted with Dneasy Blood & Tissue Kit (QIAGEN, Hilden, Germany). At first, partial mitochondrial geneCytB and 9 nuclear loci were amplified (see Table S2-S4 for PCR recipe, protocol, and primers) and sanger sequenced. Then, their whole genome DNA were sequenced with DNBSEQ-T7 platform in 150 bp pair-end runs by BGI company (Shenzhen, China). Results of the two methods were consistent. Additionally, we sampled two cubs (CAT1, 2) reportedly hybrids of male Chinese mountain cat and female domestic cat in Labu Town, Chindu County, approximately 40 km away from the Gyatong Grassland. Blood samples were collected from ears, stored in vials and preserved in ice bags, to be delivered to Berry Genomics (Biotechnical Company, Beijing, China) for DNA extraction and next-generation sequencing by Illumina NovaSeq 6000 system in 150 bp pair-end runs.
High-throughput sequencing generated 60.4G raw data for each of the two Chinese mountain cats and 53.9G, 41.5G for the two hybrid cats respectively. After filtering poor quality reads, adapter trimming and duplicates removal, clean reads were mapped to public sequences of nuclear loci on autosome and X chromosome in Johnson et al.(2006) and Y chromosome in Yu et al. (2021) for genotyping, and a complete mitochondrial genome (GenBank Accession Number MT499915) ofFelis catus in Geneious Prime® 2020.0.3. Consensus sequences were generated and annotated for bases covered by at least 2 reads with Highest Quality. Our mitochondrial genome sequences were aligned with mitochondrial fragments sequenced by Yu et al. (2021) and the phylogeny was reconstructed by MrBayes 3.2.6 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003), with GTR+I+G model and 1,100,000 iterations with beginning 100,000 burn-in. Species-diagnostic nuclear genes of 8 autosomal genes, 3 X chromosome genes, and 2 Y chromosome genes inferred from published data (Accession Numbers DQ081730-DQ082545; Johnson et al. , 2006; Yu et al. , 2021) was referred to identify genetic composition to the four cats examined in our study.