Study area and sampling
This study was conducted on the Shiretoko Peninsula (43°50´–44°20´ N, 144°45´–145°20´ E; Figure. 2), eastern Hokkaido, Japan. An area from the middle to the tip of the peninsula has been on the UNESCO World Natural Heritage List since 2005. It is valued for the unique ecosystem formed by the interrelationship of its marine and terrestrial environments. Genetic samples were collected in and around the peninsula using multiple methods that detailed in previous studies (Shimozuru, et al. , 2020; Shimozuru, et al. , 2019; Shimozuru, et al. , 2020; Shimozuru, et al. , 2017; Shirane, et al. , 2018; Shirane, et al. , 2019). In this study, the area consisting of Shari, Rausu, and Shibetsu towns was defined as being inside the peninsula (approximately 1,760 km2), with additional samples from Kiyosato and Nakashibetsu towns also included in the analysis (Figure 2). Most samples (blood and tissue) were from bears that were dead due to nuisance control (mostly outside the national park) or hunting (limited to the October to January period outside the national park), or that were captured for research purposes. For bears captured or killed between 1998 and 2020, age was estimated by counting the dental cementum annuli (Craighead, Craighead and Mccutchen, 1970; Tochigi, et al. , 2018). In addition, we also obtained hair collected by hair-traps in several locations during 2010–2020, skin tissues collected by biopsy dart sampling during 2011–2020, and fecal samples collected during 2009–2020.
During 2019–2020, we conducted intensive, noninvasive genetic sampling for hair and feces. For hair, 63 and 67 tree-rub traps (For details, see Sato, et al. , 2020; Shimozuru, et al. , 2020) were placed throughout the peninsula in 2019 and 2020, respectively, except for areas where it was difficult to gain access (Figure 2). In the tree-rub trap, the trunk was partially smeared with wood preservative (Creosote R; Yoshida refinery, Tokyo, Japan) to lure bears (Sato, et al. , 2020), and barbed wire was wrapped around the trunk between 30 and 230 cm above the ground. From late May to October, we visited each trap at approximately 2-week intervals (a total of 10 and 11 collections in 2019 and 2020, respectively), and collected hairs from individual barbs, which then were stored separately in envelopes. Samples were dried and kept at –30°C until DNA extraction. Each tree-rub was monitored by an automatic camera (HykeCam SP108-J; Hyke Inc., Asahikawa, Japan). The recording time and intervals were set to 25 and 5 s, respectively. All videos were checked to estimate the number of bears that potentially rubbed against the tree, and their sex/age status was determined visually if possible. Through a combination of genetic analysis and video data, breeding status was clarified in some females, e.g., by the accompanying presence of cubs or yearlings. For fecal samples, we collected bear scats with ages of 0–4 days as estimated by field collectors. They were stored in Inhibitex buffer (Qiagen Inc., Tokyo, Japan) and kept at –30°C until DNA extraction. Bear scats were collected every time field collectors found them during bear patrols in and around popular tourist areas and farmland, driving on forest roads, and during exploratory investigations in the forest. To collect DNA samples from the areas without tree-rub traps, field collectors periodically (1–2 times a month from June to September) made explorations on foot into those areas, e.g., high-elevation areas and the tip of the peninsula.