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.