Results
The number of samples analyzed and the number of bears identified by an intensive survey during 2019–2020 are shown in Table 1. The distribution of feces that was successfully analyzed is shown in Appendix A. From the 2-year intensive genetic survey in 2019–2020, 499 unique bears (281 females and 218 males) were identified. Among them, 172 bears (96 females and 76 males) had been genetically identified by 2018. Finally, with the samples collected between 1998 and 2020, we genotyped 1,288 bears (616 females and 672 males), including 1,221 bears from the Shiretoko Peninsula (i.e., Shari, Rausu, and Shibetsu towns), and 67 bears from areas adjacent to the peninsula (i.e., Kiyosato and Nakashibetsu towns). Approximately 58% of the sampled bears (748 bears) were confirmed to be dead, due to management culls, hunting, accidents, or natural causes. All bears were genotyped at all of the loci. We found seven haplotypes in the mitochondrial analysis, which was similar to the results of previous studies on the same population (Shirane, et al. , 2018): HB-02 (N = 37), HB-10 (N = 139), HB-11 (N = 703), HB-12 (N = 66), HB-13 (N = 122), HB-new1 (N = 107), and HB-new2 (N = 1); and one heteroplasmic pattern: HB-10/11 (N = 113). For the Y chromosomal haplotype analysis, we found four haplotypes (BR1_02, BR1_04, BR1_05, and BR1_06) that were reported in a previous study (Hirata, et al. , 2017). In addition, based on two markers, UarY369.4 and 15020.1, which were excluded in the above study due to the pseudoheterozygous genotypes identified in bears sampled outside Hokkaido, the haplotypes BR1_04 and BR1_05 were classified into two and three sub-haplotypes, respectively. Finally, we found six haplotypes, BR1_02 (N = 32), BR1_04a (N = 1), BR1_04b (N = 339), BR1_05a (N = 57), BR1_05b (N = 91), BR1_05c (N = 1), and BR1_06 (N= 149). Two samples were not available for Y chromosomal haplotypes due to an unstable amplification.
Table 2 summarizes the results of the parentage analysis with CERVUS. Among the 499 unique bears identified in 2019–2020, 7 males with the HB-02 mitochondrial haplotype were considered to be immigrated males from outside the peninsula, because no females with HB-02 were identified on the peninsula (Shirane, et al. , 2018). Therefore, those males were excluded from the candidate bears that were possibly born inside the peninsula. Both parents were assigned for over two-thirds of the remaining 492 bears, and less than 8% of the bears were unassigned to one parent. Among the 499 bears, including the seven immigrant males, 125 females and 65 males were confirmed to be breeders, due to the existence of ≥1 offspring between 1998 and 2020. In addition, 27 females and 18 males were identified as ≥4 years old as of 2019, based on the year of first visual/genetic identification (12 females and 6 males), the year of death of their parent (15 females and 8 males), or an age estimation at death by counting the cementum annuli of the teeth (4 dead males in 2019–2020), although they did not have any breeding record. Among the 499 bears, no bears were assigned as daughters/sons, or mothers/fathers of bears sampled outside the peninsula (i.e., Kiyosato and Nakashibetsu towns). Taken together, among the 499 bears identified in 2019–2020, 152 females and 83 males were confirmed to be adults (i.e., bears with reproductive experience or ≥4 years old) as of 2019.
Table 3 summarizes estimations of the breeding population by including past-identified breeders (previously existed, but not identified in 2019–2020) and hypothetical parents, based on a pedigree reconstruction by the combination of CERVUS and COLONY analyses. Among the bears identified between 1998 and 2018 but not in 2019–2020, 16 females and 10 males (identified between 2008 and 2018) were assigned as parents of bears identified in 2019–2020. Among them, one female was assigned as a mother of a bear that was born in 2018 and was dead in 2020. She was included in the minimum breeding population because it was reasonable to assume that she survived until the time of separation with the offspring in 2019. On the other hand, four females were estimated to be ≥30 years old based on the reconstructed pedigree, and one female (unidentified since 2012) and three males (unidentified since 2008–2015) were assumed to be dead due to their long-term non-identification in the Rusha area where continuous genetic monitoring had been conducted. By excluding these bears, 8.8 and 5.4 bears were included in the maximum breeding population as of 2019. Subsequently, COLONY generated 51 hypothetical mothers and 37 hypothetical fathers as potential parents of the bears (identified in 2019–2020) that remained unassigned to both or either of the parents in the CERVUS analysis. Among them, two females and one male were included in the minimum breeding population because they were assigned as parents of bears born in 2019 (two hypothetical females) and in 2020 (one hypothetical male). Among the remaining hypothetical parents, 13 females and 16 males were excluded due to the estimated age (two females and three males were estimated to be ≥30 and ≥28 years old, respectively), and due to the limitation of maximum maternal/paternal generations (9 females and 13 males were considered to be great-great-grandmothers and great-grandfathers, respectively). In addition, two females were assumed to be dead because they were mothers of resident adult females in the Rusha area, but were not observed in the past 12 years. Finally, the minimum/maximum adult populations of females and males were estimated to be 155–200 and 84–109, respectively.
The minimum bear population in 2019 in the Shiretoko Peninsula is shown in Table 4. It was found that a total of 449 (252 females and 197 males) existed as of 2019 in the Shiretoko Peninsula. Changes in the cumulative number of unique bears counted as the minimum population in 2019 are shown in Figure 3. Bears identified visually (one female) or inferred by pedigree reconstruction (one existing female, two hypothetical females, and one hypothetical male) were excluded from this analysis. Three females were counted as adults, not at the timing of first genetic identification, but when they were proven to be an adult (e.g., at a time when they were confirmed to be present with offspring).