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).