Study area, animals, and blood sampling
Captive bears
Blood samples were obtained from 34 brown bears (17 males and 17 females) kept at Noboribetsu Bear Park (Noboribetsu, Hokkaido, Japan; Figure 1) during 2020–2022. Their ages ranged from 2 to 34 years old (Supplementary Table S_M1). Four individuals were rescued in the wild at 0 years of age and were moved to the facility, while all others were born in the facility. All individuals were fed bear pellets (ZOOFOOD bear; Nosan Co., Kanagawa, Japan), concentrated feed formulated for cows (Soyokazenokaori MG; Nippon Formula Food Manufacturing Co., Ltd., Kanagawa, Japan), dog food (Maibitto; PETLINE Inc., Gifu, Japan), vegetables, and fruits once or twice daily. Water was provided ad libitum . Bears fed in winter and did not hibernate. Prior to blood sampling, bears were anesthetized via intramuscular administration of xylazine HCl (1 mg/kg; Selactar; Bayer, Tokyo, Japan) and a 1:1 mixture of zolazepam HCl and tiletamine HCl (2.0–4.0 mg/kg; Zoletil 100; Virbac, Carros, France) using a blow dart. After blood sampling was completed, atipamezole HCl (1 mg/kg; Atipame; Kyoritsu Co., Ltd., Tokyo, Japan) was injected intramuscularly to aid recovery from anesthesia. Blood samples were collected via the medial saphenous vein into vacuum tubes containing ethylenediaminetetraacetic acid disodium (EDTA-2Na) as an anticoagulant. The collected blood samples were stored at −80°C as whole blood or buffy coat until genomic DNA extraction. Buffy coat samples were obtained by centrifuging blood samples at 1,880 × g for 10 minutes.
Wild bears
We used 31 blood samples from 13 wild brown bears (1 male and 12 females) living in the Rusha area (44°11′N, 145°11′E) of the Shiretoko Peninsula, eastern Hokkaido, Japan (Figure 1). Seven bears were sampled multiple times at different ages. Their ages ranged from 1 to 26 years old (Supplementary Table S_M1). In this area, long-term bear monitoring surveys have been conducted since 1997 until the present, enabling age determination based on visual and DNA-based identification (Shimozuru et al. 2017).
Bears were anesthetized via intramuscular administration of a 1:1 mixture of zolazepam HCl and tiletamine HCl (5.5 mg/kg; Zoletil 100; Virbac) and 75 µg/kg medetomidine HCL (Dorbene Vet; Kyoritsu Co., Ltd.) based on estimated body weight using an air rifle. After immobilization, bears were weighed, and blood was collected. When blood sampling was completed, atipamezole HCl (375 µg/kg; Atipame; Kyoritsu Co., Ltd.) was injected intramuscularly to aid recovery from anesthesia. Blood samples were collected via the jugular vein into vacuum tubes containing EDTA-2Na as an anticoagulant. The collected blood samples were stored at −80°C as whole blood or buffy coat until genomic DNA extraction. Buffy coat samples were obtained by centrifuging blood samples at 1,880 × g for 10 minutes.
Additionally, two female cubs-of-the-year that were captured with their mother in a barrel trap were sampled in Shibetsu Town, located in the southeastern part of the Shiretoko Peninsula. Bears were anesthetized using a blow dart for intramuscular administration of a 1:1 mixture of zolazepam HCl and tiletamine HCl (3 mg/kg; Zoletil 100; Virbac) and 40 µg/kg medetomidine HCL (Dorbene Vet; Kyoritsu Co., Ltd.), and then were awakened using atipamezole HCl (200 µg/kg; Atipame; Kyoritsu Co., Ltd.). All other procedures were conducted as described above.
Information about the samples collected for this study is summarized in Supplementary Table S_M1. Ages were determined at the time of blood sampling based on the assumption that all bears were born on February 1 (Friebe et al. 2014).