Age is an essential trait for understanding the ecology and management of wildlife. A conventional method of estimating age in wild animals is counting annuli formed in the cementum of teeth. This method has been used in bears despite some disadvantages, such as high invasiveness and the requirement for experienced observers. In this study, we established a novel age estimation method based on DNA methylation levels using blood collected from 49 brown bears of known ages living in both captivity and the wild. We performed bisulfite pyrosequencing and obtained methylation levels at 39 cytosine-phosphate-guanine (CpG) sites adjacent to 12 genes. The methylation levels of CpGs adjacent to four genes showed a significant correlation with age. The best model was based on DNA methylation levels at just four CpG sites adjacent to a single gene, SLC12A5, and it had high accuracy with a mean absolute error of 1.3 years and median absolute error of 1.0 year after leave-one-out cross-validation. This model represents the first epigenetic method of age estimation in brown bears, which provides benefits over tooth-based methods, including high accuracy, less invasiveness, and a simple procedure. Our model has the potential for application to other bear species, which will greatly improve ecological research, conservation, and management.

Robust estimates of demographic parameters are critical for effective wildlife conservation and management, but are difficult to obtain for elusive species. We estimated the breeding and adult population sizes, as well as the minimum population size, in a high-density brown bear population on the Shiretoko Peninsula, in Hokkaido, Japan, using DNA-based pedigree reconstruction. A total of 1,288 individuals, collected in and around the Shiretoko Peninsula between 1998 and 2020, were genotyped at 21 microsatellite loci. Among them, 499 individuals were identified by intensive genetic sampling conducted in two consecutive years (2019 and 2020) mainly by noninvasive methods (e.g., hair and fecal DNA). Among them, both parents were assigned for 330 bears, and either maternity or paternity was assigned to 47 and 76 individuals, respectively. The subsequent pedigree reconstruction indicated a range of breeding and adult (≥4 years old) population sizes: 128–173 for female breeders and 66–91 male breeders, and 155–200 for female adults and 84–109 male adults. The minimum population size was estimated to be 449 (252 females and 197 males) in 2019. Long-term continuous genetic sampling prior to a short-term intensive survey would enable parentage to be identified in a population with a high probability, thus enabling reliable estimates of breeding population size for elusive species.