Less-invasive age estimation using hair based on DNA methylation in brown bears

Information on chronological age is essential for exploring the life history, conservation, and management of wildlife. Recently, DNA methylation-based methods using blood or skin have been established as alternatives to the traditional tooth-based method in bear species. However, the collection of these tissues is limited to captured or dead individuals. In the present study, we established the first hair-based age estimation model based on DNA methylation levels in brown bears, aiming for future application to less-invasively obtained hair of wild individuals. We performed bisulfite pyrosequencing and measured the methylation levels of hair root DNA. The methylation levels of cytosine-phosphate-guanine sites adjacent to the genes VGF, KCNK12, and ELOVL2 were found to be correlated with age. The best age estimation model used three cytosine-phosphate-guanine sites adjacent to two genes, VGF and KCNK12, with a mean absolute error of 3.2 years and median absolute error of 2.2 years after leave-one-out cross-validation. Our method is innovative because of the simplicity of sampling and the lack of requirement to capture bears. If this method can be widely applied to hair samples obtained in the field, the age structure of wild populations can be understood, contributing to ecological research, conservation, and management of bear species.