Establishment of a non-invasive age estimation method based on fecal DNA methylation levels in brown bears

Age is important for understanding the life history of wildlife. In this study, we established a non-invasive age estimation method based on DNA methylation levels using fecal DNA collected from 43 brown bears of known age (0–28 years; 27 females and 16 males) living in both captivity (18 bears) and the wild (25 bears). Bisulfite pyrosequencing was performed to determine the methylation levels of fecal DNA, and the best model was constructed based on four cytosine-phosphate-guanine (CpG) sites: one adjacent to DLX5 and three adjacent to SLC12A5 . The mean absolute error after leave-one-out cross-validation was 2.08 years, and the median absolute error was 0.99 years; these results demonstrate high accuracy. Furthermore, a method was implemented for quantifying host DNA copy numbers, demonstrating that analyses tend to fail or exhibit large estimation errors when the amount of host DNA is insufficient. This is a significant advancement compared to previous techniques that relied on fecal DNA methylation levels. Applying this method to field surveys will greatly contribute to ecological research and the development of appropriate conservation and management strategies for bears, while facilitating future epigenetic clock studies on other animals.