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Age structure is a key determinant of population dynamics and conservation management in wildlife, yet reliable age estimation typically depends on invasive sampling. Epigenetic clocks enable DNA-based age prediction across taxa, but their application to wildlife remains limited by reliance on blood or tissue samples. Feces offer a non-invasive alternative, although fecal-based age prediction has so far shown limited accuracy. Using captive Amur tigers ( Panthera tigris altaica ) of known ages as a model, we characterized DNA methylation patterns in fecal nuclear DNA, primarily derived from exfoliated intestinal epithelial cells, via whole-genome bisulfite sequencing. We identified 512 age-associated methylation sites and constructed age prediction models using Elastic Net regression, Support Vector Machine, and Random Forest regression based on methylation levels and methylation heterogeneity. The Random Forest model achieved the best performance, with a coefficient of determination (R²) of 0.981 and a mean absolute error (MAE) of 7.18 months in the training set, and an R² of 0.972 with an MAE of 7.37 months in the test set. Prediction error accounted for approximately 3.35% of the maximum sampled age (220 months) and 2.46–2.79% of the known lifespan of captive Amur tigers, outperforming previously reported fecal- or tissue-based epigenetic clocks in mammals. These results demonstrate that fecal nuclear DNA retains sufficient age-associated epigenetic information for accurate age estimation. We further propose a captive-to-wild equivalent age framework to facilitate application in free-ranging populations. Despite of the current constraints of sample size and validation strategy, this preliminary and exploratory study suggests the utility of feces and technical framwork for age structure inference in wildlife management and conservation practice.
Wang et al. (Thu,) studied this question.