An epigenetic DNA methylation clock for age estimates in Indo-Pacific bottlenose dolphins (Tursiops aduncus)

Knowledge of an animal's chronological age is crucial for understanding and predicting population demographics, survival and reproduction, but accurate age determination for many wild animals remains challenging. Previous methods to estimate age require invasive procedures, such as tooth extraction to analyse growth layers, which are difficult to carry out with large, mobile animals such as cetaceans. However, recent advances in epigenetic methods have opened new avenues for precise age determination. These `epigenetic clocks' present a less invasive alternative and can provide age estimates with unprecedented accuracy. Here, we present a species--specific epigenetic clock based on skin tissue samples for a population of Indo--Pacific bottlenose dolphins (Tursiops aduncus) in Shark Bay, Western Australia. We measured methylation levels at 37,492 cytosine--guanine sites (CpG sites) in 165 samples using the mammalian methylation array. Chronological age estimates with an accuracy of +/- 1 year were available for 68 animals as part of a long--term behavioral study of this population. Using these samples with known age, we built an elastic net model with Leave--One--Out--Cross--Validation, which retained 43 CpG sites, providing an r = 0.86 and median absolute age error (MAE) = 2.1 years (5% of maximum age). This model was more accurate for our data than the previously published methylation clock based on skin samples of common bottlenose dolphins (T. truncatus: r = 0.83, MAE = 2.2) and the multi--species odontocete methylation clock ( r = 0.68, MAE = 6.8), highlighting that species--specific clocks can have superior performance over those of multi-species assemblages. We further developed an epigenetic sex estimator, predicting sex with 100% accuracy. As age and sex are critical parameters for the study of animal populations, this clock and sex estimator will provide a useful tool for extracting life history information from skin samples rather than long--term observational data for free--ranging Indo--Pacific bottlenose dolphins worldwide.