Noninvasive, epigenetic age estimation in an elasmobranch, the cownose ray (Rhinoptera bonasus)

Age data are essential for estimating life history parameters and are thus critical for population assessment, management, and conservation. Traditional vertebrae-based age estimation in elasmobranchs can be costly, time intensive, of low accuracy, and is by necessity lethal. Herein, epigenetic clocks were developed for an elasmobranch, the cownose ray (Rhinoptera bonasus), using aquarium-born individuals (n = 42) with known dates of birth (age range: 7-7,878 days or 0-21 years) and two tissue types (fin clips and whole blood) that can be sampled in a relatively non-invasive manner. Enzymatically-converted restriction site-associated DNA sequencing (ECrad-seq) was used to identify CpG sites that exhibited age-correlated DNA methylation. The epigenetic clocks developed were highly accurate (mean absolute error, MAE, < 0.75 years) and precise (R² > 0.98). Age-associated CpG sites were identified across tissues, and a multi-tissue clock was also highly accurate (MAE < 1 year) and precise (R² = 0.97). Using the developed fin clip clock, three wild-caught individuals of unknown age but managed in aquariums for > 22 years were predicted to be 22.10-23.49 years old. Overall, the results have important implications for future epigenetic clock development and noninvasive age estimation in elasmobranchs.