Enhancing epigenetic aging clocks in cetaceans: accurate age estimations in small endangered delphinids, killer whales, pilot whales, belugas, humpbacks, and bowhead whales

Abstract This study presents refined epigenetic clocks for cetaceans, building on previous research that estimated ages in several species from bottlenose dolphins to bowhead and humpback whales using cytosine methylation levels. We combined publicly available data (generated on the HorvathMammalMethylChip40 platform) from skin (n = 805) and blood (n = 286) samples across 13 cetacean species, aged 0 to 139 years. By combining methylation data from different sources, we enhanced our sample size, thereby strengthening the statistical validity of our clocks. We used elastic net regression with leave one sample out (LOO) and leave one species out (LOSO) cross validation to produce highly accurate blood only (Median Absolute Error [MAE] = 1.64 years, r = 0.96), skin only (MAE = 2.32 years, r = 0.94) and blood and skin multi-tissue (MAE = 2.24 years, r = 0.94) clocks. In addition, the LOSO blood and skin (MAE = 5.6 years, repeated measures r = 0.83), skin only (MAE = 6.22 years, repeated measures r = 0.81), and blood only (MAE = 4.11 years, repeated measures r = 0.95) clock analysis demonstrated relatively high correlation toward cetacean species not included within this current data set and provide evidence for a broader application of this model. Our results introduce a multi-species, two-tissue clock for broader applicability across cetaceans, alongside single-tissue multi-species clocks for blood and skin, which allow for more detailed aging analysis depending on the availability of samples. In addition, we developed species-specific clocks for enhanced precision, resulting in four blood-specific clocks and eight skin-specific clocks for individual species; all improving upon existing accuracy estimates for previously published species-specific clocks. By pooling methylation data from various studies, we increased our sample size, significantly enhancing the statistical power for building accurate clocks. These new epigenetic age estimators for cetaceans provide more accurate tools for aiding in conservation efforts of endangered cetaceans.