Epigenetic Signatures of Ageing in Asian Elephants Revealed by Reduced Representation Bisulphite Sequencing

Accurate age estimation is essential for understanding life-history variation, modelling population dynamics and informing conservation strategies, yet remains challenging for long-lived species. Here, we developed a genome-wide DNA methylation-based epigenetic clock for Asian elephants (Elephas maximus), an endangered species, using reduced representation bisulphite sequencing (RRBS). Genome-wide methylation profiles from 91 blood samples yielded 144,611 candidate CpG sites, of which 389 CpG sites were strongly associated with chronological age. The final model predicted age with high accuracy (r = 0.96, MAE = 4.82 years), corresponding to a relative error of 6.06%, comparable to epigenetic clocks developed for humans and other non-model species. Longitudinal analyses revealed heterogeneous epigenetic ageing trajectories, with most individuals showing increases in epigenetic age over time, while others exhibited relatively slower or minimal change, indicating a potential inter-individual variation in ageing processes. Functional enrichment analyses revealed that age-associated CpGs were enriched in genes and pathways related to development, neurogenesis, metabolism and social or physiological regulation, including oxytocin, apelin and melanogenesis signalling. Age-related methylation changes were characterised by predominant hypermethylation in CpG islands and genic regions, consistent with patterns reported across mammals. Together, these findings demonstrate that epigenetic clocks capture biologically meaningful features of ageing that could be linked to life-history traits. As long-lived mammals with complex social systems and extended reproductive capacity, Asian elephants provide a valuable comparative model for investigating the molecular architecture of ageing. This study contributes to the growing field of comparative epigenetic ageing and highlights the potential of DNA methylation-based approaches to inform evolutionary and conservation-relevant research when applied in appropriate ecological and methodological contexts.