Epigenetic Aging in Brain Tissue of the Self-Fertilizing Vertebrate, Kryptolebias marmoratus

DNA methylation changes predictably with age across taxa, but in most species, these patterns are confounded by genetic variation. As a result, age-predictive methylation models have mostly been developed in genetically heterogeneous, cross-fertilizing organisms, limiting inference about epigenetic aging per se. Disentangling epigenetic and genetic effects is therefore essential for understanding aging, adaptation, and evolution. Here, we exploit the mangrove rivulus (Kryptolebias marmoratus), one of only two known self-fertilizing vertebrates (together with K. hermaphroditus), to examine epigenetic aging in a system of naturally occurring near-isogenic individuals. Using reduced-representation bisulfite sequencing of 89 brain samples spanning 60-1100 days of age, we identified 40 CpG sites whose methylation levels predict chronological age with high accuracy (R ² > 0.96, Median Absolute Error of 28.7 days). These 40 age-associated CpG sites were linked to nearby genes with known roles in cellular maintenance and neurodegeneration. These include genes implicated in aging and neurodegenerative processes across vertebrates, such as lamin-A, the aryl hydrocarbon receptor, and genes associated with Alzheimer's disease in humans. By leveraging a self-fertilizing vertebrate, this study demonstrates that DNA methylation undergoes consistent, age-associated changes across the lifespan in the near absence of genetic variation. Our results establish self-fertilizing vertebrates as powerful models for disentangling epigenetic aging from genetic effects and provide a foundation for comparative and evolutionary studies of aging.