Epigenetic Aging in Brain Tissue of the Self-Fertilizing Vertebrate, <i>Kryptolebias marmoratus</i>.

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 (<i>Kryptolebias marmoratus</i>), one of only two known self-fertilizing vertebrates (together with <i>K. hermaphroditus</i>), 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 (<i>R</i> 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.