Integrated Genomic and Methylome Profiling Reveals Promoter Repression and Age-Linked CpGs in the California Mussel

While most knowledge of animal DNA methylation comes from vertebrates, this epigenetic mark remains poorly understood in invertebrates, which comprise the majority of animal diversity. For instance, how promoter and gene body methylation contribute to gene regulation, and how methylation relates to aging, are still relatively unknown in most invertebrates. Focusing on the California mussel (Mytilus californianus), we paired whole-genome resequencing and whole-genome bisulfite sequencing from the same individuals and evaluated relationships among promoter methylation, gene body methylation, gene expression, and age. Using seven individuals spanning a range of body sizes from the Santa Barbara Channel, California, we found standing genetic variation levels similar to related species and a relatively small effective population size. CpG methylation was enriched in gene bodies, and gene body methylation was positively associated with expression. Promoter methylation was less frequent but showed a strong negative association with expression and remained the best predictor of repression after accounting for gene body methylation, aligning with patterns widely documented in vertebrates and adding to the limited evidence in invertebrates that promoter methylation can be regulatory. We identified thousands of age-associated differentially methylated loci with directional changes across age classes, providing candidate sites for epigenetic clocks that could enable assessment of biological age, health, and stress resilience in wild and cultured populations.