Sex-Specific DNA Methylation in the Liver of Ancherythroculter nigrocauda, a Fish with Female-Biased Size Dimorphism

Sexual size dimorphism (SSD) is widespread in teleost fishes, yet the epigenetic mechanisms underlying sex-specific growth remain poorly understood, particularly in non-model species. Here, we performed genome-wide DNA methylation profiling of liver tissue in Ancherythroculter nigrocauda, a species exhibiting female-biased SSD, using the MethylRAD approach. MethylRAD sequencing of five females and five males generated approximately 1.0–1.5 million unique methylated loci per sample. Comparative analysis identified 82,872 differentially methylated sites (DMSs), which clearly separated sexes and were significantly enriched in promoters and CpG islands. Gene ontology analyses revealed that sex-biased methylation preferentially affected pathways related to intracellular signaling, cell adhesion, cytoskeletal organization, and protein modification, processes closely associated with hepatic growth and metabolic regulation. Chromosomal binning uncovered large contiguous male-biased methylation domains on Superscaffold15 and Superscaffold20, consistent with recombination-suppressed regions and suggesting candidate sex-linked loci, with Superscaffold20 supported by prior sex-specific coverage differences. Integration with liver transcriptome data revealed a threshold-dependent regulatory architecture: genes harboring dense methylation clustering (>20 DMS) were approximately six-fold more likely to exhibit sex-biased expression, and directional concordance between methylation and transcription further supported functional coupling. Together, these findings demonstrate that sex-biased DNA methylation in the liver is widespread yet concentrated within regulatory hotspots and discrete chromosomal domains, highlighting epigenetic regulation as a key contributor to hepatic sexual dimorphism and providing new insights into the molecular basis of SSD in teleost fishes.