Genome-wide evolution of lineage-specific chromatin accessibility links regulatory changes to cichlid phenotypic diversity.

East African cichlids from Lakes Victoria, Malawi and Tanganyika have undergone explosive adaptive radiation, generating extensive morphological, ecological and behavioural diversity within a few million years. Understanding how genome-wide regulatory evolution contributes to this diversification requires comparative analyses of chromatin accessibility, transcription factor (TF) binding and gene regulatory networks across species and tissues. Here we combine high-coverage ATAC-seq with matched RNA-seq in forebrain, retina, liver and testis from five East African cichlid species spanning radiating and non-radiating lineages to characterise the evolution of regulatory landscapes in this radiation. Thousands of promoter-associated accessible sites are conserved across species and tissues, yet more than half of these shared promoters show signatures of accelerated nucleotide evolution, particularly in genes linked to development and sensory function. In contrast, smaller sets of lineage- and tissue-specific accessible promoters, especially in retina, exhibit strong regulatory divergence and functional enrichment for visual system processes. Integrating TF footprinting and expression data uncovers pervasive TF binding site (TFBS) turnover at active promoters and extensive, tissue-dependent rewiring of motif-supported gene regulatory networks, with 86-97% of high-confidence edges being species-specific. Extending promoter TFBS variants into broader Malawi and tilapia phylogenies shows that functional variants in promoters of visual genes, such as actr1, segregate by clade and ecological niche. Together, these results demonstrate how accelerated regulatory sequence evolution, chromatin accessibility divergence and TFBS turnover jointly reshape regulatory architecture genome-wide in an iconic vertebrate radiation, providing insights into how non-coding regulatory evolution can drive rapid phenotypic diversification.