Evolution of regulatory networks controlling adaptive traits in cichlids

Seminal studies in vertebrate protein evolution concluded that gene regulatory changes likely drive anatomical innovations. Even in the unparalleled East African cichlid fish radiation, we demonstrate cis-regulatory divergence as a contributor to phenotypic diversity. To further investigate this mechanism, we extended the Arboretum algorithm, initially designed for yeast adaptation to study the evolution of regulatory expression divergence in complex vertebrate species. For the first time, we reconstruct tissue-specific gene regulatory networks underpinning evolutionary adaptations from multiple vertebrate species in a phylogeny. Our framework consists of identifying ancestral reconstructed and extant species gene co-expression modules and integrating their associated regulators to investigate gene regulatory network evolution contributing to traits of phenotypic diversity in the East African cichlids. Along the phylogeny, we identify tissue-specific co-expression patterns across six tissues of five cichlids species that were predicted to be regulated by divergent suites of regulators. We report striking cases of rapid network rewiring for adaptive trait genes, such as the visual system. In regulatory regions of visual opsin genes e.g. sws1, in vitro assays confirm that single nucleotide polymorphisms (SNPs) in transcription factor binding sites (TFBSs) have driven network rewiring between species sharing the same visual palette. SNPs overlapping TFBSs segregate according to phylogeny and ecology suggesting ecotype-associated network rewiring in East African cichlid radiations. Our unique integrative approach inferring multi-species regulatory networks allowed us to identify regulatory changes associated with traits of phenotypic diversity in radiating cichlids.