A deep-time landscape of plant cis-regulatory sequence evolution

Developmental gene function is often conserved over deep time, but cis-regulatory sequence conservation is difficult to identify. Rapid sequence turnover, paleopolyploidy, structural variation, and limited phylogenomic sampling have impeded conserved noncoding sequence (CNS) discovery. Using Conservatory, an algorithm that leverages microsynteny and iterative alignments to map CNS-gene associations over evolution, we uncovered ~2.3 million CNSs, including more than 3000 predating angiosperms, from 284 plant species spanning 300 million years of diversification. Ancient CNSs were enriched near developmental regulators, and mutating CNSs near HOMEOBOX genes produced strong phenotypes. Tracing CNS evolution uncovered key principles: CNS spacing varies, but order is conserved; genomic rearrangements form new CNS-gene associations; and ancient CNSs are preferentially retained among paralogs but are often lost as cohorts or evolve into lineage-specific CNSs.