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

Developmental gene function is conserved over deep time, but cis-regulatory sequence conservation is rarely found. Rapid sequence turnover, paleopolyploidy, structural variation, and limited phylogenomic sampling have impeded conserved non-coding sequence (CNS) discovery. Using Conservatory, an algorithm that leverages microsynteny and iterative alignments to map CNS-gene associations over evolution, we uncovered ~2.3M CNSs, including over 3,000 predating angiosperms, from 284 plant species spanning 400 million years of diversification. Ancient CNSs were enriched near developmental regulators, and mutating these 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.