This work was supported by the European Research Council Advanced Grant(ETAP-742985 to H.T.and J.F.)and by the Ministry of Science and Technology(grant 110-2636-B-005-001 to K.-J.L.).

Plant root-nodule symbiosis(RNS)with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms,the Nitrogen-Fixing Nodulation Clade(NFNC),and is best understood in the legume family.Nodulating species share many commonalities,explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period.Regardless,comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation—what must be acquired or cannot be lost for a functional symbiosis—and the latitude for Plant Communications Genomic landscape of nodulation variation in the symbiosis.However,much remains to be learned about nodulation,especially outside of legumes.Here,we employed a large-scale phylogenomic analysis across 88 species,complemented by 151 RNA-seq libraries,to elucidate the evolution of RNS.Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key muta-tions that affect its function across the NFNC.Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants,while also identifying nodule-specific and nitrogen-response genes.Approximately 70%of symbiosis-related genes are highly conserved in the four representative species,whereas defense-related and host-range restriction genes tend to be lineage specific.Our study also identified over 900000 conserved non-coding elements(CNEs),over 300000 of which are unique to sampled NFNC species.NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions,thus representing a pool of candidate regula-tory elements for genes involved in RNS.Collectively,our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.