Summary The genus Flaveria , containing species at different evolutionary stages of the progression from C 3 to C 4 photosynthesis, is used as a model system to study the evolution of C 4 photosynthesis. Here, we report chromosome-scale genome sequences for five Flaveria species, including C 3 , C 4 , and intermediate species. Our analyses revealed that both acquiring additional gene copies and recruiting ethylene responsive factor (ERF) cis -regulatory elements (CREs) contributed to the emergence of C 4 photosynthesis. ERF transcriptional factors (TFs), especially intronless ERF TFs, were co-opted in dicotyledonous C 4 species and monocotyledonous C 4 species in parallel. These C 4 species co-opted intronless ERF TFs originated from the Late Ordovician mass extinction that occurred ∼450 million years ago in coping with environmental stress. Therefore, this study demonstrated that intronless ERF TFs were acquired during the early evolution of plants and provided the molecular toolbox facilitating multiple subsequent independent evolutions of C 4 photosynthesis.

Born with intronless ERF transcriptional factors: C4photosynthesis inherits a legacy dating back 450 million years