Integrated genomic analysis revealed a dominant role of transcriptional regulation during the evolution of C4 photosynthesis in Flaveria species

C 4 photosynthesis represents a remarkable example of convergent evolution of complex traits. In this study, we present an integrated genomic analysis of the genus Flaveria , which includes species at different evolutionary stages from C 3 to C 4 photosynthesis. The chromosome-scale genome sequences of five Flaveria species, representing C 3 , intermediate, and C 4 types, reveal a gradual increase in genome size attributed to the expansion of transposable elements (TEs). Through a combination of gene phylogeny and transcript abundance analysis, we systematically identified genes encoding C4 enzymes and transporters. We further found that C 4 species acquired additional copies of genes encoding three C4 enzymes through retrotranspositions. Moreover, we observed an increased abundance of ERF cis-regulatory elements and ERF transcriptional factors associated with the regulation of C 4 genes in C 4 species compared to non-C 4 species. Notably, C 4 genes exhibited elevated both RNA and protein levels but decreased protein-to-RNA ratios in C 4 species. Additionally, codon usage patterns are conserved among the five Flaveria species. All these results suggest a predominant role of transcriptional regulation in the evolution of C 4 photosynthesis. Our comprehensive dataset also provides valuable resources to study the evolution and regulatory mechanisms underlying C 4 photosynthesis.