Gene turnover and contingency facilitated the repeated evolution of C₄ photosynthesis in grasses

In grasses, almost all species belong to two evenly sized clades (BOP and PACMAD), yet the >20 independent origins of C4 photosynthesis in this family only occur in the PACMAD lineage. Here, we identify potential genetic precursors for C4 photosynthesis that were present at the base of the PACMAD clade, representing the last common ancestor of all C4 grasses. We generated the first reference genomes for Aristidoideae species (Aristida adscensionis and Stipagrostis hirtigluma), the sister lineage of all other PACMAD grasses. In combination with 34 other Poales genomes, we identify gene gains at the base of the PACMAD clade, and genes lost in the sister BOP lineage. Candidate C4 precursors include β-carbonic anhydrase, as well as genes involved in amino acid and nitrate transport, carbon metabolism, oxidative stress management and transcription regulation. Gene turnover created the necessary genetic contingency, facilitating the independent recruitment and refinement of C4 genes in mul..., , # Gene turnover and contingency facilitated the repeated evolution of C₄photosynthesis in grasses Dataset DOI: [10.5061/dryad.xgxd254t2](10.5061/dryad.xgxd254t2) ## Description of the data and file structure We generated HiFi reads of two Aristidoideae species, *Aristida adscensionis* and *Stipagrostis hirtigluma* (grasses from the PACMAD clade), assembled a draft genome, and performed gene annotation for each of them. ### Files and variables #### File: Stipagrostis_4n_nuclear-softMasked.gff3 **Description:** Gene annotation for *Stipagrostis hirtigluma* using Helixer. #### File: Stipagrostis_4n_nuclear-softMasked.fna **Description:** Fasta file containing all the contig sequences assembled for *Stipagrostis hirtigluma*. #### File: Aristida_nuclear-softMasked.gff3 **Description:** Gene annotation for *Aristida adscensionis* using Helixer. #### File: Aristida_nuclear-softMasked.fna **Description:** Fasta file containing all the contig sequences assembled for *Ari...,