Gene turnover in the common ancestor of all C4 grasses

Societal Impact Statement Understanding how plants evolve more efficient photosynthesis is important in a warming world where improving crop productivity and resilience is a global priority. By generating the first reference genomes for an early-diverging group of grasses called the Aristidoideae, we were able to reconstruct the genetic makeup of the last common ancestor of all grasses that use C4 photosynthesis that is more efficient in hot climates. We show that early genetic changes in this lineage may have laid the groundwork for the later evolution of C4 photosynthesis. These findings help explain how complex biological innovations can repeatedly arise yet often remain restricted to particular evolutionary lineages. Summary In grasses, almost all species belong to two evenly sized clades: BOP (Bambusoideae, Oryzoideae and Pooideae) and PACMAD (Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae and Danthonioideae). Yet the >20 independent origins of C4 photosynthesis are confined to the PACMAD lineage. This pattern suggests that PACMAD grasses may harbour genetic features that primed them for the repeated evolution of this trait. To investigate this possibility, we examine gene turnover to identify genetic components that potentially facilitated the repeated evolution of C4 photosynthesis and 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 genes gained at the base of the PACMAD clade and genes lost in the sister BOP lineage. Gene expansion at the base of the PACMAD lineage includes one with a known C4 function (β-carbonic anhydrase), as well as genes involved in amino acid and nitrate transport, carbon metabolism, oxidative stress management and transcription regulation. Gene turnover in the PACMAD stem lineage generated novel variation in the last common ancestor of all C4 grasses. Although this predates the emergence of C4 photosynthesis itself, these changes may have generated the genetic substrate and requirements for the repeated emergence of C4 photosynthesis in this clade.