Key changes in gene expression identified for different stages of C(4) evolution in Alloteropsis semialata

C(4) photosynthesis is a complex trait that boosts productivity in tropical conditions. Compared with C(3) species, the C(4) state seems to require numerous novelties, but species comparisons can be confounded by long divergence times. Here, we exploit the photosynthetic diversity that exists within a single species, the grass Alloteropsis semialata, to detect changes in gene expression associated with different photosynthetic phenotypes. Phylogenetically informed comparative transcriptomics show that intermediates with a weak C(4) cycle are separated from the C(3) phenotype by increases in the expression of 58 genes (0.22% of genes expressed in the leaves), including those encoding just three core C(4) enzymes: aspartate aminotransferase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxylase. The subsequent transition to full C(4) physiology was accompanied by increases in another 15 genes (0.06%), including only the core C(4) enzyme pyruvate orthophosphate dikinase. These changes probably created a rudimentary C(4) physiology, and isolated populations subsequently improved this emerging C(4) physiology, resulting in a patchwork of expression for some C(4) accessory genes. Our work shows how C(4) assembly in A. semialata happened in incremental steps, each requiring few alterations over the previous step. These create short bridges across adaptive landscapes that probably facilitated the recurrent origins of C(4) photosynthesis through a gradual process of evolution.