Upregulation of C 4 characteristics does not consistently improve photosynthetic performance in intraspecific hybrids of a grass.

C 4 photosynthesis is thought to have evolved via intermediate stages, with changes towards the C 4 phenotype gradually enhancing photosynthetic performance. This hypothesis is widely supported by modelling studies, but experimental tests are missing. Mixing of C 4 components to generate artificial intermediates can be achieved via crossing, and the grass Alloteropsis semialata represents an outstanding study system since it includes C 4 and non-C 4 populations. Here, we analyse F1 hybrids between C 3 and C 4 , and C 3 +C 4 and C 4 genotypes to determine whether the acquisition of C 4 characteristics increases photosynthetic performance. The hybrids have leaf anatomical characters and C 4 gene expression profiles that are largely intermediate between those of their parents. Carbon isotope ratios are similarly intermediate, which suggests that a partial C 4 cycle coexists with C 3 carbon fixation in the hybrids. This partial C 4 phenotype is associated with C 4 -like photosynthetic efficiency in C 3 +C 4  × C 4 , but not in C 3  × C 4 hybrids, which are overall less efficient than both parents. Our results support the hypothesis that the photosynthetic gains from the upregulation of C 4 characteristics depend on coordinated changes in anatomy and biochemistry. The order of acquisition of C 4 components is thus constrained, with C 3 +C 4 species providing an essential step for C 4 evolution.