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 x C-4, but not in C-3 x 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.