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

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