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C4 photosynthesis results from anatomical and biochemical characteristics that together concentrate CO2 around ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco), increasing productivity in warm conditions. This complex trait evolved through the gradual accumulation of components, and particular species possess only some of these, resulting in weak C4 activity. The consequences of adding C4 components have been modelled and investigated through comparative approaches, but the intraspecific dynamics responsible for strengthening the C4 pathway remain largely unexplored. Here, we evaluate the link between anatomical variation and C4 activity, focusing on populations of the photosynthetically diverse grass Alloteropsis semialata that fix various proportions of carbon via the C4 cycle. The carbon isotope ratios in these populations range from values typical of C3 to those typical of C4 plants. This variation is statistically explained by a combination of leaf anatomical traits linked to the preponderance of bundle sheath tissue. We hypothesize that increased investment in bundle sheath boosts the strength of the intercellular C4 pump and shifts the balance of carbon acquisition towards the C4 cycle. Carbon isotope ratios indicating a stronger C4 pathway are associated with warmer, drier environments, suggesting that incremental anatomical alterations can lead to the emergence of C4 physiology during local adaptation within metapopulations. Summary Statement: Intraspecific analyses of photosynthetically diverse Alloteropsis semialata populations show that strengthening of the C4 photosynthetic pathway is associated with changes in multiple leaf anatomical traits. Stronger C4 activity is correlated with warmer, drier habitats, indicating local adaptation. [ABSTRACT FROM AUTHOR]

C4 photosynthesis is thought to have evolved via intermediate stages, with changes towards the C4 phenotype gradually enhancing photosynthetic performance. This hypothesis is widely supported by modelling studies, but experimental tests are missing. Mixing of C4 components to generate artificial intermediates can be achieved via crossing, and the grass Alloteropsis semialata represents an outstanding study system since it includes C4 and non‐C4 populations. Here, we analyse F1 hybrids between C3 and C4, and C3+C4 and C4 genotypes to determine whether the acquisition of C4 characteristics increases photosynthetic performance. The hybrids have leaf anatomical characters and C4 gene expression profiles that are largely intermediate between those of their parents. Carbon isotope ratios are similarly intermediate, which suggests that a partial C4 cycle coexists with C3 carbon fixation in the hybrids. This partial C4 phenotype is associated with C4‐like photosynthetic efficiency in C3+C4 × C4, but not in C3 × C4 hybrids, which are overall less efficient than both parents. Our results support the hypothesis that the photosynthetic gains from the upregulation of C4 characteristics depend on coordinated changes in anatomy and biochemistry. The order of acquisition of C4 components is thus constrained, with C3+C4 species providing an essential step for C4 evolution. Summary statement: We analyse hybrids between C3 and C4, and C3+C4 and C4 accessions of the grass Alloteropsis semialata. While hybrids have upregulated C4 features, these do not consistently increase photosynthetic efficiency, highlighting the importance of the order of acquisition of components during C4 evolution. [ABSTRACT FROM AUTHOR]