Leaf anatomy explains the strength of C₄ activity within the grass species Alloteropsis semialata

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.