Phenotypic landscape inference reveals multiple evolutionary paths to C4 photosynthesis

∗ These authors contributed equally to this work. C4 photosynthesis has independently evolved from the ancestral C3 pathway in at least 60 plant lineages, but, as with other complex traits, how it evolved is unclear. Here we show that the polyphyletic appearance of C4 photosynthesis is associated with diverse and flexible evo-lutionary paths that group into four major trajectories. We conducted a meta-analysis of 18 lineages containing species that use C3, C4, or intermediate C3-C4 forms of photosynthesis to parameterise a 16-dimensional phe-notypic landscape. We then developed and experimen-tally verified a novel Bayesian approach based on a hid-den Markov model that predicts how the C4 phenotype evolved. The alternative evolutionary histories under-lying the appearance of C4 photosynthesis were deter-mined by ancestral lineage and initial phenotypic alter-ations unrelated to photosynthesis. We conclude that the order of C4 trait acquisition is flexible and driven by non-photosynthetic drivers. This flexibility will have fa-cilitated the convergent evolution of this complex trait