Phenotypic landscape inference reveals multiple evolutionary paths to C4 photosynthesis

C-4 photosynthesis has independently evolved from the ancestral C-3 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 C-4 photosynthesis is associated with diverse and flexible evolutionary paths that group into four major trajectories. We conducted a meta-analysis of 18 lineages containing species that use C-3, C-4, or intermediate C-3-C-4 forms of photosynthesis to parameterise a 16-dimensional phenotypic landscape. We then developed and experimentally verified a novel Bayesian approach based on a hidden Markov model that predicts how the C-4 phenotype evolved. The alternative evolutionary histories underlying the appearance of C-4 photosynthesis were determined by ancestral lineage and initial phenotypic alterations unrelated to photosynthesis. We conclude that the order of C-4 trait acquisition is flexible and driven by non-photosynthetic drivers. This flexibility will have facilitated the convergent evolution of this complex trait.