Evolutionary History of Blepharis (Acanthaceae) and the Origin of C4 Photosynthesis in Section Acanthodium

Premise of research.Plants with C-4 photosynthesis are able to produce carbohydrates more efficiently than plants with C-3 photosynthesis in warm climates when levels of atmospheric CO2 are reduced. The C-4 pathway has evolved multiple times in distantly related lineages, but it is not known whether the same physiological transitions occurred in all lineages. Species with intermediate C-3-C-4 physiology and anatomy offer the opportunity to study how plants transition from C-3 to C-4. It is thus vital to characterize phylogenetic relationships and photosynthetic pathways in groups with C-3-C-4 intermediate species, as well as C-3 and C-4 species.Methodology.We assessed photosynthetic pathway evolution in the Afro-Asian genus Blepharis (Acanthaceae) by sampling 99 species for carbon isotope ratios, 18 species for leaf anatomy, and 36 species for phylogenetic analysis. We estimated when Blepharis clades diverged using a BEAST molecular dating analysis, and we estimated ancestral distributions using BioGeoBEARS. We also estimated ancestral photosynthetic pathways in Blepharis, along with the rate of transitions between C-3, C-3-C-4 intermediates, and C-4 photosynthesis. Finally, we analyzed the climatic niches of 93 Blepharis taxa to better understand the current distribution patterns of species with different photosynthetic pathways.Pivotal results.Of the 99 species of Blepharis sampled for carbon isotope ratios, 13 are C-4, 2 are C-4-like, and 84 have values that indicate that they use a C-3 cycle. Nine species are putative C-3-C-4 intermediate species based on leaf anatomy. All of the C-4 and C-3-C-4 intermediate species are in section Acanthodium and are closely related. Our estimates suggest that C-4 photosynthesis evolved two or three times in southern Africa and Asia between 1 and 5 million years ago.Conclusions.We present a phylogenetic framework of Blepharis and hypotheses of where and when C-4 photosynthesis evolved in the genus. There are more than 40 species in section Acanthodium that are not C-4, some of which may be C-3-C-4 intermediate species. Blepharis thus contains many candidate C-3-C-4 intermediate species and provides an opportunity for detailed comparative analyses of the evolution of photosynthetic pathways.