How evolution repeatedly builds complexity: a case study with C₄ photosynthesis in Blepharis (Acanthaceae)

Summary With over 60 parallel origins representing evolutionary replicates, C 4 photosynthesis is well‐suited for studying complex trait evolution. However, lineages with diverse C 3 –C 4 intermediate species are scarce, leaving uncertainty in models of C 4 evolution. Phenotypic characterization of 28 living species of Blepharis (Acanthaceae) is presented, including photosynthetic gas exchange, enzyme activity assays, cell ultrastructure, and δ 13 C assays, the latter including 92 herbarium specimens from three species with phenotypic diversity. A well‐resolved transcriptome‐based phylogeny provides evolutionary context. C 3 , proto‐Kranz, C 2 , C 4 ‐like, and C 4 phenotypes occur in Blepharis sect. Acanthodium . The phylogeny supports a stepwise progression from C 3 through C 2 to C 4 states and up to five distinct origins of the C 4 cycle. Substantial intraspecific C 2 –C 4 variation is demonstrated in Blepharis mitrata , Blepharis furcata , and Blepharis macra . Blepharis gazensis is a monospecific C 4 lineage exhibiting an NADP malic enzyme C 4 pathway with features of the NAD‐ME subtype, extending the ways in which the C 4 cycle is known to function. Substantial photosynthetic diversity exists in Blepharis that rivals or exceeds the range of character states present in other C 3 to C 4 transitional lineages. This diversity in Blepharis represents a robust new model for studying convergent evolution of C 4 photosynthesis and complex traits in general.