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

With over 60 parallel origins representing evolutionary replicates, C₄ photosynthesis is well-suited for studying complex trait evolution. However, lineages with diverse C₃-C₄ intermediate species are scarce, leaving uncertainty in models of C₄ evolution. Phenotypic characterization of 28 living species of Blepharis (Acanthaceae) is presented, including photosynthetic gas exchange, enzyme activity assays, cell ultrastructure, and δ¹³C assays, the latter including 92 herbarium specimens from three species with phenotypic diversity. A well-resolved transcriptome-based phylogeny provides evolutionary context. C₃, proto-Kranz, C₂, C₄-like, and C₄ phenotypes occur in Blepharis sect. Acanthodium. The phylogeny supports a stepwise progression from C₃ through C₂ to C₄ states and up to five distinct origins of the C₄ cycle. Substantial intraspecific C₂-C₄ variation is demonstrated in Blepharis mitrata, Blepharis furcata, and Blepharis macra. Blepharis gazensis is a monospecific C₄ lineage exhibiting an NADP malic enzyme C₄ pathway with features of the NAD-ME subtype, extending the ways in which the C₄ cycle is known to function. Substantial photosynthetic diversity exists in Blepharis that rivals or exceeds the range of character states present in other C₃ to C₄ transitional lineages. This diversity in Blepharis represents a robust new model for studying convergent evolution of C₄ photosynthesis and complex traits in general.