Summary
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.
