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

Abstract

With over 60 parallel origins representing evolutionary replicates, C<inf>4</inf> photosynthesis is well-suited for studying complex trait evolution. However, lineages with diverse C<inf>3</inf>–C<inf>4</inf> intermediate species are scarce, leaving uncertainty in models of C<inf>4</inf> 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. C<inf>3</inf>, proto-Kranz, C<inf>2</inf>, C<inf>4</inf>-like, and C<inf>4</inf> phenotypes occur in Blepharis sect. Acanthodium. The phylogeny supports a stepwise progression from C<inf>3</inf> through C<inf>2</inf> to C<inf>4</inf> states and up to five distinct origins of the C<inf>4</inf> cycle. Substantial intraspecific C<inf>2</inf>–C<inf>4</inf> variation is demonstrated in Blepharis mitrata, Blepharis furcata, and Blepharis macra. Blepharis gazensis is a monospecific C<inf>4</inf> lineage exhibiting an NADP malic enzyme C<inf>4</inf> pathway with features of the NAD-ME subtype, extending the ways in which the C<inf>4</inf> cycle is known to function. Substantial photosynthetic diversity exists in Blepharis that rivals or exceeds the range of character states present in other C<inf>3</inf> to C<inf>4</inf> transitional lineages. This diversity in Blepharis represents a robust new model for studying convergent evolution of C<inf>4</inf> photosynthesis and complex traits in general.