New Insights Into the Evolution of C₄ Photosynthesis Offered by the Tarenaya Cluster of Cleomaceae

Cleomaceae is closely related to Brassicaceae and includes C₃, C₃-C₄, and C₄ species. Thus, this family represents an interesting system for studying the evolution of the carbon concentrating mechanism. However, inadequate genetic information on Cleomaceae limits their research applications. Here, we characterized 22 Cleomaceae accessions [3 genera (Cleoserrata, Gynandropsis, and Tarenaya) and 11 species] in terms of genome size; molecular phylogeny; as well as anatomical, biochemical, and photosynthetic traits. We clustered the species into seven groups based on genome size. Interestingly, despite clear differences in genome size (2C, ranging from 0.55 to 1.3 pg) in Tarenaya spp., this variation was not consistent with phylogenetic grouping based on the internal transcribed spacer (ITS) marker, suggesting the occurrence of multiple polyploidy events within this genus. Moreover, only G. gynandra, which possesses a large nuclear genome, exhibited the C₄ metabolism. Among the C₃-like species, we observed intra- and interspecific variation in nuclear genome size as well as in biochemical, physiological, and anatomical traits. Furthermore, the C₃-like species had increased venation density and bundle sheath cell size, compared to C₄ species, which likely predisposed the former lineages to C₄ photosynthesis. Accordingly, our findings demonstrate the potential of Cleomaceae, mainly members of Tarenaya, in offering novel insights into the evolution of C₄ photosynthesis.