Variation in leaf anatomical traits relates to the evolution of C₄ photosynthesis in Tribuloideae (Zygophyllaceae)

Tribuloideae (Zygophyllaceae, Zygophyllales) is a warm-climate clade of mostly creeping herbs with an un- certain number of C₄ species from at least two genera, raising the possibility of multiple C₄ origins within this lineage. To delineate C₃ and C₄ distributions within Tribuloideae, we conducted a phylogenetic analysis using four genetic markers from 78 accessions of Tribuloideae. δ¹³C values from 68 Tribuloideae members detected 42 C₄ species in the genera Kallstroemia, Tribulopis and Tribulus, and no C₄ in Balanites, Neoluederitzia, and Sisyndite. All Kallstroemia are C₄, while Tribulopis and Tribulus include C₃ species. Two independent C₄ origins are sup- ported, one in Tribulus and a second in Kallstroemia; both lineages are NADP-ME subtypes. The C₄ Tribulopis clade occurs in a conflicting position that suggests a hybrid origin between ancestral Kallstroemia and C₃ Tribulopis/Tribulus species. Anatomical assessment did not show enhanced vein density in C₃ Tribuloideae, however, relative bundle sheath area is greater in C₃ clades closely related to the C₄ lineages, supporting a hypothesis that bundle sheath enhancement in C₃ Tribuloideae facilitated C₄ evolution.