C₃ photosynthesis in Aristida longifolia: Implication for photosynthetic diversification in Aristidoideae (Poaceae)

Only a small percentage of plant species undergo C₄ photosynthesis. Despite its rarity, the C₄ pathway has evolved numerous times from C₃ ancestors, with as many as 18 independent origins in grasses alone. We report non-Kranz (C₃) anatomy in Aristida longifolia, a species in a genus of ca. 300 species previously thought to possess only Kranz (C₄) anatomy. Leaf blade transections of A. longifolia show widely spaced vascular bundles, nonradiate chlorenchyma, and few or no chloroplasts in cells of the sheaths surrounding the vascular bundle, all features indicative of C₃ photosynthesis. Carbon isotope ratios range from -27.68 to -29.71%, likewise indicative of C₃ photosynthesis. We also reconstruct the phylogeny of Aristidoideae, comprising Aristida, Sartidia (C₃), and Stipagrostis (C₄), using a sample of 11 species, including A. longifolia, and DNA sequences of the nuclear ribosomal internal transcribed spacer region and the chloroplast rpl16 intron and trnL-trnF region. Sartidia and Stipagrostis resolve as sisters, and sister to this clade is ARISTIDA: Aristida longifolia resolves as sister to the remaining species in the genus. C₃ photosynthesis is hypothesized to be ancestral in Aristidoideae, which means the C₄ pathway evolved twice in the subfamily--in Stipagrostis and early in the diversification of the Aristida clade.