Plants using the C4 pathway of carbon metabolism are marked by greater photosynthetic water and nitrogen-use efficiencies (PWUE and PNUE, respectively) than C3 species, but it is unclear to what extent this is the case in C3-C4 intermediate species. In this study, we examined the PWUE and PNUE of 14 species of Flaveria Juss. (Asteraceae), including two C3, three C4 and nine C3-C4 species, the latter containing a gradient of C4-cycle activities (as determined by initial fixation of 14 C into C-4 acids). We found that PWUE, PNUE, leaf ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) content and intercellular CO2 concentration in air (Ci) do not change gradually with C4-cycle activity. These traits were not significantly different between C3 species and C3-C4 species with less than 50% C4-cycle activity. C4-like intermediates with greater than 65% C4-cycle activity were not significantly different from plants with fully expressed C4 photosynthesis. These results indicate that a gradual increase in C4-cycle activity has not resulted in a gradual change in PWUE, PNUE, intercellular CO2 concentration and leaf Rubisco content towards C4 levels in the intermediate species. Rather, these traits arose in a stepwise manner during the evolutionary transition to the C4-like intermediates, which are contained in two different clades within Flaveria.

We demonstrate for the first time the presence of species exhibiting C3-C4 intermediacy in Heliotropium (sensu lato), a genus with over 100 C3 and 150 C4 species. CO2 compensation points (G ) and photosynthetic water-use efficiencies (WUEs) were intermediate between C3 and C4 values in three species of Heliotropium: Heliotropium convolvulaceum (G = 20 mmol CO2 mol -1 air), Heliotropium racemosum (G = 22 mmol mol -1 ) and Heliotropium greggii (G = 17 mmol mol -1 ). Heliotropium procumbens may also be a weak C3-C4 intermediate based on a slight reduction in G (48.5 mmol CO2 mol -1 ) compared to C3 Heliotropium species (52-60 mmol mol -1 ). The intermediate species H. convolvulaceum, H. greggii and H. racemosum exhibited over 50% enhancement of net CO2 assimilation rates at low CO2 levels (200-300 mmol mol -1 ); however, no significant differences in stomatal conductance were observed between the C3 and C3-C4 species. We also assessed the response of G to variation in O2 concentration for these species. Heliotropium convolvulaceum, H. greggii and H. racemosum exhibited similar responses of G to O2 with response slopes that were intermediate between the responses of C3 and C4 species below 210 mmol O2 mol -1 air. The presence of multiple species displaying C3-C4 intermediate traits indicates that Heliotropium could be a valuable new model for studying the evolutionary transition from C3 to C4 photosynthesis.

This study investigated whether Euphorbia subgenus Chamaesyce subsection Acutae contains C(3)-C(4) intermediate species utilizing C(2) photosynthesis, the process where photorespired CO(2) is concentrated into bundle sheath cells. Euphorbia species in subgenus Chamaesyce are generally C(4), but three species in subsection Acutae (E. acuta, E. angusta, and E. johnstonii) have C(3) isotopic ratios. Phylogenetically, subsection Acutae branches between basal C(3) clades within Euphorbia and the C(4) clade in subgenus Chamaesyce. Euphorbia angusta is C(3), as indicated by a photosynthetic CO(2) compensation point (Г) of 69 μmol mol(-1) at 30 °C, a lack of Kranz anatomy, and the occurrence of glycine decarboxylase in mesophyll tissues. Euphorbia acuta utilizes C(2) photosynthesis, as indicated by a Г of 33 μmol mol(-1) at 30 °C, Kranz-like anatomy with mitochondria restricted to the centripetal (inner) wall of the bundle sheath cells, and localization of glycine decarboxlyase to bundle sheath mitochondria. Low activities of PEP carboxylase, NADP malic enzyme, and NAD malic enzyme demonstrated no C(4) cycle activity occurs in E. acuta thereby classifying it as a Type I C(3)-C(4) intermediate. Kranz-like anatomy in E. johnstonii indicates it also utilizes C(2) photosynthesis. Given the phylogenetically intermediate position of E. acuta and E. johnstonii, these results support the hypothesis that C(2) photosynthesis is an evolutionary intermediate condition between C(3) and C(4) photosynthesis.

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