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This study investigated whether Euphorbia subgenus Chamaesyce subsection Acutae contains C3–C4 intermediate species utilizing C2 photosynthesis, the process where photorespired CO2 is concentrated into bundle sheath cells. Euphorbia species in subgenus Chamaesyce are generally C4, but three species in subsection Acutae (E. acuta, E. angusta, and E. johnstonii) have C3 isotopic ratios. Phylogenetically, subsection Acutae branches between basal C3 clades within Euphorbia and the C4 clade in subgenus Chamaesyce. Euphorbia angusta is C3, as indicated by a photosynthetic CO2 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 C2 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 C4 cycle activity occurs in E. acuta thereby classifying it as a Type I C3–C4 intermediate. Kranz-like anatomy in E. johnstonii indicates it also utilizes C2 photosynthesis. Given the phylogenetically intermediate position of E. acuta and E. johnstonii, these results support the hypothesis that C2 photosynthesis is an evolutionary intermediate condition between C3 and C4 photosynthesis. [ABSTRACT FROM PUBLISHER]