Three metabolic subtypes of the C4 pathway are known, defined by the decarboxylase enzyme used to release CO2 in the vascular sheath cells. The most commonly used decarboxylase is NADP-dependent malic enzyme (NADP-ME), localized in chloroplasts, followed by NAD-dependent malic enzyme (NAD-ME), localized in mitochondria. The differences in nicotinamide cofactor and subcellular localization has important consequences for other aspects of the C4 cycle: in NADP-ME species, the release of CO2 also reduces NADP, which provides for one half of the reduced NADP needed by the Calvin cycle.⁷ As a result, expression of photosystem II, responsible for reducing NADP to power photosynthetic carbon assimilation, can be decreased in the sheath cells, with the added benefit of reducing the production of O2 in the sheath cells where Rubisco functions.⁸ In NADP-ME species, malate is the primary metabolic carrier for CO2, and it also carries reducing power into the sheath cells. In contrast, the reduction of NAD during decarboxylation by NAD-ME species cannot be readily consumed by the Calvin cycle. As a result, aspartate serves as the carrier for CO2 into the sheath cells in these species, which means reducing power is not shuttled in. Instead, reducing power is used to convert aspartate to malate, and then regenerated by decarboxylation, resulting in no net redox change. Thus NAD-ME species still have normal requirements for photosystem II in the sheath cells. A third subtype which occurs mainly in grasses utilizes PEP carboxykinase (PCK) to supply CO2 to Rubisco directly from PEP.⁷ This decarboxylase typically functions alongside NAD-ME, and there is some debate as to whether it represents a true C4 subtype or just a variation of the NAD-ME subtype.⁹

It is not known what factors influence the evolutionary trajectory toward one of these three subtypes. The NADP-ME pathway may be the most common simply because it is biochemically simplest, or there may be certain features of the early upregulation of C4 biochemistry in C2 species that predisposes the evolution of one subtype over another. Additionally, all known C4 lineages utilizing PCK are phylogenetically nested within larger C4 clades¹⁰ suggesting that the PCK subtype evolves as an elaboration on an existing C4 pathway, although how and why this could occur is unknown.

Several clades include closely related C4 lineages utilizing different metabolic subtypes: the Sesuvioideae subfamily of the Aizoaceae family includes six transitions to C4 with a mix of NADP-ME and NAD-ME subtypes;¹¹ Portulaca (Portulacaceae) includes at least two NADP-ME clades with an interleaving C2 and NAD-ME clade;¹² and Allionia and Boerhavia in the Nyctaginaceae family utilize NAD-ME and NADP-ME, respectively.¹⁴ Additionally, the Cleomaceae family includes three C4 lineages, all of which utilize the less common NAD-ME pathway, suggesting there is some predisposition favouring NAD-ME over NADP-ME in this group.¹⁴ Three of the four known PCK lineages are all nested within the Chlorodoideae grass subfamily, which represents a single large C4 clade utilizing NAD-ME. The fourth PCK lineage is the Melinidinae grass subtribe, sister to the NADP-ME subtribe Cenchrinae and the NAD-ME subtribe Panicinae.¹⁰ By targeting species across these key lineages for detailed phenotypic and comparative genomic analyses we seek to understand how evolution “selects” between these three alternative C4 pathways and what ancestral factors might influence this process.