In subfamily Suaedoideae, four independent gains of C4 photosynthesis are proposed, which includes two parallel origins of Kranz anatomy (sectionsSalsinaandSchoberia) and two independent origins of single-cell C4 anatomy (BienertiaandSuaeda aralocaspica). Additional phylogenetic support for this hypothesis was generated from sequence data of the C-terminal portion of the phosphoenolpyruvate carboxylase (PEPC) gene used in C4 photosynthesis (ppc-1) in combination with previous sequence data.ppc-1sequence was generated for 20 species in Suaedoideae and two outgroupSalsolaspecies that included all types of C4 anatomies as well as two types of C3 anatomies. A branch-site test for positively selected codons was performed using the software package PAML. From labelling of the four branches where C4 is hypothesized to have developed (foreground branches), residue 733 (maize numbering) was identified to be under positive selection with a posterior probability >0.99 and residue 868 at the >0.95 interval using Bayes empirical Bayes (BEB). When labelling all the branches within C4 clades, the branch-site test identified 13 codons to be under selection with a posterior probability >0.95 by BEB; this is discussed considering current information on functional residues. The signature C4 substitution of an alanine for a serine at position 780 in the C-terminal end (which is considered a major determinant of affinity for PEP) was only found in four of the C4 species sampled, while eight of the C4 species and all the C3 species have an alanine residue; indicating that this substitution is not a requirement for C4 function.
Phylogenetic analysis of independent gains of C4 photosynthesis in Suaedoideae shows differences compared with other C4 lineages in amino acids of phosphoenolpyruvate carboxylase under positive selection and their position relative to functional residues.