Trong selective pressure for acquiring Rubisco with C4 kinetics which then evolves during the stage of optimisation of C4 photosynthesis [58].Parallel amino-acid replacements in Rubisco from phylogenetically distant Castanospermine web lineagesBayesian analyses of rbcL sequences in a phylogenetic framework allowed us to identify two residues under directional selection along C4 branches within Amaranthaceae (Table 2). There are no common trends in physicochemical properties of `C4′ amino acids with buy BTZ043 respect to properties such as 1676428 residue hydrophobicity, solvent accessibility, or location within the tertiary structure of the enzyme (Table 2). Alanine at the position 281 was replaced by serine at least eleven times within the studied species with nine of replacements taking place within C4 clades and two replacements in C3 species Chenopodium bonus-henricus and Spinacia oleracea (Fig. 1). Methionine at the position 309 was replaced by isoleucine at least four times, all of which within C4 clades (Fig. 1). Only three C4 species, Atriplex spongiosa, A. rosea and Horaninovia ulicina, had both `C4′ amino acids simulteniously. Seven C4 clades of which one was monospecific had `C4′ amino acids, while nine C4 clades of which six consisted of only one species did not have `C4′ amino acids (Fig. 1). More frequent occurrence of `C4′ amino acids in cladesconsisting of many species compared to monospecific clades corresponds to our findings of stronger positive selection within C4 clades (Table 1). Interestingly, both selected residues in C4 Amaranthaceae are among the eight residues selected in 
C4 Cyperaceae and Poaceae [26] and the `C4′ amino acid 309I is also among selected in C4 Flaveria [27]. None of the `C4′ amino acids is fixed among C4 species, but they are more frequent among C4 lineages, ranging from 17 to 35 in C4 Amaranthaceae, and from 14 to 87 in C4 Cyperaceae and Poaceae (Table 2; percentage for C4 Cyperaceae and Poaceae calculated using numbers from [26]). Although `C4′ amino acids are not fixed among all C4 species, there is a significant positive association between their presence and C4 photosynthetic type in Amaranthaceae. Given the existence of C4 species without `C4′ amino acids , it is likely that other as yet unidentified amino acids replacements may be involved in Rubisco adaptation. The model of sequence evolution used to identify Rubisco residues under positive selection within C4 lineages averages selective pressure among selected branches (C4 branches in our case) and hence allows detection only of the most typical substitutions, potentially missing ones that are unique for a particular branch. Other possible explanations are variation in Rubisco kinetic properties not only between C3 and C4 groups of species but also within these groups [3,4,5,23] and putative differences 
in other proteins which form the Rubisco complex (small subunit, Rubisco activase). Although the large subunits contain active sites, changes in small subunits may make significant contribution to kinetic properties of plant and algal Rubiscos [59], including differences observed between C3 and C4 plants [60], and the rbcS genes encoding small subunits have been shown under positive selection in C4 Flaveria [27]. Identical amino-acids in Rubisco of C4 Amaranthaceae and C4 Cyperaceae and Poaceae, representing eudicots and monocots with significantly different anatomy and ecological preferences [22], constitute a remarkable example of parallel molecular evolution in phylogen.Trong selective pressure for acquiring Rubisco with C4 kinetics which then evolves during the stage of optimisation of C4 photosynthesis [58].Parallel amino-acid replacements in Rubisco from phylogenetically distant lineagesBayesian analyses of rbcL sequences in a phylogenetic framework allowed us to identify two residues under directional selection along C4 branches within Amaranthaceae (Table 2). There are no common trends in physicochemical properties of `C4′ amino acids with respect to properties such as 1676428 residue hydrophobicity, solvent accessibility, or location within the tertiary structure of the enzyme (Table 2). Alanine at the position 281 was replaced by serine at least eleven times within the studied species with nine of replacements taking place within C4 clades and two replacements in C3 species Chenopodium bonus-henricus and Spinacia oleracea (Fig. 1). Methionine at the position 309 was replaced by isoleucine at least four times, all of which within C4 clades (Fig. 1). Only three C4 species, Atriplex spongiosa, A. rosea and Horaninovia ulicina, had both `C4′ amino acids simulteniously. Seven C4 clades of which one was monospecific had `C4′ amino acids, while nine C4 clades of which six consisted of only one species did not have `C4′ amino acids (Fig. 1). More frequent occurrence of `C4′ amino acids in cladesconsisting of many species compared to monospecific clades corresponds to our findings of stronger positive selection within C4 clades (Table 1). Interestingly, both selected residues in C4 Amaranthaceae are among the eight residues selected in C4 Cyperaceae and Poaceae [26] and the `C4′ amino acid 309I is also among selected in C4 Flaveria [27]. None of the `C4′ amino acids is fixed among C4 species, but they are more frequent among C4 lineages, ranging from 17 to 35 in C4 Amaranthaceae, and from 14 to 87 in C4 Cyperaceae and Poaceae (Table 2; percentage for C4 Cyperaceae and Poaceae calculated using numbers from [26]). Although `C4′ amino acids are not fixed among all C4 species, there is a significant positive association between their presence and C4 photosynthetic type in Amaranthaceae. Given the existence of C4 species without `C4′ amino acids , it is likely that other as yet unidentified amino acids replacements may be involved in Rubisco adaptation. The model of sequence evolution used to identify Rubisco residues under positive selection within C4 lineages averages selective pressure among selected branches (C4 branches in our case) and hence allows detection only of the most typical substitutions, potentially missing ones that are unique for a particular branch. Other possible explanations are variation in Rubisco kinetic properties not only between C3 and C4 groups of species but also within these groups [3,4,5,23] and putative differences in other proteins which form the Rubisco complex (small subunit, Rubisco activase). Although the large subunits contain active sites, changes in small subunits may make significant contribution to kinetic properties of plant and algal Rubiscos [59], including differences observed between C3 and C4 plants [60], and the rbcS genes encoding small subunits have been shown under positive selection in C4 Flaveria [27]. Identical amino-acids in Rubisco of C4 Amaranthaceae and C4 Cyperaceae and Poaceae, representing eudicots and monocots with significantly different anatomy and ecological preferences [22], constitute a remarkable example of parallel molecular evolution in phylogen.