Iversity of kinetics better related to species ecology than phylogeny [4]. All eight residues shown under selection in Amaranthaceae using SLR and PAML models M2 and M8 were already shown to be under Darwinian selection in other groups of plants [6]. Five of these residues (145, 225, 262, 279 and 439) were among twenty most commonly selected Rubisco large subunit residues [6]. Findings in Amaranthaceae are in agreement with the previously described uneven distribution of putative fine-tuning residues in Rubisco [6]. Residues 43, 145, 225, 262 and 279 had only twoResults Phylogenetic analysisThe ML phylogenetic tree (Fig. 1) for rbcL sequences from 179 Amaranthaceae species was largely congruent with previously obtained phylogenies and accepted taxonomic subdivisions of the family [19,28,29,30,45,46,47,48]; however no statistical tests for topological similarity between our tree and previously published trees were performed because of different sizes and species compositions of datasets. A minimum of 16 independent origins of C4 photosynthesis were represented in the Amaranthaceae phylogeny if conservative approach for observed polytomies had been taken (Fig. 1), which is consistent with the PZ-51 web estimate by Sage et al. [16]. The other assumption of this estimate was that no reversals from C4 to C3 were allowed. Predominance of C4 gains over reversals to C3 is supported by both empirical data and theoretical work [49].Tests for positive selectionLikelihood ratio tests (LRTs) for variation in dN/dS ratios and for positive selection [33] were applied to the dataset of rbcL sequences from 179 C3 and C4 Amaranthaceae species. LRTs that were run using two different initial dN/dS values (0.1 and 0.4) to test for suboptimal local peaks produced identical results. LRTs for positive selection [33] showed that the models assuming positive selection (M2a and M8) fit the data better than the nested models without positive selection (M1a and M8a; p-value ,0.00001;Rubisco Evolution in C4 EudicotsTable 2. Characteristics of amino-acid replacements under positive selection in the C4 lineages of Amaranthaceae.AA No.aAA order 520-26-3 changes `C3’R`C4’Type of changesbDHcDPdDVeSAf ( )DGg (kJ/mol)RFPS ( ) hC3/ C4 species iLocation of residueStructural motifs ?within 5 AInteractionsj281A MR RS IHN R UP HN R HN22.6 2.1.1 20.0.4 3.0.00 8.DS (210.6) S (21.3)2.7 19.2.1/34.5 0.0/16.Helix 4 Strand FHelices 4, 5 Strand E; Helices F,DD IDAmino acid (AA) numbering is based on the spinach sequence after [63]. Side chain type changes. Types abbreviations: H ?hydrophobic; N ?nonpolar aliphatic; P ?polar uncharged; U ?hydrophilic (after [64]). Hydropathicity difference [65]. d Polarity difference [66]. e van der Waals volume difference [67]. f Solvent accessibility calculated using the spinach structure (pdb file 1RBO) by CUPSAT [44]. g Overall stability of the protein predicted using the spinach structure (pdb file 1RBO) by CUPSAT [44]. DS ?destabilizing, S ?stabilizing. h RFPS ?relative frequency of the particular residue to be under positive selection in C3 plants. Data from 112 rbcL datasets with detected positive selection from [6]. i Percentage of C3 and C4 species that have `C4′ amino acid among the 95 C3 species and 84 C4 species of Amaranthaceae analysed. j ?Interactions in which the selected residues and/or residues within 5 A of them are involved. ID ?intradimer interactions; DD ?dimer-dimer interactions (after [63]). doi:10.1371/journal.pone.0052974.tb caalternative amino acids.Iversity of kinetics better related to species ecology than phylogeny [4]. All eight residues shown under selection in Amaranthaceae using SLR and PAML models M2 and M8 were already shown to be under Darwinian selection in other groups of plants [6]. Five of these residues (145, 225, 262, 279 and 439) were among twenty most commonly selected Rubisco large subunit residues [6]. Findings in Amaranthaceae are in agreement with the previously described uneven distribution of putative fine-tuning residues in Rubisco [6]. Residues 43, 145, 225, 262 and 279 had only twoResults Phylogenetic analysisThe ML phylogenetic tree (Fig. 1) for rbcL sequences from 179 Amaranthaceae species was largely congruent with previously obtained phylogenies and accepted taxonomic subdivisions of the family [19,28,29,30,45,46,47,48]; however no statistical tests for topological similarity between our tree and previously published trees were performed because of different sizes and species compositions of datasets. A minimum of 16 independent origins of C4 photosynthesis were represented in the Amaranthaceae phylogeny if conservative approach for observed polytomies had been taken (Fig. 1), which is consistent with the estimate by Sage et al. [16]. The other assumption of this estimate was that no reversals from C4 to C3 were allowed. Predominance of C4 gains over reversals to C3 is supported by both empirical data and theoretical work [49].Tests for positive selectionLikelihood ratio tests (LRTs) for variation in dN/dS ratios and for positive selection [33] were applied to the dataset of rbcL sequences from 179 C3 and C4 Amaranthaceae species. LRTs that were run using two different initial dN/dS values (0.1 and 0.4) to test for suboptimal local peaks produced identical results. LRTs for positive selection [33] showed that the models assuming positive selection (M2a and M8) fit the data better than the nested models without positive selection (M1a and M8a; p-value ,0.00001;Rubisco Evolution in C4 EudicotsTable 2. Characteristics of amino-acid replacements under positive selection in the C4 lineages of Amaranthaceae.AA No.aAA changes `C3’R`C4’Type of changesbDHcDPdDVeSAf ( )DGg (kJ/mol)RFPS ( ) hC3/ C4 species iLocation of residueStructural motifs ?within 5 AInteractionsj281A MR RS IHN R UP HN R HN22.6 2.1.1 20.0.4 3.0.00 8.DS (210.6) S (21.3)2.7 19.2.1/34.5 0.0/16.Helix 4 Strand FHelices 4, 5 Strand E; Helices F,DD IDAmino acid (AA) numbering is based on the spinach sequence after [63]. Side chain type changes. Types abbreviations: H ?hydrophobic; N ?nonpolar aliphatic; P ?polar uncharged; U ?hydrophilic (after [64]). Hydropathicity difference [65]. d Polarity difference [66]. e van der Waals volume difference [67]. f Solvent accessibility calculated using the spinach structure (pdb file 1RBO) by CUPSAT [44]. g Overall stability of the protein predicted using the spinach structure (pdb file 1RBO) by CUPSAT [44]. DS ?destabilizing, S ?stabilizing. h RFPS ?relative frequency of the particular residue to be under positive selection in C3 plants. Data from 112 rbcL datasets with detected positive selection from [6]. i Percentage of C3 and C4 species that have `C4′ amino acid among the 95 C3 species and 84 C4 species of Amaranthaceae analysed. j ?Interactions in which the selected residues and/or residues within 5 A of them are involved. ID ?intradimer interactions; DD ?dimer-dimer interactions (after [63]). doi:10.1371/journal.pone.0052974.tb caalternative amino acids.
