Sco 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 GNE-7915 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 phylogenetically distant groups. This example becomes even more interesting if C3 plants are considered as well. Various groups of C3 plants such as some aquatic species and C3 species from cold habitats have faster but less CO2-specific Rubisco compared with their C3 relatives from terrestrial and warm conditions, respectively [3,23]. Hence, some groups of C3 plants can arrive at the same evolutionary solutions for Rubisco fine-tuning as C4 plants. Indeed, `C4′ amino acids shown for CRubisco Evolution in C4 EudicotsAmaranthaceae in the present study and for C4 monocots and Flaveria previously [26,27], have been reported to be under positive selection in various groups of C3 plants by Kapralov and Filatov [6]. Moreover, residue 309 is among the most frequently positively selected sites in land plants, and although residue 281 itself is not, its close neighbours, residues 279 and 282, are among the most often positively selected ones [6]. Thus, we can conclude that both `C4′ amino acids, 281S and 309I, evolved in parallel in various phylogenetically distant lineages of C3 and C4 plants in which faster but less specific Rubisco was needed. The residue 309 is located on the interface of large subunits within a large subunit dimer, while the residue 281 is involved into dimer-dimer interactions (Table 2). Methionine at position 309 is replaced by the smaller and more hydrophobic isoleucine, which has a stabilising and favourable effect on overall molecule stability according to CUPSAT calculations using spinach pdb-structure [44], while A281S replacement decreases hydrophobicy and may be destabilising (Table 2). Effects of A281S replacement on kinetics of land plants Rubisco has not been studied, while recent study by Whitney et al. [61] using MedChemExpress Gilteritinib mutagenic approach showed that M309I replacement in Flaveria changed Rubisco kinetics from “C3-like” to “C4-like” making 11967625 the enzyme faster but less CO2-specific. Importance of M309I replacement for changes in kinetics of Flaveria Rubisco was predicted using in silico approach similar to one used in the present study [27] and confirmed in planta by the study of Whitney et al. [61] making it a good case in support of further application of phylogeny-based methods for detecting residues under positive selection in Rubisco and elsew.Sco 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 phylogenetically distant groups. This example becomes even more interesting if C3 plants are considered as well. Various groups of C3 plants such as some aquatic species and C3 species from cold habitats have faster but less CO2-specific Rubisco compared with their C3 relatives from terrestrial and warm conditions, respectively [3,23]. Hence, some groups of C3 plants can arrive at the same evolutionary solutions for Rubisco fine-tuning as C4 plants. Indeed, `C4′ amino acids shown for CRubisco Evolution in C4 EudicotsAmaranthaceae in the present study and for C4 monocots and Flaveria previously [26,27], have been reported to be under positive selection in various groups of C3 plants by Kapralov and Filatov [6]. Moreover, residue 309 is among the most frequently positively selected sites in land plants, and although residue 281 itself is not, its close neighbours, residues 279 and 282, are among the most often positively selected ones [6]. Thus, we can conclude that both `C4′ amino acids, 281S and 309I, evolved in parallel in various phylogenetically distant lineages of C3 and C4 plants in which faster but less specific Rubisco was needed. The residue 309 is located on the interface of large subunits within a large subunit dimer, while the residue 281 is involved into dimer-dimer interactions (Table 2). Methionine at position 309 is replaced by the smaller and more hydrophobic isoleucine, which has a stabilising and favourable effect on overall molecule stability according to CUPSAT calculations using spinach pdb-structure [44], while A281S replacement decreases hydrophobicy and may be destabilising (Table 2). Effects of A281S replacement on kinetics of land plants Rubisco has not been studied, while recent study by Whitney et al. [61] using mutagenic approach showed that M309I replacement in Flaveria changed Rubisco kinetics from “C3-like” to “C4-like” making 11967625 the enzyme faster but less CO2-specific. Importance of M309I replacement for changes in kinetics of Flaveria Rubisco was predicted using in silico approach similar to one used in the present study [27] and confirmed in planta by the study of Whitney et al. [61] making it a good case in support of further application of phylogeny-based methods for detecting residues under positive selection in Rubisco and elsew.