Ion on the rbcL gene occured on branches leading to C4 clades and/or within C4 clades. Finally, we address the following question: which amino-acid replacements were associated with transitions from C3 to C4 photosynthesis in Amaranthaceae, and are these replacements unique to this lineage or shared with C4 monocots and/or Flaveria?Materials and Methods Phylogenetic analysisWe obtained all Amaranthaceae rbcL nucleotide sequences available in GenBank and aligned them. Sequences shorter than 1341 base pairs and sequences with missing data were excluded. The resulting trimmed alignment consisted of 179 rbcL sequences of 1341 base pairs long which represented 94 of the rbcL coding region and corresponded to positions 64 to 1404 of the rbcL sequence of Spinacia oleracea (GenBank AJ400848). The analysed dataset consisted of 95 C3 and 84 C4 species (Table S1). Most of the included sequences came from four studies [19,28,29,30] and evenly represented all main lineages within the family (Fig. 1). Phylogeny was reconstructed using a maximum-likelihood inference (ML) conducted with RAxML version 7.2.6 [31] using the raxmlGUI interface [32]. We conducted five independent runs from different starting points to assess convergence within two likelihood units of the best tree, which was consistently selected. The parameters of partition were allowed to vary independently under the GTRGAMMA model of evolution as implemented in RAxML. ML nodal support was calculated by analysing 1000 Verubecestat bootstrap replicates. The best-scoring ML tree was used for tests of positive selection (see below).Tests for positive selectionPositive, neutral, or purifying selection at the molecular level can be inferred by comparing rates of non-synonymous (dN) and synonymous (dS) mutations along a phylogenetic tree [33]. Under neutrality, the two rates are expected to be equal (dN/dS = 1), while purifying (negative) or adaptive (positive) selection is expected to deflate (dN/dS,1) or inflate (dN/dS.1) this ratio, respectively. One can use likelihood ratio tests to detect positive selection that affects only a subset of codons in a protein-coding gene, with positive selection indicated by PHCCC web accelerated nonsynonymous substitutions. Models assuming positive selection along all phylogeny or prespecified branches only (e.g. C4 lineages in our case) can be employed within Phylogenetic Analysis by Maximum Likelihood (PAML) framework [33]. We used the codeml program in the PAML v.4.4 package [33] to estimate dN/dS ratio in the model M0, that allows for a single dN/ dS value across the whole phylogenetic tree obtained previously (see Phylogenetic analyses section). Further, codeml was used to perform likelihood ratio tests (LRTs) for positive selection among aminoRubisco Evolution in C4 Eudicots0.01 Polycnemum perenneNitrophila occidentalis Hemichroa diandra Bosea yervamoraCharpentiera ovata Charpentiera obovata Deeringia amaranthoides5178 89 100Hermbstaedtia glauca Celosia trigyna Celosia argentea Chamissoa altissima100 90Amaranthus greggii Amaranthus tricolorAmaranthus blitum Amaranthus 11967625 hypochondriacus Ptilotus manglesii Pupalia lappacea63Calicorema capitata Pandiaka angustifolia Sericostachys scandens Achyranthes aspera Nototrichium humile Aerva javanica Iresine palmeri96Gomphrena elegans Pseudoplantago friesii Hebanthe occidentalis Blutaparon vermiculare93 73100Guilleminea densa Gomphrena serrata Gomphrena haageana Tidestromia lanuginosa74 100Alternanthera pungens Alternanthera caracasana A.Ion on the rbcL gene occured on branches leading to C4 clades and/or within C4 clades. Finally, we address the following question: which amino-acid replacements were associated with transitions from C3 to C4 photosynthesis in Amaranthaceae, and are these replacements unique to this lineage or shared with C4 monocots and/or Flaveria?Materials and Methods Phylogenetic analysisWe obtained all Amaranthaceae rbcL nucleotide sequences available in GenBank and aligned them. Sequences shorter than 1341 base pairs and sequences with missing data were excluded. The resulting trimmed alignment consisted of 179 rbcL sequences of 1341 base pairs long which represented 94 of the rbcL coding region and corresponded to positions 64 to 1404 of the rbcL sequence of Spinacia oleracea (GenBank AJ400848). The analysed dataset consisted of 95 C3 and 84 C4 species (Table S1). Most of the included sequences came from four studies [19,28,29,30] and evenly represented all main lineages within the family (Fig. 1). Phylogeny was reconstructed using a maximum-likelihood inference (ML) conducted with RAxML version 7.2.6 [31] using the raxmlGUI interface [32]. We conducted five independent runs from different starting points to assess convergence within two likelihood units of the best tree, which was consistently selected. The parameters of partition were allowed to vary independently under the GTRGAMMA model of evolution as implemented in RAxML. ML nodal support was calculated by analysing 1000 bootstrap replicates. The best-scoring ML tree was used for tests of positive selection (see below).Tests for positive selectionPositive, neutral, or purifying selection at the molecular level can be inferred by comparing rates of non-synonymous (dN) and synonymous (dS) mutations along a phylogenetic tree [33]. Under neutrality, the two rates are expected to be equal (dN/dS = 1), while purifying (negative) or adaptive (positive) selection is expected to deflate (dN/dS,1) or inflate (dN/dS.1) this ratio, respectively. One can use likelihood ratio tests to detect positive selection that affects only a subset of codons in a protein-coding gene, with positive selection indicated by accelerated nonsynonymous substitutions. Models assuming positive selection along all phylogeny or prespecified branches only (e.g. C4 lineages in our case) can be employed within Phylogenetic Analysis by Maximum Likelihood (PAML) framework [33]. We used the codeml program in the PAML v.4.4 package [33] to estimate dN/dS ratio in the model M0, that allows for a single dN/ dS value across the whole phylogenetic tree obtained previously (see Phylogenetic analyses section). Further, codeml was used to perform likelihood ratio tests (LRTs) for positive selection among aminoRubisco Evolution in C4 Eudicots0.01 Polycnemum perenneNitrophila occidentalis Hemichroa diandra Bosea yervamoraCharpentiera ovata Charpentiera obovata Deeringia amaranthoides5178 89 100Hermbstaedtia glauca Celosia trigyna Celosia argentea Chamissoa altissima100 90Amaranthus greggii Amaranthus tricolorAmaranthus blitum Amaranthus 11967625 hypochondriacus Ptilotus manglesii Pupalia lappacea63Calicorema capitata Pandiaka angustifolia Sericostachys scandens Achyranthes aspera Nototrichium humile Aerva javanica Iresine palmeri96Gomphrena elegans Pseudoplantago friesii Hebanthe occidentalis Blutaparon vermiculare93 73100Guilleminea densa Gomphrena serrata Gomphrena haageana Tidestromia lanuginosa74 100Alternanthera pungens Alternanthera caracasana A.
