Nymphaelaes A Magnolids Monocots Ranunculales Proteales Trochodendrales Buxales Gunnerales Saxifragales Vitales Rosids Caryophyllales Cornales FUL-Like Ericales Asterids Gunnerales Saxifragales Vitales Rosids FUL Caryophyllales Cornales Ericales Asterids Gunnerales Saxifragales Vitales Rosids euAP1 Caryophyllales Cornales Ericales Asterids Amborellales Nymphaelaes B Austrobaileyales Magnolids Chloranthales Monocots Ranunculales Proteales Trochodendrales Buxales Gunnerales Saxifragales Vitales Rosids Dilleniales Santanales TM6 Caryophyllales Cornales Ericales Asterids Saxifragales Vitales Rosids Dilleniales Caryophyllales Cornales euAP3 Ericales Asterids Amborellales Nymphaelaes C Austrobaileyales Magnolids Chloranthales Monocots Ranunculales Proteales Trochodendrales Buxales Gunnerales Saxifragales Vitales Rosids Dilleniales Caryophyllales Cornales PI Ericales Asterids Amborellales D Nymphaelaes Magnolids Monocots Ranunculales Proteales Trochodendrales Buxales Gunnerales Saxifragales Vitales Rosids euAG Caryophyllales Cornales Ericales Asterids Gunnerales Saxifragales Vitales Rosids SHP Dilleniales Asterids Amborellales E Chloranthales Magnolids Monocots Ranunculales Proteales Buxales Gunnerales Saxifragales Vitales Rosids STK Caryophyllales Ericales Asterids Amborellales Nymphaelaes F Magnolids Monocots Ranunculales Proteales Trochodendrales Buxales Gunnerales Saxifragales Vitales Rosids Caryophyllales Cornales FBP9 Ericales Asterids Vitales Rosids Caryophyllales SEP4 Cornales Asterids Gunnerales Saxifragales Vitales Rosids SEP1/2 Caryophyllales Cornales Ericales Asterids Amborellales Chloranthales G Magnolids Monocots Ranunculales Proteales Trochodendrales Buxales Gunnerales Vitales Rosids Asterids SEP3 Caryophyllales Cornales Ericales Asterids Amborellales H Nymphaelaes Magnolids Monocots Ranunculales Proteales Buxales Saxifragales Vitales Rosids SVP Caryophyllales Cornales Ericales Asterids Saxifragales Vitales Rosids Dilleniales Caryophyllales StMADS11 Ericales Asterids Gunnerales Vitales Rosids AGL24 Dilleniales Caryophyllales Cornales Ericales Asterids Amborellales Nymphaelaes I Magnolids Monocots Ranunculales Proteales Trochodendrales Buxales Saxifragales Vitales Rosids Santanales Caryophyllales Cornales FBP22 Asterids Gunnerales Vitales Rosids Berberidopsidales AGL14 Cornales Ericales Asterids Saxifragales Vitales Rosids TM3 Dilleniales Berberidopsidales Cornales Asterids Supplemental Figure 1: Simplified phylogenetic trees of the MADS-box gene subfamilies used for ASR and selective pressure estima- tion. AP1- (A), AP3- (B), PI- (C), AG- (D), STK- (E), SEP1/2/4 (F), SEP3 (G), SVP/AGL24 (H), SOC1- (I) gene lineages. Arrows denote the nodes used for ASR before the γ triplication (blue) and at the Rosid/Asterid split (green). Red arrows denote the nodes for which no reliable ancestral genes could be reconstructed. ancAP1 pre-γ ancPI post-γ ancPI 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 31 41 51 61 71 81 91 1 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 11 11 11 2 2 101 121 131 141 151 161 171 181 191 201 101 121 131 141 151 161 171 181 191 201 221 231 241 251 101 121 131 141 151 161 171 181 191 201 MADS I K C MADS I K C MADS I K C anceuAP1 ancFUL-LIKE ancFUL 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 11 11 11 2 2 2 101 121 131 141 151 161 171 181 191 201 221 231 241 101 121 131 141 151 161 171 181 191 201 221 231 241 251 101 121 131 141 151 161 171 181 191 201 221 231 241 MADS I K C MADS I K C MADS I K C ancAP3 anceuAP3 ancTM6 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 11 11 11 2 2 2 101 121 131 141 151 161 171 181 191 201 221 231 101 121 131 141 151 161 171 181 191 201 221 101 121 131 141 151 161 171 181 191 201 221 MADS I K C MADS I K C MADS I K C ancAG anceuAG ancSHP 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 C 0.4 0.2 0.2 0.2 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 11 11 11 2 2 101 121 131 141 151 161 171 181 191 201 101 121 131 141 151 161 171 181 191 201 101 121 131 141 151 161 171 181 191 201 MADS I K C MADS I K C MADS I K C pre-γ ancSTK post-γ ancSTK 1 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0 0 1 1 1 1 1 1 1 1 1 1 1 1 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 11 11 2 2 101 121 131 141 151 161 171 181 191 201 221 101 121 131 141 151 161 171 181 191 201 221 MADS I K C MADS I K C ancSEP1/2/4 pre-γ ancSEP3 post-γ ancSEP3 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 1 1 21 31 41 51 61 71 81 91 11 11 2 2 11 101 121 131 141 151 161 171 181 191 201 221 231 241 101 121 131 141 151 161 171 181 191 201 221 231 241 2 101 121 131 141 151 161 171 181 191 201 221 231 241 MADS I K C MADS I K C MADS I K C ancSEP1/2 ancSEP4 ancFBP9 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 31 41 51 61 71 81 91 1 1 1 1 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 11 2 11 101 121 131 141 151 161 171 181 191 201 221 231 241 251 11 2 2 101 121 131 141 151 161 171 181 191 201 221 231 241 251 101 121 131 141 151 161 171 181 191 201 221 231 241 251 MADS I K C MADS I K C MADS I K C ancSVP/AGL24 ancSVP ancAGL24 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 31 41 51 61 71 81 91 1 1 1 1 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 11 2 11 11 101 121 131 141 151 161 171 181 191 201 221 231 2 2 101 121 131 141 151 161 171 181 191 201 221 231 101 121 131 141 151 161 171 181 191 201 221 231 MADS I K C MADS I K C MADS I K C ancSOC1 ancFBP22 ancTM3 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 31 41 51 61 71 81 91 1 1 1 1 21 31 41 51 61 71 81 91 21 31 41 51 61 71 81 91 11 2 11 11 101 121 131 141 151 161 171 181 191 201 2 2 101 121 131 141 151 161 171 181 191 201 221 101 121 131 141 151 161 171 181 191 201 221 MADS I K C MADS I K C MADS I K C Supplemental Figure 2: Ancestral sequences and their accuracy. Dots represent the posterior probabilities (y-axis) for each amino acid site (x-axis) for all reconstructed genes. Arrows denote ambiguous sites defined as sites for which the most likely amino acid has a posterior probability lower than 0.80 and that have an alternative amino acid state with a posterior probability higher than 0.2. Green arrows represent ambiguous sites of which the most likely amino acids are biochemically similar (S and T; V, L and I; D and E; N and Q; K and R were considered similar). A Gene subfamilies AP1 SQUA/AP1/FUL CAL Overlapping FUL PPIs only in de Folter et al (2005) DEF/AP3 AP3 PPIs only in this study GLO/PI PI No interactions detected AG AG SHP1 SHP2 STK STK SEP1 SEP1/2/4 SEP2 SEP4 SEP3 SEP3 SVP SVP/ StMADS11 AGL24 AGL42 SOC1/TM3 SOC1 AP1 CAL FUL AP3 PI AG SHP1 SHP2 STK SEP1 SEP2 SEP4 SEP3 SVP AGL24 AGL42 SOC1 Gene s ubfamilies B MC TM4 Overlapping SQUA/AP1/FU L SLMBP 7 PPIs only in Leseberg et al (2008) PPIs only in this study SLMBP2 0 No interactions detected TAP3 DEF/AP 3 TM6 LeP I GLO/P I TP I TAG1 AG TAGL1 SLMBP 3 STK TAGL1 1 TM29 RIN SEP1/2 /4 LeMADS1 SLMBP21 SEP3 TM5 JOINTLESS SVP/S tMADS11 SLMBP2 4 SLMBP1 8 SOC1/TM 3 TM3 MC TM4 SLMBP7 SLMBP20 TAP3 TM6 LeP I TP I TAG1 TAGL1 SLMBP3 TAGL11 TM29 RIN LeMADS1 SLMBP21 TM5 JOINTLESS SLMBP24 SLMBP18 TM3 C Arabidopsis Interaction No interaction Total Sensitivity 0,6052632 This study Interaction 23 16 39 Accuracy 0,7973856 No interaction 15 99 114 Precision 0,5897436 Total 38 115 153 Specificity 0,8608696 Solanum Interaction No interaction Total Sensitivity 0,4125 This study Interaction 33 12 45 Accuracy 0,7445887 No interaction 47 139 186 Precision 0,7333333 Total 80 151 231 Specificity 0,9205298 Arabidopsis/Solanum Interaction No interaction Total Sensitivity 0,4745763 This study Interaction 56 28 84 Accuracy 0,765625 No interaction 62 238 300 Precision 0,6666667 Total 118 266 384 Specificity 0,8947368 Supplemental Figure 3: Comparison of the PPIs obtained in this study with the determined PPIs from previous published literature.