Fig. 6: Some ipecac alkaloid biosynthetic enzymes appear to have arisen independently in C. ipecacuanha and A. salviifolium.

Maximum-likelihood phylogenetic trees of amino acid sequences of pathway enzymes and homologs from other Cornales and Gentianales species. AlphaFold3 models of representative pathway enzymes are shown alongside the different enzyme classes. a, OMTs. Analyzed sequences cluster with different known classes and subclades of OMTs. AsDOMTs form part of the clade of caffeoyl CoA 3-OMTs, which are Mg²⁺-dependent class I OMTs well known to be involved in lignin biosynthesis. All other OMTs of this study are class II Mg²⁺-independent class II OMTs. Both classes share a common ___domain with the same fold but class II OMTs contain an additional ___domain. Class II OMT sequences form two clades: a clade containing OMTs with high sequence similarity and predicted to have caffeic acid O-methylation (COMT) activity and a clade containing OMTs with less sequence similarity and diverse functions (separation of clades has been previously observed⁷⁵). AsDPOMTs are found in the COMT clade, while CiDOMTs and CiDPOMT are part of the diverse clade. A tree containing all bootstrap values is shown in Supplementary Fig. 22. MIA, monoterpendoid indole alkaloids. b, Glucosidases. The protoemetine pathway-specific AsS6DGD is a member of the GH-3 family and, thus, has a different protein fold than the other glucosidases characterized in this study, which are of the GH-1 type. The GH-1 A. salviifolium and C. ipecacuanha GH-1 sequences cluster with sequences from the Cornales or the Gentianales order, respectively; this suggests parallel evolution. A tree containing all bootstrap values is shown in Supplementary Fig. 23. Enzyme names are shown if available. ATASAMT, Arabidopsis thaliana N-acetylserotonin OMT; Ca10OMT, C. acuminata 10-hydroxycamptothecin OMT; TiN10OMT, Tabernanthe iboga noribogaine 10-OMT; Vm16OMT, Vinca minor 16-hydroxyvincadifformine 16-OMT; Cr16OMT, C. roseus 16-hydroxytabersonine OMT; SnvOMT, Strychnos nux-vomica strychnine OMT; GsRH11OMT, Gelsemium sempervirens rankinidine/humatenine-11-OMT; CpOMT1, C. pubescens quinine OMT; AtBGCL3, A. thaliana β-glucosidase 3; SBgl3, Solanum torvum furastonol glycoside glucosidase; AtBGLU21, A. thaliana β-glucosidase 21; AtBG1, A. thaliana β-glucosidase 1; SGD, strictosidine glucosidase; RsRGD, Rauvolfia serpentina raucaffricine glucosidase; CeBGlu1, Centaurium erythraea β-glucosidase.