In defense of Apocynaceae: inference on evolution of pyrrolizidine alkaloids from evolution of an enzyme in their biosynthetic pathway, homospermidine synthase

Premise When enzymes encoded by paralogous genes produce identical specialized metabolites in distantly related plant lineages, it is strong evidence of parallel phenotypic evolution. Inference of phenotypic homology for metabolites produced by orthologous genes is not so straightforward, however, since orthologs may be recruited in parallel into novel pathways. Prior research on pyrrolizidine alkaloids (PAs), specialized metabolites of Apocynaceae, reconstructed evolution of homospermidine synthase (HSS), an enzyme of PA biosynthesis, and inferred a single origin of PAs because HSS enzymes of all known PA-producing Apocynaceae species are orthologous and descended from an ancestral enzyme with the predicted motif (VXXXD) of an optimized HSS. Methods We increased sampling, tested the effect of amino acid motif on HSS function, revisited motif evolution, and tested for selection to infer evolution of HSS function and its correlation with phenotype. Key results Some evidence supports a single origin of PAs: an IXXXD HSS, similar in function to VXXXD HSS, evolved in the shared ancestor of all PA-producing species; loss of optimized HSS occurred multiple times via pseudogenization and perhaps via evolution of an IXXXN motif. Other evidence indicates multiple origins: the VXXXD motif, highly correlated with the PA phenotype, evolved two or four times independently; the ancestral IXXXD gene was not under positive selection while some VXXXD genes were; substitutions at sites experiencing positive selection occurred on multiple branches in the HSS -like gene tree. Conclusions Complexity of the genotype-function-phenotype map confounds inference of PA homology from HSS evolution in Apocynaceae. ### Competing Interest Statement The authors have declared no competing interest.