Homospermidine synthase evolution and the origin(s) of pyrrolizidine alkaloids in Apocynaceae.

Premise: Enzymes that are encoded by paralogous genes and produce identical specialized metabolites in distantly related plant lineages are strong evidence of parallel phenotypic evolution. Inference of phenotypic homology for metabolites produced by orthologous genes is less straightforward, since orthologs may be recruited in parallel into novel pathways. In prior research on pyrrolizidine alkaloids (PAs), specialized metabolites of Apocynaceae, the evolution of homospermidine synthase (HSS), an enzyme of PA biosynthesis, was reconstructed and a single origin of PAs inferred because HSS enzymes of all known PA-producing Apocynaceae species are orthologous and descended from an ancestral enzyme with the 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.
Results: Some evidence supports a single origin of PAs: an IXXXD HSS-like gene, similar in function to VXXXD HSS, evolved in the shared ancestor of all PA-producing species; loss of HSS function 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; and substitutions at sites experiencing positive selection occurred on multiple branches in the HSS-like gene tree.
Conclusions: The complexity of the genotype-function-phenotype map confounds the inference of PA homology from HSS-like gene evolution in Apocynaceae.
(© 2025 The Author(s). American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)