The de novo evolution of genes and the novel proteins they encode has stimulated much interest in the contribution such innovations make to the diversity of life. Most research on this de novo evolution focuses on transcripts, so studies on the biochemical steps that can enable completely new proteins to evolve and the time required to do so have been lacking. Sunflower PawS1 is an unusual albumin precursor because in addition to producing albumin it also yields a potent, bicyclic

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... itor called SFTI-1. Here we show how this inhibitor peptide evolved stepwise over tens of millions of years. To trace the origin of the inhibitor peptide SFTI-1 we assembled seed transcriptomes for 110 sunflower relatives whose evolution could be resolved by a chronogram, which allowed dates to be estimated for the various stages of molecular evolution. A genetic insertion event in an albumin precursor gene ~45 million years ago introduced two additional cleavage sites for protein maturation and conferred duality upon PawS1-Like genes such that they also encode a small buried macrocycle. Expansion of this region, including two Cys residues, enlarged the peptide ~34 million years ago and made the buried peptides bicyclic. Functional specialization into a protease inhibitor occurred ~23 million years ago. These findings document the evolution of a novel peptide inside a benign region of a pre-existing protein. We illustrate how a novel peptide can evolve without de novo gene evolution and, critically, without affecting the function of what becomes the protein host. One sentence summary: A peptide is born within another protein and evolves stepwise into a protease inhibitor Drummond AJ, Suchard MA, Xie D, Rambaut A. 2012. Bayesian Phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution 29:1969-1973. Dunse KM, Stevens JA, Lay FT, Gaspar YM, Heath RL, Anderson MA. 2010. Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field.