Abstract Specialized metabolite (SM) diversification is a core process to plants’ adaptation to diverse ecological niches. Here we implemented a computational mass spectrometry (MS)-based metabolomics approach to explore SM diversification in tissues of 20 species covering Nicotiana phylogenetics sections. To drastically increase metabolite annotation, we created a large in silico fragmentation database, comprising more than 1 million structures, and scripts for connecting class prediction to consensus substructures. Altogether, the approach provides an unprecedented cartography of SM diversity and section-specific innovations in this genus. As a case-study, and in combination with NMR and MS imaging, we explored the distribution of N- acyl nornicotines, alkaloids predicted to be specific to Repandae allopolyploids, and revealed their prevalence in the genus, albeit at much lower magnitude, as well as a greater structural diversity than previously thought. Altogether, the novel data integration approaches provided here should act as a resource for future research in plant SM evolution. Teaser Computational metabolomics delineates main trends in the diversification of specialized metabolism in the genus Nicotiana

Evolutionary metabolomics of specialized metabolism diversification in the genus<i>Nicotiana</i>highlights allopolyploidy-mediated innovations in<i>N</i>-acylnornicotine metabolism

Evolutionary metabolomics of specialized metabolism diversification in the genusNicotianahighlights allopolyploidy-mediated innovations inN-acylnornicotine metabolism