Comprehensive mass spectrometry‐guided phenotyping of plant specialized metabolites reveals metabolic diversity in the cosmopolitan plant family Rhamnaceae

Summary Plants produce a myriad of specialized metabolites to overcome their sessile habit and combat biotic as well as abiotic stresses. Evolution has shaped the diversity of specialized metabolites, which then drives many other aspects of plant biodiversity. However, until recently, large‐scale studies investigating the diversity of specialized metabolites in an evolutionary context have been limited by the impossibility of identifying chemical structures of hundreds to thousands of compounds in a time‐feasible manner. Here we introduce a workflow for large‐scale, semi‐automated annotation of specialized metabolites and apply it to over 1000 metabolites of the cosmopolitan plant family Rhamnaceae. We enhance the putative annotation coverage dramatically, from 2.5% based on spectral library matches alone to 42.6% of total MS/MS molecular features, extending annotations from well‐known plant compound classes into dark plant metabolomics. To gain insights into substructural diversity within this plant family, we also extract patterns of co‐occurring fragments and neutral losses, so‐called Mass2Motifs, from the dataset; for example, only the Ziziphoid clade developed the triterpenoid biosynthetic pathway, whereas the Rhamnoid clade predominantly developed diversity in flavonoid glycosides, including 7‐O‐methyltransferase activity. Our workflow provides the foundations for the automated, high‐throughput chemical identification of massive metabolite spaces, and we expect it to revolutionize our understanding of plant chemoevolutionary mechanisms. Significance Statement Tandem mass spectrometry (MS/MS) has become a popular tool for untargeted metabolomics study, but annotation, classification and identification of metabolites are still significant obstacles in MS/MS‐based plant metabolomics workflows. This study introduces a next‐generation data analysis strategy which provides structural insight into hundreds of specialized metabolites at the level of both chemical class and diversified scaffolds; the efficacy of the workflow was demonstrated by revealing the chemical space of the Rhamnaceae family.