From differentially accumulated volatiles to the search of robust metabolic classifiers: Exploring the volatome of Citrus leaves

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Although genetic differences among Citrus species are well characterized, few are known about how they may affect the small molecules profile of the Citrus leaf. In this study, we analyzed, qualitatively and quantitatively using gas chromatography coupled to mass spectrometry (GC–MS), 57 volatile chemicals in the leaves of 4 Citrus species. These compounds include 18 monoterpenes, 12 sesquiterpenes, 15 terpenic alcohols and aldehydes, 2 aliphatic alcohols and aldehydes and 5 esters. PCA and HCA, based on metabolite profiling data, and according to the chemotypes found in the leaf, revealed the existence of four distinguished groups of leaf types and species associated to specific composition patterns. Clusters based on similarity of metabolic features were largely congruent with the species phylogeny. These results clearly demonstrate that leaf metabolite profiles of Citrus species could be exploited for the taxonomical and phylogenetic studies of the genus and are complementary to the existing taxonomic evidence. A cross comparison of the leaf metabolome overlap among Citrus species revealed the presence of a core of nine metabolites systematically found in the leaf tissue. After using and comparing several chemometric methods, it was possible to narrow the number of taxonomic classifiers to eight (linalyl acetate, geranyl acetate, neryl acetate, β-cis-Ocimene, ∆-3-Carene, β-Myrcene, sabinene and α-Terpineol), while increasing the differentiation resolution. Along with morphometric and molecular genetic studies, the present metabometric approach will definitely have a high impact on further elucidation of complex relationships among taxa within the Citrus genus.