Comparative Metabolomic Reveals Chemotaxonomic Markers of Resin Fossils for Identification of Botanical Origins

The terpene compounds in amber are secondary metabolites secreted by higher plants. Due to the different secondary metabolites secreted by various plants, these abundant terpene compounds can serve as chemical fingerprints for interpreting the ancient plant sources of amber^[1-2]. However, the existing chemotaxonomy of fossil resins is limited in its ability to distinguish them at phylum, family, and genus levels using small amount of samples^[3-6]. Herein an advanced head-space solid phase microextraction-comprehensive two-dimensional gas chromatography- time-of-flight mass spectrometer platform was established to analyze the complex compositions of resin fossils derived from ancient gymnosperms (e.g., Eocene Cupressaceae, Cretaceous Araucariaceae) and angiosperms (Miocene Dipterocarpaceae and Miocene Hymenaea)^[7-9]. The secondary metabolites including 83 monoterpenes, 186 sesquiterpenes, 84 diterpenes, and 43 n-alkanes, were annotated and then chemically fingerprinted for the first time. Prior to conducting chemical composition classification, this study tested the vitrinite reflectance of resin-bearing rocks and maximum pyrolysis peak temperature (T_max) of the ambers to demonstrate their similar thermal maturity. After that, chemotaxonomic markers were disclosed using multivariate statistical analysis to identify the botanical origins of resin fossils, and four identification rules were concluded. Gymnosperm and angiosperm resins can be distinguished by the relative content of monoterpenes and diterpenes; Eocene Cupressaceae and Cretaceous Araucariaceae resins can be identified by ten chemotaxonomic markers which were screened out by orthogonal partial least-squares regression; Miocene Hymenaea and Dipterocarpaceae resins have different combinations of chemical superclasses, with sesqui- and di-terpenes being the major compositions of Hymenaea resins, and mono- and sesqui- terpenes accounting for most of the compositions of Dipterocarpaceae resins; Specific chemotaxonomic markers can identify Hymenaea mexicana and Hymenaea protera resins. As the chemical components of fossil resins were determined by the genes present in their botanical origins, the chemotaxonomic markers revealed in this study have a genetic basis^[10-12]. These findings provide a comprehensive database of secondary metabolites from ancient resin plants, which can be utilized for further research on their corresponding paleoenvironment and to clarify their phylogeny.