In this study, we examine the interface between seed dispersal, seed predation, and herbivory within the phytochemical traits of 12 sympatric species of Piper (Piperaceae). We use comparative metabolomics to explore whether and how differential selective pressures and constraints across reproductive and vegetative tissues have shaped tissue- and species-level diversification and distribution of secondary metabolites. Our specific objectives are to:
A) Describe the occurrence patterns of secondary metabolites across reproductive and vegetative tissues in the focal species. This is done using untargeted LC-MS-MS based metabolomics and molecular networking, along with in-silico fragmentation modeling, machine learning, and distance-based comparisons of tissue- and species-level patterns of metabolite occurrence and structural relationships. As exploratory work, this aims to inform our understanding of the potential functions of particular secondary metabolites or classes of metabolites, based on where they occur in the plant.
B) Test the hypothesis that animal-dispersed fruit acts as an incubator of phytochemical diversification in plants, owing to the variety of fruit-focused ecological interactions as well as the direct fitness consequences of those interactions. To accomplish this, we compare multiple dimensions of phytochemical diversity across leaves and fruit tissues, both based on secondary metabolite occurrence and on metabolite structural relationships.
Results/Conclusions
A) Among 1312 secondary metabolites found across species and tissue types, 92 were found only in fruit, while 4 were restricted to leaves. Random forest analysis indicated that the secondary metabolites most predictive of tissue type were spread across all major structural classes represented within the study. The high proportion of metabolites shared across fruits and leaves suggests that the defensive functional characteristics most often ascribed to foliar secondary metabolites may also apply in fruit. Meanwhile, the occurrence of fruit-specific metabolites across a range of structural classes suggests that modifications of function are often required to suit the fruit-specific constraints of concurrent defense against antagonists and palatability to mutualists.
B) In their alpha diversity of secondary metabolites, leaves and fruit exhibited a strong interaction (LMM; p < 0.001) between tissue type and species, illustrating the wide variation from species to species in the relative richness of each organ. Meanwhile, fruit exceeded leaves in secondary metabolite beta diversity (βw ; p < 0.001) and in chemical structural dissimilarity (cosθ ; p < 0.001). Our results point to limited physiological or evolutionary constraints on the divergence of secondary metabolomes across plant tissues, along with a peak of chemical diversity in reproductive tissues.