Shedding Light on Chemically Mediated Tri-Trophic Interactions: A 1H-NMR Network Approach to Identify Compound Structural Features and Associated Biological Activity

Abstract

Diverse mixtures of plant natural products play an important role in plant-herbivore-parasitoid interactions. In the pursuit of understanding these chemically-mediated interactions, we are often faced with the challenge of determining ecologically and biologically relevant compounds present in complex phytochemical mixtures. Using a network-based approach, we analyzed binned 1H-NMR data from 196 prepared mixtures of commonly studied secondary metabolites including alkaloids, amides, terpenes, iridoid glycosides, saponins, phenylpropanoids, flavonoids and phytosterols. The mixtures included multiple dimensions of chemical diversity, including molecular complexity, mixture complexity and differences in relative compound concentrations. This approach yielded modules of co-occurring chemical shifts that were correlated with specific compounds or common structural features shared across compounds. This approach was then applied to crude phytochemical extracts of 31 species in the phytochemically diverse tropical plant genus Piper (Piperaceae). Combining the activity of crude plant extracts in an array of bioassays with our 1H-NMR network approach, we identified a potential prenylated benzoic acid from these mixtures that exhibits antifungal properties and identified small structural differences that were potentially responsible for antifungal activity. In an intraspecific analysis of individual Piper kelleyi plants, we also found ontogenetic differences in chemistry that may affect natural plant enemies. In sum, this approach allowed us to characterize mixtures as useful network modules and to combine chemical and ecological datasets to identify biologically important compounds from crude extracts.

The many dimensions of phytochemical diversity: linking theory to practice

“…However, the reality is that the vast majority of structures of phytochemicals are undescribed, making these metrics inapplicable. Studies in ecology are just beginning to calculate the structural similarity metrics for complex mixtures of undescribed compounds using either raw MS‐MS fragmentation data (Sedio ; Sedio et al ) or NMR data (Richards et al ; Richards et al ). These methods use spectral features to generate measures of structural similarity among compounds, which can then be visualised using molecular networks (e.g.…”

“…A surge of recent research, fuelled by metabolomics and other modern approaches in analytical chemistry (Hartley et al ; Sedio ; Dyer et al ; Richards et al ), has finally begun to address the ecological and evolutionary roles of phytochemical diversity itself. This new line of research has been enabled by a key conceptual advance – the use of concepts and metrics from the species diversity literature (Iason et al ; Dyer et al ; Moore et al ; Marion et al ).…”

Evidence for both phylogenetic conservatism and lability in the evolution of secondary chemistry in a tropical angiosperm radiation

“…Piper is a hyper-diverse lineage in which phytochemical variation has influenced evolutionary and ecological processes and shaped complex tropical communities (e.g., Salazar et al, 2016;Richards et al, 2018). However, there have been limitations in both the degree of phylogenetic resolution and the understanding of phytochemical variation in this group.…”

“…Piper (Piperaceae) is a highly diverse, pantropical genus of nearly 2,600 accepted species (Callejas-Posada, 2020), with the highest diversity occurring in the Neotropics (Gentry, 1993;Martínez et al, 2015). Chemically, Piper is impressively diverse (Parmar et al, 1997;Dyer & Palmer, 2004;Richards et al, 2015): chemical profiling in a modest number of taxa has yielded 667 different compounds from 11 distinct structural classes thus far (Parmar et al, 1997;Kato & Furlan, 2007;Richards et al, 2018). This phytochemical diversity has likely contributed to the diversification of several herbivorous insect lineages that specialize on Piper, including most notably the geometrid moth genus Eois (Strutzenberger et al, 2012;Wilson et al, 2012;Jahner et al, 2017).…”

“…This phytochemical diversity has likely contributed to the diversification of several herbivorous insect lineages that specialize on Piper, including most notably the geometrid moth genus Eois (Strutzenberger et al, 2012;Wilson et al, 2012;Jahner et al, 2017). Furthermore, phytochemical variation in Piper communities has been shown to shape tri-trophic interactions and the structure of tropical communities Glassmire et al, 2016;Richards et al, 2018). As a species-rich genus with abundant and ecologically consequential phytochemical variation, Piper represents a valuable system for understanding how the history of diversification underlies the evolution of phytochemical variation.…”