Plants produce a multitude of metabolites that contribute to their fitness and survival, and play a role in local adaptation to environmental conditions. The effects of environmental variation is particularly well studied within the genus Plantago, however, previous studies have largely focused on targeting specific metabolites. Studies exploring metabolite wide changes are lacking, and the effects of natural environmental variation and herbivory on the metabolomes of plants growing in situ remain unknown. An untargeted metabolomic approach using ultra-high performance liquid chromatography-mass spectrometry, coupled with variation partitioning, general linear mixed modelling, and network analysis was used to detect differences in metabolic phenotypes of Plantago major in fifteen natural populations across Denmark. Geographic region, distance, habitat type, phenological stage, soil parameters, light levels, and leaf
area, were investigated for their relative contributions to explaining differences in foliar metabolomes. Herbivory effects were further investigated by comparing metabolomes from damaged and undamaged leaves from each plant. Geographic region explained the greatest number of significant metabolic differences. Soil pH had the second largest effect, followed by habitat and leaf area, whilst phenological stage had no effect. No evidence of the induction of metabolic features was found between leaves damaged by herbivores compared to undamaged leaves on the same plant. Differences in metabolic phenotypes explained by geographic factors are attributed to genotypic variation and/or unmeasured environmental factors that differ at the regional level in Denmark. A small number of specialised features in the metabolome may be involved in
facilitating the success of a widespread species such as Plantago major into such wide range of environmental conditions, though overall resilience in the metabolome was found in response to environmental parameters tested. Untargeted metabolomic approaches have great potential to improve our understanding of how specialised plant metabolites respond to environmental change and assist in adaptation to local conditions.