Metabolomic analysis reveals reliance on secondary plant metabolites to facilitate carnivory in the Cape sundew, Drosera capensis.

BACKGROUND AND AIMS: Secondary metabolites are integral to multiple key plant processes (growth regulation, pollinator attraction and interactions with conspecifics, competitors and symbionts) yet their role in plant adaptation remains an underexplored area of research. Carnivorous plants use secondary metabolites to acquire nutrients from prey, but the extent of the role of secondary metabolites in plant carnivory is not known. We aimed to determine the extent of the role of secondary metabolites in facilitating carnivory of the Cape sundew, Drosera capensis. METHODS: We conducted metabolomic analysis of 72 plants in a time-series experiment before and after simulated prey capture. We used ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and the retention time index to identify compounds in the leaf trap tissue that changed up to 72 h following simulated prey capture. We identified associated metabolic pathways, and cross-compared these compounds with metabolites previously known to be involved in carnivorous plants across taxa. KEY RESULTS: For the first time in a carnivorous plant, we have profiled the whole-leaf metabolome response to prey capture. Reliance on secondary plant metabolites was higher than previously thought - 2383 out of 3257 compounds in fed leaves had statistically significant concentration changes in comparison with unfed controls. Of these, ~34 compounds are also associated with carnivory in other species; 11 are unique to Nepenthales. At least 20 compounds had 10-fold changes in concentration, 12 of which had 30-fold changes and are typically associated with defence or attraction in non-carnivorous plants. CONCLUSIONS: Secondary plant metabolites are utilized in plant carnivory to an extent greater than previously thought - we found a whole-metabolome response to prey capture. Plant carnivory, at the metabolic level, likely evolved from at least two distinct functions: attraction and defence. Findings of this study support the hypothesis that secondary metabolites play an important role in plant diversification and adaptation to new environments.

The function of secondary metabolites in plant carnivory.

BACKGROUND: Carnivorous plants are an ideal model system for evaluating the role of secondary metabolites in plant ecology and evolution. Carnivory is a striking example of convergent evolution to attract, capture and digest prey for nutrients to enhance growth and reproduction and has evolved independently at least ten times. Though the roles of many traits in plant carnivory have been well studied, the role of secondary metabolites in the carnivorous habit is considerably less understood. SCOPE: This review provides the first synthesis of research in which secondary plant metabolites have been demonstrated to have a functional role in plant carnivory. From these studies we identify key metabolites for plant carnivory and their functional role, and highlight biochemical similarities across taxa. From this synthesis we provide new research directions for integrating secondary metabolites into understanding of the ecology and evolution of plant carnivory. CONCLUSIONS: Carnivorous plants use secondary metabolites to facilitate prey attraction, capture, digestion and assimilation. We found ~170 metabolites for which a functional role in carnivory has been demonstrated. Of these, 26 compounds are present across genera that independently evolved a carnivorous habit, suggesting convergent evolution. Some secondary metabolites have been co-opted from other processes, such as defence or pollinator attraction. Secondary metabolites in carnivorous plants provide a potentially powerful model system for exploring the role of metabolites in plant evolution. They also show promise for elucidating how the generation of novel compounds, as well as the co-option of pre-existing metabolites, provides a strategy for plants to occupy different environments.

N2 fixation and cycling in Alnus glutinosa, Betula pendula and Fagus sylvatica woodland exposed to free air CO2 enrichment.

We measured the effect of elevated atmospheric CO(2) on atmospheric nitrogen (N(2)) fixation in the tree species Alnus glutinosa growing in monoculture or in mixture with the non-N(2)-fixing tree species Betula pendula and Fagus sylvatica. We addressed the hypotheses that (1) N(2) fixation in A. glutinosa will increase in response to increased atmospheric CO(2) concentrations, when growing in monoculture, (2) the impact of elevated CO(2) on N(2) fixation in A. glutinosa is the same in mixture and in monoculture and (3) the impacts of elevated CO(2) on N cycling will be evident by a decrease in leaf δ(15)N and by the soil-leaf enrichment factor (EF), and that these impacts will not differ between mixed and single species stands. Trees were grown in a forest plantation on former agricultural fields for four growing seasons, after which the trees were on average 3.8 m tall and canopy closure had occurred. Atmospheric CO(2) concentrations were maintained at either ambient or elevated (by 200 ppm) concentrations using a free-air CO(2) enrichment (FACE) system. Leaf δ(15)N was measured and used to estimate the amount (N(dfa)) and proportion (%N(dfa)) of N derived from atmospheric fixation. On average, 62% of the N in A. glutinosa leaves was from fixation. The %N(dfa) and N(dfa) for A. glutinosa trees in monoculture did not increase under elevated CO(2), despite higher growth rates. However, N(2) fixation did increase for trees growing in mixture, despite the absence of significant growth stimulation. There was evidence that fixed N(2) was transferred from A. glutinosa to F. sylvatica and B. pendula, but no evidence that this affected their CO(2) response. The results of this study show that N(2) fixation in A. glutinosa may be higher in a future elevated CO(2) world, but that this effect will only occur where the trees are growing in mixed species stands.

Prey capture by the non-native carnivorous pitcher plant Sarracenia purpurea across sites in Britain and Ireland.

The carnivorous pitcher plant Sarracenia purpurea is native to North America, but has been introduced into Europe, where it is now widespread. Understanding of how this species functions in its non-native range is limited. We measured pitcher morphology and prey capture by S. purpurea in its non-native range in Britain and Ireland. Pitchers were removed from different plants at each of six bogs covering the species range in Britain and Ireland (n = 10 pitchers per site). For each pitcher we counted and identified every prey item and took measurements of morphology. We also compiled prey capture data for existing studies in Europe and North America. Prey capture characteristics varied between sites in Britain and Ireland. The amount of prey captured varied 20-fold between sites and was partially explained by differences in pitcher size; larger pitchers caught more prey. The primary prey was Formicidae, Diptera and Coleoptera. At the rank of order, prey composition varied between bogs, some contained mainly Formicidae, some mainly Diptera and some a mix. Prey capture was less evenly distributed at some bogs compared to others, suggesting more specialization. There was no overall difference in prey capture (composition or evenness) at the rank of order between plants in Europe compared to those in North America. At the rank of species, prey capture varied between populations even within the same order. This study demonstrates a large amount of variability between sites in prey capture characteristics. This may reflect different site characteristics and/or plant strategies, which will likely impact plant function, and may impact the inquiline community. In terms of prey capture at the rank of order, S. purpurea functions identically in its non-native range. This supports its use as a model system in a natural experiment for understanding food webs.

The contribution of insect prey to the total nitrogen content of sundews (Drosera spp.) determined in situ by stable isotope analysis.

• The contribution of insect prey to total N in the carnivorous plants, Drosera rotundifolia and D. intermedia, was quantified in situ and without any experimental manipulation using natural abundance stable isotope analysis. • Samples of D. rotundifolia and D. intermedia, insects and noncarnivorous reference plants were collected from three contrasting locations across Britain. The proportion of Drosera nitrogen obtained from insect prey was calculated by a mixing model using δ15 N values from the different plant groups. • The mean proportion of Drosera N derived from prey was 50%. There were significant differences in this proportion between sites, and significant differences within sites. There were significant differences between plant tissues and a significant negative relationship between the proportion of N derived from prey and the C : N ratio of Drosera tissues. • There was little evidence of differences in prey capture/utilisation in response to N availability, possibly due to a limited range in available N between the sites. However, evidence of a positive benefit of prey capture was apparent through the decrease in C : N ratio with increasing prey N concentrations in the plants.