1 Review Paper: The function of secondary metabolites in plant carnivory 2 Christopher R. Hatcher*, Dr David B. Ryves, Dr Jonathan Millett 3 Geography and Environment, Loughborough University, Loughborough, LE11 3TU 4 * Corresponding author: 5 Email: [email protected] 6 1 1 ABSTRACT 2 Background: Carnivorous plants are an ideal model system for evaluating the role of 3 secondary metabolites in plant ecology and evolution. Carnivory is a striking example of 4 convergent evolution to attract, capture and digest prey for nutrients to enhance growth and 5 reproduction and has evolved independently at least ten times. Though the role of many traits 6 in plant carnivory has been well studied, the role of secondary metabolites in the carnivorous 7 habit are considerably less understood. 8 Scope: This review provides the first synthesis of research in which secondary plant 9 metabolites have been demonstrated to have a functional role in plant carnivory. From these 10 studies we identify key metabolites for plant carnivory, their functional role, and highlight 11 biochemical similarities across taxa. From this synthesis we provide new research directions 12 for integrating secondary metabolites into understanding of the ecology and evolution of 13 plant carnivory. 14 Conclusions: Carnivorous plants use secondary metabolites to facilitate prey attraction, 15 capture, digestion and assimilation. We found ~170 metabolites for which a functional role in 16 carnivory has been demonstrated. Of these, 26 compounds are present across genera that 17 independently evolved a carnivorous habit, suggesting convergent evolution. Some secondary 18 metabolites have been co-opted from other processes such as defence or pollinator attraction. 19 Secondary metabolites in carnivorous plants provide a potentially powerful model system for 20 exploring the role of metabolites in plant evolution. They also show promise for elucidating 21 how generation of novel compounds, as well as co-option of pre-existing metabolites, 22 provides a strategy for plants to occupy different environments. 23 Key words: carnivorous plants, secondary metabolites, ecology, evolution, function, plant- 24 insect 25 2 1 INTRODUCTION 2 Secondary plant metabolites are increasingly being considered as important drivers of plant 3 diversification and evolution (Stevenson et al. 2017). This is in part due to their involvement 4 in a large number of fundamental processes associated with plant fitness: regulating growth 5 and interacting with the environment, conspecifics, competitors and symbionts and as a 6 defence against abiotic and biotic stress (Hartmann 2007; Kessler and Baldwin 2007; 7 Agrawal 2011). Plants retain a high metabolic diversity and have the genetic capacity to 8 quickly generate novel compounds due to gene duplication and divergence (Pichersky and 9 Gang 2000). Plants are therefore able to potentially adapt to new environments at higher rates 10 than explained by mutation (Moore et al. 2014). If secondary plant metabolites play an 11 important role in plant evolutionary adaptation, they should be inherently involved in 12 processes specific to the characteristics of the plant’s ecology. The role of metabolites in 13 adaptation may, therefore, be greater than previously considered, which would imply that we 14 are missing important information on plant evolutionary ecology (Nishida 2014; Moore et al. 15 2014). Consequently, similar patterns of metabolite production may emerge among close and 16 distantly related taxa that have evolved under similar environmental conditions. 17 Carnivorous plants are a diverse, polyphyletic group of flowering plants, generally restricted 18 to nutrient poor sites (Ellison and Adamec 2018). Plant carnivory has evolved independently 19 at least ten times in five orders (Fleischmann et al. 2018). Carnivorous plants represent an 20 example of convergent evolution of a single syndrome (Figure 1), to enhance growth and 21 reproduction by attaining nutrients from prey. The benefits of carnivory are numerous: 22 increased growth rate, earlier flowering, increased seed set, increased rate in photosynthetic 23 output, increased nutrient uptake via roots after prey capture and increased seedling growth 24 (Schulze et al. 2001; Adamec 2002; Pavlovič et al. 2009; Hatcher and Hart 2014). Within 3 1 carnivorous plants, there is striking evolutionary convergence (e.g. pitcher plants, Figure 2 1“c”), but also a wide diversity of morphological and physiological approaches to attract, 3 capture, digest and assimilate prey, even within single evolutionary lineages (e.g. 4 Nepenthales). It is now clear that, to some extent, plant carnivory has a biochemical basis 5 (Table 1). As such, carnivorous plants are an ideal study system to understand the role of 6 secondary plant metabolites in evolutionary ecology (Thorogood et al. 2017). 7 Determining the function of secondary metabolites in the carnivorous habit is considerably 8 more challenging and less well understood than determining morphological function (Ellison 9 and Adamec 2018). This lack of knowledge is critical because growing understanding of the 10 importance of the role of metabolites in rapid plant diversification and adaptation means that 11 we may be missing important insight into the evolution of plant carnivory (Moore et al. 12 2014). Advanced analytical capabilities and reduced costs have resulted in an increase in the 13 number of studies identifying secondary metabolites in carnivorous plants. The result is a 14 growing body of literature describing secondary metabolites, mostly in relation to their 15 anthropogenic use (Banasiuk et al. 2012; Egan and Van Der Kooy 2013). We lack, however, 16 a synthesis of current knowledge of specific biochemical function in relation to the tissue in 17 which these compounds are produced; this is required to begin to link gene expression to 18 secondary metabolite synthesis, carnivorous plant ecology and evolution. 19 In this review we synthesised current understanding of the secondary plant metabolites that 20 are involved in the carnivorous syndrome. The aim is to use this synthesis to explore the 21 extent to which secondary plant metabolites play a role in all aspects of plant carnivory 22 across independent lineages. It will also highlight comparisons of secondary plant metabolite 23 function across lineages, providing insight on the biochemical evolutionary convergence and 24 divergence of carnivorous plants. Secondary metabolites were identified through a systematic 4 1 search of ‘Google Scholar’ and ‘Web of Science’ using search terms ‘metabolite’, 2 ‘carnivorous plant’ and their synonyms, carnivorous plant genera and prominent study 3 species. We also searched the reference list of relevant journal articles in case papers were 4 not identified from the previous search terms. We did not include studies where the focus was 5 on extracting secondary metabolites, rather than considering their role in carnivory. Because 6 we are interested in the role of metabolite function in plant carnivory, we have collated 7 metabolites under the four key stages of the carnivorous habit: prey attraction, capture, 8 digestion, and assimilation of nutrients. 9 SECONDARY METABOLITES INVOLVED IN THE ATTRACTION OF PREY 10 Darwin (1875) was the first to suggest that carnivorous plants might attract their prey through 11 the production of odour. It has been demonstrated that some (but by no means all) 12 carnivorous plant species actively attract prey by mimicking fruit and flowers using olfactory 13 or visual cues, or through the production of extrafloral nectar (Bennett and Ellison 2009; 14 Kreuzwieser et al. 2014). Attraction mechanisms are still, however, relatively poorly 15 understood or inconsistent across species and the evidence is, in some cases, controversial. 16 Olfactory cues 17 Volatile organic compounds (VOCs), compounds volatile at ambient temperature, are 18 involved in the attraction of prey; such animal attraction is not uncommon elsewhere in 19 nature (Pichersky and Gershenzon 2002). VOCs can increase the frequency of prey 20 interaction but have also been demonstrated to enable carnivorous plants to select prey by 21 species and potentially by size (Di Giusto et al. 2010; Kreuzwieser et al. 2014; Hotti et al. 22 2017). VOCs are by far the most researched group of metabolites produced by carnivorous 23 plants (over half of the ~170 compounds listed in Supplementary Table 1). Nepenthes 5 1 rafflesiana, a vine species of pitcher plant, synthesises at least 54 different VOCs that, as a 2 blend of compounds, attracts their prey (Di Giusto et al. 2010). Kreuzwieser et al., (2014) 3 identified more than 60 different VOCs released by Dionaea muscipula (Venus flytrap). 4 Their comparison of VOC concentrations before and after prey capture suggested that 20 5 compounds were directly related to prey attraction, through mimicry of fruit and flowering 6 scents. They also found differences in levels of 1-(1-cyclohexen-1-yl)-ethanone and 7 dodecanoic acid, propyl ester before and after prey capture. These compounds have not 8 previously been attributed as attractants to insects, suggesting that D. muscipula produce a 9 mix of VOCs, some of which are the same as those in flowers and fruit, whilst others are 10 novel. The synthesis of fruit and flower mimicking VOCs alongside novel VOCs may be a 11 strategy for avoiding pollinator-prey conflict (El-Sayed et al., 2016). Alternatively, it might 12 be the case that novel VOCs were part of the evolution of