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 snap-traps in D. muscipula as an
13 adaptation for the capture of larger prey than co-occurring carnivorous plants (Hutchens and
14 Luken 2009).
15 Prey spectrum specialisation through the use of modified secondary plant metabolites is
16 evident in other carnivorous plant genera. Nonanal is a floral scent compound, the synthesis
17 of which is widespread in plants. This VOC is an attractant for Nematocera spp., which are
18 abundant in the natural habitats of carnivorous plant species (i.e. bogs) and a common prey
19 taxa (Ellison and Adamec 2018). This VOC is synthesised by the closely related carnivorous
20 plants; Sarracenia spp. and Darlingtonia californica and confirmed to be a prey attractant
21 through Y-tube olfactory choice assays ex situ (Di Giusto et al. 2010; Hotti et al. 2017). In
22 the pitcher plant, Nepenthes rafflesiana, synthesis of VOCs differs between upper and lower
23 pitchers; this attracts a different spectra of prey—aerial insects to upper pitchers and ants to
24 lower pitchers (Di Giusto et al. 2010). In addition, juvenile or freshly opened N. rafflesiana
6
1 pitchers that have captured few or no prey emit flower-like scents (Bauer et al. 2009);
2 putrefaction odours are produced by older pitchers that have accumulated prey. It is not clear
3 whether this is through compounds emitted by the leaf tissue, or is a function of actual prey
4 decomposition (Jürgens et al. 2009). This demonstrates the potential for prey spectrum
5 specialisation within different tissues of a single individual, controlled by secondary
6 metabolite synthesis. These results also highlight how the main biochemical drivers of
7 attraction may change throughout the life of a plant or leaf.
8 There are several instances in which a VOC linked to the attraction of organisms may also
9 serve an additional function in plant carnivory. Coniine is a similar metabolite to floral scent
10 compounds found in non-carnivorous plants (Roberts and Wink 1998). This compound has
11 been found to be produced in the lids and pitchers of Sarracenia spp., suggesting a prey
12 attraction function (Hotti et al. 2017). In addition, coniine may play a role in retaining prey if
13 concentrations are sufficient as it can act as an insect stunning agent, though actual
14 concentrations required for this to be effective have not been found in situ. Pulegone, a floral
15 scent compound also found in Sarracenia spp., functions as an attractant but may also
16 function as an insecticide (Franzios et al. 1997), demonstrating the possibility that it plays a
17 role in the killing of prey as well as attraction. Tetradecanoic acid and hexadecanoic acid
18 methyl ester are both floral scent compounds and have also been found in the traps of D.
19 muscipula and Sarracenia spp. (Kreuzwieser et al. 2014). Hexadecanoic acid production is
20 increased by D. muscipula after feeding, suggesting some carnivorous function.
21 Hexadecanoic acid is a major component of oils in plants and may act as a surfactant or be
22 involved in enzymatic activity during prey digestion, though this has not been tested.
23 Production of insect reproductive pheromones for attraction of pollinators is well established
24 in nature (Nishida 2014). Some VOCs produced by carnivorous plant species are also thought
7
1 to attract prey by mimicking insect reproductive pheromones. One individual of Sarracenia
2 flava was found to contain 14-β-Pregna, a VOC known to be a sex pheromone of the insect
3 Eurygaster maura, though whether the plant produced this cannot be confirmed (and hence
4 this is not included in Supplementary Table 1, see Hotti et al., 2017). In addition, Sarracenia
5 spp. also produce actinidine and trans-jasmone, pheromones used by species of
6 Hymenoptera. The direct role of these VOCs for prey attraction has not been tested under a
7 single compound regime, though a blend of VOCs is likely to enhance prey attraction
8 efficiency similar to floral blends (Kreuzwieser et al. 2014).
9 This co-option of existing VOCs—from fruit and floral compounds for use as a prey
10 attractant—demonstrates the versatility of secondary metabolites to provide multiple
11 functions, differentiated by the location or tissue from which the compound is emitted. The
12 potential independent evolution of VOCs similar to those used by insects to attract other
13 insects is an example of convergent evolution driven by evolutionary pressure to attract
14 insects, though for entirely different purposes. Plants’ deception of insects is well
15 documented, but carnivorous plants have adopted a multitude of strategies to lure and deceive
16 animals for the ultimate goal of nutrient acquisition, in addition to reproduction (Ellison and
17 Adamec 2018).
18 Further work is needed to identify the specific roles of these novel and pre-existing
19 compounds and to determine whether their effectiveness is dependent on the compounds
20 acting as a blend, or if each compound acts as an effective attractant to prey. To date, no
21 studies have investigated how VOC blends may change over time or growth stages i.e. longer
22 than immediately before and after prey capture. Dionaea muscipula, for example, captures an
23 entirely different spectrum of prey as seedlings compared to mature stages (Hatcher and Hart
24 2014); it seems possible that their volatile blend may change over the lifecycle of the plant to
8
1 reflect this specialisation. A research gap exists in understanding how plastic the metabolic
2 profile of carnivorous plants is in response to growth stage, nutrient status and environmental
3 stimuli.
4 Visual cues
5 Secondary metabolites constitute the pigments that plants use to create the visual cues which
6 play a key role in plant-animal communication. Anthocyanins, for example, produce a red
7 colour that advertises fruit ripeness. Anthocyanins, flavonoids, carotenoids and betalains are
8 responsible for flower colour and pattern which attracts pollen vectors for reproduction and
9 dispersal (Tanaka et al. 2008). These pigments have been assumed to attract prey to
10 carnivorous plants, due to the distinctive red trap colouration of many carnivorous plants
11 (Figure 1). The anthocyanins and co-pigments which constitute this colouration in trap leaves
12 include kaempferol, quercetin and cyanidin (Sheridan and Griesbach 2001). A variety of
13 carnivorous plant traps also display UV reflectance patterns. These patterns are also
14 suggested to have a prey-attraction function, and are attributed to the accumulation of
15 phenolics, quinones and sugars (Kurup et al. 2013) (Figure 2b). There is, however, as yet no
16 robust evidence that pigments in traps serve a prey attraction function ubiquitously (Bennett
17 and Ellison 2009). Red pigmentation is not likely to be an attractant to many invertebrate
18 species as they cannot perceive red wavelengths of light, and in some carnivorous plants red
19 pigmentation has been shown not to impact prey capture rate (Foot et al. 2014). The potential
20 for pigmentation as a prey attractant may be plant species-prey specific, or colouration may
21 indirectly attract prey due to a contrast to background colouration for insect species that do
22 not perceive light in the red frequencies (Figure 2c). Other functions for trap pigments have
23 been suggested, such as photoprotection or light stress as seen in Drosera rotundifolia
24 (Figure 2a) (Millett et al. 2018) and nutrient stress as observed in Dionaea muscipula and 9
1 Drosera spathulata (Ichiishi et al. 1999). Analysis of pigments in carnivorous plants has,
2 however, been useful in determining phylogeny. The Venus flytrap (Dionaea muscipula),
3 formerly grouped under Caryophyllales, synthesise anthocyanins rather than betalains
4 (characteristic of the majority of Caryophyllales) and provided further support for grouping
5 these plants under ‘Nepenthales’ using molecular phylogenetic data (Ellison and Adamec
6 2018). Interestingly, pigment concentrations were shown to be significantly different in
7 petioles compared to traps of D. muscipula. In particular, cyanidin was present in the
8 digestive glands, but absent from the non-carnivorous leaf tissue (Henarejos-Escudero et al.
9 2018). Given the limitations of current approaches, further work is required to determine the
10 adaptive carnivorous function, if any, of the synthesis of these pigment metabolites.
11 Extra-floral nectaries
12 Nectar is a saccharide-rich liquid produced by plants in glands or nectaries and is composed
13 of primary metabolites including sucrose, fructose and glucose, but is also often a
14 combination of many secondary compounds (Stevenson et al. 2017). Flowering plants
15 possess nectaries that function as a reward or bribe to attract pollinating insects (Roy et al.
16 2017). Many plant species also possess extra-floral nectaries that perform functions other
17 than pollinator attraction, such as recruiting animals that deter herbivores, and the nectar
18 often contains additional metabolites (Marazzi et al. 2013). Extra-floral nectaries are present
19 on the peristome of carnivorous pitfall traps in the genera Darlingtonia, Heliamphora,
20 Sarracenia, Cephalotus and Nepenthes, and on the trap lobes of the snap-trap of D.
21 muscipula (Płachno 2007) (Figure 3). Nepenthes are also known to have coprophagous
22 relationships with birds and small mammals that perch on the pitcher rim to feed on the
23 nectar (Clarke et al. 2009; Chin et al. 2010; Grafe et al. 2011). The chemical constituents of
24 the nectar produced by these extra-floral nectaries have not been closely studied in 10
1 carnivorous plants, though such nectaries have evolved independently at least three times in
2 carnivorous plants and are likely to be a combination of primary metabolites (sugars) and
3 secondary compounds such as VOCs (Figure 3) (Givnish 2015). The degree to which this
4 convergent evolution also applies to nectar chemistry would be valuable to study to explain
5 how carnivorous plants may alleviate the overlap between pollinators and prey. Currently, as
6 far as we are aware, there has been no study comparing the metabolite blend of a species’
7 floral, extrafloral and trap related extrafloral nectaries.
8 Presence of metabolites across independent lineages of plant carnivory for prey attraction
9 This review has identified 26 secondary plant metabolites involved in the attraction of prey
10 which are present across independent lineages of carnivory. These secondary metabolites are
11 the only known aspect of carnivory where function of the same compound has been
12 demonstrated across independent lineages of plant carnivory, with the exception of benzoic
13 acid, of which the function is not confirmed but is suggested to have a function in attraction
14 and digestion (Supplementary Table 1). There is also high overlap within different genera of
15 the same carnivorous lineage (boxed ticks of Table 1 and light grey bars in Supplementary
16 Table 1). These secondary metabolites are all volatile organic compounds (VOCs) and
17 provide evidence of convergent evolution of carnivory at a biochemical scale.
18 The majority of these VOCs are common across non-carnivorous plant taxa as fruit or floral
19 scents to attract pollinators. There are only a handful of carnivorous genera, however, that
20 have been studied for the presence of volatile organic compounds for prey attraction. The
21 majority of pre-attraction VOCs have been identified in a few studies on Nepenthes spp.,
22 Dionaea muscipula and the separate carnivorous lineage of Sarracenia spp. Clearly, VOCs as
23 prey attractants is the ideal focus for understanding convergent evolution of carnivory at a
11
1 biochemical scale. Future work on the role of secondary metabolites in attraction should
2 focus on determining whether other lineages of carnivorous plant attract prey through the use
3 of VOCs, particularly VOCs already known in other species. Given that most of the
4 compounds found to be used in the attraction of prey are also widespread as fruit or floral
5 scents, determining whether these compounds were present before evolutionary divergence or
6 afterwards would show the frequency of exaptation of current plant metabolites or generation
7 of novel compounds for the carnivorous syndrome.
8 SECONDARY METABOLITES INVOLVED IN THE CAPTURE OF PREY
9 Prey capture mechanisms are the most studied aspect of carnivory in plants (Ellison and
10 Adamec 2018). The different approaches to prey capture result in novel and unusual
11 morphologies and plant movements. In carnivorous plants, five distinct trapping mechanisms
12 have been identified: (a) flypaper—leaves that secrete a sticky mucilage; (b) snap-traps—
13 some of the most rapid plant movements in the kingdom; (c) pitchers/pitfall traps; (d) lobster
14 pot traps— that force prey movement in one direction away from the trap entrance using
15 downward facing hairs; and (e) suction traps/ bladder traps (Figure 1). These trapping
16 mechanisms rely on distinct morphological adaptation, but in many cases, they also require
17 the use of secondary metabolites to enhance capture efficiency and prey retention. Secondary
18 metabolites can be split into two functional groups for prey capture: those which play a
19 physical role, and those which signal prey capture.
20 Physical trapping mechanisms
21 The physical role of metabolites for trapping prey includes adhesive properties as found, for
22 example, in mucilage of Drosera spp., glue of Roridula species and the fluids of pitcher
23 plants, some of which have viscoelastic properties, or wax crystals and metabolites in nectar 12
1 within the pitcher plants relying on slippery surfaces for capture (Riedel et al. 2003; Płachno
2 2007; Bauer et al. 2008). One example of how secondary metabolites are involved in these
3 functions is the use of extra-floral nectar to decrease friction on the peristome of Nepenthes
4 spp. pitchers (Figure 3; Bauer et al., 2008). This increases prey capture efficiency by
5 reducing the ability of prey (primarily ants, Formicidae) to adhere to the pitcher lip,
6 increasing the likelihood of falling into the pitcher trap. Secondary metabolites specifically
7 implicated in this process are as yet unknown, but given the novel, mechanical function of
8 this nectar, chemical composition may differ from nectar produced specifically for attraction.
9 A similar functional role is performed by erucamide in the pitcher plants of the unrelated
10 genus Heliamphora, where it acts as a lubricating agent in the nectar produced around the
11 peristome of the traps to increase trapping efficiency (Płachno 2007). Plumbagin may also
12 play a key role in prey capture for Nepenthes khasiana. Plumbagin is found on the rim of
13 species of Nepenthes pitcher plants and is suggested to work in tandem with the slippery
14 surface by inflicting an anaesthetic or toxic influence on prey inhibiting their escape
15 responses (Raj et al. 2011). Plumbagin is a secondary metabolite found in all genera of the
16 Nepenthales and rarely outside this group of plants, though its specific function in carnivory
17 in these species is yet to be fully determined (Schlauer et al. 2005).
18 Anti-adhesive wax production used in prey capture
19 Epicuticular wax crystals are widespread in the plant kingdom and fulfil multiple functions.
20 Within carnivorous plants, formation of wax is present in pitcher forming plants; Catopsis
21 berteroniana, Brocchinia reducta, Cephalotus, Sarracenia, Darlingtonia, Heliamphora and
22 Nepenthes spp. (Riedel et al. 2003; Gaume et al. 2004; Gorb et al. 2005; Poppinga et al.
23 2010). However, the specific structure and composition of the wax has only been documented
24 in Nepenthes spp. 13
1 In pitcher plants of Nepenthes spp., the inner surface of the trap is densely covered with
2 epicuticular wax crystals (Riedel et al. 2003). This wax impedes insect adhesion reducing
3 their ability to escape from the pitcher. The wax constituents are secondary metabolites from
4 primary alcohols, fatty acids, alkyl esters and n-alkanes, with the exact composition
5 determining the specific formation pattern of the crystals. Triacontanal is the crucial
6 compound in the formation of the wax crystals. This aldehyde homologue contributes 43% of
7 the entire wax crystal mass (Riedel et al. 2003). Triacontanal is common in a variety of non-
8 carnivorous flora cuticular waxes and epicuticular wax platelets (Naeem et al. 2012). The
9 other components of Nepenthes spp. pitcher wax are currently unknown. It is, however, likely
10 that novel wax-forming metabolites are distributed throughout the crystal network or
11 segregate into individual platelets and also form wax crystals to enhance prey capture (Gorb
12 et al. 2005).
13 Mucilage and glue production for prey capture
14 Two variants of the sticky substance used in flypaper traps are produced by carnivorous
15 plants. Within the Nepenthales (Drosera, Drosophyllum, Triphyophyllum), and within the
16 Lamiales (Pinguicula and Byblis), they produce a polysaccharide-based mucilage. The
17 mucilage of Drosera is composed mostly of primary metabolites and hence not included in
18 Supplementary Table 1. Myo-Inositol represents a significant proportion of the non-
19 polysaccharide organic compounds, though is also a primary metabolite, derived from
20 glucose (Loewus and Murthy 2000; Kokubun 2017). Though no detailed analyses are
21 available, the other mucilage-producing carnivorous plants likely produce a similar product
22 to the polysaccharide-based mucilage described. The mucilage is similar in appearance and
23 characteristics, though confirmation of its chemical constituents would provide additional
14
1 insight into the evolution of strategies to capture prey as the flypaper strategy spans at least
2 five separate lineages (Figure 1 "a" annotation, Pereira et al., 2012).
3 The second type of flypaper glue is not mucilage, nor an aqueous solution of acidic
4 polysaccharides as described above; it is a lipophilic resin with high viscosity and is
5 produced by species of Roridula. The resinous glue is composed of triterpenoids and
6 acylglycerides (Simoneit et al. 2008). No saccharides or proteins have been previously
7 detected within this glue, but traces of flavonoids have been identified in two species
8 (Wollenweber 2007). This glue is found to be considerably more viscous than the
9 mucilaginous secretions of other carnivorous plants. In addition, this resin-based glue is
10 much more resilient to water submergence and does not wash away as does Drosera spp.’s
11 mucilage. Here, two morphologically similar strategies to capture prey (flypaper) have
12 evolved with distinctly contrasting chemical composition to act as a glue. One of a
13 saccharide-based (primary metabolite) substance, one of a resin based (secondary metabolite)
14 glue to achieve effective prey capture.
15 Prey capture signalling
16 Carnivorous plants have evolved strategies to detect prey capture and respond by
17 synthesising specific metabolites to advance the process of nutrient uptake from prey
18 (Nakamura et al. 2013; Krausko et al. 2017). These signals may instigate trap movement, the
19 accumulation of digestive enzymes, prey digestion and prevention of microbial decay of prey
20 (Table 1).
21 Jasmonates are usually a component of plant defence systems. In Droseraceae, however, they
22 have been co-opted to control the signalling of prey capture to instigate trap movement
23 (Krausko et al. 2017). Jasmonates are lipid-based metabolites that typically act as
15
1 phytohormones known to be involved in a wide range of regulatory processes. In particular,
2 plant responses to stress, including herbivory and other kinds of biotic and abiotic stress,
3 elicit jasmonate production, though they also serve as regulators of growth, photosynthesis
4 and reproductive development (Wasternack and Hause 2013). Accumulation of these
5 metabolites is usually a fast response, occurring within minutes or hours of wounding or
6 herbivory (Wasternack and Hause 2013).
7 The sundew Drosera capensis increases concentrations of jasmonic acid and its conjugates in
8 response to prey capture (Krausko et al. 2017). These increasing concentrations of jasmonic
9 acid instigate leaf bending, which is targeted, due to jasmonate accumulation being controlled
10 at an individual cell level, preventing a whole-plant response to jasmonates (Nakamura et al.
11 2013). This ensures that the traps curl and envelop around prey, increasing the trap surface
12 area important in prey retention, digestion and assimilation. Drosera capensis has also
13 retained the herbivory defence response instigated by levels of jasmonic acid. Drosera
14 capensis therefore differentiates between prey capture and herbivory through propagation of
15 electrical signals and consequent jasmonic acid accumulation (Krausko et al. 2017).
16 SECONDARY METABOLITES INVOLVED IN THE DIGESTION OF PREY
17 Once captured, the prey of carnivorous plants are digested. Enzymes are fundamental to the
18 digestion of prey and identification of these specialised proteins is a current and ongoing
19 endeavour (for a review see Ravee et al., 2018). Secondary metabolites can instigate enzyme
20 production, enhance the efficiency of digestion and protect the plant from infection by
21 preserving prey whilst it is being digested.
22 Three species that share the same carnivorous ancestor—Nepenthes alata and the two closely
23 related species, Drosera capensis and Dionaea muscipula—demonstrate how the same
16
1 secondary metabolites (jasmonates) can act as a biochemical signal for indicating the same
2 stimuli but instigating a range of processes. Drosera spp. and D. muscipula share a common
3 Drosera-like ancestor (Gibson and Waller 2009; Givnish 2015), but they also have different
4 prey trapping mechanisms. Drosera spp. use sticky mucilage and slow leaf movement;
5 Dionaea muscipula uses fast snap-traps (Forterre et al. 2005; Erni et al. 2008). Prey capture
6 results in the accumulation of jasmonic acid, 12-oxo Phytodienoic acid and isoleucine
7 conjugate of jasmonic acid in all three species, Nepenthes alata, Drosera capensis and
8 Dionaea muscipula. While jasmonates instigate enzyme secretion in all three species, it also
9 initiates trap movement (to slowly envelop prey) in Drosera capensis (Mithöfer et al. 2014;
10 Krausko et al. 2017) and in Dionaea muscipula the accumulation of these secondary
11 metabolites also instigates sealing of the ‘outer-stomach’ (Libiaková et al. 2014; Pavlovič et
12 al. 2017). In Dionaea muscipula, jasmonates instigate production of endopeptidase dionain, a
13 compound involved in the chemical breakdown of prey (Libiaková et al. 2014). Trap
14 movement in D. muscipula is, similarly to Drosera spp., instigated by intercellular electrical
15 signals but relies on mechanical snap-buckling instability for the initial prey capture,
16 followed by slow trap closure to form an 'outer-stomach’ instigated by phytohormones.
17 Reliance on a physical snap-buckling mechanism over phytohormone controlled leaf
18 movement in D. muscipula is presumably because of the faster reaction times required for a
19 snap-trap compared to a sticky flypaper trap for prey capture and retention. It seems likely
20 that the prey-capture signalling function is common to the three species because of their
21 shared carnivorous ancestor, but the function of this signal has somewhat diverged because a
22 different trap strategy evolved in Dionaea muscipula and Nepenthes spp.
23 Two additional groups of secondary metabolites appear to be part of the process of prey
24 digestion: naphthoquinones and phenolics. In non-carnivorous plant species these secondary
17
1 metabolites act as antibacterial protection against pathogenic attack on plant tissue (Krolicka
2 et al. 2008). In carnivorous plants they appear to have been co-opted to preserve and aid in
3 the digestion of prey by preventing decay from bacteria as well as functioning to protect the
4 plant tissue during the process of prey breakdown.
5 Naphthoquinones are a class of organic compounds derived from naphthalene. In non-
6 carnivorous plants, the naphthoquinone plumbagin is used as a deterrent against herbivorous
7 insects, and has fungicidal and microbicidal effects (Paiva et al. 2003). Similarly, juglone is
8 inhibitory to insects, and so prevents herbivory. Plumbagin and juglone accumulate in the
9 trap leaves of Drosera spp. in response to prey capture, suggesting that they play a role in the
10 process of carnivory (Egan and Kooy 2012). While their specific function has not yet been
11 tested in carnivorous plants in situ, it seems likely that they enhance prey digestion by
12 protecting the plant from pathogens that may otherwise infect the plant as a result of decaying
13 prey. Nepenthes khasiana utilises naphthoquinones as a chitin-induced antifungal agent found
14 in its specialised pitcher fluid (Eilenberg et al. 2010). It appears to be used to preserve caught
15 prey for digestion and assimilation of nutrients. Droserone and 5-O-methyldroserone have
16 also been identified specifically for antifungal abilities in Drosera spp., D. muscipula and
17 Nepenthes spp. (Table 1, Acton, 2012).
18 Presence of compounds involved in digestion within the same carnivorous lineage
19 Plumbagin is present in the trap tissue of every genus of the Nepenthales—Aldrovanda,
20 Drosophyllum, Triphyophyllum, Dionaea, Drosera and Nepenthes—which share a common
21 carnivorous ancestor (Table 1). Plumbagin is considered to function as an antimicrobial agent
22 during digestion (Aung et al. 2002). Other possible roles, however, have been suggested.
23 Plumbagin may function as an allelopathic, insecticidal, molluscidal, antifeedant, while it
18
1 also potentially may be important in attraction as a method to differentiate prey with
2 pollinators (Krolicka et al. 2008; Raj et al. 2011).
3 It is suggested that, as the potential benefits of carnivory increase, the morphology of
4 carnivorous plant traps become more complex (Ellison and Adamec 2018). Secondary
5 metabolites involved in digestion seem, however, to be fairly consistent across several
6 different trap types. If plumbagin and its isomers are critical in protecting the plant during
7 digestion or enhancing prey digestion, it would explain why these compounds have persisted
8 regardless of trap morphology. Their presence across other taxa has, in this case, been
9 investigated—7-methyl-juglone and plumbagin are not present in five other lineages of
10 carnivory (Cephalotus, Byblis, Roridula, Sarracenia and Heliamphora (of Ericales lineage)
11 and Utricularia) (Zenk et al. 1969; Culham and Gornall 1994). These findings contribute
12 important extensions to confirmation of compounds found in the Nepenthales. If preservation
13 of prey or protection of trap tissue during prey digestion are such important adaptations, to
14 the extent that they have persisted in all Nepenthales, why are these compounds not present
15 across all carnivorous lineages? Other carnivorous lineages may have evolved novel
16 compounds for the same function as the 1,4-naphthoquinones such as plumbagin in
17 Nepenthales, but to date this has not been investigated.
18 SECONDARY METABOLITES INVOLVED IN NUTRIENT ASSIMILATION AND WHOLE PLANT
19 RESPONSES
20 Currently, the metabolic role of, and responses to, assimilation of nutrients from prey is
21 vastly unexplored. Carnivorous plants balance nutrient uptake and rate of photosynthesis
22 within the same tissue—leaves—though root nutrient uptake is maintained in some species
23 (Gao et al. 2015). This suggests, for some species, an interesting leaf–root signalling
19
1 mechanism for nutrient status, distinct from typical nutrient dynamics in plants. Uptake of
2 nutrients from prey via leaves has been shown either to maintain or increase root activity, the
3 reverse response of leaf nitrogen uptake via stomata in non-carnivorous flora (Adamec 2002;
4 Gao et al. 2015). This strongly suggests a novel role of plant signalling in this complex
5 nutrient regulation system.
6 Other aspects of the assimilation of nutrients from prey are known. For example, breakdown
7 of nonabsorbable compounds by secretion of enzymes in specialised glands and
8 transmembrane transport of nutrients can occur through use of membrane carriers and
9 endocytosis (Schulze et al. 1999; Plachno et al. 2006; Adlassnig et al. 2012). Pulse–chase
10 experiments and research into metabolites that are taken up by the plant via prey digestion
11 have confirmed the uptake of carbon and nitrogenous compounds; but identification of
12 subsequent plant response has varied (Adamec 2002; Rischer et al. 2002; Karagatzides et al.
13 2009). Carbon from prey-derived protein in Nepenthes insignia was found to be utilised for
14 production of plumbagin (Rischer et al. 2002). Conversely, Drosera capensis was found to
15 have similar levels of quercetin and kaempferol before and after prey capture (Kováčik et al.
16 2012). Plant response and signalling using plant-synthesised metabolites in response to the
17 assimilation of animal metabolites remains largely unexplored. Metabolic profiling of trap,
18 non-carnivorous leaf and root tissue before, during and after nutrient uptake from prey may
19 provide insight into any biochemical signalling within the plant to control assimilation or
20 regulate nutrient balance within the whole plant. Research in this area may provide insight
21 into exaptation of current plant regulation processes or a novel method for plants to control
22 nutrient uptake via leaves.
20
1 OCCURRENCE OF SECONDARY PLANT METABOLITES INVOLVED IN CARNIVORY ACROSS
2 PLANT TAXA
3 This review has identified ~170 secondary plant metabolites that have, or are highly likely to
4 have, a role in some aspect of the carnivorous habit, some of which are isomers or derivatives
5 or select groups of compounds. Of these, 48 secondary metabolites have been found to play a
6 role in carnivory in more than one genus (Table 1, or grey bars in Supplementary Table 1).
7 Twenty-six compounds are shared across independent evolutionary lineages of plant
8 carnivory (Nepenthales and Sarraceniaceae, unboxed ticks in Table 1). However, only
9 Nepenthales and Ericales have been investigated for secondary plant metabolites from an
10 ecological function perspective (Figure 1), and of the 33 studies used for the generation of
11 Supplementary Table 1, only one examines the metabolic role in carnivory across multiple
12 evolutionary lineages (Jürgens et al. 2009). There are, however, many studies identifying
13 these and other compounds across other taxa for some kind of anthropogenic use
14 (pharmaceuticals or biotechnology), or as taxonomic markers, but their plant function is not
15 specified (e.g. Budzianowski 1996; Muhammad et al. 2013). We excluded these studies from
16 this review because these artificially derived compounds may have no function in the
17 carnivorous habit.
18 The origins of the evolution of carnivory is beginning to be determined, but there remain key
19 components of the evolution of the carnivorous syndrome that are yet to be identified.
20 Aspects of the evolution of carnivory have recently been linked to plant defence (Pavlovič
21 and Saganová 2015). This link is appropriate given the similarity in function of response to
22 and digestion of prey, compared to responses to herbivory using anti-microbial and
23 insecticidal compounds and phytohormones. Similarly, most metabolites used for attraction
24 are related to flower or fruiting scents (Jaffé et al. 1995; Kreuzwieser et al. 2014). Given 21
1 carnivorous plants, so far, are all angiosperms, it would be interesting to investigate the
2 genetic origins of these compounds, as it is currently unclear whether compounds for
3 attraction are similarly co-opted from pollinating and dispersal mechanisms, or whether novel
4 co-evolution has taken place to mimic olfactory cues.
5 The majority of metabolites, and metabolites identified so far across different taxa, are
6 involved in the attraction and or capture phases of plant carnivory, this may however, be
7 falsely disproportionate (Supplementary Table 1). Over representation of metabolites
8 involved in attraction for plant carnivory is understandable, given the relative simplicity in
9 inferring their functional role. Presence of volatiles found on traps are logically assumed to
10 have some olfactory attraction for animals and up- or down-regulation of these compounds
11 following prey capture reinforces this assumption. Similarly, composition of sticky mucilage
12 exuded on traps leads to a conclusion of a function in capture or retention (though potentially
13 also attraction). Only a handful of studies identifying compounds in attraction or capture
14 specifically test these compounds with prey capture assays (e.g. Jürgens et al., 2009).
15 Furthermore, classification of many VOCs is from a technical chemical standpoint and may
16 not have a significant impact on olfactory attraction in situ as their volatility is not realised
17 under biologically relevant temperatures. More stringent restrictions of inclusion of these
18 compounds would reduce the overall number of compounds significantly, though perhaps
19 unnecessarily. Further investigation of these compounds is required to determine their
20 function, rather than their outright exclusion as important compounds. This, coupled with the
21 challenge of identifying a carnivorous role of a compound in digestion and assimilation, may
22 have led to an overrepresentation of certain compounds in the literature and may erroneously
23 suggest that only these aspects are strongly reliant on secondary plant metabolites. There are
24 currently many secondary metabolites extracted from specifically carnivorous plants for
22
1 pharmaceutical, pseudo-medicinal or biotechnological use (see Ellison & Adamec, 2018 and
2 references therein). These could be compared with chemicals where a functional role is
3 known in order to establish metabolic similarities across taxa. Experiments testing the
4 function of these known compounds would be an effective way to rapidly grow the library
5 and taxonomic presence of compounds strictly related to plant carnivory that function in
6 processes other than attraction or capture.
7 CONCLUDING REMARKS AND FUTURE PERSPECTIVES
8 Secondary metabolites are known to be important to carnivorous plants in the attraction,
9 capture and digestion of prey for nutrient assimilation. There is a high degree of co-option as
10 well as generation of novel metabolites to aid the carnivorous syndrome. This is likely due to
11 inherently high chemical diversity and the utility of these compounds for multiple functions
12 (Moore et al. 2014). A key question is the importance of secondary metabolites in
13 carnivorous plant evolution. Have similar metabolic approaches to carnivory evolved
14 independently in different carnivorous plant lineages? There is evidence of metabolites
15 involved in carnivory present in more than one genus and, in some cases, these are found
16 across independently evolved lineages (Table 1 unboxed ticks, also highlighted dark grey in
17 Supplementary Table 1). There may be great potential in examining the role of novel
18 secondary metabolites for carnivory in different species, which differ in their morphological
19 approach to carnivory, but which share a common ancestor (e.g. Nepenthales purple block in
20 Figure 1 & dark grey block in Table 1).
21 There is growing interest in the role of secondary metabolites in the rapid diversification and
22 evolution of plants to new environments. This is due to high chemical diversity, with rapid
23 generation of novel compounds being attributed to adaptation rather than mutation (Moore et
24 al. 2014). Carnivorous plants have been found to have huge chemical diversity, of which we 23
1 know most about through their use in biotechnology (see Ellison & Adamec, 2018 and
2 references therein). We hypothesise that metabolite diversity provided a mechanism for the
3 evolution of carnivory in plants, and that this continued diversity facilitates rapid evolution
4 into new environments. Due to the multiple times carnivory has independently evolved, and
5 the general restriction of carnivorous plants to high-stress environments, these plants are an
6 ideal system to investigate whether metabolic diversity may have been or is a way for novel
7 traits to evolve and be maintained. Metabolites are clearly integral to the survival of
8 carnivorous plants in nutrient-deficient environments and further investigation of the
9 carnivorous habit will continue to provide new insights into novel pathways of plant
10 evolution.
11 FUNDING:
12 This work was supported by a Loughborough University PhD studentship to CH.
13 ACKNOWLEDGEMENTS:
14 We thank Richard Harland for his help with chemical groupings and Angus Davidson for
15 assistance with plant drawings. We also thank the reviewers for their useful comments.
16 REFERENCES:
17 Acton A. 2012. Naphthalenes—Advances in Research and Application. Scholarly Editions.
18 Adamec L. 2002. Leaf absorption of mineral nutrients in carnivorous plants stimulates root
19 nutrient uptake. New Phytologist 155: 89–100.
20 Adlassnig W, Koller-Peroutka M, Bauer S, Koshkin E, Lendl T, Lichtscheidl IK. 2012.
21 Endocytotic uptake of nutrients in carnivorous plants. The Plant Journal 71: 303–313.
22 Agrawal AA. 2011. Current trends in the evolutionary ecology of plant defence. Functional 24
1 Ecology 25: 420–432.
2 Aung H, Chia L, Goh N, et al. 2002. Phenolic constituents from the leaves of the
3 carnivorous plant Nepenthes gracilis. Fitoterapia 73: 445–447.
4 Banasiuk R, Kawiak A, Krölicka A. 2012. In vitro cultures of carnivorous plants from the
5 Drosera and Dionaea genus for the production of biologically active secondary metabolites.
6 Biotechnologia 93: 87–96.
7 Bauer U, Bohn HF, Federle W. 2008. Harmless nectar source or deadly trap: Nepenthes
8 pitchers are activated by rain, condensation and nectar. Proceedings of the Royal Society B:
9 Biological Sciences 275: 259–265.
10 Bauer U, Willmes C, Federle W. 2009. Effect of pitcher age on trapping efficiency and
11 natural prey capture in carnivorous Nepenthes rafflesiana plants. Annals of Botany 103:
12 1219–1226.
13 Bennett KF, Ellison AM. 2009. Nectar, not colour, may lure insects to their death. Biology
14 letters 5: 469–72.
15 Budzianowski J. 1996. Naphthohydroquinone glucosides of Drosera rotundifolia and D.
16 intermedia from in vitro cultures. Phytochemistry 42: 1145–1147.
17 Chin L, Moran JA, Clarke C. 2010. Trap geometry in three giant montane pitcher plant
18 species from Borneo is a function of tree shrew body size. New Phytologist 186: 461–470.
19 Clarke CM, Bauer U, Lee CC, Tuen AA, Rembold K, Moran JA. 2009. Tree shrew
20 lavatories: a novel nitrogen sequestration strategy in a tropical pitcher plant. Biology Letters
21 5: 632–635.
22 Culham A, Gornall RJ. 1994. The taxonomic significance of naphthoquinones in the
25
1 Droseraceae. Biochemical Systematics and Ecology 22: 507–515.
2 Darwin C. 1875. Insectivorous Plants. London - John Murray.
3 Egan PA, Kooy F Van Der. 2012. Coproduction and Ecological Significance of
4 Naphthoquinones in Carnivorous Sundews (Drosera). Chemistry & Biodiversity 9: 1033–
5 1044.
6 Egan PA, Van Der Kooy F. 2013. Review: Phytochemistry of the Carnivorous Sundew
7 Genus Drosera (Droseraceae) – Future Perspectives and Ethnopharmacological Relevance.
8 Chemistry and Biodiversity 10: 1774–1790.
9 Eilenberg H, Pnini-Cohen S, Rahamim Y, et al. 2010. Induced production of antifungal
10 naphthoquinones in the pitchers of the carnivorous plant Nepenthes khasiana. Journal of
11 Experimental Botany 61: 911–922.
12 El-Sayed AM, Byers JA, Suckling DM. 2016. Pollinator-prey conflicts in carnivorous
13 plants: When flower and trap properties mean life or death. Scientific Reports 6: 21065.
14 Ellison AM, Adamec L. 2018. Carnivorous Plants: Physiology, Ecology, and Evolution.
15 Oxford University Press.
16 Erni P, Varagnat M, McKinley GH, et al. 2008. Little Shop of Horrors: Rheology of the
17 Mucilage of Drosera sp., a Carnivorous Plant In: AIP Conference Proceedings. AIP, 579–
18 581.
19 Fleischmann A, Schlauer J, Smith SA, Givnish TJ. 2018. Evolution of carnivory in
20 angiosperms In: Ellison AM, Adamec L, eds. Carnivorous Plants: Physiology, ecology, and
21 evolution. Oxford University Press, 22–42.
22 Foot G, Rice SP, Millett J. 2014. Red trap colour of the carnivorous plant Drosera
26
1 rotundifolia does not serve a prey attraction or camouflage function. Biology letters 10:
2 20131024–20131024.
3 Forterre Y, Skotheim J, Dumais J, Mahadevan L. 2005. How the Venus flytrap snaps.
4 Nature 433: 421–425.
5 Franzios G, Mirotsou M, Hatziapostolou E, Kral J, Scouras ZG, Mavragani-Tsipidou P.
6 1997. Insecticidal and Genotoxic Activities of Mint Essential Oils. Journal of Agricultural
7 and Food Chemistry 45: 2690–2694.
8 Gao P, Loeffler TS, Honsel A, et al. 2015. Integration of trap- and root-derived nitrogen
9 nutrition of carnivorous Dionaea muscipula. New Phytologist 205: 1320–1329.
10 Gaume L, Perret P, Gorb E, Gorb S, Labat J-J, Rowe N. 2004. How do plant waxes cause
11 flies to slide? Experimental tests of wax-based trapping mechanisms in three pitfall
12 carnivorous plants. Arthropod Structure & Development 33: 103–111.
13 Gibson TC, Waller DM. 2009. Evolving Darwin’s ‘most wonderful’ plant: Ecological steps
14 to a snap-trap. New Phytologist 183: 575–587.
15 Di Giusto B, Bessière J-M, Guéroult M, et al. 2010. Flower-scent mimicry masks a deadly
16 trap in the carnivorous plant Nepenthes rafflesiana. Journal of Ecology 98: 845–856.
17 Givnish TJ. 2015. New evidence on the origin of carnivorous plants. Proceedings of the
18 National Academy of Sciences 112: 10–11.
19 Gorb E, Haas K, Henrich A, Enders S, Barbakadze N, Gorb S. 2005. Composite structure
20 of the crystalline epicuticular wax layer of the slippery zone in the pitchers of the carnivorous
21 plant Nepenthes alata and its effect on insect attachment. The Journal of experimental
22 biology 208: 4651–62.
27
1 Grafe TU, Schöner CR, Kerth G, Junaidi A, Schöner MG. 2011. A novel resource–
2 service mutualism between bats and pitcher plants. Biology Letters 7: 436–439.
3 Hartmann T. 2007. From waste products to ecochemicals: fifty years research of plant
4 secondary metabolism. Phytochemistry 68: 2831–46.
5 Hatcher CR, Hart AG. 2014. Venus flytrap seedlings show growth-related prey size
6 specificity. International Journal of Ecology 2014: 631–636.
7 Henarejos-Escudero P, Guadarrama-Flores B, García-Carmona F, Gandía-Herrero F.
8 2018. Digestive glands extraction and precise pigment analysis support the exclusion of the
9 carnivorous plant Dionaea muscipula Ellis from the Caryophyllales order. Plant Science 274:
10 342–348.
11 Hotti H, Gopalacharyulu P, Seppänen-Laakso T, Rischer H. 2017. Metabolite profiling of
12 the carnivorous pitcher plants Darlingtonia and Sarracenia (V Gupta, Ed.). PLOS ONE 12:
13 1–21.
14 Hutchens JJ, Luken JO. 2009. Prey capture in the Venus flytrap: collection or selection?
15 Botany 87: 1007–1010.
16 Ichiishi S, Nagamitsu T, Kondo Y, Iwashina T, Kondo K, Tagashira N. 1999. Effects of
17 macro-components and sucrose in the medium on in vitro red-color pigmentation in Dionaea
18 muscipula Ellis and Drosera spathulata labill. Plant Biotechnology 16: 235–238.
19 Jaffé K, Blum MS, Fales HM, Mason RT, Cabrera A. 1995. On insect attractants from
20 pitcher plants of the genus Heliamphora (Sarraceniaceae). Journal of Chemical Ecology 21:
21 379–384.
22 Jürgens A, El-Sayed AM, Suckling DM. 2009. Do carnivorous plants use volatiles for
23 attracting prey insects? Functional Ecology 23: 875–887. 28
1 Karagatzides JD, Butler JL, Ellison AM. 2009. The Pitcher Plant Sarracenia purpurea
2 Can Directly Acquire Organic Nitrogen and Short-Circuit the Inorganic Nitrogen Cycle (J
3 Chave, Ed.). PLoS ONE 4: e6164.
4 Kessler D, Baldwin IT. 2007. Making sense of nectar scents: the effects of nectar secondary
5 metabolites on floral visitors of Nicotiana attenuata. The Plant Journal 49: 840–54.
6 Kokubun T. 2017. Occurrence of myo-inositol and alkyl-substituted polysaccharide in the
7 prey-trapping mucilage of Drosera capensis. Die Naturwissenschaften 104: 83.
8 Kováčik J, Klejdus B, Štork F, Hedbavny J. 2012. Prey-induced changes in the
9 accumulation of amino acids and phenolic metabolites in the leaves of Drosera capensis L.
10 Amino Acids 42: 1277–1285.
11 Krausko M, Perutka Z, Šebela M, et al. 2017. The role of electrical and jasmonate
12 signalling in the recognition of captured prey in the carnivorous sundew plant Drosera
13 capensis. New Phytologist 213: 1818–1835.
14 Kreuzwieser J, Scheerer U, Kruse J, et al. 2014. The Venus flytrap attracts insects by the
15 release of volatile organic compounds. Journal of Experimental Botany 65: 755–766.
16 Krolicka A, Szpitter A, Gilgenast E, Romanik G, Kaminski M, Lojkowska E. 2008.
17 Stimulation of antibacterial naphthoquinones and flavonoids accumulation in carnivorous
18 plants grown in vitro by addition of elicitors. Enzyme and Microbial Technology 42: 216–
19 221.
20 Kurup R, Johnson AJ, Sankar S, Hussain AA, Kumar CS, Sabulal B. 2013. Fluorescent
21 prey traps in carnivorous plants (H Rennenberg, Ed.). Plant Biology 15: 611–615.
22 Libiaková M, Floková K, Novák O, Slováková L, Pavlovič A. 2014. Abundance of
23 cysteine endopeptidase dionain in digestive fluid of Venus flytrap (Dionaea muscipula Ellis) 29
1 is regulated by different stimuli from prey through jasmonates. PloS one 9: e104424.
2 Loewus FA, Murthy PPN. 2000. myo-Inositol metabolism in plants. Plant Science 150: 1–
3 19.
4 Marazzi B, Bronstein JL, Koptur S. 2013. The diversity, ecology and evolution of
5 extrafloral nectaries: current perspectives and future challenges. Annals of botany 111: 1243–
6 50.
7 Millett J, Foot GW, Thompson JC, Svensson BM. 2018. Geographic variation in Sundew
8 (Drosera) leaf colour: plant–plant interactions counteract expected effects of abiotic factors.
9 Journal of Biogeography 45: 582–592.
10 Mithöfer A, Reichelt M, Nakamura Y. 2014. Wound and insect-induced jasmonate
11 accumulation in carnivorous Drosera capensis : two sides of the same coin. Plant Biology 16:
12 982–987.
13 Moore BD, Andrew RL, Külheim C, Foley WJ. 2014. Explaining intraspecific diversity in
14 plant secondary metabolites in an ecological context. New Phytologist 201: 733–750.
15 Muhammad A, Haddad PS, Durst T, Arnason JT. 2013. Phytochemical constituents of
16 Sarracenia purpurea L. (pitcher plant). Phytochemistry 94: 238–242.
17 Naeem M, Khan MMA, Moinuddin. 2012. Triacontanol: a potent plant growth regulator in
18 agriculture. Journal of Plant Interactions 7: 129–142.
19 Nakamura Y, Reichelt M, Mayer VE, Mithöfer A. 2013. Jasmonates trigger prey-induced
20 formation of ‘outer stomach’ in carnivorous sundew plants. Proceedings. Biological sciences
21 / The Royal Society 280: 1–6.
22 Nishida R. 2014. Chemical ecology of insect–plant interactions: ecological significance of
30
1 plant secondary metabolites. Bioscience, Biotechnology, and Biochemistry 78: 1–13.
2 Paiva SR de, Figueiredo MR, Aragão TV, Kaplan MAC. 2003. Antimicrobial activity in
3 vitro of plumbagin isolated from Plumbago species. Memórias do Instituto Oswaldo Cruz 98:
4 959–961.
5 Pavlovič A, Jakšová J, Novák O. 2017. Triggering a false alarm: Wounding mimics prey
6 capture in the carnivorous Venus flytrap (Dionaea muscipula). New Phytologist 216: 927–
7 938.
8 Pavlovič A, Saganová M. 2015. A novel insight into the cost-benefit model for the evolution
9 of botanical carnivory. Annals of Botany 115: 1075–1092.
10 Pavlovič A, Singerová L, Demko V, Hudák J. 2009. Feeding enhances photosynthetic
11 efficiency in the carnivorous pitcher plant Nepenthes talangensis. Annals of Botany 104:
12 307–314.
13 Pereira CG, Almenara DP, Winter CE, Fritsch PW, Lambers H, Oliveira RS. 2012.
14 Underground leaves of Philcoxia trap and digest nematodes. Proceedings of the National
15 Academy of Sciences of the United States of America 109: 1154–1158.
16 Pichersky E, Gang DR. 2000. Genetics and biochemistry of secondary metabolites in plants:
17 an evolutionary perspective. Trends in Plant Science 5: 439–445.
18 Pichersky E, Gershenzon J. 2002. The formation and function of plant volatiles: perfumes
19 for pollinator attraction and defense. Current Opinion in Plant Biology 5: 237–243.
20 Płachno BJ. 2007. Sweet but dangerous: nectaries in carnivorous plants. Acta Agrobotanica
21 60: 31–37.
22 Plachno BJ, Adamec L, Lichtscheidl IK, Peroutka M, Adlassnig W, Vrba J. 2006.
31
1 Fluorescence labelling of phosphatase activity in digestive glands of carnivorous plants.
2 Plant Biology 8: 813–820.
3 Poppinga S, Koch K, Bohn HF, Barthlott W. 2010. Comparative and functional
4 morphology of hierarchically structured anti-adhesive surfaces in carnivorous plants and
5 kettle trap flowers. Functional Plant Biology 37: 952.
6 Raj G, Kurup R, Hussain AA, Baby S. 2011. Distribution of naphthoquinones, plumbagin,
7 droserone, and 5-O-methyl droserone in chitin-induced and uninduced Nepenthes khasiana:
8 Molecular events in prey capture. Journal of Experimental Botany 62: 5429–5436.
9 Ravee R, Mohd Salleh F ‘Imadi, Goh H-H. 2018. Discovery of digestive enzymes in
10 carnivorous plants with focus on proteases. PeerJ 6: e4914.
11 Riedel M, Eichner A, Jetter R. 2003. Slippery surfaces of carnivorous plants: composition
12 of epicuticular wax crystals in Nepenthes alata Blanco pitchers. Planta 218: 87–97.
13 Rischer H, Hamm A, Bringmann G. 2002. Nepenthes insignis uses a C2-portion of the
14 carbon skeleton of L-alanine acquired via its carnivorous organs, to build up the
15 allelochemical plumbagin. Phytochemistry 59: 603–9.
16 Roberts MF, Wink M. 1998. Alkaloids : Biochemistry, Ecology, and Medicinal
17 Applications. Springer US.
18 Roy R, Schmitt AJ, Thomas JB, Carter CJ. 2017. Review: Nectar biology: From
19 molecules to ecosystems. Plant Science 262: 148–164.
20 Schlauer J, Nerz J, Rischer H. 2005. Carnivorous plant chemistry. Acta Botanica Gallica
21 152: 187–195.
22 Schulze W, Frommer WB, Ward JM. 1999. Transporters for ammonium, amino acids and
32
1 peptides are expressed in pitchers of the carnivorous plant Nepenthes. The Plant Journal 17:
2 637–646.
3 Schulze W, Schulze ED, Schulze I, Oren R. 2001. Quantification of insect nitrogen
4 utilization by the Venus flytrap Dionaea muscipula catching prey with highly variable
5 isotope signatures. Journal of experimental botany 52: 1041–1049.
6 Sheridan PM, Griesbach RJ. 2001. Anthocyanidins of Sarracenia L. flowers and leaves.
7 HortScience 36: 384.
8 Simoneit BRT, Medeiros PM, Wollenweber E. 2008. Triterpenoids as major components
9 of the insect-trapping glue of Roridula species. Zeitschrift fur Naturforschung - Section C
10 Journal of Biosciences 63: 625–630.
11 Stevenson PC, Nicolson SW, Wright GA. 2017. Plant secondary metabolites in nectar:
12 impacts on pollinators and ecological functions (J Manson, Ed.). Functional Ecology 31: 65–
13 75.
14 Tanaka Y, Sasaki N, Ohmiya A. 2008. Biosynthesis of plant pigments: Anthocyanins,
15 betalains and carotenoids. Plant Journal 54: 733–749.
16 Thorogood CJ, Bauer U, Hiscock SJ. 2017. Convergent and divergent evolution in
17 carnivorous pitcher plant traps. New Phytologist 217: 1035–1041.
18 Wasternack C, Hause B. 2013. Jasmonates: biosynthesis, perception, signal transduction
19 and action in plant stress response, growth and development. An update to the 2007 review in
20 Annals of Botany. Annals of Botany 111: 1021–1058.
21 Wollenweber E. 2007. Flavonoids occurring in the sticky resin on Roridula dentata and
22 Roridula gorgonias (Roridulaceae). Carnivorous plant newsletter. 36: 77–80.
33
1 Zenk MH, Fürbringer M, Steglich W. 1969. Occurrence and distribution of 7-
2 methyljuglone and plumbagin in the Droseraceae. Phytochemistry 8: 2199–2200.
3
4 FIGURE LEGENDS:
5 Figure 3: Evolution and production zones of metabolites for attraction of prey via use of
6 extrafloral nectaries. Highlighted areas are highest density of extrafloral nectaries for
7 attraction of prey. Areas for secretion of secondary metabolites and nectar are important for
8 prey attraction and capture. Length of phylogenetic branches do not signify relatedness or
9 any other metric of evolution.
10 TABLE LEGENDS:
11 Table 1: Secondary plant metabolites involved in carnivory present in more than one genera.
12 Role indicates role in the carnivorous habit. Ticks represent presence across independent
13 lineages of carnivory. Ticks within boxes represent presence of compound found within
14 different genera of the same carnivorous ancestor. Dark grey are of Nepenthales, light grey
15 are of Ericales lineage of carnivory. Note that some compound isomers are identified, but in
16 some studies, this is not included. Full list and references are included in Supplementary
17 Table 1.
18 Supplementary Table 1: Secondary plant metabolites involved in plant carnivory. Light
19 grey bars signify the compound is found in more than one genus of the same carnivorous
20 lineage. Dark grey bars signify the compound is found in more than one independent lineage
21 of carnivory. No compound has been found in more than 2 separate lineages of carnivory.
22 Compounds reported from Hotti et al., 2017 and Kreuzweiser et al., 2014 have 70%
23 confidence minimum. Function of metabolite is known or highly likely. Note that some
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1 compound isomers are identified, but in some studies, this detail is not included. Volatile
2 organic compounds (VOC) is ascribed in likely function if detail on the compound’s scent is
3 not available.
4
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TABLES
Table 1: Secondary plant metabolites involved in carnivory present in more than one genera. Ticks represent the 26 compounds present across independent lineages of carnivory. Ticks within boxes represent presence of compound found within different genera of the same carnivorous ancestor. Role indicates role in the carnivorous habit. Dark grey are of Nepenthales, light grey are of Ericales lineage of carnivory. Note that some compound isomers are identified, but in some studies, this is not included. Full list and references are included in Supplementary Table 1. Triphyophyllum Drosophyllum Heliamphora Darlingtonia Aldrovanda Sarracenia Nepenthes Dionaea Drosera
Role Metabolite Group
Attraction Heptadecane Alkane Attraction Tridecane Alkane Attraction Tetradecanoic acid Alkanoic acid Attraction Hexadecanoic acid ethyl ester Alkanoic acid ester Attraction Isoamyl acetate Alkanoic acid ester Attraction Palmitic acid methyl ester Alkanoic acid ester Attraction Nonanal Alkanoic aldehyde Attraction or digestion Benzoic acid Aromatic acid Attraction Benzyl acetate Aromatic acid ester Attraction Benzyl benzoate Aromatic acid ester Attraction Ethyl benzoate Aromatic acid ester Attraction Methyl benzoate Aromatic acid ester Attraction Methyl salicylate Aromatic acid ester Attraction Phenylethyl acetate Aromatic acid ester Attraction 2-Phenylethanol Aromatic alcohol Attraction Benzyl alcohol Aromatic alcohol 36
Triphyophyllum Drosophyllum Heliamphora Darlingtonia Aldrovanda Sarracenia Nepenthes Dionaea Drosera
Role Metabolite Group
Attraction Benzaldehyde Aromatic aldehyde Attraction 6-Methyl-5-hepten-2-one Monoterpenoid Attraction Limonene Monoterpenoid Attraction Linalool Monoterpenoid Attraction Myrcene Monoterpenoid Attraction α-Pinene Monoterpenoid Attraction α-Terpinene Monoterpenoid Attraction p-Cymene Monoterpenoid (aromatic) Attraction (Z,E)-α-farnesene Sesquiterpenoid Attraction α-Copaene Sesquiterpenoid Attraction Hexadecane Alkane Attraction Pentadecane Alkane Attraction Tetradecane Alkane Attraction Decanal Alkanoic aldehyde Attraction Coniine Alkanoic secondary amine Attraction & capture Erucamide Alkenoic amide Attraction Acetophenone Aromatic ketone Digestion Myricetin Flavanoid Attraction Sarracenin Monoterpene (iridoid) Digestion 7-methyl-juglone Naphthoquinone Digestion Droserone (plumbagin derivative) Naphthoquinone Digestion Isoshinanolone Naphthoquinone Capture & digestion Plumbagin Naphthoquinone Digestion PlumbasideA Naphthoquinone 37
Triphyophyllum Drosophyllum Heliamphora Darlingtonia Aldrovanda Sarracenia Nepenthes Dionaea Drosera
Role Metabolite Group
Capture/ digestion/ retention Jasmonates Oxylipin Digestion Gallic acid Phenolic acid Digestion Protocatechuic acid Phenolic acid Attraction Caryophyllene oxide Sesquiterpene Attraction Humulene Sesquiterpene
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1 2 Figure 1: Phylogenetic tree of carnivorous plants and research and metabolites identified
3 with known function in carnivory. Branches are to emphasise the location of carnivorous taxa
4 and do not signify time since divergence or a metric of relatedness. Trap morphology is
5 grouped into: (a) flypaper; (b) snap-trap; (c) pitfall; (d) lobster pot; and (e) suction bladder.
6 Colour bands signify a common carnivorous ancestor. Tree generated using NCBI taxonomy
7 browser, iTOL and APG (APG, 2016; Letunic & Bork, 2016). Nepenthales sensu stricto
8 sister to Caryophyllales (Fleischmann et al. 2018). Twenty-six common compounds are
9 found in the Nepenthales lineage and Sarraceniaceae lineage. Note that Aldrovanda and
10 Utricularia are genera of aquatic carnivorous plants.
11
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1 2 Figure 2
3 Function of secondary metabolites in trap colouration requires further investigation; (a)
4 Drosera rotundifolia change in colour from accumulation of anthocyanin (red pigmentation)
5 in response to environmental stimuli but this is not confirmed to be related to carnivory; (b)
6 upper figure: Nepenthes species show redder locations, possibly to contrast trap peristome to
7 background for prey attraction. Lower figure: Nepenthes spp. also use UV florescence for
8 prey attraction ; (c) Sarracenia purpurea with high diversity of red and green pigmentation
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1 from secondary compounds with high background contrast in situ (Photos of Nepenthes
2 courtesy of Dr Baby in Kurup et al., 2013, Drosera rotundifolia and Sarracenia purpurea
3 photos by C. R. Hatcher).
4
5 Figure 3: Evolution and production zones of metabolites for attraction of prey via use of
6 extrafloral nectaries. Highlighted areas are highest density of extrafloral nectaries for
7 attraction of prey. Areas for secretion of secondary metabolites and nectar are important for
8 prey attraction and capture. Length of phylogenetic branches do not signify relatedness or any
9 other metric of evolution.
10
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