A novel insight into the cost–benefit model for the evolution of botanical carnivory

Abstract

Background The cost–benefit model for the evolution of botanical carnivory provides a conceptual framework for interpreting a wide range of comparative and experimental studies on carnivorous plants. This model assumes that the modified leaves called traps represent a significant cost for the plant, and this cost is outweighed by the benefits from increased nutrient uptake from prey, in terms of enhancing the rate of photosynthesis per unit leaf mass or area (A_N) in the microsites inhabited by carnivorous plants.

Scope This review summarizes results from the classical interpretation of the cost–benefit model for evolution of botanical carnivory and highlights the costs and benefits of active trapping mechanisms, including water pumping, electrical signalling and accumulation of jasmonates. Novel alternative sequestration strategies (utilization of leaf litter and faeces) in carnivorous plants are also discussed in the context of the cost–benefit model.

Conclusions Traps of carnivorous plants have lower A_N than leaves, and the leaves have higher A_N after feeding. Prey digestion, water pumping and electrical signalling represent a significant carbon cost (as an increased rate of respiration, R_D) for carnivorous plants. On the other hand, jasmonate accumulation during the digestive period and reprogramming of gene expression from growth and photosynthesis to prey digestion optimizes enzyme production in comparison with constitutive secretion. This inducibility may have evolved as a cost-saving strategy beneficial for carnivorous plants. The similarities between plant defence mechanisms and botanical carnivory are highlighted.

INTRODUCTION

Carnivorous plants have long fascinated scientists, and were described by Charles Darwin in the book Insectivorous plants (Darwin, 1875). Carnivorous plants typically attract, capture and digest animal prey by modified leaves called traps. No carnivorous plant is able to capture prey by its flower. Givnish et al. (1984) proposed that a plant must fulfil two basic requirements to be considered as carnivorous. First, it must be able to absorb nutrients from dead prey, and thereby obtain some increment to fitness in terms of increased growth, pollen production or seed set. Secondly, the plant must have some adaptation or resource allocation whose primary result is the active attraction, capture and/or digestion of prey. The first is needed to differentiate carnivory from defensive adaptation that immobilizes or kills animal enemies without leading to substantial nutrient absorption and thus increased plant survival. The second is required because many plants can passively profit by absorbing some nutrients from dead animals decomposing in the soil or on leaf surfaces. A plant must have at least one adaptation (i.e. active attraction, capture and digestion) in combination with nutrient absorption to be qualified as carnivorous, because many genera of carnivorous plants lack some of these attributes. For example, Utricularia and Pinguicula probably lack resource allocation to prey attraction. Heliamphora and Darlingtonia lack digestive glands and enzymes (except probably H. tatei; Jaffé et al., 1992) and rely on symbiotic bacteria and other organisms to break down the prey (Adlassnig et al., 2011). The genus Roridula relies on a mutualistic relationship with Pameridea roridulae, a species of capsid bug, which lives on the plant and feeds on the trapped insects. The plant obtains nutrients from the droppings of this symbiotic insect (Anderson, 2005; Plachno et al., 2009). Nepenthes bicalcarata relies on the symbiotic ants Camponotus schmitzi to catch and digest its prey (Bonhomme et al., 2011a; Bazile et al., 2012; Thornham et al., 2012). These alternative methods of prey digestion, called digestive mutualisms, may represent extremely specialized adaptation to the carnivorous syndrome because they reduce the costs of having to produce digestive enzymes (Anderson and Midgley, 2003). According to this new definition, the plant Paepalanthus bromelioides is considered as a carnivorous plant (Nishii et al., 2013; Givnish et al., 2015).

Although 19 genera in 12 families and five orders of carnivorous plants are now recognized (Aldrovanda, Brocchinia, Byblis, Catopsis, Cephalotus, Darlingtonia, Drosera, Drosophyllum, Dionaea, Genlisea, Heliamphora, Nepenthes, Paepalanthus, Philcoxia, Pinguicula, Roridula, Sarracenia, Triphyophyllum and Utricularia; Givnish et al., 2015; Fig. 1), some authors argue that there are more carnivorous plant genera than previously believed and that there is a clear continuum between carnivorous and non-carnivorous plants. Recently, Chase et al. (2009) discussed as carnivorous the sticky plants Potentilla glandulosa, Geranium viscosissimum, Petunia violacea, Petunia nyctaginiflora and Solanum tuberosum, and plants that use glandular hairs to protect their flowers, including Stylidium species, Passiflora foetida and Plumbago auriculata. They also included plants that make pitchers with their leaves including Dipsacus fullonum, plants that kill birds including Puya raimondii, and more. Although that paper attracted media attention, many authors disagree with such a broad definition of carnivorous plants, because they do not fulfil the above-mentioned criteria (Brittnacher, 2011; Rice, 2011).

Fig. 1.

The carnivorous plants. (A) Cephalotus follicularis, (B) Darlingtonia californica, (C) Dionaea muscipula, (D) Nepenthes tentaculata, (E) Heliamphora nutans, (F) Sarracenia flava, (G) Drosera roraimae, (H) Pinguicula alpina, (I) Utricularia humboldtii.

A long-standing problem in evolutionary biology, i.e. an explanation for the ecological conditions under which botanical carnivory is likely to evolve repeatedly, was resolved by Givnish et al. (1984). Several comprehensive reviews of the rise of carnivorous plants have been published over the past decade, all focusing on trade-offs among physiological and morphological traits (Ellison and Gotelli, 2001, 2009; Ellison, 2006; Ellison and Adamec, 2011). Here, we review the cost–benefit model for the evolution of botanical carnivory in view of new data on the molecular biology of trap leaves. In addition to the classical ecological intepretations of that model, we highlight the importance of energetic costs of active trapping mechanisms. We also attempt to address the similarities between carnivory and plant defence mechanisms and the role of jasmonate signalling in carnivory. Finally, we extend the intepretation of the cost–benefit model to alternative nutrient sequestration strategies in carnivorous plants.

TRAPPING MECHANISMS

The traps of carnivorous plants may be active or passive, depending on whether movement aids the capture of prey. Five basic trapping mechanisms are found in carnivorous plants (Juniper et al., 1989; Król et al., 2012).

Pitfall traps (pitcher plants)

These trap prey into a modified pitcher that contains a pool of digestive enzymes or bacteria. Special adaptation such as wax layers, anisotropy of digestive glands, slippery peristome and acidic and viscoelastic digestive fluid are involved in the capture mechanism (Bohn and Federle, 2004; Gaume et al., 2004; Gorb et al., 2004; Gaume and Forterre, 2007; Bazile et al., 2015). Prey usually lose stability on slippery surfaces and fall inside the pitcher with digestive fluid. Passive pitfall traps are thought to have evolved six times independently in the families Sarraceniaceae (Sarracenia, Heliamphora, Darlingtonia), Cephalotaceae (Cephalotus), Nepenthaceae (Nepenthes) and Eriocaulaceae (Paepalanthus) and twice in the family Bromeliaceae (Brocchinia and Catopsis) (Givnish et al., 2015).

Flypaper traps

These traps involve a sticky mucilage on the leaf surface. Flypapers have evolved at least five times independently in the following genera: Drosera (family Droseraceae), Drosophyllum (family Drosophyllaceae), Triphyophyllum (family Dioncophyllaceae), Byblis (family Byblidaceae), Pinguicula (family Lentibulariaceae), Roridula (family Roridulaceae) and the recently described genus Philcoxia (family Plantaginaceae; Renner and Specht, 2011; Pereira et al., 2012; Givnish et al., 2015). Mucilage-secreting glands may be short (like those of the Pinguicula), or long and mobile (like those of Drosera). There are active and passive traps. For example, Triphyophyllum has a passive flypaper trap that secretes mucilage, but whose leaves do not move in response to prey capture. Sundew (Drosera) has active flypaper traps with tentacles and leaf bending reactions. A special category of tentacles has recently been described: fast-moving snap tentacles in Drosera glanduligera (Poppinga et al., 2012).

Snap traps

These traps utilize rapid leaf movements. There are only two closely related active snap trap genera – Dionaea and Aldrovanda (family Droseraceae). The rapid trap closure is triggered by action potentials generated by touch of sensitive hairs on the trap lobes (Volkov et al., 2008).

Bladder suction traps

These suck in prey with a bladder that generates an internal vacuum and are exclusively found in the genus Utricularia (family Lentibulariaceae). The bladders actively pump ions out of their interiors and water follows by osmosis; this generates a partial vacuum inside the bladder. Aquatic invertebrates touch the trigger hairs and deform the door by lever action, releasing the trap wall and causing water influx. The invertebrate is sucked into the bladder, where it is digested (Vincent et al., 2011a, b; Adamec, 2012).

Eel-traps

These traps force prey to move towards a digestive organ with inward-pointing hairs. These are passive traps. They are found in the genus Genlisea (family Lentibulariaceae) and in Sarracenia psittacina (family Sarraceniaceae) (Adamec, 2003).

COST–BENEFIT MODEL FOR EVOLUTION OF BOTANICAL CARNIVORY

Today >650 species of carnivorous plants have been recognized, including monocotyledons and eudicotyledons (Ellison and Adamec, 2011). They have evolved at least nine times independently and are thus an example of convergent evolution (Albert et al., 1992; Givnish et al., 2015). Recently, the first fossilized (35–47 milion years old) carnivorous plant trap of the family Roridulaceae was found (Sadowski et al., 2015). Convergent evolution is an independent evolution of similar features in species of different lineages which grow or live in a similar environment. For example, pitcher plants Sarracenia and Nepenthes are not related and pitcher traps are analogous, not homologous, structures (i.e. they have not evolved from a common ancestor). What environmental factors are the driving forces for evolution of botanical carnivory? Ecologist Thomas Givnish was the first to recognize why carnivorous plants are mainly restricted to habitats and microsites that are not only nutrient poor, but sunny and moist as well. In January 1980, Thomas Givnish visited the Gran Sabana in Venezuela, where he observed terrestrial bromeliad Brocchinia reducta. He noticed that this bromeliad has evolved some features which resembled a carnivorous lifestyle. In contrast to related species such as B. tatei, B. reducta forms bright yellow-green leaves which are held vertically enclosing its own water tank. The fluid inside the tank is highly acidic (pH 2.8–3.0) and contains abundant remains of dead insects. The tank emits a sweet odour. The leaf surface is coated with a fine waxy layer inhibiting prey escape from the central tank. After removing this layer by brushing, attempts by ants to ascend the leaf surface succeeded. Finally, the trichomes of B. reducta can absorb ³H-labelled leucine. Based on the criteria mentioned above, B. reducta fulfils all the requirements to be classified as carnivorous. Although the evidence that absorption of nutrients increases B. reducta fitness is missing, it is very likely that it occurs, because it grows in a nutrient-poor habitat that is the home of other genera of carnivorous plants (Drosera, Heliamphora, Genlisea and Utricularia), where benefit from prey capture in terms of increased growth was documented (Ellison, 2006). Givnish et al. (1984, 1997) also proposed possible mechanisms for how the carnivory in B. reducta may have evolved. The closest relative of B. reducta is B. tatei, which is a facultatively epiphytic tank species growing in shady cloud forest. Its nearly horizontal rosette-forming green leaves are well adapted for light capture (Fig. 2). The leaves of B. tatei form a spreading rosette and capture moderate amounts of precipitation and leaf litter in their bases. Under such conditions, it probably obtains some nutrients from the breakdown of such debris, like many other tank epiphytes. Invasion of sunny sterile savannah by B. reducta’s ancestors would have favoured the evolution of steeply inclined leaves with strongly reflective waxy cuticles to reduce light capture. These traits would be a pre-adaptation for the evolution of carnivory. The crucial shift to carnivory probably involved the leakage into the tank of a volatile compound stored in the leaf base of many Brocchinia species. This would attract insects into the tank and promote the evolution of other carnivorous-related functions. Thus B. reducta was the first documented case of carnivory in Bromeliaceae and even in monocotyledons. The related species B. hectioides also growing on Gran Sabana in Venezuela is considered as the second carnivorous species in the genus Brocchinia. It has evolved traits very similar to those of B. reducta. The third species is probably Catopsis berteroniana, but more research on this species is needed (Frank and O’Meara, 1984; Givnish et al., 1984; Givnish, 1989). The fact that the genus Brocchinia also possesses other specialized adaptations to nutrient capture (myrmecophytic B. accuminata, nitrogen (N) fixation in some populations of B. tatei) in nutrient-poor habitats indicates that they have evolved in the process of adaptive radiation. Givnish et al. (1997) hypothesized that the tank habit and absorptive trichomes are the two key innovations that allowed the evolution of a great diversity of specialized mechanisms of nutrient capture including carnivory.

Fig. 2.

Two related species of Brocchinia growing in the Guiana Highlands, Venezuela. (A) The non-carnivorous species Brocchinia tatei often grows in cloud forest and forms nearly horizontal rosette-forming green leaves. (B) The carnivorous species Brocchinia reducta grows in open vegetation and forms bright yellow-green leaves which are held vertically.

The observations of Givnish in Gran Sabana in Venezuela led him to formulate a cost–benefit model for evolution of botanical carnivory. Cost–benefit models have been a hallmark of ecological analyses for >50 years, and ask what organismal form, physiology or behaviour would maximize energy capture in a particular environment and thus be likely to result in maximal competitive ability and fitness in that context. The amount of energy a given organism can allocate to different functions is fixed, so that there are inevitably trade-offs among allocations to those functions, with the optimal allocation almost certainly varying with environmental conditions. In other words, it is based on the assumption that organisms cannot do equally well and there must be some trade-off. Carnivorous plants are model systems for studying a wide range of ecophysiological and ecological processes, and the application of a cost–benefit model for the evolution of carnivory by plants has provided many novel insights. Carnivory should evolve if benefits from increased uptake of nutrients from animal prey exceed the cost of investment in carnivorous adaptations. In this case, the plants obtain an advantage in competing with other plants in nutrient-poor habitats. Givnish et al. (1984) considered that the costs of carnivory included the extra energy required to attract prey (e.g. production of lures), capture prey (e.g. production of wax or mucilage) and digest prey (e.g. production of digestive enzymes), as well as a decreased rate of photosynthesis per unit leaf mass or area (A_N). Against these, he proposed three potential benefits from carnivory:

1. Carnivory may increase a plant’s A_N through improved nutrient supply, particularly N status, in two ways: either by increased A_N per unit leaf mass or by an increase in the total leaf mass that can be supported.

2. Carnivory may result in an increased seed production through improved mineral acquisition.

3. Carnivory may replace autotrophy partly by heterotrophy.

Givnish et al. (1984) considered the second benefit as a part of the first, as increased A_N should lead to increased seed production, and the third benefit as unlikely, because experimental evidence supports the fact that carnivorous plants obtain minerals, not carbon. He concluded that the primary benefit from carnivory is increased A_N through increased nutrient supply from prey. However, the A_N does not increase equally well under all conditions. If the factors such as light or water are in short supply and limit photosynthesis, then A_N increases more slowly with improved N content than at high light or high water supply, because of light and stomatal limitation of photosynthesis. It is known that carnivorous plants give up carnivory temporarily if they grow in nutrient-rich soil or they do not have enough water and light. For example, the genera Nepenthes and Cephalotus stop producing their pitchers, Sarracenia forms non-carnivorous phyllodia instead of traps, Dionaea decreases excitability of their trap, and Drosera and Pinguicula decrease the stickness of their leaves (Zamora et al., 1998; Ellison and Gotelli, 2002; Thorén et al., 2003; Pavlovič et al., 2010b; Escalante-Pérez et al., 2011). Thus, in summary, obviously where there is a shortage of nutrients and enough water and light, there is the greatest impact on photosynthetic gains from prey capture, and such conditions favour the evolution of plant carnivory (see fig. 3 in Pavlovič et al., 2009). In contrast, the costs to carnivory are thought to exceed the benefits in shady, nutrient-rich and dry habitats, and carnivory has no adaptive value in such an environment and does not pay off. Thus the main outcome from the cost–benefit model, that energetic benefits of carnivory are likely to exceed its costs only in sunny, moist and nutrient-poor environments, became a framework for studies in carnivorous plants (Ellison and Gotelli, 2009).

Fig. 3.

Classical interpretation of the cost–benefit model for evolution of botanical carnivory in the genus Nepenthes. Comparison of photosynthetic characteristics between the pitcher (the lid as a flat part of the pitcher) and lamina. (A) The leaf of Nepenthes truncata. (B) A/Ci response curve of photosynthesis. (C) Light response curve of photosynthesis. (D) Light response curve of effective photochemical quantum yield of photosystem II (ϕ_PSII). (E) Stomatal conductance (g_s). (F) Protein content and protein gel blot analysis (the same amount of protein was electrophoresed), leaf (1), digestive zone (2), peristome (3), lid (4). (G) Elemental composition of the leaf. (H) Pigment content and ratio. Data are means ± s.e. (n = 5); different letters denote significant differences among plant tissues (one-way ANOVA followed by Tukey’s test). For details of the methods, see the Supplementary Data.

When Givnish proposed the cost–benefit model for the evolution of botanical carnivory 30 years ago, there was almost no compelling evidence bearing on whether his intepretation was right or wrong. Although Darwin’s son was the first who showed that the growth of the carnivorous plant Drosera rotundifolia was enhanced by insect feeding (Darwin, 1878), the final experimental proof that carnivory may enhance A_N was missing for another 130 years. The first study performed by Méndez and Karlsson (1999) did not show enhanced A_N in response to feeding in Pinguicula vulgaris probably due to the short feeding period. The study of Ellison and Gotelli (2002) showed that Sarracenia purpurea responded to nutrient addition by shifting from production of carnivorous pitchers to production of more photosynthetically active phyllodia. However, Wakefield et al. (2005) measured the A_N after feeding in the same species with prey but he did not find any increase of A_N. Later Ellison and Farnsworth (2005) showed that A_N correlated with foliar N content in Darlingtonia californica from different sites. Ellison (2006) summarized data from 24 studies, and his meta-analysis showed that there is a significant positive effect of prey addition on carnivorous plant growth, but data regarding photosynthesis are still lacking. The first experimental evidence that A_N increased in response to carnivory was documented at almost the same time in ten Sarracenia species (Farnsworth and Ellison, 2008), Utricularia australis (Adamec, 2008) and Nepenthes talangensis (Pavlovič et al., 2009). Later, the positive effect of feeding on A_N was found in Nepenthes ampullaria (Pavlovič et al., 2011b), Nepenthes alata (He and Zain, 2012), Drosera capensis (Pavlovič et al., 2014) and Dionaea muscipula (Kruse et al., 2014). In many of these studies the increase in A_N correlated well with the increased leaf N and/or phosphorus (P) concentrations and increased growth. The chlorophyll concentration also increased in response to feeding and it is the most sensitive indicator of nutrient stress in carnivorous plants (Moran and Moran, 1998; Farnsworth and Ellison, 2008; Pavlovič et al., 2009, 2011b, 2014; Bazile et al., 2012; He and Zain, 2012). Moreover, Adamec (1997, 2002) suggested that absorption of leaf mineral nutrients from prey stimulates root nutrient uptake. Nutrients taken up by the roots can enhance A_N and thus increase the benefit from carnivory in Nepenthes talangensis (Pavlovič et al., 2010b), indicating a high capacity for root nutrient uptake in some carnivorous plants (Gao et al., 2015). Increased flowering frequency and seed production as a secondary benefit from carnivory is also documented in carnivorous plants (Karlsson and Pate, 1992; Thorén and Karlsson, 1998; Pavlovič et al., 2009).

All these photosynthetic studies however showed another important thing: the A_N and photosynthetic nitrogen use efficiency (PNUE) in terrestrial carnivorous plants are lower than those in non-carnivorous plants, including graminoids, forbs, and deciduous and evergreen trees and shrubs. This documents the cost of carnivory in terms of a decreased rate of A_N (decreased A_N in traps) and in terms of the physiological consequences of slow growth and conditions of extremely low nutrient availability (decreased A_N in photosynthetic lamina; Mendéz and Karlsson, 1999; Ellison and Farnsworth, 2005; Ellison, 2006; Ellison and Adamec, 2011). Although the construction costs (the amount of glucose required to synthesize 1 g of carbon skeleton/biomass) of traps are not higher than the construction costs of, for example, roots or leaves, the low A_N results in long pay-back times (the time that a leaf needs to photosynthesize in order to recover the carbon investment used in its construction; Osunkoya et al., 2008; Karagatzides and Ellison, 2009). However, the construction costs do not include only construction of trap organs. Many carnivorous plants secrete sugar- (carbon) rich exudates. The mucilage of Drosera capensis comprises l-arabinose, d-xylose, d-galactose, d-mannose and d-glucuronic acid (Gowda et al., 1983). Also, the viscoelastic digestive fluid and waxy zone of the trap in the genus Nepenthes is composed of long-chain polysaccharides and aldehydes, respectively (Riedel et al., 2003; Bonhomme et al., 2011b). Similarly, rootless Utricularia plants supply easily available organic carbon (glucose, fructose and lactate) from photosynthesis to the microbial community thriving within the trap environment while benefiting from its by-products (Sirová et al., 2010, 2011). The traps of carnivorous plants are therefore probably more costly than was previously thought (Osunkoya et al., 2008).

From a morphological point of view, two types of carnivorous plants can be distinguished. Butterworts (Pinguicula), sundews (Drosera) and American pitcher plants (Heliamphora and Darlingtonia) have leaves that can both photosynthesize and capture prey. The Australian pitcher plant (Cephalotus), Venus flytrap (Dionaea), bladderworts (Utricularia), North American pitcher plants (Sarracenia), corkscrew plants (Genlisea) or Asian pitcher plants (Nepenthes) produce leaves that can photosynthesize but do not capture prey (photosynthetic laminae or phyllodes), and/or produce traps that capture prey and photosynthesize little or not at all (Ellison and Gotelli, 2001). This trait allows them to vary their investment in carnivory as a function of environmental conditions (light, water and nutrient availability sensu stricto Givnish’s cost–benefit model; Knight and Frost, 1991). These plants are suitable models for cost–benefit studies, because reworking leaf morphology and physiology to carnivory apparently reduces the efficiency of A_N in traps. Pavlovič et al. (2007, 2009) compared A_N in the Nepenthes alata, N. mirabilis and N. talangensis lamina/leaf and pitcher trap separately and described the traits responsible for the carnivorous syndrome. This is briefly summarized in Fig. 3, here in the case of Nepenthes truncata (Fig. 3A). The traps of Nepenthes have very low A_N (close to zero), lower effective photochemical quantum yield of photosystem II (ϕ_PSII) and apparent quantum yield of CO₂ fixation (ϕ_CO2, slope of the linear portion of the light response curve, 0·019 ± 0·0019 and 0·003 ± 0·0006 mol CO₂ mol photons^–1 for leaf and pitcher lid conditions, respectively), low stomatal density and conductance for CO₂ and H₂O (g_s), compact mesophyll without palisade parenchyma, and lower chlorophyll and carotenoid content but higher anthocyanin content (Fig. 3B–E, H). The pitchers, in comparison with photosynthetic leaves, also have a decreased amount of N and P (Fig. 3G). The pitchers also have lower protein content and a decreased amount of total soluble proteins invested in the Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) large and small subunits, RbcL and RbcS, respectively, but almost the same invested in the chlorophyll-binding proteins (D1, LHCI and LHCIIb; Fig. 3F). This is consistent with decreased apparent Rubisco activity (ε) in the pitcher trap expressed as the slope of the linear part of the A/C_i response curve (0·44 ± 0·04 and 0·03 ± 0·005 µmol CO₂ m^–2 s^–1Pa^–1 for leaf and pitcher lid, respectively; Fig. 3B). In the prey-deprived plants of N. ampullaria, where the N concentration in leaves and traps was <0·5 %, the Rubisco content was almost comparable between the lamina and pitcher trap, making it tempting to assume that surplus N from prey is incorporated into Rubisco in the leaves, which may explain the higher A_N in fed plants (Pavlovič et al., 2009, 2011b), an original hypothesis of Givnish et al. (1984). Rubisco is present at very high levels in photosynthesizing cells of C₃ plants and may contribute up to 50 % of soluble leaf proteins and 20–30 % of total leaf N (Feller et al., 2008). Therefore, the photosynthetic capacity of leaves is related to the N content, and the proportion of total N in Rubisco increases with increasing leaf N (Evans, 1989). Recently, Galmés et al. (2014) characterized the kinetic properties of Rubisco from 28 terrestrial plant species, representing different phylogenetic lineages, environmental adaptations and photosynthetic mechanisms, and found that carnivorous plants incorporated less total soluble proteins into Rubisco than non-carnivorous plants. As a result of extremely low protein incorporation into Rubisco, the Rubisco in carnivorous plants has evolved toward a higher maximum carboxylation rate. The higher maximum carboxylation rate probably does not compensate for lower protein investment in Rubisco, which may explain the lower PNUE in carnivorous plants. It seems that oxygen evolution (He and Zain, 2012) and electron transport from water (expresssed as ϕ_PSII) are not reduced to such an extent as dark enzymatic reactions of photosynthesis (compare the differences between the trap and lamina in A_N and ϕ_PSII in Fig. 3B–D, F), indicating that electrons from water are probably used in traps for competing processes other than photosynthesis. Pavlovič et al. (2007) predicted that the above-mentioned characteristics, which make photosynthesis in traps inefficient, may be responsible for the carnivorous function in nutrient-poor habitats (e.g. compact anatomy might serve for symplastic transport of nutrients gained from prey, N and carbon skeleton are allocated more into digestive fluid, enzymes and lures than to photosynthesis-related proteins).

A very low A_N and chlorophyll content were also found in the bladders in comparison with leaves of aquatic Utricularia by Knight (1992) and Adamec (2006). Although nobody has studied the genus Genlisea, it is almost certain that its colourless traps also have very low A_N if any. Thus, it is not surprising that such strongly modified leaves almost without photosynthetic activity are usually buried in substrate. Underground they can target a different type of prey, thus avoiding competition with sympatric terrestrial carnivorous plants. However, some other carnivorous genera have traps with not such drastically reduced A_N. For example pitchers of Cephalotus, Sarracenia or Darlingtonia have A_N almost comparable with the photosynthetic efficiency of assimilatory lamina (Ellison and Farnsworth, 2005; Farnsworth and Ellison, 2008; Hájek and Adamec, 2010; Pavlovič, 2011; Table 1). This indicates that some species indeed have reduced photosynthetic capacity in the trap organs (Nepenthes, Utricularia), but in others the A_N in the trap is only slightly, if at all, reduced in comparison with photosynthetic lamina (e.g. Sarracenia, Cephalotus, Dionaea), but is still very low in comparison with non-carnivorous plants (Méndez and Karlsson, 1999; Ellison and Farnsworth, 2005; Table 1). Altogether, these studies unequivocally showed that there is a photosynthetic cost in producing the traps in that the carbon invested in the traps does not return as much carbon as it would if it was invested in assimilatory tissue. As a result of our analyses, there is compelling evidence that traps have lower A_N than leaves (16 out of 18 cases), and that plants have higher A_N after feeding (16 out of 19 cases; Table 1).

Table 1.

Rate of net photosynthesis (A_N) in carnivorous plants with leaves differentiated into traps and photosynthetic lamina or in response to experimental feeding (fed plants in parentheses)

Species (and reference)	Leaf type	AN (nmol CO2 g–1 d. wt s–1) or ANaq (mmol O2 kg–1 f. wt h–1)	AN (µmol CO2 m–2 s–1)	Lower AN in trap	Higher AN after feeding
Aldrovanda vesiculosa (aq) (Adamec, 2008)	Shoot	34·1 ± 1·5 (21·5 ± 2·2)		N/A	–
Cephalotus follicularis (Pavlovič, 2011)	Lamina	42·8 ± 2·1		+	N/A
	Trap	27·2 ± 2·5
Dionaea muscipula (Hájek and Adamec, 2010)	Lamina	90 ± 9	4·03 ± 0·38	+	N/A
	Trap	52 ± 3	3·04 ± 0·20
Dionaea muscipula (Kruse et al., 2014)	Trap	Approx. 25 (approx. 38)		N/A	+
Drosera capensis (Pavlovič et al., 2014)	Trap		2·18 ± 0·52 (4·88 ± 0·57)	N/A	+
Nepenthes alata (Pavlovič et al., 2007)	Lamina	42·3 ± 4·5		+	N/A
	Trap	–2·4 ± 1·4
Nepenthes alata (He and Zain, 2012)	Lamina		Approx. 4 (approx. 6)*	+	+
	Trap		Approx. 1 (approx. 2)*
Nepenthes ampullaria (Pavlovič et al., 2011b)	Lamina		2·51 ± 0·55 (3·38 ± 0·37)	+	+
	Trap		0·07 ± 0·01 (0·20 ± 0·01)
Nepenthes × Coccinea (Karagatzides and Ellison, 2009)	Lamina	29·8 ± 23·1 (s.d.)		+	N/A
	Trap	2·5
Nepenthes mirabilis (Pavlovič et al., 2007)	Lamina	24·4 ± 5·5		+	N/A
	Trap	0·0 ± 1·6
Nepenthes × Miranda (Karagatzides and Ellison, 2009)	Lamina	36·0		+	N/A
	Trap	1·1
Nepenthes talangensis (Pavlovič et al., 2009)	Lamina	19·4 ± 2·0 (37·8 ± 5·4)	3·1 ± 0·3 (6·0 ± 0·5)	+	+
	Trap	2·0 ± 0·9 (3·5 ± 1·4)	0·10 ± 0·04 (0·15 ± 0·03)
Pinguicula vulgaris (Méndez and Karlsson, 1999)	Plant	55·6 ± 16·9 (s.d.) (52·1 ± 10·0) (s.d)	2·2 ± 0·6 (s.d.) (2·2 ± 0·4) (s.d.)	N/A	0
Sarracenia spp. (ten species) (Farnsworth and Ellison, 2008)	Trap	Approx. 22 (38–45)		N/A	+ (10×)
Sarracenia flava (Karagatzides and Ellison, 2009)	Lamina	45·5 ± 7·3. (s.d.)		0	N/A
	Trap	43·0 ± 10·1 (s.d.)
Sarracenia leucophylla (Karagatzides and Ellison, 2009)	Lamina	35·8 ± 24·1 (s.d.)		0	N/A
	Trap	36·5 ± 10·1 (s.d.)
Sarracenia purpurea (Hájek and Adamec, 2010)	Wing	64 ± 6	5·26 ± 0·46	N/A	N/A
	Trap	58 ± 3	3·97 ± 0·22
Sarracenia purpurea (Wakefield et al., 2005)	Trap		3·1 ± 1·2 (s.d.)	N/A	0
			(3·1 ± 1·2) (s.d.)
Utricularia australis (aq) (Adamec, 2008)	Leaf	66·7 ± 1·6 (89·2 ± 3·7)		N/A	+
Utricularia australis (aq) (Adamec, 2006)	Leaf	86·5 ± 10·1		+	N/A
	Trap	9·03 ± 1·22
Utricularia bremii (aq) (Adamec, 2006)	Leaf	40·0 ± 6·6		+	N/A
	Trap	5·24 ± 0·51
Utricularia floridana (aq) (Adamec, 2006)	Leaf	66·4 ± 3·9		+	N/A
	Trap	–5·09 ± 0·33†
Utricularia intermedia (aq) (Adamec, 2006)	Leaf	117 ± 13·1		+	N/A
	Trap	–6·77 ± 0·57†
Utricularia macrorhiza (aq) (Knight, 1992)	Leaf	0·758–0·965‡		+	N/A
	Trap	0·328–0·558‡
Utricularia ochroleuca (aq) (Adamec, 2006)	Leaf	111 ± 5·6		+	N/A
	Trap	–5·15 ± 0·42†
Utricularia vulgaris (aq) (Adamec, 2006)	Leaf	96·9 ± 5·2		+	N/A
	Trap	14·7 ± 0·64
Summary				(+) 16	(+) 16
				(–) 0	(–) 1
				(0) 2	(0) 2

A positive response is indicated by ‘+’, a negative response by ‘–’, no response by ‘0’, no data by ‘N/A’.

Note the different unit for aquatic carnivorous plants (A_Naq).

Means ± s.e. whenever possible (or s.d. if indicated).

*Values are in µmol O₂ m^–2 s^–1

^†The traps are achlorophyllous; a negative sign of the number indicates CO₂ release (i.e. R_D).

^‡Values are in mg C g tissue^–1 h^–1; values are from a population in Grassy Lake.

Carbon uptake from prey considered as the third benefit from carnivory is negligible in terrestrial carnivorous plants, but is ecologically important for aquatic carnivorous plants (see Adamec, 1997). Although some studies have documented the uptake of carbon from prey in Drosera erythrorhiza, Nepenthes insignis or Dionaea muscipula, this absorbed carbon seems more likely to be in N-bearing amino acids than in carbohydrates (Dixon et al., 1980; Rischer et al., 2002; Kruse et al., 2014). Chandler and Anderson (1976) have shown that growth of dark-grown Drosera whittakeri was not enhanced in response to feeding, confirming the suggestion that carnivory cannot replace autotrophy by heterotrophy. This is in contrast to plants with a heterotrophic mode of nutrition (e.g. parasitic plants). Although the major source of the carbon skeleton in carnivorous plants is photosynthesis, recently the loss of genes, accelerated substitution rates and relaxation of selection in plastid genomes of Utricularia, Genlisea and Pinguicula were documented in comparison with non-carnivorous plants. Almost all genes for light and dark reactions of photosynthesis are affected and resemble the obligate photosynthetic parasitic plants such as Cuscuta, indicating that alternative paths of acquiring nutrients may promote the rapid evolution of plastid genes in Lentibulariaceae or in carnivorous plants in general (Revill et al., 2005; Wicke et al., 2014).

WATER PUMPING AND ELECTRICAL SIGNALLING

There is now a significant amount of evidence that the cost–benefit model proposed by Givnish et al. (1984) is valid for terrestrial carnivorous plants with slight modifications for aquatic carnivorous plants. Ellison and Adamec (2011) suggested that nutrient limitation is more pronounced in terrestrial carnivorous plants, which also have much lower growth rates and lower A_N than aquatic carnivorous plants. Because traps of aquatic carnivorous plants are energetically very costly, it is plausible that P limitation (of, for example, ATP) might be of more consequence for aquatic than for terrestrial carnivorous plants. As demonstrated by Sydenham and Findlay (1975), ions and water pumping during the resetting of Utricularia bladders is a process requiring high amounts of metabolic energy derived from aerobic respiration. When trigger hairs situated on trap door are touched by a prey, the door opens, prey is aspirated into the trap lumen and the watertight door closes again with a speed of 5 ms. In contrast to the Venus flytrap, this process has a purely mechanical basis, as electrical signals have never been recorded in Utricularia (Vincent et al., 2011a, b; Adamec, 2012). The first attempt to include respiratory cost in the cost–benefit model was made by Knight (1992), who found at least a 10 % greater respiration rate (R_D) in Utricularia bladders than in leaves. Adamec (2006) showed that R_D in Utricularia after firing and during resetting the bladders is even 75–200 % greater than in the leaves (Table 2). This results in anoxia inside the bladders within 30 min after trap firing and causes captured prey to die of suffocation (Adamec, 2007, 2010b). These findings led Laakkonen et al. (2006) to modify the cost–benefit model including respiratory costs as an additional trade-off parameter. Jobson et al. (2004) documented that the rate-limiting enzyme in the cellular respiration pathway, cytochrome c oxidase subunit I (COX I), may be functionally altered in bladderworts. They sequenced an intron-containing gene for 21 Utricularia species and identified that the otherwise conserved Leu113–Ser114 motif in COX I is replaced by Cys113–Cys114 across all examined Utricularia and some Genlisea species. No other carnivorous plant families have the motif, and neither does it appear in any other of the >30 000 COX I sequences currently databased for eukaryotes (with one exception – Welwitschia mirabilis). This motif lies directly at the docking point of COX I helix 3 and cytochrome c. The authors suggested two possible implications for such a substitution. The first is that a disulphide bridge at the bladderwort C–C motif could lead to early termination of helix 3 and decrease the surface area for cytochrome c association–dissociation and upregulate COX I kinetics. The second is that early termination of helix 3 leads to uncoupling of proton pumping from electron transport. Such decoupling would permit bladderworts to optimize power output during times of needs, although with a 20 % decrease in total energy efficiency of respiration. Moreover, Ibarra-Laclette et al. (2011) have recently found that bladders overexpress genes involved in respiration in comparison with leaves. Considering a higher R_D, they also found a higher rate of reactive oxygen species (ROS) production, which may be responsible for high elevated nucleotide substitution rates in the Utricularia organellar and nuclear genome and the dynamic evolution of genome size (but questioned by Wicke et al., 2014). The physiological data with some molecular support confirmed that the bladders of Utricularia are expensive structures due to their high R_D/A_N ratio (Ellison and Adamec, 2011).

Table 2.

Rate of dark respiration (R_D) in carnivorous plants with leaves differentiated into traps and photosynthetic laminae or in response to experimental feeding (fed plants in parentheses)

Species (and reference)	Leaf type	RD (nmol CO2 g–1 d. wt s–1) or RDaq (mmol O2 kg–1 f. wt h–1)	RD (µmol CO2 m–2 s–1)	Higher RD in traps	Higher RD after feeding
Aldrovanda vesiculosa (aq) (Adamec, 2008)	Shoot	8·17 ± 0·81 (8·97 ± 0·72)		N/A	0
Cephalotus follicularis (Pavlovič, 2011)	Lamina	3·48 ± 0·46		0	N/A
	Trap	2·97 ± 0·66
Cephalotus follicularis (Adamec, 2010a)	Lamina	4·26 ± 0·25*		–	N/A
	Trap	2·22 ± 0·26*
Dionaea muscipula (Adamec, 2010a)	Lamina	12·3 ± 1·2*		0	N/A
	Trap	14·2 ± 2·0*
Dionaea muscipula (Hájek and Adamec, 2010)	Lamina	8·2 ± 1·8		0	N/A
	Trap	6·8 ± 0·6
Dionaea muscipula (Pavlovič et al., 2010a)	Lamina		Approx. 0·3	+†	N/A
	Trap		Approx. 3·3
Dionaea muscipula (Kruse et al., 2014)	Trap	Approx. 4·5 (approx. 8·5/4·5) (during/after feeding)		N/A	+/0 (during/after feeding)
Drosera capensis (Adamec, 2010a)	Lamina	24·9 ± 0·4*		–	N/A
	Trap	20·8 ± 0·9*
Drosera capensis (Pavlovič et al., 2014)	Trap		0·93 ± 0·10	N/A	0
			(0·74 ± 0·38)
Drosera prolifera (Adamec, 2010a)	Lamina	7·16 ± 0·70*		+†	N/A
	Tentacles	52·0 ± 7·5*
Nepenthes alata (Pavlovič et al., 2007)	Lamina	9·7 ± 0·9		–	N/A
	Trap	6·3 ± 1·1
Nepenthes alata (He and Zain, 2012)	Lamina		Approx. 2‡	–	N/A
	Trap		Approx. 1‡
Nepenthes ampullaria (Pavlovič et al., 2011b)	Lamina		0·54 ± 0·10 (0·59 ± 0·05)	–	0
	Trap		0·35 ± 0·06 (0·38 ± 0·06)
Nepenthes mirabilis (Pavlovič et al., 2007)	Lamina	8·0 ± 0·6		0	N/A
	Trap	11·6 ± 0·9
Nepenthes talangensis (Pavlovič et al., 2009)	Lamina	5·8 ± 0·5 (7·0 ± 0·21)	0·97 ± 0·02 (1·15 ± 0·06)	0/− (g–1 d. wt/m–2)	0
	Trap	6·5 ± 0·4 (9·6 ± 1·7)	0·43 ± 0·02 (0·60 ± 0·10)
Nepenthes ventricosa (Adamec, 2010a)	Lamina	5·86 ± 0·93*		0	N/A
	Trap	8·20 ± 1·47*
Pinguicula vulgaris (Méndez and Karlsson, 1999)	Plant	19·2 ± 8·1 (s.d.) (23·1 ± 8·2) (s.d.)	0·8 ± 0·3 (s.d.) (1·0 ± 0·4) (s.d.)	N/A	0
Sarracenia minor (Adamec, 2010a)	Wing	4·79 ± 0·27*		–	N/A
	Trap	3·76 ± 0·25*
Sarracenia psittacina (Adamec, 2010a)	Wing	2·28 ± 0·25*		0	N/A
	Trap	2·40 ± 0·49*
Sarracenia purpurea (Adamec, 2010a)	Wing	5·81 ± 0·62*		+	N/A
	Trap	8·38 ± 0·32*
Sarracenia purpurea (Hájek and Adamec, 2010)	Wing	9·5 ± 1·1		N/A	N/A
	Trap	12·6 ± 0·9
Sarracenia rubra (Adamec, 2010a)	Wing	5·57 ± 0·20*		0	N/A
	Trap	4·98 ± 0·41*
Utricularia australis (aq) (Adamec, 2008)	Leaf	7·89 ± 0·68		N/A	0
		(7·42 ± 0·93)
Utricularia australis (aq) (Adamec, 2006)	Leaf	4·86 ± 0·23		+†	N/A
	Trap	8·56 ± 0·35
Utricularia bremii (aq) (Adamec, 2006)	Leaf	3·07 ± 0·36		+†	N/A
	Trap	7·02 ± 0·45
Utricularia floridana (aq) (Adamec, 2006)	Leaf	2·65 ± 0·29		+†	N/A
	Trap	5·09 ± 0·33
Utricularia intermedia (aq) (Adamec, 2006)	Leaf	3·48 ± 0·34		+†	N/A
	Trap	6·77 ± 0·57
Utricularia macrorhiza (aq) (Knight, 1992)	Leaf	0·751§		0	N/A
	Trap	0·804§
Utricularia ochroleuca (aq) (Adamec, 2006)	Leaf	1·73 ± 0·17		+ †	N/A
	Trap	5·15 ± 0·42
Utricularia vulgaris (aq) (Adamec, 2006)	Leaf	3·94 ± 0·36		+†	N/A
	Trap	7·49 ± 0·21
Summary				(+) 9†	(+) 0
				(–) 7	(–) 0
				(0) 9	(0) 7

A positive response is indicated by ‘+’, a negative response by ‘–’, no response by ‘0’, no data by ‘N/A’.

Note the different unit for aquatic carnivorous plants (R_Daq).

Means ± s.e. whenever possible (or s.d. if indicated).

*Values are in nmol O₂ g^–1 d. wt s^–1.

^†Active trap (electrical signalling or water pumping).

^‡Values are in µmol O₂ m^–2 s^–1.

^§Values are in mg C g tissue^–1 h^–1 from a population in Grassy Lake.

As a result of our analysis, it is evident that the energetic or respiration costs of traps in comparison with leaves greatly depend on the type of the trap, whether it is active or passive (Table 2). In passive pitcher traps of Sarracenia, Cephalotus and Nepenthes, the difference in R_D between traps and leaves (or pitcher walls and wings) is rather small or ambiguous. In many cases (seven out of 24 cases in Table 2), the R_D in the passive traps is even lower than in the leaves, probably as a consequence of reduced A_N or due to a different leaf mass area (LMA) of these two distinct organs and not to specialization for carnivory, as is documented in the case of N. talangensis (Pavlovič et al., 2007, 2009; Adamec, 2010a, Hájek and Adamec, 2010; Table 2). Because the R_D in traps is comparable with that in leaves (Table 2) and traps have lower A_N than leaves (Table 1), traps and carnivorous plants have a higher R_D/A_N ratio (Bruzzese et al., 2010; Hájek and Adamec, 2010). In the active but resting traps of Dionaea and Drosera differences in R_D between the trap and petiole are also negligible (Adamec, 2010a; Hájek and Adamec, 2010; Table 2). However during their action, spatio-temporal changes in A_N and R_D occur. Pavlovič et al. (2010a) showed that A_N and ϕ_PSII decreased and R_D increased in response to trigger hair stimulation, simulating prey capture and retention in Dionaea muscipula. As a result of decreased photochemistry, non-photochemical quenching dissipates the absorbed light energy safely (Fig. 4A–C). Jaffe (1973) and Williams and Bennett (1982) showed that traps of Dionaea are expensive, and during trap closure about 29 % of the cellular ATP is lost; this is used for rapid transport of water, resulting in changes in turgor pressure and subsequent closure of the trap. Pavlovič et al. (2010a) showed that rapid changes of A_N and R_D in the Venus flytrap are due to trigger hair stimulation which generates electrical signals (action potentials, APs) and is independent of trap closure: ‘single hair irritation’ and the ‘prey struggle phase in the closed trap’ also resulted in reduction of A_N and stimulation of R_D (Fig. 4A–D). Volkov et al. (2007, 2008) and Pavlovič and Mancuso (2011) found that action potential signalling and changes in R_D and A_N in Dionaea are confined to the traps and are not propagated to the photosynthetic laminae, what may decrease the overall costs. In their subsequent studies, they found that mainly dark enzymatic reactions are targeted, with a negligible effect on light reactions of photosynthesis (Pavlovič et al., 2011a; Vredenberg and Pavlovič, 2013). The effect of electrical signals on photosynthesis and respiration in plants is now well recognized (for reviews, see Fromm and Lautner, 2007; Pavlovič, 2012b; Gallé et al., 2015). Increased R_D was also found in separated tentacles of Drosera prolifera in the study of Adamec (2010a), and the author suggested that such an increase is also probably due to electrical signalling in tentacles (Williams and Pickard, 1972a, b; Table 2). Because consumption of ATP during action potentials was documented (Beilby, 2007), the decrease in the ATP/ADP ratio after APs seems to be the factor increasing R_D (Pavlovič et al., 2011a). It is very likely that a similar response of APs on photosynthesis and respiration also occurs in the aquatic carnivorous plant Aldrovanda vesiculosa; however this plant has never been studied in this respect.

Fig. 4.

A novel insight into the cost–benefit model in the Venus flytrap (Dionaea muscipula). (A) Spatio-temporal changes of chlorophyll a fluorescence in response to mechanical touches of trigger hairs (at time 660–670 s) and generation of action potentials, maximum quantum yield of photosystem II (F_v/F_m), effective photochemical quantum yield of photosystem II (ϕ_PSII), photochemical quenching (qP) and non-photochemical quenching (NPQ); for detailed explanations of the kinetics of chlorophyll a fluorescence in Venus flytrap see Pavlovič et al. (2011a). (B) Simultaneous measurement of gas exchange at a light intensity of 80 µmol m^–2 s^–1 PAR. (C) Measurement of gas exchange in the dark indicating the rate of respiration (R_D). (D) Action potentials in the trap in response to mechanical stimulation of trigger hairs. (E) Light response curve of photosynthesis in open control traps and traps in the digestive period 36 h after chemical stimulation with NH₄Cl. (D) The endogenous jasmonate level (JA, jasmonic acid; JA-Ile, isoleucine conjugate of jasmonic acid; cis-OPDA, cis-12-oxophytodienoic acid) in trap tissue 36 h after induction with NH₄Cl. Data are means ± s.e. (n = 5). For details of the methods, see the Supplementary Data.

The high metabolic or energetic costs of traps also occur during prey digestion. Kruse et al. (2014) found increased R_D during the digestive period of the Venus flytrap, hence resulting in a significantly greater R_D/A_N ratio. We measured the light response curve of photosynthesis and we also found higher R_D 36 h after induction of digestion (Fig. 4E, at zero irradiance). This is in accordance with the observations of Robins and Juniper (1980), who found that the mitochondria of stimulated D. muscipula glands closely resemble mitochondria of active animal tissue. Following stimulation, the mitochondria become extended, greatly increasing their surface to volume ratio accompanied by an increase in the density of cristae. Recently, Gao et al. (2015) found that fed D. muscipula traps are frequently short of organic carbon (probably spent by R_D and enzyme production), so that they even attract carbon resources from the roots. The behaviour of glands in passive pitcher traps has never been investigated in this respect, so we cannot exclude the possibility that they also increase R_D in response to prey capture.

Besides digestion, some chemicals in insect prey can also affect carbon metabolism in carnivorous plants. Using chlorophyll fluorescence imaging, Pavlovič (2010) found that formic acid in ants, a common prey of carnivorous plants, strongly inhibits the photosynthetic reaction in PSII during digestion in Drosera capensis. Formic acid inhibits electron transport on the acceptor side of PSII, particularly from plastoquinone A to plastoquinone B.

What is the benefit of costly electrical signalling in carnivorous plants? For a successful hunt, the fast trap closure triggered by electrical signals must be faster than the prey escape reaction. Electrical signalling is carbon costly in term of decreased A_N and increased R_D; however, successful insect capture and digestion provides a significant amount of nutrients which can later stimulate photosynthesis (Pavlovič et al., 2010; Kruse et al., 2014). However, the importance of cost–benefit analysis of electrical signalling in carnivorous plants goes beyond fast prey capture. After the rapid closure secures the prey, repeated mechanical stimulation of trigger hairs by struggling prey and generation of hundreds of APs result in further closure and tightening of the trap to a tightly appressed state and secretion of digestive fluid (Affolter and Olivo, 1975). Escalante-Peréz et al. (2011) found that 30 min after prey capture in the Venus flytrap, the level of the jasmonic acid (JA) precursor, 12-oxo-phytodienoic acid (OPDA), increased. Libiaková et al. (2014) also found in the same species an increased level of jasmonates in response to mechanical and chemical stimuli (Fig. 4F). Nakamura et al. (2013) and Mithöfer et al. (2014) found an increased level of JA and its bioactive isoleucine conjugate (JA-Ile) in Drosera capensis in response to prey capture. Libiaková et al. (2014) suggested that jasmonate molecules can regulate production of digestive enzymes in response to mechanical and chemical stimuli from prey. It has been found that mechanical and subsequent electrical activity is not sufficient to trigger the full enzymatic capacity in Drosera and Dionaea, and some chemical signals must be involved (Matušíková et al., 2005; Libiaková et al., 2014; Pavlovič et al., 2014). External application of JA or coronatine, a structural mimic of JA-Ile, bypasses mechanical and chemical signalling and initiates secretion of digestive fluid with high proteolytic activity of the cysteine endopeptidase Dionain, and induces transcription of type I chitinase and ammonium channels in the Venus flytrap (Escalante-Pérez et al., 2011; Scherzer et al., 2013; Paszota et al., 2014; Libiaková et al., 2014). The link between electrical and jasmonate signalling is not novel and has been documented in the systemic response in many non-carnivorous plants (e.g. Hlaváčková et al., 2006; Mousavi et al., 2013). Jasmonates are lipid-derived compounds acting as key signalling molecules in plant stress and defence responses (Wasternack and Hause, 2013), and their accumulation is probably dependent on a cytosolic Ca²⁺ increase (Fisahn et al., 2004; Vadassery et al., 2014). In the sites that receive the electrical signals, jasmonates mediate defence-responsive gene expression (Mousavi et al., 2013). These induced defences are generally believed to have evolved as a resource-saving strategy because JA acts as a signal to redirect the gene expression and biosynthetic capacity from photosynthesis and growth to defence, and that represent a significant allocation cost for plants, which might be offset by the fitness benefit of not incurring these costs, when defence is not needed (Heil and Baldwin, 2002; Meldau et al., 2012; Vos et al., 2013; Attaran et al., 2014). Moreover, jasmonates inhibit photosynthetic reactions and repress transcription of many photosynthesis-related genes (Herde et al., 1997; Heil and Baldwin, 2002; Hlaváčková et al., 2006; Nabity et al., 2013; Attaran et al., 2014). This cost–benefit explanation has been widely applied to evolution and maintenance of inducible defence (Baldwin, 1998; Heil and Baldwin, 2002; Vos et al., 2013). The finding that jasmonates play a role in plant carnivory is not surprising, because it is now believed that carnivory has evolved from plant defence mechanisms (Juniper et al., 1989; Hatano and Hamada, 2012). The possible hierarchy of consecutive events in trap tissue which are initiated by prey capture adopted from plant defence mechanisms is suggested in Fig. 5. Thus electrical and jasmonate signalling is beneficial for regulation of enzyme and transporter expression in carnivorous plants with active trapping mechanisms, and inducibility allows carnivorous plants to forgo the allocation costs when the prey in the trap is not present. In Fig. 4E, F, the increased accumulation of jasmonates in Venus flytraps 36 h after induction of digestion and decreased A_N can be seen. Whether the reduction of A_N is the result of increased R_D (as discussed above) or reduced photosynthesis as a result of allocation costs is unclear. Venus flytraps strongly change their trap shape from the open to narrowed phase, which complicates interpretation of the data due to the changes of light interception. Nevertheless, an increased R_D/A_N ratio is an indicator of substantial cost during the digestive period in the Venus flytrap (Kruse et al., 2014). These costs are, however, offset by the benefits of not incurring these costs when digestion is not needed. It remains to be elucidated whether the jasmonates accumulate only in digestive glands or also in mesophyll tissue, and where exactly they reprogramme the gene expression.

Fig. 5.

Probable timed hierarchy of consecutive events detectable in carnivorous plants with the active trapping mechanism in response to prey capture adopted from plant defence mechanisms (Maffei et al., 2007). The earliest events measurable are action potentials generated by mechanical stimuli (Williams and Pickard, 1980; Hodick and Sievers, 1988; Krol et al., 2006; Escalante-Pérez et al., 2011) or chemical stimuli from prey (Scherzer et al., 2013), which trigger a cytosolic calcium increase (Escalante-Pérez et al., 2011) and generation of H₂O₂ (Chia et al., 2004; Ibarra-Laclette et al., 2011). Increased cytosolic Ca²⁺ is probably sensed by binding to calmodulin protein (CaM) or other calcium-sensing proteins, which can interact with mitogen-activated protein kinases (MAPKs; this part of the signaling pathway has not yet been documented in carnivorous plants). MAPKs regulate biosynthesis of jasmonates which trigger the expression of carnivory-related genes (Scherzer et al., 2013; Nakamura et al., 2013; Libiaková et al., 2014; Mithöfer et al., 2014; Paszota et al., 2014).

This may seem to be in contrast to pitcher plants, which are considered to have passive traps (Juniper et al., 1989). In contrast to Venus flytraps, the digestive fluid and enzymes in carnivorous plants of the genus Nepenthes are present even in the pitcher traps without captured prey (Eilenberg et al., 2006; Hatano and Hamada 2008, 2012). Indeed, some of the digestive enzymes are constitutively expressed (type I chitinase Nkchit2b and S-like RNAase cf-I in Nepenthes khasiana and Cephalotus follicularis, respectively; Eilenberg et al., 2006; Nishimura et al., 2013). However, transcription of some carnivory-related genes in the genus Nepenthes is also regulated by the presence of prey, such as ammonium transporter NaAMT1, H⁺-ATPase NaPHA3, type I chitinase Nkchit1b, type III chitinase Nrchit1 and thaumatin-like protein (Schulze et al., 1999; An et al., 2001; Eilenberg et al., 2006; Rottloff et al., 2011, 2013). Hatano and Hamada (2012) identified new proteins in digestive fluid of N. alata in response to chitin addition: class III peroxidase (NaPrx1a), β-1,3-glucanase (NaBGLUC2) and class III chitinase (NaCHIT3). Expression of protease, RNase, nuclease and phosphatase is also induced by the presence of appropriate chemical signals (such as nucleic acids, protein and reduced N) in the passive trap of Sarracenia purpurea (Gallie and Chang, 1997). Although the electrical signals have never been documented in passive pitcher traps, the intracellular measurements of membrane potential in the glands of Venus flytrap showed that application of NH₄⁺, the most effective inductor in carnivorous plants, resulted in strong depolarization of membrane potentials due to the action of the ammonium transporter DmAMT1 and even triggered APs (Scherzer et al., 2013). A similar ammonium transporter NaAMT1 was immunodetected in the glands of the carnivorous plant Nepenthes alata (Schulze et al., 1999), and NH₄⁺ is rapidly taken up from digestive fluid (An et al., 2001; Moran et al., 2010); however, no electrophysiological measurements have been done in this species. Also what kind of molecule or phytohormone is involved in transduction of stimuli from prey to changes of gene expression is unknown. It is tempting to assume that carnivorous pitcher traps are not so passive and electrically silent as was previously believed and more investigation in this genus is needed. In contrast, very active traps of bladderworts (Utricularia sp.) probably have constitutive enzyme production and besides enzymes also rely on microbial digestion (Sirová et al., 2003, 2010, 2011; Adamec et al., 2011).

ARE ALL CARNIVOROUS PLANTS REALLY CARNIVOROUS?

Our perception of carnivorous plants as merciless killers catching anything that is moving and careless is being changed. As the first, Cresswell (1998) reported that over half of the weight of dead matter found in the pitchers of Nepenthes ampullaria in Borneo was plant derived. This observation was supported by a study of Moran et al. (2003) who proposed that N. ampullaria exhibits a leaf litter trapping syndrome. The leaf litter trapping syndrome in plants is not novel and is very common in non-carnivorous tank epiphytic bromeliads (Givnish et al., 1997, 2014; Dézerald et al., 2013). The N. ampullaria plant has adapted to this alternative nutrition by an unusual growth pattern and pitcher morphology:

1. the pitcher lid is reflexed away from pitcher mouth and allows debris to fall directly into the pitcher;

2. the pitchers sit above the soil surface in a tightly packed ‘carpet’ and the upper pitchers are only rarely produced on the climbing stem;

3. Nepenthes ampullaria is often found growing beneath the forest canopy, whereas most other lowland species are found predominantly in open, secondary vegetation.

The ‘rain’ of debris from the canopy and tightly packed ‘carpet’ of pitchers positioned above the soil surface may intercept this source of nutrient before it reaches roots of other non-carnivorous species and may provide competitive advantage over non-carnivorous plants co-occurring in the same habitat (Fig. 6A). On the other hand, some traits involved in prey attraction (nectar glands) and retention (waxy zone and lunate cells) are reduced or absent and some are still present (large slippery peristome, chitinase in digestive fluid) (Clarke and Moran 2001; Moran et al. 2003; Rottloff et al., 2011).

Fig. 6.

Unique nutrient sequestration strategies in three species of Nepenthes from Borneo. (A) Leaf litter utilization by N. ampullaria. (B) Pitcher plants N. lowii and N. rajah (C) have modified pitcher morphology for collecting faeces from the mountain tree shrew (Tupaia montana).

Other species, such as Nepenthes lowii, produce pitchers lacking the features normally associated with arthropod prey capture – slippery peristome, waxy zone and viscoelastic digestive fluid. This species is perhaps the most unusual in the genus, being characterized by its strongly constricted upper pitchers with a reflexed lid and numerous bristles on its lower surface (Fig. 6B). Clarke et al. (1997) found that the aerial pitcher of N. lowii contained large amounts of vertebrate faeces, but no invertebrate prey. This combination of aerial pitcher characteristics and contents indicated a possible interaction with vertebrates. Clarke et al. (2009) resolved the special adaptation of this species definitively when they filmed the tree shrew Tupaia montana defecating into the pitchers after feeding on exudates that accumulate on the pitcher lid. Stable N isotope analysis revealed that tree shrew faeces account for between 57 and 100 % of foliar N in mature N. lowii plants. This unique nutrient sequestration strategy was later also found in two other species of the genus (N. rajah and N. macrophylla; Chin et al., 2010). On the basis of unique morphological characteristics of all three species, Chin et al. (2010) concluded that extraordinary modifications to nutrient acquisition strategies in carnivorous plants may occur through simple modifications of trap geometry. The pitchers of N. lowii, N. rajah and N. macrophylla with large orifices, and lids that are concave, elongated and oriented approximately at right angles to the orifice capture faeces of the tree shrew T. montana, whose body size is optimal to defecate into the pitchers (Fig. 6C). Moreover, all three Nepenthes species were shown to produce visual signals, in which the underside of the pitcher lid stood out in high contrast to the adjacent area on the pitcher, in the blue and green wavebands visible to the tree shrews (Moran et al., 2012). Analysis of volatiles extracted from the secretions of the pitcher lids by gas chromatography coupled to mass spectrometry (GC/MS) revealed 44 volatile compounds, including hydrocarbons, alcohols, esters, ketones and sulphur-containing compounds, which are commonly present in sweet fruit and flower odours (Wells et al., 2011). The body of a drowned tree shrew is occasionally found in the pitcher of N. rajah, providing much greater benefits than droppings. Recently it has been documented that beside diurnal T. montana, the nocturnal summit rat (Rattus baluensis) also defecates into the N. rajah pitchers. Temporal segregation of pitcher visits by the two mammal species enables T. montana and R. baluensis to exploit the same resource whilst largely avoiding direct conflict (Greenwood et al., 2011; Wells et al., 2011).

The pitchers of Nepenthes rafflesiana var. elongata (recently described as the new taxon Nepenthes hemsleyana; Scharmann and Grafe, 2013) is very similar to the typical form of N. rafflesiana, but is elongated in all respects. Hardwicke’s woolly bats (Kerivoula hardwickii) roost in the upper pitchers of N. hemsleyana. The elongated pitchers provide enough space for the bats and in return the plant receives additional N input in the form of faeces. It has been estimated that the plant derives 33·8 % of its total foliar N from the bat’s droppings; however, only 20·8 % of traps are occupied (Grafe et al., 2011).

The fact that the genus Nepenthes demonstrates a remarkable variety in pitcher morphology implies that it may be a candidate model for adaptive radiation with regard to N sequestration strategies (Chin et al., 2010; Pavlovič, 2012a). If the changes in pitcher morphology are adaptive, the plant will obtain some increment in fitness. Pavlovič et al. (2011b) tested the hypothesis of whether leaf litter utilization by N. ampullaria can increase photosynthetic efficiency according to Givnish’s cost–benefit model. They found that leaf litter utilization slightly increased A_N; however, the nutrient stress was not completely alleviated in comparison with experiments with insect prey (Pavlovič et al., 2009). It seems that these unique nutrient sequestration strategies make the best of a bad situation. Because the insect body contains around 9·8 % (Pavlovič et al., 2009), animal faeces 4·9 % (Chin et al., 2010) and the leaves of co-habiting plant species only 1·2 % of N (Osunkoya et al., 2007), the animal prey is still the best source of N. This might explain why the plants do not completely rely on alternative sources of nutrients and still capture some insect prey (Pavlovič et al., 2011b).

A unique sequestration strategy is not confined to Nepenthes. Roridula plants capture insects by using a sticky hydrophobic secretion on the heads of immobile tentacles. It has been hypothesized that Roridula leaves absorb N from the faeces of the obligately associated, carnivorous hemipteran bug Pameridea roridulae (Anderson, 2005; Plachno et al., 2009). Also, the non-carnivorous plants Bromelia balansae and Paepalanthus bromelioides (now considered as carnivorous) obtain a significant amount of N from spider faeces (Romero et al., 2006; Nishi et al., 2013) as does Vriesea gigantea from amphibian excrement (Inselsbacher et al., 2007). Peroutka et al. (2008) documented that algae of 45 genera were found in Utricularia bladders, which form up to 80 % of total prey. Ninety per cent of them were dead. This is the reason why the authors named their paper appropriately: ‘Utricularia – a vegetarian carnivorous plant’. The algae probably entered the traps due to an incidental and spontaneous firing (Adamec, 2011, 2012; Vincent et al., 2011a). Koller-Peroutka et al. (2015) confirmed the ecological importance of autonomous firing in Utricularia and found that the contribution of pollen grains and algae to the nutrition of Utricularia plant is comparable with the benefit gained from prey animals. Harder and Zemlin (1968) demonstrated in axenic cultures of Pinguicula lusitanica, grown on agar without N and P for 8 weeks, nutrient utilization from supplied Pinus pollen. The pollen-fed plants grew faster, contained more chlorophyll, and aged more slowly. In contrast to unfed plants, they initiated flower buds very early and flowered richly. Thus, the Pinguicula species with broad leaves (and possibly also some Drosera, e.g. Drosera schizandra growing beneath the forest canopy in Queensland) may benefit from aerial rain of pollen and probably also of spores, seeds and leaf fragments under natural conditions.

CONCLUSIONS

In 2014 the cost–benefit model for evolution of plant carnivory celebrated 30 years of existence. During that time, the cost–benefit model became a framework for interpretation of results from a wide range of experimental studies on many carnivorous plant species. The relationship between nutrients gained from prey digestion and photosynthesis has been central in cost–benefit model analyses. In the years since the cost–benefit model was initially proposed, several studies have now confirmed two of its key assumptions, showing both that traps have lower photosynthetic rates than asimilatory leaves, and that photosynthesis of carnivorous plants increases as a result of feeding (Table 1). Recent research has shown that active trapping mechanisms are costly in terms of spatial and temporal activation of respiration and inactivation of photosynthesis. Water pumping in Utricularia bladders, prey digestion, and electrical signalling in Dionaea, Drosera and probably Aldrovanda represent energetic costs for plants. Against these, jasmonate signalling has evolved as a cost-saving strategy, because instead of producing costly digestive enzymes permanently, carnivorous plants often activate their production only in response to electrical and chemical signals that implicate the presence of prey. The involvement of jasmonates and chitinases in the digestive process support the hypothesis that carnivory may have evolved from plant defence mechanisms – and it is not only the carnivory. Recent works have revealed different strategies that carnivorous plants employ in acquiring nutrients and that the cost–benefit model can also be applied here.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxfordjournals.org and give the materials and methods for the data in Figs 3 and 4.