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Carnivorous plants circumvent the nutrient shortage characterizing the habitats they colonize by deriving key nutrients from arthropods which they attract, trap and digest in specialized leaves (Juniper et al., 1989; Ellison & Gotelli, 2001). Nepenthes (Caryophyllales: Nepenthaceae) is a climbing and carnivorous plant genus characterized by leaves modified as pitcher traps (Fig. 1). It encompasses > 100 species, mainly distributed in southeastern Asia, with the islands of Borneo and Sumatra as hotspots of diversity (Clarke, 1997, 2001; Cheek & Jebb, 2001; McPherson, 2009), and colonize various habitats, including coastal lowlands, cliffs and high-altitude forests, with a high rate of endemism (Clarke, 1997; McPherson, 2009). Nepenthes species show a great diversity of pitcher morphologies, which could reflect differences in trapping strategies and stem from their adaptation to different arthropod fauna found in their habitats. But it is not known whether the trapping strategies of these pitcher plants are actually functionally diverse, and whether this diversity is linked to ecological characteristics of their environment. Nepenthes species are known to vary in their arthropod prey assemblage (Kato et al., 1993; Adam, 1997; Merbach et al., 2002) and even in their N-sequestration strategies (Moran et al., 2010), with some outlying species moving away from a purely carnivorous habit by deriving part of their nitrogen from leaf detritus (Moran et al., 2003), vertebrate faeces (Clarke et al., 2009; Chin et al., 2010), or from the nutritional service of a symbiotic hunter ant (Bonhomme et al., 2011). The trapping strategy of strictly insectivorous species (the vast majority of these pitcher plants) has never been investigated in a comparative study within the genus.
Figure 1. Experimental designs used to test the effects of pitcher waxiness and fluid viscoelasticity on ants and flies. (a) Ants were handled using a soft tube and allowed to walk freely on the pitcher rim. (b) The jar containing the experimental flies was opened and linked by a gauze mesh to a glass beaker covering the upper part of the pitcher. (c) The photograph shows a Nepenthes pitcher with a waxy zone (pale area, arrow) from which crystalline wax (also see scanning electron microscope view, inset) was extracted using hot chloroform. (d) The extensional rheometry measurements of the digestive fluid were made by high-speed video-recording and analyses of the thinning dynamics of a filament (measure of its diameter D relative to its initial diameter D0) created by vertically stretching a droplet of digestive fluid between two plots 3 cm apart. Filament lifespan was used to estimate the fluid viscoelasticity.
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Plants in the Nepenthes genus have long been thought to function as simple pitfall traps relying on slippery surfaces that decrease insect adhesion (Juniper & Burras, 1962; Juniper et al., 1989; Gaume et al., 2002, 2004; Gorb et al., 2005) and wettable surfaces that cause insect aquaplaning (Bohn & Federle, 2004; Bauer et al., 2009). But in 2007, Nepenthes rafflesiana was shown experimentally to use another mechanism. It produces a digestive liquid partly made up of long-chain polymers, the viscoelastic properties of which have a marked effect on insect retention (Gaume & Forterre, 2007; Di Giusto et al., 2008). Even when greatly diluted by water, the digestive liquid in N. rafflesiana has sufficient elastic properties to trap insects (Gaume & Forterre, 2007). This not only means that the digestive liquid might be crucial for the capture success of this tropical pitcher plant that is often subjected to heavy rains, but also that even species with a liquid viscosity similar to that of water may have unsuspected elastic properties that result in high trapping capacities. Therefore, the viscoelastic character of the digestive fluid might have remained cryptic in a number of species and could be far more widespread than expected in the Nepenthes genus. Interestingly, N. rafflesiana var. typica bears pitchers with a waxy zone and mainly traps ants during its juvenile phase; but as the plant ages, the waxy layer is lost (Gaume & Di Giusto, 2009) and the upper pitchers, which are only produced in the adult phase, contain a highly viscoelastic fluid that proves to be very efficient against flying insects (Di Giusto et al., 2008). By contrast, the elongated traps of N. rafflesiana var. elongata keep their waxy layer throughout plant ontogeny and the plant mainly captures ants (Gaume & Di Giusto, 2009).
This casts doubts on the common belief that all Nepenthes species exhibit the same trapping strategy based on the slipperiness of their pitchers. This study explores whether viscoelastic fluids are common among Nepenthes species and whether they are produced in addition to or at the expense of a slippery waxy layer. It also investigates the effects of each of these two retentive devices on the capture of different insect types.
To address these questions, we studied the functional diversity of Nepenthes pitcher plants in a sample of species differing in their geographic origins and habitats. We measured the traits directly involved in the ‘slippery’ and ‘viscoelastic’ strategies, i.e. waxiness (quantity and density of wax coating the inner pitcher walls) and viscoelasticity (relaxation time) of the digestive liquid. We used insect bioassays to compare the retentive ability of different Nepenthes species and measure how efficiently pitcher waxiness and fluid elasticity contribute to the retention of each type of prey. Finally, we investigated whether waxiness and viscoelasticity are correlated.
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Our comparative study of the trapping systems of Nepenthes pitcher plants generated three important results. First, and contrary to common belief (but see Gaume & Di Giusto, 2009; Bonhomme et al., 2011), the different species show functional diversity in their retentive devices and do not rely solely on the slipperiness of their trap to capture insects. The results of this study show that N. rafflesiana is not the only species to possess a viscoelastic fluid. This character may be widespread within the genus as found in two-thirds of the species in our study. Secondly, wax and viscoelatic fluids do not target the same type of prey: wax appears to be efficient only for ants, whereas viscoelasticity proved to be a powerful trapping device for both insect types and is more often found in mountain than in lowland species. Thirdly, our cross-species comparison suggests that investments in the ‘waxy’ trait and in ‘viscoelastic fluid’ could be made at the expense of the other. At the least, species that are very viscoelastic do produce very little wax. Altogether this suggests that there are two different trapping strategies in these pitcher plants, a ‘waxy’ strategy and a ‘viscoelastic’ strategy.
A widely shared viscoelastic trap in Nepenthes pitcher plants and the question of its origin
The fluid contained in the pitchers of most of the Nepenthes species studied was viscoelastic, and this might therefore represent the rule in the genus rather than the exception. Contrary to wax, whose efficiency as a trapping device has been shown to be quantity-dependent, even low viscoelasticity appears to contribute strongly to a plant’s trapping ability. Therefore, it is surprising to note that the fluids of some Nepenthes species are highly viscoelastic. It is possible that this viscoelasticity increases with plant age (Gaume & Di Giusto, 2009). But, for all species, the plants used in our study were carefully chosen to be of similar age. Another explanation could be that, as the plant species differ in their habitats, some are more subject than others to rainfall and humidity (e.g. those of altitudinal mossy forests or those that have a reduced lid that can protect the pitcher) and to subsequent fluid dilution by water. Greater production of the polymers that are assumed to cause the viscoelasticity of pitcher fluid might have been selected in some species, helping them to cope with the problem of daily dilution. This hypothesis is corroborated by the results of Gaume & Forterre (2007), who showed that the elastic fluid of N. rafflesiana (which here appears to be among the most viscoelastic species), when diluted in 95% of water, was still viscoelastic enough to capture all the insects dropped into the pitchers.
Since relaxation times of < 100 ms are impossible to detect without the use of a high-speed camera, viscoelastic fluids have probably gone unnoticed in many species and may be far more common than suspected. This raises the question of whether the viscoelastic fluids in Nepenthes species have a common origin. Interestingly, the glue secreted by the leaves of Drosera, another carnivorous genus, is composed of acid polysaccharides (Gowda et al., 1982) and these have been demonstrated to be viscoelastic (Erni et al., 2008). This is also probably the case for the glue secreted by Drosophyllum (V. Bonhomme, unpublished). Both of these genera are the closest relatives of Nepenthes in the phylogeny of the Caryophyllales (Heubl et al., 2006) and their traps are known to function like flypaper (Juniper et al., 1989). We can therefore advance the hypothesis that the viscoelastic polysaccharide fluids in Nepenthes and in these other carnivorous genera have a common and thus plesiomorphic origin, with the glue of the other carnivorous genera simply containing a far higher concentration of polysaccharides than the fluid in Nepenthes.
Relative investment in different trapping devices
The appearance of botanical carnivory and the evolution of specialized traps are subject to powerful cost–benefit constraints (Givnish et al., 1984; Ellison & Gotelli, 2001; Pavlovičet al., 2007). We can therefore assume that it is costly for carnivorous plants to produce modified leaves with lower photosynthetic capacities (Pavlovičet al., 2007, 2009), and that the biosynthesis of trapping features is subject to selective pressure and can be maintained throughout evolution only if the cost of these features is exceeded by the benefits they provide in terms of insect-derived nutrients. Development of the waxy zone, mainly composed of aliphatic compounds dominated by very long-chain aldehydes (e.g. triacontanal or dotriacontanal containing 30 or 32 carbon atoms, respectively Riedel et al., 2003, 2007), is metabolically costly for the plant. The molecules responsible for digestive fluid viscoelasticity are assumed to be long-chain polysaccharides (Gaume & Forterre, 2007) that must also be costly to synthesize. This could provide part of the explanation as to why none of the species we tested possesses both very viscoelastic fluids and very waxy pitchers. The inverse relationship that seems to link these two quantitative traits in our cross-species comparisons might illustrate the existence in the plant of an investment trade-off. This will need to be tested at the genus scale with an analysis that takes into account the phylogenetic relationships of the measured species. A test of this hypothesis will also necessitate studies of several populations of a given species showing some variations in these traits.
Interestingly, a few plant species in our study showed both nonviscoelastic fluids and only slightly waxy pitchers, or pitchers that contained no epicuticular wax at all, such as N. ampullaria and N. ventricosa. These plants are outliers in the Nepenthes genus. Perhaps the pitchers do not have a strictly carnivorous diet; this is the case for N. ampullaria, which obtains part of its nitrogen from leaf debris (Moran et al., 2003). An additional explanation is that they utilize other trapping strategies. It is possible that N. ampullaria relies uniquely on its peristome, which forms a steep slop, to trap its prey. Several other features, such as water-dependent structures facilitating insect-aquaplaning (Bohn & Federle, 2004; Bauer et al., 2008) or specific pitcher morphology, might favour both insect capture and retention. But as attested by the high coefficients of determination of the models testing for the effect of wax and viscoelasticity, these other features only played a minor role in insect retention.
The role of prey in the evolution of different trapping strategies
Whether wax and viscoelastic fluids are produced at each other’s expense, as these devices are necessarily costly, the question arises as to the selective factors that have favoured the evolution of these traits. Pitcher wax causes insects to slide and is thus implicated in both capture and retention (Juniper & Burras, 1962; Gaume et al., 2002), while the viscoelastic fluid acts on retention (Gaume & Forterre, 2007). The results obtained in our study show that the efficiency of these strategies is prey-dependent. Wax is more efficient on ants than on flies, whereas viscoelasticity is very efficient on both insect types and definitely more efficient than wax on flies. Winged insects are able to take off from the pitcher wall without even touching the waxy surface, and even if they do enter into contact with it, the wax acts only on their attachment systems (Gaume et al., 2004; Gorb et al., 2005) not on their flying system. By contrast, crawling insects have no other option than to cope with the wax that contaminates their pads and causes them to lose adhesion. Moreover, since winged insects have a higher surface : volume ratio than crawling insects, they offer a larger surface area for the viscoelastic fluid to exert its retentive force and this may explain why they are more often retained in Nepenthes liquids (Gaume et al., 2002; Gaume & Forterre, 2007).
Hence wax and viscoelastic fluid clearly do not have the same function and do not target the same types of insect. This suggests that they might represent adaptation to different prey spectra and that local differences in entomofauna might exert different selective pressure on the development of wax and/or viscoelasticity. For any given pitcher waxiness, a ‘viscoelastic strategy’ is needed to trap flies with the same efficiency as ants. This means that habitats dominated by ants, such as the lowland forests of Borneo (Gunsalam, 1999; Davidson et al., 2003), may favour the development of a waxy ‘slippery’ strategy. On the other hand, habitats dominated by flying insects may favour the development of a ‘sticky’, viscoelasticity-based strategy. Such habitats are generally found at higher altitudes where ants are few in number but flying insects are relatively more abundant (Collins, 1980). This can also temporarily be the case for lowland, open, and regularly flooded habitats such as those inhabited by N. rafflesiana var. typica (Gaume & Di Giusto, 2009), which is associated with a flower scent cue that more specifically targets flying insects (Di Giusto et al., 2010).
We therefore advance the hypothesis that the scarcity of ants in tropical mountains (Borneo (Collins, 1980; Clarke et al., 2009), the Philippines (Samson et al., 1997)) and the relative abundance of flying insects (Collins, 1980) provide part of the explanation for the widespread viscoelastic strategy among mountain Nepenthes species. A comparative study (Adam, 1997) corroborates this hypothesis by showing that mountain species tend to trap a larger prey spectrum, including more dipterans and coleopterans, than lowland species, which were recorded to trap mostly ants. Furthermore, at least seven species in mountain mossy forests, and known to possess a highly viscous fluid, are reported to be specialized in the capture of flying insects: N. inermis has been reported to be (under the name of N. bongso) specialized in trapping midges (Kato, 1993); N. aristolochioides is specialized in trapping midges; N. dubia, N. jamban, N. eymae and N. talangensis are specialized in trapping small dipterans (McPherson 2009); and N. jacquelinae has been observed to trap mainly larger flying prey (Clarke, 2001). Interestingly, the pitchers of such species do not contain a waxy zone and are all funnel-shaped.
Few comparative studies have been conducted on the prey spectra of Nepenthes species (but see Kato et al., 1993; Adam, 1997). To test our hypotheses on the evolution of Nepenthes trapping devices, we need to carry out studies comparing the prey spectra of Nepenthes species with the entomofauna found in their habitats, and relating this to their insect-trapping devices. This would help in understanding the ecological mechanisms underlying the evolution and diversification of these pitcher plants.