New Phytologist Research

Slippery or sticky? Functional diversity in the trapping strategy of carnivorous

Vincent Bonhomme1, Herve´ Pelloux-Prayer1, Emmanuelle Jousselin2, Yoe¨l Forterre3, Jean-Jacques Labat4 and Laurence Gaume1 1Universite´ Montpellier II, CNRS, UMR AMAP: botAnique et bioinforMatique de l’Architecture des Plantes, CIRAD – TA A51 ⁄ PS2 Boulevard de la Lironde, F-34398 Montpellier cedex 5, France; 2INRA, UMR CBGP, Campus International de Baillarguet, CS 30016, 34988 Montferrier-sur-Lez, France; 3CNRS, Universite´ de Provence IUSTI, Technopole Chaˆteau-Gombert, 13000 Marseille, France; 4Pe´pinie`re Nature et Paysages et Jardin Botanique de Plantes Carnivores, Peyrusse-Massas, France

Summary

Author for correspondence: • The pitcher-shaped leaves of Nepenthes carnivorous plants have been consid- Vincent Bonhomme ered as pitfall traps that essentially rely on slippery surfaces to capture insects. But Tel: +33 (0)4 67 61 7166 a recent study of Nepenthes rafflesiana has shown that the viscoelasticity of the Email: [email protected] digestive fluid inside the pitchers plays a key role. Received: 4 December 2010 • Here, we investigated whether Nepenthes species exhibit diverse trapping strat- Accepted: 8 February 2011 egies. We measured the amount of slippery wax on the pitcher walls of 23 taxa and the viscoelasticity of their digestive liquid and compared their retention effi- New Phytologist (2011) 191: 545–554 ciency on ants and flies. doi: 10.1111/j.1469-8137.2011.03696.x • The amount of wax was shown to vary greatly between species. Most mountain species exhibited viscoelastic digestive fluids while water-like fluids were predomi- nant in lowland species. Both characteristics contributed to insect trapping but wax Key words: biological trade-off, carnivorous pitcher , digestive fluid, leaf wax, was more efficient at trapping ants while viscoelasticity was key in trapping insects Nepenthes, trapping strategy, viscoelasticity. and was even more efficient than wax on flies. Trap waxiness and fluid visco- elasticity were inversely related, suggesting the possibility of an investment trade- off for the plants. • Therefore Nepenthes pitcher plants do not solely employ slippery devices to trap insects but often employ a viscoelastic strategy. The entomofauna specific to the plant’s habitat may exert selective pressures, favouring one trapping strategy at the expense of the other.

trapping strategies and stem from their adaptation to differ- Introduction ent arthropod fauna found in their habitats. But it is not Carnivorous plants circumvent the nutrient shortage charac- known whether the trapping strategies of these pitcher plants terizing the habitats they colonize by deriving key nutrients are actually functionally diverse, and whether this diversity is from arthropods which they attract, trap and digest in spe- linked to ecological characteristics of their environment. cialized leaves (Juniper et al., 1989; Ellison & Gotelli, 2001). Nepenthes species are known to vary in their arthropod prey Nepenthes (: Nepenthaceae) is a climbing and assemblage (Kato et al., 1993; Adam, 1997; Merbach et al., genus characterized by leaves modified as 2002) and even in their N-sequestration strategies (Moran pitcher traps (Fig. 1). It encompasses > 100 species, mainly et al., 2010), with some outlying species moving away from a distributed in southeastern Asia, with the islands of purely carnivorous habit by deriving part of their nitrogen and as hotspots of diversity (Clarke, 1997, 2001; from leaf detritus (Moran et al., 2003), vertebrate faeces Cheek & Jebb, 2001; McPherson, 2009), and colonize vari- (Clarke et al., 2009; Chin et al., 2010), or from the nutri- ous habitats, including coastal lowlands, cliffs and high- tional service of a symbiotic hunter ant (Bonhomme et al., altitude forests, with a high rate of endemism (Clarke, 1997; 2011). The trapping strategy of strictly insectivorous species McPherson, 2009). Nepenthes species show a great diversity (the vast majority of these pitcher plants) has never been of pitcher morphologies, which could reflect differences in investigated in a comparative study within the genus.

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(a) (b)

(c) (d)

Fig. 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.

Plants in the Nepenthes genus have long been thought to water may have unsuspected elastic properties that result in function as simple pitfall traps relying on slippery surfaces high trapping capacities. Therefore, the viscoelastic charac- that decrease insect adhesion (Juniper & Burras, 1962; ter of the digestive fluid might have remained cryptic in a Juniper et al., 1989; Gaume et al., 2002, 2004; Gorb et al., number of species and could be far more widespread than 2005) and wettable surfaces that cause insect aquaplaning expected in the Nepenthes genus. Interestingly, N. rafflesiana (Bohn & Federle, 2004; Bauer et al., 2009). But in 2007, var. typica bears pitchers with a waxy zone and mainly traps Nepenthes rafflesiana was shown experimentally to use ants during its juvenile phase; but as the plant ages, the waxy another mechanism. It produces a digestive liquid partly layer is lost (Gaume & Di Giusto, 2009) and the upper made up of long-chain polymers, the viscoelastic properties pitchers, which are only produced in the adult phase, con- of which have a marked effect on insect retention (Gaume tain a highly viscoelastic fluid that proves to be very efficient & Forterre, 2007; Di Giusto et al., 2008). Even when against flying insects (Di Giusto et al., 2008). By contrast, greatly diluted by water, the digestive liquid in N. rafflesiana the elongated traps of N. rafflesiana var. elongata keep their has sufficient elastic properties to trap insects (Gaume & waxy layer throughout plant ontogeny and the plant mainly Forterre, 2007). This not only means that the digestive captures ants (Gaume & Di Giusto, 2009). liquid might be crucial for the capture success of this tropi- This casts doubts on the common belief that all cal that is often subjected to heavy rains, but Nepenthes species exhibit the same trapping strategy based also that even species with a liquid viscosity similar to that of on the slipperiness of their pitchers. This study explores

New Phytologist (2011) 191: 545–554 Ó 2011 The Authors www.newphytologist.com New Phytologist Ó 2011 New Phytologist Trust New Phytologist Research 547 whether viscoelastic fluids are common among Nepenthes f. ex Burb., N. copelandii Merr. ex Macfarl., N. eymae Sh. species and whether they are produced in addition to or at Kurata, N. glabrata J. R. Turnbull & A. T. Middleton, the expense of a slippery waxy layer. It also investigates the N. gracilis Korth., N. macrophylla (Marabini) Jebb & effects of each of these two retentive devices on the capture Cheek, N. madagascariensis Poir., N. mindanaonensis Sh. of different insect types. Kurata, N. mirabilis var. typica (Lour.) Druce, N. rafflesiana To address these questions, we studied the functional var. elongata Hort., N. tenuis Nerz & Wistuba and N. vogelii diversity of Nepenthes pitcher plants in a sample of species Schuit. & de Vogel. Although, the thickness and length of differing in their geographic origins and habitats. We mea- the waxy layer do not to the naked eye seem to vary when the sured the traits directly involved in the ‘slippery’ and pitcher ages, the fluid viscoelasticities are reported to ‘viscoelastic’ strategies, i.e. waxiness (quantity and density decrease when the pitchers age, some of them becoming of wax coating the inner pitcher walls) and viscoelasticity apparently watery 11–14 d after pitcher opening in N. (relaxation time) of the digestive liquid. We used insect bio- rafflesiana (Bauer et al., 2009). To reduce as far as possible assays to compare the retentive ability of different Nepenthes development-induced heterogeneity between pitchers, waxi- species and measure how efficiently pitcher waxiness and ness and viscoelasticity were measured for each species in the fluid elasticity contribute to the retention of each type of youngest pitcher that had opened in the previous week. prey. Finally, we investigated whether waxiness and visco- elasticity are correlated. Measurement of insect retention ability Experiments comparing the trapping ability of the 12 Materials and Methods Nepenthes species were carried out in April 2007 under homogeneous temperature and hygrometry conditions (26– Studied plants 27°C, 80–90%). Retention rates in the 12 species were One of the authors (J-J. L.) owns a glasshouse collection of compared for the ant Lasius niger L. (Hymenoptera, Nepenthes pitcher plants (located in Peyrusse-Massas, Gers, Formicidae, Formicinae) and the fly Calliphora vomitoria L. France) which in 1995 was recognized as the National (Diptera, Calliphoridae). According to the prey spectra of French Conservatory of Carnivorous Plants. The insect bio- Nepenthes species published so far (reviewed by Juniper assays performed in April 2007 employed the following et al., 1989; Ellison & Gotelli, 2009), ants and flies are the subset of 12 Nepenthes species: Jack, most commonly trapped insects. Although the species used N. fusca Danser, N. longifolia Nerz & Wistuba, N. maxima in our bioassays do not occur, at least for the ants, in the Reinw. ex Nees, N. mirabilis var. echinostoma (Lour.) Druce, natural prey fauna of the Nepenthes species tested, they N. petiolata Danser, N. rafflesiana var. typica Jack, represent insects typically trapped by Nepenthes as regards N. ramispina Ridl., N. spathulata Danser, N. spectabilis their size and shape and the insect order to which they Danser, N. tobaica Danser and N. ventricosa Blanco. These belong. Our aim here was simply to compare the ability of species are representative of the ecological and geographical different Nepenthes species to retain flying insects vs crawl- diversities found within the genus (terrestrial ⁄ epiphytic ing insects. A colony of L. niger found near the glasshouses climbers; lowland ⁄ mountain species; species originating provided us with worker ants. They were transferred to a from Borneo, Sumatra, , and Peninsular plastic tube and placed on the pitcher rim, the so-called Malaysia). Nepenthes species usually produce two types of peristome (Fig. 1a). About 500 laboratory-bred pitchers: lower terrestrial pitchers, produced during the self- C. vomitoria larvae were kept at 30°C for 7 d until adults supporting ⁄ juvenile stage of the plant, and upper aerial emerged. The adults were then collected and confined to a pitchers produced during the climbing ⁄ sexually mature stage glass jar connected to a cylindrical mesh, the aperture of (Clarke, 1997). Since the glasshouse plants did not all pro- which was closed by a piece of string. Once some of the flies duce upper pitchers, only young individuals with lower emerged, the string was removed and the few emerging flies pitchers were used. One freshly opened lower pitcher (open- were collected in an inverted beaker connected to the mesh. ing dating from < 1 wk) was selected on three different The beaker and mesh were then disconnected from the jar, individuals of approximately the same size (30–60 cm) in which was rapidly closed with a temporary cap, then each of the 12 species. A total of 36 pitchers were thus used. ‘slipped’ onto a pitcher, enclosing pitcher and flies together In April 2008, a larger sample corresponding to 23 taxa (Fig. 1b) by attaching the end of the mesh around the ten- and 21 species was used for comparisons of pitcher waxiness dril sustaining the pitcher. These experimental set-ups and viscoelasticity within the genus. This sample comprised allowed ants and flies direct access to the pitcher. Each trial all the previously cited species, except N. ramispina and consisted of an insect’s fall into the pitcher and the insect’s N. maxima, which did not produce any lower pitchers at fate was recorded as the binary outcome, retained ⁄ not the time of our study, together with 13 other taxa: retained within the pitcher. We observed each insect until it N. albomarginata T. Lobb ex. Lindl, N. burgbidgeae Hook. died or escaped from the pitcher. An insect was considered

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as retained if it did not successfully escape from the pitcher. measurements were performed under homogeneous tem- We considered three different pitchers for each of the 12 perature conditions (25–26°C). Nepenthes species, and performed 10 tests per pitcher, each time with a different ant, and 10 tests, each time with a dif- Statistical analyses ferent fly, gathering a total of 720 binary responses and 72 retention rates. Logistic regressions were used to explain the variability in insect retention success. Backward procedures were adopted for model selection, starting with removal of the nonsignifi- Quantitative measurement of the characters involved cant, highest-order interactions. in trapping Logistic models were used to address the following ques- All the (intra- + epicuticular) wax in the pitchers was tions: does retention success vary between Nepenthes species, extracted using warm chloroform as described by Riedel types of insect (ant ⁄ fly) and pitchers within a given species; et al. (2003) and weighed to within 1 lg on a Sartorius and could this variability be explained by the trapping fea- MC5 balance (Gottingen, Switzerland – Fig. 1c). Because tures of the species, for example, the waxiness of pitcher the plants are fragile, only one pitcher per species was used walls and the viscoelasticity of pitcher fluid? We first per- for wax extraction and measurement. The total weight of formed a mixed logistic model on observed retention wax was used as an explanatory variable for retention rates: successes, which set ‘species’, ‘insect type’ and ‘specie- it takes account not only of the thickness but also of the s’ · ‘insect type’ interaction as fixed explanatory factors and length of the waxy zone, and both parameters are probably ‘pitcher’ as a random factor nested within species. This important characteristics of the slippery trap. Each species’ mixed model was run with SAS v. 9.2 software package relative investment in ‘wax’ and ‘liquid viscoelasticity’ was using the GLIMMIX procedure (SAS Institute, Cary, NC, determined by measuring the density of wax, estimated as USA), but all the further statistical analyses were carried out the weight of wax per cm2 of pitcher wall. This measure- using R software (R Development Core Team, 2009). As ment of wax density is relevant because the viscoelasticity the pitcher effect was not significant, we then pooled reten- measurement also reflects density (i.e. that of polymers) in tion success data for the three pitchers of each species. In the fluid. In any case, the two different measurements of order to test whether species retention rates (number of tests wax quantity plotted against fluid viscoelasticity showed per species = 30) could be explained by their trapping fea- similar statistical trends. tures, we thus performed two other logistic regression Fluid viscoelasticity was first estimated by observing the models (one for each type of insect) on retention rates with presence or absence of a filament when the fluid was ‘presence ⁄ absence of a viscoelastic liquid’ and ‘quantity of stretched between two fingers. This qualitative measure- wax in the trap’ as explanatory variables. Corrections for ment of viscoelasticity was performed on the fluid of each overdispersion were applied when necessary using the quasi- of the three pitchers in the 12 Nepenthes species studied in binomial error distribution implemented in R. April 2007. Appropriate equipment for rheological studies Relaxation times for the 23 digestive fluids measured in was acquired in April 2008 and fluid from the 23 taxa was April 2008 were compared using Student’s t-tests with the subject to a quantitative measurement of its viscoelasticity. capillary pinch-off time for water, that is, the shortest break- An estimation of pitcher fluid viscoelasticity was obtained ing time for a filament. It should be noted that elastic by measuring elastic relaxation time, defined as the time relaxation times shorter than the capillary pinch-off time required for a filament of fluid to break, as described by for water cannot be measured using this capillary break-up Gaume & Forterre (2007). The fluid was subject to vertical method (Rodd et al., 2005). A fluid was conservatively strain by rapidly lifting a thin rod (diameter D0 = 3.0 mm) qualified as viscoelastic if its relaxation time was signifi- 3 cm vertically from a 40 ll sample of liquid, thus creating cantly longer than the capillary pinch-off time for water, an elongated liquid filament. The subsequent thinning with P < 0.01. To determine whether there was a correla- speed and time to filament rupture were recorded at a high tion between quantity of wax and viscoelasticity of the spatial and temporal resolution (31 pixels mm)1 and up to digestive liquids, we selected the species shown to have a 3500 frames s)1) using a Phantom Miro IV high-speed viscoelastic fluid and tested whether the quantity of wax camera (Vision Research, Wayne, NJ, USA) and a Nikkor fitted a linear then a hyperbolic function of liquid viscoelas- 60 mm macro lens. The recordings were then analysed ticity. Goodness of fit of these two regressions was then using an Image J – R script (Abramoff et al., 2004; R compared using Akaike’s information criterion in R. Development Core Team, 2009) that we developed for this Fisher’s exact test was performed on a contingency table purpose (Fig. 1d). Each fluid was tested in triplicate and (viscoelastic ⁄ nonviscoelastic liquid vs mountain ⁄ lowland mean relaxation time calculated as an estimation of its vis- species) to statistically compare the frequencies of species coelasticity. The relaxation time of distilled water was associated with viscoelastic fluid in mountain and lowland measured six times as the nonviscoelastic reference fluid. All species. The altitude range of the studied species was

New Phytologist (2011) 191: 545–554 Ó 2011 The Authors www.newphytologist.com New Phytologist Ó 2011 New Phytologist Trust New Phytologist Research 549 obtained from different bibliographic sources (Clarke, retention success in the mixed logistic regression model; 1997, 2001; Cheek & Jebb, 2001; McPherson, 2009) and Table 1, Fig. 2) and they were on average more efficient on the rough threshold of 1000 m was applied to distinguish ants than on flies (significant insect effect, percentage of ants between lowland and mountain species. This threshold is retained = 72 ± 18%, flies = 62 ± 27%, SD given in the commonly used in tropical areas (Richards, 1996) to distin- text; Table 1). However, some species were more efficient guish between species, including Nepenthes spp. (Clarke, on ants while others retained flies more easily (significant 1997), of low and high altitudes. In our Nepenthes sample, insect · species interaction; Table 1). There was no signifi- species that were considered as lowland species generally cant difference in the retention success of pitchers of the occur from sea level up to 1000 m at the most (apart from same species (no random effect of pitcher: pitcher variance N. longifolia which is not found below 300 m and could be estimate = 1.73 · 10)19, tests of covariance based on the considered as an intermediate species), while species that residual pseudo-likelihood: P 1).  were considered as montane species occur from 700 up to 2900 m. The average altitudinal range of our lowland spe- The efficiency of pitcher wax and viscoelastic fluid is cies is 265 ± 236 m, while the average altitudinal range of insect-dependent our montane species is 1550 ± 399 m. Measurements on each Nepenthes species are not truly independent because The total weight of wax averaged 1.85 mg and ranged from species share evolutionary history, and comparative meth- 0.28 mg in N. ampullaria, which has no visible epicuticular ods that take into account the phylogenetic relatedness of wax in its pitchers, to 3.40 mg in N. maxima, which bears the studied species should be used to test the relationship a thick layer of epicuticular wax in its pitchers. Fluid between traits (Revell, 2010). But available phylogenies for viscoelasticity also varied between species. The pitchers the genus Nepenthes (Meimberg & Heubl, 2006) are only of six species (N. ampullaria, N. longifolia, N. mirabilis, poorly resolved, precluding applications of these tests. However, species used here are scattered throughout the Table 1 Results of the mixed logistic model testing for the fixed currently available phylogenetic trees of the genus, and effects ‘Nepenthes species’ and ‘insect type’ and the random effect therefore statistical biases as a result of close relatedness ‘pitcher within species’ on trapping success among species should be minimized. Variability in insect retention

Response variable: retention success ndf ddf FP(> F) Results Covariate Functional diversity in the trapping success of Species 11 24 7.29 < 0.0001 Insect 1 672 5.92 0.0152 Nepenthes Species · Insect 11 672 3.78 < 0.0001

The species studied differed significantly in terms of their The effect of pitcher was not significant (variance = 1.73 · 10)19, retention success on the insects employed in our bioassays P 1). ndf, numerator degrees of freedom; ddf, denominator  (67 ± 21% of insects retained, significant species effect on degrees of freedom.

Nonviscoelastic species Viscoelastic species

100 90 80 70 60 50 40 30 20 10

Fig. 2 Percentage of ants (open bars) and Percentageinsects of retained 0 a s a a ta a flies (closed bars) retained inside the pitchers ili ca ica foli lat s an a tab u iola si of 12 Nepenthes species. Species are ranked gi c t . fu ath maxima fle tob lon mpullari . N . according to increasing mean capture rate. . spe a sp raf N N. mirabilisN . . N N. pe . Error bars are ± SE. N. ramispinaN. ventricosa N N. N N

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Table 2 Results of logistic regression testing for wax quantity wetted by the liquid were sometimes observed to climb up and fluid viscoelasticity effects on trapping success for ants (a) and the pitcher wall and then successfully take off from there, flies (b) occasionally without even touching the waxy layer. If wetted Response variable: retention rates df v2 P (> v2) by the viscoelastic liquid, the insects had little chance of escaping. The more they struggled in the fluid, the greater it (a) Ant retention success dependent on viscoelasticity and wax 2 resisted their movements and the insects rapidly became quantity, Cox–Snell pseudo r = 0.53 exhausted and drowned. Viscoelasticity 1 8.1 0.004 Wax quantity 1 5.35 0.021 (b) Fly retention success dependent on viscoelasticity, Cox–Snell Relative investments in wax and liquid viscoelasticity pseudo r2 = 0.74 Viscoelasticity 1 25.54 < 0.0001 Wax quantities measured in April 2008 were consistent with Wax quantity 1 2.58 0.108 those obtained in 2007. Wax densities ranged from 0.022 mg cm)2 (N. ampullaria,waxquantity=0.229mg)to 0.608 mg cm)2 (N. macrophylla,waxquantity=3.180mg), N. ramispina, N. spectabilis and N. ventricosa) were shown averaging 0.124 mg cm)2 (median = 0.087 mg cm)2,SD= to contain a nonviscoelastic, apparently water-like fluid, 0.135). Thirteen of the 23 species were viscoelastic, that is, whereas the pitchers of six other species (N. fusca, their relaxation time was significantly longer than that of N. maxima, N. petiolata, N. rafflesiana, N. spathulata and water, the nonviscoelastic reference fluid (t-test results in N. tobaica) contained a fluid shown to be viscoelastic by the Supporting Information, Table S1). N. longifolia and creation of a filament when stretched between two fingers. N. spectabilis appeared to be slightly viscoelastic but were not The highest retention rates were observed in species with classified as viscoelastic in 2007, probably because they viscoelastic fluids, and the lowest were observed in species create viscoelastic filaments that are not visible by direct with water-like fluids (Fig. 2). observation. Among the 13 mountain species, 12 were found Differences in wax quantity and fluid viscoelasticity to be viscoelastic whereas only three lowland taxa (two explained most of the variations observed in the trapping species, N. longifolia, N. rafflesiana var. typica and ability of the species which also differed with regard to N. rafflesiana var. elongata) out of the 10 lowland taxa (nine insect type. For ants, retention rates increased significantly species) in our sample were found to be viscoelastic. These with wax quantity and fluid viscoelasticity (Table 2a, proportions differed significantly (Fisher’s exact test per- Fig. 3a). For flies, retention rates did not significantly formed on the 23 taxa, P = 0.006; on the 21 species, depend on the amount of wax but on fluid rheometry as P = 0.003), demonstrating that mountain species more retention rates were far higher when the fluid was viscoelas- often exhibit viscoelastic fluids. By way of comparison, the tic (Table 2b, Fig. 3b). Our observations of insect values obtained for water ranged from 0.024 to 0.028 s behaviour corroborated these results. Ants that fell into the (n = 6). None of the species with very waxy pitchers was liquid close to the pitcher wall were not extensively wetted found also to exhibit a very viscoelastic fluid, and vice versa. and were often observed to reach and climb up the pitcher Moreover, the quantity of wax produced in the pitchers of wall but they then slipped frequently when reaching the viscoelastic species were seen to better fit a hyperbolic func- waxy zone. By contrast, flies that were not extensively tion (Akaike information criterion (AIC) = )29.72) than a

(a) Ants Flies (b) 100 100 P < 0.0001 90 P = 0.40 90 80 80 P = 0.02 70 70 60 60 50 50 40 40 Fig. 3 (a) Percentage of ants retained within the Nepenthes pitchers according to wax 30 30 quantity and fluid viscoelasticity. Regression 20 20 lines are shown for viscoelastic (solid line)

Percentage of insects retained 10 10 and nonviscoelastic taxa (dotted line). (b) Percentage of flies retained within the 0 0 pitchers according to fluid viscoelasticity. Non-VE VE 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Retention rates did not depend on wax Wax quantity (mg) Viscoelasticity quantity for flies.

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0.70 0.65 Montane, viscoelastic sp. 0.60 Montane, nonviscoelastic sp.

) 0.55 Lowland, viscoelastic sp. –2 0.50 Lowland, nonviscoelastic sp. 0.45 0.40 0.35 0.30 0.25 0.20 2 Fig. 4 Plant investment in the two Wax density (µg cm 0.15 r = 0.77 trapping devices (pitcher waxiness and fluid 0.10 viscoelasticity) distinguished for lowland and 0.05 mountain species of Nepenthes. The vertical 0.00 line represents the upper value of the 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 relaxation time obtained for water. Relaxation time of the digestive liquid (s) linear function (AIC = )11.00) of the fluid viscoelasticity. some Nepenthes species are highly viscoelastic. It is possible The more wax a species produces, the less viscoelastic is its that this viscoelasticity increases with plant age (Gaume & fluid (wax density (mg cm)2) = 0.013 + 0.014⁄ relaxation Di Giusto, 2009). But, for all species, the plants used in our 2 time (s), F1,12 = 14.19,P= 0.001, R = 0.75; Fig. 4). 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 Discussion humidity (e.g. those of altitudinal mossy forests or those Our comparative study of the trapping systems of Nepenthes that have a reduced lid that can protect the pitcher) and to pitcher plants generated three important results. First, and subsequent fluid dilution by water. Greater production of contrary to common belief (but see Gaume & Di Giusto, the polymers that are assumed to cause the viscoelasticity of 2009; Bonhomme et al., 2011), the different species show pitcher fluid might have been selected in some species, help- functional diversity in their retentive devices and do not rely ing them to cope with the problem of daily dilution. This solely on the slipperiness of their trap to capture insects. hypothesis is corroborated by the results of Gaume & The results of this study show that N. rafflesiana is not the Forterre (2007), who showed that the elastic fluid of only species to possess a viscoelastic fluid. This character N. rafflesiana (which here appears to be among the most may be widespread within the genus as found in two-thirds viscoelastic species), when diluted in 95% of water, was still of the species in our study. Secondly, wax and viscoelatic viscoelastic enough to capture all the insects dropped into fluids do not target the same type of prey: wax appears to be the pitchers. efficient only for ants, whereas viscoelasticity proved to be a Since relaxation times of < 100 ms are impossible to powerful trapping device for both insect types and is more detect without the use of a high-speed camera, viscoelastic often found in mountain than in lowland species. Thirdly, fluids have probably gone unnoticed in many species and our cross-species comparison suggests that investments in may be far more common than suspected. This raises the the ‘waxy’ trait and in ‘viscoelastic fluid’ could be made at question of whether the viscoelastic fluids in Nepenthes spe- the expense of the other. At the least, species that are very cies have a common origin. Interestingly, the glue secreted viscoelastic do produce very little wax. Altogether this sug- by the leaves of Drosera, another carnivorous genus, is com- gests that there are two different trapping strategies in these posed of acid polysaccharides (Gowda et al., 1982) and pitcher plants, a ‘waxy’ strategy and a ‘viscoelastic’ strategy. 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 A widely shared viscoelastic trap in Nepenthes pitcher genera are the closest relatives of Nepenthes in the phylogeny plants and the question of its origin of the Caryophyllales (Heubl et al., 2006) and their traps The fluid contained in the pitchers of most of the Nepenthes are known to function like flypaper (Juniper et al., 1989). species studied was viscoelastic, and this might therefore We can therefore advance the hypothesis that the viscoelas- represent the rule in the genus rather than the exception. tic polysaccharide fluids in Nepenthes and in these other Contrary to wax, whose efficiency as a trapping device has carnivorous genera have a common and thus plesiomorphic been shown to be quantity-dependent, even low viscoelas- origin, with the glue of the other carnivorous genera simply ticity appears to contribute strongly to a plant’s trapping containing a far higher concentration of polysaccharides ability. Therefore, it is surprising to note that the fluids of than the fluid in Nepenthes.

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the evolution of these traits. Pitcher wax causes insects to Relative investment in different trapping devices slide and is thus implicated in both capture and retention The appearance of botanical carnivory and the evolution of (Juniper & Burras, 1962; Gaume et al., 2002), while the specialized traps are subject to powerful cost–benefit con- viscoelastic fluid acts on retention (Gaume & Forterre, straints (Givnish et al., 1984; Ellison & Gotelli, 2001; 2007). The results obtained in our study show that the effi- Pavlovicˇ et al., 2007). We can therefore assume that it is ciency of these strategies is prey-dependent. Wax is more costly for carnivorous plants to produce modified leaves efficient on ants than on flies, whereas viscoelasticity is very with lower photosynthetic capacities (Pavlovicˇ et al., 2007, efficient on both insect types and definitely more efficient 2009), and that the biosynthesis of trapping features is sub- than wax on flies. Winged insects are able to take off from ject to selective pressure and can be maintained throughout the pitcher wall without even touching the waxy surface, evolution only if the cost of these features is exceeded by the and even if they do enter into contact with it, the wax acts benefits they provide in terms of insect-derived nutrients. only on their attachment systems (Gaume et al., 2004; Development of the waxy zone, mainly composed of Gorb et al., 2005) not on their flying system. By contrast, aliphatic compounds dominated by very long-chain alde- crawling insects have no other option than to cope with the hydes (e.g. triacontanal or dotriacontanal containing 30 or wax that contaminates their pads and causes them to lose 32 carbon atoms, respectively Riedel et al., 2003, 2007), is adhesion. Moreover, since winged insects have a higher sur- metabolically costly for the plant. The molecules responsi- face : volume ratio than crawling insects, they offer a larger ble for digestive fluid viscoelasticity are assumed to be long- surface area for the viscoelastic fluid to exert its retentive chain polysaccharides (Gaume & Forterre, 2007) that must force and this may explain why they are more often retained also be costly to synthesize. This could provide part of the in Nepenthes liquids (Gaume et al., 2002; Gaume & explanation as to why none of the species we tested pos- Forterre, 2007). sesses both very viscoelastic fluids and very waxy pitchers. Hence wax and viscoelastic fluid clearly do not have the The inverse relationship that seems to link these two quanti- same function and do not target the same types of insect. tative traits in our cross-species comparisons might illustrate This suggests that they might represent adaptation to differ- the existence in the plant of an investment trade-off. This ent prey spectra and that local differences in entomofauna will need to be tested at the genus scale with an analysis that might exert different selective pressure on the development takes into account the phylogenetic relationships of the of wax and ⁄ or viscoelasticity. For any given pitcher waxi- measured species. A test of this hypothesis will also necessi- ness, a ‘viscoelastic strategy’ is needed to trap flies with the tate studies of several populations of a given species same efficiency as ants. This means that habitats dominated showing some variations in these traits. by ants, such as the lowland forests of Borneo (Gunsalam, Interestingly, a few plant species in our study showed 1999; Davidson et al., 2003), may favour the development both nonviscoelastic fluids and only slightly waxy pitchers, of a waxy ‘slippery’ strategy. On the other hand, habitats or pitchers that contained no epicuticular wax at all, such as dominated by flying insects may favour the development of N. ampullaria and N. ventricosa. These plants are outliers in a ‘sticky’, viscoelasticity-based strategy. Such habitats are the Nepenthes genus. Perhaps the pitchers do not have a generally found at higher altitudes where ants are few in strictly carnivorous diet; this is the case for N. ampullaria, number but flying insects are relatively more abundant which obtains part of its nitrogen from leaf debris (Moran (Collins, 1980). This can also temporarily be the case for et al., 2003). An additional explanation is that they utilize lowland, open, and regularly flooded habitats such as those other trapping strategies. It is possible that N. ampullaria inhabited by N. rafflesiana var. typica (Gaume & Di Giusto, relies uniquely on its peristome, which forms a steep slop, to 2009), which is associated with a flower scent cue that more trap its prey. Several other features, such as water-dependent specifically targets flying insects (Di Giusto et al., 2010). structures facilitating insect-aquaplaning (Bohn & Federle, We therefore advance the hypothesis that the scarcity of 2004; Bauer et al., 2008) or specific pitcher morphology, ants in tropical mountains (Borneo (Collins, 1980; Clarke might favour both insect capture and retention. But as et al., 2009), the Philippines (Samson et al., 1997)) and the attested by the high coefficients of determination of the relative abundance of flying insects (Collins, 1980) provide models testing for the effect of wax and viscoelasticity, these part of the explanation for the widespread viscoelastic strat- other features only played a minor role in insect retention. egy among mountain Nepenthes species. A comparative study (Adam, 1997) corroborates this hypothesis by show- ing that mountain species tend to trap a larger prey The role of prey in the evolution of different trapping spectrum, including more dipterans and coleopterans, than strategies lowland species, which were recorded to trap mostly ants. Whether wax and viscoelastic fluids are produced at each Furthermore, at least seven species in mountain mossy other’s expense, as these devices are necessarily costly, the forests, and known to possess a highly viscous fluid, are question arises as to the selective factors that have favoured reported to be specialized in the capture of flying insects:

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N. inermis has been reported to be (under the name of Chin L, Moran JA, Clarke C. 2010. Trap geometry in three giant N. bongso) specialized in trapping midges (Kato, 1993); montane pitcher plant species from Borneo is a function of tree shrew body size. 186: 461–470. N. aristolochioides is specialized in trapping midges; New Phytologist Clarke CM. 1997. . Kota Kinabalu, Sabah, Malaysia: N. dubia, N. jamban, N. eymae and N. talangensis are spe- Natural History Publications (Borneo) Sdn. Bhd. cialized in trapping small dipterans (McPherson 2009); and Clarke CM. 2001. Nepenthes of Sumatra and . Kota N. jacquelinae has been observed to trap mainly larger flying Kinabalu: Natural History Publications (Borneo). prey (Clarke, 2001). Interestingly, the pitchers of such Clarke CM, Bauer U, Ch’ien CL, Tuen AA, Rembold K, Moran JA. species do not contain a waxy zone and are all funnel-shaped. 2009. Tree shrew lavatories: a novel nitrogen sequestration strategy in a tropical pitcher plant. Biology Letters 5: 632–635. Few comparative studies have been conducted on the Collins NM. 1980. The distribution of soil macrofauna on the west ridge prey spectra of Nepenthes species (but see Kato et al., 1993; of Gunung (Mount) Mulu, . Oecologia 44: 263–275. Adam, 1997). To test our hypotheses on the evolution of Davidson DW, Cook SC, Snelling RR, Chua TH. 2003. Explaining the Nepenthes trapping devices, we need to carry out studies abundance of ants in lowland tropical rainforest canopies. Science 300: comparing the prey spectra of species with the 969–972. Nepenthes Di Giusto B, Bessie`re J-M, Gue´roult M, Lim LBL, Marshall DJ, entomofauna found in their habitats, and relating this to Hossaert-McKey M, Gaume L. 2010. Flower-scent mimicry masks a their insect-trapping devices. This would help in under- deadly trap in the carnivorous plant Nepenthes rafflesiana. Journal of standing the ecological mechanisms underlying the evolution Ecology 98: 845–856. and diversification of these pitcher plants. Di Giusto B, Grosbois V, Fargeas E, Marshall DJ, Gaume L. 2008. Contribution of pitcher fragrance and fluid viscosity to high prey diversity in a Nepenthes carnivorous plant from Borneo. Journal of Acknowledgements Biosciences 33: 121–136. Ellison AM, Gotelli NJ. 2001. Evolutionary ecology of carnivorous plants. We wish to thank B. Buatois, M. Gue´roult and R. Leclerc Trends in Ecology & Evolution 16: 623–629. for their helpful assistance in the laboratory and glasshouses, Ellison AM, Gotelli NJ. 2009. Energetics and the evolution of carnivorous G. Le Mogue´dec for statistical advice, and S. Martinez, plants - Darwin’s ‘most wonderful plants in the world’. Journal of Experimental Biology 60: 19–42. J-M. Felio and P. Cervetti (IUSTI) for technical assistance Erni P, Varagnat M, McKinley GH. 2008. 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