Journal of Ecology 2010, 98, 845–856 doi: 10.1111/j.1365-2745.2010.01665.

Flower-scent mimicry masks a deadly trap in the

carnivorous plant Nepenthes rafflesiana
Bruno Di Giusto1,2, Jean-Marie Bessière3, Michaël Guéroult1, Linda B. L. Lim4,
David J. Marshall5, Martine Hossaert-McKey6 and Laurence Gaume1*
1
UMR CNRS 5120, AMAP: botAnique et bioinforMatique de l’Architecture des Plantes, CIRAD – TA-A51 ⁄ PS2
Boulevard de la Lironde, F-34398 Montpellier Cedex 5, France; 2Ming Chuan University, ELC, 250 Zhong Shan
N. Rd., Sec. 5, Taipei 111, Taiwan; 3Laboratoire de Chimie Appliquée, Ecole Nationale Supérieure de Chimie de
Montpellier, 8 rue de l’Ecole Normale, F-34296 Montpellier Cedex 5, France; 4Chemistry Department, University of
Brunei Darussalam, Jalan Tungku Link, Gadong BE 1410, Brunei Darussalam; 5Biology Department, University of
Brunei Darussalam, Jalan Tungku Link, Gadong BE 1410, Brunei Darussala; and 6UMR CNRS 5175, Centre
d’Ecologie Fonctionnelle et Evolutive, 1919, route de Mende, F-34033 Montpellier Cedex 5, France

Summary
1. Nepenthes rafflesiana is a carnivorous vine from Borneo characterized by an ontogenetic pitcher
dimorphism with aerial (upper) and ground (lower) pitchers of different morphologies. Previous
studies have shown that fragrant upper pitchers of climbing parts of the plant are more effective in
trapping flying insects than non-fragrant lower pitchers, which are essentially restricted to an ant
diet. We tested the hypotheses that odours are effective cues for prey attraction in this carnivorous
plant and that upper pitchers biochemically mimic flowers in their olfactory cues.
2. The visitor diversity and the scent composition of each pitcher type were determined for different
sites and periods during field studies in Borneo. Olfactometer bioassays were conducted using fruit
flies and ants as models for flying flower-visitors and non-flying visitors, respectively.
3. Fifty-four volatile compounds were identified and the analysis of their relative quantities in the
blends showed significant differences between pitcher types. The blends of lower pitchers contained
some aliphatics and terpenoids but were poor in benzenoids. Upper pitchers differed from lower
ones in that they attracted a greater quantity and diversity of insects, including a guild of flower-visi-
tors absent from the visitor spectrum of lower pitchers. Upper pitchers also emitted a greater quan-
tity of odours and a larger spectrum of volatiles, including some terpenoids and benzenoids that
often characterize the sweet scents classically found in flower blends. Choice bioassays showed that,
in absence of any visual cue, the scents of the nectariferous pitcher rim (peristome) were particularly
attractive to ants and flies, and those of upper pitchers were more attractive to flies than those of
lower pitchers.
4. Synthesis. This study demonstrates the use of scent by Nepenthes carnivorous plants to mediate
prey attraction. The climbing part of the plant produces pitcher-modified leaves that mimic flower
olfactory cues and suggest an evolutionary convergent strategy with that of generalist pollination
systems.
Key-words: flower mimicry, GC–MS, headspace volatile collection, insect attraction, odour
cue, olfactometer bioassays, pitcher plant, plant–insect interaction, volatile compounds

use long-distance attracting cues as well as short-distance

Introduction
Attraction systems used by plants in pollination and carnivo-
rous syndromes have been reported to show similarities (Joel
1988). Both flowers and the modified leaves of carnivorous
plants exploit insects, which respectively provide pollination
and nutritional benefits to the plant. Both flowers and leaves
*Correspondence author. E-mail: laurence.gaume@cirad.fr
rewarding lures such as floral and extrafloral nectar, respec-
tively. There is an extensive body of literature on the role of
plant volatile emissions in mediating a variety of plant–animal
interactions (Bernays & Chapman 1994; Gershenzon &
Dudareva 2007), and much of this specifically concerns attrac-
tion in pollination systems (Dobson & Bergström 2000;
Dudareva & Pichersky 2006a; Raguso 2008). In contrast,
relatively little is known about the olfactory cues delivered by

 2010 The Authors. Journal compilation  2010 British Ecological Society

846 B. Di Giusto et al.
carnivorous plants to attract their prey (but see Joel 1988; Jaffé
et al. 1995; Moran 1996). Most studies on prey attraction by
these plants have focused on visual stimulation (Joel, Juniper
& Dafni 1985; Moran, Booth & Charles 1999; Schaefer & Rux-
ton 2008). However, a recent study focused on the American
pitcher plant, Sarracenia purpurea, has demonstrated that the
colour patterns of the pitchers do not constitute important sig-
nals for prey attraction (Bennett & Ellison 2009). In the carniv-
orous Nepenthes genus, the use of volatile compounds could be
suspected in Nepenthes albomarginata since this species attracts
termites, virtually blind insects, in mass (Merbach et al. 2002).
Leaves and petals are fundamentally similar organs and
show little divergence in their developmental processes, sug-
gesting that similar volatile compounds may occur in their
emissions (Caissard et al. 2004). For example the aliphatic
green-leaf volatile compounds classically employed in plant
defence are also often released by flowers (Paré & Tumlinson
1999). However, the odour bouquet emitted from flowers is
usually more complex and richer in volatiles than vegetative
scents (Dudareva & Pichersky 2006b; Knudsen, Eriksson &
Gershenzon 2006). Flower scents can not only be rich in ali-
phatic compounds but also often contain many aromatics and
terpenoids (Knudsen, Eriksson & Gershenzon 2006), and these
three major categories of volatile compounds are usually
simultaneously present in the flower blends of generalist polli-
nation systems (Dudareva & Pichersky 2006a). Since the pitch-
ers of Nepenthes plants are, like flowers, modified leaves, and
since they already show various similarities with flowers (Joel,
Juniper & Dafni 1985) in the mechanisms used to attract
insects [nectar (Juniper, Robins & Joel 1989; Merbach et al.
2001) and visual cues (Moran 1996; Moran, Booth & Charles
1999; Schaefer & Ruxton 2008)], we ask whether they could
also chemically mimic flowers using insect-attracting volatiles.
Like most Nepenthes species (Cheek & Jebb 2001), Nepen-
thes rafflesiana var. typica Beck exhibits heteroblastic develop-
ment characterized by ontogenetic pitcher dimorphism
(Gaume & Di Giusto 2009) with terrestrial (lower) pitchers
associated with the young self-supporting life-form of the
plant, and aerial (upper) pitchers associated with the mature,
climbing form. The life span of lower pitchers is c. 2 months
and a half (B.D.G. & L.G., pers. obs.; Moran 1996) and a little
less for upper pitchers (Bauer, Willmes & Federle 2009). Lower
pitchers are globular, winged and reddish to green, while upper
pitchers of climbing plants are more elongate, trumpet-shaped
and green to yellow in colour. These two types of pitchers trap
different quantities and spectra of prey (Moran 1996; Di Giu-
sto et al. 2008) partly because of differences in retentive power
of their viscoelastic digestive liquids (Gaume & Forterre 2007;
Di Giusto et al. 2008). Differences in visual and odour cues, as
well as the effect of the pitcher’s elevation on the plant, proba-
bly also explain prey capture differences between these pitcher
types (Moran 1996). However, because upper pitchers of
N. rafflesiana also attracted a greater richness of insects than
lower pitchers when they were cut longitudinally and placed at
ground level, the inner face – of similar colour patterns for
lower and upper pitchers – exposed to insects (Di Giusto et al.
2008), we hypothesize that odour delivered by the pitcher has
 2010 The Authors. Journal compilation  2010 British Ecological Society, Journal of Ecology, 98, 845–856
greater effect on insect capture than its visual characteristics or
its position on the plant. However, up to now, no detailed
study of chemical ecology has ever been conducted on pitcher
plants to identify the vegetative site releasing the attractive
scent, the kind of volatile compounds emitted or the effect they
have on insects.
Here, we investigated the scent composition of the two
pitcher types and analysed its effect on ants and fruit flies,
insects belonging to the two main groups of Nepenthes prey
(Hymenoptera and Diptera), and to the non-flying and flying
flower-visitor (Sakai 2002) categories. We identified the insect
guilds visiting nectar-rich pitchers and investigated whether
prey segregation between the pitcher types could be linked to
the pitcher’s odour signature and whether upper pitchers
attract flower-visitors by mimicking floral blends. To localize
the main sites of scent emission in the pitchers, the olfactory
attractiveness of different pitcher parts to insects was also
investigated by field recordings and olfactory bioassays in the
laboratory.
Materials and methods
STUDIED SPECIES
The dioecious vine Nepenthes rafflesiana Jack occurs in Southeast
Asia from Peninsular Malaysia, Singapore and Sumatra to northern
Borneo, where it is widespread. This lowland plant grows on nutri-
ent-poor white sands, in open and wet areas such as tropical heath
forests (kerangas) and peat swamp forests (Clarke 1997). Nepenthes
rafflesiana var. typica (henceforth NRT) is characterized by a strong
pitcher dimorphism (Gaume & Di Giusto 2009) and a sweet fragrance
perceptible by humans that is uniquely released by pitchers, but not
by photosynthetic parts (Moran 1996; Di Giusto et al. 2008). The
plant has got a complex trapping system that is based on a slippery
and wettable ‘peristome’, i.e. a nectar-secreting pitcher rim (Bohn &
Federle 2004; Bauer, Bohn & Federle 2008), and on viscoelastic prop-
erties of the digestive liquid (Gaume & Forterre 2007). This taxon
was identified in the field using the Flora Malesiana (Cheek & Jebb
2001).
COLLECTION, IDENTIFICATION OF VOLATILE
COMPOUNDS AND ANALYSES OF PITCHER BLENDS
Two sets of NRT plants without any trace of herbivory were selected
in Brunei Darussalam (Borneo) and one pitcher was selected from
each studied plant. Nine upper and seven lower pitchers were selected
in this way from the Badas Reserve Forest (0458¢31¢¢ N,
11453¢28¢¢ E, ‘Badas’ site) between 4 and 19 July 2004, and seven
upper and 12 lower pitchers were sampled from ‘Tungku’ site between
Pantai Tungku and Berakas Hutan (0459¢12¢¢ N, 11454¢20¢¢ E)
between 28 April and 19 May 2006.
Volatile compounds of mature open pitchers c. 2–3 weeks old were
collected in situ by headspace adsorption (Grison-Pigé et al. 2001).
Each pitcher was enclosed in a polyethylene terephtalate (Nalophan)
bag (Nalo Wursthüllen; Kalle GmbH, Wiesbaden, Germany). Pure
air, filtered by charcoal filters, was drawn into the bags at
400 mL min)1 and extracted at 300 mL min)1 through a trap con-
taining 30 mg of Alltech Super Q absorbent (ARS, Gainesville, FL,
USA). The difference in flux ensured that the system was continu-
ously purged through the inevitable leaks and that no outside air

could enter the system. For each session, blanks were collected in par-
allel as a control sample using an empty bag. After 3 h of collection,
trapped volatiles were eluted with 150 lL of dichloromethane and
preserved at )18 C until analysis. Two internal standards (nonane
and dodecane, 200 ng lL)1) were added to each sample for quantifi-
cation purposes.
The volatile compounds were analysed by injection to a Varian
CP-3800 gas chromatograph with a flame ionization detector (Varian
CP-SIL 8 CB, non-polar, length 30 m, internal diameter 0.25 mm,
film thickness 0.25 lm, with helium as the carrier gas at 1 mL min)1),
in which the oven temperature was regulated by a multi-rise pro-
gramme (see details in Grison-Pigé et al. 2001). Compounds were
identified using a gas chromatograph–mass spectrometer (GC–MS;
Varian SATURN 2000 (Varian, Palo Alto, CA, USA); Varian
CP-SIL 8 CB, length 30 m, internal diameter 0.25 mm, film thickness
0.25 lm, carrier gas He at 1 mL min)1). Volatiles were identified by
comparing their mass spectra with those of the NIST98 library and
with GC retention times and MS spectra of the authentic compounds
when possible as well as on retention indices reported in the literature
(Adams 1995). For quantification, we calculated the peak area of the
two internal standards (nonane and dodecane) corresponding to
200 ng in the sample and used this area per mass relationship to esti-
mate the quantity of each compound present in the samples, each of
them having been collected during exactly 3 h during day-time.
Using the candisc procedure, a discriminative analysis according to
pitcher type was performed on the relative abundances of the com-
pounds in the blends to test whether the combination of the com-
pound frequencies was sufficient to distinguish between lower and
upper pitchers. A manova was performed to test for the effects of
pitcher type and site (and ⁄ or year but assigned as ‘site’ factor) on the
relative abundances of compounds. Both analyses were performed
using as explanatory variables only the compounds that were present
in more than 20% of the pitchers. This was done, not only because
these were the compounds of ecological significance but also because
using all compounds would have resulted in variables too numerous
compared with the number of studied pitchers for the model to be
performed. Mixed-model anovas were used to compare the cumula-
tive quantity of all compounds in the pitcher emissions among com-
pound types (fatty acid derivatives, benzenoids and terpenoids),
pitcher types and sites (as fixed factors), and for plant nested within
pitcher type and site (as a random factor). A Poisson regression model
was carried out using the genmod procedure to analyse the total num-
ber of volatile compounds in the pitcher emissions. According to the
square root of the ratio of Pearson’s v2 to the associated number of
degrees of freedom, no correction for overdispersion was necessary.
INSECT OLFACTORY BIOASSAYS
We aimed to determine which part of the plant produces the
insect-attracting volatile compounds and to compare the olfactory
attractiveness of pitchers for two types of insects, representative of
classical Nepenthes prey in the non-flying and flying – potentially
flower-visiting – categories. For that, we carried out behavioural
assays on Oecophylla smaragdina ants and Drosophila melanogaster
fruit flies using Y-tube olfactometers. Both insect species were fre-
quently observed collecting nectar from the pitchers and flowers of
NRT plants (B. Di Giusto, pers. obs.) and are usual prey of these
plants (Di Giusto et al. 2008). Oecophylla smaragdina ants were
collected on the University of Brunei Darussalam campus and kept
for short periods in humidified Petri dishes before the trials.
Drosophila melanogaster were bred on a nutritive substratum until
emergence of imagoes. The Y-tubes measured 5 cm in diameter;
 2010 The Authors. Journal compilation  2010 British Ecological Society, Journal of Ecology, 98, 845–856
Flower-scent mimicry in Nepenthes rafflesiana 847
each arm was 14 cm long and the basal arm 8 cm long. During the
experiment, humidified air was blown into each of the two arms of
the Y-tube (30 mL min)1). In one arm of the Y-tube, the air was
blown through a bag containing freshly cut parts (peristomes, or
lids; c. 10 cm2) or digestive liquid of upper pitchers. Only humidified
air was blown into the other arm. Controls were performed with
only humidified air being passed into each arm of the Y-tube. The
pitchers used for bioassays were collected from different haphaz-
ardly selected plants in the field that emitted a detectable scent. A
total of 160 trials were performed, 20 per insect category and treat-
ment. Each plant sample was replaced every five trials. To avoid
experimental bias, the position of humidified air only and humidified
air plus plant sample was inverted between the two arms for each
trial. Before each trial, the entire olfactometer was cleaned with neu-
tral soap and organic solvent, previously checked to be neutral with
regards to insect behaviour. Control and experimental bioassays
were alternated. A vial containing the experimental insects and with
a narrow entrance hole was connected to the entrance of the Y-tube.
Once an insect had reached this entrance, its behaviour was recorded
for 3 min; preliminary trials showed that longer periods of observa-
tion provided no additional information. We calculated the proba-
bility of obtaining the patterns of frequencies of insect choices
between the two arms of the Y-tube under the null hypothesis of
‘random choice’ using a binomial test (number of events = 20,
probability of an event = 0.5). If the probability was < 0.05, then
the insect was considered not to have chosen a given arm by chance.
The total number of events was not always 20 as not all insects made
a choice (£ 3 per test) and such events were disregarded.
Using the same protocol as above, further experiments were per-
formed to compare the attractiveness of lower versus upper pitchers.
Sectioned peristomes of either a lower or an upper pitcher of NRT
plants were used for that purpose. A total of 120 trials, 20 per insect
category and treatment, were performed.
FIELD RECORDS OF INSECT VISITS ON PITCHERS AND
THEIR DIFFERENT PARTS
In July 2004, we gathered systematic records on attraction from
observations made at two sites for 37 plants and 63 pitchers (one to
two per plant). We determined the numbers of arthropod visitors and
the number of species (only morphospecies were distinguished) on the
different parts of the pitchers for 10-min periods. Observations were
made on 22 lower and 20 upper pitchers at a site near Liang
(0438¢51¢¢ N, 11430¢30¢¢ E) and on 10 lower and 11 upper pitchers
at the Badas site (0458¢31¢¢ N, 11453¢28¢¢ E). Using mixed Poisson
regression models, we analysed the effect of plant (random factor)
and the effects of the three fixed factors – site (Liang versus Badas),
pitcher type (lower versus upper) and pitcher part (lid, peristome,
external surface, conductive area) – on the total number of arthro-
pods recorded as visitors. All second- and third-order interactions
were tested. We also analysed the number of visitor species and the
numbers of terrestrial and flying insects by performing mixed Poisson
regression models. The mixed Poisson regressions were carried out
using the macro glimmix, with a Poisson error distribution.
STATISTICAL ANALYSES
Statistical analyses were carried out using the software package
sas v.9 (SAS Institute, Cary, NC, USA). For model selection, back-
ward procedures were adopted, starting with the removal of the non-
significant highest-order interactions. The third-order interactions
were never significant.
848 B. Di Giusto et al.
Results
A RICHER AND BROADER-SPECTRUM ODOUR
BOUQUET IN UPPER THAN IN LOWER PITCHERS
Fifty-four compounds were identified in the pitcher odours of
NRT (Table 1). They belong to the three main categories of
volatiles: fatty acid derivatives, benzenoids and phenylpropa-
noids, and terpenoids (including monoterpenes and sesquiterp-
enes). The canonical variable obtained from the discriminative
analysis showed a significant discrimination between pitcher
types as regards the combination of their relative quantities in
the blend (F4,30 = 13.80, P = 0.01). According to the manova,
the relative quantities of the different compounds were signifi-
cantly different between pitcher types (F4,30 = 3.80,
P = 0.01) but not significantly different between sites and ⁄ or
years (F3,30 = 4.97, P = 0.11). There was no effect of the
interaction term either (F2,30 = 16.04, P = 0.06).
Lower and upper pitcher essentially differed in the
absolute quantities of the emitted compounds (Table 1,
Figs 1 and 2a). Upper pitchers produced scents in quantities
c. 20 times greater than lower pitchers (101.9 ± 86.3 ng h)1
for upper pitchers (N1 = 16) and 4.8 ± 3.7 ng h)1 for lower
pitchers (N2 = 19)). This was a general trend (GLM:
pitcher type significant; Table 2a) regardless of the category
of volatile emitted, but the magnitude of difference between
quantities emitted by lower and upper pitchers depended
greatly on the category of compounds (significant interac-
tion between pitcher type and compound types; Table 2a).
The contrast was dramatic for benzenoids, less significant
for terpenoids and non-significant for fatty acid derivatives
(Fig. 2a). The total amount of volatile compounds emitted
for any category of compounds was unaffected by the site
and ⁄ or year of emission, but it was significantly dependent
on the plant itself (significant random factor; Table 2a), with
some plants producing greater amounts of volatiles than
others.
Qualitatively, despite some variations between sites and ⁄ or
years and plants (Table 2b), upper pitchers emitted a larger
number of volatile compounds (23.06 ± 5.60, minimum = 11,
maximum = 34) than lower pitchers (8.95 ± 4.07, mini-
mum = 3, maximum = 19; Poisson regression model:
pitcher type significant; Table 2b, Fig. 2b). The number of vol-
atiles emitted was quite different between the categories of
compounds (significant effect of compound type; Table 2b),
the terpenoids and the benzenoids being globally the richest
categories and the fatty acid derivatives the poorest. The trend
of upper pitchers to produce a greater number of volatiles than
lower ones was verified for each of the three categories of vola-
tile compounds (non-significant interaction between pitcher
type and compound type; Fig. 2b). There was a site and ⁄ or
year effect, which was especially significant through its interac-
tion with the compound category (Table 2b). Indeed, plants
from Badas 2004 differed from those from Tungku 2006
in their profile of scent emission because the odour blends of
Badas plants were poorer in fatty acid derivatives than those
from Tungku plants but richer in benzenoids and terpenoids.
 2010 The Authors. Journal compilation  2010 British Ecological Society, Journal of Ecology, 98, 845–856
The great majority of the constituents of the volatile blends
of lower pitchers were also found in the blend of upper pitch-
ers, though occurring in different amounts. In contrast, there
were many compounds found only in upper pitchers (espe-
cially some belonging to benzenoids and phenylpropanoids;
Table 1). Lower pitchers were characterized by a lower diver-
sity of volatiles emitted and by a high variability between
pitchers in the presence and amount of volatiles. The main
constituents of lower pitchers were monoterpenes such as lim-
onene (the most frequent volatile), cis- and trans-ocimenes
and linalool, aliphatics such as nonanal, decanal, 2-hepta-
none and 2-nonanone, and benzaldehyde, the sole benzenoid
compound. The main constituents of upper pitchers were
more diverse. They included, for benzenoids, compounds
classically found in flowers (Table 1) such as benzylalcohol,
benzaldehyde, methylbenzoate, 2-phenylethanol, acetophe-
none as well as very rarely found compounds, such as the
ketones 1-phenyl-2,3-butanedione and 2-hydroxy-1-phenyl-3-
butanone and the associated alcohol, 1-phenyl-2,3-butanedi-
ol. For terpenoids, they included monoterpenes such as
linalool, cis- and trans-ocimenes, cis- and trans-linalool
oxides, and sesquiterpenes such as b-caryophyllene, which
are all compounds commonly emitted by flowers. The only
fatty acid derivative that was frequently found in upper pitch-
ers was (Z)-3-hexenylbutanoate.
PERISTOMES AS THE MAIN SOURCE OF OLFACTORY
CUES PRODUCED BY PITCHERS
Peristome emits more attractive odour
The control performed for Oecophylla smaragdina ants showed
no directional trends in the olfactometer (binomial test:
N1 = 9, Ntot = 18, P = 0.185; Table 3a). In contrast, in the
olfactory test with the peristome of upper pitchers, the ants
moved significantly more often towards the experimental arm
than towards the empty one, i.e. the one with humidified air
only (N1 = 15, Ntot = 18, P = 0.003). This was not the case
for the lid (N1 = 7, Ntot = 17, P = 0.148). Interestingly, in
the test involving the digestive liquid, ants significantly ‘chose’
the empty arm (N1 = 4, Ntot = 18, P = 0.012). Compari-
sons between treatments showed that ants chose significantly
more often the arms with peristomes than those with digestive
liquids (Yates-corrected v2 = 11.14, P = 0.0008) or with lids
(Yates-corrected v2 = 4.97, P = 0.03).
The control performed on Drosophila melanogaster showed
no directional trend in the olfactometer (binomial test:
N1 = 9, Ntot = 20, P = 0.160). In contrast, the tests using
the peristome showed that fruit flies significantly preferred the
experimental arm (binomial test: N1 = 14, Ntot = 20,
P = 0.037; Table 3a), but there was no significant preference
in tests using either the lid (N1 = 12, Ntot = 20, P = 0.120)
or the digestive liquid (N1 = 9, Ntot = 20, P = 0.120).
For both insect species combined, the frequency of visits to
the arms with peristomes was significantly higher than that
to the arms with lids (v2 = 5.07, P = 0.024; Table 2a) or to
arms with digestive liquids (v2 = 7.67, P = 0.006; Table 3a).
 Flower-scent mimicry in Nepenthes rafflesiana 849

Table 1. Volatile compounds emitted by upper and lower pitchers of Nepenthes rafflesiana var. typica. The compounds are listed according to
their biosynthetic origin. ‘*’ refers to samples that have been compared with authentic standards. RI is the retention index. The mean relative
quantity (%) and the mean absolute quantity (expressed in ng for 3 h of headspace extraction) of each volatile in the blend are presented. Report
index in flowers refers to the number of citations where the compound was reported from flowers in the review of Knudsen, Eriksson &
Gershenzon (2006); compounds reported more than 20 times are highlighted in grey

Relative quantity (% in blend) Absolute quantity (ng lL)1)

Low. pitcher Up. pitcher Low. pitcher Up. pitcher

(19) (16) (19) (16)
Report ind.
Volatile compound RI Mean SE Mean SE Mean SE Mean SE in flower

Fatty acid derivatives

Nonanal 1107 5.74 1.43 0.93 0.74 0.84 0.19 0.55 0.18 57
Decanal 1208 2.16 0.76 0.45 0.40 0.38 0.13 0.17 0.09 48
2-Heptanone* 894 2.57 0.75 0.05 0.04 0.47 0.13 0.11 0.07 21
2-Nonanone* 1093 3.41 2.23 0.02 0.02 0.52 0.34 0.11 0.07 14
Tetradecane* 1400 3.38 1.02 0.18 0.12 0.55 0.22 0.22 0.09 47
Pentadecane* 1500 6.07 1.70 0.49 0.21 0.93 0.23 0.94 0.44 61
Hexadecane* 1600 0.13 0.13 0.00 0.00 0.03 0.03 0.00 0.00 48
Ethyl 2-methylbutanoate 860 0.11 0.11 0.05 0.05 0.03 0.03 0.09 0.06 9
Isoamyl acetate* 886 0.00 0.00 0.02 0.01 0.00 0.00 0.05 0.04 4
(Z)-3-hexenyl propanoate 1105 0.00 0.00 0.23 0.13 0.00 0.00 0.92 0.60 5
(Z)-3-hexenyl butanoate 1193 0.79 0.51 1.10 0.24 0.05 0.03 4.08 1.38 14
Benzenoids
Benzaldehyde* 965 5.32 1.09 10.23 2.18 0.72 0.16 33.19 9.62 164
Benzyl alcohol* 1039 3.43 1.98 30.16 4.58 0.10 0.05 98.76 25.23 143
Methyl benzoate* 1097 0.93 0.63 3.13 0.71 0.02 0.01 9.64 2.93 118
Ethyl benzoate 1173 0.00 0.00 0.10 0.07 0.00 0.00 0.24 0.18 34
Acetophenone* 1069 0.05 0.05 0.94 0.40 0.02 0.02 2.15 0.88 23
Benzyl acetate 1166 0.00 0.00 0.46 0.14 0.00 0.00 2.24 1.21 101
Benzyl benzoate 1770 0.00 0.00 0.05 0.04 0.00 0.00 0.31 0.21 58
2-Phenylethanol* 1117 0.33 0.30 1.39 0.34 0.03 0.02 6.22 2.57 134
2-Phenylethyl acetate 1235 0.00 0.00 0.03 0.03 0.00 0.00 0.27 0.27 38
1-Phenybutan-2,3-dione 1212 0.07 0.07 5.50 1.48 0.00 0.00 18.73 5.62 0
2-Hydroxy-1phenylbutan-3-one 1348 0.00 0.00 11.43 2.00 0.00 0.00 47.67 19.05 0
1-Phenylbutan-2,3-diol 1419 0.00 0.00 1.43 0.49 0.00 0.00 4.26 1.35 0
Methyl salicylate* 1195 0.00 0.00 0.30 0.11 0.00 0.00 1.03 0.52 115
Terpenoids
Monoterpenoids
6-Methyl-5-hepten-2-one 988 0.31 0.31 0.11 0.04 0.08 0.08 0.46 0.29 1
Myrcene* 991 0.00 0.00 0.02 0.02 0.00 0.00 0.07 0.06 175
(Z)-ocimene* 1037 4.18 1.60 1.68 0.83 0.68 0.36 4.45 2.25 105
(E)-ocimene* 1047 10.01 4.14 6.69 2.42 1.30 0.89 17.17 6.98 167
Cis-ocimene oxide 1139 0.03 0.03 0.08 0.04 0.01 0.01 0.30 0.18 24
Trans-ocimene oxide 1141 0.05 0.05 0.12 0.06 0.02 0.02 0.44 0.27 5
Perillene 1114 0.00 0.00 0.40 0.23 0.00 0.00 0.53 0.24 17
Linalool* 1102 9.00 2.67 11.02 2.75 1.68 0.75 26.24 7.74 219
Cis-linalool oxide (furan) 1072 0.59 0.35 0.99 0.18 0.06 0.04 2.87 0.68 33
Trans-linalool oxide (furan) 1088 0.58 0.35 0.90 0.17 0.09 0.07 2.61 0.55 38
Cis-linalool oxide (pyran) 1176 0.03 0.03 0.53 0.25 0.01 0.01 1.92 1.16 14
Trans-linalool oxide (pyran) 1178 0.03 0.03 0.34 0.08 0.01 0.01 1.18 0.33 17
Limonene* 1030 29.84 5.03 2.08 1.26 4.09 0.89 1.88 0.52 200
a-Pinene* 956 1.48 0.84 0.00 0.00 0.30 0.14 0.00 0.00 153
b-Pinene* 979 0.00 0.00 0.02 0.02 0.00 0.00 0.02 0.02 123
Sesquiterpenoids
(Z,E)-a-farnesene 1490 0.00 0.00 0.05 0.04 0.00 0.00 0.23 0.18 12
(E,E)-a-farnesene 1505 1.75 1.53 0.26 0.17 0.07 0.05 1.06 0.79 32
(Z)-dendrolasine 1555 0.21 0.21 0.11 0.08 0.05 0.05 0.24 0.17 1
(E)-dendrolasine 1573 0.00 0.00 0.33 0.24 0.00 0.00 0.73 0.51 1
(Z)-nerolidol 1531 0.00 0.00 0.04 0.03 0.00 0.00 0.09 0.06 19
(E)-nerolidol* 1561 0.50 0.40 2.26 2.26 0.15 0.13 2.46 2.46 1

 2010 The Authors. Journal compilation  2010 British Ecological Society, Journal of Ecology, 98, 845–856
850 B. Di Giusto et al.

Table 1. (Continued)

Relative quantity (% in blend) Absolute quantity (ng lL)1)

Low. pitcher Up. pitcher Low. pitcher Up. pitcher

(19) (16) (19) (16)
Report ind.
Volatile compound RI Mean SE Mean SE Mean SE Mean SE in flower

b-Caryophyllene* 1419 2.64 1.26 2.16 1.06 0.23 0.15 4.39 3.15 121
Caryophyllene oxide 1582 0.00 0.00 0.14 0.09 0.00 0.00 0.77 0.49 29
a-Humulene* 1455 0.90 0.79 0.88 0.66 0.03 0.02 3.29 2.93 61
Germacrene A 1508 0.60 0.42 0.00 0.00 0.17 0.12 0.00 0.00 4
b-Selinene 1489 0.31 0.22 0.00 0.00 0.09 0.07 0.00 0.00 11
a-Selinene 1494 0.34 0.24 0.00 0.00 0.10 0.07 0.00 0.00 2
b-Elemene 1390 0.14 0.13 0.01 0.01 0.04 0.04 0.04 0.03 21
d-Cadinene 1522 0.67 0.31 0.03 0.01 0.11 0.05 0.08 0.04 24
a-Copaene* 1375 1.31 1.26 0.08 0.03 0.21 0.19 0.35 0.14 50

We also compared the ‘responsiveness’ of ants among or the experimental arm versus ants that did not
treatments, quantified as the proportion of ants that made make any choice and stayed in the basal arm of the
a choice, i.e. ants that moved towards the empty arm Y-tube. No differences in responsiveness were recorded

(a) Blend profile of a typical upper pitcher

38:300
1.50

1.25
Dodecane

1.00
MCounts

Nonane
0.75

0.50

0.25

0.00

10 15 20 25 30 35
Min
(b)
Blend profile of a typical lower pitcher
1.50 38:300

1.25

Dodecane

1.00
MCounts

0.75 Nonane

0.50
Fig. 1. Representative gas chromatography–
flame ionization detector (GC-FID) chroma-
0.25 tograms for headspace extracts of (a) an
upper pitcher and (b) a lower pitcher of
Nepenthes rafflesiana. The two internal
0.00
standards, nonane and dodecane, served as
10 15 20 25 30 35 quantitative scales for comparative
Min purposes.

 2010 The Authors. Journal compilation  2010 British Ecological Society, Journal of Ecology, 98, 845–856
 Flower-scent mimicry in Nepenthes rafflesiana 851

(a) Quantity of volatile compounds Table 2. Richness and diversity of pitcher scent. Results of (a) the
500 mixed-model analysis of variance on the quantity of volatile
c
compounds and (b) the Poisson regression model on the number of
Total quantity of volatile compounds

450 Lower pitcher

in the pitcher blend (ng.microL–1)

Upper pitcher volatile compounds. Only the significant variables are shown after
400
backward selection, although the models initially tested the random
350
effect of plant and the fixed effects of compound type (fatty acid
300 derivatives, benzenoids and terpenoids), pitcher type (upper, lower)
250 and site (and ⁄ or year) of collection (Badas 2004, Tungku 2006) as
200 well as the effects of their interactions
150 b
Effect d.f. F P-value
100
50 a a (a) Dependent var. = total quantity of volatile compounds
a a
0 Generalized linear model
Fatty acid derivatives Benzenoids Terpenoids Compound 2 11.69 <0.0001
Pitcher 1 27.79 <0.0001
(b) Diversity of volatile compounds Compound · Pitcher 2 12.71 <0.0001
Number of volatile compounds in the blend

18
Lower pitcher
(b) Dependent var. = total number of volatile compounds
d
16 Upper pitcher Poisson regression model
c'
14 Compound 2 84.36 <0.0001
12 Pitcher 1 93.7 <0.0001
c c' Site 1 4.13 0.0422
b
10
b'
Plant(Pitcher · Site) 49 54.87 0.0071
8 b'
Compound · Pitcher 2 29.87 <0.0001
6 b' Site · Compound 2 48.63 <0.0001
4 a
a
a
2 a'
0
Fatty acid Benzenoids Terpenoids Fatty acid Benzenoids Terpenoids
derivatives derivatives Table 3. Olfactory bioassays on Oecophylla smaragdina ants and
Badas 2004 Tungku 2006 Drosophila melanogaster fruit flies. Test of the attractive properties of
the scents produced by (a) different parts of the upper pitchers and
Fig. 2. Analysis of the volatile production of pitchers during a 3-h (b) by the peristome of upper and lower pitchers
headspace extraction. Total amount of volatiles in ng lL)1 (a) and
total number of volatile compounds (b) of fatty acid derivatives, Number of insects in each arm of the Y-tube
benzenoids and terpenoids compared for the scents of upper and
lower pitchers and for the sites of Badas and Tungku (when signifi- P-value of
cant site effect). Mean ± SD are used. Different letters indicate signif- choice by
icantly different mean values. Experimental Control hazard
Treatment arm arm (binomial test)
between treatments (Fisher’s exact test, P > 0.5;
Table 2b). (a)
Oecophylla smaragdina
Peristome 15 3 0.003
Flies more attracted to odours emitted from upper Digestive liquid 4 14 0.012
Lid 7 10 0.148
pitchers
Control 9 9 0.185
In the second experiment, the control test for O. smaragdina Drosophila melanogaster
Peristome 14 6 0.037
ants showed no directional trends in the olfactometer (bino-
Digestive liquid 8 12 0.120
mial test: N1 = 9, Ntot = 18, P = 0.185; Table 3b). In olfac- Lid 12 8 0.120
tory tests with the peristomes of lower and upper pitchers, ants Control 9 11 0.160
more often chose the experimental arms with peristomes than (b)
the empty arms and this was not due to random choice (bino- Oecophylla smaragdina
Upper pitcher 16 2 0.001
mial tests: upper pitcher, N1 = 16, Ntot = 18, P < 0.001;
Lower pitcher 14 5 0.022
lower pitcher, N1 = 14, Ntot = 19, P = 0.02). The choice fre- Control 9 9 0.185
quencies of arms with peristomes of upper pitchers were not Drosophila melanogaster
significantly different from those for lower pitchers (Yates-cor- Upper pitcher 13 7 0.074
rected v2 = 0.58, P = 0.45). Again, there were no differences Lower pitcher 6 12 0.071
Control 10 10 0.176
in responsiveness among bioassays with upper pitchers, lower
pitchers and controls (one-tailed Fisher’s exact test, P > 0.5).
The control test for D. melanogaster did not show any direc- significant preference towards the arms with the peristomes of
tional trend in the olfactometer (binomial test: N1 = 10, upper pitchers over the empty arms (binomial test: N1 = 13,
Ntot = 20, P = 0.18; Table 3b). The fruit flies showed a non- Ntot = 20, P = 0.07), but this was not the case for lower

 2010 The Authors. Journal compilation  2010 British Ecological Society, Journal of Ecology, 98, 845–856
852 B. Di Giusto et al.

pitchers (N1 = 6, Ntot = 18, P = 0.07). The frequency of Table 4. Results of the mixed Poisson regression models performed
visits to arms with peristomes from upper pitchers was on dependent variables: (a) the number of species, (b) the number of
significantly greater than that to arms with peristomes from terrestrial arthropods, and (c) the number of flying arthropods that
lower ones (v2 = 3.8, P = 0.05; Table 3b). were recorded visiting the pitchers in the field. We tested for the
random effect of plant (variance ⁄ residual), and two fixed factors:
pitcher type (lower ⁄ upper) and site (Badas ⁄ Liang)
PERISTOMES OF UPPER PITCHERS ARE ‘HOTSPOTS’
OF INSECT DIVERSITY Numerator Denominator
Effect d.f. d.f. F P-value
During the diurnal 10-min observation sessions carried out in
the field, upper pitchers attracted a greater number of insects (a)
Plant (0.02 ⁄ 0.59) NS
than lower pitchers (mixed Poisson regression model: effect of
Pitcher type 1 38 11.37 0.0017
pitcher type, F1,268 = 14.99, P = 0.0001) despite non-signifi- Site 1 21 0.06 0.81
cant variation among plants (plant as a random effect: vari- (b)
ance = 0.06, residuals = 1.23) and significant variation Plant (0.005 ⁄ 1.69) NS
between sites (F1,268 = 6.70, P = 0.0101, arthropod visitors Pitcher type 1 38 11.40 0.0017
Site 1 21 1.71 0.205
were less numerous at Badas than at Liang). In addition, there
(c)
was an important effect of pitcher part on the number of insect Plant (1.43 ⁄ 0.66) S
visitors (F1,268 = 16.96, P < 0.0001): the peristome attracted Pitcher type 1 38 3.62 0.06
the greatest number of arthropods at both sites (non-signifi- Site 1 21 2.05 0.17
cant interaction between pitcher part and site) and from both
pitcher types (non-significant interaction between pitcher part pitchers in flying insects and more specifically in nectar-visiting
and pitcher type) (Fig. 3). insects such as mosquitoes, as well as by an extension of the vis-
The overall analyses of insect richness and diversity at pitch- itor spectrum to the guild of other potential flower-visitors,
ers showed that upper pitchers attracted significantly more such as butterflies, beetles and thrips, which were absent from
insect species than lower pitchers (mixed Poisson regression the visitor spectrum of lower pitchers.
models; Table 4a) as well as more terrestrial arthropods
(mainly ants; Table 4b). There was no ‘site’ or ‘plant’ effect in
Discussion
the two analyses. In contrast, there was a strong effect of plant
as random factor on the number of flying visitors, explaining For the first time in Nepenthes, we experimentally demon-
perhaps why the trend of upper pitchers to attract more flying strated that an olfactory signal mediates insect attraction to
insects than lower ones was only weakly significant (Table 4c).
Some individual plants were significantly more attractive than Visitors Prey
100
others to this category of insects.
A total of 54 different species were recorded in the spectrum 90 Flower-visiting
insects
of arthropods visiting NRT pitchers. One hundred and forty- 80
Percentage of arthropods

six arthropod visitors and 36 species were recorded on the 31 70

upper pitchers examined compared to 77 arthropod visitors 60 Butterflies & moths
and 23 species for the 31 lower pitchers examined. The differ- Beetles
50 Wasps & bees
ences in arthropod visitor spectra between lower and upper Thrips
40 Mosquitoes
pitchers (Fig. 4) are characterized by a slight increase for upper Flies & midges
30 Bugs
Ascalaphs
3.5 d 20 Grasshoppers
Termites
Cockroaches & mantis
3 10
Number of arthropod visitors

Spiders
Ants
2.5 0
Lower Upper Lower Upper
during 10 min

Lower pitcher (n = 31) pitcher pitcher pitcher pitcher

2
Upper pitcher (n = 31)
c
1.5 b Fig. 4. Arthropod proportions compared between lower and upper
bc pitchers in the visitor spectrum (present study: 31 lower pitchers and
b
1 b 31 upper pitchers) and prey spectrum (data from Di Giusto et al.
0.5
2008: 17 lower pitchers and 17 upper pitchers). The brackets comprise
a a a a the estimated percentage of flying flower-visiting insects, which are
0 more diversified for upper pitchers than for lower pitchers in the two
Peristome Lid External Conductive Liquid
surface zone
types of insect spectrum. For flies and midges, we estimated from
empirical observations, and probably conservatively, that half
belonged to flower-visiting taxa. Among the Diptera, diverse flies,
Fig. 3. Attraction of arthropod visitors to lower and upper pitchers including male mosquitoes, various midges, carrion flies, pollen-feed-
and their different parts. Mean (±SD) number of insect visitors on the ing Syrphidae and long-tong nectar feeders, such as those we
different parts of the pitchers during 10-min field-observation ses- observed visiting pitchers, are known to pollinate flowering plants
sions. Different letters indicate significantly different mean values. (Resh & Cardé 2003).

 2010 The Authors. Journal compilation  2010 British Ecological Society, Journal of Ecology, 98, 845–856

the plant’s carnivorous structures. We showed that when NRT
plants develop into the climbing form, their pitcher-modified
leaves mimic flowers in their odour blends and that this con-
tributes to the widening of their prey spectrum to the guild of
generalist flower-visitors.
FLOWER BIOCHEMICAL MIMICRY BY CARNIVOROUS
LEAVES
The analysis of volatiles shows that the upper pitchers, pro-
duced when the plant enters the climbing phase (Gaume & Di
Giusto 2009), mimic flowers in their patterns of scent emission.
First, terpenes such as limonene, (E)-ocimene, linalool, a-
pinene and caryophyllene, and benzenoids such as benzyl alco-
hol and 2-phenylethanol, major constituents of upper pitchers
scent, occur in the floral bouquets of species belonging to more
than half of the angiosperms families tested (Knudsen, Eriks-
son & Gershenzon 2006). Secondly, some of these compounds,
such as phenylacetaldehyde, 2-phenylethanol and linalool and
its oxides have been shown in numerous plant species to be
delivered exclusively by flower tissues and are supposed to
mainly serve as signals to pollinators (e.g. Andersson et al.
2002). Thirdly, the three main categories of volatiles (fatty acid
derivatives, benzenoids and terpenoids) are usually simulta-
neously present in flowers of generalist pollination systems
(Knudsen, Eriksson & Gershenzon 2006) while blends usually
emitted by leaves have a narrower odour spectrum than flow-
ers (Paré & Tumlinson 1999; Dudareva & Pichersky 2006b).
The upper pitchers of NRT produced a rich and complex mix-
ture of compounds belonging to these three categories with up
to 54 volatiles at a mean rate of 100 ng h)1, which stands
within the scale of production of flower blends (Knudsen &
Gershenzon 2006; Raguso 2008).
Monoterpenes and sesquiterpenes can also be released
together with green-leaf volatiles in leaves of certain plants
after herbivory, serving to deter herbivores or attracting their
natural enemies (Mattiacci et al. 2001; Kost & Heil 2006; Sch-
nee et al. 2006). However, the complete absence of herbivory
on the plants tested and the high frequency of these volatiles in
our sample do not suggest an emission resulting from induced
chemical defence. This indicates instead that these compounds
are constitutively produced in the scent of these trapping
leaves, play an effective role in insect attraction, and are a key
component of the carnivorous syndrome.
The upper pitchers were indeed shown to attract in natura a
larger spectrum of arthropods than lower pitchers and particu-
larly a larger spectrum of flower-visitors, including a guild of
generalist pollinating insects (e.g. Lepidoptera, Thysanoptera,
Coleoptera) which do not visit lower pitchers. Such a niche
extension in upper pitchers was more evident when prey spec-
tra including nocturnal insects (moths) and hymenopterans
(bees and wasps) were considered (Fig. 4, Di Giusto et al.
2008). Moran (1996) showed that NRT pitchers had spectral
reflectance properties that could play a role in the attraction of
the flying prey. We demonstrate that attraction is not only due
to visual cues. Our olfactory bioassays on ants and flies con-
firmed that the scents released by both pitcher types are attrac-
 2010 The Authors. Journal compilation  2010 British Ecological Society, Journal of Ecology, 98, 845–856
Flower-scent mimicry in Nepenthes rafflesiana 853
tive to insects and that the scents of upper pitchers attract a
wider insect spectrum.
Such floral mimicry, in which plants get a fitness advantage
in mimicking the flowers of other plants, has already been
reported (Roy & Widmer 1999; Benitez-Vieyra et al. 2007)
even among carnivorous plants (Joel, Juniper & Dafni 1985;
Juniper, Robins & Joel 1989) but it involved visual mimicry
only. Flower chemical mimicry is rarer (less evident and less
studied). However, palm leaves (Dufaÿ, Hossaert-McKey &
Anstett 2003) and fungi (Raguso & Roy 1998) were shown to
chemically mimic flowers as strategies to attract pollinators
and their analogues. Our study therefore adds a new case of
flower chemical mimicry that occurs among carnivorous plants
as a strategy to attract prey.
DUAL OLFACTORY SIGNATURE AND INSECT-TRAPPING
STRATEGY?
The ontogenetic pitcher dimorphism of NRT is not clearly
associated with a dual biochemistry: all the compounds emit-
ted by lower pitchers were also found in upper pitchers. Hence,
the plant does not use different biochemical pathways during
its development. Nevertheless, the olfactory signature of upper
pitchers differs from that of lower ones because (i) the com-
pounds were emitted in greater quantities, (ii) 16 out of the 54
identified compounds, benzenoids for the majority, were found
uniquely in the blends of upper pitchers, and (iii) the two types
of pitchers had very specific and discriminative combinations
of volatile compounds. Variations in the quantity of emitted
volatiles could clearly explain the observed variation in the
quantity of insect visitors (this study) and prey (Di Giusto
et al. 2008) between the two pitcher types. Variation in com-
pound identity and in the combination of shared volatiles may
account for the difference in species attracted to the two pitcher
types.
The two pitcher types were highly attractive to ants and
shared several terpenes and fatty acid derivatives in their emis-
sions. Fatty acid derivatives are very commonly used in ant
communication (Hölldobler 1995), especially in Formicinae
such as Oecophylla smaragdina (Peerzada, Pakkiyaretnam &
Renaud 1990), here shown to be attracted to the odours of
both pitcher types. Ants also use terpenes as recruitment (e.g.
limonene (Kaib & Dittebrand 1990)) or trail pheromones (e.g.
a-farnesene (Hölldobler 1995)). Benzenoids also mediate ant
recruitment (e.g. phenylethanol, Hölldobler 1995) and are
sometimes crucial for some ant–plant mutualisms (e.g. methyl
salicylate, Brouat et al. 2000). All these compounds were abun-
dant in the pitcher blends of NRT and could play a role in ant
attraction.
Lower and upper pitchers essentially differed in the diversity
of flying insect visitors and to a lesser degree in their abundance
perhaps partly because some flies and midges, important flying
visitors, might be attracted by the odour of the decomposing
prey present in the two pitcher types. However, the niche diver-
sification of upper pitchers towards the guild of flower-visiting
– often pollinating – insects can be directly linked to the
chemical identity of the volatiles involved. Indeed, syrphids,
854 B. Di Giusto et al.
pollinator dipterans (Resh & Cardé 2003) largely represented
in the NRT prey spectrum (Di Giusto et al. 2008), are known
to be attracted by linalool (Knudsen 1999), a volatile present in
greater quantities in upper pitchers. As for the drosophilid flies,
trapped by upper pitchers only (Di Giusto et al. 2008) and
known as plant pollinators (Sakai 2002), the olfactory tests
showed that they were attracted uniquely by the odour of
upper pitchers, perhaps because of their richness in 2-pheny-
lethanol, which is known to elicit a strong electroantennogram
response in fruit flies (Zhu, Park & Baker 2003). Interestingly,
among the dipterans, mosquitoes visited upper pitchers more
frequently than lower ones. Mosquitoes, particularly Culex
pipiens, are common nectar-feeding visitors of flowers and fluid
ovipositors of the NRT pitchers and have been reported to
pollinate Caryophyllales other than Nepenthes (Silene otites,
Jhumur, Dötterl & Jürgens 2008). In that study, the strongest
responses of mosquitoes were evoked by linalool and its oxides
as well as by (Z)-3-hexenyl acetate, benzaldehyde and methyl
salicylate, some of the most abundant volatiles in upper pitcher
blends. Other floral volatiles emitted specifically or in greater
quantities by upper pitchers (e.g. benzaldehyde, benzyl alco-
hol, (E)-ocimene, phenylacetaldehyde, linalool and 2-pheny-
lethanol) are typically found in Lepidopteran pollination
systems (Andersson et al. 2002; Dobson 2006). As expected
for carnivorous plants, which are nitrogen-limited (Juniper,
Robins & Joel 1989; Ellison et al. 2003), no nitrogen-bearing
compounds, often signatures of moth-pollinated plants (Dob-
son 2006), were released by NRT pitchers. Although not sub-
ject of this diurnal study, moths were nevertheless commonly
observed as prey and nocturnal visitors of NRT (B. Di Giusto,
pers. obs.), and numerous moth-attracting volatile compounds
such as linalool, ocimene, a-farnesene and nerolidol, several
benzenoids and fatty acid derivative esters (Knudsen & Toll-
sten 1993; Dobson 2006) were emitted by the upper pitchers.
Finally, methyl benzoate and linalool, which attract a wide
range of beetle pollinators (Maetô & Fukuyuma 1995), were
emitted in greater quantities by upper pitchers. Interestingly,
beetles were mainly trapped by upper pitchers (Di Giusto et al.
2008).
While most of the compounds contributing to the biochemi-
cal specificity of upper pitchers are well known (Knudsen,
Eriksson & Gershenzon 2006), three volatiles found in the
emissions of more than 80% of the upper pitchers deserve
further considerations: the two aromatic ketones, 1-phenyl-
2,3-butanedione and 2-hydroxy-1-phenyl-3-butanone, and the
associated diol, 1-phenyl-2,3-butanediol. These compounds
are close to the raspberry ketone, which is known as a sweet
and common insect attractant (Dobson 2006), but to our
knowledge, they have been reported by only a couple of bio-
logical studies to be emitted by flower by-products (D’Arcy
et al. 1997; Alissandrakis et al. 2007). According to Alissan-
drakis et al. (2007), 2-hydroxy-1-phenyl-3-butanone, which
has been isolated from thyme (Corydothymus capitatus) honey,
has an intense floral and sweet odour. Clarke et al. (2001)
related the presence of the two ketones in the body extract
of some Eucerceris decorator wasps and suggested their
implication in the synthesis of (Z)-3-hexenyl (R)-3-hydroxy-
 2010 The Authors. Journal compilation  2010 British Ecological Society, Journal of Ecology, 98, 845–856
butanoate, a territorial-marking sex pheromone. Interestingly,
(Z)-3-hexenyl butanoate was the most abundant fatty acid
derivative in the pitchers of NRT.
FACTORS OF VARIATION IN SCENT EMISSION
The fact that lower pitchers produced weaker olfactory cues
than upper ones may reflect ecological costs. Juvenile plants
are more subject to mortality than mature plants, and to invest
in a complex odour cue is likely to be less rewarding for terres-
trial pitchers confronted with a lower diversity of prey than
aerial pitchers. The poorer olfactory cue produced by juvenile
plants may also have to do with the presence in lower pitchers,
just beneath the peristome, of a waxy layer, which is absent in
upper pitchers (Gaume & Di Giusto 2009). The wax plays a
crucial role in insect capture (Gaume, Gorb & Rowe 2002;
Gaume et al. 2004) but it might also hamper the emission of
volatiles. Indeed, lipid crystals act as a cuticular barrier for the
transport of scent molecules (Jetter 2006) and scent-releasing
surfaces in plants most often lack epicuticular wax (Eveling
1984). The peristome with its smooth domed ridges was the
main site of attraction. In addition to carrying nectar glands, it
might be particularly adapted to scent emission because of its
wetting properties facilitating the spreading of nectar (Bauer,
Bohn & Federle 2008) and its corrugations likely to increase
the emission area (Jetter 2006).
We observed a significant variation in the total number of
volatiles produced between plants within a given category of
pitcher and a given site and ⁄ or date of collection. Such a varia-
tion between plants might reflect a variation in pitcher age.
Indeed the odour intensity of pitchers is, according to the smell
of humans, age-dependent (Bauer, Willmes & Federle 2009).
Interestingly, we observed some significant between-site ⁄
year variations in the number of volatiles emitted. Environ-
mental conditions (light: Paré & Tumlinson 1999; water stress:
Takabayashi, Dicke & Posthumus 1994; nitrogen supply: Van
Wassenhove et al. 1990) can influence the physiology of plants
and thus modulate their emissions. The type of entomofauna
may also explain the between-site variation as in other attrac-
tion systems (e.g. Dötterl, Wolfe & Jürgens 2005). One alterna-
tive hypothesis is that in the site of Badas, where NRT was
found in sympatry with N. rafflesiana var. elongata, the two
taxa might differentiate their diets to avoid competition
(Gaume & Di Giusto 2009).
In conclusion, the carnivorous plant Nepenthes rafflesiana
var. typica uses an olfactory cue to attract its prey, which varies
with plant ontogeny, mimicking flower scent and being more
efficient when the vine enters into the climbing phase. Whether
such flower mimicry hampers the pollination or whether barri-
ers have been erected by evolution to prevent potential pollina-
tor–prey conflicts remains to be explored.
Acknowledgements
We thank the Forestry Department of Brunei Darussalam for permission to
carry out the field research and the University of Brunei Darussalam for
financial support during B.D.G.’s post-doctorate. We also thank H. Pang and

C. Pan Kiew for their assistance in the laboratory, M. Desvoyes for her field
services and B. Buatois for his invaluable help for the use of the GC–MS at
CEFE–CNRS, Montpellier. R. Borges and D. McKey are thanked for their
helpful comments on the manuscript.
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Received 20 May 2009; accepted 16 March 2010
Handling Editor: Martin Heil