Downloaded from http://rspb.royalsocietypublishing.org/ on June 16, 2017
rspb.royalsocietypublishing.org
Research
Cite this article: Suinyuy TN, Donaldson JS,
Johnson SD. 2015 Geographical matching
of volatile signals and pollinator olfactory
responses in a cycad brood-site mutualism.
Proc. R. Soc. B 282: 20152053.
http://dx.doi.org/10.1098/rspb.2015.2053
Received: 25 August 2015
Accepted: 10 September 2015
Subject Areas:
behaviour, ecology, evolution
Keywords:
coevolution, pollination mutualism,
Encephalartos villosus
Author for correspondence:
Terence N. Suinyuy
e-mail: [email protected]
Geographical matching of volatile signals
and pollinator olfactory responses in a
cycad brood-site mutualism
Terence N. Suinyuy1,2,3, John S. Donaldson2,3,4 and Steven D. Johnson1
1
School of Life Sciences, University of KwaZulu-Natal, P/Bag X01, Scottsville, Pietermaritzburg 3201,
South Africa
2
Kirstenbosch Research Centre, South African National Biodiversity Institute, P/Bag X7, Claremont,
Cape Town 7735, South Africa
3
Department of Biological Sciences, University of Cape Town, P/Bag Rondebosch, Cape Town 7701,
South Africa
4
Research Associate, Fairchild Tropical Botanic Garden, 10901 Old Cutler Road, Coral Gables, Miami,
FL 33156, USA
Brood-site mutualisms represent extreme levels of reciprocal specialization
between plants and insect pollinators, raising questions about whether these
mutualisms are mediated by volatile signals and whether these signals and
insect responses to them covary geographically in a manner expected from
coevolution. Cycads are an ancient plant lineage in which almost all extant
species are pollinated through brood-site mutualisms with insects. We
investigated whether volatile emissions and insect olfactory responses are
matched across the distribution range of the African cycad Encephalartos
villosus. This cycad species is pollinated by the same beetle species across its
distribution, but cone volatile emissions are dominated by alkenes in northern
populations, and by monoterpenes and a pyrazine compound in southern
populations. In reciprocal choice experiments, insects chose the scent of
cones from the local region over that of cones from the other region. Antennae
of beetles from northern populations responded mainly to alkenes, while
those of beetles from southern populations responded mainly to pyrazine.
In bioassay experiments, beetles were most strongly attracted to alkenes in
northern populations and to the pyrazine compound in southern populations.
Geographical matching of cone volatiles and pollinator olfactory preference is
consistent with coevolution in this specialized mutualism.
1. Introduction
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2015.2053 or
via http://rspb.royalsocietypublishing.org.
Coevolution is one of the major processes that drive diversification and speciation among plants and animals [1]. Obligate mutualisms represent some of the
most significant coevolutionary interactions [2] and include various highly
specialized brood-site pollination mutualisms [1] that are likely to be mediated
by volatile signals [3]. Most research on coevolved mutualisms has focused on
morphological co-adaptations rather than aspects of co-adaptation of plant
chemical signals and animal perception and cognition. Floral volatiles that
mediate plant –insect pollinator interactions are usually blends of compounds
belonging to several chemical classes [4]. There is considerable variation
among plant species in the number and relative amounts of the different constituents, and in their temporal and spatial emission patterns [5,6], all factors
which can influence attractiveness to pollinators [7,8]. As for morphological
floral traits, scent properties of a species can vary geographically [9] in a
manner which can be clinal [10] or unstructured [11], continuous or discrete.
Possible explanations for this geographical variation include phenotypic
plasticity, local hybridization, neutral processes such as genetic drift, and
adaptive processes such as coevolution or pollinator shifts [4,9,12].
In reciprocally specialized plant –pollinator interactions that are mediated
by floral volatiles, both floral signals and olfactory responses in the pollinators
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
Downloaded from http://rspb.royalsocietypublishing.org/ on June 16, 2017
2. Experimental procedures
2
(a) Study area and plant material
rspb.royalsocietypublishing.org
Field bioassays were conducted between 2009 and 2010 in
natural populations of E. villosus in the southern and northern
parts of the distribution range in the Eastern Cape (EC) and
KwaZulu-Natal (KZN) provinces of South Africa. The populations were Umtiza Nature Reserve (SOUTH 1) and Ocean
View Guest Farm (SOUTH 2) near East London in EC, and
Oribi Gorge Nature Reserve (NORTH 1) and at the Kranzkloof
Nature Reserve (NORTH 2) in KZN (figure 1a–c).
(b) Collection, analysis and identification of volatile
compounds
To confirm the geographical variation of cone volatile composition documented in a previous study [34], volatile odours
were sampled from male and female cones of E. villosus in
the SOUTH 1, SOUTH 2, NORTH 1 and NORTH 2 populations
between 2012 and 2013. Volatile compounds were analysed
and identified using the same collection methods and gas
chromatography–mass spectrometry (GC–MS) protocols
described previously [34].
(c) Gas chromatography –electroantennogram detection
We used combined gas chromatography–electroantennogram
detection and a flame ionization detector (GC–EAD–FID) [37]
to identify physiologically active compounds in headspace
samples of cones of E. villosus. Porthetes sp., Erotylidae sp.
nov., M. goodei and A. zamiae associated with E. villosus cones
were tested for their electrophysiological responses to cone
volatiles sampled from the SOUTH 1, SOUTH 2, NORTH 1
and NORTH 2 populations. For each analysis, the head was
excised, and a few segments on the tip of the two antennae
were cut off and mounted onto two silver metal electrodes
using an electrical conductive gel (Spectra 360, Parker Inc,
Orange, NJ, USA).
The GC column (DB-wax) was 30 m 0.25 mm internal
diameter 0.25 mm film thickness (Alltech, Deerfield, IL,
USA) and had a Y-tube connection installed at the end to split
the stream of volatile compounds into two equal parts, one
going to the FID and one to the EAD, where a prepared antenna
had been inserted under a humidified air stream to prevent the
antennae from drying out. The GC–EAD data were analysed
using AUTOSPIKE v. 3.3 software (Syntech, Kirchzarten,
Germany). A total of 24 GC–EAD analyses (six individuals
per beetle species) were carried out to determine the responses
of beetles. A compound was considered to be physiologically
active when it consistently elicited antennal responses in at
least three runs with three different antennae.
(d) Field bioassays with bucket traps
To test the effects of the electrophysiologically active compounds on cycad insect behaviour, bioassays were carried
out in the field using bucket funnel traps obtained from
Insect Science, South Africa. Because colour influences the behaviour of several insect species [38 –40], we conducted trials
to determine whether the colour of the bucket traps would
affect the behaviour of cycad insects. Pollen-shedding cones
were used as lures in five green and five yellow bucket
traps. These were placed together with an equivalent
Proc. R. Soc. B 282: 20152053
may be shaped by coevolution. Likely examples include the
fig–fig wasp, yucca– yucca moth, Glochidion–Epicephala
moth and Lithophragma–Greya moth pollination systems
[13 –16] in which there is already some evidence that volatiles
mediate their extraordinary reciprocal specificity [17–19].
Variation in floral volatiles between species in these systems
is usually associated with different pollinators. If coevolution,
rather than unilateral pollinator shifts, has shaped these
plant –pollination mutualisms, then one would predict
that plant volatile signals and pollinator responses to those
signals will also covary among populations in early stages
of divergence.
Divergence in floral traits is often attributed to geographical mosaics or clines in pollinator composition, an idea
known as the ‘pollinator-shift’ or ‘Grant–Stebbins’ model of
divergence [20,21]. The rationale is that different pollinators
often have different floral preferences, thus geographical
variability in pollinator composition would be likely to
result in divergent selection pressures and lead to the evolution of ‘pollination ecotypes’ [22]. These ecotypes may also
evolve because pollinators exhibit geographical variation in
their preferences due to differences in conditioning [21]. Yet
another scenario is that both floral signals and pollinator
preferences coevolve at the population or regional level,
producing a pattern of geographically structured covariation
in pollinator and plant traits [23,24].
The pollination system of cycads typically involves broodsite mutualisms in which the pollinators are insect herbivores
whose larvae feed on male cone tissues and female cones are
pollinated when visited by mistake [25–29]. Pollination in
these dioecious gymnosperms is mediated by specific cone
volatile compounds [30]. The brood-site mutualisms in
cycads probably evolved from herbivore interactions in
which herbivorous insects were attracted to specific cues
associated with nutrient-rich cycad reproductive structures
[31]. The obligate nature of these brood-site mutualisms
makes it likely that cycads and their insect pollinators have
subsequently coevolved [25,32,33].
A recent study of the African cycad Encephalartos villosus
revealed remarkable intraspecific geographical variation in
the profile of volatiles emitted from cones [34]. The main
compounds emitted by cones of E. villosus from the
southern distribution range were eucalyptol and 2-isopropyl3-methoxypyrazine, whereas plants from the northern range
were characterized by emissions of (3E)-1,3-octadiene and
(3E,5Z)-1,3,5-octatriene. The pollinators Porthetes sp. (preliminary manuscript name: P. pearsonii [35]), an undescribed
species of Erotylidae and Metacucujus goodei, as well as a less
important pollinator—the seed predator Antliarhinus zamiae
[26]—were recorded from 10 populations across the distribution
range [34]. As all of these beetles use E. villosus as a brood site
and thus have potentially coevolved with the cycad, it was
hypothesized that the geographical variation in scent would
reflect corresponding differences among beetle populations in
their detection of particular compounds and behavioural
response to them. Attraction of beetles to E. villosus cone volatiles
has long been considered likely as they tend to be active on
cones at the pollen-shedding stage, when emission of volatile
compounds is at a peak [36].
In this study, we determined whether cone volatiles of
E. villosus attract the associated beetles and whether there
was geographical matching between cone volatiles and beetle
preferences, as predicted by the coevolution hypothesis.
Downloaded from http://rspb.royalsocietypublishing.org/ on June 16, 2017
4
Kranzkloof NR
response (mV)
1
2
Durban
Pietermaritzburg
3
Port Shepstone
Oribi Gorge NR
200
(3E,5Z)-1,3,5-octatriene
3.5
5.5
7.5
9.5
11.5
(e)
1
1
4
Eastern Cape
2
response (mV)
Port St Johns
Ocean View Guest Farm
(SOUTH 2)
Ocean View Guest Farm
East London
Umtiza NR
300
Umtiza NR
(SOUTH 1)
(c)
4
(3E)-1,3-octadiene
EAD
200
100
(3E,5Z)-1,3,5-octatriene
GC
3.5
1
(b)
GC
5.5
7.5
9.5
11.5
(f)
2
1
EAD
300
O
N
200
N
GC
100
7.5
2-isopropyl
-3-methoxypyrazine
8.5
10.5
11.5
9.5
GC retention time (mins)
12.5
Figure 1. (a) Map showing geographical variation of cone volatile compounds of E. villosus in the different study sites; pie charts depict the percentage of the
compounds emitted and tested in the field and other compounds in odour profile of cone volatiles. 1 ¼ 2-isopropyl-3-methoxypyrazine (active), 2 ¼ eucalyptol
(non-active), 3 ¼ (3E)-1,3-octadiene (active), 4 ¼ others (non-active): a-pinene, b-pinene, linalool, benzaldehyde, heptanal, phenol, hexanal, p-anisaldehyde.
(b) Female cone and (c) male cones of E. villosus. (d – f ) Representative GC – EAD traces showing responses of (d ) Erotylidae sp. nov. and (e) Porthetes sp. to
the volatile compounds (3E)-1,3-octadiene and (3E,5Z )-1,3,5-octatriene emitted by male cones of E. villosus from northern populations, and (f ) responses of
Porthetes sp. to 2-isopropyl-3-methoxypyrazine emitted by male cones of E. villosus from the southern populations. Total ion chromatogram (GC) represents compounds present in cone volatile profiles. Electroantennographically detected peaks represent beetle physiological responses to specific compounds (structures and
names presented in the figures). (Online version in colour.)
number of empty bucket traps of the same colour serving as
controls in the botanic garden of the University of KZN.
To test whether individual compounds attracted insects to
traps, (3E)-1,3-octadiene and 2-methoxy-3-isopropylpyrazine,
two electrophysiologically active compounds, and eucalyptol, a compound that consistently occurs in substantial
concentration in almost all populations, were used as
baits in bucket traps set-up in the SOUTH 1, SOUTH 2,
NORTH 1 and NORTH 2 populations. The electrophysiologically active alkene (3E,5Z )-1,3,5-octatriene was omitted
from this analysis as it is not commercially available. For
each treatment, we used the same proportions (50 : 50 or as
close to this as possible) of green and yellow bucket traps.
Solutions of 50 ml of either (3E)-1,3-octadiene, 2-methoxy-3isopropylpyrazine, or eucalyptol and 950 ml of liquid paraffin
(Alpha Pharma, South Africa) used as baits were put into
amber glass bottles with cotton wicks inserted through
the cork to facilitate gradual release of volatile compound.
Paraffin oil without the test compounds served as a control.
Each bait bottle or control was placed into an individual
bucket trap. The bucket traps were placed about 15 m apart
in an area with E. villosus individuals. Each trial lasted 24 h
(starting at 9.00 and ending at 9.00 the following day), after
which the positions of the traps were randomized and the
number and identity of insects were recorded. Each treatment and control had five replicates and each trap was
monitored for five days, apart from those at NORTH 2,
which were additionally sampled with seven replicates each
on day 5 and 6, and those at NORTH 1, which were additionally sampled on days 6, 7 and 8 with nine, eleven and eight
replicates each, respectively.
(e) Olfactometer tests with cycad cones
Olfactometer assays were carried out in the laboratory between
2009 and 2013 using cycad cones at the pollen-shedding stage
Proc. R. Soc. B 282: 20152053
Oribi Gorge NR
(NORTH 1)
(3E)-1,3-octadiene
300
100
2
4
EAD
rspb.royalsocietypublishing.org
3
KwaZulu-Natal
N
3
(d)
Kranzkloof NR
(NORTH 2)
response (mV)
(a)
Downloaded from http://rspb.royalsocietypublishing.org/ on June 16, 2017
Statistical analyses for bucket trap data were analysed using
restricted maximum-likelihood (REML) and generalized
linear mixed models implemented in GENSTAT v. 12.1. These
models used a negative binomial error distribution with a
log-link function and the aggregation factor was set to 1. Controls were not included in the analysis as none of the control
traps attracted any beetles, whereas all the treatments
attracted beetles irrespective of the colour combination. In
the experiment using garden plants, bucket colour had no
significant effect on the number of beetles attracted to traps
(colour: Wald x 2 ¼ 0.21, p ¼ 0.97; colour insect species:
Wald x 2 ¼ 11.37, p ¼ 0.26), and this factor was therefore not
analysed further.
The first analysis tested the effect of compound type on the
differences in the total number of beetles attracted per day to
the volatile compounds in the bucket traps in the different
study populations. The region, volatile compound and beetle
species were fitted as fixed factors, while population, trial
(bucket traps were randomly re-arranged for each trial
3. Results
(a) Volatile emissions
The volatile compounds emitted by cones from different
populations of E. villosus conformed to the same overall
geographical pattern as described in a previous study [34]
(figure 1a). The cone odour of E. villosus from SOUTH 1 and
SOUTH 2 populations was dominated by the nitrogencontaining compound 2-isopropyl-3-methoxypyrazine and
the monoterpene eucalyptol, with a-terpinene, a-pinene and
b-pinene contributing substantial amounts. The unsaturated
hydrocarbons (3E)-1,3-octadiene and (3E,5Z)-1,3,5-octatriene
characterized the cone odour of NORTH 1 and NORTH 2
plants, together with eucalyptol and other monoterpenes
also found in SOUTH 1 and SOUTH 2 plants (figure 1a).
(b) Electrophysiological response to cone volatiles
Three compounds in the odour profile of E. villosus
were consistently electrophysiologically active in repeated
GC–EAD experiments (figure 1d–f ). Two compounds—(3E)1,3-octadiene and (3E,5Z)-1,3,5-octatriene emitted by E. villosus
in its northern distribution range—elicited electrophysiological
responses in Erotylidae sp. nov. and Porthetes sp. collected
from the northern region (figure 1d,e). There were no responses
from M. goodei and A. zamiae from the northern populations.
The compound 2-isopropyl-3-methoxypyrazine emitted by
E. villosus from the southern region elicited electrophysiological
responses in Porthetes sp. from the southern region (figure 1f).
Erotylidae sp. nov., M. goodei and A. zamiae from the southern
populations did not respond to any compounds.
(c) Field trapping experiments with volatile compounds
In field trapping experiments, A. zamiae, Erotylidae sp. nov.,
M. goodei and Porthetes sp. were attracted to traps containing
4
Proc. R. Soc. B 282: 20152053
(f ) Data analysis
of 24 h) and trap number nested within population were
fitted as random factors. The subsequent analyses tested the
differences in the mean number of individual beetles of each
species attracted per volatile compound treatment per day in
the different regions. The region, volatile compound and
their interaction were fitted as fixed factors while population
and trials nested within population were fitted as random
factors. Wald x 2 statistics generated by REML were used to
determine the significance of fixed factors and their interactions. Means and standard errors were back-transformed
from the log scale. Post hoc comparisons among means were
conducted using Tukey tests.
The sources of variation in the behaviour of insects to the
treatments (cone volatiles) in the olfactometer experiments
were assessed using generalized linear models implemented
in PASW v. 18 (SPSS, Chicago, IL, USA). Each observation
for a batch of beetles represented the proportion of beetles
that entered the olfactometer (i.e. participated) or the proportion of beetles that responded to a particular type of
cone in the olfactometer. Models used a binomial error distribution with an events/trials structure where observations
consisted of the number of trials ( participating beetles in
each replicate) and events (beetles that responded). Models
had a logit-link function and significance of effects was
assessed by using likelihood ratio statistics. Means and
standard errors were back-transformed from the logit scale.
rspb.royalsocietypublishing.org
to determine how individuals of the known pollinator
Porthetes sp. collected from the southern and northern range
of E. villosus respond to cone volatiles of E. villosus from
these two regions. Two types of experiment were undertaken
under laboratory conditions (temperatures between 25.08C
and 28.08C) using male cones from plants in the southern
and northern populations. In the first, pollen-shedding cones
of plants from the southern and northern regions were offered
to Porthetes sp. individuals from these two regions in a design
where beetles had a choice between an arm containing cycad
cone sporophylls and an empty arm containing pure air. In
the second, pollen-shedding cones of E. villosus from both
regions were simultaneously offered, each in a different arm
of the olfactometer, to insects from northern and southern
populations, respectively. Each experiment was replicated
12 times using 10–20 beetles per replicate, and for each replicate in the different experiments fresh cone samples and a
new batch of insects were used.
The olfactometer used was a simple Y-tube (stem 10 cm,
arms 8 cm at 458 angle, internal diameter 2 cm) and was
placed on a light table so as to have homogeneous lighting
and one arm connected to a polyacetate bag (Nalo Bratfolie
Kalle GmbH) containing an odour source (cone sporophylls)
and the other arm connected to an empty polyacetate bag
with pure air. Air was drawn through silicon tubing by a portable battery-operated pump (Spectrex Personal Air Sampler
PAS 500) set at 200 ml min21 and was split at the Y-junction
to create equal air streams. Beetle pollinators were introduced
to the base of the stem of the olfactometer using an insect
holder. The response from the beetle was determined when
they (a) entered one of the arms and stayed there, and
(b) left the olfactometer arm and crawled into the polyacetate
bag. Beetles that remained in the holder and the stem of the
Y-maze were considered to have not made a choice. Venting
of each odour field continued for an hour and the number of
beetles collected in the different arms of the olfactometer was
counted. After each experiment, the olfactometer was cleaned
with 70% EtOH, rinsed with acetone and baked at 2508C
for 2 h. On running the next experiment, the control arm of
the olfactometer was switched from left to right or vice
versa to avoid bias.
Downloaded from http://rspb.royalsocietypublishing.org/ on June 16, 2017
(a)
5
5
regionn.s.
compound***
insects***
R × C***
R × I***
C × I***
R × C × I***
3
key
a
2
1
Antliarhinus zamiae
a
a
b b a
a
b
b a b
0
(b)
5
Eastern Cape
Erotylidae sp. nov.
no. insects trap–1 day–1
4
3
b
Metacucujus goodei
2
b
1
b
a a a a
a a a
a
b
Porthetes sp.
0
2-isopropyl3-methoxypyrazine
chemical compound
(3E)-1,3-octadiene
eucalyptol
Figure 2. Number (mean + s.e) of beetle individuals of different species attracted to (3E)-1,3-octadiene, eucalyptol or 2-isopropyl-3-methoxypyrazine in trapping
experiments in different E. villosus populations. The means and standard errors are back-transformed from the log scale. Significance values (***p , 0.001; n.s., not
significant) are given for predictors and their interactions. Letters above bars denote homogeneous groups (based on Tukey tests) for each beetle species across the
different compounds. The key shows beetle species and sexual dimorphism for one species. Scale bar, 10 mm. (Online version in colour.)
volatile compounds. Porthetes sp. at the northern sites were
attracted mainly to (3E)-1,3-octadiene (figure 2a), whereas
those at the southern sites were attracted mainly to 2-isopropyl3-methoxypyraxine (figure 2b). Erotylidae sp. nov. and
A. zamiae showed a similar pattern but were attracted in lower
numbers (figure 2a,b). Relatively few beetles were recovered
from traps baited with eucalyptol (figure 2a,b). Overall, the interaction of region and compound type had a highly significant
( p , 0.001) effect on the abundance of beetles attracted to the
traps (electronic supplementary material, table S1). For the individual beetle species, the interaction of region and volatile
compound was a strongly significant factor explaining the abundance of trapped Porthetes sp., A. zamiae, Erotylidae sp. nov. and
M. goodei (electronic supplementary material, table S1).
(d) Behavioural response of Porthetes sp. to cone
volatile compounds
In the experiments, a high proportion of beetles entered the
olfactometer and chose either the scented or control arms
(figure 3a). In olfactometer choices between a cone scent and
an unscented control, responses of beetles differed significantly
according to whether cones were from the same region as the
beetles or from a different region. When beetles from the
southern and northern populations were separately offered
cones from corresponding populations and vice versa, they
were more attracted to cones from the same region
(x21 ¼ 105:65, p , 0.0001, n ¼ 48 trials; figure 3b). When
pollen-dehiscent cones from both regions were simultaneously
Proc. R. Soc. B 282: 20152053
no. insects trap–1 day–1
4
rspb.royalsocietypublishing.org
KwaZulu-Natal
a
Downloaded from http://rspb.royalsocietypublishing.org/ on June 16, 2017
offered to Porthetes sp., responses of individuals from the
southern and northern regions differed (x21 ¼ 39:95, p ,
0.0001, n ¼ 24 trials), with each showing a significant preference
for local cones (figure 3c).
1.0
Eastern Cape cones
KwaZulu-Natal cones
0.8
0.6
4. Discussion
0.4
insects***
C × I***
0
proportion of beetles in the scented arm
(b) 1.0
0.8
0.6
0.4
conesn.s.
0.2
insectsn.s.
C × I***
proportion of visits to Eastern Cape cones
(c) 1.0
0
0.8
0.2
insects***
0.6
0.4
0.4
0.6
0.2
0.8
0
1.0
Eastern Cape
insects
proportion of visits to KwaZulu-Natal cones
0
KwaZulu-Natal
insects
Figure 3. Mean (+95% CI) proportional responses of Porthetes sp. during
olfactometer choice trials involving E. villosus cone volatiles. (a) Proportions
of beetles that responded in the trials when they entered any arm of the
olfactometer. (b) Proportions of responding beetles that chose the scented
arm of the olfactometer rather than the unscented arm when beetles
were separately offered cones from corresponding region and vice versa.
(c) Response of Porthetes sp. when E. villosus cone volatiles from EC and
KZN were simultaneously offered to the beetles from each region in a
series of olfactometer choice experiments. The means and CI values are
back-transformed from the logit scale. Significance values (***p , 0.001;
n.s., not significant) are given for predictors and their interactions. Means
with confidence intervals that do not overlap the dotted lines of equal
choice in (b,c) represent significant preference for the scented arm. (Online
version in colour.)
Proc. R. Soc. B 282: 20152053
conesn.s.
0.2
This study provides evidence for geographical matching of
plant volatiles and insect physiological and behavioural
responses. Olfactometer experiments showed that the weevil
pollinator Porthetes sp. is attracted to E. villosus cones from
its local region, rather than to E. villosus cones from a different
region (figure 3b,c). The chemical basis for this pattern is
revealed in the electrophysiological experiments in which
antennal responses by beetles were detected in response to
volatiles emitted by cones from the same region as the beetles.
This was confirmed by the field bioassays, which showed that
the main pollinators in the northern populations responded
most strongly to (3E)-1,3-octadiene (figure 2a) while those
in the southern populations responded most strongly to
2-isopropyl-3-methoxypyrazine (figure 2b). These patterns
are consistent with the idea of coevolutionary divergence in
obligate brood-site mutualisms [16,19,41].
Volatiles appear to play a central role in attraction of pollinators in specialized brood-site mutualisms. In the fig–fig wasp
mutualism, species specificity appears to be maintained by
responses of the pollinating wasps to specific fig volatiles,
although there is still uncertainty about which compounds mediate these interactions [42,43]. There is also evidence that specific
volatiles mediate the brood-site pollination mutualism between
Glochidion trees and Epicephala moths, and between Lithophragma
plants and Greya moths [16,17]. In such highly species-specific
brood-site mutualisms, plants may evolve one or a few specific
and unusual compounds that represent ‘private channels’ in the
sense that they would not be detected by other pollinators in the
same communities [4,44]. However, this idea has not received
much empirical support as most compounds reported thus far
in the floral scent of brood-site pollination mutualisms are
common volatile constituents of leaves and flowers [6]. In the
yucca–yucca moth mutualism, yucca moths are attracted to yucca floral scent [18] and the odour profiles of three
allopatric species contain a physiologically active compound
(E)-4,8-dimethyl-1,3,7-nonatriene together with similar odour
compounds that are widespread in flowering plants [11,18].
The physiologically active compound may function as a private communication channel, but its role remains to be
determined. In this study, we found that (3E)-1,3-octadiene
and (3E,5Z)-1,3,5-octatriene emitted by E. villosus from the
northern region and 2-isopropyl-3-methoxypyrazine emitted
by E. villosus from the southern region elicit electrophysiological
responses (figure 1d–f) and attract pollinators of E. villosus in
their respective regions (figure 2a,b). These compounds are
rare and have been recorded in only a few plants [45,46], and
may thus function as private channels of communication in
the communities in which these cycads occur. The only previous
report of attraction of a pollinator to a pyrazine compound
involves a sexually deceptive Australian orchid that mimics
the pyrazine sex pheromone of female thyniine wasps [47].
What makes the E. villosus cycad pollination system particularly novel is the geographical variation in key attractive
compounds and the preference of widespread pollinators for
6
rspb.royalsocietypublishing.org
proportion of trials evoking a response
(a)
Downloaded from http://rspb.royalsocietypublishing.org/ on June 16, 2017
7
Proc. R. Soc. B 282: 20152053
may be tracked by other species that interact less strongly
[1,64]. Since Porthetes sp. is the major pollinator of E. villosus
[26], we expect this species to have coevolved with E. villosus.
By contrast, the responses of Erotylidae sp. nov., M. goodei
and A. zamiae to the volatile odours of E. villosus may reflect
tracking of chemical traits that have diverged between populations, as these beetles have a near-obligate dependence on
its cones but do not contribute to pollination of E. villosus
to the same extent as Porthetes sp. [26].
The beetles Erotylidae sp. nov. and M. goodei, which
are attracted to E. villosus volatiles, also visit the cones of
E. natalensis [65], which similarly emits (3E)-1,3-octadiene
[63]. They play a small role in pollination and occur in low
numbers in male and female cones of E. villosus [26,36],
where their larvae develop in central axis and sporophyll
tissues of both male and female cones in the case of erotylids,
and in male cone tissues in the case of M. goodei [26].
Other erotylids and Metacucujus spp. occur on E. natalensis
and other Encephalartos cycads [29,66,67] that overlap with
E. villosus distribution and emit different compounds [63].
These beetles contribute to pollination of various Encephalartos
species [26,29,67] and may have coevolved with the genus.
The taxonomy of Erotylidae sp. nov. in South Africa has not
been studied and there are no molecular studies of this
beetle to provide any information on species-level variation.
It is thus difficult to confirm for this secondary pollinator
whether there is only one species that has coevolved with
E. villosus across the distribution range, and whether it has
been involved in coevolution with E. villosus or, alternatively,
has either tracked evolutionary changes in the cycad or
shows local chemical imprinting according to the brood site.
The beetle seed predator A. zamiae occurs in very low numbers in male cones and is a minor pollinator of E. villosus [26],
but occurs in large numbers in female cones during the pollination stage, where they mate and oviposit, and their larvae
develop in the seeds [26,36]. Though attracted in small numbers, there was some evidence that they could distinguish
between the alkene and pyrazine compounds offered in traps
at the southern sites (figure 2b; electronic supplementary material, table S1). These beetles also occur in other
Encephalartos species [68] that overlap in distribution and volatile compound emission in E. villosus, as well as others that emit
different compounds [63], suggesting that they may also use
additional cues for host plant location. They have a rostrum
length that varies according to the seed size of different
cycad species [69]. This phenotypic trait matching suggests
that they have coevolved with various cycads.
This study shows that plant volatiles and signal perception
and cognition by insects involved in specialized brood-site
mutualisms may reflect coevolutionary processes that drive
divergence among populations, and could ultimately lead to
speciation. The divergence in the odour profile observed
among populations of E. villosus may be viewed in the light
of the geographical mosaic theory of coevolution [23]. This
theory predicts that geographical differences in selection pressures due to interactions not just between strongly interacting
species pairs, but also with other species in local communities,
as well as genetic drift and migration, create a range of possible
outcomes [24]. These varying outcomes at the population level
lead to a geographical mosaic of coevolutionary hotspots and
cold spots [1,23,24]. Our study suggests that localized coevolution may have occurred between beetles and E. villosus despite
the lack of variation in molecular markers among populations
rspb.royalsocietypublishing.org
local plant chemotypes. The close correspondence between
chemical signals of E. villosus and the olfactory responses of
its pollinators suggests that these interactions may have been
shaped by coevolution [48]. Nevertheless, it is possible that
there has been adaptation of the cycads to cryptic weevil
species, which would be a case of divergence driven by shifts
in pollinators, rather than coevolution [49,50]. However, a
recent molecular study of phylogenetic relationships within
Porthetes spp. showed that specimens from different E. villosus
populations across the distribution range had identical
sequences for the mitochondrial cytochrome oxidase 1 (CO1)
gene [51], with no obvious morphological characters separating specimens from northern and southern E. villosus
populations. It is also possible that E. villosus consists of two
geographically separate species. Few molecular data are available for Encephalartos due to notoriously low genetic variation
within the genus [52], and among cycads in general [53,54].
There is some morphological variation in E. villosus, with
plants from the southern region having shorter, heavily
spined leaflets and cones with toothed edges, and those from
the northern region having longer, almost entire leaflets and
cones with lightly toothed edges [55]. However, this morphological variation is largely clinal and has been regarded as
insufficient to split E. villosus into different species. There is
no difference in the beetle assemblages on cones in different
populations of E. villosus [34], but the geographical differences
we observed in beetle olfactory responses may influence the
direction and intensity of selection on plant traits leading to
divergence and hence formation of chemotypes.
The cognitive basis of pollinator attraction to floral volatiles
can be innate [56] or learned [57]. The sensory preferences of
insects involved in highly specialized brood-site mutualisms
are expected to be innate [58,59], and may arise from mutual
adaptations of the pollinators’ sensory systems and floral
signals. This contention is supported by the differences in electrophysiological responses to particular compounds that we
observed for beetles from different populations (figure 1d–f ).
An alternative explanation for the finding that beetles respond
positively to scent of local cones when offered local and nonlocal cones simultaneously (figure 3c) is that beetles undergo a
chemical imprinting process during their development on
local cones [60–62]. Interestingly, we found some evidence of
active avoidance of cones that are not from the local region
(figure 3b), suggesting that there are volatiles in these cones
that affect behaviour of local beetles, even though we did
not detect antennal electrophysiological responses to volatiles
from non-local cones. Absence of detectable electrophysiological
responses of insects to volatiles can, however, be simply due to
limitations of the recording equipment.
As is the case for the fig–fig wasp mutualism [42,43],
cycad brood-site mutualisms are not always completely reciprocally specialized. Some of the beetles that pollinate E. villosus
also visit male and female cones of other Encephalartos cycads.
The Porthetes sp. in this study also occurs on cones of
E. aplanatus and E. umbeluziensis [51], which are phylogenetically related to [52] and out of the range of E. villosus.
Encephalartos aplanatus emits the same volatile compounds
as E. villosus in its northern distribution range, while
E. umbeluziensis emits compounds that are different from those
in any E. villosus populations [63]. Hence some cycad weevil
species may have coevolved with more than one cycad species.
In mutualisms, coevolution may occur mainly between
strongly interacting species, and the resulting trait evolution
Downloaded from http://rspb.royalsocietypublishing.org/ on June 16, 2017
Ethics. The experiments conducted herein comply with the ethical
8
Botanic Garden, The Andrew W. Mellon Foundation, South African
National Biodiversity Institute, South African National Research Foundation and the Universities of Cape Town and KwaZulu-Natal.
Acknowledgements. We thank Jacques De Wet Bösenberg for assistance
in the field and Andreas Jürgens for the advice on GC– EAD in the
laboratory. We thank Sandy-Lynn Steenhuisen for logistical support
and James Rodger for helpful discussions on the statistics. We are
also thankful to Ian Smith, Jan Van Ryneveld and the horticulturist
of the University of KwaZulu Natal (UKZN) Botanic Garden, Pietermaritzburg Campus for granting us access to their plants. The
Eastern Cape Parks Board and Ezemvelo KwaZulu Natal Wildlife
issued permits to work on cycads in their reserves.
References
1.
Thompson JN. 1994 The coevolutionary process.
Chicago, IL: Chicago University Press.
2. Smith CI, Godsoe WKW, Tank S, Yoder JB,
Pellmyr O. 2008 Distinguishing coevolution from
covicariance in an obligate pollination mutualism:
asynchronous divergence in Joshua tree and its
pollinators. Evolution 62, 2676 –2687. (doi:10.1111/
j.1558-5646.2008.00500.x)
3. Pellmyr O, Thien LB. 1986 Insect reproduction and
floral fragrances: keys to the evolution of the
angiosperms? Taxon 35, 76 –85. (doi:10.2307/
1221036)
4. Raguso RA. 2008 Wake up and smell the roses: the
ecology and evolution of floral scent. Annu. Rev.
Ecol. Evol. Syst. 39, 549–569. (doi:10.1146/
annurev.ecolsys.38.091206095601)
5. Raguso RA. 2004 Why do flowers smell? The
chemical ecology of fragrance-driven pollination. In
Advances in insect chemical ecology (eds RT Cardé,
JG Millar), pp. 141 –178. Cambridge, UK:
Cambridge University Press.
6. Knudsen JT, Eriksson R, Gershenzon J, Ståhl B. 2006
Diversity and distribution of floral scent. Bot. Rev. 72,
1–120. (doi:10.1663/0006-8101(2006)72[1:DADOFS]
2.0.CO;2)
7. Dodson CH, Dressler RL, Hills HG, Adams RM,
Williams NH. 1969 Biologically active compounds in
orchid fragrances. Science 164, 1243 –1249. (doi:10.
1126/science.164.3885.1243)
8. Kumano-Nomura Y, Yamaoka R. 2009 Beetle
visitations, and associations with quantitative
variation of attractants in floral odours of
Homalomena propinqua (Araceae). J. Plant Res. 122,
183–192. (doi:10.1007/s10265-008-0204-6)
9. Schlumpberger BO, Raguso RA. 2008 Geographic
variation in floral scent of Echinopsis ancistrophora
(Cataceae); evidence of constraints on hawkmoth
attraction. Oikos 117, 801–814. (doi:10.1111/j.
2008.0030-1299.16211.x)
10. Knudsen JT. 2002 Variation in floral scent
composition within and between populations of
Geonoma macrostachys (Arecaceae) in the western
Amazon. Am. J. Bot. 89, 1772 –1778. (doi:10.3732/
ajb.89.11.1772)
11. Svensson GP, Hickmann Jr MO, Bartram S, Boland
W, Pellmyr O, Raguso RA. 2005 Chemistry and
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
geographic variation of floral scent in Yucca
filamentosa (Agavaceae). Am. J. Bot. 92,
1624 –1631. (doi:10.3732/ajb.92.10.1624)
Pellmyr O. 1986 Three pollination morphs in
Cimicifuga simplex; inicipient speciation due to
inferiority in competition. Oecologia 68, 304–307.
(doi:10.1007/BF00384804)
Pellmyr O. 2003 Yuccas, yucca moths, and
coevolution: a review. Ann. MO Bot. Gard. 90,
35 –55. (doi:10.2307/3298524)
Weiblen GD. 2002 How to be a fig wasp. Annu. Rev.
Entomol. 47, 299–330. (doi:10.1146/annurev.ento.
47.091201.145213)
Kawakita A, Kato M. 2009 Repeated independent
evolution of obligate pollination mutualism
in the Phylantheae–Epicephala association.
Proc. R. Soc. B 276, 417–426. (doi:10.1098/
rspb.2008.1226)
Friberg M, Schwind C, Raguso RA, Thompson JN.
2013 Extreme divergence in floral scent among
woodland star species (Lithophragma spp.)
pollinated by floral parasites. Ann. Bot. 111,
539 –550. (doi:10.1093/aob/mct007)
Okamoto T, Kawakita A, Kato M. 2007 Interspecific
variation of floral scent composition in Glochidion
and its association with host-specific pollinating
seed parasite (Epicephala). J. Chem. Ecol. 33,
1065 –1081. (doi:10.1007/s10886-007-9287-0)
Grant V, Grant KA. 1965 Flower pollination in the Phlox
family. New York, NY: Columbia University Press.
Friberg M, Schwind C, Roark LC, Raguso RA,
Thompson JN. 2014 Floral scent contributes to
interaction specificity in coevolving plants and their
insect pollinators. J. Chem. Ecol. 40, 955–965.
(doi:10.1007/s10886-014-0497-y)
Stebbins GL. 1970 Adaptive radiation of reproductive
characteristics in angiosperms. I. Pollination
mechanisms. Annu. Rev. Ecol. Syst. 1, 307–326.
(doi:10.1146/annurev.es.01.110170.001515)
Ellis AG, Johnson SD. 2009 The evolution of floral
variation without pollinator shifts in Gorteria diffusa
(Asteraceae). Am. J. Bot. 967, 93 –801. (doi:10.
3732/ajb.0800222)
Svensson GP, Pellmyr O, Raguso RA. 2011 Pollinator
attraction to volatiles from virgin and pollinated
host flowers in a yucca/moth obligate mutualism.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
Oikos 120, 1577–1583. (doi:10.1111/j.1600-0706.
2011.19258.x)
Thompson JN. 2005 The geographic mosaic of
coevolution. Chicago, IL: Chicago University Press.
Thompson JN. 1999 Specific hypotheses on the
geographic mosaic of coevolution. Am. Nat.
153(Suppl. 5), S1 –S14. (doi:10.1086/303208)
Tang W. 1987 Insect pollination in the cycad Zamia
pumila (Zamiaceae). Am. J. Bot. 74, 90 –99.
(doi:10.2307/2444334)
Donaldson JS. 1997 Is there floral parasite
mutualism in cycad pollination? Pollination biology
of Encephalartos villosus (Zamiaceae). Am. J. Bot.
84, 1398 –1406. (doi:10.2307/2446138)
Hall JA, Walter GH, Bergstrom DM, Machin P. 2004
Pollination ecology of the Australian cycad
Lepidozamia peroffskyana (Zamiaceae). Aust. J. Bot.
52, 333 –343. (doi:10.1071/BT03159)
Terry LI, Walter GH, Donaldson JS, Snow E, Forster PI,
Machin PJ. 2005 Pollination of Australian Macrozamia
cycads (Zamiaceae): effectiveness and behavior of
specialist vectors in a dependent mutualism.
Am. J. Bot. 92, 931–940. (doi:10.3732/ajb.92.6.931)
Suinyuy TN, Donaldson JS, Johnson SD. 2009 Insect
pollination in the African cycad Encephalartos
friderici-guilielmi Lehm. S. Afr. J. Bot. 75, 682 –688.
(doi:10.1016/j.sajb.2009.08.005)
Terry I, Walter GH, Moore C, Roemer R, Hull C. 2007
Odor-mediated push-pull pollination in cycads.
Science 318, 70. (doi:10.1126/science.1145147)
Oberprieler RG. 1995 The weevils (Coleoptera:
Curculionoidea) associated with cycads. 1.
Classification, relationships, and biology. In Proc.
3rd Int. Conf. cycad biology (ed. P Vorster), pp. 295–
334. Stellenbosch, South Africa: Cycad Society of
South Africa.
Oberprieler RG. 1999 Systematics and evolution
of the cycad associated-weevil genus Apinotropis
Jordan (Coleoptera: Anthribidae). Afr. Entomol. 7,
1–33.
Norstog KJ, Stevenson DW, Niklas KJ. 1998
Pollination biology of cycads. In Reproductive
biology (eds SJ Owen, PJ Rudall), pp. 277– 294.
Kew, UK: Royal Botanic Gardens.
Suinyuy TN, Donaldson JS, Johnson SD. 2012
Geographical variation in cone volatile composition
Proc. R. Soc. B 282: 20152053
regulations of South Africa.
Data accessibility. These data are available on Dryad: http://dx.doi.org/
10.5061/dryad.t6g5t.
Authors’ contributions. T.N.S., J.S.D. and S.D.J. conceived the study; T.N.S.
carried out the research (designed the study, collected field data and
wrote the manuscript); T.N.S. and S.D.J. analysed data; T.N.S., J.S.D.
and S.D.J. contributed substantially to the revision.
Competing interests. We declare we have no competing interests.
Funding. This study was financially supported by The Fairchild Tropical
rspb.royalsocietypublishing.org
of these organisms. Further studies on chemical signal perception and cognition in the beetle pollinators together
with efforts to resolve the detailed phylogeny of cycads and
their pollinators will be likely to improve our understanding
of cycad–insect coevolution.
Downloaded from http://rspb.royalsocietypublishing.org/ on June 16, 2017
36.
38.
39.
40.
41.
42.
43.
44.
45.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58. Cunningham JP, Moore CJ, Zalucki MP, West SA.
2004 Learning, odour preference and flower
foraging in moths. J. Exp. Biol. 207, 87 –94.
(doi:10.1242/jeb.00733)
59. Cunningham JP, Moore CJ, Zalucki MP, Cribb BW.
2006 Insect odour perception: recognition of odour
components by flower foraging moths. Proc. R. Soc.
B 273, 2035 –2040. (doi:10.1098/rspb.2006.3559)
60. Proffit M, Khallaf MA, Carrasco D, Larsson MC,
Anderson P. 2015 ‘Do you remember the first
time?’ Host plant preference in a moth is
modulated by experiences during larval feeding and
adult mating. Ecol. Lett. 18, 365–374. (doi:10.
1111/ele.12419)
61. Anderson P, Anton S. 2014 Experience-based
modulation of behavioural responses to sensory
cues in insect herbivores. Plant Cell Environ. 37,
1826– 1835. (doi:10.1111/pce.12342)
62. Anderson P, Sadek MM, Larsson M, Hansson BS,
Thöming G. 2013 Larval host plant experience
modulates both mate finding and oviposition choice
in a moth. Anim. Behav. 85, 1169–1175. (doi:10.
1016/j.anbehav.2013.03.002)
63. Suinyuy TN, Donaldson JS, Johnson SD. 2013
Variation in the chemical composition of cone
volatiles within the African cycad genus
Encephalartos. Phytochemistry 85, 82 –91. (doi:10.
1016/j.phytochem.2012.09.016)
64. Anderson B, Johnson SD. 2009 Geographical
covariation and local convergence of flower depth in
a guild of fly-pollinated plants. New Phytol. 182,
533–540. (doi:10.1111/j.1469-8137.2009.02764.x)
65. Endrödy-Younga S. 1991 Boganiidae coleoptera
cucujoidea associated with cycads in South Africa:
two new species and a new synonym. Ann.
Transvaal Mus. 35, 285 –293.
66. Endrödy-Younga S, Crowson RA. 1986 Boganiidae, a
new beetle family for the African fauna (Coleoptera
Cucujoidea). Ann. Transvaal Mus. 34, 253–273.
67. Donaldson JS, Nänni I, Bösenberg De Wet J.
1995 The role of insects in pollination of
Encephalartos cycadifolius. In Proc. 3rd Int.
Conf. cycad biology (ed. P Vorster), pp. 423 –434.
Stellenbosch, South Africa: Cycad Society of
South Africa.
68. Donaldson JS. 1992 Adaptation for oviposition into
concealed cycad ovules in the cycad weevils
Antliarhinus zamiae and A. signatus (Coleoptera:
Curculioniodea). Biol. J. Linn. Soc. 47, 23 –35.
(doi:10.1111/j.1095-8312.1992.tb00653.x)
69. Donaldson JS. 1991 Adaptation to the host and
evolution of host specialisation in cycad weevils
(Coleoptera: Brentidae). PhD thesis, University of
Cape Town, Cape Town, South Africa.
9
Proc. R. Soc. B 282: 20152053
37.
46.
Biochem. Syst. Ecol. 19, 623 –627. (doi:10.1016/
0305-1978(91)90078-E)
Kaiser R. 2004 Vanishing flora—lost chemistry: the
scents of endangered plants around the world.
Chem. Biodivers. 1, 13 –27. (doi:10.1002/cbdv.
200490005)
Bohman B, Phillips RD, Menz MHM, Berntsson BW,
Flematti GR, Barrow RA, Dixon KW, Peakall R. 2014
Discovery of pyrazines as pollinator sex pheromones
and orchid semiochemicals: implications for the
evolution of sexual deception. New Phytol. 203,
939 –952. (doi:10.1111/nph.1280)
Schiestl FP, Stefan Dötterl S. 2012 The evolution of
floral scent and olfactory preferences in pollinators:
coevolution or pre-existing bias? Evolution 66, 2042–
2055. (doi:10.1111/j.1558-5646.2012.01593.x)
Johnson SD. 1996 Pollination, adaptation and
speciation models in the Cape flora of South Africa.
Taxon 45, 59– 66. (doi:10.2307/1222585)
Johnson SD, Steiner KE. 1997 Long-tongued fly
pollination and evolution of floral spur length
in Disa draconis complex. Evolution 51, 45 –53.
(doi:10.2307/2410959)
Downie DA, Donaldson JS, Oberprieler RG. 2008
Molecular systematics and evolution in an African
cycad –weevil interaction: Amorphocerini
(Coleoptera: Curculionidae: Molytinae) weevils on
Encephalartos. Mol. Phylogenet. Evol. 47, 102– 116.
(doi:10.1016/j.ympev.2008.01.023)
Treutlein J, Vorster P, Wink M. 2005 Molecular
relationships in Encephalartos (Zamiaceae,
Cycadales) based on nucleotide sequences of
nuclear ITS1 and 2, Rbcl, and genomic ISSR
fingerprinting. Plant Biol. 7, 79 –90. (doi:10.1055/
s-2004-830478)
Ellstrand NC, Ornduff R, Clegg JM. 1990 Genetic
structure of the Australian cycad, Macrozamia
communis (Zamiaceae). Am. J. Bot. 77, 677–681.
(doi:10.2307/2444814)
González D, Vovides AP. 2002 Low intralineage
divergence in the genus Ceratozamia (Zamiaceae)
detected with nuclear ribosomal DNA ITS and
chloroplast DNA trnL-F non-coding region. Syst. Bot.
27, 654– 661. (doi:10.1043/0363-6445-27.4.654)
Goode D. 1989 Cycads of Africa. Cape Town, South
Africa: Struik Publishers.
Henning JA, Peng Y-S, Montague MA, Teuber LR.
1992 Honey bee (Hymenoptera: Apidae) behavioral
response to primary alfalfa (Rosales, Fabaceae) floral
volatiles. J. Econ. Entomol. 85, 233–239. (doi:10.
1093/jee/85.1.233)
Wells PH, Wells H. 1985 Ethological isolation of
plants 2. 521 Odour selection by honeybees.
J. Apicult. Res. 24, 86 –92.
rspb.royalsocietypublishing.org
35.
among populations of the African cycad
Encephalartos villosus. Biol. J. Linn. Soc. 106,
514–527. (doi:10.1111/j.1095-8312.2012.01905.x)
Oberprieler RG. 1996 Systematics and evolution of
the tribe Amorphocerini (Coleoptera: Curculionidae),
with a review of the cycad weevils of the world.
PhD thesis, University of the Orange Free State,
Bloemfontein, South Africa.
Suinyuy TN, Donaldson JS, Johnson SD. 2013
Patterns of odour emission, thermogenesis and
pollinator activity in cones of an African cycad: do
push-pull models apply? Ann. Bot. 112, 891–902.
(doi:10.1093/aob/mct159)
Schiestl FP, Marion-Poll F. 2002 Detection of
physiologically active flower volatiles using gas
chromatography coupled with
electroantennography. In Analyses of taste and
aroma (eds JF Jackson, HF Linskens), pp. 173–198.
Berlin, Germany: Springer.
Raine NE, Chittka L. 2007 The adaptive significance
of sensory bias in a foraging context: floral colour
preferences in the bumblebee Bombus terrestris.
PLoS ONE 2, e556. (doi:10.1371/journal.pone.
0000556)
Ings TC, Raine NE, Chittka L. 2009 A population
comparison of the strength and persistence of
innate colour preference and learning speed in the
bumblebee Bombus terrestris. Behav. Ecol. Sociobiol.
63, 1207 –1218. (doi:10.1007/s00265-009-0731-8)
Gumbert A. 2000 Colour choices by bumblebees
(Bombus terrestris): innate preferences and
generalization after learning. Behav. Ecol. Sociobiol.
48, 36 –43. (doi:10.1007/s002650000231)
Thompson JN, Schwind C, Guimarães Jr PR, Friberg
M. 2013 Diversification through multitrait evolution
in a coevolving interaction. Proc. Natl Acad. Sci. USA
110, 11 487–11 492. (doi:10.1073/pnas.
1307451110)
Hossaert-McKey M, Gebernau M, Frey JE. 1994
Chemosensory attraction of fig wasps to substances
produced by receptive fig. Entomol. Exp. Appl. 70,
185–191. (doi:10.1111/j.1570-7458.1994.tb00747.x)
Song Q, Yang D, Zhang G, Yang C. 2001 Volatiles
from Ficus hispida and their attractiveness to fig
wasps. J. Chem. Ecol. 27, 1929 –1942. (doi:10.1023/
A:1012226400586)
Chen C, Song Q, Proffit M, Bessière J-M, Li Z,
Hossaert- McKey M. 2009 Private channel: a single
unusual compound assures specific pollinator
attraction in Ficus semicordata. Funct. Ecol. 23,
941–950. (doi:10.1111/j.1365-2435.2009.01622.x)
Pellmyr O, Tang W, Groth I, Bergström G, Thien LB.
1991 Cycad cone and angiosperm floral volatiles:
inferences for the evolution of insect pollination.