ORIGINAL RESEARCH
published: 28 July 2016
doi: 10.3389/fevo.2016.00081
Butterfly Learning and the
Diversification of Plant Leaf Shape
Denise D. Dell’Aglio 1, 2*, María E. Losada 2 and Chris D. Jiggins 1, 2
1
Butterfly Genetics Group, Department of Zoology, University of Cambridge, Cambridge, UK, 2 Smithsonian Tropical
Research Institute, Panama City, Panama
Edited by:
Peter Schausberger,
University of Vienna, Austria
Reviewed by:
Emilie Snell-Rood,
University of Minnesota, USA
Martha Weiss,
Georgetown University, USA
*Correspondence:
Denise D. Dell’Aglio
ddd23@cam.ac.uk
Specialty section:
This article was submitted to
Behavioral and Evolutionary Ecology,
a section of the journal
Frontiers in Ecology and Evolution
Received: 27 April 2016
Accepted: 17 June 2016
Published: 28 July 2016
Citation:
Dell’Aglio DD, Losada ME and
Jiggins CD (2016) Butterfly Learning
and the Diversification of Plant Leaf
Shape. Front. Ecol. Evol. 4:81.
doi: 10.3389/fevo.2016.00081
Visual cues are important for insects to find flowers and host plants. It has been proposed
that the diversity of leaf shape in Passiflora vines could be a result of negative frequency
dependent selection driven by visual searching behavior among their butterfly herbivores.
Here we tested the hypothesis that Heliconius butterflies use leaf shape as a cue to initiate
approach toward a host plant. We first tested for the ability to recognize shapes using a
food reward conditioning experiment. Butterflies showed an innate preference for flowers
with three and five petals. However, they could be trained to increase the frequency of
visits to a non-preferred flower with two petals, indicating an ability to learn to associate
shape with a reward. Next we investigated shape learning specifically in the context of
oviposition by conditioning females to lay eggs on two shoots associated with different
artificial leaf shapes: their own host plant, Passiflora biflora, and a lanceolate non-biflora
leaf shape. The conditioning treatment had a significant effect on the approach of
butterflies to the two leaf shapes, consistent with a role for shape learning in oviposition
behavior. This study is the first to show that Heliconius butterflies use shape as a cue
for feeding and oviposition, and can learn shape preference for both flowers and leaves.
This demonstrates the potential for Heliconius to drive negative frequency dependent
selection on the leaf shape of their Passiflora host plants.
Keywords: leaf shape, flower shape, host selection, oviposition, Passiflora, Heliconius
INTRODUCTION
Co-evolution between plants and herbivores is a major cause of both plant and insect diversity
and adaptation (Ehrlich and Raven, 1964). The role of host shifts and key innovations as a
driving force in herbivore diversification has been widely studied. Similarly, in recent years
there has been considerable interest in the role of herbivores in promoting plant diversification,
specifically through the Janzen-Connell effect (Janzen, 1970; Connell, 1971). This hypothesis states
that herbivores could exert negative frequency dependent selection by adapting to exploit the
commonest host plants in their local environment. This could in turn favor rare plant species and
promote local plant species diversity.
The Janzen-Connell hypothesis has generally been discussed in the context of specialist
herbivores preventing the local establishment of common plant species. However, an alternative
mechanism is that more generalist herbivores might learn a “search image” for locally common
plant species. This could similarly generate negative frequency dependence, but on a much shorter
timescale (Sinervo and Calsbeek, 2006). In visually searching predators, this could be driven by
learning of distinctive cues for finding host plants, such as leaf shape. Variation in the size and
shape of leaves is often considered to be mainly a result of the physiological and biomechanical
demands imposed by different habitats (Brown et al., 1991). The role of herbivores in influencing
Frontiers in Ecology and Evolution | www.frontiersin.org
1
July 2016 | Volume 4 | Article 81
Dell’Aglio et al.
Butterfly Pressure on Leaf Shape
the evolution of leaf size and shape has mainly considered in
the context of physical barriers to herbivory (Brown et al.,
1991). The role of leaf shape as an adaptation against visual
herbivores has been less well studied, but one example is leaf
mimicry in the Boquila trifoliolata vine, which mimics the leaves
of its supporting trees to avoid visual herbivores (Gianoli and
Carrasco-Urra, 2014).
In order to test the idea that herbivores might use visual
cues such as leaf shape in finding their host plants, we need
to demonstrate that the relevant herbivores can indeed use
shape cues. Shape perception in insects has primarily been
studied from the perspective of foraging bees (Anderson, 1977;
Zhang et al., 1995), which show a preference for radial patterns
when searching for nectar (Lehrer et al., 1995). Monarch
butterflies, Danaus plexippus, are capable of learning shape
only in association with color, showing that both stimuli must
appear together in the context of foraging for nectar (Cepero
et al., 2015). Furthermore, leaf shape detection and learning
has been demonstrated in oviposition preference in Battus
philenor (Rausher, 1978; Rausher and Papaj, 1983; Papaj, 1986;
Weiss and Papaj, 2003) and in Eurema, which landed more
often on leaves that resemble their host (Mackay and Jones,
1989).
Perhaps the most promising system in which visually
searching herbivores interact with a diverse community of
leaf shapes is among Heliconius butterflies and their Passiflora
host plants (Gilbert, 1982). Leaf morphology in the family
Passifloraceae, both between and within species, is among the
most variable observed in any plant group (Figure 1A). In any
locality, Passiflora species exhibit a wide variety of leaf shapes
even if they are closely related and inhabit similar physical
conditions (Benson et al., 1975; Gilbert, 1975, 1982). Some
species also show a huge range of intra-specific variation in
shapes, especially between young and old leaves (Gilbert, 1982).
For example, Passiflora suberosa shows a high degree of leaf
plasticity when raised in different light intensities (Barp et al.,
2006). In addition, some Passiflora are very similar in form and
texture to other non-host plants, which might be a form of
mimicry. Gilbert (1975) speculated that visual searching behavior
by Heliconius butterflies acts as a diversifying evolutionary force
on Passiflora vines. Heliconius larvae feed almost exclusively
on the family Passifloraceae, and can cause severe foliage
damage (Gilbert, 1982). This close insect-plant interaction has
led to the evolution of various defense mechanisms in Passiflora
plants in response to selective forces imposed by Heliconius
caterpillars.
The Heliconius-Passiflora interaction is already well
established as an example of insect-host co-evolution. Passiflora
species possess a range of defensive traits, such as production
of chemical compounds that provide feeding barriers (Smiley,
1985a; Engler et al., 2000) and mechanical protection such as
hooked trichomes that are able to pierce larvae, resulting in
death for the majority of Heliconius caterpillars on P. adenopoda
(Gilbert, 1971). In turn Heliconius charithonia has evolved
to overcome these trichomes and is the only species that can
feed on this host. In addition, extra-floral nectaries on some
Passiflora species are similar to Heliconius eggs (Gilbert, 1982).
Frontiers in Ecology and Evolution | www.frontiersin.org
FIGURE 1 | Passiflora species that occur in Gamboa or nearby
Soberanía National Park, Panama, highly differ in leaf morphology. (A)
From left to right: top, P. ambigua, P. biflora, P. edulis; bottom, P. coriacea, P.
menispermifolia, P. auriculata. (B) Heliconius erato petiverana female laying
egg on a P. biflora shoot.
In Passiflora cyanea projections on the stipules resemble, in
shape and color, eggs of Heliconius ethilla (Williams and Gilbert,
1981). Females avoid ovipositing in the presence of a conspecific
egg on the host, as young larvae are often cannibalistic
(Nardin and Araújo, 2011), and are therefore deterred by these
egg mimics. Egg-mimicry of extra-floral nectaries provides
strong evidence that ovipositing females use visual cues in
the selection of suitable Passiflora vines. Another function of
extra-floral nectaries against herbivores is the production of
nectar that attracts ants to the plant, to collect this valuable
food resource (Apple and Feener, 2001; Izaguirre et al., 2013).
Ants in turn attack Heliconius larvae and eggs (Smiley, 1985b,
1986).
Based on field observations, females of Heliconius
butterflies use visual cues while searching for host plants
(Brown, 1981). Females may inspect objects that resemble
a Passiflora structure, such as similarly shaped leaves or
vines that look like tendrils (Benson et al., 1975; Gilbert,
1982). A female searching for a specific Passiflora plant
typically flutters slowly just above the vegetation, periodically
approaching, and landing on leaves (Figure 1B). Upon
landing, she drums her forelegs and presumably stimulates
tarsal chemoreceptors, allowing the female to “taste” the
plant with her gustatory receptors (Briscoe et al., 2013). It
has been hypothesized that Passiflora leaf shape variation
might make it harder for Heliconius females to detect host
plants.
However, shape detection has not yet been demonstrated in
Heliconius butterflies. They can be trained to associate a color
stimulus with a food reward, demonstrating a high precision
of discrimination and learning (Swihart and Swihart, 1970;
Swihart, 1971; Blackiston et al., 2011). Here we extend these
experiments to show that Heliconius erato can be trained to
associate a shape cue with a food reward, demonstrating the
perceptual ability to detect and distinguish shapes. Next, we
tested shape perception for leaf morphology using ovipositing
females trained on artificial leaves. H. erato naturally feeds on
three species with diverse leaf shapes in our study area, and
our results show that learnt shape preference is therefore a
plausible selective force on Passiflora leaf morphology in this
community.
2
July 2016 | Volume 4 | Article 81
Dell’Aglio et al.
Butterfly Pressure on Leaf Shape
MATERIALS AND METHODS
Butterfly Rearing
Experiments were performed between March 2014 and August
2015 in insectary facilities located in Gamboa, Panama. Wild
adults of H. erato Linnaeus, 1758 were caught in the surrounding
areas and kept in insectary cages for egg collection. Caterpillars
were reared on Passiflora biflora Lam. leaves. Adults were fed
with sugar solution and pollen from Psiguria sp. flowers and were
around maximum 2 weeks old at the beginning of the training
period.
Flower Shape Experiment
This experiment was designed to test whether Heliconius
butterflies can perceive shapes using a learning experiment with
a food reward. As shapes are defined in terms of the luminance
contrast at their boundaries against the background (Zhang et al.,
1995), five flower shapes were chosen varying the number of
petals (zero, two, three, four, and five, Figure 2), generating
marked differences in the shape edges and perimeter. Artificial
flowers were constructed of red foam sheets (ethylene-vinyl
acetate) with a 1 ml Eppendorf tube for sugar water solution
attached in the center. The color red was chosen to facilitate
association of model flowers with food, because most of the
flowers used by Heliconius have this coloration (Estrada and
Jiggins, 2002). Prior to the experiment adult butterflies were
fed with a sugar solution presented in feeders made of red
card to increase the association of color and food. Butterflies
were subjected to over-night food deprivation to ensure they
would be willing to feed. Groups of five to six butterflies were
separated in a different cage for the experiment, both females and
males.
The first part of this experiment was designed to demonstrate
spontaneous feeding preferences and to determine innate choice
of flower shapes. A set of five shapes was presented, none
of which contained a food reward. The relative position of
flowers was randomized. The first choice of flower, and the
number of feeding attempts in which the butterfly landed
on the artificial flower and probed with its proboscis were
recorded for 30 min. Over the following 8 days, butterflies
were presented with the least preferred shape from the first
trial (the two-petal shape) with sugar solution, while the other
shapes contained only water. The shape choice trial was then
repeated by again presenting the set of five shapes without
a food reward, using the same method described above. We
aimed to determine whether feeding experience could modify
initial feeding preferences through learning. All experiments
were performed in the early morning when butterflies were active
and willing to feed and the experimental flowers provided the
first food source of the day. After the experiments, butterflies
were allowed to feed on Psiguria sp. flowers for pollen. Butterflies
were tested only once and were not re-used for the subsequent
experiment.
FIGURE 2 | Heliconius butterflies learnt to associate flower shape with
a food reward. (A) Number of individuals that selected each shape as first
choice. (B) Number of feeding attempts to the five shapes during the assays.
The five shapes correspond to no-petals, two, three, four, and five petal
artificial flowers. Trials: Innate response, gray bars; Learnt response, black
bars.
choice of plants for oviposition. Two artificial leaf models were
constructed from green foam sheet (ethylene-vinyl acetate), one
P. biflora leaf shape, which is depressed obovate with two lateral
lobes, and one non-biflora lanceolate leaf shape (Figure 3). The
shapes were generated using real leaves (approx. width × height
and area: biflora = 10 × 8 cm and 25 cm2 ; non-biflora =
6 × 12 cm and 21 cm2 ). Four artificial leaves were attached to a
metal frame onto which a young P. biflora shoot without leaves
was also attached (∼70 cm high). The shoot was placed in a
bottle of water, located on the floor and at the center of the
cage. P. biflora shoots were used, which is the most common host
plant for H. erato in Gamboa. It was anticipated that preference
for the leaf shape of this species might be the innate response
for this species (Smiley, 1978). The stimulus combination of
the green leaf with the real plant shoot odor and taste was
shown to be sufficient to stimulate oviposition by Heliconius
butterflies.
Adult females were kept in 2 m3 insectaries cages without
Passiflora plants. All females were mated prior to the
experiments. Females were randomly separated into two
different training cages: the biflora shape training with only
P. biflora artificial leaves, and the non-biflora shape training
with only lanceolate artificial leaves. Females were free to
lay eggs on the young shoots, which were replaced daily.
Eggs were counted and collected every day to confirm that
Leaf Shape Experiment
Following the results of the first experiment, we then wanted
to determine whether shape perception also functions in the
Frontiers in Ecology and Evolution | www.frontiersin.org
3
July 2016 | Volume 4 | Article 81
Dell’Aglio et al.
Butterfly Pressure on Leaf Shape
FIGURE 3 | Female butterflies were more likely to approach the leaf shape on which they had been trained. Probabilities of (A) approach and (B) landing on
the leaf shapes (mean ± SE). (C) Proportion of first leaf approach: Biflora, gray bars; Non-biflora, black bars; and *P < 0.05.
(biflora or non-biflora) and training regime (on biflora and nonbiflora). We used a Pearson’s Chi-squared test (with simulated
p-value) for first leaf approach data for given proportions.
females were actively laying eggs on the shoots with artificial
leaves.
The females were kept in the training cage for a minimum of
8 days, and then moved to a cage without plants for 2 days. Next,
a choice experiment was performed, presenting a single focal
female with a choice between two leaf shapes, biflora and nonbiflora artificial leaves in the same set up as the training period,
and placed 1 m apart. The female was observed for 30 min, and
the first leaf choice, the number of approaches (flying around
the stimulus to within a 15 cm distance), number of landings
and eggs laid on the shoot associated with each leaf shape were
recorded. Each individual butterfly was tested twice using this
choice experiment, totaling 1 h of observation. Results from the
two trials were combined for analysis.
RESULTS
Flower Shape Learning
We recorded a total of 112 feeding attempts during the innate
behavior trial and 126 feeding attempts during the learned
behavior trial of 53 butterflies. The results indicate that there was
a distinctive preference for certain flower shapes. The butterflies
showed a preference for the more flower like patterns, with the
two-petal flower chosen significantly less than three and five
petals as first choice [2 petals: z(260) = −2.957, P = 0.003; posthoc: 2–3 petals, P = 0.023; 2–5 petals, P = 0.022; Figure 2A].
In contrast, the number of feeding attempts during the innate
trial did not differ significantly [2 petals: t (237) = −1.285, P =
0.2; Figure 2B]. Neither area nor perimeter influenced number
of feeding attempts [area: t (239) = 0.195, P = 0.845; perimeter:
t (239) = 1.189, P = 0.236], but perimeter influenced first choice
[area: t (262) = 1.033, P = 0.302; perimeter: t (262) = 3.353, P =
0.0009].
We then trained the butterflies on their less preferred two
petal shape. After the training period, the frequency of visits
to the two petal artificial flower increased from 14 to 36% of
all visits (46/126). The first feeding attempt preference shifted
significantly to the two petal model flower [z(260) = 2.334, P =
0.019; Figure 2A], and number of feeding attempts also differed
significantly [t (217) = 4.218, P < 0.001; Figure 2B]. There was a
significant interaction between petals and trials for first choice
[2 petals∗ learnt: z(520) = 3.75, P = 0.0001], and for number of
feeding attempts [2 petals∗ learnt: z(485) = 3.446, P = 0.0006].
Statistical Analysis
All statistical analysis was carried out in R (R Core Team,
2015) with multcomp package (Hothorn et al., 2008). In the
flower shape analysis, we used a general linear model (GLM)
in each trial for first choice (binomial) and number of feeding
attempts (individuals as random factor), followed by post-hoc
tests for the significance of pairwise comparisons when relevant.
We calculated the effect of flower area and perimeter on the
number of feeding attempts and first choice. We also calculated
the interaction between trial (innate and learnt) and flower shape
(zero, two, three, four and five) for first choice and number of
feeding attempts. There were no differences in behavior between
females and males, so both sexes were considered together in the
analyses. In the leaf shape analysis, we used a binomial GLM
with prior weights, in which the proportion of successes was
the response factor weighted by the total number of approaches,
landing and eggs, to test for an interaction between leaf choice
Frontiers in Ecology and Evolution | www.frontiersin.org
4
July 2016 | Volume 4 | Article 81
Dell’Aglio et al.
Butterfly Pressure on Leaf Shape
the training period, in contrast to the way that the flower
shape experiment could provide a negative stimulus in terms of
the absence of sugar water. The difference in response of the
butterflies to the two leaf shape treatments is therefore the result
of a learnt association between leaf shape cue and availability of
an oviposition stimulus.
The lack of any significant difference between treatments in
egg laying perhaps indicates that shape is not the only clue
used to oviposit on host plants. Other cues such as leaf color,
plant architecture and plant odor and taste are also likely to be
extremely important (Rausher, 1978; Allard and Papaj, 1996).
Specifically, in the confined insectary space female butterflies do
not have trouble in eventually finding the shoots, irrespective
of their associated leaf shape. Thus, once they have found both
shoots the optimal strategy for a female is to distribute her
available eggs evenly between the two shoots. Nonetheless, in the
wild where long distance detection of host plants is likely to be
more challenging than in a small insectary cage, it seems likely
that leaf shape could play an important role in the location of
host plants used by female butterflies.
We can therefore speculate about the potential for this
learning behavior to influence the evolution of leaf shape in
Passiflora. It has been previously suggested that diversification
of leaf morphology might be a response to herbivore pressure
(Gilbert, 1975; Rausher, 1978). Three elements of Passiflora leaf
morphology may have evolved in response to Heliconius visual
perception: mimicry, divergence in leaf shape between species
and different adult and juvenile foliage (Gilbert, 1982). Negative
frequency dependent selection could favor leaf polymorphism,
as an unusual or rare leaf morphology would be more likely to
escape the attentions of ovipositing butterflies using shape cues.
Our results support this and we suggest that this might be an
example of “enemy free space” competition (Jeffries and Lawton,
1984; Brown et al., 1991) between Passiflora plants for survival
against Heliconius caterpillars.
Heliconius females can show strong host plant preferences
that may not be perfectly aligned with larval food preference
and survival (Copp and Davenport, 1978; Smiley, 1978; Kerpel
and Moreira, 2005; Silva et al., 2014). The ability to learn to
associate new leaf shapes with oviposition sites may allow females
to tailor their search image to the local Passiflora community.
Specifically, in the case of H. erato, there are three important host
species in the Gamboa area which have dramatically different
leaf shapes, P. biflora, P. auriculata, and P. coriaceae (Merrill
et al., 2013). Furthermore, there is considerable variation in all
three species both between individuals and between young and
old leaves. It seems plausible that visual searching behavior by
H. erato could play a role both in promoting the coexistence
of these three species, and as a selective pressure favoring
the evolution and maintenance of within species leaf shape
diversity.
The Janzen-Connell hypothesis proposes that interactions
between parasites and their host could be a driving force in
maintaining plant species diversity (Wright, 2002) and even
egg coloration polymorphism (Yang et al., 2010). Here we have
demonstrated the potential for behavioral plasticity in animal
responses to play a role in maintaining plant species diversity.
In addition, the learnt response in terms of number of feeding
attempts was influenced by flower perimeter, but not by area
[area: t (219) = −1.198, P = 0.232; perimeter: t (219) = −3.185, P
= 0.0016]. Similar results are seen for the first choice data [area:
t (262) = −0.482, P = 0.63; perimeter: t (262) = −2.286, P = 0.023].
Leaf Shape Choice
We trained 12 H. erato butterflies on biflora artificial leaves
and 14 on non-biflora artificial leaves. There was a significant
effect of both leaf shape and trial on approach probability. In
addition, there was also a significant interaction between training
regime and approach probability, demonstrating evidence for
learning. Butterflies experienced with non-biflora leaf models
were subsequently more likely to approach the non-biflora
leaf shape than butterflies experienced with biflora leaf models
[training∗ approach: z(48) = 2.592, P = 0.0095; Figure 3A]. We
also found significant differences for first leaf approach (χ 2 =
4.147, p = 0.041; Figure 3C). However, the preference for landing
did not differ between the two training groups [training∗ landing:
z(48) = −0.116, P = 0.908; Figure 3B].
Females trained on non-biflora leaves laid 46% on non-biflora,
while females trained on biflora leaves laid 47% of the eggs
on non-biflora. There was therefore no significant difference
in eggs laid on the shoots between the two training regimes
[z(49) = −0.132, P = 0.895].
DISCUSSION
Here we have shown for the first time that Heliconius butterflies
can use shape cues to search for both flowers and leaves during
feeding and oviposition. One explanation for the observed
spontaneous preference for three and five petal flower shapes is
an innate preference for radial symmetry, which corresponds to
the actinomorphic flowers used most commonly by Heliconius
(Corrêa et al., 2001). After training, we conditioned individuals
to shift their shape feeding preference to an artificial flower with
two petals, the least preferred shape initially. Previous studies
have shown that conditioned Heliconius butterflies can shift
their preference to yellow and green flowers, against their innate
preference for orange and red (Swihart and Swihart, 1970). Here
we show a similar effect for shape cues. Color is perhaps a more
reliable visual cue for finding flowers since it is not affected by the
angle of approach, as observed in Monarch butterflies (Cepero
et al., 2015). However, shape is a complementary cue and may be
important to distinguish objects that are similar in color.
The pipevine swallowtail butterfly, B. philenor, has long been
known to use leaf shape in oviposition, in experiments in which
the butterflies were trained on both real plants (Papaj, 1986)
and artificial leaf models (Rausher, 1978; Allard and Papaj,
1996; Weiss and Papaj, 2003). Here we have provided the
first evidence that Heliconius also use shape for leaf detection.
Our results therefore support field observations of Heliconius
female butterflies visually discriminating different leaves while
searching for host plants. The butterflies exposed to lanceolate
leaves approach the lanceolate shape more than those exposed
to P. biflora shape. It is worth noting that our artificial plants
did not provide a negative stimulus against laying eggs during
Frontiers in Ecology and Evolution | www.frontiersin.org
5
July 2016 | Volume 4 | Article 81
Dell’Aglio et al.
Butterfly Pressure on Leaf Shape
If generalist herbivores commonly learn a “search image” for
locally common plant species this could be an important source
of negative frequency dependent selection favoring rare plant
species. In the highly diverse and complex tropical rainforest
environment, such an effect might play a role in maintaining
species diversity and in particular in sustaining populations of
rare species.
FUNDING
AUTHOR CONTRIBUTIONS
We thank Owen McMillan and the Smithsonian Tropical
Research Institute for support while working in Panama;
Elizabeth Evans and Oscar Paneso for assistance in the
insectaries; and two reviewers for valuable comments on this
manuscript.
Cambridge Trust (UK) and CAPES (Brazil) to DD, and
Smithsonian Tropical Research Institute (Panama) to ML
supported this study.
ACKNOWLEDGMENTS
DD and CJ conceived, planned and designed the study. DD and
ML conducted the experiments. DD analyzed the data. DD and
CJ wrote the manuscript.
REFERENCES
Gianoli, E., and Carrasco-Urra, F. (2014). Leaf mimicry in a climbing plant protects
against herbivory. Curr. Biol. 24, 984–987. doi: 10.1016/j.cub.2014.03.010
Gilbert, L. E. (1971). Butterfly-plant coevolution: has Passiflora adenopoda won
the selectional race with heliconiine butterflies? Science 172, 585–586. doi:
10.1126/science.172.3983.585
Gilbert, L. E. (1975). “Ecological consequences of a coevolved mutualism between
butterflies and plants,” in Coevolution of Animals and Plants, eds L. E. Gilbert
and P. H. Raven (Austin, TX: University of Texas Press), 210–240.
Gilbert, L. E. (1982). The coevolution of a butterfly and a vine. Sci. Am. 247,
110–121. doi: 10.1038/scientificamerican0882-110
Hothorn, T., Bretz, F., and Westfall, P. (2008). Simultaneous inference in general
parametric models. Biom. J. 50, 346–363. doi: 10.1002/bimj.200810425
Izaguirre, M. M., Mazza, C. A., Astigueta, M. S., Ciarla, A. M., and Ballaré, C. L.
(2013). No time for candy: passionfruit (Passiflora edulis) plants down-regulate
damage-induced extra floral nectar production in response to light signals of
competition. Oecologia 173, 213–221. doi: 10.1007/s00442-013-2721-9
Janzen, D. H. (1970). Herbivores and the number of tree species in tropical forests.
Am. Nat. 104, 501–528. doi: 10.1086/282687
Jeffries, M. J., and Lawton, J. H. (1984). Enemy free space and the structure of
ecological communities. Biol. J. Linn. Soc. 23, 269–286. doi: 10.1111/j.10958312.1984.tb00145.x
Kerpel, S. M., and Moreira, G. R. P. (2005). Absence of learning and local
specialization on host plant selection by Heliconius erato. J. Insect Behav. 18,
433–452. doi: 10.1007/s10905-005-3701-7
Lehrer, M., Horridge, G. A., Zhang, S. W., and Gadagkar, R. (1995). Shape vision
in bees: innate preference for flower-like patterns. Philos. Trans. Biol. Sci. 347,
123–137. doi: 10.1098/rstb.1995.0017
Mackay, D. A., and Jones, R. E. (1989). Leaf shape and the host-finding behaviour
of two ovipositing monophagous butterfly species. Ecol. Entomol. 14, 423–431.
doi: 10.1111/j.1365-2311.1989.tb00944.x
Merrill, R. M., Naisbit, R. E., Mallet, J., and Jiggins, C. D. (2013). Ecological
and genetic factors influencing the transition between host-use strategies
in sympatric Heliconius butterflies. J. Evol. Biol. 26, 1959–1967. doi:
10.1111/jeb.12194
Nardin, J., and Araújo, A. M. (2011). Kin recognition in immatures of
Heliconius erato phyllis (Lepidoptera; Nymphalidae). J. Ethol. 29, 499–503. doi:
10.1007/s10164-011-0272-2
Papaj, D. R. (1986). Conditioning of leaf-shape discrimination by chemical cues in
the butterfly, Battus philenor. Anim. Behav. 34, 1281–1288. doi: 10.1016/S00033472(86)80199-3
Rausher, M. D. (1978). Search image for leaf shape in a butterfly. Science 200,
1071–1073. doi: 10.1126/science.200.4345.1071
Rausher, M. D., and Papaj, D. R. (1983). Host plant selection by Battus philenor
butterflies: evidence for individual differences in foraging behaviour. Anim.
Behav. 31, 341–347. doi: 10.1016/S.0003-3472(83)80052-9
R Core Team (2015). R: A Language and Environment for Statistical Computing.
Vienna, Austria: R Foundation for Statistical Computing. Available online at:
http://www.R-project.org/
Silva, A. K., Gonçalves, G. L., and Moreira, G. R. P. (2014). Larval feeding choices
in heliconians: induced preferences are not constrained by performance and
Allard, R. A., and Papaj, D. R. (1996). Learning of leaf shape by pipevine swallowtail
butterflies: a test using artificial leaf models. J. Insect Behav. 9, 961–967. doi:
10.1007/BF02208982
Anderson, A. M. (1977). Shape perception in the honey bee. Anim. Behav. 25,
67–79. doi: 10.1016/0003-3472(77)90068-9
Apple, J. L., and Feener, Jr., D. H. (2001). Ant visitation of extrafloral
nectaries of Passiflora: the effects of nectary attributes and ant behavior on
patterns in facultative ant-plant mutualisms. Oecologia 127, 409–416. doi:
10.1007/s004420000605
Barp, E. A., Soares, G. L. G., Gosmann, G., Machado, A. M., Vecchi, C., and
Moreira, G. R. P. (2006). Phenotypic plasticity in Passiflora suberosa L.
(Passifloraceae): induction and reversion of two morphs by variation in light
intensity. Braz. J. Biol. 66, 853–862. doi: 10.1590/S1519-69842006000500011
Benson, W. W., Brown, K. S. Jr., and Gilbert, L. E. (1975). Coevolution of
plants and herbivores: passion flower butterflies. Evolution 29, 659–680. doi:
10.2307/2407076
Blackiston, D., Briscoe, A. D., and Weiss, M. R. (2011). Color vision and learning
in the monarch butterfly, Danaus plexippus (Nymphalidae). J. Exp. Biol. 214,
509–520. doi: 10.1242/jeb.048728
Briscoe, A. D., Macias-Muñoz, A., Kozak, K. M., Walters, J. R., Yuan,
F., Jamie, G. A., et al. (2013). Female behaviour drives expression and
evolution of gustatory receptors in butterflies. PLoS Genet. 9:e1003620. doi:
10.1371/journal.pgen.1003620
Brown, K. S. (1981). The biology of Heliconius and related genera. Annu. Rev.
Entomol. 26, 427–456. doi: 10.1146/annurev.en.26.010181.002235
Brown, V. K., Lawton, J. H., and Grubb, P. J. (1991). Herbivory and the
evolution of leaf size and shape. Philos. Trans. Biol. Sci. 333, 265–272. doi:
10.1098/rstb.1991.0076
Cepero, L. C., Rosenwald, L. C., and Weiss, M. R. (2015). The relative importance
of flower color and shape for the foraging monarch butterfly (Lepidoptera:
Nymphalidae). J. Insect Behav. 28, 499–511. doi: 10.1007/s10905-015-9519-z
Connell, J. H. (1971). “On the role of natural enemies in preventing competitive
exclusion in some marine animals and in rain forest trees,” in Dynamics
of Populations, eds P. J. D. Boer and G. Gradwell (Wageningen: PUDOC),
298–312.
Copp, N. H., and Davenport, D. (1978). Agraulis and Passiflora I. control of
specificity. Biol. Bull. 155, 98–112. doi: 10.2307/1540868
Corrêa, C. A., Irgang, B. E., and Moreira, G. R. P. (2001). Estrutura floral das
angiospermas usadas por Heliconius erato phyllis (Lepidoptera, Nymphalidae)
no Rio Grande do Sul, Brasil. Iheringia 90, 71–84. doi: 10.1590/S007347212001000100008
Ehrlich, P. R., and Raven, P. H. (1964). Butterflies and plants: a study in
coevolution. Evolution 18, 586–608. doi: 10.2307/2406212
Engler, H. S., Spencer, K. C., and Gilbert, L. E. (2000). Preventing cyanide release
from leaves. Nature 406, 144–145. doi: 10.1038/35018159
Estrada, C., and Jiggins, C. D. (2002). Patterns of pollen feeding and habitat
preference among Heliconius species. Ecol. Entomol. 27, 448–456. doi:
10.1046/j.1365-2311.2002.00434.x
Frontiers in Ecology and Evolution | www.frontiersin.org
6
July 2016 | Volume 4 | Article 81
Dell’Aglio et al.
Butterfly Pressure on Leaf Shape
Williams, K. S., and Gilbert, L. E. (1981). Insects as selective agents on plant
vegetative morphology: egg mimicry reduces egg laying by butterflies. Science
212, 467–469. doi: 10.1126/science.212.4493.467
Wright, S. J. (2002). Plant diversity in tropical forests: a review of mechanisms
of species coexistence. Oecologia 130, 1–14. doi: 10.1007/s0044201
00809
Yang, C., Liang, W., Cai, Y., Shi, S., Takasu, F., Møller, A. P., et al. (2010).
Coevolution in action: disruptive selection on egg colour in an avian brood
parasite and its host. PLoS ONE 5:e10816. doi: 10.1371/journal.pone.00
10816
Zhang, S. W., Srinivasan, M. V., and Collett, T. (1995). Convergent processing
in honeybee vision: multiple channels for the recognition of shape. Proc. Natl.
Acad. Sci. U.S.A. 92, 3029–3031. doi: 10.1073/pnas.92.7.3029
host plant phylogeny. Anim. Behav. 89, 155–162. doi: 10.1016/j.anbehav.2013.
12.027
Sinervo, B., and Calsbeek, R. (2006). The developmental, physiological,
neural, and genetical causes and consequences of frequency-dependent
selection in the wild. Annu. Rev. Ecol. Evol. Syst. 37, 581–610. doi:
10.1146/annurev.ecolsys.37.091305.110128
Smiley, J. (1978). Plant chemistry and the evolution of host specificity:
new evidence from Heliconius and Passiflora. Science 201, 745–747. doi:
10.1126/science.201.4357.745
Smiley, J. T. (1985a). Are chemical barriers necessary for evolution of butterflyplant associations? Oecologia 65, 580–583. doi: 10.1007/BF00379676
Smiley, J. T. (1985b). Heliconius caterpillar mortality during establishment
on plants with and without attending ants. Ecology 66, 845–849. doi:
10.2307/1940546
Smiley, J. T. (1986). ant constancy at Passiflora extrafloral nectaries: effects on
caterpillar survival. Ecology 67, 516–521. doi: 10.2307/1938594
Swihart, C. A. (1971). Colour discrimation by the butterfly, Heliconius charitonius
Linn. Anim. Behav. 19, 156–164. doi: 10.1016/S0003-3472(71)80151-3
Swihart, C. A., and Swihart, S. L. (1970). Colour selection and learned feeding
preferences in the butterfly, Heliconius charitonius Linn. Anim. Behav. 18,
60–64. doi: 10.1016/0003-3472(70)90071-0
Weiss, M. R., and Papaj, D. R. (2003). Colour learning in two behavioural contexts:
how much can a butterfly keep in mind? Anim. Behav. 65, 425–434. doi:
10.1006/anbe.2003.2084
Frontiers in Ecology and Evolution | www.frontiersin.org
Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2016 Dell’Aglio, Losada and Jiggins. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other forums is permitted, provided the
original author(s) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use, distribution
or reproduction is permitted which does not comply with these terms.
7
July 2016 | Volume 4 | Article 81
Purchase answer to see full
attachment