Submitted 19 May 2017
Accepted 2 October 2017
Published 7 November 2017
Corresponding author
Chris D. Jiggins,
c.jiggins@zoo.cam.ac.uk
Academic editor
Chris Cutler
Additional Information and
Declarations can be found on
page 17
DOI 10.7717/peerj.3953
Copyright
2017 Darragh et al.
Distributed under
Creative Commons CC-BY 4.0
OPEN ACCESS
Male sex pheromone components in
Heliconius butterflies released by the
androconia affect female choice
Kathy Darragh1,2,*, Sohini Vanjari1,2,*, Florian Mann3,*,
Maria F. Gonzalez-Rojas4,*, Colin R. Morrison2,5, Camilo Salazar4,
Carolina Pardo-Diaz4, Richard M. Merrill1,2,6, W. Owen McMillan2,
Stefan Schulz3 and Chris D. Jiggins1,2
1Department of Zoology, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom
2 Smithsonian Tropical Research Institute, Panama
3 Institute of Organic Chemistry, Technische Universität Braunschweig, Braunschweig, Germany
4Biology Program, Faculty of Natural Sciences and Mathematics, Universidad del Rosario, Bogota, Colombia
5Department of Integrative Biology, University of Texas at Austin, Austin, TX, United States of America
6Division of Evolutionary Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Munich,
Germany
*These authors contributed equally to this work.
ABSTRACT
Sex-specific pheromones are known to play an important role in butterfly courtship,
and may influence both individual reproductive success and reproductive isolation
between species. Extensive ecological, behavioural and genetic studies of Heliconius
butterflies have made a substantial contribution to our understanding of speciation.
Male pheromones, although long suspected to play an important role, have received
relatively little attention in this genus. Here, we combine morphological, chemical
and behavioural analyses of male pheromones in the Neotropical butterfly Heliconius
melpomene. First, we identify putative androconia that are specialized brush-like scales
that lie within the shiny grey region of the male hindwing. We then describe putative
male sex pheromone compounds, which are largely confined to the androconial region
of the hindwing of mature males, but are absent in immature males and females.
Finally, behavioural choice experiments reveal that females of H. melpomene, H. erato
and H. timareta strongly discriminate against conspecific males which have their
androconial region experimentally blocked. As well as demonstrating the importance
of chemical signalling for female mate choice in Heliconius butterflies, the results
describe structures involved in release of the pheromone and a list of potential male
sex pheromone compounds.
Subjects Animal Behavior, Entomology, Evolutionary Studies
Keywords Heliconius, Pheromone, Sexual selection, Mate choice, Androconia, Lepidoptera,
Reproductive isolation
INTRODUCTION
Sex pheromones are species-specific blends of chemical compounds that mediate
intraspecific communication between males and females (Wyatt, 2003; Wyatt, 2014).
Among insects, pheromone communication can involve a single chemical, but often
How to cite this article Darragh et al. (2017), Male sex pheromone components in Heliconius butterflies released by the androconia af-
fect female choice. PeerJ 5:e3953; DOI 10.7717/peerj.3953
relies on a complex combination of multiple chemical components (Grillet, Dartevelle &
Ferveur, 2006; Nieberding et al., 2008; Symonds, Johnson & Elgar, 2012). This chemical
complexity provides the potential to convey sophisticated information, such as the
inbreeding status of the emitter (Ando, Inomata & Yamamoto, 2004; Van Bergen et al.,
2013; Menzel, Radke & Foitzik, 2016), mate quality (Dussourd et al., 1991; Ruther et al.,
2009), and species identity (Danci et al., 2006; Saveer et al., 2014). Perhaps the best studied
insect sex pheromones are those produced by female moths to attract mating partners,
often over long distances (Löfstedt, 1993; Smadja & Butlin, 2008). However, male insects
also produce sex pheromones (Eggert & Müller, 1997; Kock, Ruther & Sauer, 2007; Ruther
et al., 2009; Meinwald, Meinwald & Mazzocchi, 1969), and chemical signalling can occur
over short distances (Nishida et al., 1996; Mas & Jallon, 2005; Smadja & Butlin, 2008;
Wicker-Thomas, 2011; Grillet et al., 2012).
Sex pheromones can play a key role in determining the reproductive success of
individuals within a species, and may also result in reproductive isolation between species
if signals diverge (Johansson & Jones, 2007; Smadja & Butlin, 2008; Wyatt, 2014). Within
Lepidoptera, the importance of chemical signalling in mate choice and speciation is well
established amongmoth species (Phelan & Baker, 1987; Löfstedt, 1993;Bethenod et al., 2004;
Dopman, Robbins & Seaman, 2010; Lassance et al., 2010; Saveer et al., 2014). Mostmoths fly
at night, when visual signalling is unlikely to be as effective in attracting mates. In contrast,
butterflies are mostly diurnal and visual signals are usually important for initial mate
attraction (Vane-Wright & Boppré, 1993). However, chemical signals can play other roles in
butterfly mate choice, with evidence that close-range courtship interactions often involve
pheromones emitted by males, in contrast to the long-distance signalling with female-
emitted pheromones more commonly observed in moths (Vane-Wright & Boppré, 1993).
Acceptance behaviour in the queen butterfly Danaus berenice, for example, is regulated by
a dihydropyrrolizine alkaloid released by the male during courtship (Brower & Jones, 1965;
Meinwald, Meinwald & Mazzocchi, 1969; Pliske & Eisner, 1969). Another danaine butterfly,
Idea leuconoe, displays brush-like structures, called ‘hair-pencils’, emitting a mixture of
volatiles during courtship, which when applied to dummy males elicits an acceptance
posture in females (Nishida et al., 1996). Pieris rapae and P. brassicae both use macrocyclic
lactones as a pheromone to induce acceptance in females (Yildizhan et al., 2009). Finally,
in Bicyclus anynana males with reduced amounts of male sex pheromone have decreased
mating success, implying a direct involvement in reproductive fitness (Nieberding et al.,
2008; Nieberding et al., 2012).
Here we focus on the potential role of male pheromones in Heliconius butterflies.
Heliconius is a diverse Neotropical genus, which has been extensively studied in the context
of adaptation and speciation (Jiggins, 2008; Supple et al., 2014; Merrill et al., 2015). These
butterflies are well known for Müllerian mimicry, in which unrelated species converge
on the same warning signal to more efficiently advertise their unpalatability to predators.
Closely related Heliconius taxa, however, often differ in colour pattern and divergent
selection acting on warning patterns is believed to play an important role in speciation
(Bates, 1862; Jiggins et al., 2001;Merrill et al., 2011b).
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 2/23
MaleHeliconius display conspicuous courtship behaviours likely because the availability
of receptive females in nature is limited. Female re-mating is a rare event in Heliconius
(Walters et al., 2012) and males must compete to find virgin females within a visually
complex environment (Merrill et al., 2015). In addition, males donate a nutrient-rich
spermatophore during mating (Boggs & Gilbert, 1979; Boggs, 1981) which, together with
costs associated with extended copulation, will select for discrimination against less suitable
mates in both sexes. A combination of colour (hue) and movement stimulates courtship by
Heliconiusmales (Crane, 1955). More recently, it has repeatedly been shown across multiple
Heliconius species that males are more attracted to their own warning pattern than that of
closely related taxa (Jiggins et al., 2001; Jiggins, Estrada & Rodrigues, 2004; Kronforst et al.,
2006;Melo et al., 2009;Muñoz et al., 2010;Merrill et al., 2011a;Merrill et al., 2011b;Merrill,
Chia & Nadeau, 2014; Finkbeiner, Briscoe & Reed, 2014; Sánchez et al., 2015).
In addition to colour pattern, male Heliconius also use chemical signals to locate and
determine the suitability of potential mates. This includes the use of green leaf volatiles
during mate searching. Six-carbon alcohols and acetates are released by host plants in
larger amounts after leaf tissue damage caused by caterpillars, which adult males of the
pupal mating species H. charithonia then use to find potential mates (Estrada & Gilbert,
2010). Once males find pupae they also use chemical cues to determine sex (Estrada et al.,
2010). Supporting a further role for chemical signals, Heliconius erato males distinguish
between wings dissected from conspecific and localH. melpomene females that are virtually
identical in wing pattern, but this effect disappears after wings have been washed in hexane
(Estrada & Jiggins, 2008).
As well as attraction, chemicals can also be involved in repulsion. Males are repelled by a
strong odour released by previously mated females (Gilbert, 1976). This ‘anti-aphrodisiac’
is produced by males soon after eclosion and is then transferred during copulation (Schulz
et al., 2008). The abdominal glands of male H. melpomene, for example, contain a complex
chemical bouquet consisting of the volatile compound (E)- β-ocimene together with
some trace components and esters of common C16—and C18—fatty acids with alcohols,
where β-ocimene acts as the main antiaphrodisiac component (Schulz et al., 2008). This
antiaphrodisiac effect occurs in several Heliconius species, which show species-specific
patterns of scent gland constituents (Gilbert, 1976; Estrada et al., 2011).
Despite the focus on male mate choice, analysis of courtship in Heliconius has shown
that females can exhibit rejection behaviours, such as raising their abdomen and flattening
their wings (Mallet, 1986; Klein & Araújo, 2010). There are also a number of observations
that indicate a role for chemical recognition in female mate choice. Heliconius erato males
separate their wings during courtship to reveal the silvery overlap region, suggested to
be involved in the distribution of pheromones. This behaviour, described as androconial
exposition, occurs in every courtship that results in mating, suggesting that pheromones
influence the female response (Klein & Araújo, 2010). Additionally, direct evidence that
Heliconius females use chemical signals to distinguish conspecific males comes from studies
of the closely related species H. timareta and H. melpomene, which share the same warning
patterns in Peru (Mérot et al., 2015). Males experimentally treated with abdominal scent
gland and wing extracts of heterospecifics show a reduced probability of mating. Chemical
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 3/23
analysis of both abdominal glands and whole wings provides evidence for qualitative and
quantitative differences in the chemical signatures between these closely related species
(Mérot et al., 2015).
Here, we investigate the role of chemical signalling in female mate choice in Heliconius
at three levels. First, we investigate morphological structures potentially associated with
pheromone production. In butterflies, a variety of species-specific structures including
brushes, fans, and differentiated scales on wings, legs or abdomen are used to expose
pheromones produced in associated glands (Wyatt, 2003; Nieberding et al., 2008). In
particular, male-specific scent glands, termed androconia, are common across the
Lepidoptera. In male Heliconius, a patch of shiny grey scales is present on the overlapping
region of the hind and forewing (Fig. 1). The observed sexual dimorphism in this trait
suggests that these are androconia, and may be associated with a male sex pheromone
(Emsley, 1963). Furthermore, earlier authors have identified brush-like scales in the
hindwing androconial region that are the putative site for pheromone production and
emission (Müller, 1912; Barth, 1952). Here we investigate the structure of these scales
using scanning electron microscopy. Second, we complement recently published chemical
analysis of whole H. melpomene wings (Mérot et al., 2015) by dissecting wing regions to
identify those associated with the production of compounds and identify the potential male
sex pheromone compounds isolated from this region. AsH. melpomene rosinawas available
for more extensive behavioural experiments at the insectaries in Panama, we focused
further work on this population, including repeating chemical analyses to ensure that the
sexual dimorphism was also present. Finally, we carry out mate choice experiments in
H. melpomene rosina, H. melpomene malleti, H. timareta florencia and H. erato demophoon
to test the importance of pheromones for female choice in Heliconius.
METHODS
Individuals used for morphological and chemical analyses were from an outbred stock of
Heliconius melpomene plesseni and Heliconius melpomene malleti (sold as H. m. aglaope),
maintained at the University of Cambridge insectaries (Fig. 1A). These two races are from
the region of a hybrid zone in the eastern Andes of Ecuador, and showed considerable
inter-racial hybridization in the stocks, so are treated here as a single population and
referred to as the Ecuador samples. These stocks were established from individuals
obtained from a commercial breeder (Stratford-Upon-Avon Butterfly Farm, Swans
Nest, Stratford-Upon-Avon, UK: http://www.butterflyfarm.co.uk). Laboratory stocks
were maintained on the larval food plants, Passiflora menispermifolia and P. biflora. Adult
butterflies were fed on ∼10% sucrose solution mixed with an amino acid supplement
(Critical Care Formula R©; Vetark Professional, Winchester, UK). Further chemical and
behavioural analysis were carried out on the mimetic but distantly related H. melpomene
rosina and H. erato demophoon reared at the Smithsonian Tropical Research Institute
(STRI) facilities in Gamboa, Panama, and are referred to as the Panama samples. Both
males and females of the Panama samples were from outbred stocks established from wild
individuals collected in Gamboa (9◦7.4′N, 79◦42.2′W, elevation 60 m) within the nearby
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 4/23
Figure 1 Heliconius melpomene wings showing androconial dimorphism. (A) H. melpomene malleti
(Ecuador sample, left) and H. melpomene plesseni (Ecuador sample, right). (B) Dissected wings from spec-
imens of H. melpomene plesseni showing sexual dimorphism in the androconial region, with male (left)
and female (right). For each sex, the left set of wings shows the ventral surface and the right set the dor-
sal surface. (C) Expanded view of the male forewing overlapping region. The pale grey-brown region was
dissected for chemical analysis. (D) Expanded view of the male hindwing androconial region, with ar-
row highlight the vein Sc+ R1. The pale grey-brown region was dissected for chemical analysis. The ven-
tral side of the forewing is on the top and the dorsal side of the hindwing is on the bottom. The pale grey-
brown region in the male wing was dissected for chemical analysis.
Full-size DOI: 10.7717/peerj.3953/fig-1
Soberania National Park, and San Lorenzo National Park (9◦17′N, 79◦58′W; elevation
130 m). Larvae were reared on Passiflora williamsi and P. biflora. Adult butterflies were
provided with ∼20% sugar solution with Psychotria sp., Gurania sp., and Psiguiria sp. as
pollen sources. Finally, behavioural experiments were carried out in the mimetic and
closely related species Heliconius melpomene malleti and H. timareta florencia reared at the
insectaries of Universidad del Rosario (UR) in La Vega, Colombia. These stocks derived
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 5/23
from wild caught individuals from Sucre, Caqueta (01◦48′12′′N, 75◦39′19′′W, elevation
1,200 m). Larvae were reared on Passiflora oerstedii and adults were provided with Psiguiria
sp. as pollen source and ∼20% sugar solution.
Morphological analysis
The detailed morphology of androconial scales was determined using a Field Emission
Scanning Electron Microscope. Three males and two females of H. melpomene from
Ecuador were used for this analysis. The overlap grey scale region was dissected out
from both hind and forewings and attached to aluminium stubs with carbon tabs and
subsequently coated with 20 nm of gold using a Quorum/Emitech sputter coater. The
gold-coated regions were then viewed in an FEI XL30 FEGSEM operated at 5 kV. Images
were recorded digitally using XL30 software at 500× magnification.
Characterization of potential male sex pheromone
Wing tissue from ten males (five newly emerged and five 10-day old) and five females (10-
day old) from the Ecuador stock was collected between November 2011 and March 2012
for chemical analysis. Wings were dissected into four parts: forewing overlap, hindwing
androconia, forewing rest and hindwing rest. The ‘androconia’ and ‘overlap’ regions
corresponded to the grey-brown region (Figs. 1B, 1C and 1D), with rest corresponding
to the remaining portion of the wing which is not overlapping. In females, a region
corresponding in size and extent to the grey-brown region seen in males was dissected.
The dissected sections were then immediately placed in 200 µl hexane or dichloromethane
in 2 mL glass vials and allowed to soak for three hours. Initial analysis showed no major
differences in extracted chemicals between hexane and dichloromethane extracts (data
not shown). Therefore, the more polar dichloromethane was used in later analyses. Due
to a larger available stock of H. melpomene rosina for behavioural experiments, hindwing
androconial tissue was also then collected from 20 males and 11 females (both 10–12
days old) in Panama between February and July 2016. The tissue was soaked in 200 µl
dichloromethane in 2 ml glass vials, with PTFE-coated caps, for one hour. The extraction
time was shortened as this had no influence on the results (Fig. S1). The solvent was then
transferred to new vials and stored at −20 ◦C. Samples were evaporated under ambient
conditions at room temperature prior to analysis.
Mature male androconial extracts from the Ecuador stock were analysed by gas
chromatography/mass spectrometry (GC/MS) using a Hewlett-Packard model 5975 mass-
selective detector connected to a Hewlett-Packard GC model 7890A, and equipped with
a Hewlett-Packard ALS 7683B autosampler. All other Ecuador extracts were analysed by
comparison to the male androconial results. Extracts from the Panama stock were analysed
by GC/MS using a Hewlett-Packard model 5977 mass-selective detector connected to
a Hewlett-Packard GC model 7890B, and equipped with a Hewlett-Packard ALS 7693
autosampler. HP-5MS fused silica capillary columns (30 m × 0.25 mm, 0.25 µm; Agilent,
Santa Clara, CA, USA) were used in both GCs. In both cases, injection was performed in
splitless mode (250 ◦C injector temperature) with helium as the carrier gas (constant flow
of 1.2 ml/min). The temperature programme started at 50 , was held for 5 min, and then
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 6/23
rose at a rate of 5 ◦C/min–320 ◦C, before being held at 320 ◦C for 5 min. Components
were identified by comparison of mass spectra and gas chromatographic Kovats retention
index with those of authentic reference samples and also by analysis of mass spectra.
The double bond positions of unsaturated compounds were determined by derivatisation
with dimethyl disulfide (Buser et al., 1983). To confirm chemical structures, alcohols were
synthesised from the corresponding methyl esters by reduction according to established
procedures (Becker & Beckert, 1993, p. 570). The aldehydes were synthesised by oxidation
of the respective alcohols (More & Finney, 2002).
Compounds found in the extracts were quantified using gas chromatography with flame
ionisation detection with a Hewlett-Packard GC model 7890A or 7890B equipped with
a Hewlett-Packard ALS 7683B (Ecuador) or 7693 (Panama) autosampler. A BPX-5 fused
silica capillary column (SGE, 25 m × 0.22 mm, 0.25 µm) was used in both cases. Injection
was performed in splitless mode (250 ◦C injector temperature) with hydrogen as the carrier
gas (constant flow of 1.65 ml/min). The temperature programme started at 50 ◦C, held for
5 min, and then rose to 320 ◦C with a heating rate of 5 ◦C/min. Pentadecyl acetate (10.1 ng)
or (Z )-4-tridecenyl acetate (1 ng) were used as internal standard for Ecuador samples, and
2-tetradecylacetate (200 ng) for Panama samples. Only compounds eluting earlier than
hexacosane were considered for analysis. Later compounds were identified as cuticular
hydrocarbons, 2,5-dialkyltetrahydrofurans, cholesterol and artefacts (e.g., phthalates or
adipates). The variability in the late eluting cuticular hydrocarbons was low and did not
show characteristic differences between samples.
For the Ecuador samples, groups were visualised as boxplots, due to the high frequency
of absent compounds in the samples. We then used non-parametric Kruskal–Wallis to
test for differences between the amounts of compounds present in different wing regions
of mature males, and also between age and sex categories. This was followed up by Dunn
post-hoc testing (Dinno, 2017; Ogle, 2017), with Bonferroni correction.
For the Panama samples, due to the higher sample size, and larger number of
compounds identified we visualised the males and females as two groups using a non-
metric multidimensional scaling (NMDS ordination, based on a Bray-curtis similarity
matrix. We used the metaMDS function in the package vegan (Oksanen et al., 2017), with
visualisation using the package ade4 (Dray & Dufour, 2007). This was followed up with
ANOSIM to compare differences between groups, and non-parametric Kruskal–Wallis
tests to determine which compounds differed between sexes. All statistical analyses were
performed with R version 3.3.1 (R Core Team, 2016).
Behavioural experiments
To test female acceptance of male pheromones, behavioural experiments were conducted
in insectaries at STRI, Gamboa, Panama between February and July 2016, and also in
insectaries at UR in La Vega, Colombia between November 2015 and June 2016. One
day old virgin females were presented with a control male and a ‘pheromone blocked’
male, both of which were at least ten days old. Males from Panama were treated with
transparent nail varnish (Revlon Liquid Quick Dry containing cyclomethicone, isopropyl
alcohol, ethylhexyl palmitate, mineral oil and fragrance) applied to wings, following
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 7/23
Costanzo & Monteiro (2007). Males from Colombia were treated with transparent nail
varnish (Vogue Fantastic containing butyl acetate, ethyl acetate , nitrocellulose, adipic
acid, neopentyl glycol, trimellitic anhydride copolymer, isopropyl alcohol, acetyl tributyl
citrate, stearalkonium bentonite, styrene, acrylates copolymer, silica benzophenone-
1, calcium sodium borosilicate, synthetic fluorphlogopite, polyethylene terephthalate
and polyurethane-11). Pheromone blocked males had the dorsal side of their hindwing
androconia blocked, whilst control males had the same region on the ventral side of the
wing blocked.
Males were randomlymarked using a black Sharpie marker with an ‘x’ on either their left
or right wing for identification purposes during the experiment. In Panama, experiments
began at 8.30 am and males were left in the cage until 3 pm. During mating, Heliconius
pairs invariably remain connected for at least an hour and so observations were made
every hour to check for matings. If no mating occurred on the first day, this was repeated
the next day with the same butterflies. Behavioural observations were recorded for 17
trials with H. erato demophoon and 31 trials with H. melpomene rosina for the first two
hours of the experiment on day one. Observations were divided into one minute intervals,
during which both female and male behaviours were recorded. In Colombia, experiments
were conducted from 7 am to 1 pm, checking every 30 min for matings. As before, if
no mating occurred on the first day, the experiment was repeated the next day with
the same butterflies. Female behavioural observations were recorded for 17 trials with
H. timareta florencia and 18 trials with H. melpomene malleti for the first two hours of the
experiment on day one. Observations were divided into one minute intervals and were
recorded only when a male was actively courting the female. Four female behaviours were
recorded: ‘Flutter’ refers to a high frequency flutter of the wings with a raised abdominal
position carried out when another butterfly is in close proximity, which has typically been
interpreted as a rejection behaviour (Klein & Araújo, 2010; Jiggins, 2017). ‘Wings open’
refers to when the female is alighted with wings open and abdomen raised but without
wing fluttering; ‘Abdomen up’ refers to when the female is alighted with wings closed and
abdomen concealed within the wings; ‘Fly away’ refers to when the female flies away from
the male. Of note, ‘Flutter’ behaviour was only observed when a male was actively courting
the female. Male courtships, previously defined as hovering directly over the female (Klein
& Araújo, 2010), were recorded. Mating outcome results were analysed with binomial tests.
We used generalized linear mixed models (GLMMs) with a binomial error distribution and
logit link function to test whether females respond differently to control and experimental
males. The response variable was derived from trial minutes in which males courted where
females performed a particular behaviour (‘success’) or did not (‘failure’). Significance
was determined with likelihood ratio tests comparing models with and without male type
included as an explanatory variable. Individual female was included as a random effect
in all models to avoid pseudoreplication. All statistical analyses were performed with R
version 3.3.1 (R Core Team, 2016), along with the packages ggplot2 (Wickham, 2009), car
(Fox & Weisberg, 2011) and binom (Dorai-Raj, 2014).
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 8/23
Figure 2 SEM images of scales from overlap regions ofH. melpomene wings. (A) Male hindwing; (B)
male forewing and; (C) female hindwing at 500×magnification. (D) Magnified view of brush-like struc-
tures of the special scales in the male hindwing androconial region. Scale bars indicate 50 µm (A–C) and
2 µm (D).
Full-size DOI: 10.7717/peerj.3953/fig-2
RESULTS
Morphological analysis
We identified a marked sexual dimorphism in scale structure (Fig. 2). In the central region
of the male hindwing androconia along vein Sc + R1 we identified specialised scales
(Fig. 2A), which were absent in females and in the forewing overlap region of males
(Figs. 2B and 2C). These scales had brush-like structures at their distal end (Fig. 2D), and
were not detected in any other wing region examined. The brush-like scales were found in
alternating rows with scales with a normal structure. Moving away from the Sc + R1 wing
vein, the density and width of these scales decreased, with isolated brush-like scales found
completely surrounded by normal scales. In addition, the base of these brush-like scales
was more swollen and glandular as compared to other scales (Fig. 3).
Characterization of potential male sex pheromone
We initially investigated candidate wing pheromone composition using a stock of butterflies
from Ecuador. By use of GC/MS and synthesis, six compounds were consistently found
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 9/23
Figure 3 SEM images of scales from hindwing overlap region in female andmaleH. melpomene. (A)
Scale from wing-overlap region of female. (B) Scale from androconial region of male with brush-like
structures; the arrow highlights the bulge in the scale base in this region. Scale bars indicate 10 µm.
Full-size DOI: 10.7717/peerj.3953/fig-3
in the male wing extracts from these samples (Fig. 4) that were identified as the aldehydes
(Z )-9-octadecenal, octadecanal, (Z )-11-icosenal, icosanal, and (Z )-13-docosenal and the
alkane henicosane (C21).
Firstly, a comparison of different wing regions of 10-day old males was carried out
(Fig. 5A). Henicosane was found in all regions of the wing and was not considered
in further analysis. The amount of (Z )-9-octadecenal was not significantly different
between area categories. Octadecanal, (Z )-11-icosenal, icosanal and (Z )-13-docosenal
showed significant differences between wing areas. Post-hoc testing found that these four
compounds were significantly more abundant in the hindwing androconia than the rest of
the forewing and hindwing, but not the forewing overlap region (see Table S1 for statistical
details). We suggest that the hindwing overlap region be referred to as the androconial
region, based on morphological and chemical analyses. The potential role of the, forewing
overlap region as an androconia remains to be demonstrated.
Secondly, the hindwing overlap region of old males, old females, and young males were
compared (Fig. 5B).With the exception of henicosane, the other compounds were observed
to be age-specific and sex-specific. (Z )-9-octadecenal was found more in old males than
youngmales or old females but this was not statistically significant. In contrast, octadecanal,
(Z )-11-icosenal, icosanal and (Z )-13-docosenal showed significant differences between
age and sex categories. Post-hoc testing found that these compounds were all present in
significantly greater amounts in old males than young males or old females (See Table S2
for statistical details).
As stocks ofH. melpomene rosina from Panama were available for behavioural assays, we
then investigated the chemical composition of this population, using a larger sample size.
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 10/23
12.00
Panama sample
Ecuador sample
IS1
3
4
5
6
7
8
9
10
4
11
cuticular compounds
[min]20.00 28.00 36.00 44.00 52.00 60.00
IS
12
2I
Panama sample
Ecuador sample
A
B
Figure 4 Regional differences in male androconial extracts. Total ion chromatogram of extract from
the androconial region of (A) a Panamanian H. melpomene rosina hindwing and (B) an Ecuadorian
H. melpomene hindwing. 1, syringaldehyde; 2, (Z )-9-octadecenal; 3, octadecanal; 4, methyloctadecanals; 5,
1-octadecanol; 6, henicosane; 7, methyloctadecan-1-ols and nonadecanal; 8, (Z )-11-icosenal; 9, icosanal;
10, (Z )-11- icosenol; 11, tricosane; 12, (Z )-13-docosenal. All peaks eluting later than 44 min are cuticular
compounds consisting of larger n-alkanes, 2,5-dialkyltetrahydrofurans, cholesterol or are contaminations.
IS, internal standard.
Full-size DOI: 10.7717/peerj.3953/fig-4
These Panama samples showed some similarities to the Ecuadorean samples, although they
contained more compounds and in higher amounts (Fig. 4; Fig. S2 and Table S3). Females
andmales grouped separately with NMDS visualisation, and these groups were significantly
different (Fig. S2). In this larger dataset, (Z )-9-octadecenal, octadecanal, (Z )-11-icosenal,
icosanal, (Z )-13-docosenal and henicosane were all found in significantly larger amounts in
old males than old females, along with many other compounds (Table S3). Small amounts
of nonadecanal, methyl-branched octadecanals and their respective alcohols occurred
that had not been detected in the Ecuador samples, potentially due to the difference
in equipment sensitivity, genuine geographic variation, or the fact that the Ecuadorean
butterflies have spent more generations in captivity. Additionally, syringaldehyde was
present, which was not detected in the Ecuador samples.
Behavioural experiments
In our mate choice trials, females of all four species/races discriminated against conspecific
males in which pheromone transmission was experimentally blocked (Table 1). Across
all four taxa tested, only seven of 71 matings (9.8%) were with the pheromone blocked
male, with the remaining 64 matings (90%) being with the control (unblocked) males.
This was not due to altered male courtship attempts as control and experimental males
courted equally in three out of four species (Fig. S3). In experiments with H. timareta
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 11/23
010
20
30
40
Young Male Old Female Old Male
Age and Sex
A
m
ou
nt
(n
g)
(Z)−11−icosenal
(Z)−13−docosenal
(Z)−9−octadecenal
henicosane
icosanal
octadecanal
0
10
20
30
FW androconia FW rest HW androconia HW rest
Wing Region
(Z)−11−icosenal
(Z)−13−docosenal
icosanal
octadecanal
0
10
20
30
FW overlap FW rest HW androconia HW rest
Wing Region
A
m
ou
nt
(n
g/
ul
) (Z)−11−icosenal
(Z)−13−docosenal
(Z)−9−octadecenal
icosanal
octadecanal
A
B
0
10
20
30
40
Y ung Male Old Female Old Male
Age and Sex
A
m
ou
nt
(n
g)
( ) i l
( ) l
( ) t l
henicosane
icosanal
octadecanal
Wing Region
Figure 5 Compounds detected by GC/MS ofH. melpomene (Ecuador samples) wing extracts. (A)
Presence of compounds in different wing regions of five males (10 days post-eclosion). (B) Presence of
compounds in five females (10 days post-eclosion), five young males (0 days post-eclosion) and five old
males (10 days post-eclosion).
Full-size DOI: 10.7717/peerj.3953/fig-5
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 12/23
Table 1 Outcome of mate choice trials across different species/races. Proportion of successful copu-
lations with the control male was tested using an exact binomial test. Females mated significantly more
with the control male than the experimental (pheromone-blocked) male in all four populations. Statistical
analysis based on females which mated.
Species Mated with
control
Mated with
experimental
Did not
mate
p-value
(exact binomial test)
H. melpomene rosina 15 0 18 <0.001
H. erato demophoon 14 1 31 <0.001
H. melpomene malleti 19 3 8 <0.001
H. timareta florencia 16 3 5 <0.01
florencia, the control males courted more than experimental males (Fig. S3). When data
from H. timareta florencia are excluded, 48 out of 52 matings (92%) occurred with the
control (unblocked) males.
We observed no consistent significant differences in female behavioural responses
towards control and experimental males (Fig. 6). Some female behaviours were observed
more often towards experimental males in experiments with H. melpomene malleti and
H. timareta florencia (Fig. 6). In particular, H. melpomene malleti females were more likely
to open their wings towards experimental males (21lnL = 17.093, d.f. = 1, p< 0.001),
fly away (21lnL = 8.0356, d.f. = 1, p< 0.01) and also flutter (21lnL = 15.823, d.f. =
1, p< 0.001). Similarly, H. timareta florencia females were also more likely to open their
wings towards experimental males (21lnL = 22.909, d.f. = 1, p< 0.001), fly away (21lnL
= 6.1368 , d.f. = 1, p< 0.001), and flutter (21lnL = 26.037, d.f. = 1, p< 0.001). To
ensure that behavioural trials without successful mating were not skewing our analysis of
female behaviours, we additionally analysed differences between males that mated with
the females versus those that did not mate (including those from experiments without
matings). Differences between female behaviour towards mated versus unmated males
did not differ from differences seen in behaviour towards experimental versus control
males (Fig. S4). However, despite these differences, these behavioural responses were not
consistently observed across the four species/races tested. Some of these results are driven
by just a few individual females, with differences in behaviour no longer significant when
they are removed. Furthermore, although some significant female behavioural responses
were seen for H. melpomene malleti in Colombia, no corresponding difference was found
for H. melpomene rosina in Panama, where a larger number of courtships were observed,
so caution should be taken when interpreting these results.
DISCUSSION
Visual cues are known to be important for mate finding and courtship behaviours by male
Heliconius butterflies, with implications for reproductive isolation and speciation (Merrill
et al., 2015). Here, we have shown that female choice based on chemical signalling is also
important for reproduction.Wehave identified compounds associatedwith sexuallymature
male wings and described morphological structures putatively involved in pheromone
release. Furthermore, we have shown that chemical signalling is involved in mating in
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 13/23
0.00
0.25
0.50
0.75
1.00
H. erato
n=17
H. m. malleti
n=17
H. m. rosina
n=31
H. timareta
n=16
Pr
op
or
tio
n 
of
 C
ou
rts
hip
s Flutter
0.00
0.25
0.50
0.75
1.00
H. erato
n=17
H. m. malleti
n=17
H. m. rosina
n=31
H. timareta
n=16
Pr
op
or
tio
n 
of
 C
ou
rts
hip
s Fly away
0.00
0.25
0.50
0.75
1.00
H. erato
n=17
H. m. malleti
n=17
H. m. rosina
n=31
H. timareta
n=16
Pr
op
or
tio
n 
of
 C
ou
rts
hip
s Abdomen up
0.00
0.25
0.50
0.75
1.00
H. erato H. m. malleti H. m. rosina H. timareta
Species
Pr
op
or
tio
n 
of
 C
ou
rts
hip
s Wings open
A
B
C
D
Figure 6 Proportion of courtships which resulted in different female behavioural responses. Control
males are represented in red (left) and experimental males in blue (right). Means are marked with a black
square and boxplots mark the inter-quartile ranges. Size of datapoint is proportional to the number of
courtships by that male. Female behavioural responses (A) Flutter; (B) Fly away; (C) Abdomen up; (D)
Wings open.
Full-size DOI: 10.7717/peerj.3953/fig-6
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 14/23
Heliconius, with females from three different species across four races showing strong
discrimination against males which have had their androconia experimentally blocked.
Our results are broadly comparable with another recent analysis of wing compounds
in Heliconius (Mérot et al., 2015), although the previous study did not compare different
wing regions, or males and females of the same age. As the previous study also did not
use synthesis to identify compounds, our work is highly complementary and extends
their results to confirm region- and age-specific localization of compounds to older male
androconia. MaleHeliconius do not become sexually active until several days after eclosion,
so the absence of these compounds from females and younger males is strongly suggestive
of a role in mating behaviour. Future experiments will be required to determine if these
compounds are sequestered from larval host plants, and if there is genetic control of the
production of these compounds, both of which could facilitate a role in reproductive
isolation.
The restriction of these five putative male sex pheromones, (Z )-9-octadecenal,
octadecanal, (Z )-11-icosenal, icosanal, and (Z )-13-docosenal, to the hindwing androconia
of mature males (Fig. 5A) suggests that pheromone storage or production is restricted to
the hindwing. This is supported by the scanning electron microscope images which show
special brush-like scales in the androconial region (Fig. 2A), located primarily around and
along the hindwing vein Sc + R1, similar to the depiction in Fig. 73 of Emsley’s previous
morphological analysis (Emsley, 1963). Similar scales have been described from light
microscopy in other Heliconius species, but not previously in H. melpomene (Müller, 1912;
Barth, 1952). The base of these special brush-like scales was more swollen and glandular
as compared to other scales (Fig. 3), perhaps indicating a role in storage or production of
pheromones by these scales. Trace amounts of chemicals on the forewing overlap region
may be due to contact in the overlapping portion of the fore- and hindwings, and both
wings may play a role in dispersal of the compounds during courtship.
Samples from Panama showed both a greater diversity and amounts of compounds
(Fig. S2 andTable S3). Thismight reflect an issuewith inbreeding in the Ecuador population
because they were obtained from a commercial breeder, or technical differences between the
two locations where the analysis was performed. However, it could also reflect differences in
rearing conditions or genuine variation between geographic populations of H. melpomene.
Further work will be needed to confirm the nature and extent of geographic and individual
variation in pheromone composition.
Females exhibited a strong preference for males which did not have their androconia
blocked. This suggests that, as in other butterfly systems (Costanzo & Monteiro, 2007),
female Heliconius are actively involved in mating decisions. Nonetheless, there were no
consistent differences in the female behaviours we recorded in our experiments. It is
possible that the important female preference behaviours are subtle and were missed in
our study, or that our sample size may be too small to detect behavioural differences due
to individual variation. Alternatively, female acceptance of a male may instead simply
represent a decision to stop rejection behaviours, and therefore not be associated with any
particular characteristic behavioural response.
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 15/23
It remains unclearwhich compounds are biologically active and exactlywhat information
is being conveyed. The signal clearly influences female mating decisions, and these
compounds may convey complex information about male species identity, quality, age
etc. that are interpreted by females. It is also unknown whether females, like males, use
visual cues in courtship. The use of multiple signals is common in animal communication
(Candolin, 2003). Whilst butterflies primarily use visual cues to locate mates (Kemp &
Rutowski, 2011), it has been shown in B. anynana that in addition to visual cues, chemical
cues also play a role and are equally important in sexual selection by female choice (Costanzo
& Monteiro, 2007). Our work establishes the potential for similar multimodal signalling in
Heliconius butterflies.
This study provides evidence for the importance of pheromones in intraspecific
mate choice in Heliconius butterflies. Evolution of cues within populations could lead
to reproductive isolation between populations if both cues, and their corresponding
preferences, diverge (Ptacek, 2000). In other butterflies, male wing compounds contribute
to reproductive isolation between closely related species (Grula, McChesney & Taylor,
1980; Phelan & Baker, 1987; Bacquet et al., 2015). Evidence suggests that strong pre-mating
barriers in addition to mate preference based on colour wing pattern exist between
Heliconius cydno andH. melpomene (which differ in colour pattern) and between the latter
and H. timareta (which are mimetic) (Mérot et al., 2017; Giraldo et al., 2008). For example,
H. cydno males show a preference for their own pattern over that of the closely related H.
melpomene, but will court wing pattern models of H. melpomene. Heliconius cydno males,
however, have virtually never been observed mating with H. melpomene females (Naisbit,
Jiggins & Mallet, 2001). On the other hand, although males of the mimetic H. timareta
florencia and H. melpomene malleti equally court female wing models of both species,
interspecific matings occur in very low frequency (Sánchez et al., 2015; Mérot et al., 2017).
Furthermore, male androconial compounds differ between species (Mérot et al., 2015;
Mann et al., 2017), suggesting that they could play a role in reproductive isolation. Future
work will allow us to explore the multidimensional aspect of speciation by understanding
both male and female choice, and the role that multiple modes of signalling could play in
reproductive isolation.
ACKNOWLEDGEMENTS
We acknowledge the support of Caroline Nieberding and Christer Löfstedt who provided
early encouragement to pursue this project, and Kelsey Byers who gave useful advice on data
analysis and manuscript preparation. We also thank Andrew Mongue and an anonymous
reviewer who provided helpful comments on the manuscript. We thank our team at the
insectaries in Panama, including Oscar Paneso, Sylvia Fernanda Garza Reyes and Rachel
Crisp. We also thank Oscar Penagos for his technical support breeding butterflies at
insectaries in La Vega, Colombia.
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 16/23
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
KD is funded by a Natural Environment Research Council Doctoral Training Partnership.
SJ was funded by a Manmohan Singh studentship from St John’s College. RMM was
funded by a Junior Research Fellowship at King’s College, Cambridge. CDJ and RMM are
supported by a European Research Council grant number 339873 Speciation Genetics.
MFG, CS and CPD were funded by the Universidad del Rosario FIUR grant QDN-DG001
and COLCIENCIAS (Grant FP44842-5-2017). WOM was supported by the Smithsonian
Tropical Research Institute and NSF grant DEB 1257689. CRM was supported by a STRI
Predoctoral Fellowship. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
Natural Environment Research Council.
Doctoral Training Partnership.
Manmohan Singh studentship.
Junior Research Fellowship.
European Research Council: 339873.
Universidad del Rosario FIUR: QDN-DG001.
COLCIENCIAS: FP44842-5-2017.
Smithsonian Tropical Research Institute and NSF: DEB 1257689.
Competing Interests
The authors declare there are no competing interests.
Author Contributions
• Kathy Darragh and Sohini Vanjari conceived and designed the experiments, performed
the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote
the paper, prepared figures and/or tables, reviewed drafts of the paper.
• Florian Mann conceived and designed the experiments, performed the experiments,
analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or
tables, reviewed drafts of the paper.
• Maria F. Gonzalez-Rojas conceived and designed the experiments, performed the
experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
• Colin R. Morrison performed the experiments, reviewed drafts of the paper.
• Camilo Salazar, Carolina Pardo-Diaz and W. Owen McMillan conceived and designed
the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the
paper.
• Richard M. Merrill conceived and designed the experiments, analyzed the data,
contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the
paper.
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 17/23
• Stefan Schulz conceived and designed the experiments, analyzed the data, contributed
reagents/materials/analysis tools, reviewed drafts of the paper.
• Chris D. Jiggins conceived and designed the experiments, analyzed the data, contributed
reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables,
reviewed drafts of the paper.
Data Availability
The following information was supplied regarding data availability:
Raw data can be found in the Supplemental Information.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.3953#supplemental-information.
REFERENCES
Ando T, Inomata S, YamamotoM. 2004. Lepidopteran sex pheromones. In: Schulz
S, ed. The chemistry of pheromones and other semiochemicals I. Topics in current
chemistry. Berlin Heidelberg: Springer, 51–96.
Bacquet PMB, BrattströmO,Wang H-L, Allen CE, Löfstedt C, Brakefield PM,
Nieberding CM. 2015. Selection on male sex pheromone composition contributes to
butterfly reproductive isolation. Proceedings of the Royal Society B: Biological Sciences
282:Article 20142734 DOI 10.1098/rspb.2014.2734.
Barth R. 1952. Os órgãos odoriferos masculinos de alguns Heliconiinae do Brasil.
Memórias do Instituto Oswaldo Cruz 50:355–367.
Bates HW. 1862. Contributions to an insect fauna of the Amazon valley. Lepi-
doptera: Heliconidae. Transactions of the Linnean Society of London 23:495–566
DOI 10.1111/j.1095-8312.1981.tb018S2.x.
Becker HG, Beckert R. 1993.Organikum-organisch-chemisches praktikum. Weinheim:
Wiley-VCH.
BethenodM-T, Thomas Y, Rousset F, Frérot B, Pélozuelo L, Genestier G, Bourguet D.
2004. Genetic isolation between two sympatric host plant races of the European corn
borer, Ostrinia nubilalisHübner. II: assortative mating and host-plant preferences for
oviposition. Heredity 94:264–270 DOI 10.1038/sj.hdy.6800611.
Boggs CL. 1981. Selection pressures affecting male nutrient investment at mating in
heliconiine butterflies. Evolution 35:931–940 DOI 10.2307/2407864.
Boggs C, Gilbert LE. 1979.Male contribution to egg production in butterflies: evidence
for transfer of nutrients at mating. Science 206:83–84
DOI 10.1126/science.206.4414.83.
Brower LP, Jones MA. 1965. Precourtship interaction of wing and abdominal sex glands
in male Danaus butterflies. Proceedings of the Royal Entomological Society of London.
Series A, General Entomology 40:147–151 DOI 10.1111/j.1365-3032.1965.tb00302.x.
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 18/23
Buser HR, Arn H, Guerin P, Rauscher S. 1983. Determination of double bond position
in mono-unsaturated acetates by mass spectrometry of dimethyl disulfide adducts.
Analytical Chemistry 55:818–822 DOI 10.1021/ac00257a003.
Candolin U. 2003. The use of multiple cues in mate choice. Biological Reviews
78:575–595 DOI 10.1017/S1464793103006158.
Costanzo K, Monteiro A. 2007. The use of chemical and visual cues in female choice in
the butterfly Bicyclus anynana. Proceedings of the Royal Society B: Biological Sciences
274:845–851 DOI 10.1098/rspb.2006.3729.
Crane J. 1955. Imaginal behaviour of a Trinidad butterfly, Heliconius erato hydara
Hewitson, with special reference to the social use of color. Zoologica 40:167–196.
Danci A, Schaefer PW, Schopf A, Gries G. 2006. Species-specific close-range sexual
communication systems prevent cross-attraction in three species of Glyptapanteles
parasitic wasps (Hymenoptera: Braconidae). Biological Control 39:225–231
DOI 10.1016/j.biocontrol.2006.07.009.
Dinno A. 2017. dunn.test: Dunn’s test of multiple comparisons using rank sums. R
package version 1.3.4. Available at https://CRAN.R-project.org/package=dunn.test .
Dopman EB, Robbins PS, Seaman A. 2010. Components of reproductive isolation
between North American pheromone strains of the European corn borer. Evolution
64:881–902 DOI 10.1111/j.1558-5646.2009.00883.x.
Dorai-Raj S. 2014. binom: binomial confidence intervals for several parameterizations. R
package version 1.1-1. Available at https://CRAN.R-project.org/package=binom.
Dray S, Dufour AB. 2007. The ade4 package: implementing the duality diagram for
ecologists. Journal of Statistical Software 22:1–20 DOI 10.18637/jss.v022.i04.
Dussourd DE, Harvis CA, Meinwald J, Eisner T. 1991. Pheromonal advertise-
ment of a nuptial gift by a male moth (Utetheisa ornatrix). Proceedings of the
National Academy of Sciences of the United States of America 88:9224–9227
DOI 10.1073/pnas.88.20.9224.
Eggert A-K, Müller JK. 1997. Biparental care and social evolution in burying beetles:
lessons from the larder. In: Choe JC, Crespi BJ, eds. The evolution of social behavior
in insects and arachnids. Cambridge: Cambridge University Press, 216–236.
Emsley MG. 1963. A morphological study of imagine Heliconiinae (Lep., Nymphalidae)
with a consideration of the evolutionary relationships within the group. Zoologica
48:85–130.
Estrada C, Gilbert LE. 2010.Host plants and immatures as mate-searching cues in Heli-
conius butterflies. Animal Behaviour 80:231–239 DOI 10.1016/j.anbehav.2010.04.023.
Estrada C, Jiggins CD. 2008. Interspecific sexual attraction because of conver-
gence in warning colouration: is there a conflict between natural and sex-
ual selection in mimetic species? Journal of Evolutionary Biology 21:749–760
DOI 10.1111/j.1420-9101.2008.01517.x.
Estrada C, Schulz S, Yildizhan S, Gilbert LE. 2011. Sexual selection drives the evolution
of antiaphrodisiac pheromones in butterflies. Evolution; International Journal of
Organic Evolution 65:2843–2854 DOI 10.1111/j.1558-5646.2011.01352.x.
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 19/23
Estrada C, Yildizhan S, Schulz S, Gilbert LE. 2010. Sex-specific chemical cues from
immatures facilitate the evolution of mate guarding in Heliconius butterflies. Proceed-
ings of the Royal Society. Biological Sciences 277:407–413 DOI 10.1098/rspb.2009.1476.
Finkbeiner SD, Briscoe AD, Reed RD. 2014.Warning signals are seductive: relative
contributions of color and pattern to predator avoidance and mate attraction in
Heliconius butterflies. Evolution 68:3410–3420 DOI 10.1111/evo.12524.
Fox J, Weisberg S. 2011. An {R} companion to applied regression. Thousand Oaks: Sage..
Gilbert LE. 1976. Postmating female odor in Heliconius butterflies: a male-contributed
antiaphrodisiac? Science 193:419–420 DOI 10.1126/science.935877.
Giraldo N, Salazar C, Jiggins CD, Bermingham E, Linares M. 2008. Two sisters
in the same dress: Heliconius cryptic species. BMC Evolutionary Biology 8:324
DOI 10.1186/1471-2148-8-324.
Grillet M, Dartevelle L, Ferveur J-F. 2006. A Drosophilamale pheromone affects female
sexual receptivity. Proceedings of the Royal Society B: Biological Sciences 273:315–323
DOI 10.1098/rspb.2005.3332.
Grillet M, Everaerts C, Houot B, Ritchie MG, CobbM, Ferveur J-F. 2012. Incipient
speciation in Drosophila melanogaster involves chemical signals. Scientific Reports
2:224 DOI 10.1038/srep00224.
Grula JW,McChesney JD, Taylor OR. 1980. Aphrodisiac pheromones of the sulfur
butterflies Colias eurytheme and C. Philodice (Lepidoptera, Pieridae). Journal of
Chemical Ecology 6:241–256 DOI 10.1007/BF00987543.
Jiggins CD. 2008. Ecological speciation in mimetic butterflies. BioScience 58:541–548
DOI 10.1641/B580610.
Jiggins CD. 2017. The ecology and evolution of Heliconius butterflies. Oxford: Oxford
University Press.
Jiggins CD, Estrada C, Rodrigues A. 2004.Mimicry and the evolution of premating
isolation in Heliconius melpomene Linnaeus. Journal of Evolutionary Biology
17:680–691 DOI 10.1111/j.1420-9101.2004.00675.x.
Jiggins CD, Naisbit RE, Coe RL, Mallet J. 2001. Reproductive isolation caused by colour
pattern mimicry. Nature 411:302–305 DOI 10.1038/35077075.
Johansson BG, Jones TM. 2007. The role of chemical communication in mate choice.
Biological Reviews 82:265–289 DOI 10.1111/j.1469-185X.2007.00009.x.
KempDJ, Rutowski RL. 2011. The role of coloration in mate choice and sexual interac-
tions in butterflies. In: Advances in the study of behavior. San Diego: Elsevier Science
Publishing Co Inc., 55–92.
Klein AL, Araújo AM de. 2010. Courtship behavior of Heliconius erato phyllis
(Lepidoptera, Nymphalidae) towards virgin and mated females: conflict
between attraction and repulsion signals? Journal of Ethology 28:409–420
DOI 10.1007/s10164-010-0209-1.
Kock D, Ruther J, Sauer KP. 2007. A male sex pheromone in a scorpionfly. Journal of
Chemical Ecology 33:1249–1256 DOI 10.1007/s10886-007-9304-3.
Kronforst MR, Young LG, Kapan DD,McNeely C, O’Neill RJ, Gilbert LE. 2006. Linkage
of butterfly mate preference and wing color preference cue at the genomic location
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 20/23
of wingless. Proceedings of the National Academy of Sciences of the United States of
America 103:6575–6580 DOI 10.1073/pnas.0509685103.
Lassance J-M, Groot AT, LiénardMA, Antony B, Borgwardt C, Andersson F,
Hedenström E, Heckel DG, Löfstedt C. 2010. Allelic variation in a fatty-acyl
reductase gene causes divergence in moth sex pheromones. Nature 466:486–489
DOI 10.1038/nature09058.
Löfstedt C. 1993.Moth pheromone genetics and evolution. Philosophical Transactions of
the Royal Society B: Biological Sciences 340:167–177.
Mallet J. 1986. Gregarious roosting and home range in Heliconius butterflies. National
Geographic Research 2:198–215.
Mann F, Vanjari S, Rosser N, Mann S, Dasmahapatra KK, Corbin C, Linares
M, Pardo-Diaz C, Salazar C, Jiggins C, Schulz S. 2017. The scent chemistry
of Heliconius wing androconia. Journal of Chemical Ecology 43:843–857
DOI 10.1007/s10886-017-0867-3.
Mas F, Jallon J-M. 2005. Sexual isolation and cuticular hydrocarbon differences be-
tween Drosophila santomea and Drosophila yakuba. Journal of Chemical Ecology
31:2747–2752 DOI 10.1007/s10886-005-7570-5.
Meinwald J, Meinwald YC, Mazzocchi PH. 1969. Sex pheromone of the queen butterfly:
chemistry. Science 164:1174–1175 DOI 10.1126/science.164.3884.1174.
MeloMC, Salazar C, Jiggins CD, Linares M. 2009. Assortative mating preferences
among hybrids offers a route to hybrid speciation. Evolution 63:1660–1665
DOI 10.1111/j.1558-5646.2009.00633.x.
Menzel F, Radke R, Foitzik S. 2016. Odor diversity decreases with inbreeding in the ant
Hypoponera opacior . Evolution 70:2573–2582 DOI 10.1111/evo.13068.
Mérot C, Frérot B, Leppik E, JoronM. 2015. Beyond magic traits: multimodal mating
cues in Heliconius butterflies. Evolution 69:2891–2904 DOI 10.1111/evo.12789.
Mérot C, Salazar C, Merrill RM, Jiggins CD, JoronM. 2017.What shapes the continuum
of reproductive isolation? Lessons from Heliconius butterflies. bioRxiv 107011.
Merrill RM, Chia A, Nadeau NJ. 2014. Divergent warning patterns contribute to
assortative mating between incipient Heliconius species. Ecology and Evolution
4:911–917 DOI 10.1002/ece3.996.
Merrill RM, Dasmahapatra KK, Davey JW, Dell’Aglio DD, Hanly JJ, Huber B, Jiggins
CD, JoronM, Kozak KM, Llaurens V, Martin SH, Montgomery SH, Morris J,
Nadeau NJ, Pinharanda AL, Rosser N, ThompsonMJ, Vanjari S, Wallbank RWR,
Yu Q. 2015. The diversification of Heliconius butterflies: what have we learned in 150
years? Journal of Evolutionary Biology 28:1417–1438 DOI 10.1111/jeb.12672.
Merrill RM, Gompert Z, Dembeck LM, Kronforst MR, McMillanWO, Jiggins CD.
2011a.Mate preference across the speciation continuum in a clade of mimetic
butterflies. Evolution 65:1489–1500 DOI 10.1111/j.1558-5646.2010.01216.x.
Merrill RM, Van Schooten B, Scott JA, Jiggins CD. 2011b. Pervasive genetic associations
between traits causing reproductive isolation in Heliconius butterflies. Proceedings of
the Royal Society B: Biological Sciences 278:511–518 DOI 10.1098/rspb.2010.1493.
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 21/23
More JD, Finney NS. 2002. A simple and advantageous protocol for the oxidation
of Alcohols with o-Iodoxybenzoic Acid (IBX). Organic Letters 4:3001–3003
DOI 10.1021/ol026S27n.
Müller F. 1912. X. The scent-scales of the Male ‘‘Maracujá butterflies’’. In: Longstaff GB,
ed. Butterfly hunting in many lands. New York: Longmans, Green & Co., 655–659.
Muñoz AG, Salazar C, Castaño J, Jiggins CD, Linares M. 2010.Multiple sources of
reproductive isolation in a bimodal butterfly hybrid zone. Journal of Evolutionary
Biology 23:1312–1320 DOI 10.1111/j.1420-9101.2010.02001.x.
Naisbit RE, Jiggins CD, Mallet J. 2001. Disruptive sexual selection against hybrids
contributes to speciation between Heliconius cydno and Heliconius melpomene.
Proceedings of the Royal Society B: Biological Sciences 268:1849–1854.
Nieberding CM, De Vos H, Schneider MV, Lassance J-M, Estramil N, Andersson J,
Bång J, Hedenström E, Löfstedt C, Brakefield PM. 2008. The male sex pheromone
of the butterfly Bicyclus anynana: towards an evolutionary analysis. PLOS ONE
3:e2751 DOI 10.1371/journal.pone.0002751.
Nieberding CM, Fischer K, SaastamoinenM, Allen CE,Wallin EA, Hedenström E,
Brakefield PM. 2012. Cracking the olfactory code of a butterfly: the scent of ageing.
Ecology Letters 15:415–424 DOI 10.1111/j.1461-0248.2012.01748.x.
Nishida R, Schulz S, Kim CS, Fukami H, Kuwahara Y, Honda K, Hayashi N. 1996.
Male sex pheromone of a giant danaine butterfly, Idea leuconoe. Journal of Chemical
Ecology 22:949–972 DOI 10.1007/BF02029947.
Ogle DH. 2017. FSA: Fisheries Stock Analysis. R package version 0.8.13. Available at
https://CRAN.R-project.org/package=FSA.
Oksanen J, Guillaume Blanchet F, Friendly M, Kindt R, Legendre P, McGlinn D,
Minchin P, O’Hara R, Simpson G, Solymos P, Stevens H, Szoecs E, Wagner H.
2017. vegan: community ecology package. R package version 2.4-2. Available at
https://CRAN.R-project.org/package=vegan.
Phelan PL, Baker TC. 1987. Evolution of male pheromones in moths: reproductive
isolation through sexual selection? Science 235:205–207
DOI 10.1126/science.235.4785.205.
Pliske TE, Eisner T. 1969. Sex pheromone of the queen butterfly: biology. Science
164:1170–1172 DOI 10.1126/science.164.3884.1170.
PtacekM. 2000. The role of mating preferences in shaping interspecific diver-
gence in mating signals in vertebrates. Behavioural Processes 51:111–134
DOI 10.1016/S0376-6357(00)00123-6.
R Core Team. 2016. R: A language and environment for statistical computing. Vienna: R
Foundation for Statistical Computing. Available at http://www.r-project.org .
Ruther J, MatschkeM, Garbe L-A, Steiner S. 2009. Quantity matters: male sex
pheromone signals mate quality in the parasitic wasp Nasonia vitripennis. Proceedings
of the Royal Society B: Biological Sciences 276:3303–3310 DOI 10.1098/rspb.2009.0738.
Sánchez AP, Pardo-Diaz C, Enciso-Romero J, Muñoz AG, Jiggins CD, Salazar C,
Linares M. 2015. An introgressed wing pattern acts as a mating cue. Evolution;
International Journal of Organic Evolution 69:1619–1629 DOI 10.1111/evo.12679.
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 22/23
Saveer AM, Becher PG, Birgersson G, Hansson BS,Witzgall P, BengtssonM. 2014.
Mate recognition and reproductive isolation in the sibling species Spodoptera lit-
toralis and Spodoptera litura. Frontiers in Ecology and Evolution 2:18
DOI 10.3389/fevo.2014.00018.
Schulz S, Estrada C, Yildizhan S, Boppré M, Gilbert LE. 2008. An antiaphrodisiac
in Heliconius melpomene butterflies. Journal of Chemical Ecology 34:82–93
DOI 10.1007/s10886-007-9393-z.
Smadja CM, Butlin RK. 2008. On the scent of speciation: the chemosensory system and
its role in premating isolation. Heredity 102:77–97 DOI 10.1038/hdy.2008.55.
Supple M, Papa R, Counterman B, McMillanWO. 2014. The genomics of an adaptive
radiation: insights across the Heliconius speciation continuum. Advances in Experi-
mental Medicine and Biology 781:249–271 DOI 10.1007/978-94-007-7347-9_13.
SymondsMRE, Johnson TL, Elgar MA. 2012. Pheromone production, male abundance,
body size, and the evolution of elaborate antennae in moths. Ecology and Evolution
2:227–246 DOI 10.1002/ece3.81.
Van Bergen E, Brakefield PM, Heuskin S, Zwaan BJ, Nieberding CM. 2013. The scent
of inbreeding: a male sex pheromone betrays inbred males. Proceedings of the Royal
Society B: Biological Sciences 280(1758):20130102 DOI 10.1098/rspb.2013.0102.
Vane-Wright RI, Boppré M. 1993. Visual and chemical signalling in butterflies: func-
tional and phylogenetic perspectives. Philosophical Transactions of the Royal Society B:
Biological Sciences 340:197–205 DOI 10.1098/rstb.1993.0058.
Walters JR, Stafford C, Hardcastle TJ, Jiggins CD. 2012. Evaluating female remating
rates in light of spermatophore degradation in Heliconius butterflies: pupal-mating
monandry versus adult-mating polyandry. Ecological Entomology 37:257–268
DOI 10.1111/j.1365-2311.2012.01360.x.
Wicker-Thomas C. 2011. Evolution of insect pheromones and their role in reproductive
isolation and speciation. Annales de la Société Entomologique de France 47:55–62
DOI 10.1080/00379271.2011.10697696.
WickhamH. 2009. ggplot2: elegant graphics for data analysis. New York: Springer-
Verlag..
Wyatt TD. 2003. Pheromones and animal behaviour: communication by smell and taste.
Cambridge: Cambridge University Press.
Wyatt TD. 2014. Pheromones and animal behavior: chemical signals and signatures.
Cambridge: Cambridge University Press.
Yildizhan S, Van Loon J, Sramkova A, Ayasse M, Arsene C, Ten Broeke C, Schulz S.
2009. Aphrodisiac pheromones from the wings of the small cabbage white and
large cabbage white butterflies, Pieris rapae and Pieris brassicae. ChemBioChem
10:1666–1677 DOI 10.1002/cbic.200900183.
Darragh et al. (2017), PeerJ, DOI 10.7717/peerj.3953 23/23