© 2009 The Authors Entomologia Experimentalis et Applicata 131: 109–114, 2009
Journal compilation © 2009 The Netherlands Entomological Society 109
DOI: 10.1111/j.1570-7458.2009.00837.x
Blackwell Publishing Ltd Prenuptial agreements: mating frequency predicts
gift-giving in Heliconius species
Márcio Zikán Cardoso1*, James J. Roper2 & Lawrence E. Gilbert3
1Departamento de Botânica, Ecologia e Zoologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte,
Natal-59072-970, Brazil, 2Programa de Pós-Graduação em Ecologia e Conservação, Universidade Federal do Paraná,
Curitiba, Brazil, and 3Section of Integrative Biology, University of Texas, Austin, TX, USA
Accepted: 6 January 2009
Key words: spermatophore, sexual selection, cyanogenic glycoside, reproduction, Lepidoptera,
Nymphalidae, Heliconini
Abstract Theory predicts that when males provision females with nuptial gifts that include nutrients, the
degree of polyandry should be positively correlated with the size or quality of the gift. This is because
larger and more nutritious gifts tend to increase female refractory period, reducing the chances the
female will remate soon. This decreases the likelihood of sperm competiton and consequently
increases the donor male fitness. Butterflies in the genus Heliconius Kluk (Lepidoptera: Nymphalidae:
Heliconini) exhibit variable mating systems that include monandry and polyandry. In addition to
protein in the spermatophore, males increase gift quality by providing females with cyanide, which
may contribute to protection of the female or her eggs. We tested whether degree of polyandry and
gift quality (spermatophore weight and cyanide content) were correlated in nine Heliconius species
from greenhouse populations. As predicted, both spermatophore weight and cyanide content were
correlated with mating frequency. This is the first report to show that degree of polyandry correlates
with allocation of defensive chemical as part of a nuptial gift.
Introduction
Butterfly mating systems are highly variable and range
from strict monandry to polyandry (Drummond, 1984;
Svärd & Wiklund, 1989; Wedell et al., 2002). For species
in which males provide females with nuptial gifts (e.g.,
proteinaceous spermatophores; Bissoondath & Wiklund,
1995), female fecundity has been shown to increase with
multiple matings (Oberhauser, 1989; Wiklund et al., 1993;
Karlsson, 1998; Arnqvist & Nilsson, 2000). Ideally, a male will
maximize his reproductive success by being the last to mate
with a polyandrous female, as last copulations gain sperm
precedence in egg fertilization (Drummond, 1984; Bissoo-
ndath & Wiklund, 1997; Wedell & Cook, 1998) although
male size may also influence sperm precedence (LaMunyon
& Eisner, 1993, 1994; Bissoondath & Wiklund, 1997).
Repeated matings may lead to sperm competition
(Arnqvist & Nilsson, 2000; Torres-Vila & Jennions, 2005)
and males of polyandrous species tend to provide females
with larger or higher quality ejaculates (Svärd & Wiklund,
1989; Gage, 1994; Bissoondath & Wiklund, 1995, 1997;
Karlsson, 1996; Wiklund, 2003), probably in response to
the correlation between ejaculate size and refractory
period (Sugawara, 1979; Silberglied et al., 1984; Oberhauser,
1989; Cook & Wedell, 1999). Females that receive larger
gifts will tend to remate later than females that receive
smaller gifts. Thus, larger gifts would reduce the chances
of females accepting a second mating soon and arrest
competition with a second male ejaculate. Also, better
quality gifts reflect in better female nutrition and/or higher
egg production, increasing male fitness (Wiklund, 2003).
Quality in a nuptial gift may be more than just nutrition
and may include defensive compounds that protect both
the female and her eggs (Dussourd et al., 1988, 1989).
The influence of mixed currency (such as defense and
nutrition) on fitness is poorly understood. For instance,
chemical defenses in Utetheisa ornatrix (L.) are derived
from their larval host plant (reviewed in Eisner &
Meinwald, 1995). Females may lose defensive chemicals
acquired as larvae through egg-laying (Dussourd et al.,
1988) and males may then replenish the supply by including
*Correspondence: Márcio Zikán Cardoso, Departamento de
Botânica, Ecologia e Zoologia, Centro de Biociências, Universidade
Federal do Rio Grande do Norte, Natal-59072-970, Brazil.
E-mail: mzc@cb.ufrn.br

110 Cardoso et al.
defense chemicals in the nuptial gift during copulation
(González et al., 1999; Rossini et al., 2001). Yet, mating success
in males is strongly correlated with both spermatophore
size and body size (LaMunyon & Eisner, 1993, 1994;
LaMunyon, 1997). For females, multiple matings increase
fecundity due to the accumulating contribution of the
nuptial gifts (LaMunyon, 1997).
Here, we examine whether different mating systems are
associated with spermatophore size and defensive chemicals
in the nuptial package of the unpalatable butterfly genus
Heliconius Kluk (Lepidoptera: Nymphalidae: Heliconini).
These aposematic butterflies use cyanide-releasing
cyanogenic glycosides for defense (Nahrstedt & Davis,
1983, 1985). Specifically, we ask the following questions:
(i) Are the degree of polyandry and spermatophore size
positively correlated? (ii) Are the degree of polyandry and
cyanide content of the spermatophore positively correlated?
Materials and methods
Study organisms
Heliconius is an ideal system to investigate whether
polyandry affects spermatophore traits (Bissoondath &
Wiklund, 1995) because of the variation present within
their mating systems. Nearly half of the species belong to
the pupal mating clade (where the adult mates with the
female while she is in or has just left the pupa), wherein
females are presumably monandrous (Deinert et al., 1994;
Deinert, 2003). The remaining species do not mate as
pupae. Rather, similar to other butterfly species, males
court females and females are presumably polyandrous.
Because spermatophores have been counted in only some
species, the degree of polyandry in Heliconius remains
uncertain. Heliconius butterflies produce defensive
cyanogenic compounds from amino acid precursors
obtained while feeding as larvae and adults (Nahrstedt &
Davis, 1985) or directly from cyanogens in the host plants
(Engler-Chaouat & Gilbert, 2007). Both proteins (Boggs &
Gilbert, 1979) and cyanides (Cardoso & Gilbert, 2007) are
included in the nuptial gifts that males give to females.
We used butterflies from six non-pupal (NP) and three
pupal (PU) mating species in greenhouse populations
[(NP: Heliconius cydno Doubleday, Heliconius ethilla Godart,
Heliconius hecale (Fabricius), Heliconius ismenius Latreille,
Heliconius melpomene (L.), and Heliconius numata (Cramer);
PU: Heliconius erato (L.), Heliconius hewitsoni Hewitson,
and Heliconius charithonia (L.)]. Stocks descended from
wild-collected individuals from Costa Rica, except
H. ethilla (Brazil) and H. numata (Ecuador). Populations
were housed in glass greenhouses (4 × 6.5 × 3 m) or in
screened insectaries (3.7 × 7.4 × 2 m) within a large green-
house (11 × 20 m), both provided with host plants. Stocks
arising from one to few females have been maintained up
to 15 years, with occasional bottlenecks during winter.
Mating studies
During June–August of 1998 and 1999, mating pairs were
sought in the greenhouse populations. Pairs found were
carefully induced to enter a cylindrical cloth cage (30 in
diameter × 45 cm high), where they remained undisturbed
while mating. Cages were periodically inspected and
females were collected and frozen as soon as mating had
ended. This procedure ensured that butterflies engaged in
normal mating behavior and that it remained as natural
as possible. Because specimens came from free-flying
populations, we were unable to control for mating history
in most cases, except in H. charithonia, where virgin males
and females were mated in small cages inside a glass
greenhouse in Stanford University, CA, USA. Mated
females were frozen and shipped to Austin, TX, USA.
Dissections
Females were dissected in Ringer’s solution under a
dissecting microscope. Fresh spermatophores were
easily recognized by their hard consistency, pearly color,
and short cola. Some females had more than one
spermatophore, and older spermatophores were shrunken
and stacked at one end of the bursa. Following dissection,
spermatophores were kept in Eppendorf tubes with
methanol for cyanide extraction (see below). Subsequently,
spermatophores were oven dried at 68 °C for 72 h and
weighed to the nearest 0.01 mg.
Cyanide content
Cyanide content data of the spermatophores were
obtained from Cardoso & Gilbert (2007). In brief, cyanide
content was estimated from the methanolic extract of the
dissected spermatophores. The extract was air-dried and
cyanide released by adding 10 μl of flax β-glycosidase
(donated by H. Engler-Chaouat, University of Texas,
Austin) and trapped by NaOH, becoming NaCN.
Colorimetric analysis on NaCN permitted the conversion
to cyanide weight (μg) based on a standard curve of known
concentrations of NaCN. Colorimetric analysis is very
sensitive and capable of detecting cyanide concentrations
as low as 5 p.p.b. (Lambert et al., 1975), considerably lower
than those obtained in our samples. Further details can be
found in Cardoso & Gilbert (2007).
Statistical analysis
Mating frequency was determined by the average count of
spermatophore per female per species. Mating frequency
was tested for correlation with spermatophore mass and
cyanide content. To control for phylogeny, we used

Heliconius mating system 111
comparative analysis based on independent contrasts
(Harvey & Pagel, 1991), using the option ‘independent
contrasts’ in Compare 4.6b (Martins, 2004). We tested for
correlation (Pearson) between either cyanide content
or spermatophore mass with mating frequency index.
Contrasts were generated using a phylogeny for Heliconius
proposed by Beltrán et al. (2007) (Figure 1).
Results
Heliconius spermatophore counts, mass, and cyanide
Seventy-three Heliconius females were dissected, 16 from
the pupal mating group and 57 from non-pupal mating
species. All females of the pupal mating species had a single
spermatophore, along with H. ethilla, a non-pupal mating
species. The remaining five non-pupal mating species
showed at least one instance of multiple mating (Table 1).
The maximum number of spermatophores was five, in
H. hecale and H. numata. Average mating frequency for
our greenhouse populations varied from 1 to 2.2 (Table 1).
Based on captive individuals, our mating frequency estimates
are in agreement with previous published spermatophore
counts with free-flying (Ehrlich & Ehrlich, 1978) and
captive butterflies (Boggs, 1990).
The largest spermatophores were found in H. hecale
(mean ± SD: 1.23 ± 1.06 mg; n = 4), whereas the smallest
were found in H. charithonia (0.21 ± 0.11 mg; n = 9; Table 1).
Average cyanide content varied from 0.08 ± 0.06 μg in
H. hewitsoni to 2.32 ± 4.18 μg in H. cydno (Table 1; Cardoso
& Gilbert, 2007).
Mating frequency, spermatophore mass, and cyanide
Mating frequency index and spermatophore mass were
positively correlated across the phylogeny (r = 0.73, P<0.05;
n = 8; Figure 2). Mating frequency was also correlated with
cyanide content (r = 0.72, P = 0.05; n = 8; Figure 3).
Discussion
Mating frequency and the gift-giving system of Heliconius butterflies
As expected, all pupal mating species had no indication of
multiple matings. Polyandry was confirmed among all
non-pupal mating species with one exception: H. ethilla
had a low mating frequency index. At the other end of the
spectrum, H. cydno had an average of greater than two
matings. The correlation found between mating frequency
and spermatophore mass supports the prediction that
larger spermatophores are expected when sperm
competition is more likely to occur (Svärd & Wiklund,
1989). Given the widespread occurrence of sperm precedence
Figure 1 Topology for the Heliconius tree used for analysis of
independent constrasts, derived from the phylogenetic
hypothesis following Beltrán et al. (2007). The numbers indicate
independent contrasts used in comparative analyses and in
Figures 2 and 3.
Table 1 Summary of samples sizes, average (± SD) spermatophore mass, cyanide content, mating system, and mating frequency index for
the nine Heliconius species studied. Numbers in parentheses refer to realized sample sizes for mass or cyanide estimates. Realized samples,
when smaller than n, imply loss of material or cyanide not detected. Cyanide content data derive from Cardoso & Gilbert (2007).
PU, pupal mating; NP, non-pupal mating
Species n
Spermatophore
mass (mg)
Cyanide
content (μg)
Mating
system
Mating
frequency index
Heliconius charithonia 10 0.21 ± 1.11 (9) 0.66 ± 0.40 (9) PU 1
Heliconius erato 3 0.66 ± 0.28 (2) 0.31 ± 0.50 (2) PU 1
Heliconius hewitsoni 3 0.31 ± 0.18 (3) 0.08 ± 0.06 (3) PU 1
Heliconius cydno 5 0.86 ± 0.52 (5) 2.32 ± 4.18 (4) NP 2.2
Heliconius ethilla 6 0.34 ± 0.04 (6) 0.55 ± 0.35 (6) NP 1
Heliconius hecale 10 1.23 ± 1.06 (6) 0.38 ± 0.64 (9) NP 1.6
Heliconius ismenius 12 0.82 ± 0.50 (10) 0.47 ± 0.48 (12) NP 1.25
Heliconius melpomene 5 0.71 ± 0.25 (5) 0.43 ± 0.24 (5) NP 1.4
Heliconius numata 19 0.47 ± 0.47 (17) 0.26 ± 0.21 (17) NP 1.5

112 Cardoso et al.
in Lepidoptera, larger gifts may serve as a means of
delaying remating in butterflies. Indeed, evidence shows
that females that receive a larger gift take longer to remate
than females that receive smaller ones (Kaitala & Wiklund,
1994; Cook & Wedell, 1999).
The amount of spermatophore protein, a measure of gift
quality, and polyandry have been found to be correlated
(Bissoondath & Wiklund, 1995). We did not examine
protein content as other studies have confirmed this
relationship (Boggs, 1981a,b, 1990). Boggs (1990) found
that the nitrogen portion of the spermatophores of poly-
androus H. cydno is greater than that in the monandrous
H. charithonia. Also, Dryas iulia (Fabr.), another polyandrous
species related to Heliconius (Penz & Peggie, 2003), contains
a larger spermatophore with a greater proportion of
nitrogen than that in the monandrous H. charithonia
(Boggs, 1981a,b). Thus, larger gifts are also apparently of
better quality in polyandrous Heliconius (and related) species.
An analysis of how protein concentration in spermatophores
varies along a gradient of polyandry within the Heliconius
genus should be ideal to test this prediction.
Do cyanides play a role in gift quality?
Cyanide content in the spermatophore correlated with the
mating index in the same fashion as spermatophore mass,
corroborating our suggestion that these may also play a
role in the nuptial package received by females during
mating (Cardoso & Gilbert, 2007). Despite extensive
Figure 2 Plot of phylogenetic contrasts for
mating frequency and spermatophore mass
for nine Heliconius species (for numbers see
Figure 1).
Figure 3 Plot of phylogenetic contrasts for
mating frequency and spermatophore
cyanide content for nine Heliconius species
(for numbers see Figure 1).

Heliconius mating system 113
research on the biology of Heliconius and related genera
(Brown, 1981; Gilbert, 1991), the relationship of cyanogenic
glycosides with differing mating strategies in Heliconius
remains unknown. Ample evidence demonstrates that
cyanogenic insects are unpalatable (Boyden, 1976; Muhtasib
& Evans, 1987; Chai, 1990), suggesting that cyanides in the
nuptial package would benefit the female either by offering
protection to her or to her eggs. Such a benefit should be
tested experimentally in a study of allocation of cyanogenic
compounds by the female.
Cyanide concentrations are relatively high in eggs of
Heliconius (Nahrstedt & Davis, 1983, 1985), demonstrating
that females allocate some or all of their reserves to their
eggs. Thus, the nuptial gift may replenish the female’s supply.
Suggestively, in H. cydno females the number of matings
and pollen feeding are inversely correlated, indicating that
the nuptial gift may partly replace the need to feed (Boggs,
1990). Other studies have shown similar nutritional benefits
of multiple matings in gift-giving species (Kaitala &
Wiklund, 1994; Arnqvist & Nilsson, 2000; Wedell et al.,
2002). Even more suggestively, multiple mating in
U. ornatrix seems to increase both nutrition and defensive
compound accumulation by the female (LaMunyon, 1997;
Rossini et al., 2001).
Thus, evidence suggests that cyanide concentrations
influence spermatophore quality, and, as we show here,
that polyandry is positively correlated with cyanide
concentration in the spermatophores of Heliconius
butterflies. An alternative hypothesis states that cyanide in
the spermatophores is simply inadvertent, that is, males
cannot avoid ‘contamination’. However, cyanides are more
prevalent in the accessory glands (and directly involved
in spermatophore production) than in the testes of
H. hewitsoni males (Cardoso & Gilbert, 2007). This suggests
that cyanide allocation is directed and not due to ‘inadvertent
contamination’ and thus is a mechanism of increasing
spermatophore quality.
It is increasingly clear that, along with male age and
access to pollen resources, male mating history affects the
outcome of mating (Torres-Vila & Jennions, 2005). If these
interacting effects were confounding, then we would have
expected the relationship between spermatophore mass
and polyandry to be less clear. Thus, this and the relation-
ship between cyanide content and mating frequency
support the hypothesis that spermatophores offer more
than just nutritional benefits to females. It remains to be
seen just how the female reaps these benefits, whether in
her own survival or the survival of her offspring. The natural
variation in mating systems within the genus Heliconius
provides an ideal laboratory for this and other questions
into the evolution of mating systems and the consequent
contribution of males to female survival and fecundity.
Acknowledgements
We would like to thank Carol Boggs for providing the
H. charithonia individuals, H. Engler-Chaouat for pro-
viding the β-glycosidase, and Emilia Martins and Wendy
Hodges for clarifying contrast analysis. We also thank
Christer Wiklund, Nina Wedell, Helene Engler-Chaouat,
Philip Schappert, and Krushnamegh Kunte for comment-
ing on earlier drafts of this article, and Letty Brown for
reviewing it. We thank MINAE, SINAC (Costa Rica),
IBAMA (Brazil) for collecting permits, and USDA APHIS
for importation permits. Márcio Zikán Cardoso thanks
CAPES (Brazil) for a doctoral fellowship at University of
Texas, Austin. Greenhouse facilities were provided by NSF
(DEB 790633 and 8315399) and University of Texas grants
to Lawrence E. Gilbert.
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