Behavioral Ecology

doi:10.1093/beheco/arp003
Advance Access publication 30 January 2009

Sperm precedence in monarch butterflies

(Danaus plexippus)
Michelle J. Solenskya and Karen S. Oberhauserb
Department of Biology, The College of Wooster, 931 College Mall, Wooster, OH 44691, USA and
b
Department of Fisheries, Wildlife and Conservation Biology, University of Minnesota, 1980 Folwell
Avenue, St. Paul, MN 55108, USA

n polyandrous species in which females store sperm, selection

should favor males that gain sperm precedence over other
males. Factors that might affect the relative number of fertilizations gained by a male include the number of sperm transferred
(Parker 1970), sperm motility, longevity, or ability to penetrate
the ovum (e.g., Lanier et al. 1979; Gomendio and Roldan 1991;
Briskie and Montgomerie 1992; Birkhead et al. 1995; Radwan
1996; LaMunyon and Ward 1998; Garca-Gonzalez and
Simmons 2007), and cryptic female choice during genitalic
coupling, intromission, insemination, or fertilization (e.g.,
Walker 1980; Simmons 1987; Eberhard 1991, 1996, 1998;
Birkhead and Mller 1993; LaMunyon and Eisner 1994; Ward
1998, 2000; Simmons et al. 1999; Bloch Qazi 2003; Bussiegre
et al. 2006; Fedina and Lewis 2006; Luck et al. 2007).
Sperm precedence in insects ranges from complete first-male
to complete last-male precedence (e.g., Simmons and SivaJothy 1998; Simmons 2001). Although last-male sperm precedence is most common in Lepidoptera, first-male precedence
and mixed paternity also occur (reviews in Drummond 1984;
Silberglied et al. 1984; Simmons and Siva-Jothy 1998). The preponderance of last-male precedence in the Lepidoptera probably results from displacement of the first males sperm in the
elongate spermatheca; after a pair separates, sperm move from
the bursa copulatrix, through the ductus seminalis and into the
spermatheca, where they are stored until being released to
fertilize eggs. Because the opening from which sperm are released is the same opening into which sperm move, sperm from
the last male are likely to comprise most of the fertilization set
from which sperm are drawn (Parker 1970). However, some
sperm from previous males may remain in the fertilization
set, and mixing of sperm is likely to occur.
To better understand potential factors affecting the outcome
sperm competition in Lepidoptera, we studied patterns of
sperm precedence in monarch butterflies (Danaus plexippus)
in a series of three experiments. The first experiment was

Address correspondence to K. Oberhauser. E-mail: oberh001@umn.

edu.
Received 21 April 2008; revised 13 November 2008; accepted
1 December 2008.
 The Author 2009. Published by Oxford University Press on behalf of
the International Society for Behavioral Ecology. All rights reserved.
For permissions, please e-mail: journals.permissions@oxfordjournals.org

designed to determine how male mating history affects sperm

precedence in this polygynandrous species. Subsequent experiments tested potential mechanisms for our initial results, with
explicit tests of how relative sperm numbers affected sperm precedence patterns. We also analyzed the effects of relative male
size and our genetic marker on sperm precedence patterns.
GENERAL METHODS
Rearing techniques and manipulation of mating history
Experimental butterflies were the second-fourth generations
of lab colonies derived from wild adults. We reared larvae in
screen cages (0.5 m 3 0.3 m 3 0.6 m) on milkweed (potted
Asclepias curassavica and cut stems of Asclepias syriaca). Larvae
developed at room temperature (;24
C) under natural light
conditions during the summer in either Minnesota or Ohio
(;18L:6D). Five to nine hours after eclosion, we placed adults
in glassine envelopes, and in experiment 1 only, weighed
them to the nearest 0.01 mg 24 h after eclosion. We checked
them for Ophryocystis elektroscirrha spores (a neogregarine protozoan parasite, Altizer et al. 2000) and euthanized infected
individuals by freezing them. We labeled all healthy butterflies
with a unique number on the hind wing discal cell using
a fine-point permanent marker and measured both forewings
from the point of attachment to distal wing tip.
Experiments took place in mesh cages (flight cages 2 m3,
oviposition cages 0.7 m3) in a greenhouse with natural summer light conditions or outdoors. Butterflies had continuous
access to milkweed and sponges saturated with honey water. We
released males into the cages within 13 days of eclosion, and
47 days before their first experimental mating and females on
the morning of their first assigned mating. Males and females
were held separately except on assigned mating days. After
their first mating, we kept experimental females in individual
oviposition cages with potted A. curassavica plants, and their
second mates were introduced to these cages on the assigned day
(see details below). In experiment 1, cages were on a residential
lawn; in experiment 2, on a fifth floor roof patio; and in experiment 3, in a temperature-controlled greenhouse (;24 to 28
C).
Monarchs usually remain coupled overnight (Oberhauser
1988; Solensky and Oberhauser 2009) and do not initiate

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We characterized sperm precedence in monarch butterflies (Danaus plexippus), using a series of experiments in which we
manipulated male mating histories to vary spermatophore size and the number of sperm transferred. Several factors affected
the outcome of sperm competition. There was a pattern of second-male sperm precedence, but second-male precedence was
rarely complete, and several other factors had significant effects on paternity patterns. Larger males outcompeted smaller males
when they were not matched for size. Phosphoglucose isomerase (PGI) genotype affected the outcome of sperm competition
under very hot conditions. When sperm from the same pair of males competed in different females, males fared better when they
transferred more sperm. These results demonstrate that sperm precedence within a species can be affected by many factors,
including the circumstances under which it is measured. Key words: monarch butterflies, PGI genotype, sperm competition,
sperm transfer. [Behav Ecol 20:328334 (2009)]

Solensky and Oberhauser Sperm precedence in monarch butterflies

Paternity assessment
We used variation at the phosphoglucose isomerase (PGI) locus to determine egg paternity. We used standard electrophoretic methods (Hebert and Beaton 1993) to determine
genotype and assigned matings in the prior generation to
obtain individuals homozygous for medium (m) and fast (f)
alleles in experiments 1 and 2 and an additional allele (slow
[s]) in experiment 3 (names based on electrophoretic mobility). Within each experiment, all females were homozygous
for the same allele.
We sampled adult genotypes by drawing ;5 ll of hemolymph from a small incision in the abdomen that healed
quickly (personal observation). We mixed the hemolymph into
two drops of deionized water and centrifuged the solution
before applying the sample to a Helena cellulose acetate
gel. We stored eggs in liquid nitrogen (196
C) until analysis,
crushed them with a fine-point forceps into 10 ll of Tris Borate
buffer (McClellan 1982) held in a Helena well plate, and applied
to a gel without further dilution. We ran gels at 300 V for 25 min.
To ensure that eggs were fertile, we allowed them to develop
for 34 days before freezing. In experiment 1, we did not have
enough ff males to assign each female two males of opposite
genotypes, so some first and second mates had genotypes of
mm and mf or mf and mm, respectively. To assign paternity in
these cases, we assumed that heterozygous males produced
equal numbers of sperm with m and f alleles and that these
two genotypes had equal fertilization success.
After determining the number of eggs fertilized by each
male, we calculated 95% confidence intervals for the proportion of eggs fertilized by the second male (P2) based on the
binomial distribution. We concluded that sperm precedence,
or nonrandom fertilization, had occurred if these intervals
did not include 0.5. Our use of heterozygous males in experiment 1 introduced error into our assignments of paternity,
because the two males shared an allele. To account for this
error, we determined the confidence interval for P2/2, based
on the actual number of eggs that contained the unique allele. We then doubled the endpoints of this interval to obtain
the confidence interval for P2 itself.
Individual experiment methods and results
Experiment 1: effects of male size and mating history on sperm
precedence
Methods: Males had either mated 1 day prior to their experimental mating and delivered small (S) spermatophores or
were virgins (V). Virgin males either had no prior access to

females or had been in cages with females but had not mated.
The first experimental matings took place in outdoor flight
cages, after which we randomly selected 24 females that had
mated to S males and 24 females that had mated to V males.
We then randomly assigned half of each group of females to
either a V or S second mating, generating four mating treatments designated by the order and type of spermatophores
each received: SS, SV, VS, and VV. No males were used for more
than one experimental mating, and male genotypes were
equally divided among treatments and mating order.
One day after their first mating, we put females into separate
oviposition cages, and collected 1030 eggs from each to assess
their fertility and genotype. Beginning on the third day, we put
a male of the assigned genotype and mating history for the second mating into the cage with each female. We replaced the
male if the pair did not mate on a given day. We removed
10 females that failed to remate within 7 days or lay eggs after
the second mating. Four of the 38 successful second matings
were the result of hand pairing, but we did not record which
pairings required this intervention, so could not assess its
effects.
Results: Females (n 38) laid an average of 700 eggs (range
1521179), and we analyzed the genotype of an average of
301 eggs (range 26616) from each female. There was strong
second-male precedence; 71% of the 38 females had P2 values
significantly more than 0.5, 11% had values for which the
95% confidence interval included 0.5, and 18% had values
significantly less than 0.5 (Figure 1a).
Male mass ranged from 437 to 629 mg, with mass differences
between a females two mates ranging from 2 to 162 mg. In
a linear regression of arcsine-transformed P2 values weighted
by egg number, the mass difference between the males was
a significant predictor of P2 (Table 1a, Figure 1b). The negative coefficient for M1M2 indicates that larger males did
better in sperm competition. The effect of being in the SV
treatment had a marginally negative effect; virgin second
males tended to fare poorly against previously mated first
males (Figure 1c). Male genotypes, intermating interval
(3- to7-day range), and total fecundity did not affect P2.
To further analyze the effect of male mating history, we did
a post hoc comparison of P2 between S and V second males.
There was a trend for an effect of treatment on rank, with
higher P2 when the second male had mated 1 day prior
(MannWhitney U 120, n 38, one-tailed P 0.0519,
Figure 1c).
Experiment 2: effect of spermatophore size on sperm precedence
Rationale: Experiment 2 explored mechanisms for the increased advantage enjoyed by second males mated 1 day prior
(S) in experiment 1. The two male groups in the first experiment were not random subsets of our experimental population; some V males had not mated despite having access to
females, whereas all S males had mated. Three hypotheses
for the advantage of S males include 1) females preferentially
used sperm from S males, perhaps using small spermatophores
to indicate previous mating success (Oberhauser 1989), 2)
females use a cue other than spermatophore size to bias
sperm use in favor of males that are better maters, or 3) males
that were more successful at mating transferred more competitive sperm. The first two hypotheses suggest cryptic female
choice, whereas the third suggests that male competition determines sperm precedence patterns. We rejected a fourth
possible hypothesis that spermatophores from the S males
contained more sperm; S males transfer fewer eupyrene (nucleated) sperm than V males (Solensky and Oberhauser
2009).
To test the first three hypotheses, we set up situations in
which sperm from the same two males competed in different

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mating after dark, so all matings can be recorded by checking

cages once at dusk. In some cases, we hand-paired males that
had not yet mated by 1700h; we held their wings together
above their bodies, then rubbed the end of the males abdomen near the genital opening at the ventral end of the females abdomen until he coupled with the female.
The mating history of the two males mated to each experimental female, and thus the size of the ejaculates transferred
(Oberhauser 1988; Solensky and Oberhauser 2009), were independent variables in all experiments. We manipulated male
mating histories to generate three categories of experimental
males: mated 1 day prior to experimental mating that transfer
small spermatophores (710 mg: S), mated 4 days prior to
experimental mating that transfer large spermatophores (22
25 mg: L), and virgins that transfer large spermatophores
(mean mass increases with age, . ;24 mg: V). After mating,
we maintained females in oviposition cages, collecting up to
40 eggs/female/day for genetic analysis, until they had laid
no eggs for a week or died.

329

Behavioral Ecology

330

Table 1
Factors affecting P2 values
Parameter
a. Experiment 1
Constant
M1M2
1DNV, V treatment
b. Experiment 2
Constant
Second male ff
First female 3 second male ff
c. Experiment 3
Constant
Only first male hand paired
Second male ff

Coefficient

Std error

0.8492
20.00279
20.2579

1.550
9.779E-04
0.1397

1.415
21.262
0.564

0.904
0.170
0.168

0.131
,0.001
0.003

0.663
0.267
0.293

0.713
0.146
0.105

0.355
0.071
0.006

P-value

0.587
0.0072
0.0732

Figure 1
P2 in experiment 1. Significant sperm precedence is defined by
a value whose binomial 95% confidence interval does not include
0.5. (a) P2 ranged from 0 to 1, with second-male precedence more
common. (b) P2 was negatively correlated with the difference in mass
between the two males (male 12 male 2). (c) P2 separated by mating
treatment. Triangles are not significantly different from 0.5, and
numbers indicate that more than one female had that value.

females. By manipulating male mating history, we could determine if males fared better when they had mated more recently.
Although not part of the experimental design, conditions

during the experiment led to a test of an interaction between

PGI genotype and temperature.
Methods: We created 12 pairs of males, matched for wing
length, to mate to a series of females. One male of the pair
was always the first mate, with an equal number of ff and
mm males assigned to this role. We manipulated male mating
history so that males had either mated 1 (S) or 4 (L) days
previously. Each female received two spermatophoresLS,
SL, or SSwith 1 day between matings. Four male pairs mated
to three females, seven mated to two females, and one mated
to one female.
On the first 3 days of this study, temperatures on the concrete rooftop patio where cages were placed reached or
exceeded 40
C each day (high daily temperatures near the
concrete roof ranged from 40 to 42
C), resulting in some mortality or subsequent failure to mate or lay eggs. Males first experienced the high temperatures on the day of their first
experimental mating (matings to manipulate spermatophore
size occurred in the temperature-controlled greenhouse), so
sperm received by the first female to which each male pair
mated were only exposed to high temperatures for a few hours
before mating. Subsequent females to which the male pairs
mated received sperm from males that had been exposed to
hot conditions for more than 1 day.
Results: Females laid an average of 177 eggs (range 40353)
over the 10 days after their second mating, and P2 values
ranged from 0 to 1 (Figure 2a). Complete sperm precedence
was common; only 44% of females laid eggs fertilized by
both males. First- and second-male sperm precedences were
equally common, and only one 95% confidence interval
included 0.5.
Sperm precedence was influenced by male PGI genotype
and an interaction between female number (first or subsequent) and genotype, but not by hand pairing, male mating
history treatment, male or female wing length, male pair
identity, or the difference in wing length between the two
males (Table 1b). Second males that were mm fertilized more
eggs than ff males, and this pattern was weaker for matings to
first females (Table 1b [positive coefficient for first female 3
second male ff], Figure 2b).
Arcsine-transformed P2 values for the regression reported
in Table 1b failed to meet assumptions of normality

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Weighted (by number of eggs analyzed) stepwise linear regression

models (backward elimination criterion: P . 0.10). P2 values were
arcsin transformed; mating and genotype treatments were included in
models as indicator variables. (a) Experiment 1 (n 38, R2 0.2255).
(b) Experiment 2 (n 27, R2 0.698). The positive interaction term
for first female 3 second male ff indicates that the advantage of mm
males was weaker for first experimental matings. (c) Experiment 3
(n 88, R2 0.123).

Solensky and Oberhauser Sperm precedence in monarch butterflies

331

Experiment 3: effect of spermatophore size on sperm precedence

(controlled temperatures)
Rationale: Because the effect of genotype, likely exacerbated
by extreme heat, outweighed all other effects in experiment 2,
we conducted a third experiment in which we kept adults in
a temperature-controlled greenhouse (;24 to 28
C) throughout the experiment.
Methods: We used 60 male pairs, each of which consisted of
one mm and one ff male, and ss experimental females. Each
male pair was scheduled to mate with two females, with preassigned mating history treatments: 1) both females, SS; 2) first
female, SL; second female, SS; or 3) first female, LS; second
female, LL. Female intermating intervals were all 1 day. Of the
60 male pairs, 28 mated to two females as planned, and
32 mated to one female.
Results: Females laid an average of 187 eggs in the 10 days
after their second mating (range 22450). P2 values ranged
from 0 to 1, with mixed paternity (82%) and second-male
precedence (70%) common (Figure 3a). P2 was higher when
the second male was ff (Figure 3b), and was not influenced
by male mating history, male or female wing length, hand
pairing or the difference in male size (Table 1c).
We used a paired t-test to compare the outcome of sperm
competition when the same two males competed in two different females with different mating sequences (SL and SS or
LL and LS) in the two females. In these cases, the second but
not the first males mating history changed from one female
to the next. P2 values tended to be higher when the second
male delivered a large spermatophore than when he delivered
a small spermatophore (mean difference 0.14, t 1.71,
df 22, P 0.0504 [t-test done on arcsine-transformed values]). Figure 3c compares the difference in P2 values between
the two females mated to the same pair of males for all mating
treatments. The values illustrated in the first two columns of
Figure 3c represent the mean difference between P2 values
when the second male is S versus when he is L, respectively;
positive values indicate that the second male does better when
he delivers a large spermatophore, regardless of the mating
history of the first male. When the mating history combinations were the same in both females (third column, shown for
comparison), the mean difference between the two values was
smaller.

Figure 2
P2 in experiment 2. (a) P2 ranged from 0 to 1, with first- and secondmale sperm precedence equally common. (b) Males homozygous for
the m allele at the PGI locus fertilized more eggs than males
homozygous for the f allele. This pattern was weaker in the first
experimental matings than subsequent matings. Median values are
shown with error bars extending to upper and lower quartile values.

DISCUSSION
We observed a general pattern of second-male sperm precedence, except in one experiment in which the effect of genotype outweighed any other effects. This pattern was affected by
the mating histories and sizes of the two males, and genotype
by environment interactions. Clearly, an understanding of sexual selection in polyandrous species requires knowledge of
events that affect sperm use by females, and these events are
likely to be complicated and variable. Here, we discuss the factors that affected the outcome of sperm competition in monarchs and suggest possible mechanisms for our observations.
Overall sperm precedence patterns
Second-male sperm precedence was more common than firstmale or no sperm precedence in monarch butterflies in two of
the three experiments reported here. The advantage of second
males suggests that sperm in the elongate spermatheca are
pushed back by incoming sperm, resulting in stratification
of sperm from different males. The finding that second-male

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(KolmogorovSmirnov test: P 0.001), so we verified significant parameters identified by regression analysis using the
nonparametric MannWhitney U test. Male genotype at the
PGI locus influenced sperm precedence, with mm males fertilizing more eggs than ff males (MannWhitney U 161, n
27, P , 0.001). We tested the significance of the interaction
between male genotype and female number using separate
analyses for first and subsequent females. The positive effect
of the mm male genotype on P2 was weaker in matings to
the first female than to subsequent females (first female:
MannWhitney U 18, n 9, P 0.020; subsequent females:
MannWhitney U 72, n 18, P , 0.001).
For six of the male pairs that mated with multiple females,
95% confidence intervals of P2 values from all of their mates
overlapped (data not shown). For the five that did not overlap, no mating history combination resulted in consistently
higher P2 values; in three cases, the male pairs had mated
with three different females, and in two cases, they had mated
with two different females. The mating history combinations
and significant differences in P2 values for these five pairs
are as follows: SS . LS SL, SS . LS SS, SS . LS SL,
SS . LS, and SL . SS. Thus, relative ejaculate size did not
appear to affect the outcome of sperm competition in this
experiment.

Behavioral Ecology

332

sperm precedence is rarely complete indicates that sperm from

the first male remain in the fertilization set (Parker et al.
1990). Spermatophores delivered by the first male may serve
as a barrier for subsequent males, because the spermatophore
must align correctly with the ductus seminalis for sperm to
move into the spermatheca (Drummond 1984). Variation in
the ability to generate or overcome this barrier could cause
the bimodal distribution in sperm precedence; 5%, 33%, and
9% of females exhibited complete first-male precedence in
experiments 1, 2, and 3, respectively.
Male size

Figure 3
P2 in experiment 3. Medians are shown for each group, with error
bars from the first quartile to the third quartile. (a) P2 ranged from
0 to 1, with second-male precedence more common than first male
precedence. (b) ff males fertilized more eggs than mm males (Mann
Whitney U 719.0, P 0.047). (c) Mean differences between P2
values when the same pair of males competed in two females. Solid
gray columns: second male transferred a large (L) spermatophore
for one mating and a small (S) spermatophore for the other;
difference P2 when second male was L 2 P2 when second male was
S. Hatched column: mating histories were SS for both females;
difference between values P2 for first female 2 P2 for second female.

Male mating history

Previously mated second males tended to fare better in sperm
competition than virgins in experiment 1, even though they
transferred smaller spermatophores and probably fewer sperm
(Oberhauser 1988; Solensky and Oberhauser 2009). Experiments 2 and 3 were designed to determine if this advantage
was due to mating history per se, perhaps because females
used spermatophore size to indicate past mating success and
biased sperm use in favor of these males. We hypothesized
that spermatophore size is likely to be an honest indicator
of mating history; large spermatophores result in longer delays before female remating (Oberhauser 1992), and males

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In experiment 1, when males were not matched for size, larger

males tended to fare better in sperm competition (Figure 1b).
This effect of male size on the outcome of sperm competition
is common in insects (Lewis and Austad 1990; Simmons and
Parker 1992; LaMunyon and Eisner 1993, 1994; Gwynne
and Snedden 1995; Simmons et al. 1996; Bissoondath and
Wiklund 1997; Wedell and Cook 1998; Arnqvist and Danielsson 1999) and could be a numerical effect if larger males
transfer more sperm, as they do in the dung fly Scatophaga
stercoraria (Simmons and Parker 1992; Simmons et al. 1996).
Female choice could also mediate a numerical effect. For
example, male field crickets (Gyllus bimaculatus) allow longer
spermatophore attachments and multiple inseminations from
larger males (Simmons 1986), resulting in the transfer of
more sperm and higher success in sperm competition.
A numerical advantage is unlikely to explain the effect
of male size in the outcome of sperm competition in monarch
butterflies. Male size does not appear to affect the number of
eupyrene or apyrene sperm transferred in monarchs (Solensky
and Oberhauser 2009), and the large male advantage occurred across treatments in experiment 1, even when the
larger male had mated previously and thus transferred fewer
sperm (Solensky and Oberhauser 2009).
Other mechanisms could lead to a large male advantage in
sperm competition. For example, female dung flies store
sperm from larger males where they are probably more likely
to be used to fertilize eggs (Ward 1998, 2000). A large male
advantage could also be due to differential fertilizing capacity
of different-sized sperm. In mammals and birds, longer
sperm swim faster (Gomendio and Roldon 1991; Briskie and
Montgomerie 1992), and faster sperm may be more likely to
fertilize eggs (Birkhead et al. 1995). Male bulb mites Rhizoglyphus robini (Radwan 1996) and nematodes Caenorhabditis elegans (LaMunyon and Ward 1998) that transferred larger
sperm fared better in sperm competition. However, in the
dung beetle Onthophagus taurus, shorter sperm had higher
fertilization success in a way that depended on the size and
shape of the spermatheca (Garca-Gonzalez and Simmons
2007). We are not aware of studies showing within-species
correlations between male size and sperm size, but this correlation exists across butterfly species (Gage 1994).

Solensky and Oberhauser Sperm precedence in monarch butterflies

PGI genotype
Male PGI genotype affected sperm precedence in experiments
2 and 3, but not experiment 1. In experiment 2, mm males
fertilized significantly more eggs than ff males, and the reverse
was true in experiment 3. Males and females were reared
under similar laboratory conditions and were of similar ages
at the start of the experiments. However, butterflies in experiment 2 were exposed to very hot conditions, whereas those
in experiment 3 were kept in a temperature-controlled
greenhouse.
PGI (an enzyme used in glycolysis, Watt 1992) genotypes
affect fecundity and mate choice in Colias butterflies (Watt
et al. 1986; Watt 1992) and monarch butterfly flight ability
at low temperatures (Hughes and Zalucki 1993). In this study,
PGI may have affected sperm function or transfer, and the mm
allele appears less susceptible to denaturation or loss of activity at high temperatures. The extreme heat occurred only
during the first matings, but the genotype effect was even
stronger in subsequent matings (Figure 2b). The effect of
temperature appears to be lasting and less severe after a short
exposure. An alternative explanation stems from our use of
a relatively small number of population founders. Matings
that established the experimental population were designed
to generate adults of known genotypes, but not maximize
genetic diversity. PGI genotype may have been confounded
with a heritable trait that we did not assess.
FUNDING
National Science Foundation (IBN-0205766, DEB-9220820,
and 9442165), a University of Minnesota Graduate School,
and the Dayton and Wilkie Natural History Funds administered by the Bell Museum of Natural History at the University
of Minnesota.
We thank Don Alstad for sharing his equipment, supplies, time, and
expertise in assisting us with paternity assignment using allozyme electrophoresis and for comments on the manuscript. Erek Lam, Sonja
Lin, Rachel Hampton, Brenda Jensen, and Deborah Huddleston
ran hundreds of gels to assign paternity to tens of thousands of eggs;
Rachel Hampton, Sonja Lin, Erek Lam, Trisha Dwivedi, De Cansler,
Ann Feitl, Christine Jessup, Leah Alstad, and Michael Hektner reared
adults, set up matings, and collected eggs; Sandy Weisberg provided

statistical help; and two anonymous reviewers suggested helpful

improvements to the manuscript.

REFERENCES
Altizer SM, Oberhauser KS, Brower LP. 2000. Associations between
host migration and the prevalence of a protozoan parasite in natural populations of adult monarch butterflies. Ecol Entomol. 25:
125139.
Arnqvist G, Danielsson I. 1999. Postmating sexual selection: the effects
of male body size and recovery period on paternity and egg production rate in a water strider. Behav Ecol. 10:358365.
Birkhead TR, Fletcher F, Pellatt EJ, Staples A. 1995. Ejaculate quality
and the success of extra-pair copulations in the zebra finch. Nature.
377:422423.
Birkhead TR, Mller A. 1993. Female control of paternity. Trends Ecol
Evol. 8:100104.
Bissoondath CJ, Wiklund C. 1997. Effect of male body size on sperm
precedence in the polyandrous butterfly Pieris napi L. (Lepidoptera:
Pieridae). Behav Ecol. 8:511523.
Bloch Qazi MC. 2003. A potential mechanism for cryptic female
choice in a flour beetle. J Evol Biol. 16:170176.
Briskie JV, Montgomerie R. 1992. Sperm size and sperm competition
in birds. Proc R Soc Lond B. 247:8995.
Bussiegre LF, Hunt J, Jennions MD, Brooks R. 2006. Sexual conflict
and cryptic female choice in the black field cricket, Teleogryllus commodus. Evolution. 60:792800.
Drummond BA. 1984. Multiple mating and sperm competition in the
Lepidoptera. In: Smith RL, editor. Sperm competition and the evolution of animal mating systems. London: Academic Press.
p. 547572.
Eberhard WG. 1991. Copulatory courtship and cryptic female choice
in insects. Biol Rev. 66:131.
Eberhard WG. 1996. Female control: sexual selection by cryptic female choice. Princeton (NJ): Princeton University Press.
Eberhard WG. 1998. Female roles in sperm competition. In: Birkhead T,
Moller A, editors. Sperm competition and sexual selection. London:
Academic Press. p. 91116.
Fedina TY, Lewis SM. 2006. Proximal traits and mechanisms for biasing paternity in the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae). Behav Ecol Sociobiol. 60:844853.
Gage MJG. 1994. Associations between body size, mating pattern, testis
size and sperm lengths across butterflies. Proc R Soc Lond B.
258:247254.
Garca-Gonzalez F, Simmons LW. 2007. Shorter sperm confer higher
competitive fertilization success. Evolution. 61:816824.
Gomendio M, Roldon ERS. 1991. Sperm size and sperm competition
in mammals. Proc R Soc Lond B. 243:181185.
Gwynne DT, Snedden AW. 1995. Paternity and female remating in
Requena verticalis (Orthoptera: Tettigoniidae). Ecol Entomol. 20:
191194.
Hebert PDN, Beaton MJ. 1993. Methodologies for allozyme analysis
using cellulose acetate electrophoresis. Beaumont (TX): Helena
Laboratories.
Hughes JM, Zalucki MP. 1993. The relationship between the Pgi locus
and the ability to fly at low temperatures in the monarch butterfly,
Danaus plexippus. Biochem Genet. 31:521532.
LaMunyon C, Eisner T. 1993. Postcopulatory sexual selection in an
arctiid moth (Utetheisa ornatrix). Proc Natl Acad Sci USA. 90:
46894692.
LaMunyon C, Eisner T. 1994. Spermatophore size as determinant of
paternity in an arctiid moth (Utetheisa ornatrix). Proc Natl Acad Sci
USA. 91:70817084.
LaMunyon C, Ward S. 1998. Larger sperm outcompete smaller sperm
in the nematode Caenorhabditis elegans. Proc R Soc Lond B.
265:19972002.
Lanier DR, Estep DQ, Dewsbury DA. 1979. Role of prolonged copulatory behavior in facilitating reproductive success in a competitive
mating situation in laboratory rats. J Comp Physiol Psychol.
93:781792.
Lewis SM, Austad SN. 1990. Sources of intraspecific variation in sperm
precedence in red flour beetles. Am Nat. 135:351359.
Luck N, Dejonghe B, Fruchard S, Huguenin S, Joly D. 2007. Male and
female effects on sperm precedence in the giant sperm species
Drosophila bifurca. Genetica. 130:257265.

Downloaded from http://beheco.oxfordjournals.org/ at Emory University Libraries on May 27, 2012

probably transfer the largest spermatophore allowed by their

mating history and age (Oberhauser 1988). Alternatively, the
advantage could have been due to something inherent in the
males or their sperm, because previously mated males were
not a random subset of our population. Experiment 2 was
inconclusive because of the large genotype effect, but experiment 3 showed that females do not preferentially use sperm
from males that transfer small spermatophores. When the
second male transferred a larger spermatophore than he
had in another competition with the same male, he did better,
rather than worse. Results of experiment 1 are thus likely to
be due to something inherent in this nonrandom subset of
males, perhaps the result of cryptic female choice based on
something other than spermatophore size or a correlation
between male mating ability and sperm competitiveness. Experiment 3 does not allow us to distinguish unequivocally
between these two hypotheses. However, when the same male
pairs competed in different females, the outcome of sperm
competition varied in ways predicted by the number of sperm
transferred. This result does not rule out cryptic female
choice, but consistent patterns between females in experiment 3 would provide a stronger argument for this mechanism.

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Simmons LW, Parker GA, Stockley P. 1999. Sperm displacement in the

yellow dung fly, Scatophaga stercoraria: an investigation of male and
female processes. Amer Nat. 153:302314.
Simmons LW, Siva-Jothy MJ. 1998. Sperm competition in insects:
mechanisms and the potential for selection. In: Birkhead TR, Moller
AP, editors. Sperm competition and sexual selection. London: Academic Press. p. 341434.
Simmons LW, Stockley P, Jackson RL, Parker GA. 1996. Sperm competition or sperm selection: no evidence for female influence over
paternity in yellow dung flies Scatophaga stercoraria. Behav Ecol Sociobiol. 38:199206.
Solensky MJ, Oberhauser KS. 2009. Male monarch butterflies (Danaus
plexippus) adjust their ejaculates in response to risk and intensity of
sperm competition. Anim Behav. 77:465472.
Walker WF. 1980. Sperm utilization strategies in nonsocial insects. Am
Nat. 115:780799.
Ward P. 1998. A possible explanation for cryptic female choice
in the yellow dung fly, Scathophaga stercoraria (L). Ethology. 104:
97110.
Ward P. 2000. Cryptic female choice in the yellow dung fly Scathophaga
stercorarla (L). Evolution. 54:16801686.
Watt WB. 1992. Eggs, enzymes and evolution: natural genetic variants
change insect fecundity. Proc Natl Acad Sci USA. 89:1060810612.
Watt WB, Carter PA, Donohue K. 1986. Females choice of good
genotypes as mates is promoted by an insect mating system. Science. 233:11871190.
Wedell N, Cook PA. 1998. Determinants of paternity in a butterfly.
Proc R Soc Lond B. 265:625630.

Downloaded from http://beheco.oxfordjournals.org/ at Emory University Libraries on May 27, 2012

McLelland T. 1982. Electrophoretic buffers for polyacrylamide gels at

various pH. Anal Biochem. 126:9499.
Oberhauser KS. 1988. Male monarch butterfly spermatophore mass
and mating strategies. Anim Behav. 36:13841388.
Oberhauser KS. 1989. Effects of spermatophores on male and female
monarch butterfly reproductive success. Behav Ecol Sociobiol.
25:237246.
Oberhauser KS. 1992. Rate of ejaculate breakdown and intermating
intervals in monarch butterflies. Behav Ecol Sociobiol. 31:367373.
Parker GA. 1970. Sperm competition and its evolutionary consequences in the insects. Biol Rev. 45:525567.
Parker GA, Simmons LW, Kirk H. 1990. Analysing sperm competition
data: simple models for predicting mechanisms. Behav Ecol Sociobiol. 27:5565.
Radwan J. 1996. Intraspecific variation in sperm competition success
in the bulb mite: a role for sperm size. Proc Biol Sci. 263:855859.
Silberglied RE, Shepherd JG, Dickinson JL. 1984. Eunuchs: the role of
apyrene sperm in Lepidoptera? Amer Nat. 123:255265.
Simmons LW. 1986. Female choice in the field cricket, Gryllus bimaculatus. Anim Behav. 34:14631470.
Simmons LW. 1987. Sperm competition as a mechanism of female
choice in the field cricket, Gryllus bimaculatus. Behav Ecol Sociobiol.
21:197202.
Simmons LW. 2001. Sperm competition and its evolutionary consequences in the insects. Princeton (NJ): Princeton University Press.
Simmons LW, Parker GA. 1992. Individual variation in sperm competition success of yellow dung flies, Scatophaga stercoraria. Evolution.
46:366375.

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