Behavioral
Ecology
The official journal of the
ISBE
International Society for Behavioral Ecology
Behavioral Ecology (2013), 24(5), 1218–1228. doi:10.1093/beheco/art056
Original Article
Parental effects on early development: testing
for indirect benefits of polyandry
Sheri L. Johnson and H. Jane Brockmann
Department of Biology, University of Florida, PO Box 118525, Gainesville, FL 32611, USA
Received 11 December 2012; revised 2 June 2013; accepted 5 June 2013; Advance Access publication 6 July 2013.
Females of many species mate with multiple males even when it is costly. Multiple mating may allow females to exploit postcopulatory mechanisms to ensure that their eggs are fertilized by high quality (good genes) and/or genetically compatible males. We conducted a series of
noncompetitive in vitro fertilization experiments to evaluate the benefits of polyandry in naturally occurring pairs of horseshoe crabs, Limulus
polyphemus. In this externally fertilizing species, attached pairs migrate to shore to spawn; unpaired males are attracted to spawning pairs
by visual and chemical cues and become satellites of some (polyandrous) females while ignoring others (monandrous). When present, satellites may fertilize a high proportion of the female’s eggs, but their presence is costly to female nesting success. We measured developmental
success for monandrous and polyandrous females crossed with attached and satellite males. We found that satellite males increased the
success of polyandrous but not monandrous females. We then examined the effect of good genes and genetic compatibility on developmental success and offspring size using a North Carolina II breeding design. Results indicate that both incompatibilities between males and
females and paternal good genes effects may provide a selective advantage that offsets the costs of multiple mating in this species.
Key words: genetic compatibility, good genes, Limulus polyphemus, mate choice.
Introduction
Females mating with more than 1 male, that is, polyandry, is
taxonomically widespread (Birkhead and Møller 1998; Birkhead
et al. 2009), but the maintenance of the behavior is puzzling. In
most cases, females can fertilize all of their eggs with the sperm
from a single male and in many species mating with multiple
males can be costly for females (Thornhill and Alcock 2001;
Arnqvist and Rowe 2005), suggesting that there must be some
compensating benefits. For some species, males provide direct
benefits to the female or her offspring, such as resources or
paternal care; for others, females mate with multiple males to
ensure that all their eggs are fertilized (Ridley 1988), considered
to be a direct benefit for internal fertilizers (Jennions and
Petrie 2000). But for many others, including most externally
fertilizing species (Byrne and Roberts 2004), males provide no
benefits to females other than their genes (Jennions and Petrie
2000; Tregenza and Wedell 2000; Hosken and Stockley 2003;
Zeh and Zeh 2003; Neff and Pitcher 2005; Simmons 2005;
Kempenaers 2007; Slatyer et al. 2012). These females may gain
from increasing offspring diversity (Yasui 1998; Johnson and
Yund 2008), improving male quality (“good genes”; Kempenaers
Address correspondence to S.L. Johnson, who is now at Department of
Zoology and Department of Anatomy, University of Otago, PO Box 913,
Dunedin 9054, New Zealand. E-mail: [email protected].
© The Author 2013. Published by Oxford University Press on behalf of the
International Society for Behavioral Ecology. All rights reserved. For permissions,
please e-mail: [email protected]
et al. 1992; Wedell and Tregenza 1999) or increasing genetic
compatibility (Wedekind 1994; Zeh and Zeh 1996, 1997;
Newcomer et al. 1999; Ivy 2007). Benefits from good genes
depend solely on male genetic quality, and directional
selection favors intrinsically high-quality males through sperm
competition or cryptic female choice (Eberhard 1996; Hosken
and Stockley 2003; García-González and Simmons 2005, 2007;
García-González and Evans 2011). In contrast, benefits from
compatible genes depend on the interacting effects of male and
female genotypes (either at fertilization or subsequently during
embryo or offspring development), and nondirectional selection
favors compatible male by female crosses (Neff and Pitcher
2005; Kempenaers 2007; Neff and Pitcher 2008). Although
many studies have suggested that genetic compatibility may be
important in the evolution of multiple mating, until recently
few studies have explicitly tested the importance of genetic
compatibility in a way that separates the effects of good genes
from compatible genes (but see Wedekind et al. 2001, 2008;
Evans and Marshall 2005; Marshall and Evans 2005; Rudolfsen
et al. 2005; Trippel et al. 2005; Evans et al. 2007; Ivy 2007;
Pitcher and Neff 2007; Dziminski et al. 2008; Rodriguez-Munoz
and Tregenza 2009).
To understand the benefits of polyandry, we take advantage
of the unique mating system of the American horseshoe crab,
Limulus polyphemus. In this species, females that are ready to lay eggs
migrate toward shore during a limited period of time when high
tides are exceptionally high due to a new or full moon (Brockmann
Johnson and Brockmann • Indirect benefits of polyandry in horseshoe crabs
1219
and Johnson 2011). Males are attracted to these females visually
(Barlow and Powers 2003), and as the female swims by, a male
simply attaches to her posterior spines with his modified pedipalps
and the attached pair swims to shore where they spawn. The male
fertilizes the female’s eggs externally with free-swimming sperm as
the eggs are being laid in the sand (Brockmann 2003b). Little is
known about the behavior of pairs prior to arriving on the beach,
but there is no evidence and seemingly little opportunity for female
mate choice prior to mating (Brockmann 2003a), particularly given
the time constraints under which females are nesting (at our site
<3 h at the time of the maximum high tide). Unattached males
join some spawning pairs (polyandrous), whereas other nearby
pairs spawn without satellites (monandrous). When present, satellites fertilize 1–80% of the female’s eggs depending on their position (Brockmann et al. 1994, 2000). Recent research, using the
same study population, has established that nesting with satellites
(i.e., polyandrous nesting) is costly to females in this system: when
satellite males were experimentally removed from around polyandrous pairs, they continued to nest longer and the females laid
more eggs than control females that continued to nest with their
satellites in place (but were otherwise treated the same; Johnson
and Brockmann 2010). In spite of these costs in nesting time and
egg-laying success, the frequency of polyandrous females is high in
this system, particularly when nesting densities are high (20–80%
when more than 50 pairs are present; Johnson and Brockmann
2012). It could be that this costly nesting with satellites is due to
competitive male behavior, but there is also some evidence that
polyandrous females (or pairs) attract satellites using chemical and
visual cues (Hassler and Brockmann 2001; Schwab and Brockmann
2007; Saunders et al. 2010). What makes this system particularly
puzzling is that some females that are nesting on the same beach
and at the same time as the polyandrous females do not have satellites (monandrous) and do not attract nearby males even when
densities are high (Johnson and Brockmann 2012). These monandrous females do not pay the nesting costs associated with being
surrounded by competing satellite males, so why are not all females
monandrous? In some externally fertilizing species, polyandrous
mating increases the number of fertilized eggs, but in this system
nearly all eggs develop whether satellites are present or not (96.4%
for monandrous and 96.8% for polyandrous manipulated pairs;
Johnson and Brockmann 2010). Taken together, our previous studies suggest the hypothesis that there are postmating, indirect genetic
benefits accruing to polyandrous females that compensate for the
costs of nesting with satellites.
Using eggs and sperm collected from wild, naturally occurring
pairs, we conducted laboratory crosses to examine the hypothesis
that polyandrous females benefit from multiple mating. Using a
quantitative genetics breeding design, we further evaluated whether
those benefits came from good genes or genetic compatibility. In
Experiment 1, we conducted crosses (in vitro fertilization from collected eggs and sperm) between females and their attached males
(i.e., the male that was attached to that female when she arrived
on the beach) or females and a random selection of satellite males.
We used both kinds of females, those observed to be polyandrous
in the field and those that were collected at the same time but were
monandrous. Under our hypothesis, we predicted that polyandrous
females would show greater success (measured as developmental
success of the embryos and larvae) when their eggs were fertilized by sperm from satellite males compared with their attached
males, whereas monandrous females would show greater success
when fertilized by their attached males. Previous work (Johnson and
Brockmann 2010) demonstrated that although monandrous females
do not attract males, some would tolerate the presence of a satellite male and continue nesting with a satellite present (“tolerant”
females) if one arrived, whereas other females would not lay eggs
and would leave the beach as soon as a satellite arrived at the nesting pair (“intolerant” females). We hypothesized that these 2 kinds
of monandrous females would differ in the indirect benefits they
derived if their eggs were fertilized by sperm from satellite males. In
Experiment 2, we compared the success of eggs from “tolerant” and
“intolerant” monandrous females when they were crossed using
sperm from their attached males compared with random satellite
males. We predicted that tolerant females should have the same
or greater success when mated with satellite males and intolerant
females should show decreased success when mated with satellites
compared with their attached males.
To determine the source of postmating, genetic benefits from
multiple mating (i.e., good genes or compatibility effects), we
conducted a third experiment. We used a cross-classified breeding design (North Carolina II [NCII]) and quantitative genetics
analysis to partition phenotypic sources of variance in offspring
fitness among additive (male and female intrinsic quality) and
nonadditive (male × female interaction) effects (Evans and
Marshall 2005; Evans et al. 2007; Pitcher and Neff 2007; Dziminski
et al. 2008). We measured fitness traits for eggs from monandrous
and polyandrous females crossed with sperm from random satellite males from the population.
Our results indicate that the attached males of monandrous
and polyandrous females differ in quality because polyandrous
females have lower success with their attached males than
monandrous females have with their attached males. Further, we
find evidence for both good genes and incompatibility effects,
which may offset the costs of multiple mating in this system. We
add to the diverse list of animals that are known to show good
genes and incompatibility effects (Evans and Marshall 2005;
Rudolfsen et al. 2005; Bang et al. 2006; Evans et al. 2007; Marshall
and Evans 2007; Pitcher and Neff 2007; Rodriguez-Munoz and
Tregenza 2009), but our study is novel in that we used naturally
occurring monandrous and polyandrous females to evaluate the
role of indirect genetic benefits in this horseshoe crab mating
system.
Material and Methods
Study organism
We studied a wild population of horseshoe crabs, L. polyphemus, at
Seahorse Key, Florida (SHK), an island along the northern Gulf of
Mexico coast near Cedar Key, Levy County, Florida (Brockmann
et al. 1994). During the highest tides in March, April, and May
2008 and 2009, we observed and collected males and females
along the island’s sandy south beach. Each female arrived at the
beach with 1 male attached to her posterior opisthosomal spines
by his modified pedipalps. She dug into the sand where she usually
laid eggs in a series of batches. We defined polyandrous females
as those pairs that were joined by unattached, satellite males when
the satellite was in contact with the female, her attached male,
or another satellite. Monandrous females were defined as females
that were dug into the sand with their attached male (i.e., nesting)
but had no unattached, satellite males in contact with the pair.
Eggs are fertilized externally as they are being laid in the sand by
free-swimming sperm from the attached male or, if satellites are
Behavioral Ecology
1220
present, also by satellites (Brockmann et al. 1994, 2000). Fertilized
eggs remain in the sand for 2–4 weeks where embryos develop
and hatch into free-swimming (nonfeeding) trilobite larvae that
metamorphose into juveniles 2–4 weeks later in inshore areas
near the breeding beach. The juveniles molt several times over
the next 6 months and take 9–10 years to become adults (Shuster
and Sekiguchi 2003).
In vitro fertilization
Monandrous and polyandrous females were picked up from the
nesting beach at SHK, along with their attached males and random satellite males; they were marked uniquely with tags and taken
to the University of Florida Marine Laboratory at SHK where they
were maintained in flowing seawater tables for 1–2 days before
being returned to the same nesting beach. Prior to release, all individuals were measured for size (width of carapace at the widest
point) and condition. The condition index (ranging from 4 to 20,
with the highest condition individuals receiving a 20) included evaluations of carapace color, the surface condition of the carapace,
the condition of the eyes, and the condition of the posterior spines
(see Johnson and Brockmann 2010) for specifics.
Sperm and eggs were collected using an electrical stimulation
protocol adapted from Brown and Clapper (1980) and Sasson
et al. (2012). The animals were removed from the water and
placed on their dorsal surface on a rubber mat, and the genital
operculum was lifted to expose the gonopores. Excess water was
wiped away, and an electrical stimulus (males: 10 V; females:
15–20 V) was applied below and slightly lateral to the gonopores.
After stimulation, eggs were collected using a plastic spoon, and
approximately 30 eggs were counted out and placed in each plastic dish (11.3 cm diameter × 3.9 cm height) containing 40 mL of
clean seawater (average salinity 34 ppt). The University of Florida
obtains seawater from Whitney Marine Laboratory for use in the
laboratory at the Gainesville campus, where we reared the eggs, so
we also used this water for the fertilizations that were conducted
in the laboratory at SHK. After stimulation, 5–30 µL of concentrated sperm was collected using a pipette, placed in a microcentrifuge tube, and kept on ice until needed (1 h maximum). When we
were ready to conduct the noncompetitive in vitro fertilizations, we
diluted the sperm using filtered, 34 ppt seawater at room temperature (seawater does not activate sperm motility in Limulus; Clapper
and Brown 1980a, b). In each experiment, eggs and sperm were
combined within 5–10 min of egg collection by gently pipetting
60 µL of diluted sperm suspension (see below for specifics) onto
the eggs in each dish and gently and briefly swirling the dishes.
After 60 min, excess sperm was washed away. The in vitro fertilized eggs were taken from SHK to the laboratory at the University
of Florida in Gainesville, FL, where they were held at room temperature (22–25 °C). The water in each dish was changed daily
for the first 3–4 days and then weekly for the remainder of the
developmental period.
We had previously determined that the eggs of monandrous
and polyandrous females in this population did not differ in size
(Johnson and Brockmann 2012); hence, we did not measure egg
size for our experiments. Further, the amount of oviducal fluid was
not controlled for in our study as little measureable oviducal fluid is
released with the eggs during collection.
Although horseshoe crabs do not fall under animal ethics regulations in the United States, we were always careful to ensure animal
welfare throughout all experiments. All animals were returned to
the beach, and many were seen spawning again at a later date.
Sperm concentration measures
In all experiments, sperm were diluted using serial dilutions.
Male horseshoe crabs from our population release sperm at a
concentration of 2–9.5 × 109 sperm mL−1 (Sasson et al. 2012). In
Experiment 1, sperm were diluted to ~108 sperm mL−1 (10−1
dilution), but for Experiments 2 and 3, sperm were diluted to ~107
sperm mL−1 (10−2 dilution). The rationale behind switching to a
more dilute sperm concentration resulted from a serial dilution
experiment that we performed after starting Experiment 1. We
compared the developmental success of eggs (N = 10 pairs of
monandrous females with their respective attached males, mean
sperm concentration of 6.57 × 109 sperm mL−1 ± 4.50 × 108
standard error [SE]) fertilized with 5 dilutions of sperm (108 to 101
sperm mL−1). We found that 107 sperm mL−1 resulted in asymptotic
rates of development, and there was no significant difference
in development rates among the 108, 107, and 105 sperm mL−1
concentration (Figure S1). For Experiments 2 and 3, we opted to
use the 107 sperm mL−1 concentration because it ensured that we
had plenty of sperm, particularly for Experiment 3 where we had
to allocate sperm from a single male across 10 sets of eggs. We also
calculated exact sperm counts for each male by diluting sperm to
~106 sperm mL−1 (10−3 dilution) and fixing with a 2:1 ratio of 2%
gluteraldehyde. Sperm concentrations (sperm mL−1) were calculated
using an improved Neubauer hemocytometer (see Table 1 for
descriptive statistics). Because sperm concentrations varied
somewhat from our expected 108 sperm mL−1 or 107 sperm mL−1,
we included sperm concentration as a covariate in all analyses.
Measure of developmental success
It was not possible to assay true fertilization success (whether a
zygote had been formed or not) in our in vitro fertilization experiments because horseshoe crab eggs have a tough, opaque chorion
surrounding the egg and early embryo. This outer membrane ruptures by the 7th day at embryonic stage 19-2, and the developing
embryo is then visible inside a balloon-like inner embryonic membrane (Shuster and Sekiguchi 2003). Because of this limitation, we
measured the proportion of eggs that developed by day 20 (day
20 was chosen to ensure that we counted all eggs that were going
to develop). Note that our serial dilution experiment (Figure S1)
indicates that our sperm:egg ratios were not too high, as we see
higher fertilization success at higher concentrations, but it is unclear
whether we were using a sufficient amount of sperm to achieve fertilization of 100% of the eggs. The highest development achieved
was 88% at 107 sperm mL−1, with a mean of 64% (a result similar
Table 1 Mean sperm concentration (sperm mL−1) of the males used in
Experiments 1–3
Variable
Mean
SE
Sample size,
N (missing)
Attached males monandrous
Satellite males monandrous
Attached males polyandrous
Satellite males polyandrous
Attached males intolerant
Attached males tolerant
Satellite malesa
Satellite malesb
9.2 × 109
1.0 × 1010
9.2 × 109
8.1 × 109
1.7 × 1010
1.3 × 1010
1.9 × 1010
6.56 × 109
9.0 × 108
1.0 × 109
7.4 × 108
8.9 × 108
5.0 × 109
4.0 × 109
5.5 × 109
1.21 × 108
18 (2)
14
20 (1)
14
25
25
24 (1)
176
aSatellite
bSatellite
males used in Experiment 2.
males used in Experiment 3.
Johnson and Brockmann • Indirect benefits of polyandry in horseshoe crabs
1221
to that reported in previous studies using similar methodology;
Brown and Knouse 1973). Whether our measure of “developmental success” represents viability (i.e., all eggs were fertilized but
only a proportion of those developed) or true fertilization success is
unclear. Therefore, we take the conservative approach and refer to
this measure as developmental success, recognizing that the variance in “developmental success” could be due to factors that affect
zygote formation during the fertilization phase or genetic elements
during the early developmental phase (García-González 2008).
with 4 randomly chosen satellite males in 16 pairwise combinations,
with replicate crosses performed for each (i.e., a total of 32 crosses
per block). Eleven of these blocks were completed yielding 176 families for our genetic analysis. The eggs from each female were divided
into 4 pairs of containers with ~30 eggs (mean = 30.05 ± 0.18 SE;
median = 30; range 18–38; N = 352) in each. The eggs were fertilized
with 60 µL of ~107 sperm mL−1 of ejaculate from each male. The
sperm from each male were divided between each of the 4 females
(2 replicates each). Response variables included 1) egg development
(defined above) and 2) “early metamorphosis” (i.e., the proportion of
eggs that developed and that then reached the juvenile stage (metamorphosis) by 45 days (from the time the cross was made). Most fertilized eggs take longer than 45 days to reach metamorphosis, so this
is a measure of early metamorphosis, and 3) juvenile size at 60 days.
Juvenile size (prosoma width [mm] measured at the widest point) was
measured with calipers for 3 random juveniles from each cross.
Note that for all experiments described above, if a female or
male did not produce enough gametes for a complete block,
they were not used in the experiment.
Experiment 1: postmating benefits of multiple
mating for monandrous and polyandrous
females
We compared egg development for both female types (monandrous
and polyandrous) when crossed with their respective attached males
to their developmental success when crossed with 2 randomly chosen satellite males (Figure S2-A; conducted 3–8 May 2008 and
12–26 March 2009). The eggs from each female were divided
into replicate containers with ~30 eggs (mean = 27.5 ± 0.26 SE;
median = 29; range 16–37; N = 315) in each. The eggs were fertilized with 60 µL of ~108 sperm mL−1 from each male. Three
females were included in each block, and a total of 7 blocks were
completed for each female type. We made 2 (2008) or 3 (2009) replicates for each of the 3 crosses with the different males (attached
male, 2 satellite males). We measured the mean egg development
(as defined above) for each female with each male.
Experiment 2: postmating benefits of multiple
mating for tolerant and intolerant monandrous
females
To identify monandrous females as tolerant or intolerant, we
located a monandrous pair nesting on the beach (i.e., the female
was well buried in the sand) and allowed them to nest for an additional 2 min to confirm that they were nesting and that no unattached males were going to spontaneously approach the pair. We
then guided a nearby, unattached male toward the nesting pair
from 1–10 m away. Once this male joined the nesting pair as a satellite, we observed the behavior of the nesting female. If she left
the beach in less than 8 min after the addition of the satellite then
she was considered “intolerant” and if she stayed past 8 min she
was considered “tolerant.” We chose the 8-min threshold because
a previous study at the same site demonstrated that 8 min was the
median time for a monandrous female to leave the beach when
a satellite approached (Johnson and Brockmann 2010). We compared egg development (defined above) from each intolerant and
tolerant monandrous female when crossed with her attached male
compared with a random satellite male (Figure S2-B; conducted 13
March to 25 April 2009). The eggs from each female were divided
into replicate containers with ~30 eggs (mean = 29.6 ± 0.18 SE;
median = 30; range 19–38; N = 200) in each. The eggs were fertilized with 60 µL of ~107 sperm mL−1 from each male. A total of 25
blocks were conducted, with 2 replicate fertilizations performed for
each cross.
Experiment 3: breeding design—good genes
and/or compatible genes
The breeding design (Figure S2-C; conducted 9–26 March
2009) involved blocks of 4 × 4 factorial crosses (North Carolina Type
II breeding design; Lynch and Walsh 1998). In each block, 4 randomly chosen females (2 monandrous; 2 polyandrous) were crossed
Statistical analysis
All analyses were conducted using R. v. 2.15.2 (R Core
Development Team 2013) unless otherwise specified. To investigate how female status and male status influenced developmental success in Experiments 1 and 2, we used a generalized linear
mixed-effects model (GLMM) with a logit-link function and binomial error structure. GLMMs were implemented in the lme4 package using the lmer function (Bates et al. 2009) in R. The response
variable was a binomial vector where the binomial numerator was
the number of developed eggs and the denominator the number of
undeveloped eggs. We fitted an interaction between female status
(monandrous or polyandrous) and male status (attached or satellite)
as fixed effects, along with sperm concentration as a covariate (centered to facilitate the interpretation of interaction terms; Schielzeth
2010). We included female ID, male ID, year, block, and an additive dispersion parameter (to account for overdispersion in our
data; Nakagawa and Schielzeth 2010) as random factors in both
models. We looked for differences in male and female carapace size
or condition using linear models and Wilcoxon tests. Note that we
were missing sperm concentration data or size/condition data for a
few individuals.
For Experiment 3, we fitted a GLMM with a logit-link function to
partition variance of each response variable (eggs developed, early
metamorphosis, and mean juvenile size) into male, female, and their
interaction. The “eggs developed” response variable was a binomial
vector where the binomial numerator was the number of developed
eggs and the denominator the number of undeveloped eggs. The
“early metamorphosis” response variable was a binomial vector
where the binomial numerator was the number of juveniles and the
denominator the number of developed eggs. We included sperm
concentration (centered by scaling) as a covariate and female status
as a fixed effect. We fitted male, female, male × female, and block
as random factors in the model and estimated variance components
using a Laplace approximation (binomial response variables) or
restricted maximum likelihood procedure. We used log likelihood
ratio testing to evaluate the significance of the random effects.
Standard errors of the variance components were estimated using
ASReml v. 3.0.5 (VSN International, Hemel Hempstead, UK).
We further examined relationships between the proportion of eggs
developed, proportion of early metamorphosis, and juvenile size
using correlation coefficients (following a similar method to Evans
Behavioral Ecology
1222
et al. 2007). Correlations of the entire data set are subject to pseudoreplication (because there were only 4 uniquely independent male
× female combinations per block). We averaged response variables
across the 2 replicate samples for each male × female combination
and conducted 20 000 randomizations in which we randomly drew
4 uniquely independent male × female combinations from the 11
blocks (a total of 4 194 304 possible combinations, n = 44 for each
combination). We calculated the mean and 95% confidence limits for
the correlation coefficients generated by the possible combinations.
Finally, we tested whether there were differences in carapace size
or condition between the different male and female types.
Results
Experiment 1. Postmating benefits for monandrous
and polyandrous females
The difference in developmental success between attached and satellite males was greater for polyandrous females than for monandrous
females (GLMM logit estimate ± SE = 0.759 ± 0.191, z = 3.977,
P < 0.0001; Figure 1). This difference is driven by the significant
difference in developmental success for eggs from polyandrous
females that were fertilized by sperm from their attached males or
from satellite males (GLMM logit estimate ± SE = −0.804 ± 0.141,
z = −5.719, P < 0.0001; Figure 1). In contrast, there was no significant difference in developmental success of eggs from monandrous
females that were fertilized by sperm from their attached male or
from satellite males (GLMM logit estimate ± SE = 0.045 ± 0.129,
z = 0.352, P = 0.72; Figure 1). Hence, we found a marginally
nonsignificant difference between monandrous and polyandrous
females when crossed to their attached males (GLMM logit estimate ± SE = −0.679 ± 0.361, z = −1.880, P = 0.06; Figure 1) but
no significant difference when crossed to satellite males (GLMM
logit estimate ± SE = −0.080 ± 0.343, z = −0.233, P = 0.816;
Figure 1). There was no significant effect of sperm concentration
on developmental success in our model (GLMM logit estimate ±
SE = 0.007 ± 0.058, z = 0.124, P = 0.902). See Table S1 for estimates of random effects that were included in the model.
Figure 1 Experiment 1: Mean (± SE) developmental success with attached (black
bars) and satellite (white bars) males for monandrous (left) and polyandrous
(right) females. A total of 21 females of each type were crossed to their
respective attached males and to 2 random satellite males.
The difference in carapace size between satellite males and the
attached males of monandrous females was not significantly different than the size difference between satellites and the attached
males of polyandrous females (generalized linear model [GLM]
estimate ± SE = 0.42 ± 0.53, t = 0.802, P = 0.42, df = 67). Likewise,
there was no significant difference in condition between satellites
and the attached males of monandrous females (Wilcoxon Test
Statistic = 205.5, P = 0.12, N = 34) or satellites and the attached
males of polyandrous females (Wilcoxon Test Statistic = 196,
P = 0.21, N = 35). There was also no significant difference in carapace size (GLM estimate ± SE = 0.34 ± 0.55, t = 0.63, P = 0.54,
df = 41) or condition (Wilcoxon Test Statistic = 232, P = 0.77,
N = 42) between monandrous and polyandrous females. See
Table 2 for descriptive statistics.
Experiment 2. Postmating benefits for tolerant
and intolerant females
The developmental success of eggs from intolerant females was not
significantly different when using sperm from their attached males
(45.2 ± 3.43% SE, N = 25) compared with sperm from random
satellite males (48.1 ± 4.39% SE, N = 25; GLMM logit estimate
± SE = −0.143 ± 0.120, z = −1.197, P = 0.231). Likewise, there
was no significant difference when eggs from tolerant females
were fertilized by their attached males (48.1 ± 3.42% SE, N = 25)
compared with random satellite males (43.4 ± 3.34% SE, N = 25;
GLMM logit estimate ± SE = −0.188 ± 0.122, z = −1.534,
P = 0.125). The difference in developmental success between eggs
fertilized by attached versus satellite males was marginally greater
(though nonsignificantly) for tolerant females than for intolerant
females (GLMM logit estimate ± SE = 0.330 ± 0.170, z = 1.939,
P = 0.052). Overall, we found no significant difference between
tolerant and intolerant females when their eggs were fertilized by
attached males (GLMM logit estimate ± SE = −0.144 ± 0.238,
z = −0.603, P = 0.546) or satellite males (GLMM logit estimate
± SE = −0.188 ± 0.241, z = −0.775, P = 0.438). There was a
Table 2 Mean (± SE) carapace and median condition of horseshoe crabs
used in each experiment
Variable
Carapace
width (cm),
Mean ± SE
Condition
indexa,
Median
N
(missing)
Monandrous attached males
Monandrous satellite malesb
Polyandrous attached males
Polyandrous satellite males
Monandrous females
Polyandrous females
Intolerant attached males
Tolerant attached males
Satellite malesc
Tolerant females
Intolerant females
Monandrous females
Polyandrous females
16.25 ± 0.24
15.75 ± 0.31
16.45 ± 0.23
16.37 ± 0.27
21.62 ± 0.30
21.96 ± 0.45
15.77 ± 0.26
16.09 ± 0.17
16.43 ± 0.17
21.2 ± 0.22
21.57 ± 0.28
21.80 ± 0.30
21.8 ± 0.29
17.5
16
17
16
15
16
18
16
15
16
14
16
17
20 (1)
14 (1)
21
14
21
21
24 (1)
25
22 (3)
24 (1)
25
22
22
The sample size, N, for each descriptive statistic and the number of missing
observations (missing) are provided.
aCondition index ranging from 4 to 20, with the highest condition individuals
receiving a 20.
bSatellite males that were compared with monandrous females in this
experiment.
cSatellite males that were used in Experiment 2.
Johnson and Brockmann • Indirect benefits of polyandry in horseshoe crabs
1223
marginally nonsignificant effect of sperm concentration in our
model (GLMM logit estimate ± SE = 0.114 ± 0.067, z = 1.703,
P = 0.089), suggesting that developmental success tended to increase
with increasing sperm concentration, though the mean difference
in sperm concentration between male types was minor (Table 1).
See Table S2 for estimates of random effects that were included in
the model.
There was no significant difference in carapace size (GLM estimate ± SE = 0.37 ± 0.36, t = 1.03, P = 0.31, df = 48) or condition
(Wilcoxon Test Statistic = 367, P = 0.18, N = 42) between tolerant and intolerant females. In contrast, we detected a marginally
significant difference in carapace size between the attached males
of intolerant females and the random satellite males used in this
design (Student’s t-test, P = 0.03) but no significant difference in
carapace size between these same satellite males and attached
males of tolerant females (P = 0.26). Similarly, we found a significant difference in condition between the attached males of intolerant females and the random satellite males we used (Wilcoxon test,
Z = −2.74, P = 0.006) but no significant difference between the
attached males of tolerant females and these same satellite males
(Z = 1.30, P = 0.19). Finally, as in our previous work (Johnson
and Brockmann 2012), we found a significant difference in condition between the attached males of tolerant and intolerant females
(Z = −2.00, P = 0.046). See Table 2 for descriptive statistics.
success (measured at 20 days) ranged from 0% to 84.5%
(mean = 42.8 ± 1.42 SE, N = 176). Metamorphosis by 45 days
ranged from 0% to 63.6% (mean = 6.45 ± 0.76 SE, N = 171).
Juvenile size (carapace width in millimeter) ranged from 4.51 to
6.10 mm (mean = 5.05 ± 1.49 SE, N = 166). Sperm concentration
of the males used in the experiment ranged from 2.84 × 109 to
9.41 × 109 sperm mL−1 (mean = 6.56 × 109 ± 1.21 × 108 SE,
N = 176). There was no significant effect of sperm concentration
on developmental success or juvenile size, but we did detect a
marginally nonsignificant, positive effect on early metamorphosis
(Table 3). There was no significant effect of female status on
developmental success or juvenile size, but we did detect a
marginally nonsignificant effect on early metamorphosis (Table 3),
with a mean of 8.02 ± 1.24% of offspring from monandrous
females (N = 85) offspring reaching early metamorphosis compared
with only 4.70 ± 0.72% of the offspring of polyandrous females
(N = 86).
For developmental success, Male, Female, and Male × Female effects
explained 24.22% of the phenotypic variation (Table 3). The factorial analysis revealed a particularly strong and significant Female
effect on developmental success (Table 3), accounting for 20.85%
of the variance for this trait, which includes both potential additive genetic effects and maternal effects such as egg nutrients
(Pitcher and Neff 2007). The Male variance component was nonsignificant, indicating that there were no additive genetic effects
on developmental success. The Male × Female variance component
was significant, accounting for 2.68% of the phenotypic variance in
developmental success, suggesting there were nonadditive genetic
effects on developmental success.
Experiment 3. Breeding design: good genes and/
or compatible genes
In Experiment 3, we had 3 measures of developmental success
for the 10 578 eggs assayed from 176 crosses. Developmental
Table 3 Experiment 3: sources of variation in 3 measures of reproductive success from in vitro fertilization experiments: fertilization (and
early developmental) success, proportion of embryos that metamorphosed into juveniles within 45 days, and juvenile size measured
at 60 days
Developmental
success (eggs
developed by
day 20)
Early metamorphosis (juveniles
by day 45)
Mean juvenile
size (mm)
Variance components
Likelihood ratio test
Effect
Variance
LogLik
Sperm concentration
Female status
Block (11)
Male (44)
Female (44)
Male × Female (176)
Error
Sperm concentration
Female Status
Block (11)
Male (44)
Female (44)
Male × Female (171)
Error
Sperm concentration
Female status
Block (11)
Male (44)
Female (42)
Male × Female (166)
Error
GLMM estimate = 0.062 ± 0.052, z = 1.219, P = 0.233
GLMM estimate = 0.298 ± 0.295, z = 1.008, P = 0.313
0.020
0.142
−430.17
0.026
0.030
0.021
−431.69
3.05
0.908
0.245
−493.25
126.19
0.117
0.028
−458.12
55.926
π2/3
GLMM estimate = 0.272 ± 0.142, z = 1.920, P = 0.055
GLMM estimate = −0.580 ± 0.296, z = −1.959, P = 0.050
0.046
0.287
−290.73
0.059
0.425
0.240
−297.28
13.146
0.676
0.333
−304.08
26.744
0.310
0.162
−299.58
17.758
π2/3
0
GLMM estimate = −0.008 ± 0.033, t = −0.63, P = 0.533
GLMM estimate = 0.0137 ± 0.0375, t = 0.37, P = 0.717
0.0042
0.0038
72.784
2.012
0.0008
0.0014
73.652
0.276
0.0105
0.0038
63.476
20.629
0.0017
0.0031
73.733
0.116
0.0283
0.0034
SE
χ2
P
0.872
0.081
<0.0001
<0.0001
0.808
0.0003
<0.0001
<0.0001
0.156
0.599
<0.0001
0.734
Phenotypic
variation
(%)
0.69
20.85
2.68
75.77
9.02
14.35
6.58
70.05
1.94
25.42
4.12
65.52
Developmental success and early metamorphosis were modeled as binomial data. Male, female, and block were treated as random effects in the model. Variance
components were estimated using Laplace approximation (developmental success and early metamorphosis) or restricted maximum likelihood (juvenile size).
Standard errors (SE) are square roots of the large samples’ variances calculated using ASReml. Significance of variance components was tested using likelihood
ratio tests. Phenotypic variance (%) was based on the total phenotypic variance excluding block as this was considered a nongenetic/design factor. Note that π2/3 ≈
3.3 is the underlying residual variance of a binomial model with logit link function (Nakagawa and Schielzeth 2010). Values in boldface represent significant effects.
Behavioral Ecology
1224
For early metamorphosis (the number of individuals that metamorphosed by 45 days), Male, female, and Male × Female component
explained 29.95% of the phenotypic variance (Table 3). The Female
and Male and Male × Female components were all significant, representing 14.35%, 9.02%, and 6.58% of phenotypic variance, respectively, in early metamorphosis.
Finally, for juvenile size, Male, Female, and Male × Female explained
31.47% of the phenotypic variance (Table 3). The Male component and the Male × Female component were nonsignificant, indicating no or little additive or nonadditive genetic effects on juvenile
size. Female effects accounted for ~26% of the phenotypic variance
in juvenile size.
We also examined the relationships between developmental success, early metamorphosis, and juvenile size. We found a positive
relationship between developmental success and early metamorphosis (Table 4). In contrast, we found no significant relationship
between developmental success and juvenile size or between early
metamorphosis and juvenile size (confidence interval at α = 0.05
included zero; Table 4).
There was no significant difference between monandrous
and polyandrous females in carapace size (GLM estimate ±
SE = 0.26 ± 0.42, t = 0.63, P = 0.53, df = 43) or condition
(Wilcoxon Test Statistic = 215.5; P = 0.54, N = 44). See Table 2 for
descriptive statistics.
Discussion
Experiment 1 demonstrated that polyandrous females had lower
developmental success with their attached male compared with random satellite males, whereas monandrous females had equal success with their attached male and random satellite males (Figure 1).
This result supports our hypothesis that there are postmating, indirect genetic benefits accruing to polyandrous females from multiple
mating through improvements in offspring development, whereas
monandrous females would have no such gains were they to mate
with satellite males. This result helps to explain why polyandrous
females attract satellite males when the presence of those males is
costly, that is, when females nest longer and lay more eggs per nesting attempt without satellites (Johnson and Brockmann 2010). The
previous study also demonstrated experimentally that multiple mating would be costly for monandrous females if they were to attract
Table 4 Experiment 3: mean correlation coefficients for relationships
between proportion of eggs developed, proportion of early
metamorphosis, and mean juvenile size from NCII breeding
design
Variables
Proportion eggs developed and
proportion early metamorphosis
Proportion eggs developed and
juvenile size
Proportion early metamorphosis and
juvenile size
Mean correlation coefficient
(95% confidence limitsa)
0.279 (0.055, 0.444)
−0.086 (−0.374, 0.199)
0.088 (−0.126, 0.307)
Mean correlation coefficients from 20 000 randomizations of unique male
× female crosses. Each correlation coefficient has a sample size of 44,
the number of independent values that can be extracted from the 4 × 4
factorial units across 11 blocks.
aConfidence intervals of mean correlation coefficients that include zero are
nonsignificant.
satellites and here we show that they derive no additional, compensatory benefits through improved developmental success of their
offspring as do the polyandrous females when nesting with satellites.
We conducted Experiment 2 because we thought that monandrous females might be a heterogeneous grouping in which tolerant
monandrous females might be polyandrous females that had not as
yet attracted satellite males. We found, however, that tolerant and
intolerant monandrous females did not differ in success when their
eggs were fertilized by their attached male compared with random
satellite males. If anything, the trend was in the opposite direction:
developmental success tended to be higher when eggs were fertilized
by satellite males compared with attached males of intolerant females,
whereas developmental success tended to be lower for satellite males
compared with the attached males of tolerant females. Taken together
then, this may mean that only truly polyandrous females (those that
have attracted satellites) gain from satellite male fertilizations and that
intolerant females are somehow better able to resist sexual harassment
during nesting than tolerant females (Johnson and Brockmann 2010).
Alternatively, there may be some other compensating benefit, other
than developmental success for tolerant females.
Our results from Experiment 3 demonstrated that both genetic
compatibility and good genes may be important factors in the
benefits that polyandrous females derive from mating with multiple
males. This is particularly interesting as our breeding design was
conducted with random satellite males and not with the female’s
attached males (NCII design assumes individuals are randomly
selected from the population; Lynch and Walsh 1998). Experiment
3 focused on 3 potential fitness traits in the development of
horseshoe crab offspring: egg development by day 20 (i.e.,
developmental success), successful metamorphosis by 45 days
(i.e., early metamorphosis), and early juvenile size. Our analyses
revealed highly significant Female variance components for all 3
measures, with the largest variance observed for developmental
success (Table 3). The strong influence of female effects observed
for developmental success is consistent with a large body of work on
other marine broadcast spawners (Marshall and Evans 2005; Evans
et al. 2007; Levitan 2008) and may be due to consistent differences
in egg size, egg quality, or egg maturity. In turn, such differences
may affect both the likelihood that an egg will be fertilized and
the chance that a fertilized egg will complete the early stages of
development. There is no evidence that the eggs from monandrous
and polyandrous females differ in size (Johnson and Brockmann
2012), and we have no reason to believe that the eggs used in our
experiments were not mature. Females were collected while nesting;
hence, the small subset of eggs collected from their gonopores was
presumably the next batch ready to be spawned by these females.
It is likely that these large maternal effects are due to differences
in egg quality between females. Variation in egg quality may have
several proximate explanations, such as differences in resources
invested in egg production between females (Rudolfsen et al.
2005), but nothing is known about the effects of such variation on
fertilization and/or developmental success in Limulus.
As in other studies (Wedekind 1994; Evans et al. 2007; Ivy
2007), the influence of nonadditive and additive sources of variation depended on which trait was measured. We found a significant
Male × Female interaction and a lack of a Male effect for developmental success, suggesting that no single male is best for all females;
rather, there is individual compatibility resulting in enhanced
developmental success. This finding is consistent with the genetic
compatibility hypothesis because it suggests that postmating sexual
selection favors compatible male–female crosses (García-González
Johnson and Brockmann • Indirect benefits of polyandry in horseshoe crabs
1225
and Simmons 2005; Evans et al. 2007). That said, only 2.68% of
phenotypic variation was attributed to nonadditive variation in this
trait for females crossed to satellite males, and the main Male effect
was marginally nonsignificant. These effects may have been stronger had we included attached males in our breeding design.
Our analysis of the number of offspring that reached metamorphosis in 45 days (i.e., those that showed rapid development and
early metamorphosis) revealed that the component of variance
associated with Males was significant, accounting for ~9% of the
observed phenotypic variance for this trait (Table 3). Likewise, we
identified a significant component of variance associated with Male
× Female interaction, accounting for 6.58% of the variation in this
trait. We also found that early metamorphosis (by 45 days) and
developmental success (at 20 days) were positively correlated; thus,
male × female combinations that were most likely to result in successful egg development provided offspring with the fastest development through to metamorphosis (Table 3). This positive relationship
suggests further support for postcopulatory female choice for compatible males and/or high-quality males (good genes). If our measure of developmental success represents true fertilization success,
then this would mirror similar findings in other broadcast spawning
species (Dziminski et al. 2008; Evans et al. 2007).
It is important to note that although it is not known whether
early metamorphosis has fitness implications for horseshoe crabs,
it is reasonable to assume that the heavily armored juveniles that
feed are more likely to survive than the nonfeeding, unarmed larvae. Consequently, variation in settlement rates is likely to be an
important target for natural selection in horseshoe crabs.
The significant Male effect on early metamorphosis suggests that
postmating sexual selection is acting on females when they are
paired with a low-quality male. This result is particularly interesting as we only used satellite males in our crosses; hence, we would
expect to see even larger male effects had we conducted crosses
with attached males as in Experiment 1. Unfortunately, we have
little information about what constitutes a “good quality” male
horseshoe crab. We did observe a marginally nonsignificant effect
of sperm concentration on early metamorphosis, such that early
metamorphosis tended to increase with increasing sperm concentration. Sperm concentration could be an indication of quality in
horseshoe crabs but could also reflect the degree of sperm competition a male encounters (Simmons et al. 1999; Neff et al. 2003;
Sasson et al. 2012).
Interestingly, we also found a marginally significant effect of
female status on early metamorphosis (P = 0.05), with polyandrous
females having a somewhat lower proportion of their offspring
achieving early metamorphosis than monandrous females (8% vs.
4.7%). This difference is consistent with our previous research that
demonstrated condition-dependent differences between monandrous and polyandrous females and context-dependent differences
depending on the male that was attached to the female (Johnson
and Brockmann 2012).
Finally, our analysis of juvenile size revealed no significant Male
or Male × Female interaction effects, suggesting that neither good
genes nor compatible genes effects are important for this trait.
However, we only measured 3 individuals per male–female cross,
which may have obscured our ability to detect variation in this trait.
It is unclear why our mean developmental rate is only ~50% in
our in vitro fertilization experiments, but this value is consistent
with other studies using similar methods (Brown and Knouse 1973).
If all eggs were fertilized, then developmental success reflects offspring viability, and we presume that genetic elements during the
early developmental phase are responsible for the variation that we
observed in developmental success (García-González 2008). If on
the other hand, developmental success represents true fertilization
success, then gamete quality or gamete interactions at zygote formation are likely responsible for the variation in egg development
that we observed. Moreover, differential viability and fertilization
success are not mutually exclusive; hence, our measure of developmental success could be due to genetic elements during the early
developmental phase and to factors that affect zygote formation
during the fertilization phase (García-González 2008).
If developmental success does reflect fertilization success, then
possible explanations for a low fertilization rate include sperm–
egg ratio, maturity of eggs or sperm, lack of oviducal fluids, the
electrical stimulation used to obtain sperm and eggs, timing of
fertilizations, or salinity. We used sperm concentrations that were
optimum based on our initial serial dilution experiment (Figure S1).
There is no evidence for polyspermy in this species (Brown and
Knouse 1973), and fertilization increased with increasing sperm
concentrations in our serial dilution experiment (Figure S1). Sperm
may have been limiting, though we did observe development
between 80% and 90% in a number of crosses. Further, we would
have expected to see a stronger influence of sperm concentration on
developmental success if sperm were limiting in our experiments.
There is no reason to think that the small amount of eggs and
sperm obtained for our in vitro fertilization experiments would
be immature as they are nearest to the gonopore and presumably
would have been naturally spawned if the animals had been allowed
to continue nesting. It is possible that the electrodes may have
damaged eggs during collection, but there is no evidence to suggest
that this is an issue (Clapper and Brown 1980a, b). The timing of
fertilizations could lead to lower fertilization success if there were
a narrow window for egg receptivity (Brown and Clapper 1980),
but our experiments were conducted within the established range
for fertilizing eggs (Brown and Knouse 1973). Salinity is known to
influence development in horseshoe crabs (Ehlinger and Tankersley
2004), with the optimal range of 25 and 30 ppt (Ehlinger and
Tankersley 2003). Our experiments were conducted with 33–35 ppt
seawater, so this may have contributed to lower fertilization success.
Regardless, our protocol was the same across all experiments, so
this could not be an explanation for the differences we found.
Our in vitro fertilization methods result in oviducal fluids being
removed or diluted, though we did not observe any appreciable
amount of fluid, except in a few older females that were in poor
condition (personal observation). Studies have previously demonstrated that oviducal fluids affect sperm motility in both competitive
and noncompetitive situations, primarily in fish (Rosengrave et al.
2008; Gasparini and Pilastro 2011). Horseshoe crab sperm are
not activated by seawater (Clapper and Brown 1980a), but rather
likely by a low molecular weight molecule that diffuses from the
egg (Clapper and Brown 1980b); hence, it is unclear whether oviducal fluids play a role in sperm dynamics in this system. It would,
however, be difficult to measure the importance of ovarian fluid on
sperm dynamics and fertilization success in a biologically meaningful way as horseshoe eggs are fertilized externally in a sand-water
slurry under the nesting pair.
A potential issue with the interpretation of indirect genetic benefits in our experiments is that we were unable to measure fertilization success at the very early stages of egg development; hence, our
presumed indirect genetic benefits might actually reflect direct fertilization benefits (Evans and Marshall 2005; García-González and
Simmons 2007). Horseshoe crab eggs are surrounded by opaque egg
Behavioral Ecology
1226
membranes making it impossible to see the earliest stages of development without the use of dyes or dissection (Sekiguchi et al. 1982);
hence, we can only infer fertilization success based on whether the
eggs develop or not. We have several reasons for favoring indirect
genetic effects of multiple mating over direct fertilization success.
First, our field data suggest that the presence or addition of multiple
males does not influence the number of eggs that develop (Johnson
and Brockmann 2010). Second, there was no effect of sperm concentration on developmental success in any of our experiments.
Third, we have conducted experiments where we fertilize eggs with
1 versus 2 male’s sperm and have found no difference in the proportion of eggs that developed (unpublished data), suggesting that there
is no evidence that multiple mating alone directly increases fertilization success in our study system. Nonetheless, it is possible that some
of the differences in our measures of developmental success may be
due to differences in the direct benefit, fertilization success.
Implications for evolution of polyandry in
horseshoe crabs
It is not clear what if any precopulatory mate choice occurs at the
time of amplexus (attachment of male to female, which occurs
offshore). Amplexus could simply be driven by male choice, and
because females are limited in the time available for spawning and
because they have to have an attached male in order to spawn
(Brockmann 2003b), they may have little opportunity to engage in
premating mate choice. If this is the case, as seems likely, then the
best way for females to control mate choice may be after pairing
through their control of polyandry. Because the eggs of polyandrous females have greater developmental success when fertilized
by satellites rather than by their attached males (Experiment 1) and
because we found significant evidence for potential indirect benefits
(Experiment 3), this suggests that females may actively discriminate
and choose to be monandrous or polyandrous based on the quality
of the male that is attached to them or based on their own genotype. If so, how is this achieved? How could females acquire information about the quality of their attached males? We did not detect
size or condition differences between the attached males of monandrous and polyandrous females in this study or in our previous work
(Johnson and Brockmann 2012), but the attached males of intolerant females were in significantly better condition than attached
males of tolerant females (Johnson and Brockmann 2012), although
we found no difference in the developmental success of eggs fertilized by these males (Experiment 2). Perhaps females are able to
assess quality based on a male’s sperm traits. Although we found no
significant differences in sperm concentration in this study, recent
work using a larger sample size of all males from the same population has found that the attached males of polyandrous females have
a lower sperm concentration on average than the attached males
of monandrous females (Sasson et al. unpublished data). Is it possible that females know the quality of their attached male’s sperm
and based on that information she attracts satellites or not? Schwab
and Brockmann (2007) showed that satellites often join polyandrous
females before the first eggs were laid (and therefore before sperm
were ejaculated), but these could have been pairs that had nested
together previously. Competitive fertilization experiments to assess
whether sperm quality and/or quantity influence developmental
success for monandrous and polyandrous females would be informative and provide a means to test whether a good sperm process
is important in this system (Evans et al. 2007).
Can females discriminate among attached males based on
their own or the male’s genotype? Mechanisms underlying
compatible gene effects are largely unknown but may include
postcopulatory mate choice to prevent inbreeding or selfing,
to increase heterozygosity or major histocompatibility complex compatibility, or to avoid selfish genetic elements, such as
Wolbachia (Jennions and Petrie 2000; Tregenza and Wedell 2000;
Zeh and Zeh 2003). Limulus polyphemus populations are highly
heterozygous, and there is very little evidence of inbreeding
(King et al. 2005), suggesting that compatible gene effects might
be operating to limit inbreeding in this system. Additional studies assaying the relatedness of monandrous and polyandrous
females compared with their attached males would shed further
light on this hypothesis.
Conclusion
We have shown that polyandrous females crossed with their
attached males have lower developmental success of their eggs
than monandrous females crossed with their attached males.
This suggests that the attached males of polyandrous females
are of lower quality than the attached males of monandrous
females, which may include intrinsic quality and/or compatibility.
Our quantitative genetic breeding design identified significant
nonadditive (compatibility) variation in developmental success and
in early metamorphosis. We further identified significant additive
(good genes) variation in early metamorphosis, suggesting that
male intrinsic quality (good genes) may be an important source
of variation in this trait. Because females come ashore with an
attached male, and because females appear to be consistently
monandrous or polyandrous across multiple nesting periods
(suggesting females have some control over whether they are
polyandrous or monandrous; Johnson and Brockmann 2012), we
were able to conduct our experiments with naturally occurring
monandrous and polyandrous females and natural mating pairs. If
females have knowledge about the quality or compatibility of their
attached males and control over polyandry, then we would expect
that a female should solicit additional mates when her attached
male’s sperm are not compatible with her eggs or when he is of low
quality. Our results suggest that incompatibilities between males
and females and good genes may provide a potential selective
advantage that offsets the costs of multiple mating (Johnson and
Brockmann 2010) in this system. Whether a mating system is
dominated by mate choice for good genes or by compatibility will
have a profound effect on the course of sexual selection and the
maintenance of genetic variation in the population. Like other
studies (Wedekind et al. 2001, 2008; Bang et al. 2006; Evans et al.
2007; Ivy 2007; Pitcher and Neff 2007), we have found that both
good genes and compatibility play a role in the horseshoe crab
mating system, but we know little about how the 2 interact over
time (Neff and Pitcher 2005; Puurtinen et al. 2009). Additional
in vitro studies assaying sperm performance under competitive
conditions combined with estimating male fertilization success
would be useful in evaluating the contribution of both additive
and nonadditive genetic variation to offspring success and the
maintenance of polyandry in this system. Finally, the considerable
female effects on development that we observed in Experiment 3
deserve further consideration.
SUPPLEMENTARY MATERIAL
Supplementary Figures S1 and S2 and Table S1 can be found at
http://www.beheco.oxfordjournals.org/.
Johnson and Brockmann • Indirect benefits of polyandry in horseshoe crabs
Funding
This research was supported by the National Science Foundation
(IOB 06-41750), the University of Florida Division of Sponsored
Research, the Department of Biology, and the UF Marine
Laboratory at Seahorse Key.
We thank our field assistants: N. Montes, N. Williams, S. Kromrey,
N. Eliazar, L. Riegler, S. Steininger, and T. Sentner. We also thank
D. Sasson, M. Smith, and K. Saunders for their scholarly input and assistance in the field. Numerous undergraduate volunteers assisted with field
work and rearing. We thank K. Dodds, S. Nakagawa, and A. Senior for
help with statistical analyses. We also thank our editor and two anonymous
reviewers whose comments considerably improved this manuscript. Our
research was conducted under a permit from the Lower Suwannee National
Wildlife Refuge, J. Kasbohm, refuge manager. We thank H. Lillywhite,
H. Coulter, and Al Dinsmore of the UF Marine Laboratory at Seahorse
Key for their help with field logistics.
Handling editor: Paco Garcia-Gonzalez
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