Determinants of female fecundity in a simultaneous hermaphrodite: the role of polyandry and food availability

Abstract

Classical sexual selection theory assumes that the reproductive success of females is primarily limited by the resources available
for egg production rather than by the number of mating partners. However, there is now accumulating evidence that multiple
mating can entail fitness costs or benefits for females. In this study we investigated the effect of polyandry (i.e., the
mating with different mating partners) and food availability on the reproductive output of the female sex function in an outcrossing
simultaneous hermaphrodite, the free-living flatworm Macrostomum lignano. We exposed virgin worms to different group sizes, a treatment that has previously been shown to affect the level of polyandry
in this species. Moreover, we manipulated the food availability throughout the subsequent egg laying period, during which
the worms were kept in isolation. The number of offspring produced was used as an estimate of female fecundity. We found that
food availability, but not group size, had a significant effect on female fecundity. Additionally, female fecundity was positively
correlated with the number of stored sperm in the female sperm-storage organ at the time of isolation, but it was not correlated
with body or ovary size of the worms. Our results suggest that female fecundity in M.lignano is primarily determined by the resources available for egg production, and not by the level of polyandry, confirming classic
sexual selection theory for simultaneous hermaphrodites.

KeywordsBody size-Food availability-Group size-
Macrostomum lignano
-Multiple mating-Polyandry-Sperm storage

Full text

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RESEARCH ARTICLE
Determinants of female fecundity in a simultaneous
hermaphrodite: the role of polyandry and food
availability
Tim Janicke•Peter Sandner•Lukas Scha ¨rer
Received: 5 February 2010/Accepted: 17 June 2010/Published online: 29 June 2010
? Springer Science+Business Media B.V. 2010
Abstract
females is primarily limited by the resources available for egg production rather than by
the number of mating partners. However, there is now accumulating evidence that multiple
mating can entail fitness costs or benefits for females. In this study we investigated the
effect of polyandry (i.e., the mating with different mating partners) and food availability
on the reproductive output of the female sex function in an outcrossing simultaneous
hermaphrodite, the free-living flatworm Macrostomum lignano. We exposed virgin worms
to different group sizes, a treatment that has previously been shown to affect the level of
polyandry in this species. Moreover, we manipulated the food availability throughout the
subsequent egg laying period, during which the worms were kept in isolation. The number
of offspring produced was used as an estimate of female fecundity. We found that food
availability, but not group size, had a significant effect on female fecundity. Additionally,
female fecundity was positively correlated with the number of stored sperm in the female
sperm-storage organ at the time of isolation, but it was not correlated with body or ovary
size of the worms. Our results suggest that female fecundity in M. lignano is primarily
determined by the resources available for egg production, and not by the level of
polyandry, confirming classic sexual selection theory for simultaneous hermaphrodites.
Classical sexual selection theory assumes that the reproductive success of
Keywords
Multiple mating ? Polyandry ? Sperm storage
Body size ? Food availability ? Group size ? Macrostomum lignano ?
Introduction
Classical sexual selection theory assumes that the reproductive output of females is pri-
marily limited by the resources available for egg production rather than by the number of
mating partners (Bateman 1948). Consequently, females are expected to copulate only
once or a few times to obtain sufficient sperm to fertilize their eggs, especially if mating
T. Janicke (&) ? P. Sandner ? L. Scha ¨rer
Evolutionary Biology, Zoological Institute, University of Basel, Vesalgasse 1, 4051 Basel, Switzerland
e-mail: tim.janicke@gmx.de
123
Evol Ecol (2011) 25:203–218
DOI 10.1007/s10682-010-9402-5

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entails costs, such as time and energy expenses (Daly 1978), a higher predation risk (Rowe
1994), an increased exposure to parasites and infections (Thrall et al. 1997), a reduced
immune function (Rolff and Siva-Jothy 2002), physical injuries (Crudgington and Siva-
Jothy 2000) or a reduced lifespan caused by male accessory gland products (Chapman et al.
1995; Green and Tregenza 2009; but see Priest et al. 2008; Reinhardt et al. 2009). How-
ever, over the last decades empirical studies have revealed that multiple mating in females
is widespread across many animal taxa. There is now accumulating evidence that multiple
mating in females is not only driven by male promiscuity, but may in fact represent an
adaptive behavioural strategy that increases female fitness and often outweighs the costs of
mating (for reviews see Arnqvist and Nilsson 2000; Jennions and Petrie 2000; Hosken and
Stockley 2003; Andersson 2005; Simmons 2005).
Hypotheses for the evolution and maintenance of multiple mating in females either rely
on material (direct) or genetic (indirect) benefits (Reynolds 1996). Material benefits refer to
fitness gains mediated by resources that are provided by males and can therefore be
obtained both from multiple mating with different males (hereafter called polyandry) or
with the same male (hereafter called repeated matings), as long as males do not become
depleted in the commodity that is beneficial to females. In contrast, genetic benefits are
related to genetic diversity among male gametes, which can only be augmented consid-
erably by polyandry (but see Yasui 1997 for an argument that considers genetic diversity
within ejaculates). To avoid confusion we here use ‘multiple mating’ as a general term for
individuals that mate more than once, irrespective of whether this involves ‘repeated
matings’ or ‘polyandry’.
Material benefits of multiple mating encompass resources that females obtain from
males, such as nutrients (e.g., prey items, seminal products), parental investment and/or
protection against conspecifics or predators (e.g., Gwynne 1984; Vahed 1998; Engqvist
2007; reviewed in Reynolds 1996; Arnqvist and Nilsson 2000). Furthermore, the receipt of
sperm can represent a material benefit, if female reproductive success is limited by the
number of sperm that is available to fertilize all the eggs (e.g., Pitnick 1993; Levitan and
Petersen 1995; Fjerdingstad and Boomsma 1998; Diaz et al. 2010). Several empirical
studies across a wide range of taxa have demonstrated that repeated matings are advan-
tageous to females (e.g., Wagner et al. 2001; Fedorka and Mousseau 2002; Schwartz and
Peterson 2006; Klemme et al. 2007), which suggests that direct benefits may promote the
evolution of multiple mating. Likewise, a meta-analysis of 122 studies focussing on female
fitness consequences of multiple mating indicated that direct benefits alone can explain the
evolutionary maintenance of multiple mating in insects (Arnqvist and Nilsson 2000).
Genetic benefits derived from polyandrous mating can also be manifold (for reviews on
genetic benefits see Yasui 1998; Jennions and Petrie 2000; Zeh and Zeh 2001; Simmons
2005). For instance, females might benefit from polyandry by means of a bet-hedging
strategy that lowers the probability of mating only with inferior males (in case of error-
prone mate choice abilities) or with males that carry genes that are maladapted to future
environments (in case of unpredictable environments) (Watson 1991). Furthermore, it has
been argued that females are polyandrous in order to minimize the risk and the associated
fitness cost of fertilization by genetically incompatible sperm (Zeh and Zeh 1996, 1997).
Contrary to the other genetic benefit models the ‘genetic incompatibility hypothesis’
assumes that the interaction between maternal and paternal haplotypes involves fitness
consequences that are non-additive. Given that genetic compatibility is expected to be
required for a normal embryogenesis, the ‘genetic compatibility hypothesis’ not only
predicts a positive effect of polyandry on the quality of the offspring, but also on the
number of viable offspring produced (Zeh and Zeh 1996).
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Empirical studies indicate that there is no consistency in the effect of polyandry on
female fecundity across taxa. For instance, mating with multiple males has been shown to
be beneficial to females in echinoderms (Evans and Marshall 2005), insects (Tregenza and
Wedell 1998; Fedorka and Mousseau 2002; Dunn et al. 2005), fishes (Evans and Magurran
2000) and reptiles (LaDage et al. 2008), but it can also be associated with a reduction of the
female’s reproductive output, as reported for many insect species (e.g., Orsetti and
Rutowski 2003; Bybee et al. 2005; Ronkainen et al. 2010). At the same time, several
studies found no effect of polyandry on female fecundity in insects (e.g., Baker et al. 2001;
Schwartz and Peterson 2006; House et al. 2009).
Compared to this large body of empirical studies on separate-sexed organisms, very
little attention has been placed on the fitness consequences of polyandry in simultaneous
hermaphrodites, i.e. organisms that produce sperm and eggs at the same time. In his
seminal paper on sexual selection in simultaneous hermaphrodites, Charnov (1979)
assumed that Bateman’s principle (Bateman 1948) is also valid for simultaneously her-
maphroditic animals. However, his line of reasoning that ‘‘fertilized egg production by an
individual is limited not by the ability to get sperm, but by resources allocated to eggs’’
(Charnov 1979) differs slightly from Bateman’s principle, because it only refers to an
effect of sperm availability on female fecundity, which is not necessarily related to the
number of mating partners (unless sperm donors get sperm depleted). Therefore, Charnov
(1979) primarily made a prediction for the effect of repeated matings, but not for the effect
of polyandry on female fecundity. Charnov (1979) did not clarify if this difference was in
any way intentional or just the result of a slightly different phrasing. Either way, empirical
tests of the validity of both Bateman’s principle and Charnov’s hypothesis are still scarce
in simultaneous hermaphrodites (but see e.g., Marshall and Evans 2007; Sprenger et al.
2008).
Studying the costs and benefits of multiple mating in simultaneous hermaphrodites
reveals several interesting differences to separate-sexed organisms. First, theoretical
analyses suggest that matings in simultaneous hermaphrodites may often be more harmful
to the female function than expected for separate-sexed animals (Michiels and Koene
2006). This is because in contrast to females, simultaneous hermaphrodites should remate
even if mating entails severe fitness costs for the female function, as long as they can
compensate these costs by a sufficiently high male fitness benefit (Michiels and Koene
2006). Second, in many simultaneously hermaphroditic species mating occurs reciprocally
(i.e., each partner both donates and receives sperm during each mating), which inevitably
links the mating strategies of both sex functions (Michiels 1998). Given that multiple
mating might be beneficial for one sex function, but costly for the other function, a trade-
off between the optimal male and female mating strategies might arise within one indi-
vidual, i.e. being eager to mate in one sex function versus being reluctant in the other sex
function (Bedhomme et al. 2009; Janicke and Scha ¨rer 2009b). Third, empirical data on
female fitness consequences of multiple mating are crucial to resolve a longstanding debate
on the preferred mating role in simultaneous hermaphrodites. Here, the preferred mating
role normally refers to the sex function that on average yields a higher expected benefit
from an additional mating. Contrary to Charnov’s (1979) prediction that simultaneous
hermaphrodites ‘‘copulate not so much to gain sperm to fertilize their eggs as to give sperm
away’’, it has also been hypothesised that hermaphrodites mate preferentially in the female
sex function (Leonard 2005, 2006). To date, empirical tests of these hypotheses have
primarily aimed to demonstrate whether individuals trade male or female gametes, in order
to infer indirectly in which sex role they mate preferentially. On the one hand, there are
studies indicating that simultaneous hermaphrodites trade eggs during mating, which
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suggests an overall preference to donate sperm to fertilize the partner’s eggs, i.e. to mate in
the male role (e.g., Fischer 1980; Sella 1985). But on the other hand, there are also studies
that provide evidence for sperm trading, which may suggest that they copulate primarily in
order to receive sperm, i.e. to mate in the female role (e.g., Leonard and Lukowiak 1991;
Vreys and Michiels 1998; Anthes et al. 2005).
In order to shed light on the importance of Bateman’s principle for simultaneous
hermaphrodites, we studied the effect of polyandry and food availability on female
fecundity in the outcrossing simultaneously hermaphroditic flatworm Macrostomum lig-
nano. Copulations in this species are reciprocal (Scha ¨rer et al. 2004) and it has been shown
that worms are highly promiscuous when they are exposed to multiple potential mating
partners (Janicke and Scha ¨rer 2009a). Furthermore, worms that allocate relatively more
resources towards the male sex function mate more frequently and it has been argued that
multiple mating in this species may primarily be driven by the male sex function (Janicke
and Scha ¨rer 2009b). However, whether multiple mating causes either benefits or costs for
the female sex function is currently unknown. We manipulated the group size and the food
availability of worms to infer how both factors affect female fecundity (measured as the
number of offspring produced). Furthermore, we studied how morphological traits of
the worms and the amount of received sperm in storage relate to female fecundity.
Methods
Study organism
The free-living flatworm Macrostomum lignano (Macrostomorpha, Platyhelminthes) is an
outcrossing simultaneous hermaphrodite of the intertidal meiofauna of the Northern
Adriatic Sea (Ladurner et al. 2005). In mass cultures worms are maintained at 20?C in glass
Petri dishes filled with f/2 medium (Andersen et al. 2005) and fed with the algae Nitzschia
curvilineata. Under these conditions, body length of a fully grown worm reaches on
average 1.5 mm and the generation time is about 18 days. The worms are transparent
allowing non-invasive measurement of internal morphological traits, such as testis size,
ovary size and the size of the seminal vesicle (which stores the produced sperm before it is
transferred to the mating partners). The transparency also makes it possible to count the
sperm that is stored in the female sperm-storage organ (hereafter called ‘antrum’, plural
‘antra’) in vivo. The antrum contains the fertilized egg until it is released through the
ciliated vagina (Ladurner et al. 2005; Vizoso et al. 2010).
Experimental design
To test for an effect of polyandry and food availability on female fecundity we used a
fully factorial design, in which we manipulated both the group size and the food level.
On day 1 we pooled 1,200 adult worms from mass cultures and distributed them equally
to 6 Petri-dishes filled with f/2 medium and a dense algae layer and allowed them to lay
eggs. On day 3 we removed all adults from the Petri-dishes, which assured that their
offspring did not differ by more than 2 days in age. On day 8 we pooled all hatchlings
from the 6 Petri-dishes and isolated 840 individuals in wells of 24 well-plates, which
were filled with 1.5 ml f/2 medium and 0.1 ml of a concentrated algae solution that
guaranteed ad libitum food conditions. On day 29, when all individuals were at least
26 days old, we used these virgin worms to assemble different group sizes in order to
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manipulate the level of polyandry. Specifically, we placed virgin worms for 24 h into
groups of two individuals (hereafter called pairs), groups of three individuals (hereafter
called triplets) and groups of 16 individuals in 24 well-plates under ad libitum food
conditions. For each well-plate we balanced the number of treatments and their positions
on the plate.
On day 30 (i.e., 1 day after group formation), we randomly selected one worm out of
each group, took morphological measurements and assessed the number of stored sperm in
the antrum (see paragraph ‘‘Morphological measurement and counts of stored sperm’’ in
the ‘‘Methods’’ section). Next, each worm was isolated in a well of a 24 well-plate. In order
to manipulate the food availability, worms were randomly assigned either to wells with a
dense algae layer or wells without any algae. Therefore, our manipulation of the food
availability consisted of ad libitum food conditions and a complete lack of any food
resources after mating. Again, the number of treatments and their positions on the well-
plates were balanced among the different well-plates that were used. On days 32, 34, 36, 38
and 44 we transferred all worms to fresh wells, which allowed us to determine the number
of the produced offspring on a temporal scale and which prevented the interaction of adult
worms with their offspring (embryonic development takes 5 days). After transferring
worms to fresh wells we added 0.1 ml of a concentrated algae solution to each old well to
guarantee ad libitum food conditions for the developing offspring after hatching. The
whole experiment was split into two blocks that were temporally separated by 24 h. Each
block initially comprised six replicates for all factor combinations. Blocking had no effect
on any of the parameters that we measured (t-tests and Wilcoxon rank sum tests: all
P[0.4) and it was therefore ignored in all further analyses.
Female fecundity was defined as the number of offspring produced per day after the
isolation of the worms from the group until day 50. Offspring counts were carried out
always 10 days after removal of the parental worm from the well, which ensured that all
offspring had hatched but that none had matured yet to produce their own offspring.
Rationale for group size manipulation
In an earlier study we had placed sperm-labelled focal worms together with one, two or 15
unlabelled individuals and these focal worms were allowed to mate for 24 h under similar
conditions as in the current study (e.g., ad libitum food conditions, same enclosure size).
We could demonstrate that the number of mating partners of the focal worms was posi-
tively affected by the group size. Specifically, the average number of individuals that had
received labelled sperm from the focal worm was 0.9, 1.5 and 5.4 in pairs, triplets and
groups of 16 individuals, respectively (Janicke and Scha ¨rer 2009a). Given that copulations
in M. lignano are always reciprocal, the number of individuals that had received labelled
sperm should correspond exactly to the number of individuals with which focal individuals
copulated in their female sex function. However, the inferred numbers of mating partners
probably represent conservative estimates of the level of polyandry since the presence of
labelled sperm in the antrum of a worm might underestimate the amount of actually
received sperm due to cryptic female choice (Thornhill 1983), sperm displacement (e.g.,
Waage 1979) or passive sperm loss (e.g., Birkhead and Biggins 1998). Furthermore, one
difference in the experimental setup between this study and Janicke and Scha ¨rer (2009a) is
that we here used virgin worms instead of mated individuals. Given that virgins copulate
more frequently than already mated individuals (T. Janicke; unpublished data), we expect
that the difference in the level of polyandry between the different group sizes was, if
anything, higher in the present study than that reported by Janicke and Scha ¨rer (2009a).
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Based on these earlier findings it is very likely that our group size treatment affected the
level of polyandry in the present study. However, it is unclear to which extent our
manipulation also had an effect on the number of repeated matings with the same
individuals.
A number of previous studies on M. lignano showed that these worms are capable of
adjusting their sex allocation (i.e., the resource allocation towards the male versus the
female function) in response to the group size (e.g., Scha ¨rer and Ladurner 2003; Janicke
and Scha ¨rer 2009b; reviewed in Scha ¨rer 2009). In larger groups worms invest more
resources into the male function (in terms of larger testes) at a cost to the female function
(in terms of smaller ovaries), which reduces the female reproductive output (Scha ¨rer et al.
2005; Janicke and Scha ¨rer 2009b). However, a recent study indicated that it takes several
days of exposure to a specific group size to observe a sex allocation response (Brauer et al.
2007), which suggests that the group size manipulation of only 24 h in the current study is
unlikely to have an effect on the sex allocation of the worms.
Morphological measurement and counts of stored sperm
We took morphological measurements 24 h after group formation (directly before isolating
worms into single wells) to test whether worms in different treatment groups were similar
with regard to their morphology and to examine whether the morphology of a worm
correlates with its female fecundity. In particular, we measured body size, testis size and
ovary size. We also measured the size of the seminal vesicle, which can be used as an
estimate of the number of sperm allocated by worms in previous matings (Scha ¨rer and
Ladurner 2003). Moreover, we assessed the number of stored sperm in the antrum to test
whether this parameter was affected by our group size manipulation and if it was related to
female fecundity. On day 50 (20 days after the mating trials) we again counted the number
of stored sperm to confirm that all worms had run out of sperm and therefore where unable
to produce any further offspring.
All morphological measurements were obtained in vivo in a standardized way (Scha ¨rer
and Ladurner 2003; Janicke and Scha ¨rer 2009b). First, worms were anesthetized by
immersing them in a 5:3 mixture of 7.14% MgCl2and f/2 medium for 10 min. Overview
pictures of the entire body, the testes, ovaries and the seminal vesicle were taken after
compressing worms dorsoventrally to a fixed thickness of 35 lm between a microscope
slide and a cover slip of a haemocytometer (Scha ¨rer and Ladurner 2003). We used a Leica
DM 2500 microscope (Leica Microsystems, Germany) to which we connected a digital
video camera (DFK 41AF02, The Imaging Source Europe GmbH, Germany) and took
digital micrographs at 409 for body size and 4009 for testis size, ovary size and seminal
vesicle size. For image acquisition we used the software BTV Pro 6.0b1 (http://www.
bensoftware.com/) and we analysed micrographs using ImageJ 1.42k (http://rsb.info.nih.
gov/ij/). All these morphological measurements are repeatable within individuals (Scha ¨rer
and Ladurner 2003).
Counts of the number of stored sperm in the antrum were carried out directly after
morphological measurements using the same optical devices. First, we gently compressed
worms between a 24 9 50 mm and a 21 9 26 mm cover slip using small plasticine feet on
each corner of the smaller cover slip as a spacer. Then, we mounted the cover slips with the
worm on a modified microscope slide fitted with two raised supports on which the cover
slips could be placed. Thereby, the observer could easily flip the compressed worm from
the dorsal to the ventral view, which is required to properly count all the sperm that are
stored in the antrum. We then recorded QuickTime movies of each antrum at 1,0009
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magnification (using immersion oil) by focussing two times slowly through the entire
organ. Using these movies we later counted the number of stored sperm. All counts were
done by the same observer (T.J.) who was blind with regard to the treatment group. In
order to assess the repeatability of the sperm counts, the number of stored sperm was
assessed twice for all antrum movies except the ones that were recorded 20 days after the
mating trials. The analysis of these repeated sperm counts confirmed a high repeatability of
the number of stored sperm in the antrum using the method described above (intraclass-
correlation coefficient: ri= 0.89, F36,37= 17.66, P\0.001).
Statistical analyses
Initially we aimed to replicate each factor combination 12 times. However, losses during
measuring and pipetting errors reduced our final sample size to an average of 9.3 ± 1.0
replicates per factor combination (pairs/no food, N = 12; pairs/ad libitum, N = 8; triplets/
no food, N = 9, triplets/ad libitum, N = 6; groups of 16 individuals/no food, N = 12;
groups of 16 individuals/ad libitum, N = 9).
We tested whether worms exposed to different group sizes were morphologically
similar with respect to body size, testis size and ovary size, as intended by our experimental
setup. Furthermore, we assessed the effect of group size on the size of the seminal vesicle
(our estimate of the amount of sperm allocated during the mating trials) and the number of
received sperm. This was done using one-way ANOVAs (or Kruskal–Wallis ANOVAs in
case the assumptions for parametric tests were not met). Additionally, we tested whether
body size was correlated with the number of stored sperm in order to explore if this trait
affected the amount of sperm that an individual is capable of storing or able to obtain from
mating partners.
The determinants of female fecundity were assessed using Generalized Linear Mixed
Models (GLMMs) with Poisson error distributions and log-link functions (Venables and
Ripley 2002) to account for deviations from normality and unequal variances between the
treatment groups. In a ‘basic model’, we included group size, food availability and time
since mating as fixed factors, the individual as a random factor and body size and ovary
size as covariates. In addition, we fitted an ‘extended model’, in which we added the
number of stored sperm in the antrum (counted 24 h after group formation) as an additional
covariate to the ‘basic model’. The reason for running two separate models was that the
number of stored sperm could only be assessed from a fraction of all individuals con-
sidered in the ‘basic model’. This was because many antra contained a ripe egg, which
made it impossible to reliably count the number of stored sperm. Therefore, the sample size
in the ‘extended model’ (number of observations = 222, number of individuals 37) was
considerably lower compared to the ‘basic model’ (number of observations = 336, number
of individuals = 56), which means that we presumably had more statistical power to
explain variation in female fecundity using the ‘basic model’. None of all possible two-way
and three-way interaction terms in both the ‘basic model’ and the ‘extended model’
explained a significant amount of variation in female fecundity. Therefore, we excluded all
interaction terms in the final analyses.
All statistical analyses were carried out in SPSS 17.0 (SPSS Inc. 2008) or R v. 2.10.1 (R
Development Core Team 2009). We applied the penalized quasi-likelihood method (PQL)
for both GLMMs (Breslow and Clayton 1993) by using the glmmPQL function imple-
mented in the package MASS for R (Venables and Ripley 2010). Values are given as
means ±1 SE.
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Results
Worms exposed to different group sizes did not differ significantly in body size, testis size
and ovary size when measured directly after the mating trials (one-way ANOVAs: body
size, F2,53= 0.02, P = 0.977; testis size, F2,53= 0.52, P = 0.600; ovary size, F2,53=
0.06, P = 0.943; Fig. 1). This suggests that worms in the different groups were initially
similar with regard to these morphological traits as intended by our random assignment to
the treatment groups. Moreover, this also confirms that the group size manipulation for
24 h had no significant effect on the sex allocation of the worms. In contrast, seminal
Fig. 1 Comparison of a body
size, b testis size and c ovary size
between individuals exposed for
24 h to different group sizes, i.e.
pairs, triplets and groups of 16
individuals. Bars indicate means
±1 SE. Body size, testis size and
ovary size do not statistically
differ between treatment groups
(P[0.05). See text for statistics
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vesicle size was affected by group size (Kruskal–Wallis ANOVA: v2= 10.52, df = 2,
P = 0.005; Fig. 2a). Post hoc comparisons using Mann–Whitney U tests with Bonferroni
correction revealed that worms from groups of 16 individuals had smaller seminal vesicles
compared to worms from pairs (z = -3.23, P = 0.004, N = 41), but that there was no
statistically significant difference between pairs and triplets (z = -1.77, P = 0.232,
N = 35) or between triplets and groups of 16 individuals (z = -1.14, P = 0.764,
N = 36).
Sperm counts conducted directly after the mating trials revealed a high between-
individual variation in the number of stored sperm in the antrum (mean: 28.6 ± 2.1, range:
0–52, N = 37). There was only one individual that did not have any sperm in storage.
The number of stored sperm did not differ between individuals that were exposed to
different group sizes (Kruskal–Wallis ANOVA: v2= 1.02, df = 2, P = 0.601; Fig. 2b)
and it was not correlated with the body size of the recipient (Pearson correlation coeffi-
cient: r = 0.07, P = 0.688, N = 37). Sperm counts that were carried out 20 days after the
mating trials showed that not a single individual still had sperm in storage.
The sum of the offspring produced per worm was on average 5.2 ± 0.7 (range: 0–20).
In total, there were 11 individuals that did not produce any offspring. Excluding these
individuals from the analyses did not qualitatively change the results. We, therefore, report
the statistical results from analyses that include these 11 individuals. GLMMs revealed that
female fecundity was significantly affected by food availability, the time since mating and
the number of stored sperm (Table 1). Specifically, female fecundity was higher in worms
Fig. 2 Comparison of a seminal
vesicle size and b the number of
stored sperm in the antrum (i.e.,
the female sperm-storage organ)
between individuals exposed for
24 h to pairs, triplets and groups
of 16 individuals. Boxes show the
25th percentile, the median and
the 75th percentile, whiskers
denote the 10th and the 90th
percentiles and open circles
indicate outliers. Different letters
indicate significantly different
groups (P\0.01). See text for
statistics
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that were fed ad libitum and that had more sperm in storage after mating (Table 1; Fig. 3a).
Moreover, female fecundity decreased rapidly over time (Table 1; Fig. 3a, b). Group size,
body size and ovary size had no significant effect on female fecundity (Table 1; Fig. 3b).
Discussion
The results of our study suggest that it is not the number of mating partners but the food
availability that has an effect on female fecundity in the simultaneously hermaphroditic
flatworm M. lignano. Worms that were allowed to mate with several different sperm
donors produced a similar number of offspring via their female sex function as individuals
that could mate with only one sperm donor. In contrast, food resources that were available
for egg production had a strong positive effect on female fecundity as predicted by sexual
selection theory for simultaneous hermaphrodites (Charnov 1979).
Given that we used only the number of offspring produced under lab conditions as an
estimate of female fitness, our results are limited to this measure of hatchling production.
It has been argued that non-additive genetic benefits of polyandry are more likely to affect
the number of offspring produced rather than offspring quality (Zeh and Zeh 1996; see
‘‘Introduction’’), and we thus should have been able to detect such genetic benefits.
However, there might also have been genetic benefits of polyandry that we were not able to
detect in this study. Specifically, genetic benefits derived from mating with many
(including overall superior) mating partners might only be apparent when measuring
offspring performance, e.g. offspring size, growth rate, or the offspring’s own reproductive
success (e.g., Ojanguren et al. 2005; Fisher et al. 2006).
In this study we manipulated the level of polyandry by exposing worms to different
group sizes, which has been previously shown to affect the number of mating partners in
M. lignano. Based on these earlier findings, we expect that the average number of mating
partners in pairs, triplets and groups of 16 individuals was at least 0.9, 1.5 and 5.4 indi-
viduals, respectively (Janicke and Scha ¨rer 2009a; see also paragraph on the ‘‘Rationale for
Table 1 Summary of generalized linear mixed models testing the effect of group size, food availability,
time since mating, body size, ovary size and the number of stored sperm on female fecundity
ModelSource Estimate ± SE
dfF-value
P-value
Basic modelGroup size –2,50 0.440.647
Food availability–1,50 10.140.003
\0.001
0.713
Time since mating–5,275 26.79
Body size0.32 ± 1.111,50 0.14
Ovary size-43.29 ± 27.601,502.460.123
Extended modelGroup size–2,300.130.880
Food availability–1,307.360.011
\0.001
0.627
Time since mating–5,18016.80
Body size 0.49 ± 1.30 1,300.24
Ovary size-30.52 ± 31.94 1,300.440.510
\0.001Number of stored sperm0.06 ± 0.02 1,3014.66
Given thatthe numberof stored sperm couldonlybe assessedfor a fractionofall replicates,we report a ‘basic
model’, which does not include the number of stored sperm and an ‘extended model’ in which the number of
stored sperm was added as a covariate (see paragraph ‘‘Statistical analyses’’ in the ‘‘Methods’’ section)
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group size manipulation’’ in the ‘‘Methods’’ section). Therefore, we are confident that we
managed to manipulate the level of polyandry substantially in the current study. Never-
theless, the group size manipulation might not only have influenced the number of mating
partners, but also the number of repeated matings with the same partners, for which we
were unable to control with our experimental setup. Therefore, we can not exclude that our
manipulation of polyandry was confounded by an additional effect of group size on the
number of matings. Until now, we have no data on the effect of group size on the mating
rate for the particular situation in which worms were kept in our experiment. Previous
mating experiments, which were conducted in so-called observation chambers (in which
worms are allowed to copulate in very small drops of culture medium; for details see
Scha ¨rer et al. 2004), revealed no difference in the per capita mating rate between groups of
2, 3 and 4 individuals (T. Janicke, unpublished data). On the one hand, this previous study
shows that repeated matings with the same mating partner clearly do occur in small groups.
On the other hand, the results suggest that the group size has no direct effect on the mating
number of offspring
0.0
0.5
1.0
1.5
2.0
no food
ad libitum
days after mating
246814 20
number of offspring
0.0
0.5
1.0
1.5
2.0
2 individuals
3 individuals
16 individuals
(a)
(b)
Fig. 3 Daily female fecundity after mating shown for a the feeding levels and b the different group sizes.
Data shown are lumped across group sizes in panel a and across feeding levels in panel b. Note that x-axes
are not linearly scaled. Bars indicate means ±1 SE. See text for statistics
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rate in M. lignano. However, we have to clarify that in the current study all mating trials
were carried out in much larger enclosures and the maximum number of worms was
considerably higher, which limits the comparability of both studies.
In the current study we found that the size of the seminal vesicle was smaller in worms
that were exposed to larger groups, which means that worms spent more sperm when they
had the opportunity to mate with more mating partners. The most parsimonious expla-
nation for this effect is a higher mating rate in larger groups. However, the observed effect
could also indicate that worms allocated more sperm per mating in more competitive
situations, as predicted by sperm competition theory (e.g., Parker 1998). Consequently, it is
not possible to infer from our data, whether the group size manipulation had an effect on
the mating rate and therefore on the number of repeated matings. Irrespective of possible
effects on the frequency of repeated matings, we are certain that our experimental setup
induced variation in the level of polyandry between the different group sizes. On the
assumption that the mating rate was constant across all groups, our data indicate that
polyandry has no effect on female fecundity in M. lignano. If the worms mated more
frequently in larger groups, our results suggest that both polyandry and repeated mating
have no positive or negative effect on our measure of female reproductive output. Only if
polyandry and repeated matings have opposing fitness consequences for the female sex
function (e.g., mating with different partners is beneficial but mating several times with the
same partner incurs fitness costs) and if the per capita mating rate differed between groups,
we might have been unable to detect an effect of polyandry on the female fecundity with
our experimental setup.
Studies on the fitness consequences of multiple mating for the female sex function in
simultaneously hermaphroditic animals have primarily focused on the role of repeated
matings rather than the level of polyandry. For instance, in the land snail Arianta
arbustorum repeated matings in the female role lead to an increased number of eggs laid,
but not to a difference in the number of hatchlings produced (Chen and Baur 1993). In
contrast, in the hermaphroditic freshwater snail Lymnea stagnalis it has been shown that
individuals that were allowed to mate in groups lay fewer eggs than isolated individuals
and an experimental manipulation of the number of copulations indicated that this dif-
ference was due to costs associated with mating (van Duivenboden et al. 1985; but see
Koene et al. 2006). Recently, it has been demonstrated that at least a part of the negative
effect of mating rate on female fecundity in L. stagnalis is due to the receipt of seminal
fluids containing male accessory gland products, which presumably suppresses egg laying
(Koene et al. 2009). Although these studies on L. stagnalis did not explicitly differentiate
between effects of polyandry and repeated matings, they suggest that mating with the same
partner can be costly for the female sex function in this simultaneously hermaphroditic
snail.
Probably the most conclusive study on the influence of polyandry on female
reproduction has been carried out in a simultaneously hermaphroditic opisthobranch
Chelidonura sandrana (Sprenger et al. 2008). Similar to our findings, polyandry had no
effect on the total number of egg masses produced and the proportion of fertile eggs.
However, egg capsule volume and larval length was higher in individuals that mated once
with four different sperm donors compared to individuals that mated four times with the
same sperm donor, suggesting that the level of polyandry affected maternal provisioning
(Sprenger et al. 2008). A positive effect of polyandry on female fecundity has also been
reported for the broadcast spawning hermaphroditic ascidian Pyura stolonifera, in which a
mixture of ejaculates from different donors increased the hatching success as a result of an
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elevated fertilization success compared to ejaculates from single sperm donors (Marshall
and Evans 2007).
The positive effect of food availability on female fecundity found in our study is not
very surprising, since resources available for egg production are expected to be crucial for
the reproductive output of the female sex function (Charnov 1979). More surprising is that
the initial body size and ovary size did not significantly predict the female reproductive
output. This might either mean that both traits are indeed not correlated with female
fecundity or that the variation in both morphological traits was too small to find an effect.
The latter explanation is supported by a previous study in M. lignano, which suggests that
if one induces variation in ovary size experimentally, a positive effect on the female
reproductive output becomes apparent (Scha ¨rer et al. 2005).
The only covariate that was correlated with female fecundity was the number of stored
sperm. Individuals that managed to store more sperm produced more offspring. However,
whether this effect is relevant under more natural conditions is questionable, since
fecundity was assessed in isolated worms, which could not replenish their sperm reserves
after the mating trials. Given that the mating rate in M. lignano is relatively high (Scha ¨rer
et al. 2004) and that worms can occur at relatively high densities in the field (K. Sekii et al.,
unpublished data) it remains unclear whether access to received sperm can constrain
female fecundity under natural conditions, a question that should be studied in the field.
Remarkably, the number of stored sperm was unaffected by the group size. Therefore,
the female sex function does not seem to gain direct benefits from multiple mating in terms
of replenishing the own sperm reserves. Instead, variation in the number of stored sperm
was presumably induced by factors that are not linked to multiple mating. For instance, the
size of the female sperm-storage organ might constrain the amount of sperm an individual
is capable of storing and may thereby affect the number of offspring produced. Moreover,
quality traits of the sperm recipient might have an effect on the number of sperm that
sperm donors transfer during copulations as predicted by theoretical models on strategic
sperm allocation (e.g., Reinhold et al. 2002). So far, very little is known about how
intrinsic traits of recipients influence the number of stored sperm in M. lignano. In this
study, we found no correlation between the body size and the number of stored sperm,
which suggests that body size itself and other traits that are size-dependent do not affect the
number of sperm an individual is able to store or obtain from its mating partners.
Although the number of stored sperm was unaffected by group size, individuals in larger
groups allocated more sperm during the mating trials (as inferred from the size of the
seminal vesicle). This clearly suggests that not all sperm that are transferred during mating
are finally stored in the partner. First of all, this could simply be due to passive sperm loss
during egg laying, because fertilized eggs have to pass through the antrum before they are
laid. Similarly, the capacity of the antrum is finite and therefore some of the transferred
sperm may never become stored and therefore get lost passively. Another potential
explanation is sperm displacement, in which individuals actively displace some of the
stored sperm from previous mates. Alternatively, the recipient itself may remove the sperm
out of its own antrum in order to digest them or to bias paternity towards favoured mating
partners. Indeed, after copulating, worms often bend themselves in order to touch their
female genital opening with their pharynx and then appear to suck sperm out of the antrum
(Scha ¨rer et al. 2004). So far we know very little about sperm displacement and cryptic
female choice in M. lignano (for a description of morphologies and behaviours that might
facilitate both processes see Vizoso et al. 2010).
To summarize, in our study food availability but not group size (used as a proxy for the
level of polyandry) had an effect on female fecundity in the hermaphroditic flatworm
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M. lignano. This finding is consistent with classical sexual selection theory (Bateman
1948), which predicts that the female reproductive output primarily depends on the
resources that are available for egg production rather than on the number of mates.
Therefore, our results support the hypothesis formulated by Charnov (1979), that
Bateman’s principle can also be applied to simultaneously hermaphroditic animals.
However, in order to provide an ultimate test of Bateman’s principle for M. lignano, one
needs to assess the fitness benefits of multiple mating for both the female and the male sex
function.
Acknowledgments
referees provided constructive comments on an earlier version of the manuscript. We also thank Lucas
Marie-Orle ´ach and Matthew D. Hall for statistical advice. Finally, we are grateful to Ju ¨rgen Hottinger,
Viktor Mislin and Urs Stiefel for technical support. This study was funded by grants from the Swiss National
Science Foundation to L.S. (3100A0-113708 and 3100A0-127503).
We thank Dita B. Vizoso for technical assistance. Ralph Dobler and two anonymous
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