Blackwell Publishing Ltd Gregariousness and protandry promote reproductive insurance in the invasive gastropod Crepidula fornicata: evidence from assignment of larval paternity potx

Molecular Ecology (2006)

15

, 3009–3021 doi: 10.1111/j.1365-294X.2006.02988.x
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd

Blackwell Publishing Ltd

Gregariousness and protandry promote reproductive
insurance in the invasive gastropod

Crepidula fornicata

:
evidence from assignment of larval paternity

L. DUPONT,

*‡

J. RICHARD,

†

Y-M. PAULET,

†

G. THOUZEAU

†

and F. VIARD

*

*

Evolution et Génétique des Populations Marines, UMR ADMM 7144 CNRS-UPMC, Station biologique, place Georges Teissier, BP
74, 29682 Roscoff cedex, France,

†

Institut Universitaire Européen de la Mer — Université de Bretagne Occidentale, LEMAR UMR 6539
CNRS, Technopôle Brest-Iroise, Place Nicolas Copernic, 29280 Plouzané, France

Abstract
According to the size-advantage hypothesis, protandric sequential hermaphroditism is
expected when the increase in reproductive success with age or size is small for males but
large for females. Interestingly, some protandrous molluscs have developed gregarious
strategies that might enhance male reproductive success but at the cost of intraspecific
competition. The gastropod

Crepidula fornicata

, a European invading species, is ideal for
investigating mating patterns in a sequential hermaphrodite in relation to grouping
behaviour because individuals of different size (age) live in perennial stacks, fertilization

is internal and embryos are brooded. Paternity analyses were undertaken in stacks sampled
in three close and recently invaded sites in Brittany, France. Paternity assignment of 239
larvae, sampled from a set of 18 brooding females and carried out using five microsatellite
loci, revealed that 92% of the crosses occurred between individuals located in the same
stack. These stacks thus function as independent mating groups in which individuals may
reproduce consecutively as male and female over a short time period, a pattern explained
by sperm storage capacity. Gregariousness and sex reversal are promoting reproductive
insurance in this species. In addition, females are usually fertilized by several males (78%
of the broods were multiply sired) occupying any position within the stack, a result
reinforcing the hypothesis of sperm competition. Our study pointed out that mating
behaviours and patterns of gender allocation varied in concert across sites suggesting that
multiple paternities might enhance sex reversal depending on sperm competition intensity.

Keywords

: larvae, microsatellites, paternity analyses, sequential hermaphrodite, social groups

Received 27 January 2006; revision accepted 30 March 2006

Introduction

Sex-allocation theory predicts that sequential hermaphroditism
is expected when the increase in reproductive success with
age or size is faster for one sex than for the other (i.e. the
‘size-advantage’ hypothesis, Ghiselin 1969; Charnov 1982).
Despite theoretical advantages either over gonochorism
(because of a higher lifetime reproductive potential) or
simultaneous hermaphroditism (because of inbreeding
avoidance), only the Gastropoda and Bivalvia have
sex-changing species among the eight molluscan classes

and almost all of these are protandrous (i.e. change sex
from male to female; Wright 1988; Heller 1993). According
to Wright (1988), the fact that most molluscan species are
patchily distributed over space and/or have limited adult
mobility would tend to select for protandry because males
would have limited opportunity for mating (Ghiselin 1969;
Hoagland 1978). Moreover, because fecundity in females
generally increases with body size, Warner

et al

. (1975)
asserted that protandry might be expected to be found in
randomly mating populations, such as group spawners,
where males of all body sizes have similar chances of
fertilizing eggs; while protogyny would be found in the

Correspondence: L. Dupont and F. Viard. Fax: +44 (0) 1752 633102;
E-mail: ;
‡Present address: Marine Biological Association, The Laboratory,
Citadel Hill, Plymouth PL1 2PB, UK.

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L. DUPONT

ET AL.

© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd

case of more selective matings, such as occur in pair formation.
Nevertheless, it is noteworthy that several species of
protandrous molluscs with internal fertilization have
developed strategies of conspecific association (e.g. Hoagland
1978; Collin

et al

. 2005) that most notably enhance male
reproductive success (Levitan 1993) but at the cost of
increased intraspecific competition (Toonen & Pawlik
1994).
As the direction of sex change is closely linked to the
mating system, a better understanding of the evolution of
sex-changing strategies requires knowing more about
mating behaviours and reproductive success in sequential
hermaphrodites (Wright 1988). In their review, Munday

et al

. (2006) have recently shown that sex-changing species
use a great diversity of strategies to increase their repro-
ductive success. For instance, the study by Munoz &
Warner (2004) revealed that multiple paternity and sperm
competition influence patterns of sex change. While
sequential hermaphroditism has been extensively studied
in fishes (e.g. Hoffman

et al

. 1985; Allsop & West 2003;
Munoz & Warner 2003, 2004), rules governing sex reversal
according to reproductive behaviour in molluscs have
received less attention (but see Hoagland 1978; Collin 1995;
Warner

et al

. 1996).
Among gregarious protandrous mollusc species, the
slipper limpet (

Crepidula fornicata

) is ideal for investigating-
reproductive patterns in relation to sequential hermaph-
roditism. The patterns of gender allocation of this gastropod
have been investigated for almost an entire century
(e.g. Coe 1936, 1938); an interest largely due to its grouping
behaviour. This long-lived species (lifetime of a maximum
of 10 years, Blanchard 1995) is typically found in stacks (i.e.
groups of individuals attached to each other, with larger
(older) individuals, usually females, at the base and smaller
(younger) individuals, usually males, at the top; Coe 1936).
Because fertilization is internal and these groups are
perennial, they are likely to constitute independent mating
groups in which sex change occurs according to various
factors including sex ratio, number and size of individuals
within the stack (Coe 1938; Hoagland 1978). The location of

the individuals within a stack remains unchanged over time
except for small individuals (male or immature), poten-
tially imparting an advantage to males directly attached
above a female. Coe (1938) nevertheless speculated on the
importance of small mobile males that might obtain most
of the fertilizations. While it is well established that
sequential hermaphroditism in

C. fornicata

is characterized
by a strong social control of sex change (Collin 1995),
questions concerning mating success and sex reversal are
still largely open (Gaffney & McGee 1992; Collin 1995). For
instance, further studies are required to examine the
timing of sex change, to measure male reproductive success,
to determine the importance of small males and to investi-
gate the possibility of sperm storage and competition.
In marine species, effective (realized) fertilizations are
difficult to observe directly in the field and are often
inferred from copulatory behaviour only, thus neglecting
postcopulatory events (e.g. sperm competition). Mating
outcomes can nevertheless be deduced from molecular
information. The use of microsatellites in parentage analyses
(Jarne & Lagoda 1996) can extend to cases in which a small
amount of tissue is available, as with juveniles (e.g. Viard

et al

. 1997) or larval (e.g. Selvamani

et al

. 2001) gastropods.
So far, paternity analyses have been rare in marine species
except in mammals (e.g. Clapham & Palsboll 1997; Coltman

et al

. 1998; Hoffman

et al

. 2003; Krutzen

et al

. 2004; Garrigue

et al

. 2005), turtles (e.g. Fitzsimmons 1998; Hoekert

et al

.
2002; Moore & Ball 2002; Ireland

et al

. 2003) and fishes (e.g.
Martinez

et al

. 2000; Pitcher

et al

. 2003; Soucy & Travis 2003;
Chapman

et al

. 2004; Naud

et al

. 2004; Petersen

et al

. 2005).
In particular, very few assignments of larval paternity have
been achieved in natural populations of benthopelagic
invertebrates (but see Coffroth & Lasker 1998). Because
larvae of

C. fornicata

are brooded into the pallilal cavity of
the mother before being released as planktonic larvae, they
can be easily retrieved for paternity analyses. To our
knowledge, only one set of paternity analyses, based on an
exclusion procedure, has been carried out on

C. fornicata

in
one native (American) population from Delaware (Gaffney
& McGee 1992). Although it was concluded that multiple
paternity was likely to occur, the lack of power of the
markers used (allozyme loci) prevented a precise assign-
ment of paternity, and thus the extent of multiple paternity
within a stack, reproductive success of young mobile
males and the relation between mating success and sex
reversal could not be assessed in this species.
A second interest in studying mating strategy in

C. fornicata

relates to its successful colonization of Europe. Out of the
104 exotic species that have been introduced along the
Channel and Atlantic coasts of France (Goulletquer

et al

.
2002), only a few are invasive (i.e. exotic species with
significant side effects);

C. fornicata

is prominent among
these emblematic species. Native to western North Atlantic
coasts, this gastropod was repeatedly introduced into
Europe during the 19th and 20th centuries (Blanchard
1997). Sex reversal and social grouping could have partici-
pated to its success as a colonist, for instance by increasing
effective population size as well as the probability of
finding mates (Blanchard 1995; Dupont

et al

. 2003); yet its
mating behaviour has never been directly investigated in
European populations.
Based on five microsatellite loci and a maximum-likelihood
categorical analysis, we performed paternity assignment
of larvae in three French sites invaded by

C. fornicata

. This
study aimed at addressing the following questions and
hypotheses: (i) Does paternity come mainly from the
closest male to the study mother or from the largest male
within the stack? (ii) What is the contribution of young

LARVAL PATERNITY ASSIGNMENT IN SLIPPER LIMPETS

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© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd

mobile males (i.e. proportion of paternity assigned outside
the stack)? (iii) Is there any evidence for sperm storage and
sperm competition, two important features that might
influence reproductive success and sex-reversal strategies?
The inferred mating patterns are discussed in light of the
individual features (size, position and sex at the time of
sampling) of the assigned fathers and the population
characteristics at the sites (sex ratio and size at sex change).

Materials and methods

Collection of adults and larvae

Sampling of adults was conducted by dredging during
the breeding season in June 2003 in three sites (called
populations in the following text; Table 1), separated by
less than 10 km, in the Bay of Brest (Brittany, France), a
semi-enclosed French marine ecosystem where

Crepidula
fornicata

covered 61% of the seabed in 1995 (18 500 tonnes
wet weight; Chauvaud 1998). Fifteen, 11 and 16 stacks

(comprising 86, 87 and 112 adults) were sampled in the
Roscanvel, Keraliou and Rozegat populations, respectively
(Table 1). Six, five and seven stacks in the three populations
were dismantled respectively and, except for the individual
at the base (i.e. the study mother, see below), all the
sampled adults were preserved in 96% ethanol for genetic
analyses. From our previous surveys, the individuals located
at the base of each of these 18 stacks were known to be
females (

c

. 100% of the stacks; L. Dupont & J. Richard,
personal observation, unpublished data) and most probably
with broods because of the sampling season (i.e. in the bay
of Brest the maximum brooding activity occurred in May
to June, with

c

. 80% of females carrying eggs, Richard

et al

.
in press). These females at the base of each stack are
attached to a substratum (e.g. dead shell of

C. fornicata

) and
incubate the capsules between the propodium and neck,
with embryos packed in thin-walled capsules attached to
the substratum by a peduncle (Hoagland 1986). To avoid
disturbances that might alter embryo development within
the capsules, the study females were kept isolated in
aquaria with filtered sea water. A sand filter was used to
avoid the circulation of

C. fornicata

larvae (400–1200

µ

m,
Table 1 Study populations and paternity analyses results. Location, density (L. Guérin, personal communication), sex ratio (F: M;
b
inomial test in parenthesis) and mean number of adults per stack are given. Results of the modal decomposition of size-frequenc
y
distributions are summarized by the number of age groups (i.e. cohorts; N
AG
) and the averaged size and standard deviation (SD) o
f
each age group. Average relatedness within each population (R
pop
), between each parental pair (R
p
) and between each nonparental pair
within stacks (R

np
) are given for each population as well as the percentage of brooding females, the size at sex change (L
50
) and the number
of larvae and broods used in paternity analyses. A synthesis of the results of the paternity analysis is given by (i) the percentage of larvae
with unassigned paternity; (ii) the percentage of larvae for which all individuals in the stack were excluded as potential father; (iii) the
percentage of multiply sired broods; and (iv) the mean number of fathers per brood (N
fathers
; including external fathers). SD, standard
deviation
Roscanvel Keraliou Rozegat
Population characteristics
Location 48°19′810”N 48°22′328”N 48°19′300”N
04°30′700”W 04°25′694”W 04°31′700”W
Density (gm
−2
) 2000 100 600
N adults analysed 86 87 112
Sex ratio 0.96 : 1 (P = 0.489) 0.75 : 1 (P = 0.115) 0.69 : 1 (P < 0.001)
N
adults/stack
± SD 6.86 ± 2.26 8.64 ± 4.05 7.31 ± 1.86
N
AG
24 4
Mode (cm) 6.58–9.32 3.40–6.36–8.86–11.25 4.52–7.02–9.12–10.76
SD (cm) 0.51–1.02 1.29–0.67–1.24–0.79 1.36–0.65–0.61–0.52
R
pop
−0.009 −0.010 − 0.008

R
p
0.023 −0.130 0.107
R
np
−0.023 0.052 0.045
L
50
(cm) 9.1 9.4 9.6
Brooding females 77% 81% 57%
Paternity analyses
N
larvae
(N
broods
) 77 (6) 72 (5) 90 (7)
Unassigned paternity 5.2% 19.4% 23.3%
External paternity 1.4% 8.6% 15.9%
Multiple paternity 50% 80% 100%
N
fathers
± SD 2.2 ± 1.6 2.2 ± 1.1 3.1 ± 0.7

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ET AL.

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Journal compilation © 2006 Blackwell Publishing Ltd

Pechenik

et al

. 2004) but allowed the passage of small
particles (= 30

µ

m) for feeding. After 31 days (i.e. mean
time between egg laying and hatching in

C. fornicata

,
Richard

et al

. in press), all the 18 females had released
larvae. These females and a sample of 239 larvae (77, 72
and 90 larvae from the Roscanvel, Keraliou and Rozegat
populations, respectively) were preserved in 96%
ethanol for subsequent analyses. All the study individuals
were sexed: following Hoagland (1978), the sexual morphs
were determined morphologically, in particular according
to presence or absence of penis. Besides the study mother,
the presence of eggs was also recorded for the other

females of the stacks. In addition, the curvilinear shell
length and the location in the stack of each adult were
registered.

Sex and size analyses at the population level

Percentage of brooding females (calculated over the total
number of females) and female : male ratio were estimated
for each population. Departure from a 1 : 1 sex ratio was
tested using a binomial test (Wilson & Hardy 2002, p. 54).
Effects of sex and population on the size of individuals
were investigated using a mixed-model

anova

using the
general linear model procedure of

minitab

® release 14.1
with the sexual morphotype and the population effect
incorporated into the model as fixed factors.
Size at sex change (

L

50

= size at which 50% of the individ-

uals are female) was calculated for each population using
a logistic regression following Allsop & West (2003). To
analyse the population age structure, a crude demographic
structure analysis was carried out by modal decomposi-
tion of size-frequency distribution using the

mix

2.3
program package (MacDonald & Pitcher 1979; see Dupont

et al

. in press for details).

DNA extraction and microsatellite typing

Total genomic DNA was extracted from all adults and
larvae using Nucleospin®Multi-96 Tissue Kit (MACHEREY-
NAGEL). Samples were genotyped at five loci: four,
namely CfCA2, CfCA4, CfCATGT and CfGT14, as defined
by Dupont & Viard 2003) and one (CfH7) newly developed
locus (Kruse & Viard, unpublished data; forward and
reverse primer sequences: F: 5

′

-GGTAACGTATTGCT-
ACCGAAAG-3

′

and R: 5

′

-TCATGCGGGTTTGGTGG-3

′

).
Loci [including CfH7 (annealing temperature of 54

°

C and
1.5 m

m

of MgCl

2

)] were amplified by polymerase chain
reactions (PCR) according to Dupont & Viard (2003). For
larvae (which yielded a small amount of extracted DNA)
the only modification compared to Dupont & Viard (2003)
was a pre-amplification step with the same protocol before
final amplification. To avoid scoring error, each larva was

genotyped twice.

Paternity and relatedness analyses

At the population level, number of alleles and gene
diversity were estimated using

genetix

version 4.02 (Belkhir

et al

. 2004). Tests for genotypic linkage disequilibria among
loci were computed with

genepop

version 3.3 (Raymond &
Rousset 1995).
The paternity analysis was performed using a maximum-
likelihood-based categorical analysis following Meagher
(1986) with the software

cervus

version 2.0 (Marshall

et al

. 1998). Given the genotypes of offspring, their known
mothers and the candidate fathers, the paternity was
assigned to the most likely father, i.e. the individual with
the highest log-likelihood ratio (LOD score as defined in
Meagher 1986). Computer simulations were used to assess
the statistical significance of LOD scores (10 000 iterations,
based on allelic frequencies of the entire population):
paternity was assigned to the most likely father if the
difference between the LOD score of the most likely father
and that of the second most likely father was statistically
significant (here with an 80% confidence level; Marshall

et al

. 1998). All the sampled individuals of a given population
were considered as candidate fathers. Given the possib-
ility for change of sex in the time interval between copulation
and sampling, this list comprised all the mature individuals
(i.e. males, females and individual in sexual transition),
including the mother, to take into account the possibility
for self-fertilization (as hypothesized by Orton 1950).
The proportion of multiply sired broods was compared
between populations by a Fisher test using the program

struc

(500 000 iterations) of the software

genepop version
3.3. The relation between paternity status and size of

individuals at the population level was investigated using
a mixed-model anova, with the paternity status (father vs.
not father) and the population effect incorporated to the
model as fixed factors. Because the size of the individuals
in a stack depends on the size of the individual at the base
(Coe 1938), individual size was weighted by the size of the
individual at the base of the same stack. To analyse the
genetic relatedness between mates within maternal stacks,
a pairwise microsatellite-based relatedness coefficient R
xy
(Queller & Goodnight 1989) was calculated using identix
software (Belkhir et al. 2002). R
xy
values were compared
among two categories: parental pairs (mother and assigned
father) and nonparental pairs (mother and individuals of
the maternal stack that have not been assigned as father).
Results
Sex ratio and comparison of size within and between
populations
At the level of the Bay, the three populations did not share
common features in terms of gender allocation and
LARVAL PATERNITY ASSIGNMENT IN SLIPPER LIMPETS 3013
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
reproductive status (Table 1). A male-biased sex ratio is
expected in protandrous species (Allsop & West 2004)
including Crepidula fornicata (Hoagland 1978; Collin 1995),
but in our study such a pattern was observed in Rozegat
only (Table 1, binomial test, P < 0.001). This cannot be

explained by the sampling procedure (dredge effects), as
every population was sampled in the same way. Moreover,
only intact stacks (i.e. stacks without mark left by unstuck
individual) were collected. Previous studies carried out in
another Breton population (Morlaix Bay) and aiming at
comparing different sampling methods (scuba diving vs.
dredging) also did not show differences for the mean
number of individuals per stack and sex ratio according to
the sampling procedure (L. Dupont & F. Viard, unpublished
data). This suggests that if some individuals were lost
during dredging, they should be mainly immature
individuals that are small and not firmly attached to the
stack. Although the sex ratio may change during the year
(Richard et al. in press), the relatively high number of
females in Roscanvel is thus likely to be due to a low
recruitment in this population, as revealed by the
demographic analysis: the smallest size-class observed in
the two other populations is absent in Roscanvel (Table 1).
The Rozegat population displayed the lowest number of
brooding females, with only 57% of the females possess-
ing egg capsules (Table 1), suggesting asynchrony in
reproduction at the level of the Bay, population density
effects or different environmental forcing (e.g. contaminants).
As also expected in this protandrous species, a significant
size difference was observed between individuals of the
two sexes (d.f. = 1, F = 141.34, P = 0.000, Fig. 1) over all
populations although varying according to the population
(d.f. = 2, F = 18.49, P = 0.000), with a significant sex–population
interaction (d.f. = 2, F = 8.43, P = 0.000). We also observed
a slight variation of size at sex change (L

50
) across
populations (Table 1). Between Rozegat and Keraliou, this
difference is congruent with the difference of mean size of
males (5.3 ± 2.1 and 5.7 ± 2.1 cm, respectively). However,
Roscanvel showed the lower size at sex change together
with the higher mean size of males (7.9 ± 2.40 cm); a result
due to the lack of small (young) males in the population
and to the strong overlap of male and female size
distributions (Fig. 1).
Paternity analysis
The five loci did not exhibit any linkage disequilibria. Four
of them were highly polymorphic with 10–32 alleles over
the 285 adults analysed, while the fifth locus CfCA2
exhibited three alleles. The three populations displayed a
similar mean number of alleles (from 11.7 to 12.2) and gene
diversity (from 0.768 to 0.777) over loci. This high
polymorphism explained the very high value for exclusion
probability estimated over the whole study (i.e. 285 adult
individuals) reaching 99.5% over the five loci. As regularly
observed with microsatellite loci (e.g. McCracken et al.
1999; Davis et al. 2001; Spritzer et al. 2005) and easily
recognized with paternity analysis when the mother is
genotyped, null alleles were noticed at two loci (CfCATGT
and CfGT14). Taking advantage from the procedure
implemented in cervus, we specified a non-null error rate
so that a true father that mismatched at one or two loci
could still be identified as the most likely parent. This
procedure is reliable, provided that the exclusion power of
the loci is reasonably high (Marshall et al. 1998), which is

the case with our set of loci. As we were interested first in
excluding as many fathers as possible, this procedure was
also conservative. Note that the final assignment made
with the five loci was always confirmed with the three loci
that did not exhibit null alleles and for which maternal
alleles always segregated in accordance with expected
Mendelian proportions.
Results of maximum-likelihood-based paternity analyses
are summarized in Table 1 and detailed in the Appendix.
None of the larvae exhibited a genotype compatible with
maternal self-fertilization. Over the 239 larvae analysed, 39
could not be unambiguously assigned (i.e. with an 80%
Fig. 1 Curvilinear length frequency distribution in the three stud
y
populations. Black, white and grey bars are featuring males,
females and ‘fathers’, respectively, as assigned by paternit
y
analysis.
3014 L. DUPONT ET AL.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
confidence level). They were classified as larvae with
unassigned paternity and removed from subsequent
analyses. Out of the 200 larvae unambiguously assigned to
a father, few involved a male external to the maternal stack
(Table 1). The largest proportion of external-assigned
paternity was found in Rozegat (15.9%). Interestingly,
multiple paternities were revealed in almost all the broods
(Table 1), although the mean number of larvae analysed
per female (12.8 ± 1.9, 14.4 ± 1.9 and 12.9 ± 1.7 in Roscanvel,

Keraliou and Rozegat, respectively) was low compared to
the approximately 25 000 larvae potentially released per
female (Richard et al. in press). The maximum number of
fathers assigned for a given brood was 5 (in Roscanvel, see
Appendix). Differences were nevertheless observed across
populations multiple paternities were observed in all
the broods in Rozegat but in only half of the broods in
Roscanvel. Paternity patterns thus appeared to differ
according to population, although the associated Fisher
test was not significant (P = 0.093).
Size, position within stacks and sex of fathers
Figure 2 shows the sex of the assigned fathers at the time of
sampling. Surprisingly, depending on the population, 13%
to 35% were females, some of them with egg capsules,
suggesting not only that sex changes had occurred since
the time of copulation but also that some of these
individuals had subsequently reproduced as a female.
Another surprising result was the location of the assigned
fathers as compared to the position of the mother within
the stack (Fig. 3A): individuals occupying any position in
the maternal stack can be a father, including males that
were not the first above the mother (Fig. 3B). Figure 3A2
showed, however, that more larvae were sired by larger
males than small males in the Rozegat population.
Analysis of variance showed that fathers are, on average,
significantly taller than nonfathers within a stack (d.f. = 1,
F = 5.21, P = 0.024). In the same way, ‘male fathers’ (i.e.
fathers that were males at time of sampling) are, on
average, significantly taller than ‘male nonfathers’ (i.e.
males of the maternal stack that were not fathers; d.f. = 1,

F = 8.76, P = 0.004). Pairwise average relatedness (R
xy
)
values were not different between parental and nonparental
pairs when considering each population separately (Table 1)
or across the overall study (H = 0.10, d.f. = 1, P = 0.749).
Discussion
Of 200 larvae unambiguously assigned, 183 (91.5%) were
assigned to an individual belonging to the maternal stack.
This pattern holds in the three Breton populations studied,
confirming previous expectations based on observations
of copulatory behaviour (Hoagland 1978). The mating
patterns revealed by the present paternity analysis thus
ruled out the hypothesis that mobile males might obtain
most of the fertilizations by crawling among stacks (Coe
1936, 1938; Wilczynski 1955). In their paternity exclusion
analysis on Crepidula fornicata, Gaffney & McGee (1992)
suspected genetic contributions by individuals not pres-
ent in the stack at the time of collection; a conclusion
unfortunately hampered by the low exclusion power of the
enzymatic loci used. Here, microsatellite loci afforded
considerable improvement over allozyme markers: only
8.5% of the larvae in the study were estimated to be sired
by individuals not present in the stack at the time of
sampling. The true percentage of external fathers might
be even lower as we cannot exclude the possibility of
having lost some ‘candidate fathers’ from the stack since
copulation (for instance during the sampling, i.e. dredging
effects). Besides, the mobile males are young individuals
expected to be in a side position. Seven percent of the

larvae were assigned to four individuals located in a
side position (out of 37 identified fathers) and three of these
were old (i.e. large; one large male, one female and one
individual in sexual transition). Mobile males, if any, are thus
exceptions and mating between individuals within a stack
is the rule.
Our study highlights the importance of gregariousness
in the reproductive success of C. fornicata. The species
forms social groups, with a perennial assemblage of
individuals of various ages and sexes that reproduce with
each other and are not genetically related. Social groups
are common among animals (e.g. Wilson 1975), including
benthic marine invertebrates (e.g. annelids, bivalves and
barnacles; Toonen & Pawlik 2001). Social interactions are
known to have major influence on reproductive strategies
as is well documented for harem systems in protogynous
fishes (Munoz & Warner 2003). Adult aggregations, which
occur in numerous gastropod species (reviewed in Baur
1998), may enhance reproductive success in species with
internal fertilization and low mobility like C. fornicata by
Fig. 2 Percentage of larvae, for which the assigned father was a
male (M), an individual in sexual transition (T), a female (F) or a
b
rooding female [F(b)] at the time of sampling, in each population.
LARVAL PATERNITY ASSIGNMENT IN SLIPPER LIMPETS 3015
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
enhancing the probability for individuals to meet and
realize mating (Baur 1998). The gregarious behaviour is
thus increasing the long list of traits that make C. fornicata

a good colonizer. As an introduced species, the combination
between social grouping, repeated introductions (Blanchard
1997), dispersal ability conferred by pelagic larvae release
(Viard et al. 2006) and a long season of reproduction
(Richard et al. in press) is congruent with a rapid increase
in population density and range expansion following
primary introductions (Sakai et al. 2001). In addition, age
segregation of sex allows reproduction between different
age groups, a pattern favouring outbreeding as well as
temporal genetic homogeneity, as described in Dupont
et al. (in press).
This aptitude to find a mate, in such a species with low
mobility in the adult phase, is exemplified by a striking
pattern observed within stacks: a significant contribution
to paternity by individuals collected as transitional
individuals and females in the three study populations.
Two hypotheses could explain such a result: (i) bisexuality
of the assigned fathers; and (ii) sperm storage by the study
mothers. The former hypothesis (i.e. occurrence of transi-
tory simultaneous hermaphrodites) is highly unlikely for
three reasons: (i) none of the assigned fathers that were
females at time of collection exhibited a penis and thus
none of them could have been a functional male; (ii)
although functional hermaphroditism was once suggested
to occur exceptionally (Coe 1938), to our knowledge, bisexual
individuals have never been documented by histology
studies (see Le Gall 1980; Martin 1985 and references
therein; J. Richard, unpublished data); and (iii) the sequential
changes in morphology and anatomy during sex reversal
in C. fornicata prevent bisexuality (Orton 1909; Coe 1938;

Chipperfield 1951; Martin 1985); for example, the first step
Fi
g. 3
Di
str
ib
ut
i
on
(
percentage
)
o
f
t
h
e
different positions occupied within the
maternal stack by the assigned fathers. (A1)
Position of the father is given relative to the
position of the mother (0 is the mother).
′Side position′ is used for single individuals
recorded in a side position relative to the
main stack. ′Secondary stacks’ refer to a
situation where several individuals are
forming a secondary chain branched on the
primary stack. (A2) Same figure as A1, but
weighted by the percentage of larvae sired.
(B) Position of the father is given relative to
the position of the male (position 0), which

is the closest to the mother at the time of the
collection within the main stack. For
example, positions (−1) and (−2) refer to
females situated in between the studied
mother and the male closest to the mother.
3016 L. DUPONT ET AL.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
is the cytolysis of the spermatogenic tissues; also the distal
part of the gonoduct can develop into a prominent uterus
with folded walls, into which a number of seminal recep-
tacles opens, only when the penis degenerates, allowing
the width of the gonoduct to increase and the inner walls
to become folded longitudinally (Orton 1909; Chipperfield
1951). Conversely, several lines of evidence support the
hypothesis of sperm storage, which was also suspected in
the study of Gaffney & McGee (1992): (i) histological obser-
vations (Martin 1985; J. Richard personal observation); (ii)
experimental data: Hoagland (1978) asserted that ‘females
can store sperm for at least one year’ according to one of
her previous study (Hoagland 1975). In our experiment,
one of the study mothers also produced a second set of
broods after the first hatching indicating that sperm was
stored for at least one month. Finally, even if a rare event
of bisexuality was occurring in the study populations, this
could not explain the large proportion of assigned fathers
(32%, 13% and 35%, respectively, in the Roscanvel, Keraliou
and Rozegat populations) that were females at time of
collection. Sperm storage is thus the most likely explanation
for the observed results. That some fathers are brooding

females at time of collection demonstrates that an individual
can effectively reproduce as both a male and a female over
a relatively short time interval and contribute as both
father and mother to larvae of a given cohort. Sexual trans-
formation lasts 61 days (Coe 1938); a much longer time
than in many protogynous fish species (Reavis & Grober
1999; Sunobe et al. 2005) but similar to other protandrous
species of calyptraeids (Warner et al. 1996; Collin et al.
2005). Egg production lasts 14 days (Chipperfield 1951),
whereas hatching occurs in a minimum of 21 days
(Chipperfield 1951). Consequently, individuals that were
both females with eggs and assigned fathers had been
males and transmitted sperm at least 54 days before.
Sperm storage and gregarious behaviour might thus
work in concert to maximize individual reproductive
success in C. fornicata. However, aggregations might also
reduce individual reproductive success, especially among
males, because of competition for mates (Toonen & Pawlik
1994).
Another clear-cut result of our study is the remarkable
level of multiple paternity, which was observed in 14 out
of 18 broods. On average, two to three fathers, with a maxi-
mum of five fathers, were identified in subsamples of only
11–16 larvae per brood. Although investigations of pater-
nity among gastropods have been largely restricted to
pulmonates (e.g. Baur 1998) with few studies within marine
prosobranchs (Gaffney & McGee 1992; Paterson et al.
2001), multiple paternity has been reported in several
gastropod families, suggesting that multiple copulations
and fertilizations by different males are common (see Baur

1998). In terms of population effective size, multiple paternity
coupled with sex reversal is an advantageous breeding
tactic: the increased number of reproducing males and the
sex-ratio adjustment both enhance the effective population
size (Sugg & Chesser 1994; Martinez et al. 2000). Benefits of
multiple paternity, sex change and social structure are
combined, thus maintaining large genetic diversity as well
as large effective size over time in C. fornicata populations
(Dupont et al. 2003).
As a result of multiple copulations, sperm from different
males may compete to fertilize a single brood (Parker 1970).
In addition, the occurrence of sperm storage increases the
probability of biased paternity. Thus, as a result of multiple
paternity and sperm storage, male–male competition might
indeed occur. When sperm competition occurs, paternity is
frequently determined by the relative number of compet-
ing sperm present from rival males but also by sperm qual-
ity (i.e. sperm size, viability and mobility; review in Snook
2005). In C. fornicata, the quantity of sperm transferred to
the female could be related to the size of the male or to ease
of access to the female. Interestingly, a significant size
difference was observed between fathers and nonfathers in
the three C. fornicata populations, and there was still a size
difference when comparing only males, suggesting that
the most successful males are the largest. In addition,
Fig. 3A2 shows that more larvae have been sired by larger
males than by small males in Rozegat population. This
result means that male fertility is expected to increase with
size, a pattern expected in protandrous species exhibiting
a sex ratio biased towards the first sex (Charnov & Bull

1989). We indeed observed a male-biased sex ratio in
Rozegat population. However, because of the grouping
behaviour of C. fornicata, there is a strong interaction
between age, size, sex and position in a stack. Hence the
largest male was also often the closest to the mother, a
position that may facilitate the copulation. This study was
not designed to distinguish between the effects of size and
position. In addition, only the offspring of females at the
base of the stacks were examined, so that the male con-
tribution to the broods of other females in the stack is
unknown. Further analysis is needed to investigate precisely
the components of male reproductive success.
Recently, the study by Munoz & Warner (2003) of pro-
togynous fish gave new insight into sex-change theory: the
authors showed that social conditions indicative of sperm
competition may cause sex reversal to be deferred because
intense competition can substantially lower the expected
reproductive success of males. Considering that the
likelihood of sperm competition might influence the
timing of sex change, it is likely that population character-
istics such as sex ratio and optimal size at sex change vary
with the intensity of sperm competition. Here, we showed
that sex ratio, demographic structure and mating patterns
varied across the three study populations. In particular,
Rozegat displayed the highest incidence of external assigned
paternity, the highest frequency of multiple paternities
LARVAL PATERNITY ASSIGNMENT IN SLIPPER LIMPETS 3017
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
together with the largest male-biased sex ratio, the largest

size at sex change and the lowest proportion of brooding
females. In this population, sperm competition should increase
as the number or eggs available to fertilize decrease. Under
the assumption that the intense male–male competition
causes sex change to be delayed, it is not surprising to
observe a large optimal size at sex change and a sex ratio
largely male-biased. Conversely, levels of multiple pater-
nity and external paternity were found to be the lowest in
Roscanvel, for which the smaller size at sex change and
a sex ratio close to 1:1 were noticed (Table 1). The fact
that mating patterns and gender allocation patterns varied
in concert across sites suggests that multiple paternities,
reflecting perhaps differential sperm competition
intensity, might enhance sex reversal in C. fornicata. The
occurrence of male–male competition influencing sex
change in protandrous gregarious species might also
explain that species-forming mating groups have more
variation in size at sex change within a population than
solitary species do (Collin 2006). Detailed investigations
of male–male competition and factors determining the
timing of sex reversal in protandrous species are needed to
better elucidate the evolution of sex reversal strategies
especially in gregarious species.
Acknowledgements
This project is part of the 2001 INVABIO program of the Ministère
de l’Ecologie et du Développement Durable (MEDD; project no.
D4E/SRP/01115). Additional support for genotyping and iso-
lation of new markers was obtained from the Network of Excellence
‘Marine Genomics Europe’ (contract n°505403). F.V. acknow-
ledges support from the CNRS (ATIP program). L.D. benefited

from a PhD grant from the Region Bretagne. The authors are also
grateful to J.D.D. Bishop and M. Valero and three anonymous referees
for critical readings, editing and improvements to the manuscript.
References
Allsop DJ, West SA (2003) Constant relative age and size at sex
change for sequentially hermaphroditic fish. Journal of Evolu-
tionary Biology, 16, 921–929.
Allsop DJ, West SA (2004) Sex-ratio evolution in sex changing
animals. Evolution, 58, 1019–1027.
Baur B (1998) Sperm competition in molluscs. In: Sperm Competi-
tion and Sexual Selection (eds Birkhead TR, Moeller AP), pp. 255–
305. Academic Press, London.
Belkhir K, Castric V, Bonhomme F (2002) identix, a software to
test for relatedness in a population using permutation methods.
Molecular Ecology Notes, 2, 611–614.
Belkhir K, Borsa P, Goudet J, Chikhi L, Bonhomme F (2004) GENETIX
4.05, logiciel sous Windows pour la génétique des populations. Lab-
oratoire Génome, Population, Interactions. CNRS UMR 5000,
Université Montpellier II, Montpellier, France.
Blanchard M (1995) Origine et état de la population de Crepidula
fornicata (Gastropoda Prosobranchia) sur le littoral français.
Haliotis, 24, 75–86.
Blanchard M (1997) Spread of the slipper limpet Crepidula fornicata
(L. 1758) in Europe. Current state and consequences. Scientia
Marina, 61, 109–118.
Chapman DD, Prodohl PA, Gelsleichter J, Manire CA, Shivji MS
(2004) Predominance of genetic monogamy by females in a
hammerhead shark, Sphyrna tiburo: implications for shark
conservation. Molecular Ecology, 13, 1965–1974.
Charnov EL (1982) The Theory of Sex Allocation. Princeton University

Press, Princeton, New Jersey.
Charnov EL, Bull JJ (1989) Non-fisherian sex ratios with sex change
and environmental sex determination. Nature, 338, 148–150.
Chauvaud L (1998) La coquille St Jacques en rade de Brest; un modèle
biologique d’étude des réponses de la faune benthique aux fluctuations
de l’environnement. Thèse, Université de Bretagne Occidentale,
France.
Chipperfield PNJ (1951) The breeding of Crepidula fornicata in the
river Blackwater, Essex. Journal of the Marine Biological Association
of the United Kingdom, 30, 49–71.
Clapham PJ, Palsboll PJ (1997) Molecular analysis of paternity
shows promiscuous mating in female humpback whales
(Megaptera novaeangliae, Borowski). Proceedings of the Royal
Society of London. Series B, Biological Sciences 264, 95–98.
Coe WR (1936) Sexual phases in Crepidula. Journal of Experimental
Zoology, 72, 455–477.
Coe WR (1938) Conditions influencing change of sex in molluscs
of the genus Crepidula. Journal of Experimental Zoology, 77, 401–
424.
Coffroth M, Lasker HR (1998) Larval paternity and male repro-
ductive success of a broadcast-spawning gorgonian, Plexaura
kuna. Marine Biology, 131, 329–337.
Collin R (1995) Sex, size, and position: a test of models predicting
size at sex change in the protandrous gastropod Crepidula forni-
cata. American Naturalist,
146, 815–831.
Collin R (2006) Sex ratio, life history invariants, and patterns of sex
change in a family of protandrous gastropods. Evolution, 60,
735–745.
Collin R, McLellan M, Gruber K, Bailey-Jourdain C (2005) Effects

of conspecific associations on size at sex change in three species
of calyptraeid gastropods. Marine Ecology Progress Series, 293,
89–97.
Coltman DW, Bowen WD, Wright JM (1998) Male mating success
in an aquatically mating pinniped, the harbour seal (Phoca
vitulina), assessed by microsatellite DNA markers. Molecular
Ecology, 7, 627–638.
Davis LM, Glenn TC, Elsey RM, Dessauer HC, Sawyer RH (2001)
Multiple paternity and mating patterns in the American alligator,
Alligator mississippiensis. Molecular Ecology, 10, 1011–1024.
Dupont L, Viard F (2003) Isolation and characterization of highly
polymorphic microsatellite markers from the marine invasive
species Crepidula fornicata (Gastropoda: Calyptraeidae).
Molecular Ecology Notes, 3, 498–500.
Dupont L, Jollivet D, Viard F (2003) High genetic diversity and
ephemeral drift effects in a successful introduced mollusc
(Crepidula fornicata: Gastropoda). Marine Ecology Progress Series,
253, 183–195.
Dupont L, Bernas D, Viard F (in press) Sex and genetic structure
across age groups in populations of the European marine
invasive mollusc, Crepidula fornicata L. (Gastropoda). Biological
Journal of the Linnean Society.
Fitzsimmons NN (1998) Single paternity of clutches and sperm
storage in the promiscuous green turtle (Chelonia mydas).
Molecular Ecology, 7, 575–584.
3018 L. DUPONT ET AL.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
Gaffney PM, McGee B (1992) Multiple paternity in Crepidula
fornicata (Linnaeus). Veliger, 35, 12–15.

Garrigue C, Dodemont R, Steel D, Baker C (2005) Organismal and
‘gametic’ capture-recapture using microsatellite genotyping
confirm low abundance and reproductive autonomy of
humpback whales on the wintering grounds of New Caledonia.
Marine Ecology Progress Series, 274, 251–262.
Ghiselin MT (1969) The evolution of hermaphroditism among
animals. Quarterly Review of Biology, 44, 189–208.
Goulletquer P, Bachelet G, Sauriau PG, Noel P (2002) Open
Atlantic coast of Europe — a century of introduced species into
French waters. In: Invasive Aquatic Species of Europe (eds
Leppäkoski E, Gollash S, Olenin S), pp. 276–290. Kluwer
Academic Publishers, Dordrecht, The Netherlands.
Heller J (1993) Hermaphroditism in molluscs. Biological Journal of
the Linnean Society, 48, 19–42.
Hoagland KE (1975) Reproductive Strategies and Evolution in the
Genus Crepidula (Gastropoda: Calyptraeidae). PhD Thesis, Harvard
University Press, Cambridge, Massachusetts.
Hoagland KE (1978) Protandry and the evolution of environmentally-
mediated sex change: a study of the mollusca. Malacologia, 17,
365–391.
Hoagland KE (1986) Patterns of encapsulation and brooding in
the Calyptraeidae (Prosobranchia: Mesogastropoda). American
Malacological Bulletin, 4, 173–183.
Hoekert WE, Neufeglise H, Schouten AD, Menken SB (2002)
Multiple paternity and female-biased mutation at a microsatellite
locus in the olive ridley sea turtle (Lepidochelys olivacea). Heredity,
89, 107–113.
Hoffman SG, Schildhauer MP, Warner RR (1985) The costs of
changing sex and the ontogeny of males under contest
competition for mates. Evolution, 39, 915–927.

Hoffman JI, Boyd IL, Amos W (2003) Male reproductive strategy
and the importance of maternal status in the Antarctic fur seal
Arctocephalus gazella. Evolution, 57, 1917–1930.
Ireland JS, Broderick AC, Glen F et al. (2003) Multiple paternity
assessed using microsatellite markers, in green turtles
Chelonia mydas (Linnaeus, 1758) of Ascension Island, South
Atlantic. Journal of Experimental Marine Biology and Ecology, 291,
149–160.
Jarne P, Lagoda J-L (1996) Microsatellites, from molecules to
populations and back. Trends in Ecology and Evolution, 11, 424–
429.
Krutzen M, Barre LM, Connor RC, Mann J, Sherwin WB (2004) ‘O
father: where art thou?’ — Paternity assessment in an open
fission-fusion society of wild bottlenose dolphins (Tursiops sp.)
in Shark Bay, Western Australia. Molecular Ecology, 13, 1975–
1990.
Le Gall P (1980) Etude expérimentale d’association en chaîne et de
son influence sur la croissance et la sexualité chez la crépidule
(Crepidula fornicata Linné 1758). PhD Thesis, University of
Caen, France.
Levitan DR (1993) The importance of sperm limitation to the
evolution of egg size in marine invertebrates. American Naturalist,
141, 517–536.
MacDonald PDM, Pitcher TJ (1979) Age-groups from size-
frequency data: a versatile and efficient method of analysing
distribution mixtures. Journal of the Fish Resource Board of Canada,
36, 987–1001.
Marshall TC, Slate J, Kruuk LEB, Pemberton JM (1998) Statistical
confidence for likelihood-based paternity inference in natural
populations. Molecular Ecology, 7, 639–655.

Martin M-C (1985) Etude expérimentale de l’inversion sexuelle et de la
morphogénèse génitale femelle chez un mollusque hermaphrodite
protandre Crepidula fornicata. PhD Thesis, University of Caen,
France.
Martinez JL, Moran P, Perez J et al. (2000) Multiple paternity
increases effective size of southern Atlantic salmon populations.
Molecular Ecology, 9, 293–298.
McCracken GF, Burghardt GM, Houts SE (1999) Microsatellite
markers and multiple paternity in the garter snake Thamnophis
sirtalis. Molecular Ecology, 8, 1475–1479.
Meagher TR (1986) Analysis of paternity within a natural population
of Chamaelirium luteum. 1. Identification of most-likely male
parents. American Naturalist, 128, 199–215.
Moore MK, Ball RM (2002) Multiple paternity in loggerhead turtle
(Caretta caretta) nests on Melbourne Beach, Florida: a microsatellite
analysis. Molecular Ecology, 11, 281–288.
Munday PL, Buston PM, Warner RR (2006) Diversity and
flexibility of sex-change strategies in animals. Trends in Ecology
& Evolution, 21, 89–95.
Munoz RC, Warner RR (2003) A new version of the size-advantage
hypothesis for sex-change: incorporating sperm competition
and size-fecundity skew. American Naturalist, 161, 749–761.
Munoz RC, Warner RR (2004) Testing a new version of the
size-advantage hypohesis for sex change: sperm competition
and size-skew effects in the bucktooth parrotfish, Sparisoma
radians. Behavioural Ecology, 15
, 129–136.
Naud M-J, Hanlon RT, Hall KC, Shaw PW, Havenhand JN (2004)
Behavioural and genetic assessment of reproductive success in
a spawning aggregation of the Australian giant cuttlefish, Sepia

apama. Animal Behaviour, 67, 1043–1050.
Orton JH (1909) On the occurence of protandric hermaphrodism
in the mollusc Crepidula fornicata. Proceedings of the Royal Society
of London. Series B, Biological Sciences, 81, 468–484.
Orton JH (1950) Recent breeding phenomena in the American
slipper-limpet, Crepidula fornicata. Nature, 165, 433–434.
Parker GA (1970) Sperm competition and its evolutionary
consequences in the insects. Biological Review, 45, 525–567.
Paterson IG, Partridge V, Buckland-Nicks J (2001) Multiple
paternity in Littorina obtusata (Gastropoda, Littorinidae) revealed
by microsatellite analyses. Biological Bulletin, 200, 261–267.
Pechenik JA, Blanchard M, Rotjan R (2004) Susceptibility of larval
Crepidula fornicata to predation by suspension-feeding adults.
Journal of Experimental Marine Biology and Ecology, 306, 75–94.
Petersen CW, Mazzoldy C, Zarella KA, Hale RE (2005) Fertiliza-
tion mode, sperm characteristics, mate choice and parental care
patterns in Artedius spp. (Cottidae). Journal of Fish Biology, 67,
239–254.
Pitcher TE, Neff BD, Rodd FH, Rowe L (2003) Multiple mating and
sequential mate choice in guppies: females trade up. Proceedings
of the Royal Society of London. Series B, Biological Sciences, 270,
1623–1629.
Queller DC, Goodnight KF (1989) Estimating relatedness using
genetic markers. Evolution, 43, 258–275.
Raymond M, Rousset F (1995) genepop (version 1.2): population
genetics software for exact tests and ecumenicism. Journal of
Heredity, 86, 248–249.
Reavis RH, Grober MS (1999) An integrative approach to sex
change: social, behavioural and neurochemical changes in
Lythrypnus dalli (Pisces). Acta Ethologica, 2, 51–60.

Richard J, Huet M, Thouzeau G, Paulet Y-M (in press) Reproduc-
tion of the invasive slipper limpet, Crepidula fornicata, in the Bay
of Brest, France. Marine Biology
.
LARVAL PATERNITY ASSIGNMENT IN SLIPPER LIMPETS 3019
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
Sakai AK, Allendorf FW, Holt JS et al. (2001) The population
biology of invasive species. Annual Review of Ecology and Systematics,
32, 305–332.
Selvamani MJP, Degnan SM, Degnan BM (2001) Microsatellite
genotyping of individual abalone larvae: parentage assignment
in aquaculture. Marine Biotechnology, 3, 478–485.
Snook RR (2005) Sperm in competition: not playing by the
numbers. Trends in Ecology & Evolution, 20, 46–53.
Soucy S, Travis J (2003) Multiple paternity and population genetic
structure in natural populations of the poeciliid fish, Heterandria
formosa. Journal of Evolutionary Biology, 16, 1328–1336.
Spritzer D, Solomon NG, Meikle DB (2005) Influence of scramble
competition for mates upon the spatial ability of male meadow
voles. Animal Behaviour, 69, 375–386.
Sugg DW, Chesser RK (1994) Effective population sizes with
multiple paternity. Genetics, 137, 1147–1155.
Sunobe T, Nakamura M, Kobayashi Y, Kobayashi T, Nagahama Y
(2005) Immunoreactivity in the gobiid fish Trimma okinawae
during bidirectional sex change. Ichthyological Research, 52, 27–32.
Toonen RJ, Pawlik JR (1994) Foundations of gregariousness.
Nature, 370, 511–512.
Toonen RJ, Pawlik JR (2001) Foundations of gregariousness:
a dispersal polymorphism among the planktonic larvae of a

marine invertebrate. Evolution, 55, 2439–2454.
Viard F, Doums C, Jarne P (1997) Selfing, sexual polymorphism
and microsatellites in the hermaphroditic freshwater snail
Bulinus truncatus. Proceedings of the Royal Society of London. Series
B, Biological Sciences, 264, 39–44.
Viard F, Ellien C, Dupont L (2006) Dispersal ability and invasion
success of Crepidula fornicata in a single gulf: insights from genetic
markers and larval-dispersal models. Helgoländer Marine Research,
60, 144–152.
Warner RR, Robertson DR, Leigh EG Jr (1975) Sex change and
sexual selection. Science, 196, 633–638.
Warner RW, Fitch DL, Standish JD (1996) Social control of sex
change in the shelf limpet, Crepidula norrisiarum: size-specific
responses to local group composition. Journal of Experimental
Marine Biology and Ecology, 204
, 155–167.
Wilczynski JZ (1955) On sex behaviour and sex determination in
Crepidula fornicata. Biological Bulletin, 109, 353–354.
Wilson EO (1975) Sociobiology: The New Synthesis. Belknap Press,
Cambridge, Massachusetts.
Wilson K, Hardy ICW (2002) Statistical analysis of sex ratios:
an introduction. In: Sex Ratios. Concepts and Research Methods
(ed. Hardy ICW), pp. 48–92. University Press, Cambridge,
UK.
Wright WG (1988) Sex change in the Mollusca. Trends in Ecology &
Evolution, 3, 137–140.
This work is part of L. Dupont’s doctoral research on the dispersal
ability and reproduction of the invasive species C. fornicata along
the European coasts. The work was supervised by F. Viard, a
population geneticist with interests in studies of reproductive

system and historical vs. contemporary dispersal of coastal marine
algae and invertebrates, including introduced species. The LEMAR
group is involved in studies concerned with the interactions
between marine invertebrate populations and environmental
variability. Its main goal is to understand the dynamics of
invasions in coastal marine systems, by invertebrate alien species
(bivalves and gastropods), together with the possible consequences
on natural habitat alterations and nutrient fluxes (sedimentation/
mineralization) in a context of global change.
3020 L. DUPONT ET AL.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
Appendix
Results of the paternity analysis for each family in each population. For each stack, adults are characterized by their sex when sampled [I,
immature; M, male; T, in transition; F, female; and F(b), brooding female], size, position within the stack relative to the mother and
relatedness coefficient with the mother. The number of assigned larvae is shown for each studied mother (N
L
). For each brood, the
percentage of larvae sired by individuals within the maternal stack is given. Note that for some broods, the total is not 100%: this is indicative
of external paternity (percentage not given in the Appendix)
Population Mother Adults % larvae Sex Size (cm) Position* R
Roscanvel fR2 N
L
= 13 R2i1 100% F 9.7 1 0.080
N
L
= 73 9.6 cm R2i2 — F(b) 8.2 2 −0.280
R2i3 — M 8.8 3 −0.133
R2i4 — M 8.2 4 −0.222
R2i5 — M 9.2 5 −0.336

R2i6 — M 7.6 6 0.174
R2i7 — M 6.3 7 −0.035
fR3 N
L
= 15 R3i1 — F(b) 10.0 1 0.382
9.7 cm R3i2 — F(b) 9.9 2 −0.149
R3i3 100% M 8.9 3 0.458
R3i4 — M 8.8 4 0.101
R3i5 — M 7.3 5 −0.071
fR5 N
L
= 9 R5i1 F 10.1 1 0.160
8.3 cm R5i2 100% F(b) 10.3 2 −0.143
R5i3 — F(b) 9.7 3 0.022
R5i4 — M 8.6 4 −0.083
R5i5 — M 7.0 5 −0.338
R5i6 — M 3.2 6 −0.014
R5i7 — I 2.7 7 0.450
R5i8 — I 1.1 8 −0.241
fR8 N
L
= 11 R8i1 73% F 11.4 1 0.167
9.1 cm R8i2 18% M 11.5 2 0.020
R8i3 9% M 9.3 3 0.193
fR10 N
L
= 12 R10i1 17% F(b) 9.8 1 −0.116
8.9 cm R10i2 25% M 9.2 2 − 0.183
R10i3 42% M 10.0 3 −0.088
R10i4 8% M 8.7 4 0.102

R10i5 — M 8.9 5 0.092
R10i6 — M 3.2
fR11 N
L
= 13 R11i1 — F 10.8 1 0.141
10.1 cm R11i2 — F(b) 9.6 2 −0.220
R11i3 — M 7.2 3 0.134
R11i4 23% M 6.3 4 − 0.097
R11i5 77% M 6.5 5 − 0.114
Keraliou fK1 N
L
= 12 K1i1 — F(b) 9.9 1 0.161
N
L
= 58 7.8 cm K1i2 — F 11.0 2 0.001
K1i3 100% T 8.3 3 −0.475
fK3 N
L
= 8 K3i1 — T 9.6 1 0.229
9.5 cm K3i2 — F(b) 8.9 2 0.067
K3i3 — M 7.4 3 0.280
K3i4 — M 6.7 4 0.404
K3i5 — M 3.4 5 0.040
K3i6 — M 1.7 6 −0.158
K3i7 — T 6.3 Sec −0.009
K3i8 — M 4.6 Sec −0.248
K3i9 87% M 4.4 Sec 0.220
K3i10 — M 3.2 Sec 0.035
K3i11 — M 3.0 Sec − 0.096
fK5 N

L
= 12 K5i1 — F 9.4 1 −0.121
10.6 cm K5i2 — F(b) 6.7 2 0.386
K5i3 92% M 2.4 Side 0.020
K5i4 — M 2.6 Side −0.422
LARVAL PATERNITY ASSIGNMENT IN SLIPPER LIMPETS 3021
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
fK9 N
L
= 15 K9i1 — F 9.6 1 −0.206
8.7 cm K9i2 53% F(b) 6.8 2 − 0.393
K9i3 47% M 5.8 3 −0.288
K9i4 — M 3.7 4 0.401
K9i5 — M 2.8 5 −0.411
fK11 N
L
= 11 K11i1 45% M 11.0 1 −0.299
11.1 cm K11i2 — M 8.7 2 −0.042
K11i3 9% M 7.3 3 0.209
K11i4 — M 7.0 4 0.226
K11i5 — M 4.5 5 0.202
K11i6 18% T 8.4 Side −0.035
Rozegat fZ1 N
L
= 13 Z1i1 54% T 10.9 1 0.006
N
L
= 69 10.0 cm Z1i2 38% F(b) 9.1 2 0.063
Z1i3 — M 9.3 3 −0.038

Z1i4 — M 9.3 4 0.130
Z1i5 — M 8.0 5 0.051
Z1i6 — M 6.7 6 0.074
Z1i7 — M 4.0 7 0.118
Z1i8 8% M 5.4 Side 0.134
fZ2 N
L
= 10 Z2i1 20% F 6.9 1 0.392
11.2 cm Z2i2 60% M 6.1 2 −0.037
Z2i3 — M 5.1 3 0.100
fZ3 N
L
= 7 Z3i1 14% F 8.9 1 0.054
11.0 cm Z3i2 57% M 7.1 2 0.175
Z3i3 14% F 7.5 Sec 0.125
Z3i4 — M 4.4 Sec 0.199
Z3i5 — M 3.8 Side −0.089
Z3i6 — M 2.1 Side −0.037
fZ4 N
L
= 10 Z4i1 — F 9.4 1 −0.165
9.6 cm Z4i2 — F(b) 9.1 2 −0.089
Z4i3 — F 9.9 3 0.422
Z4i4 80% M 9.5 4 −0.162
Z4i5 10% F 6.2 Side −0.027
fZ8 N
L
= 11 Z8i1 18% M 7.2 1 0.410
9.0 cm Z8i2 46% M 7.5 2 −0.023
Z8i3 27% M 6.6 3 0.305

Z8i4 — F 5.4 Sec 0.552
Z8i5 9% M 5.1 Sec 0.187
Z8i6 — M 4.9 Sec −0.001
Z8i7 — M 4.6 Sec −0.131
Z8i8 — M 3.5 Sec 0.030
Z8i9 — M 5.0 Sec 0.403
fZ9 N
L
= 7 Z9i1 — F 9.3 1 −0.201
10.3 cm Z9i2 14% F(b) 8.5 2 −0.122
Z9i3 — M 8.8 3 0.261
Z9i4 — M 5.6 Sec −0.192
Z9i5 — M 4.0 Sec −0.208
Z9i6 — M 4.6 Sec 0.237
Z9i7 — M 3.8 Sec 0.325
Z9i8 — M 2.0 Sec −0.223
Z9i9 — M 3.1 Side 0.020
fZ12 N
L
= 11 Z12i1 — F 9.2 1 0.244
9.0 cm Z12i2 82% M 8.4 2 0.326
Z12i3 9% M 5.6 3 0.009
Z12i4 — M 4.0 4 −0.256
Z12i5 — F(b) 6.8 Sec − 0.192
Z12i6 — M 6.7 Sec 0.059
*Positions are numbered from 1 to 5 (0 is the mother as in Fig. 3A); ‘sec’ is for secondary stack and ‘side’ for side position.
Population Mother Adults % larvae Sex Size (cm) Position* R
Appendix Continued