V I A R D* *Evolution et Génétique des Populations Marines, UMR ADMM 7144 CNRS-UPMC, Station biologique, place Georges Teissier, BP 74, 29682 Roscoff cedex, France, †Institut Universita
Trang 1Molecular Ecology (2006) 15, 3009–3021 doi: 10.1111/j.1365-294X.2006.02988.x
Blackwell Publishing Ltd
Gregariousness and protandry promote reproductive
evidence from assignment of larval paternity
L D U P O N T ,*‡ J R I C H A R D ,† Y - M P A U L E T ,† G T H O U Z E A U† and F V I A R D*
*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: lidu@mba.ac.uk; viard@sb-roscoff.fr
‡Present address: Marine Biological Association, The Laboratory,
Citadel Hill, Plymouth PL1 2PB, UK
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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
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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 µm,
Table 1 Study populations and paternity analyses results Location, density (L Guérin, personal communication), sex ratio (F: M; binomial test in parenthesis) and mean number of adults per stack are given Results of the modal decomposition of size-frequency
distributions are summarized by the number of age groups (i.e cohorts; NAG) and the averaged size and standard deviation (SD) of
each age group Average relatedness within each population (Rpop), between each parental pair (Rp) and between each nonparental pair
within stacks (Rnp) are given for each population as well as the percentage of brooding females, the size at sex change (L50) 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 (Nfathers; including external fathers) SD, standard deviation
Population characteristics
Paternity analyses
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Pechenik et al 2004) but allowed the passage of small
particles (= 30 µm) 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 (L50 = 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 distribudecomposi-tion 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 mm of MgCl2)] 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
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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), suggestpossess-ing 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 (L50) 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 study populations Black, white and grey bars are featuring males, females and ‘fathers’, respectively, as assigned by paternity analysis
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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
brooding female [F(b)] at the time of sampling, in each population
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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
Fig 3 Distribution (percentage) of the 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
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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, viabilqual-ity and mobilqual-ity; 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
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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 paterpater-nity 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
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