Sex and genetic structure across age groups in populations of

Biological Journal of the Linnean Society

2007

90

365ndash374 With 4 figures

copy 2007 The Linnean Society of London


2007

90

365ndash374

365

Blackwell Publishing LtdOxford UKBIJBiological Journal of the Linnean Society0024-4066copy 2007 The Linnean Society of London 200790365374Original Article

DEMOGRAPHY AND GENETICS IN A PROTANDROUS MOLLUSCL DUPONT

ET AL

Corresponding author Current address Laboratoire de Biol-ogie des Sols et des Eaux Faculteacute des Sciences et Technologies Universiteacute Paris XII ndash Val de Marne 61 Avenue du Geacuteneacuteral de Gaulle 94010 Creacuteteil cedex France E-mail lisedupontuniv-paris12fr

Sex and genetic structure across age groups in populations of the European marine invasive mollusc

Crepidula fornicata

L (Gastropoda)

LISE DUPONT DAMIEN BERNAS and FREacuteDEacuteRIQUE VIARD

Evolution et Geacuteneacutetique des Populations Marines Station Biologique de Roscoff UMR ADMM 7144 CNRS-UPMC Place Georges Teissier BP 74 29682 Roscoff Cedex France

Received 4 April 2005 accepted for publication 1 May 2006

In long-lived species variance in allele frequencies over time may vary according to the number of generations con-tributing to progeny Here we investigate the temporal stability of genetic diversity and structure in relation to sexand age in introduced populations of

Crepidula fornicata

an exotic gastropod that successfully invaded Europe

Thisprotandrous species has the potential to change sex from male to female according not only to age but also to localsex ratio (social environment) This mechanism may adjust the reproduction efficiency across different cohorts andthus decrease the likelihood of genetic drift in the following generations Based on crude demographic structure anal-ysis in two spatially closed introduced French populations we demonstrate that recruitment is discontinuousAlthough timing of sex change is different across populations both populations have a similar age structure char-acterized by distributions of males and females changing across generations Using five microsatellite loci we showthat both populations display a temporal genetic homogeneity and a stability in genetic diversity indices across agegroups examined Our results highlight that the social control of sex change in

C fornicata

has strong implicationsto the maintenance of high genetic diversity by enhancing breeding across several generations at each reproductiveseason copy 2007 The Linnean Society of London


2007

90

365ndash374

ADDITIONAL KEYWORDS

age structure ndash exotic species ndash genetic drift ndash microsatellites ndash recruitment ndash

sex reversal

INTRODUCTION

Empirical population genetics studies generally inferthe influence of various forces on the evolution of nat-ural populations from the distribution of gene fre-quencies among local demes under the assumptionthat the opposing forces of genetic drift and migrationhave reached equilibrium However this assumptionis unlikely to be met in natural populations (Whitlock

amp McCauley 1999) Variation in the origin of recruitsindividual reproductive success and relatednessbetween successful reproductive individuals all con-tribute to increase variance in gene frequencies ateach generation (Whitlock amp McCauley 1990) In agestructured populations with overlapping generationsonly some age classes generally participate in breed-ing and the contribution to the progeny of the nextyear may differ among these age classes Thus theparents of a particular year class do not represent arandom sample from the previous generation As aconsequence populations are expected to displayallele frequency fluctuations over short time periods(ie a few generations Ryman 1997) in addition torandom genetic drift generated over longer time

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icoupcombiolinneanarticle9023652701119 by guest on 13 Septem

ber 2022

366

L DUPONT

ET AL



2007

90

365ndash374

periods (Waples 1990 Jorde amp Ryman 1995) Thus inlong-lived species with separate male and female func-tions both the sex ratio and the number of genera-tions contributing to a given reproductive eventstrongly influence effective population size and thusgenetic drift (Hedrick 2000) In species with sequen-tial hermaphroditism and social control of sex changelocal sex ratio may be modulated in particular accord-ing to local density (Charnov 1982) modifying theeffective size of populations In addition the distribu-tion of males and females may change across genera-tions (Charnov 1982) Such a system has thus thepotential to limit genetic drift by promoting reproduc-tion across several distant generations

In the present study we used the invasive marinemollusc

Crepidula fornicata

as a model to investigatethe role of sequential hermaphroditism in bufferingthe temporal evolution of genetic diversity in popula-tions This gastropod native to the West Atlantic coastalong North America was accidentally introduced intoEurope at the end of the 19th Century As a successfulinvasive species

C fornicata

has many consequenceson the native community along the Atlantic andEnglish Channel coasts (Thieltges Strasser amp Reise2003) Contrary to the pattern of founder effect fre-quently reported for invaders (Sakai

et al

2001) aprevious study showed high and similar levels ofgenetic diversity in introduced and native populationsof

C fornicata

(eg with allozyme data Dupont Jol-livet amp Viard 2003) Such a high genetic diversity canbe related to many factors (Dupont Jollivet amp Viard2003 Dupont

et al

2006 Viard Ellien amp Dupont2006) among them is a large local effective size inrelation to its high fecundity rates dispersal ability(ie 2ndash4 weeks as pelagic larvae Le Gall 1980) andbreeding system

C fornicata

is a long-lived species(life time of maximum 10 years Blanchard 1995) anda protandrous gastropod (ie mature individuals arefirst male and then change into female Coe 1936)with internal fertilization Individuals are typicallyfound in permanent stacks groups of individualsattached to each other with larger (older) individualsusually females at the base and smaller (younger)individuals usually males at the top (Coe 1936) Inrelation to the timing of sex-reversal strongly beinginfluenced by sexual morphs of neighbouring individ-uals (Coe 1938 Collin 1995) the number of ageclasses contributing to a given reproductive event mayvary according to the distribution of sex across ageclasses

To investigate the role of protandry on the distribu-tion of genetic diversity across generations in localpopulations of

C fornicata

we coupled temporal pop-ulation genetics with demographic analyses which isan approach known to have the potential for under-standing microevolutionary mechanisms in particu-

lar dispersal mating system and age structurebehind the observed spatial patterns (Lessios Wein-berg amp Starczak 1994 Charbonnel

et al

2002 Palm

et al

2003) We first carried out a crude demographicanalysis using the conventional modal decompositionmethod (Thieacutebaut

et al

2002) Then using five mic-rosatellite loci a joint analysis of sex ratio and geneticdiversity across the age groups identified was carriedout in two French populations of the Mont St MichelBay where the species was introduced more than30 years ago and is now an abundant species

MATERIAL AND METHODS

S

TUDY

SITE

AND

SAMPLING

Specimens of

C fornicata

were collected during theApril 2002 BENTHOMONT 2 cruise onboard of thelsquoNO Cocircte de la Manchersquo Two populations lsquoBayrsquo(48

deg

40

prime

55 N 01

deg

45

prime

92 W) and lsquoGulf rsquo (48

deg

45

prime

95 N01

deg

41

prime

84 W) separated by 11 km were sampled in theMont St Michel Bay (Fig 1) Four replicate samplesper site were collected using a 025-m

2

Hamon grabTwo replicates were preserved with 90 ethanol fordemographic and population genetic analysis and forthe two other replicates the number of stacks wascounted on-board The density per site was calculatedas the number of individuals sampled in 1 m

2

of sedi-ment (ie four replicates)

Figure 1

Study area and location of Gulf and Bay popu-lations sampled in 2002 in the Mont St Michel Bay France

GRANVILLE

Gulf

FRANCE

5000 m

BayCANCALE

Mont St Michel Bay

FRANCE

Dow



ber 2022

DEMOGRAPHY AND GENETICS IN A PROTANDROUS MOLLUSC

367



2007

90

365ndash374

S

EX

AND

AGE

STRUCTURE

ANALYSIS

The sexual phenotype was determined for a total of844 individuals of the Bay population and 1974 indi-viduals of the Gulf population according to presence orabsence of penis (Hoagland 1978) The sex ratio in

C fornicata

populations demonstrating good annualrecruitment has been shown to equal to 60 malesand 40 females rather than a 1 1 sex ratio (Wilc-zynski 1955 Hoagland 1978 Le Gall 1980) Thusboth departures from a 1 1 female male ratio and a067 1 sex ratio in the populations were tested usinga binomial test following Wilson amp Hardy (2002 54)Finally the size at sex change (

L

50

=

size at which 50are female) was calculated for each population accord-ing to Allsop amp West (2003a) The maximum size

L

max

was recorded for each population to calculate the rel-ative size at sex change (

L

50

L

max

)

To analyse the population age structure shells cur-vilinear length correlated to age (Le Gall 1980Deslous-Paoli 1985) were measured with a curvome-ter (Run-Mate) and size-frequency histograms wereplotted using 5-mm size-class intervals To ensure thatrecruitment is not continuous in populations the sizedistributions were compared with a normal distribu-tion using the ShapirondashWilk one-sample test The dis-tributions were compared between the two studypopulations using the MannndashWhitney test with thesoftware Minitab Size-frequency histograms weresmoothed using a weighted moving average at thethird order (Frontier amp Pichod-Viale 1991) Assumingthat the sizes follow a Gaussian distribution withineach cohort modal decomposition of size-frequencydistribution was performed using the MIX 23 pro-gram package (MacDonald amp Pitcher 1979) Accordingto the precision of the measures and the true growthmodel a Gaussian curve can be representative of sev-eral closed cohorts in particular for older (largest)individuals Nevertheless this method has beenproved to be efficient to identify major discontinuitiesin age structure (Thieacutebaut

et al

2002) Because this isour sole point of interest we refer to lsquoage groupsrsquorather than lsquocohortsrsquo For the lsquoGulf rsquo population thelarge number of immature individuals (lack of penisand size

lt

28 mm) prevented the detection of othermodes in the size-frequency distribution the modaldecomposition was thus carried out on mature indi-viduals after removing the immature individualsComparisons of homologous modes (ie approximatelyat the same position in the size-frequency distribu-tion) between populations were performed usingStudentrsquos

t

-tests employing JMP version 501a

A

GE

GROUPS

AND

POPULATIONS

GENETIC

ANALYSES

Respectively 235 and 219 individuals were selected inthe Bay and the Gulf populations for genetic analyses

A subsample of 39ndash74 individuals (Fig 2) was chosennearest the mode of each age group as defined by themodal decomposition analysis for each of the twostudy populations We avoided the subsampling ofindividuals with size corresponding to the overlappingzones between two consecutive Gaussian curves Thesize variation between the smallest and the largestindividuals in age groups was less than 09 cm and06 cm in the Bay and Gulf populations respectively

All the individuals were then genotyped at five locifour namely CfCA2 CfCA4 CfGT9 and CfGT14 asdefined by Dupont amp Viard (2003) and CfH7 (Viard

et al

2006) Loci were amplified by polymerase chainreactions (PCR) following protocols detailed in Dupontamp Viard (2003) and Viard

et al

(2006)The null hypothesis of independence between loci

was tested from statistical genotypic disequilibriumanalysis using Genepop version 33 (Raymond ampRousset 1995) For each age group and populationallele frequencies total number of alleles averagenumber of alleles (

N

all

) observed (

H

o

) and expected(

H

e

) heterozygozities were estimated using Genetixversion 402 (Belkhir

et al

2000) To take into accountvariation in sample size allelic richness (

Ar

ElMousadik amp Petit 1996) was estimated for each agegroup and populations using FSTAT version 293(Goudet 2000) Genetic diversity and allelic richnessestimates between immature individuals and adultswere compared using a paired

t

-testTo investigate to what extent a temporal Wahlund

effect accounts for heterozygote deficiencies in thisspecies population heterozygote deficiencies werequantified within each population and each age groupby calculating (Weir amp Cockerham 1984) an estima-tor of the fixation index

F

is

with Genepop version 33Tests for deviations from HardyndashWeinberg expecta-tions were carried out within each population andeach age group using Genepop version 33 (5000batches and 5000 iterations per batch)

Null alleles with non-negligible frequencies havebeen repeatedly reported in some species of marineinvertebrates (Foltz 1986) For loci for which failedamplification occurred (ie suggestive of null homozy-gotes) the number of null homozygotes was recordedand the expected frequency of null alleles was esti-mated using HardyndashWeinberg expectations (Brook-field 1996)

Genetic differentiation among populations was esti-mated using Genepop version 33 by calculating (Weir amp Cockerham 1984 an estimator of

F

st

Wright1951) Exact tests of allelic differentiation were car-ried out between populations and among age groupsTo test for the relative importance of age groups vspopulation effects on the genetic structure an

F

st

-based hierarchical analysis (ARLEQUIN version200 httpanthrounigecharlequin) was used

f

q

Dow



ber 2022

368

L DUPONT

ET AL



2007

90

365ndash374

Genetic variance was partitioned into three compo-nents variation among populations among agegroups within populations and among age groupsoverall populations

RESULTS

S

EX

RATIO

AND

AGE

GROUPS

ANALYSES

Density estimates were similar 1832 and2077 individualsm

minus

2

for the Bay and the Gulf popula-tions respectively The overall age structure was alsovery similar in each population These similaritieswere not expected because the two study populationswere recorded at very different dates the lsquoBayrsquo popu-lation is located in Cancale Bay where the slipper lim-pet is present at least after the 1970s (Blanchard ampEhrold 1999) whereas the lsquoGulf rsquo population was sam-pled in a site where the slipper limpet was not

qCT

qSC

qST

reported by a previous survey made in 1997 (Blan-chard amp Ehrold 1999)

The individual shell length was in the range 09ndash108 cm in the Bay population and 02ndash96 cm inthe Gulf population The size-frequency distributionsignificantly differed from a normal distribution(ShapirondashWilk one-sample test

P

lt

0000) in both pop-ulations The modal decompositions of size-frequencyhistograms showed four modes for the Bay population(

χ

2

=

515

P

=

096 df

=

12) and three modes for theGulf population but without immature individuals(

χ

2

=

497

P = 099 df = 14) When including imma-ture individuals four age groups were thus alsoobserved in Gulf population In both populations theyoungest age group (age group 1) was exclusively com-posed of immature individuals whereas age groups 2ndash4 were exclusively composed of adults Size-frequencyhistograms are presented in Figure 2 and the charac-teristics of each component of the modal decomposi-

Figure 2 Size-frequency histograms of the shell curvilinear length from the Bay and Gulf populations collected in April2002 A distribution of sexual morphs Respectively NI NM NT and NF are the number of immature individuals malesindividuals in transition and females in the whole sampled population B modal decomposition Mx shows the mean ofeach normal component Gaussian curves do not adjust exactly to the effective size frequency distribution because themodal decomposition was achieved on a smoothed size-frequency histogram using MIX software Individuals selected forgenetic analysis are shown in black

A Distribution of sexual morphs in size frequency histograms

0

20

40

60

80

100

05 15 25 35 45 55 65 75 85 95 105

Num

ber

ofin

divi

dua l

s

NI= 679NM = 903NT = 42NF = 350

Size classes (cm)

B Modal decomposition of size frequency distributions

N = 844

0

20

40

60

80

100

05 15 25 35 45 55 65 75 85 95 105

0

100

200

300

400

500

05 15 25 35 45 55 65 75 85 95 105

NI= 90NM = 442NT = 24NF = 288

N = 1974

0

20

40

60

80

100120

140

05 15 25 35 45 55 65 75 85 95 105

Size classes (cm)

420

440

Num

ber

ofin

divi

dual

s

Gulf populationBay population

M1

M2 M3

M4

M1

M2

M3M4

2= 515

p = 096

2= 497

p = 099

i s

1

eof

Imm tu in transi iona re individuals

Males

IndividualsIndividuals in transitionFemales

c )

065

NI= 90NM = 442NT = 24NF = 288

N = 1974

Size classes (cm)

Gulf population

X2= 515

p = 096

X

Dow



ber 2022

DEMOGRAPHY AND GENETICS IN A PROTANDROUS MOLLUSC 369

copy 2007 The Linnean Society of London Biological Journal of the Linnean Society 2007 90 365ndash374

tion of the size-frequency histograms are detailed inTable 1 The fact that the overall size distributions canbe split in several Gaussian curves highlight theoccurrence of several age groups and thus an irregularrecruitment through time in these populations Asspecified in the Material and methods section only aminimum number of age groups was obtained withthis analysis Nevertheless we unambiguously iso-lated groups in which individuals have been recruitedover a short period of time compared with individualsthat belonged to two different age groups Inter-estingly the size-frequency distributions differedsignificantly between Bay and Gulf (MannndashWhitneytest W = 16662955 P lt 00001) The differencesbetween the modal values in the two populations weresignificant for all age groups (Studentrsquos t-testP lt 0001) In particular the Bay population exhibitedhigher modal values than the Gulf population More-over the most-represented age group in the Bay pop-ulation was the fourth age group (51) whereas itwas the first (immatures 34) in the Gulf population(Table 1)

The distribution of sexual morphs was investigatedin both populations and is shown in Figure 2 The rel-ative proportions of immature individuals individualsin transition and males and females were signifi-cantly different between the two populations (χ2 = 812P lt 00001) This difference is due to the larger num-ber of females in the Bay population and the largernumber of immature individuals in the Gulf popula-tion Both populations showed significant deviationsfrom a 1 1 female male ratio (P lt 00001) in favourof males No deviation was shown for the Bay popula-tion from a 067 1 sex ratio towards males (N = 753Nfemale = 295 P = 0365) whereas the Gulf population

showed a significant deviation (N = 1253 Nfemale = 350P lt 00001) Figure 3 illustrates the proportions ofimmature individuals males individuals in transi-tion and females in the subsamples used for geneticanalyses of each age group As expected in protan-drous hermaphrodites the number of males consis-tently decreased from the second age group to the lastwhereas the number of females increased untilobtaining an inversion of male and female propor-tions Few individuals were transitional and most ofthese were found in the fourth age group in both pop-ulations (Fig 3) The relative size at sex change wasdifferent for the two populations Individuals arechanging sex when they reach 74 (L50 = 8Lmax = 108) and 65 (L50 = 62 Lmax = 96) of theirmaximum size in the Bay and Gulf populationsrespectively

POPULATIONS AND AGE GROUPS GENETIC ANALYSIS

Genetic diversity was variable among the microsatel-lite loci examined Over the 610 individuals analysedin the two populations the number of alleles per locusvaried from four for the locus CA2 to 85 for the locus

Table 1 Size distributions in the two study populations

Population

Age groups

M1 M2 M3 M4

Bay (N = 844)Proportion 008 013 028 051Mode (cm) 146 302 545 773SD (cm) 041 080 105 113

Gulf (N = 1974)Proportion 034 014 026 026Mode (cm) 080 218 416 649SD (cm) 032 067 100 118

Results of modal decomposition of size-frequency distribu-tions of the curvilinear length are summarized the modeof each identified age group is indicated together with stan-dard deviation (SD) Age group 1 represents immatureindividuals

Figure 3 Percentage of sexual morphs of each age groupsub-sample (the number of individuals of each sub-sampleis given in parentheses)

A Bay population (N = 376)

0

20

40

60

80

100

Age-groups

B Gulf population (N = 285)

Immature individuals

Males

Individuals in transitionFemales

0

20

40

60

80

100

(77) (60) (83) (83) (73)

(59) (59) (60) (60) (47)

Age-groups

1 2 3 4 5

1 2 3 4 5

p

Immature individuals Individuals in transitionFemales

i v d Individuals in transitionFemales

i i n

Dow



ber 2022

370 L DUPONT ET AL


GT9 (Table 2) Expected heterozygosity (He) for CA2CA4 GT9 GT14 and H7 were 0485 0810 09820954 and 0879 respectively No statistical linkagedisequilibrium was detected indicating independencebetween loci

Descriptive statistics are presented for each agegroup and over all age groups in Table 2 The meangene diversity was very high over all the age groups(He = 0823 and 0819 in Bay and Gulf respectively)and varied only slightly among age groups in eachpopulation [0811 (age group 3) to 0838 (age group 4)in lsquoBayrsquo 0806 (age group 4) to 0838 (age group 1) inlsquoGulf rsquo] No genetic drift effects were found in juvenilescompared with adults gene diversity values were notstatistically different between immature individuals(age group 1) and adults (paired t-test across locirespectively P = 0701 and P = 0593 for Bay and Gulfpopulations) nor allelic richness estimates (pairedt-test across loci respectively P = 0430 and P = 0996for Bay and Gulf populations)

Significant heterozygote deficiencies were observedat the population level ( = 0117 in the Bay popula-tion and = 0122 in the Gulf population) and at theage group level (Table 2) All age groups except agegroup 1 of the Bay population exhibited a significantdeparture from HardyndashWeinberg equilibrium due toheterozygote deficiency (Table 2) Significant devia-tions occurred at four loci (CfCA4 CfGT9 CfGT14and CfH7)

In this bentho-pelagic species heterozygote defi-ciencies may be explained by a temporal Wahlundeffect resulting from possible differences in allelefrequencies between consecutive settling cohorts A

ff

characteristic evolution of the deficit of heterozygotesunder a temporal Wahlund effect is an increase of theheterozygote deficit in relation to increasing numbersof cohorts pooled together (David et al 1997)Figure 4 clearly shows that the heterozygote deficien-cies observed in pooled samples are unaltered withintemporal subdivisions of the sample This result high-lights a temporal stability of the genetic pool acrossage groups

Heterozygote deficiencies might be explained by thepresence of null alleles suggested by a lack of ampli-fication observed for two loci (one individual atCfGT14 and four individuals at CfGT9 out of 454 indi-viduals) Under the strong assumption of Hardyndash

Table 2 Genetic diversity and heterozygote deficiency at age group and population levels at five microsatellite loci

N He plusmn SDAllellicrichness CfCA2 (4) CfCA4 (20) CfGT9 (85) CfGT14 (59) CfH7 (17) All loci

BayB1 54 0819 plusmn 0217 203 0043 minus0013 0136 0105 minus0032 0052B2 39 0821 plusmn 0198 202 0163 0281 0192 0119 minus0014 0146B3 68 0811 plusmn 0228 187 0046 0204 0176 0284 minus0008 0154B4 74 0838 plusmn 0185 201 0014 0153 0164 0209 minus0012 0117Population 235 0823 plusmn 0207 308 0055 0149 0165 0192 minus0015 0117

GulfG1 46 0838 plusmn 0164 195 0097 0118 0160 0161 0002 0110G2 56 0823 plusmn 0193 199 0075 0127 0145 0201 0012 0118G3 59 0811 plusmn 0194 194 0057 minus0030 0277 0220 minus0085 0102G4 58 0806 plusmn 0225 206 0112 0013 0329 0234 0034 0156Population 219 0819 plusmn 0194 310 0087 0057 0232 0206 minus0012 0122

Total 454 0822 plusmn 0199 35 0073 0109 0198 0198 minus0013 0121

The expected heterozygosities (He) with standard deviation (SD) and allelic richness are indicated over all loci For eachlocus the total number of alleles and the estimator of the fixation index Fis ( ) are indicated with probability of the exacttest for deviations from HardyndashWeinberg expectations P lt 005 P lt 001 P lt 0001

f

Figure 4 Multi-locus heterozygote deficiency in each agegroup and in each population The mean Fis over the fourage groups is also shown (standard deviation representedby vertical bars)

0

005

010

015

020

025

Age-group

1

Age-group

2

Age-group

3

Age-group

4

Mean Population

Bay

Gulf

Fis

e- p

3

lf

Dow



ber 2022



Weinberg equilibrium in each of the populations nullallele frequencies were equal to 131 and 9 forCfGT9 and 115 and 106 for CfGT14 for Gulf andBay populations respectively Tests for temporalWahlund effect were then achieved without these twolocus but the results remained unaltered Conse-quently the effects of null alleles could be neglectedfor CfGT9 and CfGT14 Nevertheless further analy-ses were conducted both with and without these loci

The global test for genetic differentiation amongpopulations revealed a slight but significant differ-ences of the distribution of allele frequencies betweenthe two populations ( = 0002 P = 0000) Whenexcluding CfGT9 and CfGT14 from the analysis thevalue of was higher ( = 0003) and remained statis-tically significant (P = 0047) even though removingthe most polymorphic loci decreased statistical powerof test for genetic differentiation A hierarchical anal-ysis was carried out to test for the origin (among pop-ulations vs among age groups) of this overallsignificant genetic structure This analysis showedthat the two populations were statistically different( = 0002 P = 0037) whereas neither the differenti-ation between the eight age groups ( = 0003P = 0094) nor the differentiation within age groupsamong populations ( = 0001 P = 0431) was signif-icant highlighting the temporal genetic stabilityacross age groups

This last result was confirmed by the pairwise exacttests of allelic differentiation among the four agegroups presented for each population in Table 3 Forthe Bay population none of the multilocus pairwisecomparison was significant In the Gulf population asignificant difference was observed for one comparisonbut did not remain significant when CfGT9 wasexcluded of the analysis ( = 0004 P = 0004) Con-versely between populations ten (out of 16) multilo-cus pairwise comparisons of the allelic frequencies

q

q q

qCT

qST

qSC

q

among age groups were significant Four exact testsremained significant when CfGT14 and CfGT9 wereexcluded from the analysis Thus no clear genetic dif-ferentiation was shown between age groups withinpopulations whereas a slight but significant overallgenetic differentiation was observed betweenpopulations

DISCUSSION

AGE AND TEMPORAL GENETIC STRUCTURE

In the two study populations of C fornicata size-frequency histograms demonstrated discontinuousrecruitment and identified separate groups of individ-uals each of them having been recruited at differenttimes Despite very different dates for the first recordof the two populations they both exhibited four mainage groups a result close to those from previous stud-ies by Deslous-Paoli (1985 four lsquocohortsrsquo Marennes-Oleacuteron Bay France) and Grady et al (2001 fivelsquocohortsrsquo Pleasant Bay MA USA) This suggests thatno major shift of the recruitment patterns occurredduring the invasion process A rigorous comparativeanalysis of introduced and native populations is nev-ertheless needed to ascertain this hypothesis

Across age groups in each of the two populations wefailed to demonstrate temporal variation in allelic fre-quencies even between juveniles (ie age group 1) andgroups of mature individuals This was supported by(1) the lack of increase of Fis values after sequentialpooling of age groups (2) the nonsignificant valuesassociated to exact tests of differentiation between agegroups and (3) the hierarchical analysis of geneticstructure Whereas the two study populations ofC fornicata are genetically different from each otherthey are both characterized by a strong temporalgenetic homogeneity between different groups of indi-viduals that settle over several years Interestingly

Table 3 Pairwise multilocus estimates (below diagonal) and probability values of the pairwise exact test of allelicdifferentiation among age groups of the Bay population [age group 1 (B1) to age group 4 (B4)] and the Gulf population[age group 1 (G1) to age group 4 (G4)] above diagonal

B1 B2 B3 B4 G1 G2 G3 G4

B1 (54) ndash 0161 0384 0071 0161 0437 0012 0612B2 (39) 0000 ndash 0124 0828 0028 0034 0023 0270B3 (68) minus0003 minus0001 ndash 0054 0004 0060 0001 0340B4 (74) 0001 minus0003 0002 ndash 0011 0010 0004 0017G1 (46) 0002 0002 0005 0003 ndash 0284 0062 0035G2 (56) minus0002 0000 0001 0001 minus0002 ndash 0121 0154G3 (59) 0003 0001 0004 0006 0001 0001 ndash 0072G4 (58) minus0001 minus0001 minus0002 0003 0004 0000 0001 ndash

Study area and location of Gulf and Bay populations sampled in 2002 in the Mont St Michel Bay FranceStatistically significant Number of individuals analysed per age group are in parentheses

q

Dow



ber 2022

372 L DUPONT ET AL


this pattern also holds for the population only recentlyrecorded (maximum age 6 years) This is an intriguingresult because temporal variation was documented fornumerous marine bentho-pelagic species (Johnson ampBlack 1984 Gosling amp Wilkins 1985 Watts Johnsonamp Black 1990 Hedgecock 1994 David et al 1997Moberg amp Burton 2000) This variation was usuallyrelated to the settlement of genetically differentiatedpool of recruits a process that enhances the variancein gene frequencies across generations In this con-text Hedgecock (1994) proposed the lsquosweepstakehypothesisrsquo predicting that marine species with highfecundity and high early mortality have extremelylarge variance in individual reproductive success Theoccurrence of genetically differentiated groups of lar-vae during a single spawning season has indeed beendocumented in marine invertebrates (Li amp Hedgecock1998) and fishes (Ruzzante Taggart amp Cook 1996) Inthe present study a significant genetic differentiationwas only observed between the youngest age group(age group 1) and the oldest (age group 4) in the Gulfpopulation suggesting a settlement of genetically dif-ferentiated pool of recruits Can this observation berelated to the recent settlement of the population Onone hand pairwise values across age groupsdecreased from G4ndashG1 to G2ndashG1 and thus support thehypothesis of founders originating from geneticallydifferent populations and then breeding occurring inthe next generations between local individuals On theother hand we did not observe a higher Fis value in G4compared with G3 or G2 so that Walhund effects dueto the mixing of genetically different founders in old-est cohorts if any are minimized

Altogether no temporal Wahlund effect due to mix-ing of genetically different age groups at a given siteappears to strongly alter genetic structure inC fornicata populations of the Mont St Michel Bay overthe short time-scale investigated (ie a few genera-tions) In addition although the sweepstake reproduc-tive hypothesis postulates lower genetic diversity incohorts of newly-recruited juveniles compared with theadult stock (Hedgecock 1994) our data indicate thatgenetic diversity of immature individuals were consis-tently high and not reduced compared with adults

ROLE OF GENETIC DIVERSITY STABILITY AND PROTANDRY ON C FORNICATA INVASIVENESS

This long-lived protandrous species is characterizedby proportions of males and females changing acrossgenerations In the study populations we observed avery low number even an absence of females in agegroups 2 and 3 Mating between largely different gen-erations is thus obligatory For example males in theage group 2 are obliged to mate with females of the agegroup 4 According to Ryman (1997) and under the

q

assumption of similar effective size a populationwhere approximately equal proportions of the off-spring are produced from each of the adult age classeswill exhibit less temporal genetic variation than a pop-ulation where the reproduction is restricted to individ-uals of only some age classes Thus independently ofthe global effective size only a slight amount of tem-poral allele variation may be expected in such a spe-cies Comparing two species of pelagic fish withdifferent life histories Gaggiotti amp Vetter (1999) haveshown that the larger the generation overlap thesmaller is the impact of environmental fluctuations onthe level of genetic variability maintained by a popu-lation This lsquostorage effectrsquo (Warner amp Chesson 1985)has been previously described by Ellner amp Hairston(1994) and defined as the capacity of long-lived organ-ism with strongly overlapping generations to main-tain genetic variation in temporally fluctuatingenvironment Crepidula fornicata fits well with thisdefinition This high genetic diversity maintained overtime in populations adds to the list of traits that makeC fornicata an efficient colonizer and invasive speciesnew populations can rapidly recover from genetic bot-tlenecks thanks to the combination of larval dispersaland protandry

The two study populations have different coloniza-tion histories In 1997 no C fornicata was reported atthe location of the Gulf population whereas the Baypopulation is situated in the Bay of Cancale where thespecies has rapidly spread and has reached high den-sity (Blanchard amp Ehrold 1999) In the Bay popula-tion individuals change sex when they reached 74 oftheir maximum size a value in agreement with othervalues from literature By comparing the relative sizeof sex change in a variety of animals (ie fish echin-oderms crustaceans molluscs and polychaete worms)Allsop amp West (2003b) showed that the timing of sexchange is surprisingly invariant across all animalsTheir results indicated that individuals change sexwhen they reach 72 (95 confidence interval = 67ndash77) of their maximum size By contrast to theseobservations a slightly lower value was observed inthe Gulf population (65) Thus sex-reversal is occur-ring earlier in the Gulf population In addition weshowed that the Gulf population exhibited a highernumber of males than the 067 1 sex ratio generallyobserved in natural population of C fornicata (Hoag-land 1978 Le Gall 1980 Dupont et al 2003) Accord-ing to sex ratio data and timing at sex change only154 of the Gulf population individuals haveexceeded 65 of the maximal size in the populationThis is in agreement with the large proportion ofimmature individuals (34) highlighting an activerecruitment together with the high proportion ofmales in the Gulf population and suggesting that sex-ual maturity is rapidly reached by newly-recruited

Dow



ber 2022



juveniles Such behaviour contributes to an effectivesex ratio adjustment (ie the number of reproducingfemales relative to the total number of individualscontributing to the next generation) in response toactive recruitment This functioning is congruent withboth study populations exhibiting the same popula-tion dynamics and genetic diversity The number ofsource populations contributing to a new population islikely to be increased due to long larval-dispersal andtiming of sex reversal rapidly decreasing any foundereffects

The present study emphasizes the importance oflife-history traits in determining the level of geneticvariability maintained by an exotic species over ashort-time scale The study shows that protandry andoverlapping generations buffer the effect of sweep-stake recruitment that could have been generated inC fornicata by long larval phase and fluctuatingenvironment

ACKNOWLEDGEMENTS

This project was funded by the PNEC-Site AtelierlsquoBaie du Mont St Michelrsquo and the 2001 INVABIO pro-gram of the Ministegravere de lrsquoEcologie et du Deacuteveloppe-ment Durable (MEDD project no D4ESRP01115)This project obtained additional support for genotyp-ing at new loci from the European Network of Excel-lence lsquoMarine Genomicsrsquo L D acknowledges theRegion Bretagne for her PhD grant The authors arealso grateful to T Comtet C Engel and M Valero forstimulating discussions andor improvements to themanuscript

REFERENCES

Allsop DJ West SA 2003a Constant relative age and size atsex change for sequentially hermaphroditic fish Journal ofEvolutionary Biology 16 921ndash929

Allsop DJ West SA 2003b Changing sex at the same rela-tive body size Nature 425 783ndash784

Belkhir K Borsa P Goudet J Chikhi L Bonhomme F2000 GENETIX 402 logiciel sous Windows pour lageacuteneacutetique des populations Montpellier CNRS UPR 9060Universiteacute Montpellier II

Blanchard M 1995 Origine et eacutetat de la population de Crep-idula fornicata (Gastropoda Prosobranchia) sur le littoralfranccedilais Haliotis 24 75ndash86

Blanchard M Ehrold A 1999 Cartographie et eacutevaluation dustock de creacutepidules (Crepidula fornicata L) en baie du MontSaint-Michel Haliotis 28 11ndash20

Brookfield JFY 1996 A simple new method for estimatingnull allele frequency from heterozygote deficiency MolecularEcoology 5 453ndash455

Charbonnel N Quesnoit M Razatavonjizay R BreacutemondP Jarne P 2002 A spatial and temporal approach to micro-

evolutionary forces affecting population biology in thefreshwater snail Biomphalaria pfeifferi AmericanNaturalist 160 741ndash755

Charnov EL 1982 The theory of sex allocation PrincetonNJ Princeton University Press

Coe WR 1936 Sexual phases in Crepidula Journal of Exper-imental Zoology 72 455ndash477

Coe WR 1938 Conditions influencing change of sex in mol-luscs of the genus Crepidula Journal of Experimental Zool-ogy 77 401ndash424

Collin R 1995 Sex size and position a test of models pre-dicting size at sex change in the protandrous gastropod Crep-idula fornicata American Naturalist 146 815ndash831

David P Perdieu M-A Pernot A-F Jarne P 1997 Fine-grained spatial and temporal population structure in themarine bivalve Spisula ovalis Evolution 51 1318ndash1322

Deslous-Paoli JM 1985 Crepidula fornicata L (gasteacuteropode)dans le bassin de Marennes-Oleacuteron structure dynamique etproduction drsquoune population Oceanologica Acta 8 453ndash460

Dupont L Jollivet D Viard F 2003 High genetic diversityand ephemeral drift effects in a successful introducedmollusc (Crepidula fornicata Gastropoda) Marine EcologyProgress Series 253 183ndash195

Dupont L Richard J Paulet Y-M Thouzeau G Viard F2006 Gregariousness and protandry promote reproductiveinsurance in the invasive gastropod Crepidula fornicata evi-dence from assignment of larval paternity Molecular Ecol-ogy 15 3009ndash3021

Dupont L Viard F 2003 Isolation and characterization ofhighly polymorphic microsatellite markers from the marineinvasive species Crepidula fornicata (Gastropoda Calyp-traeidae) Molecular Ecology Notes 3 498ndash500

El Mousadik A Petit RJ 1996 High level of genetic differ-entiation for allelic richness among populations of the argantree [Argania spinosa (L) Skeels] endemic to Morocco The-oretical and Applied Genetics 92 832ndash839

Ellner S Hairston NG Jr 1994 Role of overlapping gener-ations in maintaining genetic variation in a fluctuating envi-ronment American Naturalist 143 403ndash417

Foltz DW 1986 Null alleles as a possible cause of heterozy-gote deficiency in the oyster Crassostrea virginica and otherbivalves Evolution 40 869ndash870

Frontier S Pichod-Viale D 1991 Ecosystegravemes structurefonctionnement evolution Paris Masson

Gaggiotti OE Vetter RD 1999 Effect of life history strategyenvironmental variability and overexploitation on thegenetic diversity of pelagic fish populations Canadian Jour-nal of Fisheries and Aquatic Sciences 56 1376ndash1388

Gosling EM Wilkins NP 1985 Genetics of settling cohortsof Mytilus edulis (L) preliminary observations Aquaculture44 115ndash123

Goudet J 2000 FSTAT a program to estimate and test genediversities and fixation indices Available at httpwwwunilchizeasoftwaresfstathtml

Grady SP Rutecki D Carmichael R Valiela I 2001 Agestructure of the Pleasant bay population of Crepidula forni-cata a possible tool for estimating horseshoe crab age Bio-logical Bulletin 201 296ndash297

Dow



ber 2022

374 L DUPONT ET AL


Hedgecock D 1994 Does variance in reproductive successlimit effective population sizes of marine organisms InBeaumont AR ed Genetics and evolution of aquatic organ-isms London Chapman amp Hall 122ndash134

Hedrick PW 2000 Genetics of populations Boston MAJones and Bartlett Publishers Inc

Hoagland KE 1978 Protandry and the evolution ofenvironmentally-mediated sex change a study of themollusca Malacologia 17 365ndash391

Johnson MS Black R 1984 Pattern beneath the chaos theeffect of recruitment on genetic patchiness in an intertidallimpet Evolution 38 1371ndash1383

Jorde PE Ryman N 1995 Temporal allele frequency changeand estimation of effective size in populations with overlap-ping generations Genetics 139 1077ndash1090

Le Gall P 1980 Etude expeacuterimentale drsquoassociation en chaicircneet de son influence sur la croissance et la sexualiteacute chez lacreacutepidule (Crepidula fornicata Linneacute 1758) PhD ThesisFaculteacute des Sciences de Caen

Lessios HA Weinberg JR Starczak VR 1994 Temporalvariation in populations of the marine isopod Exirolana howstable are gene frequencies and morphology Evolution 48549ndash563

Li G Hedgecock D 1998 Genetic heterogeneity detected byPCR-SSCP among samples of larval Pacific oysters (Crassos-trea gigas) supports the hypothesis of large variance inreproductive success Canadian Journal of Fisheries andAquatic Sciences 55 1025ndash1033

MacDonald PDM Pitcher TJ 1979 Age groups from size-frequency data a versatile and efficient method of analysingdistribution mixtures Journal of the Fisheries ResearchBoard of Canada 36 987ndash1001

Moberg PE Burton RS 2000 Genetic heterogeneity amongadult and recruit red sea urchins Strongylocentrotus fran-ciscanus Marine Biology 136 773ndash784

Palm S Laikre L Jorde PE Ryman N 2003 Effective pop-ulation size and temporal genetic change in stream residentbrown trout (Salmo trutta L) Conservation Genetics 4 249ndash264

Raymond M Rousset F 1995 GENEPOP version 12 pop-ulation genetics software for exact tests and ecumenicismJournal of Heredity 86 248ndash249

Ruzzante DE Taggart CT Cook D 1996 Spatial and tem-poral variation in the genetic composition of a larval cod(Gadus morhua) aggregation cohort contribution andgenetic stability Canadian Journal of Fisheries and AquaticSciences 53 2695ndash2705

Ryman N 1997 Minimizing adverse effects of fish culture

understanding the genetics of populations with overlappinggenerations ICES Journal of Marine Science 54 1149ndash1159

Sakai AK Allendorf FW Holt JS Lodge DM Molofsky JWith KA Baughman S Cabin RJ Cohen JE EllstrandNC McCauley DE OrsquoNeil P Parker IM Thompson JNWeller SG 2001 The population biology of invasive speciesAnnual Review of Ecology and Systematics 32 305ndash332

Thieacutebaut E Huther X Shillito B Jollivet D Gaill F 2002Spatial and temporal variations of recruitment in the tubeworm Riftia pachyptila on the East Pacific Rise (9deg50primeN and13degN) Marine Ecology Progress Series 234 147ndash157

Thieltges DW Strasser M Reise K 2003 The Americanslipper limpet Crepidula fornicata (L) in the northern Wad-den Sea 70 years after its introduction Helgoland MarineResearch 57 27ndash33

Viard F Ellien C Dupont L 2006 Dispersal ability andinvasion success of Crepidula fornicata in a single gulfinsights from genetic markers and larval-dispersal modelsHelgoland Marine Research 60 144ndash152

Waples RS 1990 Temporal changes of allele frequency inpacific salmon implications for mixed-stock fishery analysisCanadian Journal of Fisheries and Aquatic Sciences 47 968ndash976

Warner RR Chesson PL 1985 Coexistence mediated byrecruitment fluctuations a field guide to the storage effectAmerican Naturalist 125 769ndash787

Watts RJ Johnson MS Black R 1990 Effects of recruit-ment on genetic patchiness in the urchin Echinometramathaei in Western Australia Marine Biology 105 145ndash151

Weir BS Cockerham CC 1984 Estimating F-statistics forthe analysis of population structure Evolution 38 1358ndash1370

Whitlock MC McCauley DE 1990 Some population geneticconsequences of colony formation and extinction genetic cor-relations within founding groups Evolution 44 1717ndash1724

Whitlock MC McCauley DE 1999 Indirect measures ofgene flow and migration Fst=1(4Nm+1) Heredity 82117ndash125

Wilczynski JZ 1955 On sex behaviour and sex determina-tion in Crepidula fornicata Biological Bulletin 109 353ndash354

Wilson K Hardy ICW 2002 Statistical analysis of sex-ratios an introduction In Hardy ICW ed Sex-ratios Con-cepts and research methods Cambridge Cambridge Univer-sity Press 48ndash92

Wright S 1951 The genetical structure of populationsAnnals of Eugenics 15 323ndash354

Dow



ber 2022

366

L DUPONT

ET AL



2007

90

365ndash374

periods (Waples 1990 Jorde amp Ryman 1995) Thus inlong-lived species with separate male and female func-tions both the sex ratio and the number of genera-tions contributing to a given reproductive eventstrongly influence effective population size and thusgenetic drift (Hedrick 2000) In species with sequen-tial hermaphroditism and social control of sex changelocal sex ratio may be modulated in particular accord-ing to local density (Charnov 1982) modifying theeffective size of populations In addition the distribu-tion of males and females may change across genera-tions (Charnov 1982) Such a system has thus thepotential to limit genetic drift by promoting reproduc-tion across several distant generations

In the present study we used the invasive marinemollusc

Crepidula fornicata

as a model to investigatethe role of sequential hermaphroditism in bufferingthe temporal evolution of genetic diversity in popula-tions This gastropod native to the West Atlantic coastalong North America was accidentally introduced intoEurope at the end of the 19th Century As a successfulinvasive species

C fornicata

has many consequenceson the native community along the Atlantic andEnglish Channel coasts (Thieltges Strasser amp Reise2003) Contrary to the pattern of founder effect fre-quently reported for invaders (Sakai

et al

2001) aprevious study showed high and similar levels ofgenetic diversity in introduced and native populationsof

C fornicata

(eg with allozyme data Dupont Jol-livet amp Viard 2003) Such a high genetic diversity canbe related to many factors (Dupont Jollivet amp Viard2003 Dupont

et al

2006 Viard Ellien amp Dupont2006) among them is a large local effective size inrelation to its high fecundity rates dispersal ability(ie 2ndash4 weeks as pelagic larvae Le Gall 1980) andbreeding system

C fornicata

is a long-lived species(life time of maximum 10 years Blanchard 1995) anda protandrous gastropod (ie mature individuals arefirst male and then change into female Coe 1936)with internal fertilization Individuals are typicallyfound in permanent stacks groups of individualsattached to each other with larger (older) individualsusually females at the base and smaller (younger)individuals usually males at the top (Coe 1936) Inrelation to the timing of sex-reversal strongly beinginfluenced by sexual morphs of neighbouring individ-uals (Coe 1938 Collin 1995) the number of ageclasses contributing to a given reproductive event mayvary according to the distribution of sex across ageclasses

To investigate the role of protandry on the distribu-tion of genetic diversity across generations in localpopulations of

C fornicata

we coupled temporal pop-ulation genetics with demographic analyses which isan approach known to have the potential for under-standing microevolutionary mechanisms in particu-

lar dispersal mating system and age structurebehind the observed spatial patterns (Lessios Wein-berg amp Starczak 1994 Charbonnel

et al

2002 Palm

et al

2003) We first carried out a crude demographicanalysis using the conventional modal decompositionmethod (Thieacutebaut

et al

2002) Then using five mic-rosatellite loci a joint analysis of sex ratio and geneticdiversity across the age groups identified was carriedout in two French populations of the Mont St MichelBay where the species was introduced more than30 years ago and is now an abundant species

MATERIAL AND METHODS

S

TUDY

SITE

AND

SAMPLING

Specimens of

C fornicata

were collected during theApril 2002 BENTHOMONT 2 cruise onboard of thelsquoNO Cocircte de la Manchersquo Two populations lsquoBayrsquo(48

deg

40

prime

55 N 01

deg

45

prime

92 W) and lsquoGulf rsquo (48

deg

45

prime

95 N01

deg

41

prime

84 W) separated by 11 km were sampled in theMont St Michel Bay (Fig 1) Four replicate samplesper site were collected using a 025-m

2

Hamon grabTwo replicates were preserved with 90 ethanol fordemographic and population genetic analysis and forthe two other replicates the number of stacks wascounted on-board The density per site was calculatedas the number of individuals sampled in 1 m

2

of sedi-ment (ie four replicates)

Figure 1

Study area and location of Gulf and Bay popu-lations sampled in 2002 in the Mont St Michel Bay France

GRANVILLE

Gulf

FRANCE

5000 m

BayCANCALE

Mont St Michel Bay

FRANCE

Dow



ber 2022


367



2007

90

365ndash374

S

EX

AND

AGE

STRUCTURE

ANALYSIS


C fornicata


L

50

=


L

max


L

50

L

max

)


et al


lt


t


A

GE

GROUPS

AND

POPULATIONS

GENETIC

ANALYSES




et al


et al



N

all

) observed (

H

o

) and expected(

H

e


et al


Ar


t



F

is




F

st


F

st


f

q

Dow



ber 2022

368

L DUPONT

ET AL



2007

90

365ndash374


RESULTS

S

EX

RATIO

AND

AGE

GROUPS

ANALYSES


minus

2


qCT

qSC

qST



P

lt


χ

2

=

515

P

=

096 df

=


χ

2

=

497




0

20

40

60

80

100

05 15 25 35 45 55 65 75 85 95 105

Num

ber

ofin

divi

dua l

s

NI= 679NM = 903NT = 42NF = 350

Size classes (cm)


N = 844

0

20

40

60

80

100

05 15 25 35 45 55 65 75 85 95 105

0

100

200

300

400

500

05 15 25 35 45 55 65 75 85 95 105

NI= 90NM = 442NT = 24NF = 288

N = 1974

0

20

40

60

80

100120

140

05 15 25 35 45 55 65 75 85 95 105

Size classes (cm)

420

440

Num

ber

ofin

divi

dual

s


M1

M2 M3

M4

M1

M2

M3M4

2= 515

p = 096

2= 497

p = 099

i s

1

eof


Males


c )

065

NI= 90NM = 442NT = 24NF = 288

N = 1974

Size classes (cm)

Gulf population

X2= 515

p = 096

X

Dow



ber 2022









Population

Age groups

M1 M2 M3 M4






0

20

40

60

80

100

Age-groups



Males


0

20

40

60

80

100

(77) (60) (83) (83) (73)

(59) (59) (60) (60) (47)

Age-groups

1 2 3 4 5

1 2 3 4 5

p



i i n

Dow



ber 2022

370 L DUPONT ET AL






ff









f


0

005

010

015

020

025

Age-group

1

Age-group

2

Age-group

3

Age-group

4

Mean Population

Bay

Gulf

Fis

e- p

3

lf

Dow



ber 2022






q

q q

qCT

qST

qSC

q


DISCUSSION





B1 B2 B3 B4 G1 G2 G3 G4



q

Dow



ber 2022

372 L DUPONT ET AL






q



Dow



ber 2022





ACKNOWLEDGEMENTS


REFERENCES


























Dow



ber 2022

374 L DUPONT ET AL






























Dow



ber 2022


367



2007

90

365ndash374

S

EX

AND

AGE

STRUCTURE

ANALYSIS


C fornicata


L

50

=


L

max


L

50

L

max

)


et al


lt


t


A

GE

GROUPS

AND

POPULATIONS

GENETIC

ANALYSES




et al


et al



N

all

) observed (

H

o

) and expected(

H

e


et al


Ar


t



F

is




F

st


F

st


f

q

Dow



ber 2022

368

L DUPONT

ET AL



2007

90

365ndash374


RESULTS

S

EX

RATIO

AND

AGE

GROUPS

ANALYSES


minus

2


qCT

qSC

qST



P

lt


χ

2

=

515

P

=

096 df

=


χ

2

=

497




0

20

40

60

80

100

05 15 25 35 45 55 65 75 85 95 105

Num

ber

ofin

divi

dua l

s

NI= 679NM = 903NT = 42NF = 350

Size classes (cm)


N = 844

0

20

40

60

80

100

05 15 25 35 45 55 65 75 85 95 105

0

100

200

300

400

500

05 15 25 35 45 55 65 75 85 95 105

NI= 90NM = 442NT = 24NF = 288

N = 1974

0

20

40

60

80

100120

140

05 15 25 35 45 55 65 75 85 95 105

Size classes (cm)

420

440

Num

ber

ofin

divi

dual

s


M1

M2 M3

M4

M1

M2

M3M4

2= 515

p = 096

2= 497

p = 099

i s

1

eof


Males


c )

065

NI= 90NM = 442NT = 24NF = 288

N = 1974

Size classes (cm)

Gulf population

X2= 515

p = 096

X

Dow



ber 2022









Population

Age groups

M1 M2 M3 M4






0

20

40

60

80

100

Age-groups



Males


0

20

40

60

80

100

(77) (60) (83) (83) (73)

(59) (59) (60) (60) (47)

Age-groups

1 2 3 4 5

1 2 3 4 5

p



i i n

Dow



ber 2022

370 L DUPONT ET AL






ff









f


0

005

010

015

020

025

Age-group

1

Age-group

2

Age-group

3

Age-group

4

Mean Population

Bay

Gulf

Fis

e- p

3

lf

Dow



ber 2022






q

q q

qCT

qST

qSC

q


DISCUSSION





B1 B2 B3 B4 G1 G2 G3 G4



q

Dow



ber 2022

372 L DUPONT ET AL






q



Dow



ber 2022





ACKNOWLEDGEMENTS


REFERENCES


























Dow



ber 2022

374 L DUPONT ET AL






























Dow



ber 2022

368

L DUPONT

ET AL



2007

90

365ndash374


RESULTS

S

EX

RATIO

AND

AGE

GROUPS

ANALYSES


minus

2


qCT

qSC

qST



P

lt


χ

2

=

515

P

=

096 df

=


χ

2

=

497




0

20

40

60

80

100

05 15 25 35 45 55 65 75 85 95 105

Num

ber

ofin

divi

dua l

s

NI= 679NM = 903NT = 42NF = 350

Size classes (cm)


N = 844

0

20

40

60

80

100

05 15 25 35 45 55 65 75 85 95 105

0

100

200

300

400

500

05 15 25 35 45 55 65 75 85 95 105

NI= 90NM = 442NT = 24NF = 288

N = 1974

0

20

40

60

80

100120

140

05 15 25 35 45 55 65 75 85 95 105

Size classes (cm)

420

440

Num

ber

ofin

divi

dual

s


M1

M2 M3

M4

M1

M2

M3M4

2= 515

p = 096

2= 497

p = 099

i s

1

eof


Males


c )

065

NI= 90NM = 442NT = 24NF = 288

N = 1974

Size classes (cm)

Gulf population

X2= 515

p = 096

X

Dow



ber 2022









Population

Age groups

M1 M2 M3 M4






0

20

40

60

80

100

Age-groups



Males


0

20

40

60

80

100

(77) (60) (83) (83) (73)

(59) (59) (60) (60) (47)

Age-groups

1 2 3 4 5

1 2 3 4 5

p



i i n

Dow



ber 2022

370 L DUPONT ET AL






ff









f


0

005

010

015

020

025

Age-group

1

Age-group

2

Age-group

3

Age-group

4

Mean Population

Bay

Gulf

Fis

e- p

3

lf

Dow



ber 2022






q

q q

qCT

qST

qSC

q


DISCUSSION





B1 B2 B3 B4 G1 G2 G3 G4



q

Dow



ber 2022

372 L DUPONT ET AL






q



Dow



ber 2022





ACKNOWLEDGEMENTS


REFERENCES


























Dow



ber 2022

374 L DUPONT ET AL






























Dow



ber 2022









Population

Age groups

M1 M2 M3 M4






0

20

40

60

80

100

Age-groups



Males


0

20

40

60

80

100

(77) (60) (83) (83) (73)

(59) (59) (60) (60) (47)

Age-groups

1 2 3 4 5

1 2 3 4 5

p



i i n

Dow



ber 2022

370 L DUPONT ET AL






ff









f


0

005

010

015

020

025

Age-group

1

Age-group

2

Age-group

3

Age-group

4

Mean Population

Bay

Gulf

Fis

e- p

3

lf

Dow



ber 2022






q

q q

qCT

qST

qSC

q


DISCUSSION





B1 B2 B3 B4 G1 G2 G3 G4



q

Dow



ber 2022

372 L DUPONT ET AL






q



Dow



ber 2022





ACKNOWLEDGEMENTS


REFERENCES


























Dow



ber 2022

374 L DUPONT ET AL






























Dow



ber 2022

370 L DUPONT ET AL






ff









f


0

005

010

015

020

025

Age-group

1

Age-group

2

Age-group

3

Age-group

4

Mean Population

Bay

Gulf

Fis

e- p

3

lf

Dow



ber 2022






q

q q

qCT

qST

qSC

q


DISCUSSION





B1 B2 B3 B4 G1 G2 G3 G4



q

Dow



ber 2022

372 L DUPONT ET AL






q



Dow



ber 2022





ACKNOWLEDGEMENTS


REFERENCES


























Dow



ber 2022

374 L DUPONT ET AL






























Dow



ber 2022






q

q q

qCT

qST

qSC

q


DISCUSSION





B1 B2 B3 B4 G1 G2 G3 G4



q

Dow



ber 2022

372 L DUPONT ET AL






q



Dow



ber 2022





ACKNOWLEDGEMENTS


REFERENCES


























Dow



ber 2022

374 L DUPONT ET AL






























Dow



ber 2022

372 L DUPONT ET AL






q



Dow



ber 2022





ACKNOWLEDGEMENTS


REFERENCES


























Dow



ber 2022

374 L DUPONT ET AL






























Dow



ber 2022





ACKNOWLEDGEMENTS


REFERENCES


























Dow



ber 2022

374 L DUPONT ET AL






























Dow



ber 2022

374 L DUPONT ET AL






























Dow



ber 2022

Documents

Sex and genetic structure across age groups in populations of