Evolution of genetic diversity in metapopulations: Arabidopsis thaliana as an experimental model

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Evolution of genetic diversity in metapopulations:

Arabidopsis thaliana as an experimental model

Claire Lavigne, X. Reboud, Madeleine Lefranc, Emmanuelle Porcher, Fabrice

Roux, Isabelle Olivieri, B. Godelle

To cite this version:

Claire Lavigne, X. Reboud, Madeleine Lefranc, Emmanuelle Porcher, Fabrice Roux, et al.. Evolution

of genetic diversity in metapopulations: Arabidopsis thaliana as an experimental model. Genetics

Selection Evolution, BioMed Central, 2001, 33 (Suppl. 1), pp.S399-S423. �halsde-00335116�

c

INRA,EDPSciences,2001

Original article

Evolution of genetic diversity

in metapopulations: Arabidopsis

thaliana as an experimental model

Claire Lavigne

∗a

,Xavier Reboud

b

,Madeleine Lefranc

a

, Emmanuelle Porcher

a

Fabrice Roux

b

IsabelleOlivieri

c

Bernard Godelle

d

a

Laboratoireécologie, systématiqueetévolution,UniversitéParis-XI/CNRS UPRES-A8079,bâtiment362,91405OrsayCedex,France

b

Laboratoiremalherbologie etagronomie,Institutnationaldelarecherche agronomique,BP86510,21065DijonCedex,France

c

Institutdessciencesdel'évolution,UniversitéMontpellier2, PlaceEugèneBataillon,34095MontpellierCedex05, France

d

Laboratoiregénome,populations,interactions,UniversitédeMontpellierII, casecourrier063,bâtiment13,34095MontpellierCedex05,France

Abstract Two experiments were set up to investigate how to maintain or cre-ategeneticdiversityinarticialormanagedpopulationsofplants. UsingArabidopsis thaliana,weestablished18metapopulationsof20populationseach,allwiththesame initialgeneticcomposition. Wetestedtheeectsofthepopulationsize,thearticial selection regimeand the extinction/recolonisationregime. Wereportthe resultsof therst fourgenerations ofevolutionfor atraitunderselection (precocity)and for allozymediversity. Asexpected,overalldiversity decreasedineachmetapopulation, anddierentiationamongpopulationsincreased. Asexpected,thedierentiationwas weakerforlargerpopulationsizesandinthetreatmentwithextinctionand recoloni-sation with no bottleneck. Articial selection was eective because the life cycle durationwasmuchreduced. However,mostofthereductionoccurredduringtherst generation. Weobservedanincreaseofoneallele attheLAP-2locusinall metapop-ulations, breachingneutralassumptions forthis locus. Finally,the selection regime madelittledierenceforsmallpopulationsizes,whereaslargemetapopulationswere more dierentiated whenarticial selection was heterogeneous among populations. Altogether, our results agree with theoretical expectations, and provide some new results, whichcould not havebeen anticipated. In particular, the overall decrease in genetic diversity was very large (of the order of 20% in4 generations) even for metapopulationsof2000individuals.

genetic diversity / experimental evolution / conservation / small populations / precocity

∗

Résumé Évolutiondeladiversitégénétiqueenmétapopulation: Arabi-dopsis thaliana comme modèleexpérimental. Deuxexpérimentations,visant àétudiercommentmainteniroucréerdeladiversitédanslespopulationsarticielles ougéréesdeplantes,ontétémenéesenutilisantArabidopsisthalianacommeespèce modèle.Nousavonscréé18métapopulationsde20populationsdemêmecomposition initiale. Nousavonstestéleseetsdelatailledespopulations,durégimedesélection etdu régime d'extinction/recolonisation. Les résultats surl'évolution du caractère sélectionné (durée du cycle de vie) et sur la diversité enzymatique sont présentés pour les quatrepremières générations. Conformément aux attendus théoriques, la diversitéaglobalementdiminuédanslesmétapopulationsetlespopulationssesont diérenciées. Ladiérenciationestplusfaiblepourlesgrandespopulationsetdansle traitementavecextinctionsetrecolonisationssurunebasegénétiquelarge. La sélec-tionarticiellearéduit ladurée ducycledesplantes. Cetteréductiona essentielle-menteulieupendantlapremièregénération. Nousavonsobservéuneaugmentation d'un des allèles au locus LAP-2 dans toutesles métapopulations, en contradiction aveclaneutralité présupposée decelocus. Enn, lasélection aeupeud'eet dans lespetites populationsalorsquelesgrandespopulationssontplusdiérenciéespour les allozymesquandlasélectionesthétérogène entrepopulations. Globalement,nos résultatssontplutôtenaccordaveclesprédictionsthéoriques;certainsrésultatssont néanmoinsinattendus. Enparticulier,lapertedediversitéglobaleaététrès impor-tante(del'ordrede20%enquatregénérations),mêmedanslesmétapopulationsde 2000individus.

diversité génétique / évolution expérimentale / conservation / petites populations / précocité

1. INTRODUCTION

Genetic resourcesof crop plants and of their wild relativeshave been col-lected and stored in gene banks forseveral decades [9]. Some ofthese banks containverylargenumbersofsamplesandtheirevaluation, managementand utilisationraiseanincreasingnumberofquestions. Fromabiologicalpointof view,themainquestionsconcern(i)thetoolstoevaluatebothneutraland po-tentiallynon-neutralquantitativegeneticdiversity,and(ii)regeneration meth-odsallowingmaintenanceoftheinitialdiversity,preventionoftheaccumulation of deleterious mutations, andavoidance of the evolutionary freeze of popu-lations removed from theirnatural environment [18]. Similar questions arise regardingtheconservation ofpopulations ofrare species. Thelackof genetic variabilityassociatedwithsmall populationsizesand,possibly,the accumula-tionof deleteriousmutationsare, indeed, thoughtto bemajorfactorsdriving smallpopulationsto extinction,althoughtheseissuesarecontroversial[11].

Toaddress some of these questions a dynamic management of genetic di-versityhas been suggested[1,18,33]. Such amanagement involvesin situor ex situ maintenance of evolving metapopulations. The metapopulation con-cept wasintroduced byLevins [23] as apopulationof populations connected bymigration eventsandsubjectto extinctionand recolonisationevents. The

aimofsuchamanagementistomaintainasmuch diversityaspossibleat the metapopulationlevel,and even tocreate newgenetic combinationswhile let-tingthepopulationsevolveandadapttotheirlocalenvironment[18]. Forcrop species,articialpopulationsneedtobecreated,whereasforwildspeciesboth in situandexsitumanagementcouldbeconsidered. Forinstance, adynamic management ofgenetic resourceswas establishedin 1984for winter wheat, a selngspecies[6,18,21,22,25,30,31].

Geneticdiversityroughlycomprisestwocomponents: neutraldiversity,which, bydenition,isnotunder selectioninthestudyenvironmentat agiventime, and non-neutraldiversity. Potentialusers ofgenetic resourcesare mainly in-terested in maintaining and evaluating diversityfor agronomictraits such as morphology, seedproductionorpest resistances,whichin most environments areunder selection. These traitsaremostlikelyalsorelatedtotnessin wild populations. The measurement of these traits, however, is time-consuming and thetrait valuescanbeverydependentontheenvironmentin which they are measured. In contrast, neutral diversity is easier to assess since numer-ousmolecular markersare available andtheir expressionis notdependent on the environment. The theory about the structure of neutral diversity in a metapopulationisfurthermorewellestablished(reviewedin[14])andthe neu-tral diversity of a population can be thought of as a reservoir for potential futureadaptation[11].However,therelationshipbetweenthepatternsof neu-tralandselecteddiversitiesin ametapopulationdependsonnumerousfactors suchaspopulationsizes,eectiverecombinationandtypeof selection[17]. In this paper,wereport on anexperiment under controlled conditionsthat was designedtoinvestigatetheeectofdierentmetapopulationmanagementson theevolutionofbothallozymeandquantitativegeneticvariability.

Twoexperimentsweresetuptoinvestigatehowtomaintainorcreategenetic diversityin articialand/or managedpopulationsofplants. Our goalwasto testtheeectsofpopulationsizes,selectionregimeandextinction/recolonisation eventsinaselngspecies. Wesetupanextinction/recolonisationexperiment tomimicthreecontrastedsituationsofinsitumanagement: ahabitatwithno disturbance, compared with twothat experience disturbances; onewith high likelihoodofrecolonisation,andtheotherwithlowlikelihoodofrecolonisation. The comparison of the evolution of diversity among the various treatments couldhelpus designandmaintainpopulationsforconservationpurposes.

2. MATERIAL ANDMETHODS

The study species is the smallannual Arabidopsis thaliana (Brassicaceae). It ismainly selng withnaturallevelsofoutcrossingof 1.22.2%[39]. It was chosenforitsshortlife cycle,whichinthegreenhousecanlast aslittleastwo months, and for the largeamount of knowledge available on its geneticsand physiology[34].

F22

(1)

F28

(2)

_F78a

₍₁₎

F78b

(1)

F06

(4)

F37

(1)

F45

(1)

F69

(1)

Figure1. Geographicoriginoftheparentallines. Thenamesoftheparental popu-lationsareinbold,followedbythenumberofparentalindividualscomingfromthese populations (in brackets). TheGBpopulation (twoparentallines) originates from Wales(UK).

2.1. Initialcompositionofthe metapopulations

Fourteenlines ofA. thaliana collectedin nine naturalpopulations(Fig. 1) and the nw77 male sterile mutant from the Nottingham Arabidopsis Stock Centre

1

were used as parental lines of the metapopulations. Male sterility is due to a singlepoint nuclear recessivemutation, which causes theabsence of petals, stamensandpollenproduction. Themalesterilemutantwas intro-duced in the populations to maintain some level of outcrossing(see below). The natural parental lines were chosen for their oweringsynchronyand for theirdierencesinelectrophoreticpatternsatveallozymeloci. Tocreatethe initialpopulations,parentallines(Tab.I)werecontrol-crossedbyhand follow-ing theprotocol described onTable II. The resultofthe crosseswaschecked byanalysingthepatternsoftheF1individualsattheveallozymelociwhich

1

Malesterilemutant: mutant NW77 Pistillata, Background Ler. Mutagen EMS, locus pi, allele pi1, Map position 5-23. The NottinghamArabidopsis stock centre, DepartmentofLifeScience,UniversityofNottingham,UniversityPark,Nottingham

Table I. Allozymiccompositionoftheinitial parentallines(foragivenlocus,each numberrepresentsaparticularallele).Indicesindicatethedierentindividualswithin apopulation.

Parentallines IDh LAP-2 AcPh1 AcPh2 SkDh

F22 2 4 2 2 1 F28

1,2

2 4 1 1 1 GB

1

1 4 2 2 1 GB

2

2 4 3 2 1 F06

1,2,3,4

2 1 1 2 2 F78a 2 4 2 2 1 F78b 2 4 2 1 1 F37 2 2 2 1 1 F45 2 3 2 2 1 F69 2 4 2 2 1 NW77 2 4 2 2 1

Table II. Crosses attheorigin oftheF2andtheircontributiontotherstgeneration.

Cross Contribution Cross Contribution

GB

1

×

F78b 10% F45

×

F28

2

10%

(GB

1

×

F06

1

)

×

NW77 10% (F45

×

F06

3

)

×

NW77 15%

GB

1

×

F28

1

5% F37

×

F06

4

5%

F37

×

F28

2

10% F22

×

F28

1

10%

F69

×

F06

2

5% GB

2

×

F78a 20%

distinguished theparents(Tab. I). F1 individuals were selfed. The resulting F2 seeds werepooledaccordingto theproportionsgiven in TableIIand this poolof seeds wasused to sowthe rstgeneration ofeach local populationof eachmetapopulation.

2.2. Experimentaldesigns

2.2.1. Treatments

Two experiments were set up (Tab. III). The rst one (hereafter experi-ment 1, set up at Orsay), aimed to explore the eect of population size and selectionregimeontheevolutionofthediversityinthemetapopulations. The secondone(hereafterexperiment2,set upat INRADijon),wasaimed at un-derstandingthe eect ofdierent extinction/recolonisationregimes. Inboth experiments,migrationrateamongextantpopulationswasxedat 2%.

For experiment 1, twelve metapopulations were set up, each containing 20 populations. The two treatments were populationsize (10, 25 or100 in-dividualsperpopulation) andselectionregime (directionalorheterogeneous).

Table III. Numberof metapopulations studiedfor eachtreatmentof each experi-ment. Eachmetapopulationwasmadeof20localpopulations.

Totalnumberof Selectionregime Experiment metapopulations Directional Heterogeneous Orsay(Noextinction) 12

Populationsize(

N

) -100 2 2 -25 2 2 -10 2 2 Dijon(

N = 100

) 6 Extinctionregime -Noextinction 2 --Migrantpool 2 --Propagulepool 2

-plants in pots of areas 26.4cm

2

(10 plants), 86.25 cm

2

(25 plants) and 350 cm

2

(100 plants). Twometapopulationsweregrown for each populationsize

×

selection regime combination. The selection for a short life cycle was ap-pliedby stoppinganywateringassoonas the rstfruitswere maturein one ofthetworeplicatemetapopulations. Inthedirectionalselectionregime,each populationofeachreplicatemetapopulationwasselectedforashortlifecycle. Intheheterogeneousselectionregime,eachmetapopulationwassplitintotwo groupsoften populations: onegroupbeingselectedforashort life cycle, the otherbeingallowedto growandoweraslongasnecessary. Populationswere assigned toeither groupat therstgeneration. Withineach metapopulation, all populations were allowed to exchange genes randomly by way of the 2% migrants.

Forexperiment2,metapopulationscontained20populationsof100 individ-ualsgrowninthesamelargepotsasinexperiment1andallselectedforashort lifecycle. Thetreatmentsconsistedofthreedierentextinction-recolonisation regimes: 1) noextinction (as in experiment 1), 2) extinction and recolonisa-tionfromalargegeneticbasis(100individualsfrom vedierentpopulations drawn at random within the metapopulation), corresponding to a migrant-poolpattern[38],3)extinctionandrecolonisationfrom anarrowgeneticbasis (ve individuals from one population drawn at random), corresponding to a propagule-poolpattern [38]. Thelocalextinction ratewas25%for metapopu-lationswithextinctions,i.e. eachgenerationvepopulationsrandomlychosen from20wereeliminated,andreplacedbyrecolonisers. Tworeplicate

metapop-2.2.2. Experimentalconditionsof eachgeneration

Intheabsenceofanyfemaleadvantage,andasmale-sterilityisdetermined byasinglerecessivegene,theproportionofmale-sterileindividualsisexpected todecreasebyhalfeachgeneration. Inordertoensureaminimumoutcrossing rate of 10%, foreach non-extinct population, and wheneverpossible, 10%of the seeds used to grow the following generation were harvested from male-sterile individuals of that population. Thus, the average frequency of male-sterile individuals among newly produced seeds was expected to be kept at 5%. About88%oftheseeds forthenextgenerationwereharvestedfromlocal hermaphrodites. The remainingaverage 2%were migrantsdrawn at random in another single, randomly chosen, population of the metapopulation. In experiment2,extinctpopulationswererecreatedasdescribedabove.

Seedsweresownonaregulargrid,wateredwithasolutioncontaining0.15% fungicide(Dericlor, CibaGeigy) andleft for oneweek in thedarkat 4

◦

Cto break dormancy. This coldtreatment wasstopped in experiment 2after the third generation. After germination, the plants were grown in a controlled compartmentofagreenhouseundera16hlight/8hdarkphotoperiod,a tem-peraturevaryingbetween15

◦

Cnightand20

◦

C,dayandwerewateredtwice aweek. Inorder toavoidlocal environmental eects, thepots were regularly moved around during thegrowingperiod. Twoorthree times during ower-ing, owersof male-sterileindividualsweregentlyrubbedwith themaximum numberofmale-fertileowersofthepopulationtoensureseedproduction.

Once watering of the plants wasstopped, theywere left to dry. Fruits of male-sterileindividualswereharvestedseparatelyandtherestofthepopulation washarvested as awhole. Seeds were then stored in the darkat 4

◦

C, 10% humidity.

2.3. Measurements

2.3.1. Allozymicdiversity

InthefourteenparentallinesweobservedfourallelesfortheLeucineAmino Peptidaselocus(LAP-2),threeallelesfortheAcidPhosphatase1locus(AcPh1) andtwoallelesfortheAcidPhosphatase2locus(AcPh2),theShikimic Dehy-drogenaselocus(SkDh)andtheIsocitricDehydrogenaselocus(IDh). Enzyme polymorphismwasassessedon IDh, LAP-2, AcPh 1 and SkDhat thefourth generationforexperiment1,andontheLAP-2, AcPh1and2locievery gen-erationforexperiment2.

Inexperiment 1,wesampled tenindividuals from vepopulationsin each metapopulationwith25or100individualsperpopulation,andseven individu-alsfromsixpopulationsineachmetapopulationwith10individualsper popu-lation. Inexperiment2,10individualsperpopulationweresampledexceptfor populationsundernarrow-basisrecolonisationforwhichthetotalpopulationof veindividualswasanalysed. Theoriginandnumberofpopulationsanalysed

variedfromgenerationtogeneration(38populationsinGeneration1(G1),44 in G2,40inG3, 56inG4).

Enzyme extracts were obtained from leaves of four week-old plants. For experiment 1 (performed in Orsay), extracts were migrated on a13% starch gelinaLithiumborate buer(adaptedfrom[40])torevealthepolymorphism for the LAPand AcPhenzymes, and ona 12.5% starch gel in a Tris citrate buer (adaptedfrom [32]) for theSkDhand IDh enzymes. Forexperiment2 (performedinDijon),foliarisoenzymeswereseparatedbyelectrophoresisona polyacrylamidegel usingthe methoddescribed byOrnstein[29]and Gasquez andCompoint[13]inadiscontinuoussystemwithpulsepower. Detailed spec-icationsoflaboratorytechniquesaregivenin[3].

2.3.2. Geneticanalyses

Nei'sunbiasedestimatorofgenediversity[28]wascalculatedforpopulations andmetapopulationsusingtheFstatsoftware[15].

Within-populationdiversitiesperlocuswereestimatedas:

H

s

=

_{˜n − 1}

˜n



1 −

X

p

2

i

−

H

_2˜n

o



,

where

˜n

is the harmonicmean of thenumbersof individuals perpopulation,

p

2

i

istheaverageoverpopulationsofthesquaredfrequencyofeachalleleat a given locus,and

H

o

istheaverageobservedheterozygosityatthis locus. The averagediversityiscalculatedastheaverage

H

s

overloci.

Overallgenediversitiesperlocusareestimated as:

H

t

= 1 −

X

(p

_i

)

2

₊

H

s

˜nn

p

−

H

o

2˜nn

p

,

where

n

p

isthenumberofpopulations,

p

i

theaveragefrequencyof eachallele atagivenlocusoverpopulations,and

H

o

istheobservedheterozygosityatthis locus. Theaveragediversityiscalculatedas theaverage

H

t

overloci.

Weirand Cockerham'sestimatorsof

F

is

and

F

st

[45] (hereafternamed

F

ˆ

is

and

F

ˆ

st

)were alsocalculatedwiththeFstat software. Thetestforsignicant dierenceofthese

F

-valuesfromzerowasperformedbypermutingindividuals overpopulations. Nostandarderrorswerecalculatedduetothesmallnumber oflocistudied.

Given theinitial composition of the metapopulations, theexpected diver-sities were

Ht

0

= Hs

0

= 0.35

for experiment 1 and

Ht

0

= Hs

0

= 0.47

for experiment 2 at the rst generation. The dierence between experiments is due tothedierencein theset oflocistudied. Expected

F

st

valueswerezero sincepopulationswerecreatedfromthesameseedpool,andtheexpected

F

is

2.3.3. Precocity

As conditions in the greenhouse may vary throughout the year, a direct comparisonofoweringphenologyinnumberofdaysbetweengerminationand owering could be misleading. To minimise the environmental eect, popu-lations from several generations were compared simultaneously. Thus, in ex-periment 2, the eciency of the selection for precocity was assessed for the rst three generationson asub-sampleof three populations per metapopula-tion leadingto sixmetapopulations X three populations X three generations

+10

samplesforgeneration

1 = 64

samples. Fromeachsample,50seeds were sownin plastic pots(

17.5 × 13 × 5.5

cm). Everydayfor90days,the propor-tionsofplantsinthefollowingstageswere noted: cotyledons,rosette,bolting, owering,greenpodsandmaturefruits.

Thecumulativeevolutionof oweringfrequencyovertimein days(D) was thenttedtothefollowinglogisticmodel:

cumevolfl =

K

[1 + (K − 1) exp(−r(D − i)), ]

where

i

istheestimatedparameterforthetimelagbeforerstowering,

r

,the synchronyofoweringand

K

,theproportionofoweringwhentheexperiment wasstopped. Thegoodnessoftwasassessedusinga

R

2

. Applyingthismodel to each ofthe64samples, all

R

2

valuesrangedbetween0.873 and0.997with ameanvalueof

0.972 ± 0.023

andwereallhighlysignicant.

Thegeneration eect was thentested usingone-wayANOVA applied indi-viduallyto eachofthethreeestimatedparametersdescribedabove.

2.4. Theoreticalpredictions

2.4.1. Eect of selection regime on local neutral diversity (Experiment1)

Selection increasesthe variancein ospring number[16],thus reducing ef-fectivepopulationsize. Thereductionin eectivepopulationsize should only depend on the strength of selection, not on it being homogeneousor hetero-geneous. Thus, forthose lociunlinkedtoselectedones,wedonotexpectany eect of the selectionregime onlocal diversity. Alternatively, onecould con-siderthat whenallpopulationsareselectedforprecocity,selectionisstronger and thus eective population sizes are smaller in the homogeneous selection treatmentcomparedwiththeheterogeneoustreatment. Inthiscase,weexpect less genetic diversity in the homogeneous selection regime. Overall, and for agiven populationsize,weexpect localgenetic diversityin thehomogeneous selection regime to be either the same or smaller than in the heterogeneous

2.4.2. Eect of selection regime on dierentiation among populations forneutral markers (Experiment1)

For a given population size, eective migration rate should be lower in the heterogeneousselection regime,as onaverage immigrants arenot locally adapted [7]. Althoughdierentiationamong populationsfor unlinkedneutral lociis notexpectedto behighly inuencedby thetypeofselection,ifit hap-penstobeitshouldbesuchthatpopulationsaremoredierentiatedunderthe heterogeneousselectionregime.

Hitch-hikingmightalso aect theevolution at neutrallociin linkage dise-quilibriumwithselected ones. Directionalselectiontendsto homogenise pop-ulations for those loci undergoingselection and linked neutral loci, whereas heterogeneousselection promotesdierentiation. Forthose loci, wethus also expect tondmoredierentiationunder theheterogeneousselectionregime.

Overall, for a given population size, we thus expect more dierentiation undertheheterogeneousselectionregime.

2.4.3. Eect of selection regime on global neutral diversity (Experiment1)

Assuming that the strength of selection is the same in the two selection regimes, the sole eect of selection is on dierentiation among populations, expectedto bestrongerunderheterogeneousselection. Wethusexpectglobal diversitytobelargerunderthisregime. Thepatternisreinforcedifweassume thatthestrengthofselectionisgreaterunderhomogeneousselectionsincelocal diversitywoulddecreasesimilarly inallpopulations.

Wethusexpectglobaldiversityto besmallerinthehomogeneousselection regime. Thisisparticularlyexpectedforthosegeneslinkedtothose determin-ingprecocity.

2.4.4. Eectofpopulationsizeonneutraldiversity(Experiment1)

Aslocalpopulationsizesincrease,geneticdriftisweakerlocally. Therefore, atthelocallevel,weexpectgeneticdiversitytoincreasewithlocalpopulation size. Assuming anequilibrium betweenmigrationand drift, theshapeof

F

st

as a function of

N

∗

_m

indicates that above the value

N

∗

_{m = 1}

, the level of dierentiation among populations is expected to be low [5]. Fora complete selfer, this threshold value is

N

∗

_{m = 3}

. In experiment 1, the rate of seed immigrationis 2%,thusthenumbers

Nm

of migrantspergeneration are0.2, 0.5and2forthethreepopulationsizes. Thisisprobablylessthanthethreshold valueabovewhichmigrationisexpectedtocounteractdriftinourexperimental system,asselngratewasinfact large, albeitmale-sterility. Thus weexpect large dierentiation among populations to build up, all the more so for low eectivesizes.

Innitepopulations,selectionhasmoreimpactonthefrequencyofafavoured allele thandrift onlyif

Ne

∗

_{s > 1}

where s istheselection coecient[16]. We thereforeexpectresponsetoselectiontobestrongerforlargerpopulationsizes, at least in the directional selectionregime. Theincrease in diversity as pop-ulationsbecome largershould thusbesmaller thanexpected with thepurely neutralmodel. In theheterogeneousselectionregime,the eectivenumberof migrantsshould also increase lessthan with thepurely neutralmodel, since, asalreadystatedabove,migrantsonaveragearenotlocallyadapted. Overall, thereductionineectivesizeforneutralgenesduetoselectionatotherlociis expectedtoincreasewithpopulationsize,atleastinthehomogeneousselection regime, reducingthe direct eect of diminishingdrift with increased popula-tion size. We thus expect a slight decrease in dierentiation with increased populationsize.

2.4.5. Eect of local extinctions and recolonisation treatment on neutraldiversity (Experiment2)

Localextinctionshaveintheorytwoeects[43,46]. First,theydecreasethe totalmetapopulationsizeandthustheoverallgeneticdiversityshouldbelower comparedto thenoextinction treatment. Second,theycreateabottleneckat recolonisation, and thus increase the amount of dierentiation among popu-lations. At the sametime, they increase gene owamong populations, thus actingagainstdierentiation. Intherstextinctiontreatment,recolonisations occur by sampling 100 migrants from several populations, so that the main eectofextinctionistoincreasegeneow. Inthesecondextinctiontreatment, only ve individuals, sampled from asinglepopulation,are allowed toact as recolonisers. Thus themain eect of this treatmentis abottleneck. We thus predict that thestrongerdierentiationat equilibrium should be observedin thesecond(narrow-basis)extinctiontreatment,followedbythenoextinction treatment, followed by the large-basis extinction treatment. Total diversity should belargerin thenoextinctiontreatment.

2.4.6. Eect of local extinctions and recolonisation treatment on response to selection(Experiment2)

Response to selection isexpected to increasewith totaleective metapop-ulationsize. Itshould thusbelargerin thenoextinction treatment, followed bytheextinction treatmentwithalargegeneticbasisrecolonisation,followed bythebottlenecktreatment. Note howeverthat enhancedgene ow,as expe-rienced in the extinction regime with large base recolonisation, might at the same time locally increase the evolutionary potential, and thus favour local adaptation[12]. Thus, noclearpredictioncanbemadewithoutfurther mod-elling.

0

1

2

3

4

4-Exp1

Generation

0.0

0.2

0.4

0.6

0.8

1.0

Frequency

of

LA

P

allele

4

Figure 2. Box plot graph representing the evolution of the frequency of allele 4 of the LAP-2 during the rst four generations in experiment 2 and at the fourth generation inthe largemetapopulationsof experiment 1. Thecircle onthe Y-axis indicates the initial frequency of the allele. The boxes represent the interquartile range, and asterisks represent outliers. Data are represented on a per population basis,independentlyofthemetapopulation.

3. RESULTS

Wepresentresultsfortherstfourgenerationsoftheexperiments.

3.1. Allozymevariability

3.1.1. Allelicand genotypic frequencies

Considering the two experiments, allele 4 of the LAP-2 increased in fre-quencybetweentherstandthefourthgenerationin16outofthe18 metapop-ulations. This is signicantly dierent from the 50%increase, 50%decrease expected withdrift(rank test

P < 0.001

). Its increasein frequencyis signi-cantin allmetapopulations (Kruskal-Wallisstatistics=31.21,

P < 0.0001

for a generation eect) in experiment 2 where it was estimated at each genera-tion. The frequency of this allele was notsignicantly dierent between the twoexperimentsatthefourthgenerationwhenonlymetapopulationswith100 individualsperpopulationwere considered(

P = 0.72

)(Fig. 2). There wasno signicanteectofthetreatmentonthefrequencyofthisalleleineither experi-ment(

P = 0.56

inexperiment1and

P = 0.95

inexperiment2). Thisallelewas

in ourlaboratory(datanotshown),and waspresentin sixoutof nine popu-lations initially used asabasis for the metapopulations (Tab. I). Signicant changes in allele frequencies were observed for the other enzymatic systems, but there wasnoobviousrelationshipbetweenthe level ofsignicance of the tests, the direction of change and the population size or selection treatment (datanotshown).

RemovingtheLAP-2locusfromthefollowinganalysesincreasedslightlythe valuesof

F

ˆ

st

,butitdidnotmodifythegeneralpattern(datanotshown). The resultsoveralllociwillthereforebediscussed.

3.1.2. Within-populationdiversity

Within-population diversity decreased in all metapopulations. After four generations,theaveragedecreasewasabout66%inexperiment1(Fig.3)and 55%inexperiment2(Fig.3,G4),assumingtheexpectedinitialdiversities.

Inexperiment1,anANOVAperformedon

H

s

, thelocaldiversityaveraged perlocusandperpopulationforeachmetapopulationsuggestsnoeect of ei-therpopulationsize

×

selectionregime(

p = 0.15

,one-wayANOVA).However, aDuncan'sMultipleRangeTestperformedonaverage

H

s

permetapopulation suggeststhat thelowestdiversitywasretainedin thetreatmentwith homoge-neousselectionandsmallestpopulationsize(

H

s

= 0.16

forthistreatment,and

H

s

= 0.23

to 0.25fortheothertreatments).

Inexperiment2,whiletheeect ofgenerationonlocaldiversitywashighly signicant(

P = 0.0001

),therewasnosignicanteect oftheextinction treat-ment (

P = 0.75

) (two-way ANOVA with generation and treatment as inde-pendentvariables). Thedecrease occurred during the rstthree generations, as averagelocaldiversitiesofgenerationsthreeandfourwerenotsignicantly dierent, whereas

H

s

of generationonewassignicantlylargerthan diversity ofgeneration two,itselfsignicantlylargerthandiversityofgenerationsthree andfour(Duncan's MultipleRangeTest ofprocedureANOVA,SAS).

3.1.3. Populationstructure:

F

ˆ

st

and

F

ˆ

is

Populationstructureincreasedovertimeand washighly dependent onthe treatment (Fig. 4). Two-wayANOVA with generation and treatment eects as independent variables in experiment 2 showed that

F

ˆ

st

was signicantly inuencedbyboththegenerationandtheextinctiontreatment(

P = 0.003

for generationand

P = 0.016

fortreatment). Inthisexperiment,theincreasewas greatestin theno extinctiontreatment (average

F

ˆ

st

= 0.30

), and smallestin metapopulationswithextinction andlargebase recolonisation(average

F

ˆ

st

=

0.13

).

Inexperiment1,theamountofdierentiationwasexplainedbylocal popu-lationsizes(

P < 0.0001

,two-wayANOVAonaverage

F

ˆ

st

permetapopulation, with population size and selection regime as independent variables). Thus,

G1

(Theor.)

Narrow Basis

No extinction

Theor.

Large basis No extinction Narrow Basis

Large basis

No extinction Narrow Basis

Large basis No extinction Narrow Basis

Large basis

Disruptive

Directional

100 Individuals

Directional

25 Individuals

Disruptive

Directional

10 Individuals

D

isruptive

0

0.5

0.4

0.3

0.2

0.1

0

0.5

0.4

0.3

0.2

0.1

0

0.5

0.4

0.3

0.2

0.1

G1

G2

G3

G4

G4 - Exp 1

M

is

sing

Figure 3. Estimates of gene diversity during the rst four generations of experi-ment2 (G1toG4)and atthefourthgenerationinexperiment1. Dataare missing forthesecondgenerationinthenoextinctiontreatment. Within-population diver-sity

H

s

; Totaldiversity

H

t

:

T heor

: theoreticallyexpectedattherstgeneration.

metapopulations with large population sizes were slightly dierentiated (av-erage metapopulation

F

ˆ

st

valueof0.03),whereasmetapopulations withsmall populationsizeswerehighlydierentiated(averagemetapopulation

F

ˆ

st

valueof 0.45). Therewasnosignicanteectwithtreatmentasmaineect(

P = 0.99

) buttheinteractionbetweentreatmentandlocalpopulationsizewassignicant (

P = 0.03

). Forpopulationsizesof100

F

ˆ

st

waslargerundertheheterogeneous selectionregime(averagemetapopulation

F

ˆ

st

= 0.12

and

F

ˆ

st

= 0.02

inthe het-erogeneousandin thehomogeneousselectionregimes, respectively;

P = 0.04

, one-wayANOVAon

F

ˆ

st

permetapopulationwithpopulationsizeof100,with selectionregimeastheindependentvariable).

All

F

ˆ

is

valueswerelargeandhighly signicant. Themean

F

ˆ

is

was

0.788 ±

0.03

across metapopulations. There wasa tendency for

F

ˆ

is

to increaseover

No extinction Narrow Basis

Large basis No extinction Narrow Basis

Large basis

0

1

0.8

0.6

0.4

0.2

0

1

0.8

0.6

0.4

0.2

G1

G2

0

1

0.8

0.6

0.4

0.2

Disruptive

Directional

100 Individuals

25 Individuals

10 Individuals

G3

G4

G4 - Exp 1

No extinction Narrow Basis

Large basis No extinction Narrow Basis

Large basis

Disruptive

Directional

Directional

D

isruptive

***

***

*

**

***

***

***

***

***

ns

***

***

***

***

***

***

***

***

***

_***

_***

***

***

*

***

***

***

_**

***

***

***

***

ns

ns

ns

M

is

sing

Figure4. Estimatesofmultilocus

F

ˆ

st

( )

and

F

ˆ

is

(



)

duringtherstfourgenerations ofexperiment2(G1toG4)andatthefourthgenerationinexperiment1.

F

ˆ

is

values are all highly signicant. Levels of statistical signicance for

F

ˆ

st

are noted as ns:

P > 0.05

,*:

P < 0.05

,**:

P < 0.01

,***:

P < 0.001

.

generationsasshownforgenerationsonetofourin Figure4forexperiment2. Two-wayANOVAofaverage(local)

F

ˆ

is

permetapopulationshowsnoeectof treatment in either experiment (

P = 0.28

for selection regime and

P = 0.26

forlocal populationsize in experiment1and

P = 0.146

forexperiment2). It conrms the generation eect on

F

ˆ

is

for experiment 2(

P < 0.0001

), but no evolutionof

F

ˆ

is

isobservedaftergenerationtwointhisexperiment(

P = 0.24

). Assumingthat

F

ˆ

is

valuesarethereforeclosetoequilibriumatthefourth gener-ation weestimated theoutcrossingratesin bothexperiments[16]. Estimated outcrossing rates are consistent between experiments (

0.12 ± 0.03

in experi-ment1and

0.12 ± 0.02

inexperiment1).

3.1.4. Globalpopulationdiversity

Global metapopulationdiversitydecreasedin all metapopulations(Fig. 3). After four generations, the average decrease was 20% in experiment 1 and 17%in experiment2. Consistently, in experiment2thegeneration eect was signicant (

P = 0.0007

) in a two-way ANOVA with extinction regime and generationasindependentfactors. Theextinctiontreatmentcontrarilywasnot signicant(

P = 0.60

). Therewasnoeectofthetreatmentonmetapopulation diversity in experiment 1either (

P = 0.30

and 0.46 forselection regime and localpopulationsizetreatmentsintwo-wayANOVA).

3.2. Precocity

The results of the selectionexperiment 2 are given in Figure 5. Selection wasecientsince50%oftheplantswerestillattherosettestage52daysafter sowing for plants of the rstgeneration, whereas this period wasreduced to 36 daysafter three generations of selection. The overall shapeof the graphs howeversuggeststhattheresponse toselectionmostlyoccurredafter therst generation of selection. Most reduction in the life cycle wasdue to a short-ening of therosette stage. Two-wayANOVA with generation and extinction treatment as independent variables shows astrong generation eect on syn-chrony (parameter

r

,

P < 0.001

)as well asasignicant increasein thenal proportionof oweringplants(parameter

K

,

P = 0.036

), whilethere wasno signicantchangeintimetoappearanceoftherstoweringplant(parameter

i

,

p = 0.29

). Theonlysignicanteect of treatment onresponse to selection wasthat extinction withlarge base recolonisationretained signicantlymore non-oweringplantsthanthetwoothertreatments.

4. DISCUSSION

Thetwoexperimentsweredesignedtoinvestigatechangesin levelsand dis-tribution of geneticdiversity under contrastedmetapopulationregimes. This knowledge may prove useful for the management of metapopulationsfor the conservationof geneticvariation. Themaintenanceofgeneticdiversityat the populationandthemetapopulationlevelsdependsoneectivepopulationand metapopulationsizes. Theeectivesize ofapopulationisusually lowerthan its censussize. Frankham [10] providesaverygeneral gureof 0.11for wild populations. Thecorrelationbetweentheeectivesizeofapopulationandits censussizedependsonfactorssuchasselngrate,selectionregimeand migra-tion. Theeects of thefactors acting in ourexperiments are detailed in the materialand methodssection. Ingeneral,weexpectedmoregeneticdiversity tobemaintainedforlargeeectivesizes.

4.1. Evolution ofallelic frequencies

Our estimation of neutral genetic diversity is based on polymorphism at fourallozymicloci. Itisgenerallyconsideredthatallozymesbehaveasneutral markers.Thisissue,however,issomewhatcontroversial: apositivecorrelation betweenheterozygosityforallozymic lociandtness-relatedtraits such as vi-abilityorgrowthratesisreportedfororganismsasdierentaswild oat[4],a numberoftreespecies(e.g. [8,41]),ash[27] andshellsh[19,42]. Similarly, thedeclineintheproportionofheterozygoteswithinbreedingisusuallylower thanexpected(e.g. Rumballetal.[36]in Drosophila).

In our experiment, we observed an increase in the frequency of allele 4of LAP-2 in mostmetapopulations. This mightsuggestthat this alleleis under selectioninourexperiment. LAPisanenzymeactiveinthecytosolwhich par-ticipatesintheturnoverofproteins[2]. Koehnetal.[19]observedacorrelation betweenallelefrequenciesattheLAP-2locusandsalinityand/ortemperature in oysterand suggestedthat oneallelemightbeunder selectionat thislocus. However, thecorrelation was no longer signicant in thesame organism in a later study [37]. Another explanation would be that allele 4 of LAP-2 is in linkagedisequilibrium with determination of precocity. It is easy to see how subsamplingat the foundation ofthe linescould havegenerated such linkage disequilibrium. Nocorrelation wasdetectedbetweenthe frequencyofallele 4 and precocity in the ten F2 lines used to found all metapopulations (data notshown). However,becauseof thehigh frequency ofthat allele in founder lines, our ability to detect linkage disequilibrium with genes for precocity is diminished. Allozymeloci havebeen convincingly shown to be under direct and directional selection in a few cases (e.g., Watt [44] for selection favour-ingparticularallelesofphospho-glucoisomerasesin butteries). Furtherwork is needed in our experimental system before we can suggest the most likely explanationforthepatternobservedwithLAP-2.

Belowwesummarisetheresultsandcomparethemwithouroriginal predic-tions.

4.2. Neutraldiversity

4.2.1. Within-populationdiversity

After four generations of management, we observedan overall decrease in localdiversityfortheallozymicmarkers.

4.2.1.1. Eect oflocal populationsizes(Experiment 1)

Prediction: Thelocal genetic diversity should decrease with the sizes of localpopulationsunder noselection.

because the reduction of eective size by selection is greater for such sizes, this eect partlycompensates theeect of decreasingpopulationsize sothat overall thereis little eect ofpopulationsizes. The onlyvisible eect is that thesmallestlocalgeneticdiversitywasobservedforoneoftheselectionregimes with the smallestpopulationsize,and this size

×

selectionregime treatment wasfoundtobedierentfromtheothersusingtheDuncanmultiplerangetest. Moreover,thelargestrangeofvariabilitywasobservedamongmetapopulations of small populations,indicating that the level oflocal diversitywasproneto larger variations from generation to generation when local populations were smaller.

4.2.1.2. Eect ofselection regime(Experiment 1)

Prediction: thelocalgeneticdiversityinthehomogeneousselectionregime should belessthanorequaltothat intheheterogeneousselectionregime.

Test:Overall,therewasnosignicanteectoftheselectionregimeonlocal diversity. However,thesmallestlocalgeneticdiversitywasobservedforoneof the three metapopulationtypes with the homogeneous selection regime,and this treatment was found to be dierent from the others using the Duncan multiple rangetest (sameasabove).

4.2.1.3. Eect oflocal extinctions(Experiment 2)

Prediction: localdiversitiesshouldbelargerinthenoextinctiontreatment Test: Unexpectedly, there was no signicanteect of local extinctions on localdiversity. Inparticular,populationsofmetapopulationsundergoinglocal extinctions and recolonisation witha narrow-basiswere noless variable than populationsexperiencingastable environment,anunexpected result. Wewill discussthisresultwhenconsideringglobaldiversity.

4.2.2. Populationdierentiation

4.2.2.1. Eect oflocal populationsizes(Experiment 1)

Prediction: Large dierentiation among populations should build up, all themoresoforloweectivesizes.

Test: Weindeedobservedahighly signicantdierentiationamong popu-lations at the fourth generation. The inuence ofpopulationsize on popula-tion structurewasverystrong andin the expected direction,suggestingthat selection did not signicantly decrease eective size at neutral loci in large populations.

4.2.2.2. Eect ofselection regime(Experiment 1)

Prediction: Foragiven populationsize,dierentiation under the hetero-geneous selection regime should be stronger compared to the homogeneous selection.

Test:Therewaslittleeectofselectionregime,exceptformetapopulations with largepopulationsizes, for which dierentiation under theheterogeneous selection regime wasindeed strongercompared to the homogeneousselection regime. Most likelyselection wasmoreecient at large populationsizes, so that the eect of theselection regime was more obviousfor such population sizes. Thelargerdierentiationin these metapopulations with heterogeneous selectioncanbeexplainedbothbylinkagedisequilibriaandbyaweaker adap-tation of the migrants, although we have no experimental evidence for this latter hypothesis. Decayof linkagedisequilibriumwould beretarded byhigh selnginourpopulations.

4.2.2.3. Eect oflocal extinctions(Experiment 2)

Prediction: Thestrongerdierentiationatequilibriumshouldbeobserved in thenarrow-basisextinctiontreatment,followed bythenoextinction treat-ment,followedbythelarge-basisextinction treatment.

Test: Thesmallestdierentiationwasindeed observedin metapopulations withalargebasisforrecolonisation,aresultconsistentwithpredictions. How-ever,andunexpectedly,signicantlylessdierentiationamongpopulationswas observedin thenarrow-basisrecolonisationtreatmentcompared totheno ex-tinctions. This could be explained by the fact that high rates of extinction andrecolonisationincreasemigrationratesandmorethancompensateforthe foundereectsatrecolonisation.

4.2.3. Globaldiversity

4.2.3.1. Eect oflocal populationsizes(Experiment 1)

Predictions: Becausethemetapopulationsizeisproportionaltolocalsize, themostobviouspredictionisthat globaldiversityshould increasewithlocal populationsize.

Test: Thelevelofglobaldiversitywasindependentofthelocalpopulation size. Asthemigrationrateisconstant,adecreaseinlocalpopulationsizeboth decreases local eectivesize and increases dierentiation among populations. Overall,thiscouldexplainwhyglobaldiversityisnotaectedbylocal popula-tionsize. Moreover,weshowedthatwhiledierentiationwasindeedlargerfor smallerpopulationsize,localdiversitywasindependentonlocalsize(possibly becauseof selectionbeing moreecientin large populations). Soit maynot besurprisingthatglobaldiversityisindependentofpopulationsize.

4.2.3.2. Eect ofselection regime(Experiment 1)

Predictions: Global diversity should be smaller in the homogeneous se-lection regime, as the local genetic diversity is expected to be less than or equal to that in theheterogeneous selectionregime, and so is the amountof dierentiationamong populations.

Test: This eect of the selection regime is not observed, as the level of diversityis notlargerunder heterogeneousselection. This istrueeven in the largemetapopulationswheredierentiationwaslargerundertheheterogeneous selection. Moregenerationsmightbeneededforthiseect tobuildup.

4.2.3.3. Eect oflocal extinctions(Experiment 2)

Predictions: Total diversity should be larger in the no extinction treat-ment.

Test: Contrary to our predictions, the global diversity was not aected byextinctions. Itcouldbethat becausealldiversitywaspresentwithin each populationatthebeginningoftheexperiment,thelossofsomepopulationshad no immediateeect on diversity. This would also explainwhy local diversity isnotinuencedbylocalextinctions.

4.3. Evolution of precocity (eect of local extinctions, Experiment2)

Ourresultsonprecocitysuggestthatweeectivelyselectedformore preco-ciousgenotypes. Theexistenceofgeneticvariationforprecocityamong popu-lationsof A.thalianahasbeenreportedin manystudies(e.g. [20])andmajor genes [24,35] and QTLs [26] responsible for this variation are known. In a comparison among 13 populations of A. thaliana, Zhang and Lechowicz [47] observed that rosette diameter and its growth rate contributed most to ex-plaining the total variation in owering time. Here we observed a similar correlated response, sinceselecting for short life cycles (i.e. early owering) mainlydecreasedthelengthoftherosettestage(Fig.5). Thesignicanteect of generation on parameter

r

of the logistic curve, showing that individuals weremoreandmoresynchronousin theiroweringbehaviour,alsoillustrates thattheeciencyofselectioncouldbelinkedtoadecreaseingeneticdiversity forthistrait.

Ingeneralitwasdiculttoassesswhatimpactselectionforprecocityhadon thelevelofneutraldiversityinourmetapopulations. Althoughmostvariation forprecocitydisappearedinthesecond generation,there wasnotendencyfor neutraldiversityto decreasemarkedlyatthesametimein experiment2.

Response to selection was not inuenced by the extinction-recolonisation treatment,suggestingthat enhancedgene ow dueto local extinctions might bringnewvariationlocally,andcompensatefortheoveralldecreased

metapop-0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

6

10

15

20

24

29

33

38

41

45

51

55

61

67

79

Pe

rcentage

of

eac

h

gr

ow

in

g

stag

e

cotyledons

rosette

bolting

flowering

green siliques

mature siliques

Generation 1

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

6

10

15

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38

41

45

51

55

61

67

79

Pe

rcentage

of

eac

h

gr

ow

in

g

stag

e

_{Generation 2}

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

6

10

15

20

24

29

33

38

41

45

51

55

61

67

79

Pe

rcentage

of

eac

h

gr

ow

in

g

stag

e

Generation 3

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

6

10

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24

29

33

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51

55

61

67

79

DAY

P

er

cen

ta

ge

of

each

gr

ow

in

g

st

ag

e

Generation 4

Figure 5. Evolutionovertimeofthe percentageof sixgrowing stages(cotyledons, rosette, bolting, owering, green pods and mature fruits) at the rst generation (500 plants) and atthe threesubsequent generations (900plantseach,pooled over treatments).

asmostofselectionresponseoccurred duringtherst generation,itcouldbe that no variation was left after this generation, so that there would be no furthereect ofdecreasedeectivesize.

5. CONCLUSION

Results are only available for the four rst generations and it is tooearly todrawgeneralconclusionsusefulforconservationofgeneticdiversityincrop or ex situ conserved species. However, despite the small number of studied loci, severaltrends emergefrom these preliminaryresults. First,weobserved a general decrease (of the order of 20%) of neutral genetic diversity in the metapopulations(Fig.3). Thisshowsthat thepopulationsizeswechosewere notlargeenoughto maintainecientlytheinitiallevelofdiversity,especially regarding thefact that articial selectionwasapplied. Inpractise, conserva-tionistsoftenhavetotolerateasmalldecreaseofdiversity,becausemaintaining largeenoughpopulationsis toocostly in termsof spaceand money. Surpris-ingly,thelossofvariabilitydidnotdependonpopulationsizeinourexperiment (Fig.3)afterfourgenerations. Thus,apoolof2000individuals(

100×20

forthe largestmetapopulation) didnot seem sucientto maintainneutral diversity

precocityactedtodecreasetheeectivesize,sothatifonecouldprevent nat-uralor articialselectionfrom acting, 2000 individualsmight be sucientto preserveneutraldiversity. As theobjectiveofsuch managementprogrammes is toletpopulationscoevolvewiththeirenvironment,itis likely(andhoped) that naturalselectionwouldoccurinanyexperimentaldesign.

In ourexperiment, aminimum of100 individualsperpopulationwas nec-essarytoobserveaneectof thetypeofselectivepressureonmetapopulation structure. Moreover,therewasnoeect ofselectionregimeonlocal diversity. Thisresultconrmsthat,evenwithlargeselectivepressures,driftoverrides se-lection insmallpopulations. Agoalofadynamicmanagementisto maintain diversityatselectedtraits,forexamplebygrowingtheplantsindierent envi-ronments. Thelargemetapopulationsin experiment2appearedlargeenough to respond to selection but larger metapopulations might prove necessary if weakerselectivepressuresareacting.

Finally, populationsize and selectionact together to determine the main-tenance of neutral diversity. Both selection and drift decrease the within-populationneutraldiversity,selectionbeingmoreecientinlargepopulations while drift is more ecient in small populations. Maintaining local natural selection in a dynamic management will thus accelerate the loss of neutral geneticdiversity,comparedtopopulationsundergoingnoselection. Heteroge-neousselectionisexpectedtocounteractthislossofdiversity,throughitseect on increased dierentiation among populations for locally adapted traits. In a selngspecies, linkage disequilibriaexist at the whole genome level. Thus, the increasein global adaptivediversitythroughincreased dierentiation be-causeofheterogeneousselectionisalsoexpectedtooccurforneutraldiversity. Assuming populationsare connected, local evolutionarypotential is restored throughraremigrationevents. Inanoutbreedingspecies,selectionindierent environmentswill allow the maintenance of adaptive variability, but neutral diversitywill be lesseasilymaintained,especiallyifselectionis strong. Thus, amilderselectionshouldbeappliedinsuchspecies.

ACKNOWLEDGEMENTS

ThisworkwasnancedbytheBureaudesressourcesgénétiquesaswellasthe RégionBourgogne. WewouldliketothankL.Saunois,G.Félix,A.Matejicek andM.Schoutithfortechnicalassistance.WearealsogratefultoP.H.Gouyon and I.Till-Bottraud forinitialideasonthese experimentsand P.Englandfor input on the nal manuscript. This is publication N

◦

ISEM2000-XX of the Institut desSciences del'Évolution.

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