Contents lists available atScienceDirect
Aquatic
Botany
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / a q u a b o t
Analysis
of
genetic
structure
of
Ranunculus
baudotii
in
a
Mediterranean
wetland.
Implications
for
selection
of
seeds
and
seedlings
for
conservation
A.
Coppi
a,∗,
L.
Lastrucci
a,
A.
Carta
b,
B.
Foggi
aaDepartmentofBiology,UniversityofFlorence,Italy
bDepartmentofBiology,UniversityofPisa,Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received12January2015
Receivedinrevisedform2June2015
Accepted6June2015
Availableonline17June2015
Keywords: Seedbank Restorationgenetics ISSRanalysis Mediterraneanisland Naturalpond
Ranunculuspeltatussubsp.baudotii
a
b
s
t
r
a
c
t
Seedcollectionandstorageofwildspeciesinex-situseedbanksshouldbecontinuedasanintegratedtool fortheconservationofplantsintheirhabitats.Althoughseed-bankfacilitiesarewidelyusedtoday,their seedsamplesoftensufferlowgeneticdiversity.Consequently,reintroducedseedsandplantmaterial maynothavetheresiliencetocopewithfutureenvironmentalstresssoleadingtocompletewastageof seeds.Moleculartechniquesallowthebenefitofquantificationofthegeneticdiversityofaseedcollection incomparisonwiththatofthenaturalpopulation.Inthisstudywefocusonex-situseedbanksamples andlivingcollectionsofRanunculuspeltatussubsp.baudotii.Wecomparetheirgeneticdiversityand structurewiththatofthenaturalpopulationbeforeandafterundertakingarestorationprojectona naturalpondintheTuscanArchipelagoNationalPark.ISSRanalyses,carriedoutonatotaloffivesampling groups,showsarelativelyhighlevelofgeneticdiversityfortheex-situcultivatedgroups.Theanalysis ofmolecularvariance,inagreementwithclusteringobtainedintheneighbour-joiningdendrogramand withthepatternfromclusteranalysis,suggestsdividingthesamplesanalysedintotwogroups:one formedbyindividualssampledbeforethepondrestorationandtheotherformedbythesubsequent pondpopulation.Theresultshighlighttheimportanceofplanningmixedpropagationlineswhichcan beobtainedthroughtheuseofarangeofgerminationconditionstoexploitanovelsourceofgenetic variabilitywhichmayotherwiseremainhiddenwithintheseedcollection.
©2015ElsevierB.V.Allrightsreserved.
1. Introduction
Biodiversitydeclineisofprimaryconcernthroughouttheworld andits conservationpresentsacritical challengetothenatural sciences(Barnoskyetal.,2011).Thebestplacetoconserve bio-diversity is in the wild, through maintenance or restoration of habitats.Such interventions aresometimessufficient for plants torecoverwithoutthereintroductionofpropagules(Bunceetal., 2013).However,thespontaneousrecoveryofrare-plant popula-tionsmaynotoccurundertheseconditions,assomespeciesare severelydispersal-limited(Clarketal.,2007),and/orhave tran-sientseedbanks(Thompsonetal.,1997)and/orareunabletocope forlongwiththenaturaldynamicsinthehabitat(Stockwelland Ashley,2004).Thus,restorationplansgenerallyincludeanumber ofstrategiesthatcanbestbeconsideredinthecontextof contem-poraryevolution(Stockwelletal.,2006).
∗ Correspondingauthor.Fax:+39055282358.
E-mailaddress:andrea.coppi@unifi.it(A.Coppi).
Restorationisrecognisedasacomplexandhigh-riskactivity whichshouldincludeplantpropagationandcultivationaswellas habitatmanagement.Priortoanyrestorationactivity,theorigin, variabilityandrelatednessofthesourcematerialsshouldbe con-sideredcarefullytoavoidsignificantdisadvantagesandafurther lossofbiodiversity(FensterandDudash,1994).However,itisnot alwaysthecasethatthedesiredplantmaterialsareavailableinthe wild.Thus,thecollectionofseedsofwildspecies,andtheir main-tenanceinex-situseedbanks,shouldbeincludedasanintegrated toolalongwithinhabitatplantconservation.Seedbanksare facili-tieswithahightechnologicalcontent(RaoandRiley,1994),which shouldtakeresponsibilityforthewholelineofseedmanagement allthewaythroughtothereintroductionstep(HayandProbert, 2013).
Toconserveseedsamplesabletobeusedmanyyearsintothe future,muchefforthasbeenmadetomaximisethetoleranceof des-iccationandtopredictseedstoragebehaviour(Merrittetal.,2014). Thishasimportantimplicationsforthesuccessfulmanagementof seedconservationcollectionsthat,alongwiththeextensive liter-atureonthedormancy-breakingandgerminationrequirementsof http://dx.doi.org/10.1016/j.aquabot.2015.06.002
26 A.Coppietal./AquaticBotany126(2015)25–31
wildspecies,shouldleadtothedevelopmentofeffective propaga-tionprotocolsforecologicalrestorationandspeciesreintroduction (MerrittandDixon,2011).However,seed-banksamplesoften suf-ferlowgeneticdiversity(BediniandCarta,2010;Godefroidetal., 2011)Consequently,introducedseedsandotherplantmaterials maynothavesufficientresiliencetocopewithfuture environmen-talstressessopossiblyleadingtoatotalwastageofseeds(Merritt andDixon,2011).
Theidentificationofsourcematerialtoensuregenetic similar-ityoftheintroduced,tothelocalpopulations,maybeacquiredby conductinganecogeographicsurvey(Guarinoetal.,2011). How-ever,geographical distanceisnotalwaysthebestindicatorof a population’sgeneticsimilarity(AllendorfandLuikart,2007).Inthis context,ageneticmolecularapproachcanprovidetheopportunity toidentifythesourceofplantmaterialandalsotounderstandthe effectivelevelofgeneticdiversityofseedcollectionsinrespectto thenaturalpopulation(Lauterbachetal.,2012).Furthermore,this approachshouldbeappliedatallstagesofplantregenerationfrom seeds,measuringthegeneticvariationoflivingcollectionsobtained throughdifferentlaboratorygerminationconditionsand/or prop-agationmethods,andeventuallyprovidinganassessmentofthe likelysuccessofthewholerestorationprotocol(Alonsoetal.,2014). Inthisstudy,werefertoarestorationexperienceundertakenin anaturalpondintheTuscanArchipelagoNationalParkinvolving habitatmanagement(Foggietal.,2011,2014)andthe constitu-tionofseedbankcollectionsandthecultivationoflivingplantsin theBotanicGardensofFlorenceandPisa(Cartaetal.,2012).We focusontheseex-situsamples,bycomparingtheirgenetic diver-sityandstructureswiththoseofthenatural populationsbefore andafter restoration.Therestoration of thepondwasurgently requiredsincetheinvasionbytallhelophytes(suchasTypha lat-ifoliaL.,TyphaangustifoliaL.and Phragmitesaustralis(Cav.)Trin exSteud.)hadsubstantiallydestroyedthisfresh-waterecosystem andthreatened,atthelocalscale,species suchasMyriophyllum alterniflorumDc.andRanunculuspeltatusSchranksubsp.baudotii (Godron)MeikleexC.D.K.Cook(Ranunculaceae;hereinafterR. bau-dotii)(Foggietal.,2011,2014).
Restoration activities which included removal of these tall, expansivehelophytes(seeFoggietal.,2014fordetails),could per-hapshavenegativelyaffectedthesoilseedbank.
Tounderstand theimpact of pond restoration onthe natu-ralpopulationofR.baudotii, itsgeneticstructurewasestimated onthebasis ofInterSimple Sequence Repeatsmarkers (ISSRs). ThismethodisaPCR-basedfingerprintingtechniquethatinvolves amplificationofaDNAsegmentbetweentwoinversely-oriented and closely-spaced microsatellite repeat regions (Reddy et al., 2002).Thismethodiswidelyused inplantpopulationgenetics duetothereliabilityandreproducibilityoftheresults (Rakoczy-TrojanowskaandBolibok2004).Inparticular,themethodhasbeen usedtoassessgeneticvariationandpopulationdifferentiationina numberofendangeredplantspeciesfromarangeoflocationsand climates(Luoetal.,2007).
Thus,ourgoalistoelucidatethefollowingtwoquestions: 1.Aretheregeneticdifferencesordivergencesbetweensamplings
attwotemporalstages:beforeandafteroftheimplementation ofrestorationactivitiesofthepond?
2.Canamoleculargeneticapproachbeeffectivelyusedto evalu-atewhetherselectedlaboratorygerminationconditionscould beresponsibleforgeneticdifferencesamongpropagationlines? Withtheseinformations,therelatednessforreintroductionor reinforcementof theex-situ propagated plantmaterial and the practical implication a wheneverecological restoration are dis-cussed.
2. Materialsandmethods
2.1. Plantmaterialsandsamplingprotocol
The plants usedin this studywere sampledby theauthors fromthenaturalpopulationofR.baudotiilocatedintheStagnone pondsituatedonCapraiaIsland(TuscanArchipelago,north-central MediterraneanSea;Fig.1)atanaltitudeof330ma.s.l.Thecurrent populationofR.baudotiioccupiesanareaofca.0.4ha.Since1991 thispopulationhasundergoneadrasticreductioninsizereaching aminimumofca.0.03hain2009.
R.baudotiiis ahydrophytebelongingtotheRanunculus sub-gen.Batrachium.Itisdistributedmainlyinbrackishwatersnear thewesternandsoutherncoastsofEuropeandoftheBalticregion, and,locally,inland inwesternand centralEurope(Cook,1993). Thisspeciescan beannualorperennial(Cook,1966), it is self-compatibleandmainlyself-pollinated(Dahlgren,1995).Vegetative propagationcanalsoplayanimportantroleinpopulation mainte-nance/growthassexualreproduction(Dahlgren,1995).R.baudotii establishescommunitiesconsideredof interesttowards conser-vation according to the Habitats Directive 92/43/CEE. Because thespeciesformsclumpsbyvegetativepropagation,inorderto avoidthesamplingof“ramets”ratherthan“genets”,werandomly selected10–15plantsgrowingatleast10mapartfromoneanother. Thesampleswerecollectedin2010(St1;pre-restoration)and2014 (St2;post-restoration).Asecondcollection(Ta)wasalsosampled in2014,whichrepresentedanephemeralpopulationlivinginan artificialreservoirabout1.5kmawayfromStagnonepond(Fig.1). Toestablishex-situcollectionsforconservationpurposes,the originalmaterialfromStagnonewascollectedin2009. Approx-imately20 plantswerecollectedand cultivatedin theFlorence BotanicalGardenandseedsampleswerestoredinthePisaSeed Bank(Cartaetal.,2009,2012).
Inadditiontothesenaturalpopulations,samplesfor genetic analysiswere alsocollectedfrom theex-situ collections in line withthefollowingscheme(Fig.2):plantscultivatedinFlorence weresampledin2010,representingthesecondgenerationofthe wildpopulationsampled in2009 (Fi);seedlings obtainedfrom laboratory-germinatedseedscollectedinthewildweresampled in2009andusedtoestablishalivingcollectioninthePisaBotanic Garden(Pi1);theseedlingsconstitutingPi1wereobtainedby ger-minatingseedsatboththenear-optimaltemperature(10◦C;PiTA) andatasupra-optimaltemperature(20◦C;PiH)asindicatedby Cartaetal.(2012);seedlingsobtainedfromgerminatedseeds pro-ducedin2010byPi1werealsosampled(Pi2).ForPi2,seedswere germinatedinthelaboratoryonlyatthenear-optimaltemperature (10◦C).
Thegroupsofsamplesanalysedandbelongingtothesevarious categories(reportedinTable1)arehereafterdefinedasSampling Groups(SG).
2.2. IsolationofgenomicDNA
GenomicDNA wasextracted fromsilica-geldried leaftissue usingtheCTABprotocol(Mengonietal.,2006).Thequantityand qualityofDNAwasestimatedspectrophotometricallyusinga Bio-Photometer(Eppendorf).
2.3. ISSRprofilingandanalysis
Twoofthesixprimerspreliminarilyscreened,yieldedclearand reproduciblebandingpatterns.Totestreproducibilityasubsetof sampleswasselectedatrandomfromthewholedataset.Primer sequences,sizerangeandnumberofpolymorphicsitesdetectedin theentiredatasetarereportedinTable2.
Fig.1. Thestudyareasamplingsites.Stagnonepondandtheartificialreservoirareshownwithblackdots.
Fig.2. Samplingdesignflowchart.Greyboxesindicateactivitiesinthewild,whiteboxesindicateactivitiesinthelaboratoryorbotanicalgardens.Thesamplinggroupcode
28 A.Coppietal./AquaticBotany126(2015)25–31
Table1
Listofexaminedaccessionandyearofsampling.Thefollowingparametersareshown:percentagesofpolymorphicsites(Ps%).Numberofrarebands(Nrf).Numberofprivate
bands(Npf).Averagegenediversityoverloci(geneticdiversity),meanpopulationheterozygosity(Hs)andtotalvariation(Ht).
SGcode Yearofsamplingandlocation Pol.(%) Nrf Npf Geneticdiversity
Pi1 2009;Pisa 85.4 23 2 0.33
Pi2 2010;Pisa 52.1 13 0 0.24
St1 2010;Stagnone 66.7 18 0 0.26
Fi 2010;Florence 72.9 20 1 0.29
St2 2014;Stagnone 82.6 9 2 0.18
Ta 2014;Reservoirforrainwater 22.9 7 0 0.12
Hs 0.24
Ht 0.34
Table2
Primersequences,sizerangeandnumberofpolymorphicsitesdetectedbyeach primerinthewholedataset.
Primer Primer sequence Size range(bp) Polymorphic sites(no.) ISSR1 (GT)7-YR 500–3000 23 CA6 (CA)6.RG 250–2200 25
PCRwerecarriedoutintotalvolumesof20lcontaining:10ng
ofDNA, 2l of reactionbuffer(Dynazyme II,Finnzyme, Espoo,
Finland),1.5mMMgCl2,200Mdeoxynucleosidetriphosphates,
2lof10mMPrimerand1.4UofTaqDNAPolymerase(Dynazyme
II,Finnzyme,Espoo,Finland).Thermocyclingwascarriedoutafter
aninitialdenaturingphaseof5minat94◦Cfollowedby35cycles
eachof40sat94◦C,45sat43◦Cand90sat72◦C.Afinalcycle
wassetfor45sat94◦C,45sat42◦Candafinalextensionstepof
5minat72◦C.Amplificationproductswereseparatedon2%(W/v)
agarosegelelectrophoresiscontaining1gml−1ofethidium
bro-mide.Electrophoresiswascarriedoutataconstant75Wfor2h.The
resultingbandswerevisualisedbyaUVtransilluminatorand
ana-lysedbyImageJsoftwarevs.1.43f(ImageProcessingandAnalysis
inJava,http://rsbweb.nih.gov/ij/index.html). 2.4. Dataanalysis
Allamplifiedbandsweretreatedasdominantmarkersandall ISSRprofilesobtainedweretranslatedina 1-0binarymatrix.A matrixofgeneticsimilaritybetweenplantswascomputedusing Jaccard’scoefficientofsimilarity.Thistakesintoaccountband shar-ingbetweenindividualsandiscommonlyusedfortheanalysisof dominantmarkers(Loweetal.,2004)suchasAFLPorISSR.
Thepercentage ofpolymorphic lociin thedatasetwasfirst determined.Thenumberofrarebands(Nrf),presentinlessthan 20individualsinthewholedataset,andofprivatebands(Npf), namelythoseuniquetoonesamplinggroupwascalculated fol-lowingStehliketal.(2001).Anintrapopulationmeasureofgenetic diversitywasthencomputedasthe“averagegenediversityover loci”(Nei,1987).ThemeangeneticdiversitybetweenSG(Hs)and thetotalgeneticdiversity(Ht)werealsocomputed.Allanalyses attheintra-SGlevelwerecarriedoutusingtheprogramArlequin 2.000(Schneideretal.,2000).
Theproportion of genetic variation explained by the differ-encesamongSGwasestimatedbymeansoftheSlatkin’slinearised FST’smatrix(Slatkin,1995P-valueinAppendix1ofSupplementary information).
The Slatkin’s matrix generated by Arlequin 2.000 software (Schneideretal.,2000)wasusedtogenerateaneighbour-joining dendrogram(Saitouand Nei, 1987)using thesoftware Mega 6 (Tamuraetal.,2013).
The binarymatrix obtainedfrom the ISSR profilewas used toimprove a bayesian phylogram by MrBayes. The best fitting modelsofbinary matrixwere“Binary Model”withcoding bias option“noabsencesites”assuggested fromMrBayes3.1 manual
(RonquistandHuelsenbeck,2003).Theanalyseswereperformed usingfourincrementallyheated Markovchains(onecold,three heated)simultaneouslystarted fromrandom trees, andrun for onemillioncyclessamplingatreeeverytengenerations.The sta-tionaryphasewasreachedwhentheaveragestandarddeviation ofsplitfrequenciesreached0.01. Treesthatprecededthe stabi-lizationofthelikelihoodvalue(theburn-in)werediscarded,and theremainingtreeswereusedtocalculateamajority-rule consen-susphylogram.ThetreewereshowedwithindicationofBayesian PosteriorProbabilities(PP)valuesfortheinternaltreenodes.
Analysisofmolecularvariance(AMOVA;Excoffieretal.,1992)as implementedinArlequin2.000(Schneideretal.,2000)wascarried outtoinvestigategeneticstructureatdifferenthierarchicallevels: withinSGandbetweenSG.TheAMOVAwascomputedthreetimes separatelyforeachofthreehypotheticalclustersofSGformations. Thestatisticalsupportfortheanalysiswastested through1023 permutations.
3. Results
ISSRanalysiswascarriedoutonatotalof57samples.Thetwo selectedprimercombinationsproducedatotalof48analysableloci (100%polymorphic).WithineachSG,thepercentageof polymor-phiclocirangedfrom22.9%(Ta)to85.4%(Pi1).Thenumberofrare andprivatefragmentsisreportedinTable1.Thethreesampling groupsPi1,St1andFishowedahighernumberofrarefragments (23,18and20,respectively)whereas,thesecondgenerationfrom Pisa(Pi2),St2andTashowedareducednumberofrarefragments. PrivatefragmentswereobservedonlyfortwosamplesofPi1,one sampleforFiandtwosamplesforSt2.Averagegenediversityover locirangedfrom0.12(Ta)to0.33(Pi1);totalgeneticdiversity(Ht) was0.34,whereasaveragegenediversitywithinSG(Hs)was0.24 (Table1).Otherwise,thegeneticdiversityofthecurrentpopulation inthepond(St2)waslower(0.18).ForPi1thegeneticdiversities ofsamplesgerminatedatnear-optimaltemperature(10◦C,PiTA) andatsupra-optimaltemperature(20◦C,PiTH)were0.40and0.18, respectively.
3.1. InterSGdiversityandclusteranalysis
The neighbour-joining dendrogram of R. baudotiiaccessions basedonSlatkin’slinearisedpairwiseFSTmatrixsuggeststhe exis-tenceoftwoclusters,namelyAandB(Fig.3a).ClusterAconsistsof Pi1accessionsandasubclusterincludingSt2andTa.ClusterB com-prisesthesecondgenerationofPisa(Pi2)andasubclusterformed bythenaturalpopulationof2010fromStagnone(St1)andFi.A sec-onddendrogramisshownwherethetwosubsamplesPiHandPiT areseparatedinthetwoclustersBandArespectively(Fig.3b).The levelofgeneticsimilarityobtainedfromISSRprofilecomparison wasusedtocreateaBayesianphylogram(Fig.4).Bayesiananalysis partiallyconfirmsthepatternsreportedbytheneighbour-joining dendrogramandonlythesamplesfromSt2andTa(bothsampled
Fig.3.(a)Neighbour-JoiningdendrogramofsamplinggroupsbasedononSlatkin’s
linearisedpairwiseFSTmatrixand(b)Neighbour-Joiningdendrogramwherethe
samplesconstitutingPi1weresubdividedinPiTAandPiH.Thescalebarindicates
Slatkin’slinearisedFSTvalueofgeneticdistance.Thetwomainclustersareindicated.
SeeTable1andFig.2forthecodesfortheaccessionsexaminedandtheirpositions
inthesamplingdesign.
in2014)formedaweaklyseparatedcluster(0.54PP)withrespect totherestofdataset.
3.2. GeneticvariationamongSG
AMOVAanalysis(Table3)revealedthatthegreatest portion ofgeneticvariationwasduetowithin-SGdifferences(71.99%of variance) rather than toamong-SGdifferences (28.01% of vari-ance). Totalamong-SG differentiation (FST) was0.280 (p-value <0.001).AmongallhypotheticalgroupingsofSGtested,onlytree showedhighly-significantresults(see Table3).Thegroups that explainedthehighestpercentageofvariation(22.07%)consisted oftwogroups,thefirstwithSGsampledin2014(St2andTa)and thesecondconstitutedbyPiH,PiTA,Pi2,St1andFi.
4. Discussion
4.1. Populationgeneticdiversityinthenaturalenvironment Stagnonepondhostsasmall,isolatedpopulationofR.baudotii whichissufferingseverecontractionduetohabitatchange.The alteringconditionslikelyaffectedthereproductivebiologyofthis speciesthat,similarlytootherRanunculusspeciesofthesubgen. Batrachiumisprobablyabletoadoptamixedbreedingsystemi.e. inbreedingoroutbreeding(Dahlgren,1995).
As suggested by Nybom (2004), species with self or mixed breedingsystemsshouldshowmeanvaluesofwithin-population geneticdiversityof0.12and0.18,respectively.Thepopulationof R.baudotiiexaminedhereexhibitsamoderatelyhighermeanlevel ofgeneticdiversity(Hs=0.24)suggestingasignificantpopulation recruitmentfromcross-pollinatedseeds.Whilstthemeanvalueis moderatelyhigher,thegeneticdiversityattheintra-SGlevelforTa, islowerthantheNybomvalue(seeTable1)whichsuggestsgenetic driftand,mostprobably,afoundereffectfortheephemeral popu-lationgrowingintheartificialreservoir.Thisphenomenonoccurs reasonablyoftenwithinthegroupofsubgen.Batrachiumduetothe capacityofitsachenestobedispersedbybirds(Dahlgren,1995).
Afirstevaluationofthevegetationresponseaftertherestoration hasshownaself-recoveryoftheformeraquaticvegetation(Foggi etal.,2014).ThepopulationofR.baudotii(St2)hasrecolonisedthe Stagnonepondwithoutanyreintroductionand/orreinforcement activity.Theabilityofthis speciestoformasoilseedbank has
Fig.4. Bayesian50%majority-ruleconsensustreefromtheISSRbinarymatrix,
withposteriorprobabilityvalues(PP)shownnearstatisticallysupportednodes.See
Table1andFig.2forthecodesoftheaccessionsanalysedandtheirpositionsinthe
30 A.Coppietal./AquaticBotany126(2015)25–31
Table3
ResultsofAnalysisofmolecularvariance(AMOVA)performedtotestthedifferentiationamong7samplinggroupsandthepartitionamongthreehypotheticalgroup
formations.Thetableshows:degreesoffreedom(df),sumofsquareddeviations,variancecomponentestimates,percentagesoftotalvariancecontributedbyeachcomponent,
andtheprobabilityofobtainingamoreextremecomponentestimatebychancealone(p).P-valueswereestimatedwith1023permutations.
Sourceofvariation df Sumofsquares Variancecomponent Percentageofvariation P-value
Withinsamplinggroups 51 312.45 6.13 71.99 <0.001
Amongsamplinggroups 5 141.38 2.38 28.01 <0.001
Total 56 453.83 8.51
StructureofhypoteticalgroupingsofSG
(PiH;St1;Fi;Pi2)–(PiTA;St2;Ta) 1 47.31 0.90 10.14 <0.001
(PiH;PiTA;St1;Fi)–(Pi2)-(St2;Ta) 2 98.40 1.82 20.20 <0.001
(PiH;PiTA;Pi2;St1;Fi)–(St2;Ta) 1 64.67 2.13 22.07 <0.001
beenlargelyreportedforothergeographicalareas(Grillasetal.,
1993).However,sincethetop10–20cmofsoilwasremoved dur-ingtherestorationwork(Foggietal.,2014),wesuggestthatthe reappearanceofR.baudotiiinthepondwasderivedfromaresilient seedbankofthesoil,probablynotcompletelydamagedduringthe restorationoreven encouragedbya mixingofthesubstrateby theearthmovingmachinery.Thishypothesisfindsfurthersupport inevidencefromtheanalysesofgeneticdiversityofSGcollected before(Pi1,Pi2,St1andFi)andaftertherestoration(ST2).Indeed, theSGcollectedin2009(Pi1)and2010(Pi2,St1andFi),showa meanvalueforgeneticdiversity(0.28)slightlyhigherinthanthat observedforSt2(sampledin2014;averagegenediversity:0.18). Futuremonitoringwilllikelydeterminewhethertheformerlevel ofgeneticdiversitywillbere-established.
4.2. Geneticdiversityanddifferentiationamongnatural populationsandex-situplantmaterial
Theanalysisof molecularvariance (AMOVA) shows thatthe largepercentage of variance wasdue to within-SG differences ratherthantointer-SGdifferences(72%and28%,respectively).It isclearthatgeneticdifferentiationofSGwithrespecttothe com-plexofgermplasmanalysed,isnegligiblebutnotentirelyabsent. AmongallthehypotheticalgroupingsofSGtestedtoexplainthe observedproportionofmolecularvariation,threeyieldedhighly significantresults(seeTable3).Theonethatexplainedthehighest percentageofvariationconsistedoftwogroups,thefirstformed byPiTA-PiH-St1-FiandthesecondwiththetwoSGof2014(St2 andTa).Thisresultisinagreementwiththeclusteringobtained intheneighbour-joiningdendrogramandwiththepatternfrom Bayesiananalysisdescribedabove(Figs.3and4)suggestingthat theSGshouldbeanalysedastwoseparategroups:thefirst con-stitutedbythesamplescollectedduringthepre-restorationperiod andthesecondbytheSGof2014(St2andTa).Theseresultssupport thehypothesisthatSt2andTaareundergoinggeneticdrift.These SGlikelyoriginatedintheimpoverishedsoilseedbanksubjected toa“bottle-neck”forSt2and/ora“foundereffect”eventforTa.
The ex-situ cultivated populations showed relatively high geneticdiversityandthusshouldbeassessedashighly-valuable material,fromaconservationpointofview.Interestingly,the pop-ulationcultivatedinFlorenceexhibitedarelativelyhighlevelof geneticdiversityafterthefirstgenerationandinPisathispattern wasmaintainedthroughthesecondgenerationalso,suggesting that a mid-term cultivation should not lead to severe genetic inbreedingprocesses.Indeed,itcanbenotedthatinthewild(and alsoundercultivatedconditions)R.baudotiibehavesasanannual hydrophytethatregenerateseachyearthroughthegerminationof seeds(A.Carta,unpublisheddata).
Amongthepropagatedlinesobtainedinthelaboratorythrough seedgermination,PiTAshowedthehighestgeneticdiversityvalue, whilethePiH lineshowedone ofthelowest although exhibit-inggenotypesapparentlynotpresent inthecurrentpopulation (see Figs. 3 and 4).This result suggests that the two different
germinationtemperaturesusedmayhavebeenresponsiblefora genotype-selectionprocess.Thatistosay,attemperaturesreported asoptimalforgerminationforthisspecies(Cartaetal.,2012)many seedsgerminated and thus the wholerange of genotypes was expressed(PiTA)whereasatthesupra-optimaltemperature, ger-minationwasstronglysuppressedsoonlyafewseedsgerminated andamorelimitedrangeofgenotypeswasexpressed(PiH).By con-sideringthegeneticvariabilityexpressedbyPiTAwealsoconfirm thatourex-situseedcollectiondoesnotsufferlowgeneticdiversity. However,tomaximisetheexpressionofthisdiversitywhenin-situ speciesrecoveryisplanned,theoptimumgerminationconditions shouldbeused.
4.3. Insightintopopulationconservation
TheresultscorroboratetheusefulnessofISSRmarkersin set-tingprioritiesandplanningoptimalactionsfortheconservation ofendangeredplantspeciesorpopulationsinthreatenedbiotopes (Thompson,2005;Lopesetal.,2014).
The monitoring of the pond after the restoration shows widespreadrecolonisationof R.baudotii.However,ifthe diver-sity level will not be re-established autonomously, it may be appropriatetore-introduceindividualsduringsubsequentyears. Twostrategiescanbeconsideredforreinforcementsinthissite. A breeding-oriented perspective suggests reinforcement of the impoverishedpopulationbyintroducingplantsfromothersitesto enrichthegeneticbasisofthepopulation.Thisstrategymayreduce theriskofgeneticdriftand,ultimately,mayhelpoffsetthe nega-tiveeffectsofhabitatchange(Coppietal.,2010).Ontheotherhand, fromaconservationperspective,thereintroductionwouldbe bet-terundertakenusingthelocalseedsourcesintheex-situseedbank whicharemorelikelytoexhibitincreasedfitnessovernon-local genotypes(Xiaoetal.,2006).Sincetheoriginalmaterialisavailable, ourdatawouldsuggestthatthesecondoptionshouldbepreferred. Inaddition,wealsosuggesttoincreasethegeneticdiversityby mixingpropagationlinesobtainedusingarange ofgermination conditionsbasedonthehypothesis,heredemonstrated,thatthis shouldincreasethegeneticvariabilityofthisspecies.
Ex-situmanagementinevitablyimpliesselectionpressures dif-ferentfromthenaturalconditions(Lauterbachetal.,2012).
Ingeneral,itispreferabletoreintroduceplantsinthewildusing seedlings(Godefroidetal.,2011).Inthecontextofourstudy,there is evidencethat increasedgeneticdiversity canbeobtainedby selectingarangeofgerminationconditions.Thus,whenecological restorationsareplanned,werecommendtoconsiderethepossible negativeeffectsofgerminating seedsunderlaboratory environ-mentswhichmayselectadifferentrangeofgenotypesthanthose expressedunderanaturalenvironment.Sinceitmaynotalways bepossibletohavegeneticdata,anadditionalrecommendation forex-situhandlingistoundertakebothcultivationand propaga-tioninanear-naturalenvironment (VolisandBlecher,2010).In linewiththeviewofSmithetal.(2011),ourresultshighlightthe importanceofconsideringex-situconservationasa
complemen-taryapproachtoin-situconservation,additionallysupportedbythe geneticmonitoring.
Acknowledgements
TheresearchwasfundedbytheTuscanArchipelagoNational Park.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.aquabot.2015.06. 002
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