Does water facilitate gene flow in spore-producing plants? Insights from the fine-scale genetic structure of the aquatic moss Rhynchostegium riparioides.

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Aquatic

Botany

j o u r n al ho me 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

Does

water

facilitate

gene

flow

in

spore-producing

plants?

Insights

from

the

fine-scale

genetic

structure

of

the

aquatic

moss

Rhynchostegium

riparioides

(Brachytheciaceae)

V.

Hutsemékers

_,

_O.J.

_Hardy

_,

_A.

_{Vanderpoorten}

a_{UniversitédeLiège,InstitutdeBotanique,B22SartTilman4000Liège,Belgium}

b_{UniversitéLibredeBruxelles,FacultédesSciences,EvolutionBiologiqueetEcologie,CP160/12,avenueF.D.Roosevelt50,1050Bruxelles,Belgium}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received7June2012

Receivedinrevisedform1February2013

Accepted11February2013

Available online 18 February 2013 Keywords:

Hydrochory

Spatialgeneticstructure

Isolation-by-distance

Geneflow

Bryophyte

a

b

s

t

r

a

c

t

Hydrochoryplaysakeyroleinthemaintenance,diversityandevolutionofaquaticplantsandhas tradi-tionallybeenthoughtto(i)decreaseorerasepatternsofisolation-by-distance,(ii)increaseoutbreeding, and(iii)resultinadownstreamincreaseofgeneticdiversity.Thesehypotheses,whichareespecially relevantinorganismswithwater-mediatedfertilizationandahighabilitytodispersevegetatively,are testedherefromfine-scalespatialanalysesofgeneticvariationatboththehaploidanddiploidphases intheaquaticmossRhynchostegiumriparioides(Hedw.)Cardot.Asubstantialgeographicalpartitioning ofgeneticvariationwasfoundatthescaleoftheriverbasinandindirectmeasuresofdispersalpoint toanoverallweakerdispersalabilityofthemossdiasporesthanpollenorwind-dispersedseeds.These observations,aswellasthehighFisobservedatthediploidstageandtheverycloseproximityofpotential

fathersoftheheterozygousdiploidgenotypes,stronglychallengethehypothesisthatwaterenhances spermanddiaspore-mediatedgeneflow.InR.riparioides,theseveredispersallimitationsrevealedby thespatialanalysesofgeneticstructuresuggestthatshootfragmentsratherthansporesareinvolvedin localdispersal,whichisfullyconsistentwiththelowlevelsofgeneticdiversityobservedwithin popula-tions.Extremelylimitedroutinedispersalbyunspecializedvegetativediaspores,coupledwithdiscrete episodesoflocalpopulationextinctionsandcolonizationsbyspores,accountfortheabsenceofa down-streamincreaseofgeneticdiversity.Asaresult,althoughspore-producingplantsaretypicallyseenas mostefficientdispersers,andalthoughstreamsaretraditionallyseenasdispersalcorridors,severe lim-itationsofclonaldispersalatthelocalscaleanddiscreteopportunitiesofrandomsporedispersaland establishmentamongcolonies,bothexplainwhytypicalmetapopulationprocessesalsoapplytoaquatic mosses.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Hydrochory,thepassivedispersaloforganismsbywater,plays akeyroleinthemaintenance,diversityandevolutionofaquatic plants (Nilsson et al., 2010). Because it is considered to facili-tatethedownstreammigrationofdiaspores(Polluxetal.,2009;

Triestetal.,2010),hydrochoryhastraditionallybeenthoughtto

enhancethespatial shuffling ofgeneticvariation (Kudohetal., 2006).Asaresult,lowlevelsofgeneticdifferentiation(seeNilsson etal.,2010forreview),andtheerasureofanysignalof isolation-by-distance(Ruckelshaus,1998; Chenetal., 2007;Polluxetal., 2009), have been recurrently reported among populations of aquaticplants.Thereduction ofthespatialaggregationof close

∗ Correspondingauthor.Tel.:+3243663842;fax:+3243662925.

E-mailaddresses:virginie.hutsemekers@gmail.com(V.Hutsemékers),

ohardy@ulb.ac.be(O.J.Hardy),a.vanderpoorten@ulg.ac.be(A.Vanderpoorten).

relatives due tothe passivedownstreamdispersal of diaspores furtherlikelypromotesoutbreeding(Kudohetal.,2006).Infact, comparatively high levelsof heterozygosity were foundwithin populations of aquatic plants (Lloyd et al., 2011; Tanakaet al., 2011).Finally,thedownstreammigrationofpropagules theoret-ically resultsinadownstreamgradientofgeneticdiversity(the unidirectional diversity hypothesis, seeHonnay etal., 2010 for review).

An increasing number of population genetic studies have addressedthesehypotheses,buttheirconclusionsremain

equiv-ocal(Honnayetal.,2010;Nilssonetal.,2010).Honnayetal.(2009)

suggestedthattherelationshipsbetweenthedirectionandthe dis-tanceofgeneflowontheonehand,andmeasuresofisolationby distanceandpopulationgeneticdifferentiationontheother,may beconsiderablyconfoundedbyrecurrentpopulationextinctions andrecolonizations.Geneticdifferentiationbetweenpopulations mayhenceincreaseconsiderablyinresponsetometapopulation dynamicsandfoundereffects(PannellandCharlesworth,2000).

0304-3770/$–seefrontmatter © 2013 Elsevier B.V. All rights reserved.

2 V.Hutsemékersetal./AquaticBotany108 (2013) 1–6 Inthepresentpaper,wedeterminewhetherfreshwater

con-nectivityaffectsspatialpatternsofgeneticstructureanddiversity inaquaticmosses.Mossesdispersebymeansofspores,specialized vegetativepropagulesandgametophytefragments.Theyrequire waterforfertilizationasavestigefromaquaticheritage.Although theprocessmaybefacilitatedbymicro-arthropods(Cronbergetal.,

2006a),fertilizationrangesareextremelylimited.Malegamete

dis-persaldistancesrangebetweenafewcentimeters(Rydgrenetal., 2006)toafewmeters(vanderVelde etal.,2001).Interrestrial environments,thedensityofsporesdecreaseswiththedistance fromthemother gametophyte(Pohjamo et al.,2006).Trapping experimentssuggestthatamajorityofsporesdisperses,however, beyondthenearestvicinityofthesource.Regularestablishment occursatthekm-scale(Lönneletal.,2012),while1%oftheregional sporerainisassumedtohaveatrans-orintercontinentalorigin

(Sundberg, 2012).Indirect estimates of migrationderived from

theanalysisofthespatialgeneticstructureneverthelessremain controversial(Korpelainenetal.,2012).Somestudiespointtothe significanceofrandomfoundingeffectsbyoneorafew individ-ualsfollowedbyclonalgrowth(Cronbergetal.,2006b;Gunnarsson etal.,2007).Others demonstratethecrucial role ofcontinuous establishmentopportunities,givingrisetohighgenotypic diver-sitywithinsites(e.g.Cronberg,1996,2002;StenøienandSastad,

1999;vanderVeldeetal.,2001).Whiletheviabilityofmoss

dias-poresinwaterwaspreviouslyinvestigated(DalenandSöderström, 1999),nostudyhasyetdeterminedwhetherstreamsandrivers mightindeedserveasdispersalcorridors.Giventhehighpotential forshootfragmentationinbryophytesandthelikelihoodof sub-sequentpassivedownstreamdispersal,HeinoandVirtanen(2006)

proposedthataquaticmossspeciesaremorewidelydistributed andshowastrongerdistribution-abundancerelationshipwithina riverbasinthannon-aquaticspecies.

Fromthefine-scalespatialanalysisofgeneticvariationatboth thehaploid anddiploidphasesintheaquaticmoss Rhynchoste-giumriparioides(Hedw.)Cardot,weassesswhethergrowthwithin anaquaticenvironmentenhancesthelikelihood offertilization, andhence,ratesofheterozygosity.Wefurtherevaluatethedegree ofpopulationgeneticdifferentiationandexaminewhetherwater connectivity reduces spatial aggregation of close relatives and henceerases anysignalof isolation by distanceat thescale of theriverbasin.Finally,wetesttherelevanceofthe‘unidirectional diversityhypothesis’,whichpredictsincreasinggeneticdiversity indownstreampopulationsdue toconstant driftof allelesin a downstreamdirection.

2. Materialsandmethods

2.1. Samplingandmolecularmethods

R.riparioidesisarobustmonoicouspleurocarpousmossforming extensivematscoveringsubmergedoroccasionallyemergedrocks infast-flowingstreams.Itfrequentlyandmassivelyproduces emer-gentsporophytes.It doesnotproduce specializedgemmae, but wholegametophytefragmentscaneasilydetachanddisperse clon-ally.Itwasselectedhereasamodelbecause(i)thespecieshasbeen mostrecentlyrecircumscribedtaxonomically(Hustemékersetal., 2012);(ii)itiswidespreadacrossEurope,NorthAfricaandwestern Asia,whereitislocallydominantinrunningwatercommunities andishenceusedasabioindicator(Ceschinetal.,2012;Manolaki

andPapastergiadou,2013);(iii)andspecificnuclearmicrosatellite

markershavebeendesigned(Hutsemékersetal.,2008),facilitating theanalysisofitsgeneticstructure.

R. riparioideswassampledat9localities alonga 12km-long transectwithintheLienne(Meusehydrographicnetwork, south-ern Belgium), a fast-flowing stream with an average oxygen

Fig. 1.Sampling designof the aquaticmossR. riparioides within theLienne

hydrographicnetwork(southernBelgium).Piediagramsrepresentthemultilocus

genotypicfrequenciesatsixSSRmarkerswithineachofthe9sampledlocalities.

saturationof95%andanaverageannualdischargeof2.9m3_s−1_at

thejunctionwiththeAmbleveriver(Fig.1).Withineachlocality, 5–36specimenswerecollectedeverymeteralonga5–40mlong transect(Table1).Altogether,156gametophytes(haploidstage) were sampled.Allthepopulations but 2and 5 includedfertile specimens.Our samplingincluded119fertile gametophytes.All ofthesporophytes(diploidstage,1–6pergametophyte)foundon thosegametophytesweresampledforatotalof169sporophytes. Individual gametophytes and sporophytes were genotyped at six variable nuclear microsatellite loci following the protocols

of Hutsemékerset al. (2008). Since the observed frequency of

individuals with identicalgenotypes was systematically higher thantheprobabilitythattwoindividualsfromatheroretical non-clonalpopulationexhibitthesamemultilocusgenotypebychance (homoplasticmutations)(Hutsemékersetal.,2010),thisprotocol allowedtodescribethespatialclonalstructureofthepopulations. 2.2. Statisticalanalyses

Uni-andmultilocusWright’sfixationindices(FIS)were

com-puted for the diploid sporophytic populations to analyze the mating system. Assuming the absence of selection and null alleles, FIS provides an estimate of the inbreeding coefficient

andcanbeusedtoinferanamalgamofbothintergametophytic (amongindividualsfromdifferentsporeswithinthesame sporo-phyte)andintragametophytic(amongindividualsfromthesame spore after clonal fragmentation of the protonema) selfing in

Table1

Geneticdiversityof9populationsofthemossR.riparioidesintheLienneriver(southernBelgium).N,samplingsize;Ng,numberofgenotypes(numberofprivategenotypes).

Ar,allelicrichness(correctedforsamplesize);G,genediversity(correctedforsamplesize).

Population 1 2 3 4 5 6 7 8 9 N 20 14 5 21 5 29 36 20 6 Ng 4(1) 3(1) 1(0) 6(2) 2(1) 2(0) 4(1) 3(1) 2(0) Ng/N 0.20 0.21 0.20 0.29 0.40 0.07 0.11 0.15 0.33 Ar 1.47 1.62 1.00 1.49 1.49 1.07 1.65 1.21 1.16 G 0.214 0.295 0.000 0.232 0.294 0.023 0.272 0.080 0.089

haploid-dominant species with combined sexes (Eppley et al., 2007).ThestandarderrorofthemultilocusFISwascomputedby

jackknifeoverlociwithSpagedi1.3(HardyandVekemans,2002). Thegenetic diversity of each haploid population (i.e. game-tophytes only) was expressed in terms of unbiased expected heterozygosity(He)andallelicrichnesscorrectedforsamplesize (Ar) with GenAlex (Peakall and Smouse, 2006) and Fstat 2.9.3

(Goudet,1995).Totestthehypothesisofadownstreamgradient

ofdiversity,variationofHeandArwascorrelatedwithgeographic distancealongtheriverfromthedownstreampopulation1(Fig.1). Thespatialgeneticstructurewasexaminedbycomputingthe globalmultilocusFST amongpopulations.SignificanceofFST was

assessedby999randompermutationsofindividualsamong popu-lationsanditsstandarderrorwasobtainedbyjackknifeoverloci asimplemented by Spagedi 1.3.We furtherinvestigated varia-tioninpairwisekinshipcoefficients(Fij)betweengametophytes

along gradientsof geographic distance. In order to investigate whetherdispersalprimarilyoccursbyhydrochoryoranemochory (i.e. dispersed by wind), two types of distance matrices were computed:onerecordingthenearestaerialdistanceamongsites, theotherrecordingpairwisedistancesfollowingtherivercourse fromupstreamtodownstreampopulations,withotherpairwise distancescoded asmissing. Thesignificanceof theslopeof the regressionofFijonthelogarithmofthespatialdistancebetween

individualswastestedbymeansof999randompermutationsof populationlocations(Manteltest)withSpagedi1.3.MeanFij

val-ueswerealsoplottedagainstagradientofpredefinedgeographic distanceintervalsrangingbetween0and12,000m,thefirst inter-valcorrespondingtopairsofindividualsfromthesamepopulation. SignificanceofthemeanFij perdistanceclasswastestedby999

randompermutationsofindividuals.TheSpstatistics,ameasure ofthedecreaseofkinship coefficientsbetweenindividuals with thelogarithmofthedistanceseparatingthem,wasmeasuredas −ˆb/





,where ˆbistheslopeoftheregressionbetweenall pairwiseFij comparisonsandthelogarithmofthedistance

sepa-ratingeachpairofindividual,and ˆF(1) isthemeanFijamong all

pairsofneighbors(VekemansandHardy,2004).

3. Results

Tenand twelvemultilocusgenotypeswerefoundamongthe 156gametophytes and 119 sporophytes, respectively. Wright’s global fixation index was FIS=0.95±0.02. Within populations,

FIS=1.00±0.00atallthelocalitieswheresporophyteswere

sam-pled,withtheexceptionoflocality7,whereFIS=0.90±0.01.Infact,

only3ofthe169diploidgenotypeswereheterozygous(oneattwo lociandtwoatthreeloci),andallwerefoundatthesame local-ity(7).Twopaternalalleleswereinvolvedinthoseheterozygous combinations,andbothofthemwerealsofoundatthehomozygous stateinthesamepopulation.Table1summarizesthegenetic diver-sitymetricsofeachofthe9populations.Multilocusgenotypeswere foundtoberandomlydistributedalongtherivercourse(Fig.1).

UnbiasedHeinthehaploidpopulationrangedfrom0.08±0.04 to0.295±0.13(Fig.2).HeandArwerenotsignificantlycorrelated

togeographicdistancealongtheriverfromlocality1(r=−0.35and −0.36,respectively,p>0.05).

TheglobalmultilocusFSTamongpopulationswas0.43±0.05

(p<0.001).Fij values quicklydecreasedalongagradientof

geo-graphic distance and the Manteltest between pairwise Fij and

geographic distance revealed a significant signal of isolation-by-distance,both employingshortestlineardistances (r=−0.19, p=0<0.01) and distances following the river course (r=−0.27, p<0.001)aspredictors(Fig.3).Theslopeoftheregressionbetween allpairwiseFijcomparisonsandthelogarithmofthedistance

sepa-ratingeachpairofindividualfollowingtherivercoursewas−0.019. ˆF(1)equaledto0.33,andtheSpstatisticswas0.029.

4. Discussion

4.1. Selfingandclonalpatternsatthelocalscale

The globalmultilocus FIS observed inthe sporophyticphase

(FIS=0.95)lieswithintheupperboundofFISvaluesreportedfor

Fig.2. ExpectedheterozygosityHeandallelicrichnessArwithinpopulationsofthe

aquaticmossR.riparioidessampledat9localitiesalongtheLienneriver(southern

Belgium)alongagradientofaerialgeographicdistancesfromLocality1(seeFig.1).

Fig.3. Mean(andstandarddeviationbars)kinshipcoefficientsFijbetween

individ-ualsforasetofspatialdistanceintervalsintheaquaticmossR.riparioideswithin

theLiennehydrographicnetwork(southernBelgium),consideringaerialorriver

4 V.Hutsemékersetal./AquaticBotany108 (2013) 1–6 monoicousmosses(0.62–0.98,Eppleyetal.,2007).Thisconfirms

thenotionthatselfingoccursfrequentlyinmonoicousmoss popu-lations(Eppleyetal.,2007).Populationgeneticstheorypredicts thatselfingmaybeselectedagainstbecauseitincreaseslevelsof homozygosityinoffspring, whichinturn,allowstheexpression ofrecessive,deleteriousalleles(CharlesworthandCharlesworth, 1998).Whileoutcrossingand,inparticular,polyandry(Szovenyi etal.,2009)areselectedforindioicousmossestoavoid sporo-phyteinbreedingdepression,recessivedeleteriousmutationsare, incontrast,rapidlypurgedinautoicousmossesthrough intraga-metophyticselfing(Tayloretal.,2007).Oncedeleteriousalleles arepurgedfromthegenome,selfingcanbeadaptiveandgeneral surveysinangiospermsconfirmthatcompleteselfingcommonly occurs(BarrettandEckert, 1990).Complete selfingasobserved inallbutoneofthepopulationssurveyedheremighthenceplay akeyroleinmaintaininglocaladaptationsalongenvironmental clinesbecauseofthelargehitchhikingeffectsexpectedunderhigh selfing(VekemansandHardy,2004).Infact,thesignificant correla-tionfoundbetweengeneticandecologicalvariationinR.riparioides attheregionalscaleissuggestiveofreproductiveisolationamong ecotypes(Hutsemékersetal.,2010).

4.2. Dispersallimitations

Atthehaploidlevel,asubstantialgeographicalpartitioningof geneticvariationwasfoundatthescaleoftheLienneriverbasin. ThustheFSTamonghaploidgametophytepopulationsofR.

ripar-ioides(0.43)issignificantandcomparabletotherangeofvalues reportedfor terrestrialmonoicous mossspecies attheregional scale(0.36–0.78,Eppleyetal.,2007).TheSpstatisticsofR. ripar-ioides in the Lienne basin (0.029) further demonstrates severe routinedispersallimitations. TheSpofR. riparioidesindeed lies withintherangeofvaluesreportedforterrestrialangiosperm self-ers(0.14±0.08,Vekemans and Hardy,2004).Itis muchhigher than that reported for spermatophytes with wind pollination (0.006±0.003),wind-dispersedseeds(0.01±0.01)(Vekemansand

Hardy,2004),andeven specializedvegetativegemmae in

liver-worts(0.01,Korpelainenetal.,2011).Theseobservations,aswell asthehighFISobserved atthediploidstageandtheveryclose

proximityofpotentialfathersofthethreeobservedheterozygous diploidgenotypes(infact,withinthesamepopulation),strongly challengethehypothesis that water might enhance sperm and diaspore-mediatedgeneflow.

Thefundamentalquestionofhowfarhydrochorescanbe dis-persedinariverandwheretheyarefinallydepositedandgerminate dependsonavarietyofintrinsicandextrinsicfactors(Nilssonet al.,2010).Extrinsicfactorsincludechannelsize,boundary condi-tionsandhydraulicroughness,andmorphologyoftheriver,which determineflowhydraulicsatdifferenttimesduringtheseason.The Lienneriveris,however,a typicalfast-flowingmountainstream whosehydrauliccharacteristicsdonothamperdownstream migra-tions.

Intrinsicfactorsinclude attributesof theseed,fruit,capsule, orvegetativepropaguleitselfsuchassize,shape,floatingability, longevity,andotherinherentfactorsthatmayfundamentally con-strainorproduceopportunitiesforlong-distancedispersal(Nilsson et al., 2010). In R. riparioides, the severe dispersal limitations revealedbythespatialanalysesofgeneticstructuresuggestthat shootfragmentsratherthansporesareinvolvedinlocaldispersal. Theshapeoftherelationbetweenkinshipcoefficientsand geo-graphicdistanceismostsimilartothatobservedinmossspecies reproducingasexually(Snäll etal., 2004).In addition,although shortestaerialdistancesandlineardistancesalongthewatercourse aresignificantlycorrelated,thematrixoflineardistancesfollowing thewatercourseaccountsforasignificantlyhigherpercent varia-tionofgeneticvariationthanthematrixofshortestaerialdistances,

asshownbythedifferencesincorrelationcoefficientswiththetwo typesofdistances.Clonalfragmentationofshootfragmentsisfully consistentwiththelowlevelsofgeneticdiversityobservedwithin populations,withHevaluesrangingfrom0.08to0.295.These val-ues,which fitwiththosereportedfor nuclearmicrosatellites or ISSRsinotherclonallydispersedmacrophytes(e.g.,in Potamoge-tonandOttelia,whereinHe<0.2,Chenetal.,2008,2009),arein factsubstantiallylowerthanthosereportedforpopulationsof out-breedingaquaticmacrophytes,whereintherangeofHewasfor example0.44–0.62and0.58–0.65amongpopulationsofVallisneria andZoostera,respectively(Lloydetal.,2011,Tanakaetal.,2011).

Sincediasporesizeisafactorofprimeimportancefordispersal abilityinthecaseofanemochory(Pohjamoetal.,2006),a straight-forwardinterpretationofthepoordispersalabilityofR.riparioides atthebasinscalewouldbethatlargeshootfragmentsexhibitlow dispersalabilities.Whilesuchaninterpretationpotentiallyholds inaterrestrialenvironmentandmightexplain,forinstance,the unexpectedlowdispersalabilityofobligateepiphyticbryophytes

(Devosetal.,2011),dispersaldistanceisunrelatedtodiasporemass

inwater(IkedaandItoh,2001).

4.3. Relativeimportanceofhydrochoryandanemochory

Species with predominantly self-fertilizing and vegetative clonalreproductionusuallydisplayahigherpercentageofgenetic variation among populations than outcrossing species (

Black-SamuelssonandAndersson,1997;Gaudeuletal.,2000),suggesting

morelimitedgeneflowamongpopulations.Thisalonemay, how-ever,notexplainthestronggeographicpatternsofgeneticvariation andlowmigrationabilityofRhynchostegium’sshootfragments.In anexperimentalstudyinfact,Surenet al.(2000)demonstrated thatpopulationsofaquaticmossessubmittedtoarangeof stream-flowintensitieslose10–12%ofbiomassaftertheflood,resultingin thereleaseofasubstantialnumberofshootfragments.Shoot frag-mentsmaybebettersuitedforroutinedispersalwithintheriver basinthananemochorousspores.Indeed,asexualpropagulesallow forearlyreproduction,arelesssensitivetohabitatquality(Löbel, 2009),resultintheproductionofnewgametophytesatafasterrate

(MishlerandNewton,1988)andincreaseestablishmentfrequency

ascomparedtospores(Löbeletal.,2009;LöbelandRydin,2010). Intypicalhydrochorousangiosperms,fruitsandseedsdisplay morphologicalandanatomicaladaptations,suchasair-filledtissue, whichenablethemtofloatforlongperiodsoftime,andothertraits, suchasdormancy,whichenhancelong-distancedispersal.Trapped air,corkytissue,andhighsurfaceareatovolumeratiosmaybe seenasexaptationsenhancinghydrochorousdispersal(seeNilsson etal.,2010for review).Theshootsof R.riparioides,incontrast, donotexhibitanyspecificfeaturesthatwouldenhanceflotation. Seedswithlowbuoyancyareverylikelytogerminate underwa-teranddevelopanewplant.Thisis,however,notpossibleinthe caseofaquaticmosses,whichdisplayanarrowecologicalniche regarding substrate quality (Glime and Vitt, 1987;Muotka and

Virtanen,1995;HylanderandDynesius,2006)andrequire

appro-priaterocksurfacestoestablishandresumegrowth.Hence,the resultspresentedheresuggestthat,intheabsenceofany morpho-logicaladaptation,shootfragmentsofR.riparioidestendtosink afterafewtenstohundredsofmeters,especiallyintranquilriver reacheswheretheycannotfindtheappropriatehabitat.

Thisraisesthequestion,however,whyaquaticmossesfailto develop adaptations toflotationif, asthepatternsof isolation-by-distance observed in R. riparioides unambiguously suggest, hydrochoryisindeedthemaindispersalstrategy.Althoughthe rel-ativelylownumberofpopulationssampledmayhavepreventeda statisticallysignificantdownstreamincreaseingeneticdiversityto emerge,thepatternofgenotypediversityalongtherivercourse (Fig.1)contrasts,however,withtheideaofaone-waydispersal

ofdiasporesanddoesnotfitwiththeexpectationsofthe unidi-rectionaldiversityhypothesis.Likeinmanyotheraquaticmosses infact,thesporophytestructureofR.riparioidesisidenticaltothat ofmanyterrestrialspecies,andsporesareassumedtodisperseby

air(VittandGlime,1984).Therandomgeneticstructureobserved

atthescaleof thehundred ofmeterscanhencebeinterpreted intermsofdiscreteepisodesoflocalpopulationextinctionsand upstreammigrationsbyspores.Thisinterpretationfitswith previ-ousobservationsonthegeneticstructureofclonalmosses,wherein spatialpatternsofgeneticvariationareexplainedbyclonal repro-ductionwithinpopulations,andstructuredbysexualreproduction amongpopulations(Cronbergetal.,2006b).Aclearerdownstream increasein geneticdiversitycouldhence beexpectedinpurely clonalspecies.Theabsenceofanyrelationshipbetweengenetic similarityanddistanceafterafewhundredsofmetersinsterile aquaticmossessuggests,however,thatrareeventsofupstream dispersal ofgametophyte fragmentsby waterfowlsfollowedby clonalpropagationmayalsotakeplace(Korpelainenetal.,inpress). Asaresult,althoughspore-producingplantsaretypicallyseenas mostefficientdispersers(seeHutsemékersetal.,2011forreview) and although streams are traditionally seen as dispersal corri-dors,severelimitationsofclonaldispersalatthelocalscale,and discreteopportunitiesofrandomdiasporedispersaland establish-mentamongcolonies,bothexplainwhytypicalmetapopulation processesalsoapplytoaquaticmosses.

Acknowledgements

ManythanksareduetoJ.Vermaatandtworeviewersfortheir commentsonthemanuscript.

FinancialsupportfromtheBelgianFundsforScientificResearch (FNRS,grants1.5036.11and2.4557.11)andtheUniversityofLiège (grantC11/32)isgratefullyacknowledged.

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