Plant Traits, EnvironmentalFactors, andPollinatorVisitationin
Winter-floweringHelleborusfoetidus(Ranunculaceae)
ALFONSOM.SA´NCHEZ-LAFUENTE1,*, JAVIERGUITIA´N2,MO‘NICAMEDRANO1, CARLOSM.HERRERA1,PEDROJ.REY3 andXIMCERDA´1
1Estacio´nBiolo´gicadeDo~nana(CSIC),Avda.deMariaLuisas/n,Pabello´ndelPer‘u,E-41013Sevilla,Spain,
2DepartamentodeBota´nica,UniversidaddeSantiago,CampusSur,E-15706SantiagodeCompostela,Spainand
3DepartamentodeBiolog´ıaAnimal,Vegetal yEcolog´ıa,UniversidaddeJae´n,E-23071Jae´n,Spain
•BackgroundandAimsThisstudyexaminedtheeffectofplanttraitsandenvironmentalfactorsonpollinator visitationinthewinter-floweringHelleborusfoetidus(Ranunculaceae)inthreedistantregionsintheIberian Peninsula.
•MethodsGeographicalvariation infloralvisitorassemblage,planttraits andenvironmentalfactorswereanalysed duringthefloweringseason.
•KeyResultsDifferenceswerefoundinallplanttraitsmeasured(numberofopenflowers,flowersize,number of stamensperflower,andnumberofnectaries)bothwithinandamongregions,anddifferencesamongregionsinall
theenvironmentalfactorsconsidered(airtemperature,exposuretosunlight,canopycover,anddistancetothe nearestneighbour). Differenceswerealsofoundamongregionsintheprobability thatplantswouldbevisitedby pollinators.
•ConclusionsTheresultsshowthat,althoughfloraldisplay(i.e.numberofopenflowersonaplantonagivenday) consistentlyexplainedamong-plantdifferencesinvisitationrateinallregions, visitationratewasnotsignificantly affectedbyanyotherbiologicalorenvironmental variable.InHelleborusfoetidus,then,‘how’theplantiswould seemtobemoreimportantthan‘where’isit.
Keywords: Environmentalfactors,floraldisplay,Helleborus foetidus,plant traits,pollinator visitation,winterflowering.
INTRODUCTION
Recentstudiesontheevolutionary ecologyofplant– pollinatorinteractions arebased onthepremisethat variation in plant traits influencesreproductive success byaffectingsuchinteractions(e.g.SchemskeandHorvitz,
1989;Campbelletal.,1991;Herrera,1993;Mitchell,1993; O’ConnellandJohnston, 1998;Guitia´n etal.,1999;Maad,
2000).Aconsiderablenumber ofstudieshaveshownthat individualvariationinflower and/orinflorescencetraits frequentlytranslatesintoindividualfitnessdifferences,as a result of effects on pollinator behaviour, visitation
frequencyand/orpollinatorspectrumcomposition (e.g. WaserandPrice,1981;Klinkhameretal.,1989;Robertson andWyatt,1990;Cresswell andGalen,1991;Connerand Rush, 1996; Go´mez, 2000; Philipp and Hansen, 2000; Gigordetal.,2001).Plantcharacteristicsinfluencingpol- linatorvisitationrateincludethoserelatedtofloraldesign
(e.g. flower colour, size or number), and the spatial andtemporalarrangementofflowers (seereferencesin Thompson, 2001;Mitchelletal.,2004).However,other studieshaveshownthatindividualdifferencesinflower orinflorescence traitsareeitherinconsequentialorquant- itativelynegligibleinexplainingindividualfitnessdiffer- ences(Herrera,1996).
* For correspondence at: Departamento de Biolog´ıa Vegetal y Ecolog´ıa, Facultad de Biolog´ıa, Universidad de Sevilla, Spain.
Twocategoriesoffactors mayaccountforthelatter observations:(1)variationsinflowerorinflorescencetraits mayhavelittleornoinfluenceonpollinationsuccess(e.g. Andersson,1994; Wilson, 1995; Wilson and Thomson,
1996;Herrera,2001);(2)evenifpollinatorsdiscriminate amongindividualplantsinresponsetofloralphenotypes,
eventualfitness differencesmaybeblurredbyfactors unrelatedtotheplant–pollinatorinteraction (e.g.post- pollination events on developing fruits; Herrera, 1993,
2000a). But site-specific effects may be as, or even more,importantthanplantphenotypic characteristicsas determinants of plant–pollinator interactions. O’Connell
andJohnston (1998), forexample,foundthatmicrohabitat characteristics(e.g.presenceofericaceous shrubs oropen canopy) had a larger effect on pollination success in
theorchidCypripedium acaulethanfloraltraits.Inthe MediterraneanshrubLavandulalatifolia,Herrera(1995a) found thatindividual variationinpollinatorcomposition depended on the sunlight regime associated with each
plant’slocation,ratherthanonintrinsic plant characterist- ics.Site-specificeffectsofthiskind(seealsoLaverty,1992; Niesenbaum,1994),whereby‘wheretheplantis’maybe
more importantthan‘how theplantis’,inexplainingindi- vidualdifferencesinplants’reproductive performance,are probablyquitecommonamongforestunderstoreyplants
(Herrera, 1995a,andreferencestherein),butfewstudies havedirectlyaddressedsucheffects.
Theaimofthispaperistoevaluatetheimportanceof
intrinsicfactors(i.e.plant-andflower-relatedphenotypic
1
32
200 km1000 km
FIG. 1.LocationofthestudyregionsintheIberianPeninsula: 1,Caurel,2,Cazorla,3,Ma´gina.
traits) and extrinsicfactors(i.e.site- andenvironment- relatedfactors)inexplaining individualdifferences in pollinatorvisitationinthewinter-floweringherbHelleborus foetidus (Ranunculaceae).Toexplorewhetherpatterns remainedconsistentamongregions,the studywasconduct- ingatthreewidelyseparated areasofthespeciesrangein theIberianPeninsula.Helleborusfoetidusiswellsuitedto investigatethe relative importanceofintrinsicandextrinsic factorsasdeterminants ofpollination-relatedindividual differencesinreproductivesuccess.Thespeciesflowers inwinterindifferent habitattypesThus,unfavourable weatherfrequentlylimitspollinatoractivity;further,differ- encesinenvironmentbetweenH.foetidus growingsites couldinturnaffecttheforagingbehaviourandvisitation rates of insect pollinators (e.g. Beattie, 1971; Herrera,
1995b).Specifically, thisstudyaimstoassess(a)whether floraltraitsandenvironmentalfactorsvaryamongthestudy
regions, and (b)whether such variationsaccountfordiffer-
encesinpollinatorvisitationrates.
MATERIALSANDMETHODS
Studysystemandsites
HelleborusfoetidusL.isaperennialherbwidelydistributed inwestern,centralandsouth-western Europe(Wernerand Ebel,1994).InSpainitmaybefoundinclearings, forest edgesandunderstorey ofmixedforests.Everyseason, plantsproduceonetoseveralinflorescences(range1–9) with25–100flowers each.Flowersareapocarpous(no.of carpels range1–5),withthestigmasborneattheendofa longstyle.Theanthers(range25–60)starttodehiscewhen flowersare6–8dold,producingpollenforapprox.2weeks. The floralnectariesarehiddeninsideaglobosecorolla, and produceabundantnectar(HerreraandSoriguer, 1983; Vesprinietal.,1999).Functionally,flowersarehermaph- roditic,protogynous andself-compatible,butsubstantial seedproductionrequires insectpollination.Theextremely long-livedflowers(upto20d;Vesprinietal.,1999)are mainlypollinatedbybumblebees(Herreraetal.,2001).
Thisstudywasconductedin1998inthreemountain regions in Spain (Caurel, Cazorla and Ma´gina, Fig. 1;
seealsoHerreraetal.,2001,2002). Theclosestregions (CazorlaandMa´gina)were50kmapart;themostdistant regions(CaurelandCazorla)were850kmaway.Ineach regiontwopopulations wereselectedand192plantswere taggedoverall(Caurel,n=31andn=33ineachpopulation; Cazorla,n=34andn=34;Ma´gina, n=30andn=30). Plantsgrewisolatedoringroupsofthreetofiveindividuals, scatteredthroughoutthestudyareas.InMa´gina,H.foetidus istheonlyfloweringplantduring mostofthestudyperiod. InCazorlaitcoexists withDaphnelaureola(Thymelea- ceae),andinCaurelwithPrimulaacaulissubsp. acaulis (Primulaceae).
Pollinatorcensuses
Geographicalvariationinfloral visitorassemblage, abundances, andpreferencesfortaggedplants,were assessed bycensusesconductedinallregionsduringthe floweringseason(late Februaryto late April).Between
20–25 censuses (3min each) were conducted for each plant(3262censusesoverall).Ineachcensusthetaxonomic
identityofallfloralvisitors,andthenumberofflowers
visitedonthefocalplant,werenoted.Adetaileddescription oftheprocedureisgivenelsewhere(Herreraetal.,2001).
Planttraits
Toinvestigatepossiblerelationships between visitation ratesandplanttraits,severalvariables relatedtofloral advertisementandrewardwereestimated.Asameasure of the plants’ floweringpattern, the numbers of open, fully functional flowers(NOF), irrespective of whether theymightbeinthefemaleormalephase, wererecorded everyday,beforecensuses werestartedandforeachplant. Attheendofthefloweringseason,plantsize(PLSZ)was estimatedasthetotalnumberofflowersproducedbyeach plant. Five fully functional flowers,in the early male stage,werecollectedrandomly fromeachplant.Flowers were preservedin a 2•5:2•5:95% formaldehyde–acetic acid–ethanol(FAA)solution. Fromthese,corollalength (CorLen;60•01mm;measuredwithadigitalcaliper),num- berofstamens(NStams)andnumberofnectaries(NNect)
wereestimated.NOFandCorLenmayberelatedtopollin- ators’perceptionoffloraldisplay,thustothedecisionto visitorrejectaplant.NStamsandNNectdescribe the amountofrewardthatvisitorsmayfindoncetheyprobe aflower.Overall,820flowerswerecollected.
Environmentalfactors
Tocharacterizetheplants’lightenvironment, hemi- spheric photographs of the forest canopy were taken above each tagged plant in all regions, using a Nikon SLRcamerawithaNikkorfisheye 16-mmlens,placed horizontallyandorientednorth–south byeachplant.All photographs were taken under similar light conditions (i.e.uniformlyovercastsky,lateintheafternoon;Steege,
1996).Photographs werescannedandthenanalysedwith WinPhot 5.0(Steege,1996;availableat uu.nl/’boev/staff/personal/htsteege/winphot/wp_index.htm) forestimationofdirectlight,diffuselight,totallightand
percentagevegetationcover(%Cover) abovetheplant.In addition, thesunlightregimeofeachplant(Sun)waschar- acterizedbeforeeachcensus,byscoringplantsaccordingto thefollowing scale:iftheplantwasunderdirectsunlight duringthecensusitwasscored1;ifitwaspartiallyshadedit wasscored 0•5;ifitwasinfullshadeitwasscored0.The averagevalueofthescoresforallcensusesofagivenplant providesaglobalmeasureofthatplant’ssunlight regime. Finally,temperature(Temp)intheimmediatevicinityof eachplantwasrecordedbeforeeachcensus,andthedis- tancebetween eachtaggedplantanditsclosestflowering conspecific(Dist)wasalsonoted,inordertoassesswhether thelocationofaplant relativetoother plantsaffectedthe likelihoodofitsvisitation.
Dataanalyses
Apreliminaryanalysis indicatedthatnoneofthe variablesconsideredshowedsignificant variationamong populations withineachregion,sothedatawerepooled within each region. One of the populations in Ma´gina was excluded due to the very low pollinator visitation rate(sixvisitsin626censuses).
Among-region variabilityinplanttraitsandenviron- mental factors was analysed using generalized linear mixedmodels(GLIMMIX;SASInstitute,1996),onefor eachvariabletested,accordingtotheirerrordistributionand usingthedefaultlinkfunction.Variables replicatedwithin plants(i.e.obtainedfromcensusesorflowers)werenested withinplants, andthesewithinregion.These includedall floraltraits,TempandSun.Inthecaseofvariablesobtained
‘perplant’ (i.e.nowithin-plantreplicates),plantswerenes- tedwithinregions.ThesewereDistand%Cover.Variations amongregionsinpollinatorabundanceweretestedbyfit-
tingageneralizedlinearmodel(GENMOD;SASInstitute,
1996).Allvisitors recordedduringcensuses weregrouped into fiveclasses: Andrena spp., Anthophora spp., Apis
mellifera, Bombus spp. and other. Region, pollinator class and their interaction were considered as factors. The responsevariable was modelled as binomial (ratio
betweentheabundanceofeachpollinatorgroupandthe
overallpollinatorabundanceineachregion). Variations amongregionsinpollinatorvisitationratesweretestedin asimilarway,treatingeach3-mincensusasasamplingunit. Abinomialresponsevariable(whethertheplantwasvisited ornotduringeachcensus) wasusedastheestimatorof visitationrates,becausethelargenumber ofzerovalues recordedinsomeregions(seeResults)precludedtheuse ofdirectcounts.
Toinvestigatetherelationshipsbetweenvisitationprob- abilityandplants’bioticandenvironmentaltraits,indi-
vidualplantswereconsideredasstudysubjects;thusdata
obtainedfromcensusesorflowerswereaveragedforeach
plant,whilemeasurestakenperplantwereusedassuch.The dependentvariablewasmodelledasbinomial (ratio between the number of censuses yielding at least one
visitandthetotalnumberofcensusesonthatplant),thus ageneralizedlinearmodel(multipleregression;GENMOD) wasused.As predictorsforintrinsictraitsthefollowing wereused:meannumberofopenflowers(NOF),mean
corollalength(CorLen), meannumberofstamensper flower(NStams),andmeannumberofnectariesperflower (NNect).Toselect predictorsamongextrinsictraits the
following procedurewasused.Informationobtainedfrom photographsmainlyaccountfortheenvironmental light conditionsaroundeachstudyplant.Aprincipalcomponent
analysis(notshownhere)waspreviouslycarriedoutonthe variablesobtainedfromhemisphericalphotographs(separ- atelyforeachregion),toidentifyorthogonalfactorsthat
mightreducethenumber oforiginalvariables. Inallcases, theanalysesrevealedtheexistenceofasingleaxisaccount- ingformostofthevariance.Thus,tosimplifytheinter- pretation of the results, only %Cover was retained as
predictorfortheenvironmental variablesextractedfrom hemisphericalphotographs, givenitsstraightforwardinter- pretationinthecontextofthisstudy.Further,meanair
temperature(Temp) andaveragesunlightregime(Sun), bothaccountingfortheenvironmental conditionssuitable for pollinators, and distance to the closest flowering
neighbour(Dist),were also included. A quadraticterm forTempwasalsoconsidered,todetectpossiblenon-linear relationships.
RESULTS
Variationinplantsize,floraldisplayandbiotictraits
Allfloraltraitsshowedsignificantamong-regionvariation (Table1A).PlantsinMa´ginahadthelargestflowersand higheststamennumber, whilenectarynumberwashighest inCaurel(Table1B).Significant variationswerealso observed bothwithinandamongplants,butwithin-plants variationwasgreaterthanamong-plantsvariation.Further, significantamong-regionvariationinplantsize(PLSZ,total number offlowersproduced byeachplantovertheseason; Table1A)wasalsofound.PlantsinMa´ginawerelargerthan plantsinCaurelandCazorla(Table1B).Similarly, signi- ficant among-regionvariationinfloral display(numberof flowersopenonacensusday,NOF)wasfound.Again, plants in Ma´ginahad significantlyhigherfloraldisplay thanplantsinCaurelandCazorla(Tables1Aand1B).
PLSZNOFClenNNectarsNStams
Fixedfactorsc2Pc2Pc2Pc2Pc2P
Amongregions / 23.26 / 0.001 / 68.89 / 0.001 / 31.03 / 0.001 / 60.05 / 0.001 / 158.97 / 0.001Randomfactors / Z / P / Z / P / Z / P / Z / P / Z / P
Amongplants / 9.62 / 0.001 / 9.59 / 0.001 / 5.50 / 0.001 / 4.82 / 0.001 / 5.33 / 0.001
Withinplants / – / – / – / – / 19.00 / 0.001 / 19.09 / 0.001 / 19.01 / 0.001
Individualplants werenestedwithin region, and,whereappropriate,individualflowerswerenestedwithin plants.
TABLE 1B.Plant means(61 s.e.)offloraltraits among regions
Caurel(n=64)Cazorla(n=67)Ma´gina(n=30) PLSZ31.3761.56 30.8962.67 83.5867.14
NOF9.4160.514.6460.3931.6662.90
CLen15.3860.1215.4360.1216.4160.15
NNectars5.6960.094.8960.045.0960.09
NStams35.1060.4041.3160.4943.6860.62
SeeMaterialsandmethodsforadescriptionofthevariables.
n,thenumberofplants usedintheanalyses.
Variationinenvironmentaltraits
Allenvironmentalvariablesshowedamong-region vari- ation (Table2A).Significantvariationswerealsoobserved, both within plants (Temp and Sun) and among plants (allvariables). Again,forTempandSun,thewithin-plants variation(i.e.theday-to-daypatternofdifferences among censuses)wasmoremarkedthantheamong-plants vari- ation.Overall,Cazorlawasthecoldest region(Table2B). PlantsinCazorlawerealsotheleastexposedtodirectsun- light.Ma´gina wasthewarmest regionandhadthemost sunlight-exposedplants.
Planttraitsandenvironmentalfactorsinrelationto pollinatorvisitationrates
Overallpollinatorabundance differedamong regions (Region:c2 =121•78,P0•001),andtherelativeabund- anceofthedifferent pollinatorgroups alsodifferedamong regions(Region·PollClasseffect:c2 =89•76,P0•001). Thus,Bombus speciesaccountedfor85•79%ofvisitsin Cazorla, 61•26% in Caurel and 50•77% in Ma´gina (Table3).Significantdifferenceswerefoundamongregions intheprobabilitythataplantwasvisited(Region:c2 =7•64, P0•02).PlantsweremorelikelytobevisitedinCaurel (11•64%ofthecensusesyieldedatleastonevisit)thanin Cazorla(6•46%) and Ma´gina(2•10%). Some plantsin CaurelandCazorlawerevisitedregularly(i.e. showedcon- sistentlyhighervisitationrates);inMa´gina,however, no suchpatternwasobserved. Overall, visitationrateswere rather low, even in the regions where pollinators were moreabundant.
Table4shows theresultsoftheregression modelsof plant and environmentaltraits on the likelihood that a plantwasvisited.Plantswithalargerfloraldisplay(i.e. higher NOF) tended to receive more visits than those with a smaller display (parameter estimates6standard
error:Caurel,0•0960•04;Cazorla,0•1460•05;Ma´gina,
0•0260•01;Fig.2).Noneoftheotherplantorenviron- mentaltraitswasaconsistentlysignificantdeterminantof
likelihoodofvisitation.
DISCUSSION
Theresultsshowsignificantvariation:(a)inallplanttraits withinplants,amongplantsandamongregions,confirming thefindingsbyHerreraetal.(2001,2002)forawiderrange ofH.foetiduspopulationsthroughouttheIberianPeninsula; (b)in allenvironmentalfactorswithinplants,amongplants andamongregions(seeHerrera, 1995a,andreferences therein);and(c)inpollinatorabundance andinthelikeli- hoodthataplantwillbevisitedamongregions,asfoundby manyother authors(seeHerrera,1988,1995a;Horvitzand Schemske, 1990;Eckhart,1992;Waseretal.,1996). Finally,theanalysesshowthatfloraldisplay(i.e.number ofopenflowersonaplant)wastheonlyoneofthefactors consideredthathadaconsistentsignificant effecton pollinatorvisitlikelihoodinallthestudyregions.
Consideringtherelativeimportance ofbioticversusabi- oticfactorsasdeterminantsfor a successfulpollination,the influenceofthelattermaybepresumablymoreimportantin plantsflowering duringperiodsofadverseweather(e.g. Herrera,1995a;Aizen,2003),ashasbeenobserved,both atamacro- andmicro-climaticlevel,forsome winter- flowering herbs(seeHerrera,1995b,andreferences therein).However,thereareexamplesofwinter-orearly spring-flowering plantspeciesthatshowhighnatural, although slightly pollen-limited, fruit set (see Herrera,
2002),andithasbeendocumented, forseveralearly- bloomingspecies,thattheinfluenceofabioticfactorsis notrelevantwhencomparedwiththatofbiotictraitsrelated topollinatorattraction(e.g.floraldisplayorflowerduration; seeMotten,1986;TotlandandMatthews,1998;Herrera,
2002).Inthepresentstudy,onlyfloraldisplayhadasig- nificanteffectonpollinatorvisitsinallthestudyregions.
Althoughflower size(corollalength)andpotentialreward
(numberofstamens andnectaries)maybepotentially importanttraitsforexplainingdifferencesbetweenplants
invisitationrates(e.g.YoungandStanton,1990;Eckhart,
1991;Burd,1995;ConnerandRush,1996;Totlandetal.,
1998), none of them were significantpredictors in the
presentstudy(seealsoTotlandandMatthews,1998). Numerousauthorshavefoundthatpollinatorattractionis
affectedbyfloraldisplay,whetherdefinedasplantsize,
numberofflowersperinflorescence,orintermsofthe
TABLE 2A.Resultsofthegeneralizedlinearmixedmodelstestingthedifferencesinenvironmentaltraitsamongstudyregions
TempSunDist%Cover
Fixedfactorsc2Pc2Pc2Pc2P
Amongregions / 928.23 / 0.001 / 43.22 / 0.02 / 16.80 / 0.001 / 160.78 / 0.02Randomfactors / Z / P / Z / P / Z / P / Z / P
Amongplants / 2.59 / 0.005 / 5.43 / 0.001 / 9.59 / 0.001 / 9.70 / 0.001
Withinplants / 30.39 / 0.001 / 28.24 / 0.001 / – / – / – / –
Individualplants werenestedwithin region,and,whereappropriate,individualflowerswerenestedwithin plants.
TABLE 2B.Plant means(61 s.e.)ofenvironmentaltraits amongregions
TABLE 4.Resultsofthegeneralizedlinearmodelstestingthe relationshipbetweenfloralandenvironmentaltraitsandthe probabilitythattheplantswerevisited,ineachstudyregion
Caurel(n=64)Cazorla(n=67)Ma´gina(n=30)
Temp.15.7760.248.6160.0813.6960.21
Sun0.3160.030.1760.020.5260.04
Caurel
(n=64)
Cazorla
(n=67)
Ma´gina
(n=30)
Dist228.59625.55211.97622.03113.58613.61
%Cover38.1661.7260.1361.2125.8162.75
Floraltraits
c2Pc2Pc2P
SeeMaterialsandmethodsforadescriptionofthevariables.
n,thenumberofplantsusedintheanalyses.
TABLE 3.Numberofvisitsandrelativeabundance ofthe differentpollinatortaxainthecensusesineachofthethree
NOF7.180.0076.920.0099.070.003
Clen0.650.420.100.750.010.95
Nnectars0.080.783.120.080.150.70
Nstams0.010.940.330.572.500.11
Environmentaltraits
Temp0.430.510.170.682.460.12
regionsstudied
(Temp)2
0.310.560.090.772.630.10
......
Caurel(985) Cazorla(1065) Ma´gina(569)
Pollinatorspecies / n / % / n / % / n / %Andrenaspp. / 7 / 1.83 / 10 / 5.08 / 9 / 13.85
Anthophoraacervorum / 91 / 23.82 / 11 / 5.58 / 8 / 12.31
Apismellifera / 45 / 11.78 / 0 / 0 / 0 / 0
Bombuspascuorum / 28 / 7.33 / 0 / 0 / 0 / 0
Bombuspratorum / 201 / 52.62 / 72 / 36.55 / 0 / 0
Bombusterrestris / 5 / 1.31 / 97 / 49.24 / 33 / 50.77
Others / 5 / 1.31 / 7 / 3.55 / 15 / 23.07
Total / 382 / 197 / 65
Valuesinbracketsarenumbersofcensuses.
spatiotemporalpatternof presentation (Cruzanetal.,1988; Eckhart,1991;deJongandKlinkhamer,1994).Thepresent resultsagreewiththesefindings,suggestingthatpollinators ofH.foetidusselectplantswithalargerfloraldisplay,and areconsistentwiththeviewthat,inwinter-floweringspe- ciesinareaswithadverseclimaticconditions,abetterfloral displayimproves attractivenesstopollinators(seerefer- encesinTotlandand Matthews,1998).Theway and extent towhichdifferentpollinatorsmayrespond tovariationsin floraldisplayhasrecentlybeenthesubjectofconsiderable attention(seeThompson, 2001).InthecaseofH.foetidus theeffectoffloraldisplayonvisitratewasconsistentamong regions,suggestingthatthedifferentpollinatorsrespondin asimilarwaystovariationsinfloraldisplay.
Noneoftheenvironmental factorsconsideredweresig- nificantpredictorsofvisitationrates,despitethevariability foundinthesefactorsatthespatialscaleanalysed. The importanceofsunlighttovisitorshasbeensuggested,as itmaymodifytheirselectionpreferencesandabilityto
Sun0 330040100750080 78
Dist1.120.290.080.7811.640.001
%Cover0.220.640.010.912.740.10
Dependentvariablewasmodelledasbinomial(no.censuseswithatleast onevisit/totalno.ofcensuses).SeeMaterialsandmethodsforadescription ofthevariables.
n,thenumberofplantsusedintheanalyses.
manipulatecertainplantsdepending onlocation(e.g. Beattie,1971;Chazdon,1988;Herrera,1997).Furthermore, theirradiancemosaicmayinfluencenotonlytheplants’ likelihoodofbeingvisited,butalsothetaxonomiccomposi- tionofthevisitorassemblage(Herrera,1997),thusaffecting plantfitness indemographicandevolutionaryterms (Herrera,2000b).Finally,relatedvariables,suchasairtem- perature orplantcover,mayalsoimposerestrictionson flowervisitors(e.g.Murcia,1990;Herrera,1995a;Totland,
2001).Nevertheless, noconsistenteffectsofanyofthese factors on visitation rates in any regionwere detected.
Similarly, visitationrateswerenotaffectedbythespatial distribution ofplants,whichmayalsoaffectthelikelihood ofsuccessfulpollination(e.g. Sih and Baltus,1987;Sowig,
1989; Laverty, 1992; Karban, 1997; O’Connell and
Johnston,1998;butseeJennerstenandNilsson,1993).
Pollinatorpreferencesaccordingtobioticandabiotictraits
Thelackofeffectsofthefactorsanalysed, otherthan floraldisplay,onpollinatorvisitationlikelihoodmightbe attributableto(a)alackofvariabilityinthefactorsanalysed atthespatialscaleconsideredhere,whichisobviously not thecase(seeResults andHerrera etal.,2002)or(b)vari- abilityinthesefactorsatalevelbelow thatdetectableby pollinators.Inthissense,pollinationservicesthroughoutthe
0·40
0·30
0·20
0·10
0·00
0·32
0·24
0·16
0·08
0·00
0·16
0·12
0·08
0·04
0·00
Caurel
n=64
036912151821
Cazorla
n=67
02468101214
Mágina
n=30
fallswithinthepreferencesandabilitiesofbumblebees,and ifthescattereddistributionofplantsinthestudypopulations isabletoneutralizeanydirectionalbehaviour ofthebees (seee.g.Cresswell, 2000),aselectionbythemainpollin- atorsofcertainplantsorfloral phenotypes,oralimitation duetoenvironmental conditionswouldnotnecessarilybe expected.Nevertheless, thepresentresultsindicatethat pollinatorsofH.foetidusselectplantsprimarilyinview of their floraldisplay (number of open flowerson the plantonagivenday),whichsuggests thattheremaybe selectionforgreaterwithin-plantfloweringsynchrony.In H. foetidus,then,‘how’theplantiswouldseemtobe more importantthanwhereitis.
Helleborusfoetidusisself-compatible,thuspollinators visitingplantswithalargefloraldisplaymayvisitseveral
flowerssequentially,presumablyfavouringgeitonogamous
self-pollination.Additionally,changesinflight distancein responsetofloraldisplaysmayinfluencematingpatterns
withinpopulations(seeMitchelletal.,2004,andreferences therein). Fromadifferentperspective, ithasbeenpredicted (seeCruzanetal.,1988;Mitchell,1993)thatselectionfor
floral traitsthatincreaseattractivenessforpollinatorswill actprimarilythroughmalefunction, i.e.pollendonation (seeWillson,1979;Queller,1997).Asaresult,itisdifficult
topredicttheextenttowhichdifferencesintherateofvisits toH.foetidusplantswilltranslateintofitness differences. Further, geographical variation captured in this study
involvesdifferencesbetweenareaswithaMediterranean (CazorlaandMa´gina) andAtlanticclimate(Caurel),with contrastingconditions. Althoughsuchclimaticdifferences donotinfluencetheflowering phenologyofthestudy species(H.foetidusstartsfloweringroughlyatthesame time in allstudyregions;pers.obs.),their contribution maymodulateinadifferentwaythepollinationprocesses
considered.Thus,amorecompleteunderstanding ofthe consequences of environmental factors and plant traits forreproductivesuccessinH.foetiduswillrequireaddi- tionalexplorationoftheextenttowhichvariationinenvir- onmentalfactorsmayleadtoamong-plant variationin vegetativecharactersinfluencingplantfitness.
020406080100120
Floral display
FIG.2.Scatterplotsofvisitationrates(numberofcensuseswithvisits/total numberofcensuses)versusmeanfloraldisplay(averagedforallcensuses fromeachregion)inthethreestudyareas.n,thenumberofplantsconsidered ineachregion.
rangeofH.foetidus aremainlyprovidedbybumblebees (Bombusspp.;ProctorandYeo,1973;Vesprinietal.,1999), namelyB. terrestrisandB. pratoruminthepopulations studied.Bumblebeescanbeconsidered aseffective pollinatorsoverawiderangeoffloralphenotypesandenvir- onmentalconditions(e.g.Mayfield etal.,2001),andtheir thermoregulatoryabilitiesfacilitatetheirroleaspollinators bothforplantsfloweringinwinter(likeH.foetidus)and plantsflowering insummer(e.g.Lavandulalatifolia; Herrera,1995a),whenweatherconditionsimposelimita- tionsonotherinsectgroupsintheMediterraneanarea.Thus,
ACKNOWLEDGEMENTS
WethankB.Garc´ıa,J.L.GarridoandA.Manzaneda for fieldassistance;M.Carrio´n, A. Prieto,and R. Requereyfor technical assistance; and Ø. Totland, J. Cresswell and J.D.Thompson forcommentsonthemanuscript. This studywassupportedbygrantPB96-0856 (SpanishMinis- terio de Educacio´n y Cultura). During the preparation of the manuscript J.G. received funding from the
‘Fundacio´nRamo´nAreces’andA.M.S.-L.wassupported by grantBOS2003-00292(SpanishMinisteriodeCienciay
Tecnolog´ıa).
LITERATURE CITED
AizenMA.2003.Influencesofanimalpollinationandseeddispersalon winter-floweringinatemperatemistletoe.Ecology84:2613–2627.
AnderssonS.1994.Floralstability,pollinationefficiency,andexperimental manipulationofthecorollaphenotypeinNemophila menziesii (Hydrophyllaceae).AmericanJournalofBotany81:1397–1402.
BeattieAJ.1971.Itinerantpollinatorsinaforest. Madro~no21:120–124.
BurdM.1995.PollinatorbehaviouralresponsestorewardsizeinLobelia deckenii:noescapefrompollenlimitationofseedset.Journal of Ecology3:865–872.
Campbell DR,Waser NM,Price MV,LynchEA,MitchellRJ.1991.
Componentsofphenotypicselection:pollenexportandflowercorolla width inIpomopsisaggregata.Evolution45:1458–1467.
Chazdon RL.1988.Sunflecksandtheirimportancetoforestunderstorey plants.AdvancesinEcologicalResearch18:1–63.
ConnerJK,RushS.1996.Effectsofflowersizeandnumberonpollinator visitationtowildradish,Raphanusraphanistrum. Oecologia105:
509–516.
CresswellJE.2000.Acomparisonofbumblebees’movementsinuniform and aggregated distributions of their forage plant. Ecological
Entomology25:19–25.
CresswellJE,GalenC.1991.Frequency-dependentselectionandadaptive surfacesforfloral charactercombinations:thepollinationof Polemoniumviscosum.AmericanNaturalist138:1342–1353.
Cruzan MB,NealPR,WilsonMF.1988.FloraldisplayinPhylaincisa:
consequencesformaleandfemalereproductivesuccess.Evolution
42:505–515.
deJongTJ,KlinkhamerPGL.1994.Plantsizeandreproductivesuccess throughfemaleandmalefunction.JournalofEcology82:399–402. EckhartVM.1991.Theeffectsoffloraldisplayonpollinatorvisitation vary among populations of Phacelia linearis (Hydrophillaceae).
EvolutionaryEcology5:370–384.
EckhartVM.1992.Spatio-temporalvariationinabundanceandvariationin foragingbehaviorofthepollinatorsofgynodioeciousPhacelialinearis
(Hydrophyllaceae).Oikos64:573–586.
GigordLDB,Macnair MR,SmithsonA.2001.Negative frequency- dependentselectionmaintainsadramaticflowercolorpolymorphism intherewardlessorchidDactylorhizasambucina(L.).Proceedingsof theNationalAcademyofSciencesoftheUSA98:6253–6255.
Go´mez JM. 2000. Phenotypicselection and responseto selection in Lobulariamaritima:importance ofdirect and correlationalcompon- entsofnaturalselection.JournalofEvolutionaryBiology13:689–699.
Guitia´nJ,Guitia´nP,Medrano M,Sa´nchezJM.1999.Variationinfloral morphology andindividualfecundityinErythroniumdens-canis (Liliaceae).Ecography22:708–714.
HerreraCM.1988.Variationinmutualisms:thespatio-temporalmosaicof
apollinatorassemblage.BiologicalJournaloftheLinneanSociety35:
95–125.
HerreraCM.1993.Selectiononfloralmorphologyandenvironmental determinantsoffecundityinahawkmoth-pollinatedviolet.Ecological Monographs63:251–275.
HerreraCM.1995a.Microclimateandindividualvariationinpollinators:
floweringplantsaremorethantheirflowers.Ecology76:1516–1524.
Herrera CM.1995b.Floralbiology, microclimate,andpollinationby ectothermicbeesinanearly-bloomingherb.Ecology76:218–228.
HerreraCM.1996.Floraltraitsandplantadaptationtoinsectpollinators:a devil’sadvocateapproach. In:Lloyd,DG,Barrett, SCH,eds.Floral biology—studieson floral evolutionin animal-pollinated plants.
NewYork, NY:ChapmanHall,65–87.
HerreraCM. 1997.Thermalbiologyandforagingresponsesofinsect pollinatorstotheforest floorirradiancemosaic.Oikos78:601–611. HerreraCM.2000a.Measuringtheeffectsofpollinatorsandherbivores: evidence for non-additivity in a perennial herb. Ecology 81:
2170–2176.
HerreraCM.2000b.Flower-to-seedlingconsequencesofdifferentpollina- tionregimesinaninsect-pollinatedshrub.Ecology81:15–29.
HerreraCM. 2001.Deconstructingafloralphenotype:dopollinatorsselect forcorollaintegrationinLavandulalatifolia?JournalofEvolutionary Biology14:574–584.
HerreraCM. 2002.Censusingnaturalmicrogametophytepopulations:
variable spatial mosaics and extreme fine-graininess in winter- floweringHelleborusfoetidus(Ranunculaceae).AmericanJournal
ofBotany89:1570–1578.
HerreraCM, Soriguer RC. 1983.Intra-andinter-floralheterogeneity ofnectarproductioninHelleborusfoetidus L.(Ranunculaceae). BotanicalJournaloftheLinneanSociety86:253–260.
HerreraCM,Sa´nchez-LafuenteAM,Medrano M,Guitia´nJ,Cerda´ X, ReyPJ.2001.Geographicalvariationinautonomousself-pollination levelsunrelatedtopollinatorservice inHelleborusfoetidus (Ranunculaceae).AmericanJournalofBotany88:1025–1032.
HerreraCM,Cerda´X,Garc´ıaMB,Guitia´nJ,MedranoM,ReyPJ,etal.
2002.Floralintegration,phenotypic covariancestructureand pollinatorvariationinbumblebee-pollinated Helleborusfoetidus. JournalofEvolutionaryBiology15:108–121.
HorvitzCC,SchemskeDW.1990.Spatiotemporalvariationininsectmutu- alisms ofaNeotropicalherb.Ecology71:1085–1097.
JennerstenO,NilssonSG.1993.Insectflowervisitationfrequencyand seedproduction inrelationtopatchsizeofViscariavulgaris (Caryophyllaceae).Oikos68:283–292.
KarbanR.1997.Neighbourhoodaffectsaplant’sriskofherbivoryand subsequentsuccess.EcologicalEntomology22:433–439.
KlinkhamerPGL,deJongTJ,DeBruynGJ.1989.Plant sizeand pollinatorvisitationinCynoglossumofficinale.Oikos54:201–204.
Laverty TM.1992.Plantinteractionsforpollinatorvisits:atestofthe magnetspecies effect.Oecologia89:502–508.
Maad J.2000.Phenotypicselectioninhawkmoth-pollinatedPlatanthera bifolia:targets andfitnesssurfaces.Evolution54:112–123.
Mayfield MM,WaserNM,PriceMV.2001.Exploringthe‘mosteffective pollinatorprinciple’withcomplexflowers:bumblebees andIpomopsis
aggregata.AnnalsofBotany88:591–596.
MitchellRJ.1993.AdaptivesignificanceofIpomopsisaggregata nectar production:observationandexperimentinthefield.Evolution47:
25–35.
MitchellRJ,KarronJD,HolmquistKG,BellJM.2004.Theinfluenceof Mimulusringensfloral displaysizeonpollinatorvisitationpatterns. FunctionalEcology18:116–124.
Motten AF.1986.Pollination ecologyofthespringwildflower communityof atemperate deciduousforest.EcologicalMonographs
56:21–42.
Murcia C.1990.Effectoffloralmorphologyandtemperatureonpollen receiptandremovalinIpomoeatrichocarpa.Ecology71:1098–1109. Niesenbaum RA. 1994.Spatialandtemporalvariationinpollentube
numbersinLinderabenzoin(Spicebush).CanadianJournalofBotany
72:268–271.
O’ConnellLM,JohnstonMO.1998.Maleandfemalepollinationsuccess inadeceptiveorchid:aselectionstudy. Ecology79:1246–1260.
PhilippM,HansenT.2000.Theinfluenceofplantandcorollasizeonpollen deposition andseedsetinGeraniumsanguineum(Geraniaceae). NordicJournalofBotany20:129–140.
Proctor M,YeoP.1973. Thepollinationofflowers.London:Collins.
Queller DC.1997.Pollenremoval,paternity,andthemalefunctionof flowers.AmericanNaturalist149:585–595.
Robertson JL,Wyatt R.1990.Evidenceforpollinationecotypesinthe yellow-fringedorchid,Platanthera ciliaris.Evolution44:121–133.
SASInstitute. 1996.SAS/STAT software: changes and enhancements
throughRelease6•11.Cary,NC:SASInstitute.
SchemskeDW,Horvitz CC.1989.Temporalvariationinselectionona floralcharacter.Evolution43:461–465.
SihA,Baltus MS.1987.Patchsize,pollinatorbehaviorandpollinator limitationinCatnip.Ecology68:1679–1690.
SowigP.1989.Effectsoffloweringplant’spatchsizeonspeciescomposition
ofpollinatorcommunities,foragingstrategies,andresourcepartition- inginbumblebees(Hymenoptera:Apidae).Oecologia78:550–558. SteegeHT.1996.WinPhot5:aprogrammetoanalyzevegetationindices, lightandlightqualityfromhemisphericalphotographs.Tropenbos GuyanaReports95-2,TropenbosGuyanaProgramme,Georgetown,
Guyana.
ThompsonJD.2001.Howdovisitationpatternsvaryamongpollinatorsin relationtofloral displayandfloral designinageneralistpollination
system?Oecologia126:386–394.
TotlandØ.2001.Environment-dependentpollenlimitationandselectionon floraltraitsinanalpinespecies. Ecology82:2233–2244.
Totland Ø,Matthews I.1998.Determinantsofpollinatoractivityand flowerpreferenceintheearlyspringbloomingCrocusvernus.Acta
Oecologica19:155–165.
Totland Ø,AndersenHL,BjellandT,DahlV,EideW,HougeS,etal.
1998.Variationinpollenlimitationamongplantsandphenotypic selectiononfloraltraitsinanearly-springfloweringherb.Oikos82:
491–501.
patternsandnectarcompositionintwoHelleborusspecies.Plant
Biology1:560–568.
WaserNM,PriceMV.1981.Pollinatorchoiceandstabilizingselectionfor flowercolor inDelphiniumnelsonii.Evolution35:376–390.
Waser NM,Chittka L,Price MV,Williams NM,Ollerton J. 1996.
Generalizationinpollinationsystems,andwhyitmatters.Ecology
77:1043–1060.
Werner K,EbelF. 1994.ZurLebensgeschichtederGattungHelleborusL. (Ranunculaceae).Flora189:97–130.
777–790.
WilsonP.1995.Selectionforpollinationsuccessandthemechanicalfitof
Impatiensflowersaroundbumblebeebodies.BiologicalJournalofthe
LinneanSociety55:355–383.
WilsonP,Thomson JD.1996.Howdoflowersdiverge?In:Lloyd,DG, Barrett,SCH,eds.Floral biology—studiesonfloralevolutionin animal-pollinatedplants.NewYork,NY:ChapmanHall,88–111. YoungHJ, Stanton ML. 1990.Influencesoffloralvariationonpollen
removalandseedproductioninwildradish.Ecology71:536–547.