ContentslistsavailableatScienceDirect
Flora
jou rn a l h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / f l o r a
Reproductive
biology
of
Cipocereus
minensis
(Cactaceae)—A
columnar
cactus
endemic
to
rupestrian
fields
of
a
Neotropical
savannah
Cristiane
Martins
a,
Reisla
Oliveira
b,
Carlos
Victor
Mendonc¸
a
Filho
c,
Liliane
Teixeira
Lopes
c,
Rodrigo
Assunc¸
ão
Silveira
a,
Juliana
Aparecida
Pereira
de
Silva
a,
Ludmilla
M.S.
Aguiar
d,
Yasmine
Antonini
a,b,∗aProgramadePósGraduac¸ãoemEcologiadeBiomasTropicais,UniversidadeFederaldeOuroPreto,MG,Brazil bDepartamentodeBiodiversidadeeEvoluc¸ão,UniversidadeFederaldeOuroPreto,OuroPreto,MG,Brazil cDepartamentodeBiologia,UniversidadeFederaldosValesdoJequitinhonhaeMucuri,Diamantina,MG,Brazil dDepartamentodeZoologia,UniversidadedeBrasília,CampusUniversitárioDarcyRibeiro,Brasília,DF,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received22August2015 Receivedinrevisedform 25November2015 Accepted26November2015 EditedbyStefanDötterl Availableonline4December2015
Keywords: Cactaceae Endemicspecies Chiropterophily Self-incompatibility
Nocturnalanddiurnalpollinators Pollenlimitation
Brazil
a
b
s
t
r
a
c
t
WestudiedthereproductivebiologyofCipocereusminensis,anendemiccolumnarcactusoftheEspinhac¸o MountainRange,SoutheasternBrazil,focusingonfloralbiology,breedingsystem,andpollination.We describedfloralmorphologyandevaluatedtheroleofnocturnalanddiurnalpollinatorsonthe repro-ductivesuccessintwopopulations.C.minensishaslarge,horizontal,cream-colored,chiropterophilous flowerswithrigidpetalsthatopenatduskandcloseonthefollowingmorning.Flowersproduceda hugeamountofpollengrainsandnectarproductionwasnocturnal.Controlledpollinationexperiments revealedthatthecactusisanobligatexenogamousspecies.Visitor-exclusionexperimentsrevealedthat thenocturnalvisitors(bats)aretheprominentpollinatorswhereashummingbirdsandsocialbees,which visitedtheflowersearlyinthemorning,contributedlittletofruitset.Weconcludethatthereproductive successofthisendemiccolumnarcactusisthreatenedintheabsenceoftheeffectivepollinatingbats.
©2015ElsevierGmbH.Allrightsreserved.
1. Introduction
Cactaceaeisadistinctivefamilyofplantsnativetothe Amer-icaswithapproximately1600species.Brazil isthethird global centerofcactidiversitywith200species,ofwhich78%are con-sidered endemic (Taylor and Zappi, 2004; Taylor, 1997; Zappi et al.,2010).Our understandingof thereproductive biology of theseplantsisseverelylimitedcomparedtootheraspectsoftheir biologysuchasmorphology,physiology, biochemistry,and eth-nobotany(Nobel,2002).Thisisespecially trueinBrazil,where, thereproductivebiologyoflessthan10%ofthespecieshasbeen studied(Zappietal.,2011).Thesestudieshaveincludedspecies ofPilosocereus(Locatelliet al.,1997; Rochaetal., 2007), Opun-tia(Schlindweinand Wittmann, 1997), Parodia,Gymnocalycium
∗ Correspondingauthorat:LaboratoryofBiodiversity,FederalUniversityofOuro Preto.CampusMorrodoCruzeiro,s/n-Bauxita-OuroPreto,ZIPCode:34500-000, MG,Brazil.
E-mailaddresses:antonini.y@gmail.com,antonini@iceb.ufop.br(Y.Antonini).
(SchlindweinandWittmann,1995),Tacinga palmadora(Locatelli andMachado,1999a),Cereus(LocatelliandMachado,1999b;Silva andSazima,1995),Micranthocereus(Aonaetal.,2006), Melocac-tus(Colac¸oetal.,2006;Gomesetal.,2014;LocatelliandMachado, 1999a),andCipocereus(Regoetal.,2012).
Self-incompatibilitysystemsincactiarecommonandoccurin atleast30%ofthegenera(Boyle,1997;Mandujanoetal.,2010; StrongandWilliamson,2007).Studiesonthepollinationbiology ofcolumnarcactihave shownthatmostspeciesareadaptedto nectar-feedingbats (Fleming et al.,2001; Locatelli etal., 1997; Munguía-Rosasetal.,2010;Nassaretal.,1997;Rochaetal.,2007; Valiente-Banuetetal., 1997a).Amongcolumnarcactiwith bat-pollinationsystems,therelative contributionofdiurnal visitors tofruitsetseemstoincreasewithincreasinglatitude.Thus,itis expectedthatinpopulationsofextra-tropicalregions,daytime vis-itorssignificantlycontributetoproductionofseeds(Flemingetal., 2001;Munguía-Rosasetal.,2009).Someofthesespeciesarealso effectivelypollinatedbydiurnalflowervisitorssuchasbirdsand bees(Flemingetal.,2001;Munguía-Rosasetal.,2009).In addi-tiontonectar-feedingbats,somecolumnarcactiwithnocturnal http://dx.doi.org/10.1016/j.flora.2015.11.010
flowersare pollinated byhawkmoths (Clark-Tapiaand Molina-Freaner,2004;Flemingetal.,2001;LocatelliandMachado,1999b). Allsix species of the genusCipocereus are endemic torock outcropsofthestateofMinasGerais,Brazil.Westudiedthe repro-ductivebiologyofC.minensisN.P.Taylor&Zappi,acolumnarcactus withnocturnalflowers,endemictotheEspinhac¸oMountainRange, describedfloralmorphology,andevaluatedthereproductive sys-temintwopopulations.Weaddressedthefollowingquestions:IsC. minensispollinator-dependentforreproduction?Whatistherole ofnocturnalanddiurnalpollinatorsforitsreproductivesuccess?
2. Materialsandmethods
2.1. Studyarea
WestudiedtwopopulationsofC.minensisapartabout80km fromeachother(Fig.1)intheEspinhac¸oMountainRage,Minas Gerais. Onepopulation waslocatedon theDiamantina Plateau (18◦1148.23S,43◦348.74W),inanareaofexpansiverocky out-cropssurroundingthetownofDiamantina.TheotherwasinRio PretoStatePark (18◦0712.9S, 43◦2036.9W), anaturereserve locatedinthemunicipalityofSãoGonc¸alodoRioPreto.The cli-mateofbothareasischaracterizedbyawell-definedrainyseason from November to March, and a cooler dry season from June to September. Bothpopulations of C. minensis were located in quartziticopengrasslandsataltitudesbetween1020(Diamantina) and950ma.s.l.(RioPretoStatePark).Themainvegetationwithin thestudyareais‘camporupestre’(rupestrianfields)(Piraniand Harley, 1997), forminga mosaic withriparian forestsand cer-rado.ThestudiesinbothareaswerecarriedoutfromMay2011 toDecember2012.
2.2. Studiedspecies
ThegenusCipocereusdiffersfromothersinthetribeCereeae inhavingblue,globose,indehiscentfruitswithtranslucentflesh (Fig.2A).Theflowershavecream-coloredpetalswithbluesepals (Zappietal.,2010)(Fig.2B).Thecactioccuronrockyquartzitic out-cropsandtheirrootsusuallyextendintothefissuresofrocksorare associatedwithtermitemounds.Twosubspeciesarerecognized withinC.minensis,subspeciesleiocarpus,thetaxonstudiedherein, andsubspeciesminensis.Thefirsthaslargerflowersandnon-ribbed smooth,andbluefruit.Thoseofsubspeciesminensisareribbedwith afewspine-bearingareoles,andarebrownish,palegreen,whitish, orbluish(TaylorandZappi,2004).
2.3. Floralmorphologyandbiology
Forbothpopulationswerecordedcolor,odorandtimeof anthe-sisoftheflowers.Todescribefloralmorphologyweused25flowers from15 plant individuals fromtheDiamantina populationand eightflowersfromeightplantindividualsfromtheRioPretoState Park.Thediameterofthecorollaandthelengthoftheflower,nectar chamber,andstigma+styleweremeasuredwithdigitalcalipers.
Wecollected30flowersinpre-anthesisfrom16individualsof theDiamantinapopulationandcountedthenumberofstamensand pollengrainsperflower.Wethenmultipliedthemeannumberof pollengrainsperantherbythemeannumberofanthersperflower (Dafnietal.,2005).
Toassesstherateofnectarsecretionandthesugar concentra-tionofnectar,webaggedeightflowerbudsofsevenindividuals. Wethenemptiedallnectarfromeachflowerintwo-hour inter-vals from19:00hto 09:00h. Theflowers werere-baggedafter eachmeasurementtoexcludeflowervisitors.Wemeasured nec-tarvolumeusinggraduatedmicrocapillarytubesandnectarsugar
concentrationwithapocketrefractometer(Atago®N1,Brixscale 0–32%).
2.4. Breedingsystem
TodeterminethebreedingsystemofC.minensis,weperformed fourtreatments:(1)non-manipulatedselfpollination—flowersin pre-anthesisweremaintainedbaggedwithoutfurther manipula-tion;(2)handself-pollination—flowerswerehand-pollinatedwith theirownpollen;(3)handcross-pollination—flowerswere emas-culatedandpollinatedwithpollengrainsfromatleasttwoflowers ofdifferentindividuals;and(4)naturalpollination—flowers acces-sibletopollinatorswereindividuallymarked(control).
Thetreatmentswereconductedon126flowersfrom5 indi-viduals oftheDiamantina populationand 202flowersfrom31 individualsoftheRioPretoStateParkpopulation.Withthe excep-tionofthecontrols,flowerbudswerebaggedwithvoilebags.We baggedtheflowersofalltreatmentsaftersenescencetoprotectthe fruitsfrompossiblepredationanddeterminedthefruitsetineach. 2.5. Floralvisitors
Inordertorecordflowervisitsbynocturnalanimals,wesetsix cameratraps(Tigrinus®andBushnell®),eachoneinfrontofa cac-tus,duringthreebloomingseasonsofC.minensisinDiamantina.The trappingeffortwasof528h(April2011),4320h(AugusttoOctober 2011)and3600h(JanuarytoFebruary2012).Diurnalfloralvisitors wererecordedadlibitum,(nosystematicmethod;Altman,1974) andthroughphotographicrecordsforDiamantina,throughoutthe studyperiod.
2.6. Visitor-exclusionexperiments
Toevaluatetheeffectivenessofdiurnalandnocturnal pollina-tors,weconductedexclusionexperimentsduringfourconsecutive days/nightsfor RioPretoand sixdays/nightsforDiamantina.In ordertoexcludediurnalvisitors,flowerswerebaggedataround 5:30hinthemorninguntiltheendofanthesis(5individualsand33 flowers–Diamantina;20individualsand51flowers–RioPreto). Toexcludenocturnalvisitors,flowerswerebaggedfrom17:30h to5:30hthenextmorning(5individualsand33flowers– Dia-mantina;20individualsand56flowers–RioPreto).
2.7. Statisticalanalysis
Generalizedlinearmodels(GLM)usingquasi-poisonerror dis-tribution with log link function (after residual analyses) were constructedtocomparefruitsetsinthevisitor-exclusion experi-mentsandamongthepollinationtreatmentsusedtodeterminethe breedingsystemoftheplant.Thetheresponsevariablewasfruitset andtheexplanatoryvariablesweretreatmentandplantindividual (usedasarandomfactor).Contrastanalyseswereperformedafter theconstructionofthemodels.Amultivariateanalysisofvariance (MANOVA)wasperformedtoassess differences inflower mor-phologybetweenthetwopopulations.Analyseswereperformed inStatistica8.0(MANOVA)andR(GLM).
3. Results
3.1. Floralmorphologyandbiology
C. minensis showed large, robust, cream colored flowers, with a large number of stamens (303±42) and pollen grains (417.865±3.345). They have a wide nectar chamber, which is approximatelyonethirdofthetotallengthoftheflower.Flower
Fig.1.LocationofthetwostudiedpopulationsofCipocereusminensis:SquarerepresentsDiamantinaPlateauandBlacktriangleStateParkofRioPreto.ModifiedfromMeyer andFranceschinelli(2011).
Fig.2.Cipocereusminensis.(A)Flowerbudsand(B)flower.
Table1
FloralmorphologyofCipocereusminensis(mean±SD).
Size(cm) Diamantina(n=15) Range RioPreto(n=8) Range
Diameterofthecorolla 3.28±0.37a 2.69–3.97 3.06±0.26a 2.69–3.74
Flowerlength 4.61±0.66a 3.54–6.34 4.90±0.44a 4.46–5.47
Lengthofthenectarchamber 1.30±0.22a 0.91–1.89 1.16±0.09b 1.04–1.26
Lengthofstigma+style 2.74±0.47a 2.34–3.33 3.31±0.34b 2.73–3.68
Statisticaldifferencesarerepresentedbydifferentletters. Table2
FruitsetafterdifferenttreatmentsofflowersofCipocereusminensisoftheSerradoEspinhac¸o,MinasGerais,Brazil.
Diamantina RioPreto
Treatment Plants Flowers Fruits % Plants Flowers Fruits %
Spontaneousselfpollination 5 33 0 – 19 84 0 –
Manualselfpollination 5 20 0 – 12 28 0 -–
Manualcrosspollination 5 23 19 83 7 16 16 100
Naturalpollination(control) 5 50 40 80 21 74 46 62
Nightpollination 5 33 11 33 14 51 27 53
Fig.3. Averageproductionandconcentration(givenasproductionpertwohours)of nectarfromCipocereusminensis(eightflowersfromsevenindividuals)throughout theanthesis.Whiskersindicatemean±standarderror.
morphologyonlydiffersbetweenpopulationsbythenectar
cham-ber(F3,22=−3.67,p<0.001)andstigma+stylelength(F3,22=−3.05,
p<0.01)(Table1).
Atthebeginningofanthesis,atdusk,theflowersalreadyemitted amildsweetodor.IntheDiamantinapopulation,flowersstarted openingfrom 17:30hto 18:30hand were fullyopen between 19:00hand21:00h.IntheRioPretopopulation,thebeginningof anthesiswasonetotwohoursdelayed.Theflowersremainedopen untillatemorningandcloseddefinitivelyaround11:00hinboth populations.
Themeantotalproductionofnectarperflowerwas260Land themeansugarconcentration20±0.5%(n=8).Nectarproduction perflowerwashighinthefirsthoursofanthesis,between19:00h and21:00h(60L/2h),anddiminishedcontinuouslyuntildawn (10L/2h).Sugarconcentrationofnectarremainedstable through-outanthesis(Fig.3).
3.2. Breedingsystem
C.minensiswasself-incompatibleandfruitsetoccurredonly afterhandcross-pollination and naturalpollination(control) at arateof83%and 80%,respectively,fortheDiamantina popula-tionand100%and62.2%,respectively,fortheRioPretopopulation (Table2).Fruit setwasdifferentamongthetreatmentsboth in Diamantina (F3,17=22.97, p<0.001) and Rio Preto (F3,51=5.218,
p<0.004).Fruit setbetweenindividualswasnot different,both in Rio Preto (F25=0.794, p=0.710) and Diamantina (F4=0.932,
p=0.230).Handcross-pollinationandnaturalpollinationdidnot differintheDiamantinapopulation(F1,9=0.234,p>0.05),whereas
intheRioPretopopulationhandcross-pollinatedflowersset sig-nificantlymorefruitsthanthoseaccessibletopollinators(control) (F1,24=13.909,p<0,005)(Table2).
3.3. Visitor-exclusionexperiment
Flowersavailableexclusivelytonocturnal visitors set signif-icantly more fruits than those available exclusively to diurnal visitorsinbothpopulations(Diamantina—F1,8=5.69,p=0.04/Rio
Preto—F1,27=9.64,p<0.001).Fruitsetbynocturnalvisitorswas33%
and 53%inDiamantina andRio Preto populations,respectively. Fruitsetinflowersexclusivelyaccessibletodiurnalvisitorswas only12%and16.7%,respectively(Table2).
3.4. Floralvisitors
IntheDiamantinapopulation, tennocturnal visitsbybatsof Anourasp.(Phyllostomidae)andsixdiurnalvisitsbyhummingbirds
ofPhaethornispretreiandEupetomenamacroura(Trochilidae)were recordedintheC.minensisflowersusingcameratraps.Workerbees ofApismelliferaandTrigonasp.(Apidae),aswellassmallbeetles (Nitidulidae)wereobservedvisitingflowersearlyinthemorning.
4. Discussion
OurstudyshowsthattheendemiccolumnarcactusC. minen-sisisaself-incompatiblespecies,pollinatedalmostexclusivelyby nocturnalflowervisitorsinbothstudiedpopulations.Thediurnal generalistvisitors,highlysocialhoneybeesandstinglessbees,and nectarseekinghummingbirds,providedonlyaminorcontribution tofruitset.Floraltraitssuchaslarge,robustand fleshy cream-coloredflowerswithoutnectarguides,ashortflowertubewith numerousstamenswithahugeamountofpollen,andthehighrates ofnectarproductionduringthenightdecreasinguntildawn,areall typicaladaptationstobatpollination(chiropterophily)(Faegriand VanDerPijl,1979;Vogel,1968).
Theextendedperiodofanthesisandthecontinuous,butlow, nectarsecretionduringthefollowingmorning,alsopermits vis-itstotheflowersbygeneralisteusocialbeeslookingforpollen,and sporadicvisitsbyhummingbirdswhichtake-uptheremaining nec-tar.However,consideringtheresultsoftheexclusionexperiment, bats,butnotbeesandhummingbirds,arethebestpollinatorsofC. minensis,inspiteoftheirlowfrequencyofflowervisits. Further-more,floralbiologyandmorphologyaswellasnocturnalanthesis areconsistentwithchiropterophily,reinforcingthestatementthat onlypartofthepollinatorspeciesorfunctionalgroupsofthem(bats inourcase)exertstrongselectivepressuresonfloraltraits(Fenster etal.,2004;Reynoldsetal.,2009).
Forsomecolumnarcacti,anocturnalfloralcyclethatextends intothefollowingdayhasbeensuggestedtobeastrategytoensure sexualreproductionwhenthereisspatialandtemporalvariation in thefrequencyof nocturnalpollinators (Fleminget al.,2001). Pollination studies withcolumnar cactiin Mexico suggest that specializedpollinationsystemslikethatofchiropterophilyprevail intropicalregions,whereasinextra-tropicalregions,insectsand hummingbirdswouldfrequentlybecomplementarypollinatorsin batpollinatedspecies(Flemingetal.,2001;Valiente-Banuetetal., 1996,1997a,b).
Inonlysixoftheapproximately100speciesofCereeae colum-narcacti(sensuZappietal.,2010)hasthepollinationsystembeen studiedindetail[CereushorrispinusBackeb,CereusrepandusMill. (Nassaret al.,1997)Pilosocereuschrysacanthus (Weber)Byles & Rowley(Valiente-Banuetetal.,1997b),Pilosocereuslanuginosus(L.) Byles&Rowley(Nassaretal.,1997),Pilosocereusroyenii(L.)Byles &Rowley(Rivera-MarchandandAckerman,2006)andPilosocereus tuberculatus(Werdermann)Byles&Rowley(Rochaetal.,2007)]. Allofthemaretropicalandexhibitabat-specializedpollination system,evenifdiurnalvisitorsaccountforaminorcontribution tofruitset.However,informationonreproductivebiologyofthe Southernhemispherecactiisscarce,preventingconclusionsabout theoccurrenceofthispatternforthisregion(Munguía-Rosasetal., 2009).
Fruit setexclusively fromcross-pollination is widespreadin cactispecies(seereviewMandujanoetal.,2010).Fruitset exclu-sivelyfromcross-pollinationinC.minensisaswellasdemonstrated for co-generic species (Cipocereus laniflorus—Rego et al., 2011, Cipocereus crassissepalus—Martins et al.unpublished) and other representativesofCereeae(Clark-TapiaandMolina-Freaner,2004; Ibarra-Cerde ˜naetal.,2005;StrongandWilliamson,2007; Valiente-Banuetetal.,1997).
Ourresultsindicatetheoccurrenceofpollinatorlimitationon fruitsetintheRioPretopopulationbutnotinDiamantina,whereas the Diamantina but not the Rio Preto population seems to be
resourcelimited.Lowvisitationfrequency,variationinpollination efficiency,andpollinatorssharinghasbeenidentifiedaspossible causesofpollinatorlimitation(Ashmanetal.,2004;Heglandand Totland,2007;PintoandSchlindwein,2015).IntheRioPretoPark (andnotinDiamantina),C.minensisoccurswithfurthertwo colum-narcacti,Pilosocereusaurisetus(Werderm.)Byles&G.D.Rowleyand Cipocereuscrassisepalus(BuiningandBrederoo)Zappi&N.P. Tay-lor.Bothspeciesarechiropterophilousandtheirbloomingperiods overlaplargely(P.aurisetus)orpartially(C.crassissepalus)withthat ofC.minensis(Martinsetal.,pers.obs.).
PopulationsofC.minensisoccurinrockyhabitats,fromc.750m to1500mofaltitude(Taylorand Zappi,2004),which is a con-strainedhabitatwithahuge thermicamplitude,lowwater and resourceavailability,andsporadicfire(TaylorandZappi,2004). Althoughbothpopulationsoccurinsuchahabitat,thefruitsetin manualcrosspollinationtreatmentswasonlyless than100%in DiamantinaindicatingthatinthisbutnotintheRioPreto popula-tionthereareinsufficientresourcestoproducefruits.Furthermore, inbreedingdepressionmightconstrainfruitsetinthesmall-sized populationofDiamantina.
TheflowersofthetwostudiedpopulationsofC.minensisdiffer onlyinthesizeofthenectarchamberandinthestigmalength. Becausebothpopulationsdependonbatpollination,such differ-encesdonotresultinashiftfromnocturnaltodiurnalpollinators. Animportantimplicationforconservationofendemicand endan-geredspecies,suchasC.minensis,isthelossofmainpollinators.The entireCactaceaefamilyislistedasendangeredintheConvention onInternationalTradeinEndangeredSpecies(CITES).Forcolumnar cacti,studiesonpossiblepopulationdecreasesofthebat pollina-torsandtheirviabilityarerequiredtobetterunderstandthecacti’s reproductiveconstraints.
Acknowledgements
We aregrateful tothefollowing for providing scholarships: ConselhoNacional dePesquisa(CNPq) toY.A., Coordenac¸ãode Aperfeic¸oamento de Pessoal de Nível Superior (CAPES) to C.M. and R.O., Universidade Federal de Ouro Preto (UFOP) to J.P., andFundac¸ãodeAmparoàPesquisado EstadodeMinasGerais (FAPEMIG)toL.L.WethanktheInstitutoEstadualdeFlorestas(IEF) forpermissiontoworkinthenaturereserveandthedirectorof thereserve,AntônioAlmeidaTonhão,forlogisticsupport.CAPES supportedthestudy.ClemensSchlindweinforhelpfulsuggestions andErikWildforEnglishrevision.
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