Are UNDERC microbial communities from different streams functionally unique?

Transcription

1 AreUNDERCmicrobialcommunitiesfromdifferent streamsfunctionallyunique? NicholasW.Grady UniversityofNotreDame MentoredbyChristopherJ.Patrick UniversityofNotreDameBIOS35502 PracticuminFieldBiology UNDERCEast2010 Director:Dr.GaryE.Belovsky AssistantDirector:Dr.MichaelJ.Cramer

2 ABSTRACT:Untilveryrecently,microbialcommunitiessharingcommon environmentswerethoughttobefunctionallyequivalentregardlessofspecies composition.recentworkhasshownthatmicrobialcommunitiesexhibitvariationin respirationratesinshort(48hour)incubationexperimentsondissolvedorganic Carbon,indicatingthatmicrobialcommunitiesmayvaryinregardstodecomposition. Wetestedthefunctionalequivalenceofmicrobialcommunitiesacrossthreestreams bycomparingthetotalorganicmatterconsumedaswellastherespirationwithin mesocosmsinoculatedwiththreedistinctmicrobialcommunitiesfromthreedifferent UNDERCstreamsacrosstwodifferentexperiments.Bothexperimentstested microbialabilitytodecomposesenescedandgreenspeckledalderleaves,buteach respectiveexperimentfilledthemesocosmswithdifferentgardensofwater (ReddingtonStreamandDIwater).Wefoundthatforbothexperimentstherewasno correlationbetweenleaftypeorinoculumregardingbothorganicmatter decompositionandrespiration,withoneexception.therespirationdataforleaf packsindiwaterindicatesthatrespirationissignificantlyhigherongreenleaves. ThetotalratesofdecompositionwerefasterfortheexperimentwithDIwater.Our resultsseemtosuggestthatthemicrobialcommunitieslivingamongthesethree UNDERCstreamsarenotfunctionallydifferent,butmoretestsshouldbeconductedin ordertosolidifyourfindings. INTRODUCTION:Decompositionisanimportantecosystemfunctionacrossmany environments.thebreakdownoforganicmatterisakeystepinthecyclingof nutrientsthroughoutecosystems(flanaganetal.1983).decompositionalsoprovides

3 foodforplants,thushavinganindirecteffectonprimaryproduction(suberkroppand Klug1976,Brinsonetal.1981).Inaquaticnetworks,decompositiondrivesmany abioticandbioticfactorssuchastheprovidingoffoodformanyinvertebratesandthe loweringofph(brinsonetal.1981).allocthonousinput(leaffall)intoaquatic systemsisthemainprovideroftherawmaterialfordecomposition.important factorsthatcontroldecompositionincludeahostofbioticandabioticfactors. Dochertyetal.(2006)haveshownthatpH,temperature,anddissolvedoxygenareall importantwhenconsideringdecompositionrates.bioticfactorsincludeleaftype, invertebrates,andmicrobialcommunitiesresidingwithinthewater. Microbialcommunitieshavealargeimpactonthedecompositionofleafmatter (SuberkroppandKlug1976,GessnerandChauvet1994,Findlay2010).Many physicalcharacteristicsofaquaticmicrobialhabitatsaffectthecommunities.ph, dissolvedoxygen,andtemperaturecanallaffecttherateatwhichmicrobial communitiesdecomposeplantmaterial.manycharacteristicsoftheplantitselfcan influencemicrobedecompositionrates(harropetal.2009,suberkroppetal.1976). Forinstance,theamountofnutrientsavailableorthelignincontentwithinthe materialbeingdecomposedhasalargeimpact(gessnerandchauvet1994).ithas beenshownthatinmanysystems,plantmaterialhighinnutrientcontenthasbeen brokendownfasterandmorecompletelythanmaterialthatisnutrientpoor. FenchelandFinlay(2004)showedthatmicrobialcommunitiesexhibita cosmopolitandistribution.thisassertionwasappliedtomicrobesthatarelessthan onemillimeterinsize,thusbeingabletoovercomegeographicbarriersthatwould otherwiseinhibitthedistributionoflargerorganisms.thisdistributionhasledto

4 muchworkonmicrobialfunctionbasedoncommunity,withthegeneralassumption thatthemicrobialcommunitiesarethesameeverywhere.thishadbeena controversialsubject,formanystudieshavefoundevidencetothecontrary.for example,stricklandetal.(2009)foundthatcommunitiesthatsharedacommon historywithagivenfoliarlitterexhibitedhigherdecompositionrateswhencompared tocommunitiesforeigntothathabitat.also,ithasbeenshownthattherolesof aerobicaquaticheterotrophicbacteriaincarboncycling,aswellastheimportanceof high molecular weightdissolvedorganicmatterasacarbonsource,maybemore complexthanisconventionallyrecognized(dochertyetal.2006). In2009,astudentinvestigatedwhethermicrobialcommunitiesfromthree differentstreams(withacontrol)brokedowndifferentleavesatdifferentratesina commongardenofwaterfromtenderfootlakethathadbeenfiltered(63um)and sterilizedviaboiling.themicrobestakenfromtenderfootcreekbrokedownthe naturallysenescedleavesatamuchhigherratethananyoftheothermicrobes.i hypothesizethatthemicrobialcommunityfromtenderfootlakewasexhibiting home fieldadvantage becausethebasewaterwastakenfromtenderfootlake.this home fieldadvantage whichhasbeendemonstratedinothermicrobial decompositionexperiments,(reedandmartiny2007,stricklandetal.2009,and Dochertyetal.2006),didnotoccuronthegreenleavesindicatingthattheadvantage maybecontextdependent. Aremicrobialcommunitiesfromdifferentstreamsfunctionallydifferent?Do microbialcommunitiesshowincreasedfunction,relativetoothercommunities,in theirhomestream?thisexperimenttestsmicrobial homefieldadvantage further.

5 IplacedmicrobialcommunitiesfromTenderfoot,Brown,andReddingtonCreeksinto mesocosmsthatwerefilledwithwaterfromreddingtoncreek.myhypothesisisthat duetothe home fieldadvantage concept,themicrobialcommunitiesfrom ReddingtonCreekwillbreakdownthenaturallysenescedleavesfasterandmore completely,buttheeffectwillnotoccurongreenleaves,similartothe2009 experiment.inacommongardenofdiwater(aneutralsource)theexperimentwas runagain.forthisexperiment,ihypothesizethatallbacteriawillhaveequalratesof decompositionandwithequaleffectiveness. MATERIALSANDMETHODS:Thisexperimentexaminedthedifferencesinthe microbialcommunitiesofthreeundercstreams(brown,tenderfoot,and Reddington)withrespecttotheirabilitiestodecomposebothnaturally(brown)and manually(green)senescedspeckledalder(alnusincana)leaves.speckledalderisthe dominantriparianspeciesatundercandthereforegavethebestrepresentationof fieldhabitat.five galloncarboyssterilizedwith10%bleachsolutionwereusedto gatherthewaterforboththecommonwatergardenandforthemicrobialinoculums. Foreachexperiment,atotalofsixcarboyswereused:threecontainingthewater gardenandthreecontainingtheinoculums.forthecommonwatergarden (ReddingtonandDIwater),thewaterwasrunthrougha.2 µmporeinorderto removeallmicrobesandbacteria.thisdifferedfromlastyear,whenthewater gardensweresterilizedviaboiling.tocreatethebacterialinoculums,waterfromthe Reddington,Brown,andTenderfootcarboyswasagitatedtoensurehomogeneity,and thenpassedthrougha125 µmsieve.thisporesizeremovedfineparticulates,but

6 allowedmicroorganismstopassthrough.400mlwasthenextractedandpouredinto eightseparate50 mlfalcontubesthatwereplacedintoacentrifuge(fouratatime) andrunat1000xgravityfor30seconds.thisproducedapelletthatcontained concentratedamountsofmicrobes.fivemlwereextractedusingamicropipette.my techniqueremovedasmuchofthepelletaspossiblewithaslittlestreamwater.this wasappliedtobetterequalizetheamountofstreamnutrientsineachmesocosm. Eachinoculumwasstoredina50 mlbottle. 32two litermesocosmsweresterilizedwith10%bleachsolutionandfilled withapproximatelyoneliterofthecommonwatergarden(reddingtonordi).five leafpackswerethenaddedtoeachmesocosm.theleafpackwasthefunctionalunit forthisprojectandwaspackedwitheitherbrownorgreenleaves.topreparealeaf pack,iobtained.3gramsofdryleafmass(+.01g)andplaceditinameshcitrussack. Theinoculumswerethenadded.Allmesocosmswereaeratedwithairfromthe UNDERCcompressedairsystemviaanairstoneandgivenlightat12 hourintervalsto mimicnaturalconditions.thelidsofthemesocosmswereclosedtohelpprevent contamination.inordertopreventclustering,themesocosmswereplacedevenly apartfromeachotherandinapredeterminedorder(chart1). Aftertheexperimenthadbegun,leafpackswerepulledondays2,6,12,18, and24.onetheseselectdays,leafpackswereremovedbyapairofforcepssterilized with20%bleachsolutionandplacedintoa50 mlfalcontube.thetubewasthen filledwithwaterofaknownvolume,temperature,anddissolvedoxygen.thetime wasnotedwhenaddingthiswater.theleafpackswerethenincubatedfortwohours inarefrigerator;lightwasabsentandthetemperaturewascontrolled.dissolved

7 oxygenandtemperaturewerethenmeasuredagainwithinthetubetomeasure respirationoverthattimeperiod.respirationwasexpressedaschangeindoperunit timeperliterwaterperdryweight(g)leafmater.theleaveswereremovedfromthe meshsack,placedonatinofaknownweight,anddriedinanovenat60 Cfortwo days.aftertwodays,thetinswereweighedagain,yieldingthedrymassoftheleaf matterandthetin.thetinswerethenbakedinamufflefurnaceat550 Cfortwo hoursthenremovedandweighed,givingustheashfreedrymass(afdm).the AFDMiscalculatedbysubtractingtheremainingashfromthedryweightoftheleaf matterandgaveusameasurementoftheamountoforganicleafmatterconsumed. WelogtransformedtheAFDMofremainingleafmatterandcalculatedtheKvalueasFORMULA = wherekistheconstantrateofdecompositionforeach replicate.wethenperformeda2x2anovausingk valueasthedependentvariable andleaftype(2levels,brownandgreen)andmicrobialinoculums(4levels, Reddington,Tenderfoot,Brown,andControl)asthefactors(8treatments,n=4 replicates)foreachexperiment.weperformedarepeatedmeasuresanovaonthe respirationdatathroughtime(8treatments,6timepoints,n=4replicates). RESULTS:TheANOVAofexperimentone(Reddingtonwater)K Valuesyieldedno significantdifferencebetweeneitherleaftreatment(df=2,f=2.425,p=0.132)or streaminoculum(df=3,f=0.193,p=0.900)(figure1).a2x2anovawithk Values asthedependentvariableandandleaftype(2levels,brownandgreen)and microbialinoculums(4levels,reddington,tenderfoot,brown,andcontrol)asthe factors(8treatments,n=4replicates)showednosignificantrelationship(df=3,

8 F=0.334.P=0.801).ArepeatedmeasuresANOVAontherespirationdatafor experimentoneshowedasignificantdifferencebetweenleaftreatments(df=4, F=2.642,P=0.07).Nosignificantdifferencewasfoundbetweeninoculums(df=12, F=0.912,P=0.553)(figuretwo). TheANOVAofexperimenttwo(DIwater)K Valuesagainyieldedno significantdifferencebetweeneitherleaftreatment(df=1,f=0.001,p=0.982)or streaminoculum(df=3,f=0.873,p=0.469)(figure3).a2x2anovawithk Values asthedependentvariableandleaftype(2levels,brownandgreen)andmicrobial inoculums(4levels,reddington,tenderfoot,brown,andcontrol)asthefactors(8 treatments,n=4replicates)showednosignificantrelationship(df=3,f= P=0.690).ArepeatedmeasureANOVAontherespirationdataforexperimenttwo showednosignificantdifferencebetweenleaftreatments(df=3,f=1.533,p=0.234) orbetweeninoculums(df=9,f=0.901,p=0.531)(figure4). DISCUSSION:Myhypotheseswerepartiallybasedupontheresultsofan experimentperformedlastyear,inwhichastudentfoundthattherewasa significantdifferenceinbothdecompositionandrespirationratesbetweenleaves collectedofftheforestfloorandgreenleaves.ithasbeenshownthatnutrition contentisanimportantfactorwhenconsideringleafdecompositionbymicrobes. Leavesthatarehighinnutrientsarebrokendownfasterandmorecompletelythan leavespoorinnutrients(websterandbenfield,1986).thisdidnotoccurinmy experiments.whencomparingtheleavesusedbetweenthe2009experimentand mine,itwasfoundthatthebrownleavesfromthepastyeardifferedfromthebrown

9 leavesfromthisyear.theleavesusedlastyearwerepickedupoffoftheforestfloor inthespringandwerethussubjecttosixmonthsofdecomposition.bythetime theywereusedintheexperiment,theleavesweresurelydevoidofmuchoftheir nutrientcontentandprobablyconsistedofmaterialdifficulttodecompose (cellulose).thiscouldexplainthedifferenceinboththedecompositionand respirationratesforthatexperimentinregardstoleaftype.incontrast,myleaves werecollectedduringleaffallinoctober2009andsubjecttoalmostno decomposition.therefore,itispossiblethatthenutritioncontentsbetweenthetwo leaftreatmentsweretoosimilartohaveshownanysignificantdifferencesinthe decompositionandrespirationdata.ialsoassertthatthewayinwhichmy experimentwasperformedinregardstoleaftypewasmoreecologicallyaccurate forundercstreamsystems.asleavesfallfromtheirtreesandlandinstreams, theyshouldcontainmorenutrientsthanthosethathadbeendecomposingonthe forestfloorallwinter.leaveswiththenutrientcontentfromthe2009experiment shouldmatchsaidleaftype.thoseleaveswouldalsobesubjecttodecomposition frommicrobesnotfoundwithinstreams.therefore,leavestakenjustbeforefalling inautumnshouldbebetterrepresentativesoftheleavesfounddecomposingin streams. Aspreviouslymentioned,thestatisticalanalysisofthedecompositionand respirationdatashowednodifferencesbetweenstreamsorbetweenleaf treatments.assumingthatthemethodsusedforthisexperimentweregood indicatorsofstreamsinthefield,itislogicaltoassumethatthemicrobesin UNDERCstreamsshownoecologicallyimportantfunctionaldifferences.Thus,

10 home fieldadvantage forundercstreammicrobialcommunitiesdoesnotappear toexistinabiologicallyimportantway.thiscouldbebecausethestreamsshare similarriparianenvironmentsandareusedtodecomposingsimilartypesofleaves. Also,thissimilaritycouldbeattributedtothewidedistributionofthesemicrobial communities,asdemonstratedbyfenchelandfinlay(2004). Asstatedabove,Dochertyetal.showedthatpHisacriticalfactorwhen examiningleafdecompositionrates(2006).whenexaminingtheleafmatter decompositiongraphsforexperimentsoneandtwo,whilewenoticethattherewere nosignificantdifferencesbetweenleaftreatmentorstreaminoculumsforeither experiment,wedoseeaninterestingtrend.theratesofdecompositionfor experimentone(reddingtonwater)weremuchslowerthanthoseforexperiment two(diwater);approximatelyhalf(graph3).thephforreddingtonstreamwas 4.3,whiletheDIwatergardenwas7.1.ThisstarkdifferenceinpHcouldpotentially explainthelargedifferenceintotaldecompositionratesforthetwoexperiments. Everyscientificexperimentcanbeimproveduponandthisoneisno different.first,contaminationfromthelaboratoryenvironmentwasnearly impossibletoprevent.changesinmethodstoincludednatestingmayhelpto assurenocontaminationoccurred.also,theissueofmicrobesalreadypresenton theleafmayhaveconfoundedourresults.theleavesthemselveswerenot sterilizedbeforetheexperimentinordertopreservetheintegrityoftheleaf.thus, thecontrolmesocosmswouldbesubjecttothosemicrobesalreadyontheleaf.due tospatialconstraints,aerationandlightingwasinconsistent.duringboth experiments,itwasnotedthatsomemesocosmsreceivedmorelightingandairthan

11 others.thischangebetweenmesocosmswasindeedsmall,butnonethelessmay havecontributedtosomesignificantchangesindata,aslightandchemicalnutrients aremajorfactorsaffectingdecompositionrates(dochertyetal.2006,gessnerand Chauvet1994,Findlay2010). Theresultsobtainedfromthesetwoexperimentshelpgiveusabetter ideaofhowthemicrobialcommunitiesinundercstreamsfunction.forexample, wemightexpectdifferentoutcomes,whenexaminingleafdecompositioninregards todifferentfactors.phcouldpotentiallylimittheamountofdecompositiontaking place(graph3).theamountofnutrientsfoundwithintheleafmattercouldalsobe alargefactorinmicrobialdecomposition.theundercstreammicrobial communitiesasawholeshouldbeconsideredanintricatecomplexoforganisms thatareaffectedbymanydifferentabioticandbioticfactors,mostprominentlyph andnutrientavailability. ACKNOWLEDGMENTS:Firstandforemost,Iwouldliketooffermysincerestthanks tomymentor,chrispatrick,whoseguidanceandexperiencemadethisproject possible.iwouldliketothankthedirectorandassistantdirectoroftheuniversity ofnotredameenvironmentalresearchcenter,dr. sgarybelovskyandmichael Cramer,forimpartingtheirinvaluableknowledgetomeformanyaspectsofmy research.iwouldliketothankourtechnician,heidimahon,forherexcellentadvice onanythingtodowiththelabortheproperty.ithankbothtasfortheunderc 2010class,MaggieManganandCollinMcCabe,forassistingmewithdifferenttasks throughoutmyproject.iwouldliketothankmyfellowclassmatedylanph

12 Fernandezforhelpinggetthisexperimentoffthegroundontime.Finally,Iwould liketoextendmydeepestgratitudetothehankfamilyandtheuniversityofnotre Dameformakingthistremendousresearchopportunitypossibleformyselfandso manyothers. LITERATURECITED: Boulton,A.J.,andP.I.Boon.1991.Areviewofmethodologyusedtomeasureleaf litterdecompositioninloticenvironments:timetoturnoveranoldleaf?australian JournalofMarineandFreshwaterResearch42:1 43. BrinsonMM,AELugoandSBrown.1981.Primaryproductivity,decompositionand consumeractivityinfreshwaterwetlands.theannualreviewofecologicalsystems. 12: Docherty,K.M.,K.C.Young,P.A.Maurice,andS.D.Bridgham.2006.Dissolved organicmatterconcentrationandqualityinfluencesuponstructureoffreshwater microbialcommunities.microbialecology52: Fenchel,T.,andB.J.Finlay.2004.Theubiquityofsmallspecies:Paternsoflocaland globaldiversity.bioscience54: Findlay,Stuart.2010.Streammicrobialecology.29(1): FlanaganP.W.andK.VanCleve.1983.Nutrientcyclinginrelationtodecomposition andorganic matterqualityintaigaecosystems.canadianjournalofforest Research13(5): Gessner,MarkO.andEricChauvet.1994.ImportanceofStreamMicrofungiin ControllingBreakdownRatesofLeafLitter.Ecology:Vol.75,No.6,pp GessnerMO,EChauvet,MDobson.1999.Aperspectiveonleaflitterbreakdownin streams.oikos:85:2 Green,J.,andB.J.M.Bohannan.2007.Biodiversityscalingrelationships:are microorganismsfundamentallydifferent?pages inD.Storch,P.A.Marquet, andj.h.brown,editors.scalingbiodiversity.cambridgeuniversitypress, Cambridge. Harrop,B.L.,J.C.Marks,andM.E.Watwood.2009.Earlybacterialandfungal colonizationofleaflitterinfossilcreek,arizona.journalofthenorthamerican BenthologicalSociety28:

13 Kominoski,J.S.,T.J.Hoellein,J.J.Kelly,andC.M.Pringle.2008.Doesmixinglitterof differentqualitiesalterstreammicrobialdiversityandfunctioningonindividual litterspecies?oikosev:1 7. Langenheder,S.,E.S.Lindstrom,andL.J.Tranvik.2006.Structureandfunctionof bacterialcommunitiesemergingfromdifferentsourcesunderidenticalconditions. Appliedandenvironmentalmicrobiology72: Reed,H.E.,andJ.B.H.Martiny.2007.Testingthefunctionalsignificanceofmicrobial compositioninnaturalcommunities.femsmicrobiologyecology62: Suberkropp,K.,Chauvet,E.(1995)RegulationofLeafBreakdownbyFungiin Streams:InfluencesofWaterChemistry.Ecology,Vol.76,No.5,pp SuberkroppKandMJKlug.1976.Fungiandbacteriaassociatedwithleavesduring processinginawoodlandstream.ecology57: SuberkroppK,GLGodshalk,andMJKlug.1976.Changesinthechemical compositionofleavesduringprocessinginawoodlandstream.ecology57: Webster,J.R.,andE.F.Benfield.1986.Vascularplantbreakdowninfreshwater ecosystems.annualreviewofecologyandsystematics17:

14 0 K ValuesforReddingtonExperiment b C R T K Value Brown Green Figure1:ComparingtheK Valuesfromexperimentone(Reddingtonwater)againstleaftype(brownvgreen)andinoculums(Brown,Control,Reddington,Tenderfoot).No significantdifferencewasfoundbetweenanyofthetreatmentsorleaf types. RespirationinchangeinDO/l/hour/g dryleafmass REDDINGTONRESPIRATION D2 D6 D12 D18 D24 Day Figure2:Respirationdataovertimebetweentreatmentsforexperimentone(Reddington water).nosignificantdifferenceswerefound. BC BT BR Bb GC GT GR Gb

15 K ValuesforDIExperiment b C R T 0.01 K Value Brown Green Figure3 ComparingtheK Valuesfromexperimenttwo(DIwater)againstleaf type(brown vgreen)andinoculums(brown,control,reddington,tenderfoot).nosignificantdifference wasfoundbetweenanyofthetreatments.asignificantdifferencewasfoundforleaftype however. RespirationinchangeinDO/l/hour/g dryleafmass DIRespiration D2 D6 D12 D18 Day Figure4:Respirationdataovertimebetweentreatmentsforexperimenttwo(DIwater). Leaf typewastheonlysignificantdifferencefound. BC BT BR Bb GC GT GR Gb