university of copenhagen Microbial diversity in a permanently cold and alkaline environment in Greenland Glaring, Mikkel Andreas; Vester, Jan Kjølhede; Lylloff, Jeanette Eva; Abu Al-Soud, Waleed; Sørensen, Søren Johannes; Stougaard, Peter Published in: PLoS ONE DOI: 10.1371/journal.pone.0124863 Publication date: 2015 Document version Publisher's PDF, also known as Version of record Document license: CC BY Citation for published version (APA): Glaring, M. A., Vester, J. K., Lylloff, J. E., Abu Al-Soud, W., Sørensen, S. J., & Stougaard, P. (2015). Microbial diversity in a permanently cold and alkaline environment in Greenland. PLoS ONE, 10(4), [e0124863]. https://doi.org/10.1371/journal.pone.0124863 Download date: 10. feb.. 2023 RESEARCHARTICLE Microbial Diversity in a Permanently Cold and Alkaline Environment in Greenland MikkelA.Glaring1,JanK.Vester1,JeanetteE.Lylloff1,WaleedAbuAl-Soud2,Søren J.Sørensen2,PeterStougaard1* 1 DepartmentofPlantandEnvironmentalSciences,UniversityofCopenhagen,Frederiksberg,Denmark, 2 DepartmentofBiology,UniversityofCopenhagen,Copenhagen,Denmark * [email protected] Abstract ThesubmarineikaitecolumnslocatedintheIkkaFjordinSouthernGreenlandrepresenta unique,permanentlycold(lessthan6°C)andalkaline(abovepH10)environmentandare hometoamicrobialcommunityadaptedtotheseextremeconditions.Thebacterialandar- chaealcommunityinhabitingtheikaitecolumnsandsurroundingfjordwascharacterisedby OPENACCESS high-throughputpyrosequencingof16SrRNAgenes.Analysisoftheikaitecommunitystruc- Citation:GlaringMA,VesterJK,LylloffJE,Abu turerevealedthepresenceofadiversebacterialcommunity,bothinthecolumninteriorand Al-SoudW,SørensenSJ,StougaardP(2015) atthesurface,andveryfewarchaea.Acleardifferenceinoveralltaxonomiccompositionwas MicrobialDiversityinaPermanentlyColdand AlkalineEnvironmentinGreenland.PLoSONE10(4): observedbetweencolumninteriorandsurface.Whereasthesurface,andinparticularnewly e0124863.doi:10.1371/journal.pone.0124863 formedikaitematerial,wasprimarilydominatedbyCyanobacteriaandphototrophicProteo- AcademicEditor:PaulJaakJanssen,Belgian bacteria,thecolumninteriorwasdominatedbyProteobacteriaandputativeanaerobicrepre- NuclearResearchCentreSCK(cid:129)CEN,BELGIUM sentativesoftheFirmicutesandBacteroidetes.Theresultssuggestastratificationofthe Received:November6,2014 ikaitecolumnssimilartothatofclassicalsodalakes,withalight-exposedsurfaceinhabited byprimaryproducersandananoxicsubsurface.Thiswasfurthersupportedbyidentification Accepted:March9,2015 ofmajortaxonomicgroupswithcloserelativesinsodalakeenvironments,includingmembers Published:April27,2015 ofthegeneraRhodobaca,Dethiobacter,ThioalkalivibrioandTindallia,aswellasveryabun- Copyright:©2015Glaringetal.Thisisanopen dantgroupsrelatedtouncharacterisedenvironmentalsequencesoriginallyisolatedfrom accessarticledistributedunderthetermsofthe MonoLakeinCalifornia. CreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninany medium,providedtheoriginalauthorandsourceare credited. DataAvailabilityStatement:Thecompletedataset ofquality-filteredsequencesinQIIME-compatible Introduction formatisavailablefromtheMG-RASTserver(http:// Extremophilesareorganismsthatgrowandreproduceoptimallyatorneartheextremeranges metagenomics.anl.gov/;ID4587481.3). ofenvironmentalvariables.Thiscanbeextremesoftemperature,pressure,pH,salinity,aridity Funding:ThisworkwassupportedbytheDanish orradiationandenvironmentsrepresentingoneormoreofthesevariablesarecommonon EnvironmentalProtectionAgency(grantno.MST- earth.Thelargestproportionofsuchextremehabitatsiscomprisedofpermanentlycoldareas, 112-00272)andtheDanishCouncilforStrategic includingpolarregions,permafrost,deepoceans,andalpineregions.Similarly,naturallyoc- Research,projectNOVENIA.Thefundershadno roleinstudydesign,datacollectionandanalysis, curringstablealkalineenvironmentssuchassodalakesanddeserts,andalkalinegroundwater decisiontopublish,orpreparationofthemanuscript. aredistributedglobally,althoughtheseoccurmuchlessfrequently.Alargenumberofbacteria havebeenisolatedfromtheseenvironmentsandtheyoftenshowadaptationstooptimal CompetingInterests:Theauthorshavedeclared thatnocompetinginterestsexist. growthundertheprevailingconditions[1–3]. PLOSONE|DOI:10.1371/journal.pone.0124863 April27,2015 1/22 MicrobialDiversityinaColdandAlkalineEnvironment Sodalakesarethemostthoroughlystudiednaturalalkalineenvironmentintermsofmicro- bialdiversity.Theyaremainlyconfinedtoaridregionsattemperateandtropicallatitudesand arecharacterisedbyhighconcentrationsofsodiumcarbonateandsodiumchloride,whichis reflectedintheadaptationsofmicroorganismsisolatedfromtheseenvironments[4–6].Prima- ryproductionfromcyanobacteriaandanoxygenicphototrophicbacteriaisveryhighandsup- portsadiversealkaliphilicmicrobialcommunitywithrepresentativesofthemajortrophic groupsofarchaeaandbacteriaparticipatingincyclingofcarbon,nitrogen,andsulphurunder aerobicandanaerobicconditions[2,5–7].Terrestrialserpentinisingsitesrepresentanother stablealkalineenvironmentwithlow-salinity.Serpentinisationisthereactionofwaterwithul- tramaficmineralstoformahighlyalkalineenvironmentrichinhydrogenandmethaneand lowininorganiccarbon,makingthegeochemistrydistinctfromthecharacterisedsodalakes. Themicrobialdiversityatafewsuchsiteshavebeendescribed,includingthesubterrestrial CabeçodeVideAquiferinPortugal[8,9]andserpentinitespringwaterfromTheCedarsin northernCalifornia[10],theTablelandsOphioliteinNewfoundland,Canada[11]and Maqarin,Jordan[12],andbacteriaoftheclassClostridiaandhydrogen-utilisingBetaproteo- bacteriarelatedtothegenusHydrogenophagaareabundantinserpentinitewater[9–11,13]. Permanentlycold,stablealkalineenvironmentsareaveryrareoccurrenceandonlyafew suchenvironmentshavebeendescribed:Theupperlayersofthepermanentlyice-coveredLake UnterseeineastAntarctica[14],aseriesofalkalinepondsintheMcMurdoDryValleyRegion intheAntarctica[15],andtheuniquesubmarineikaitetufacolumnslocatedintheIkkaFjord inSouthernGreenland[16].Theikaitecolumnsrepresentapermanentlycold(lessthan6°C) andalkaline(pH>10)environmentwithasalinityoflessthan10‰[16–18].Thecolumns arecomposedofametastablehexahydrateofcalciumcarbonate,calledikaite,ararelow-tem- peraturemineralnamedafterthelocationwhereitwasfirstdescribed.Althoughikaitecanbe foundasmicroscopicandmacroscopiccrystalsinmanycoldmarineenvironments[19–21], theIkkaFjordistheonlyknownlocationwherelargerstructuresofikaiteareformed.Thecol- umnsgrowfromthebottomoftheshallowinnerfjordandareformedwherealkalinesubma- rinespringwaterrichinsodiumcarbonatemixeswiththecoldandcalcium-richseawaterof theIkkaFjord.Ikaiteprecipitationisfavouredbythelowtemperaturesandhighphosphate contentofthespringwater.Columngrowthisprimarilyverticalandtheymayreachheightsof upto20mandseveralmetersindiameter[16,18].Apartfromsodium,phosphate,anddis- solvedinorganiccarbon,thecolumnseepwatercontainslowconcentrationsofinorganiccom- poundscomparedtothesurroundingseawater[18].Thelevelofdissolvedorganiccarbonis unknown,buttherichfaunaandfloracoveringthecolumnscombinedwiththeirvertical growthhasledtospeculationsthattheheterogeneousappearanceofsomecolumncrosssec- tionscouldbecausedbytrappedorganicmatter[22–24].Accordingtopreviousreports,thein- terioroftheikaitecolumnsishometoavarietyofcold-andalkaline-adaptedbacteria[25,26] includingthetodateonlycharacterisedbacteriumdisplayingbothpsychrophilicandalkaliphi- licgrowthproperties,Rhodonellumpsychrophilum[27,28],andthecolumnshavebeenthetar- getofrecentbioprospectingstudiesaimedatidentifyingcold-and/oralkaline-adapted enzymes[29–32]. Thereissignificantbiotechnologicalinterestinmicroorganismsandenzymesfromcold andalkalineenvironmentsandnumerousstudieshavefocussedontheisolationandcharacter- isationofnovelenzymesforlowtemperatureand/orhighpHapplications[33–36].Inaddition tothebiotechnologicalpotential,studiesofthemicrobialcommunityinhabitingtheikaitecol- umnsmayyieldinsightsintothemechanismsandadaptationsthatallowlifetothriveunder theseconditionsandspecifically,howtheydifferfromthetemperateand/orhigh-salinealka- lineenvironments.Inthisstudy,weusedpyrosequencingof16SrRNAgenestocarryoutan extensivecharacterisationofthebacterialandarchaealcommunitiesinhabitingthecoldand PLOSONE|DOI:10.1371/journal.pone.0124863 April27,2015 2/22 MicrobialDiversityinaColdandAlkalineEnvironment alkalineikaitecolumnsandthesurroundingIkkaFjord.Theresultsdemonstratethatthecol- umnsareinhabitedbyadiversebacterialcommunityandidentifyseveralcloserelativesofbac- terialgroupsandcharacterisedalkaliphilesknowntoinhabitsodalakeenvironments.Together withtheunusualenvironmentalconditions,thesefindingshighlighttheikaitecolumnsasa uniqueresourceforbothevolutionarystudiesandfuturebioprospectingprojects. MaterialsandMethods SamplecollectionintheIkkaFjord SamplesofikaitecolumnswerecollectedfromtwolocationsintheIkkaFjord,theAtollField andtheCampField[18],duringthesummerof2006,2007,2010and2011.Permissiontosam- pleintheIkkaFjordwascoveredbyasurveylicensegrantedbytheMinistryofIndustryand MineralResources,GovernmentofGreenland.Columnswereselectedbasedondifferencesin sizeandapparentageandikaitematerialwascollectedatdepthsfrom5–10mbysawingoff thetop20–50cmfromindividualcolumns.Columnmaterialwaskeptat4°Cforupto48h untilsubsamplingcouldbecarriedout.Additionalsamplesweretakenfrompreviouslycollect- edfrozensectionskeptat-20°C.SamplesofikaitematerialforDNAextractionweretaken fromthesurfaceandcross-sectionsofcolumnsbydrilling2–3cmdeepholeswithasterile 5mmdrill.Forsamplestakenfromcross-sections,thefirst5mmwerediscardedtominimise contaminationfromseawaterintroducedduringtheunderwatersampling.Theexpectedhigh pHinthecross-sectionswasconfirmedbypHstripsbeforesampling.Surfacesamplesofnewly formed,softikaiteweretakenwithaspatulabyscrapingofthetop5mmofa1–2cmdiameter area.Allsampleswerehomogenisedaspartofthedrillingprocedureorbystirringwithaspat- ula.Seawatersamplesweretakenat8–10mdepthand1lofwaterwasfilteredthrougha 0.22μmfilter,whichwassubsequentlyfrozenat-20°C.Arecentlong-termunderwaterstudy measuredasummertemperatureof3–5°CandameanpHof8.1atthisdepth[17].Sediment materialwascollectedasasinglesampleofthetop10cmlayerattheAtolandCampField. ThreeseparatesubsamplesweresubsequentlytakenfromeachlocationbeforeDNAextraction. Allsampleswerestoredat-20°CuntilDNAextraction. DNAextraction,PCRamplificationof16SrRNAgenesand pyrosequencing Atotalof70ikaitesamples,3seawatersamples,and6sedimentsampleswereusedforDNA extraction.DNAwasextractedfrom0.5gikaiteorsedimentmaterialusingaceramicbead beatingprocedurewiththeMO-BIOPowersoilDNAExtractionKit(MO-BIOLaboratories, Carlsbad,CA,USA)modifiedwithG1blocker(CarstenSuhrJacobsen,GEUS,Denmark). DNAfromseawatersampleswereextractedusingthesamemethodafterhomogenisationof filtersinliquidnitrogen.A466bpfragmentcoveringtheV3andV4hypervariableregionsof the16SrRNAgenefrombacteriaandarchaeawasPCRamplifiedusingtheprimers341F(5’- CCTAYGGGRBGCASCAG-3’)and806R(5’-GGACTACNNGGGTATCTAAT-3’)[37].The amplificationefficiencyofthisprimerpair,evaluatedusingtheTestPrimefunctioninthe SILVAribosomalRNAdatabase(http://www.arb-silva.de/;databaseversionSSUr121),was 70.7%and68.8%foracompletematchand77.6%and76.3%allowingonemismatch(exclud- ingthethreenucleotidesinthe3’endoftheprimers)fordomainBacteriaandArchaea,respec- tively.ThePCRamplificationandpreparationofampliconlibrarieswereperformedessentially asdescribed[38].Briefly,PCRreactions(50μl)included5ngoftemplateDNA,1UofPhusion HotStartDNApolymerase(Finnzymes,Vantaa,Finland),1xPhusionHFBuffer,200μMof eachdNTPand0.5μMofeachprimerandwereamplifiedwiththefollowingcycleconditions: PLOSONE|DOI:10.1371/journal.pone.0124863 April27,2015 3/22 MicrobialDiversityinaColdandAlkalineEnvironment 98°Cfor30s,followedby30cyclesof98°Cfor5s,56°Cfor20sand72°Cfor20sandafinal extensionof72°Cfor5min.PCRproductswereseparatedona1%agarosegelandpurified usinganE.Z.N.A.GelExtractionKit(OmegaBio-Tek,Norcross,GA,USA)andquantified usingtheQuant-iTdsDNAHSAssayKit(Invitrogen,LifeTechnologiesEurope,Naerum, Denmark).PCRproductsweresuccessfullyobtainedforallseawaterandsedimentsamples andfor48ofthe70ikaitesamples(S1Table).ThePCRproductsfromthethreesediment DNAextractionsfromeachlocationwerepooledinequalamountstogiveonesampleperlo- cationbeforefurtherprocessing.TwotechnicalcontrolswerepreparedbythreeseparatePCR amplificationsofDNAfromsamplesI11andI39. Adaptersandtagsforpyrosequencingwereaddedinasecond10-cyclePCRon5ngofpuri- fiedPCRproductusingtheconditionsdescribedabovewithprimers341Fand806Rcarrying sequencingadaptersandtagsformultiplexing.Theamplifiedfragmentsweregel-purified, quantified,andmixedinequalamountsbeforesequencingonaGenomeSequencerFLXpyro- sequencingsystem(454LifeSciences,Roche,Branford,CT,USA). Sequenceanalysis Trimmingandquality-filteringof16SsequenceswasperformedusingthesoftwaresuiteBio- pieces(www.biopieces.org).Initially,tagsformultiplexingandprimersequencesusedinthe initialPCR(341Fand806R)wereremoved,discardinganysequencesthatdidnotcontain bothamultiplexingtagandtheforwardprimersequence.Sequenceswerethentrimmedfrom bothendsatthefirsthigh-qualitybase(Phredqualityscoreof25)andsequenceswithanaver- agePhredqualityscorelowerthan25werediscarded.Finally,sequencesshorterthan250 basesandcontainingmorethanoneambiguousnucleotidewerediscarded.Thecompletedata- setofquality-filteredsequencesinQIIME-compatibleformatisavailablefromtheMG-RAST server(http://metagenomics.anl.gov/;ID4587481.3). OTUclusteringwasperformedusingUSEARCH[39],whichincluded:1.Dereplicationand subsequenterror-correctionbyoutputtingtheconsensussequencesofaninitialclustering-step at97%identity.2.RemovingchimericsequencesusingUCHIME[40]bycomparisontothe chimera-freeGreengenesdatabase(version13_05;http://greengenes.lbl.gov/)[41]clusteredat 97%identity.3.AfinalOTUclusteringstepat97%identity.Theparametersforchimeradetec- tionweresettoignorelow-divergencechimerasbydisregardingOTUswithatleast97%identi- tytoasequenceinthedatabaseandincreasesensitivitytohigher-divergencechimeras (UCHIMEparameters:-minh0.22-mindiv3-mindiffs5). PhylogeneticanalysiswasperformedusingtheQuantitativeInsightsintoMicrobialEcology (QIIME)pipelineversion1.7(www.qiime.org)[42].TheUSEARCHOTUswereusedasaref- erencesetforUSEARCH-basedOTUpickinginQIIMEusingtheoriginalsequencesasinput. SinceOTUscontainingonlyonesequence(singletons)haveamuchhigherchanceofrepre- sentingPCRandsequencingerrorsorlow-levelcontaminants,andarelesslikelytobeecolog- icallyimportant,thesewereremovedbeforesubsequentanalysistoimprovetheoverallquality ofthedataset.ThemostabundantsequencefromeachOTUwaschosenasarepresentative datasetandalignedwithPyNASTinQIIME.Sequencesaligningwithlessthan70%identity werediscardedaslikelynon-16Scontaminantsandaphylogenetictreewasconstructed. TaxonomywasassignedusingtheRDPclassifierwithaconfidencethresholdof50%[43] andatrainingsetfromtheGreengenesdatabase(version13_05).Rarefactioncurves,theShan- nondiversityindex,andclusteringanalysiswereperformedusingtheQIIMEscriptsalpha_di- versity.pyandbeta_diversity_through_plots.pywithdefaultoptions. PLOSONE|DOI:10.1371/journal.pone.0124863 April27,2015 4/22 MicrobialDiversityinaColdandAlkalineEnvironment ResultsandDiscussion Samplingtheikaitecolumns Sectionsofindividualikaitecolumnswerecollectedduringthesummerfromtwolocationsin theIkkaFjord(Fig1).Columnswereselectedbasedondifferencesinsizeandapparentageas estimatedbyboththecolourandhardnessoftheikaitematerial.Oldercolumnsareaffectedby partialrecrystallisationofikaiteintomonohydrocalciteandcalcite,whichformsahardenedce- ment-likematerial,whereasikaiteissoftandporous[22].Althougholdercolumnsarehard- enedstructures,whichcouldpotentiallytrapandisolatedeadordormantmicroorganisms, mostwerestillvisiblyporoustosomeextentandprecipitationofnewikaitewasobservedat thesurfaceofallcollectedcolumns,suggestingactivetransportofspringwater(datanot shown).Thecross-sectionsofcolumnshadaheterogeneousappearance,rangingfromwhiteto lightgreywithpatchesofdarkgrey,brown,blackandgreeninbothnewandoldcolumns(Fig 1).Thesurfaceoftheoldercolumnswassimilarlyheterogeneousandshowedextensivegrowth ofcorallineredalgaeaswellaspatchesofnewlyprecipitatedikaiterangingfromwhiteto green.Newercolumnsurfaceswerelightgreyorbrowntogreen,suggestingthatnewlyformed ikaitecanbecolonisedbyphototrophicorganisms.Forpracticalreasons,andduetothepro- tectednatureoftheIkkaFjord,onlythetipsofcolumnswerecollected.Sincetheikaitecol- umnsarecontinuouslygrowingstructures,thesamplesusedinthisstudyarethereforelikelyto representikaitethatisyoungerthantheaverageikaitecolumn. Somecolumnsgaveofadistinctiveodourofhydrogensulphide(H S)whenabovewater 2 immediatelyaftercollection.Thiswasalsonotedonapreviousexpedition[26],andisusually associatedwiththeactivityofsulphate-reducingbacteria(SRB)[4].Thesulphideconcentra- tioninikaitecolumnshasnotbeenmeasured,buttheobservationthatthecolumninterioris oftenblackened,couldbeanindicationofprecipitatedmetalsulphides.Similarly,theblack andanoxicsedimentsobservedinsomesodalakeshavebeentakenasanindicatorofthepres- enceofsulphideandanaerobicpopulationsofSRB[4,6,44]. SamplesforDNAextractionweretakenfromcolumnsurfacesandcross-sectionsandclassi- fiedaseither‘old’or‘new’materialbasedontheobservationsdetailedabove.Furthermore, samplesweredefinedasbeingrepresentativeofthesurfacecommunitywhentakeneitherdi- rectlyfromthesurfaceorfromcross-sectionsadjacenttothesurface.Approximately50%ofall samplestakenfromolder,hardenedcolumnsfailedtoyieldanyDNAasjudgedbyspectropho- tometricmeasurementsandPCRamplificationof16SrRNAgenes,suggestingaverylowlevel ofbiomassinthesesamples(datanotshown).Atotalof30interiorand18surfacesamples,as wellasthreeseawaterandtwosedimentsamples,wereobtainedforpyrosequencing.Detailsof individualcolumnsandsamplesusedforpyrosequencingaregiveninS1Table. Pyrosequencingof16SrRNAgenes Partial16SrRNAgenesequenceswereamplifiedusingprimersflankingtheV3andV4hyper- variableregionsofbothbacteriaandarchaea.Twotechnicaltriplicateswherepreparedasse- quencingcontrolsbyseparateamplificationandpyrosequencingofsamplesI11andI39. Pyrosequencingofthe57samplesgenerated654,635high-qualitysequencesaftertrimming andqualityfiltering.Filteringofchimericsequencesandclusteringat97%identityresultedin 452,477sequencesin18,410operationaltaxonomicunits(OTUs).Ofthese,9,407OTUswere representedbyonlyasinglesequence(singletons)andwerediscardedbeforefurtheranalysis leaving9,003OTUsinthedatasetcoveringikaite,seawaterandsedimentsamples(S2Table). PhylogenywasassignedtoeachOTUbycomparisontotheGreengenesdatabase(http:// greengenes.lbl.gov/).Anoverviewofthecombinedfrequencyofallphylogeneticgroupsandof PLOSONE|DOI:10.1371/journal.pone.0124863 April27,2015 5/22 MicrobialDiversityinaColdandAlkalineEnvironment Fig1.MapoftheIkkaFjordandimagesofikaitecolumnsusedinthisstudy.A,mapofSouthern GreenlandshowingthelocationofIkkaFjordandthetwoareas,AtolandCampField,wherematerialwas collectedforthisstudy.B,C,D,surface(left)andcross-section(right)viewofikaitecolumns#1,#9and#5, respectively(seeS1Tablefordetails).Allthreeharvestedcolumnsareapproximately40–60cminheight. doi:10.1371/journal.pone.0124863.g001 PLOSONE|DOI:10.1371/journal.pone.0124863 April27,2015 6/22 MicrobialDiversityinaColdandAlkalineEnvironment eachidentifiedOTUinbothindividualsamplesandincombinationsofrelatedsamplescanbe foundinS3andS4Tables,respectively. Microbialdiversityandrichnessintheikaitecolumns ThegeneratedsequencesandOTUswereusedtoestimatetherichnessanddiversityofthe ikaitecommunity.Itisworthnotingthatsequencedataalonedoesnottakeintoaccountthe proportionofdeadordormantmicroorganismsandmaythusoverestimatethenumberofac- tivebacteriaparticipatingincommunityfunctions.Bacteriahavepreviouslybeencultivateddi- rectlyfrombothnewandoldikaite[26,29],butthecultureddiversityonlycoversasmallpart ofthetotaldiversityandtheproportionofinactivemicroorganismsinoldikaiteisunknown. Regardless,therelativelyyoungmaterialusedinthisstudy(seeabove)wouldsuggestthatthe detectedmicroorganismshavehadarecentfunctioninthecolumncommunity. Rarefactionanalysisofspecies(OTU)richnessinacombineddatasetofallsamplestaken fromtheikaitecolumnsresultedinacurveclosetosaturationatthe8,590OTUsidentified, suggestingthatfurthersequencingeffortisunlikelytosignificantlyincreasethenumberofob- servedOTUsinthesesamples.Rarefactioncurvesofinteriorandsurfacesampleswereap- proachingsaturationatthe7,798and4,252OTUsidentified,respectively,indicatinga significantOTUoverlapbetweenthesetwodatasets(Fig2).Atotalof1,189and1,023OTUs wereidentifiedfromtheseawaterandsedimentsamples,respectively(Table1). ThetotalmicrobialdiversitywasestimatedusingtheShannondiversityindexforvarious combinationsofsamples(Table1).Assuggestedbytherarefactionanalysis,thediversitywas higherintheinteriorsamplesthaninthesurfacesamples,withShannonindicesof9.300and 7.907,respectively.AsimilarincreaseindiversitywasobservedinthealkalineMonoLake,Cal- ifornia,andSoapLake,Washington,whengoingfromthelight-exposed,oxicsurfacewatersto theanoxicbottom[45,46].Inaddition,bothdiversityandOTUrichnesswashigherinold ikaite,bothinternallyandatthesurface,butitispossiblethatthisisaconsequenceofanin- creasedproportionofdeadordormantmicroorganismsinolderikaite,ratherthananindica- torofamorecomplexcommunity.Themicrobialcommunityintheikaitecolumnswasmore diversethanboththeseawaterandsedimentcommunities,challengingthenotionthatappar- entlyhostileenvironmentsshouldbelessdiverse.ArecentanalysisoffiveEthiopiansodalakes Fig2.Rarefactionanalysisofsequencesfromikaitecolumns.OTUsweregeneratedfromthecomplete datasetbysequenceclusteringat97%identity.RarefactioncurvesweregeneratedfromOTUspresentin samplesofikaitecolumninteriororsurface,aswellasinbothsampletypes(combined).Eachdatapointis theaverageof10calculationsbasedonseparatesubsamplingstotheindicatednumberofsequences. doi:10.1371/journal.pone.0124863.g002 PLOSONE|DOI:10.1371/journal.pone.0124863 April27,2015 7/22 MicrobialDiversityinaColdandAlkalineEnvironment Table1. ShannondiversityindexandthenumberofobservedOTUsforcombinationsofsampletype andikaiteage. Sample Seqs. Shannon OTUs Interior 10,354 8.967 2,050 All 7,798 Surface 10,354 7.690 1,449 All 4,252 Seawater 10,354 6.508 962 All 1,189 Sediment 10,354 7.764 1,023 All 1,023 Ikaite,allsamples 134,578 9.243 6,717 All 8,590 Interior 134,578 9.300 6,515 Surface 134,578 7.907 4,252 Interior,oldikaite 62,361 9.070 4,727 Interior,newikaite 62,361 8.683 3,906 Surface,oldikaite 62,361 8.200 3,508 Surface,newikaite 62,361 6.712 1,974 Allcombinationsofsamplesweresubsampledtoanevendepth(sequences)beforeanalysis.Valuesare theaverageof10separatesubsamplings. doi:10.1371/journal.pone.0124863.t001 foundthatOTUrichnesswashighestinthemostalkalineandsalinelake[7]andOTUrichness intheikaitecolumnsappearedtobeevenhigher.Althoughdifferencesinsamplingandtechni- calandanalyticapproachesmakeadirectcomparisondifficult,theresultssuggestthatthe ikaitecolumnsarehometoaremarkablydiversemicrobialcommunity. Surfaceversusinteriorcommunity Differencesinoverallcommunitycomposition(beta-diversity)wereinvestigatedusingaverage linkageclusteranalysis(UPGMA)andtheweightedUniFracdistancemetric[47].Theresult- ingdendrogramshowedaclearseparationbetweeninteriorandsurfacesamples,although someoverlapwasobserved(Fig3).Specifically,oneinteriorsamplefromcolumn#4(sample I38)clusteringwiththesurfacesampleswastakennearthetipofanewcolumn,whichislikely tobeactivelygrowing,andthesamplemightthusbetterrepresentthesurfacecommunity. Phototrophicorganisms,whichwouldnaturallybepresentatthelight-exposedsurface,have previouslybeenobservedtobelivinginsidetheikaitecolumns,concentratednearthesurface [24].Agradientoflight,pH,salt,oxygenandnutrientsbetweenikaitecolumnwaterandthe surroundingseawatermightexistintheouterlayersofanikaitecolumnmakingitdifficultto preciselydefineaboundarybetweenthesurfaceandinteriorcommunity.Asurfacesample fromcolumn#1clusteredwithinteriorsamplesfromthesamecolumn(sampleI24).Thissam- plewastakenfromanareaencrustedbycorallineredalgae,whichislikelytoinhibitboththe penetrationoflightandthemixingofwaters,leadingtonear-surfaceconditionssimilarto thoseinthecolumninterior.Thetightclusteringofthetwotechnicaltriplicates(samplesI11 andI39)indicatesthattheobservedvariationamongtheothersamplesisprimarilybiological (Fig3). Despitethecleardifferencesinoverallcommunitystructure,rarefactionanalysis(see above)suggestedasignificantoverlapinthepresenceofOTUsbetweensurfaceandinterior PLOSONE|DOI:10.1371/journal.pone.0124863 April27,2015 8/22 MicrobialDiversityinaColdandAlkalineEnvironment Fig3.Dendrogramofallsamples.Analysisoftheoverallcommunitystructure(beta-diversity)wascarried outusingaveragelinkageclusteranalysis(UPGMA)withtheweightedUniFracdistancemetric.The separateclusteringofsamplestakenfromikaitecolumncross-sections(interior)andsurfacesareindicated bythegreybars. doi:10.1371/journal.pone.0124863.g003 samples.ThiswasconfirmedbyananalysisoftheOTUdistributionbetweenthesetwosample types,whichidentified3,460sharedOTUsrepresenting81.4%and44.4%ofallOTUsfromthe surfaceandinteriordatasets,respectively.Whilethisdoesnottakeintoaccounttheabun- dancesofindividualOTUs,itisinagreementwiththeobservationthatikaitecolumnscan growupto50cmperyear[16],whichwouldleadtorapidenvelopmentandinternalisationof thesurfacecommunity.Theconstantflowofspringwaterthroughthecolumnscouldalsobe responsibleforcarryingbacteriafromtheinteriortothesurface.Despitetheinterveningsea- water,thisisalsothemostlikelyroutebywhichnewcolumnsarecolonisedbythealkaline- adaptedcommunity.Theprominentcommunityoverlapcouldalsobearesultofthesampling methodandclassificationinrelationtothegeologicaldevelopmentofthecolumnsasdiscussed above.Somecaremustthusbetakenwheninterpretingcommunitydata,especiallywhen PLOSONE|DOI:10.1371/journal.pone.0124863 April27,2015 9/22