Microbial diversity in a Venezuelan orthoquartzite cave is dominated by the Chloroflexi (Class Ktedonobacterales) and Thaumarchaeota Group I.1c


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Microbial diversity in a Venezuelan orthoquartzite cave is dominated by the Chloroflexi (Class Ktedonobacterales) and Thaumarchaeota Group I.1c

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Microbial diversity in a Venezuelan orthoquartzite cave is dominated by the Chloroflexi (Class Ktedonobacterales) and Thaumarchaeota Group I.1c
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Barton A., Hazel
Giarrizzo G., Juan
Suarez, Paula
Robertson E., Charles
Broering J., Mark
Banks D., Eric
Vaishampayan A., Parag
Venkateswaran, Kasthisuri
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Venezuela ( local )
Orthoquartzite ( local )
Cave ( local )
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serial ( sobekcm )

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The majority of caves are formed within limestone rock and hence our understanding of cave microbiology comes from carbonate-buffered systems. In this paper, we describe the microbial diversity of Roraima Sur Cave (RSC), an orthoquartzite (SiO4) cave within Roraima Tepui, Venezuela. The cave contains a high level of microbial activity when compared with other cave systems, as determined by an ATP-based luminescence assay and cell counting. Molecular phylogenetic analysis of microbial diversity within the cave demonstrates the dominance of Actinomycetales and Alphaproteobacteria in endolithic bacterial communities close to the entrance, while communities from deeper in the cave are dominated (82–84%) by a unique clade of Ktedonobacterales within the Chloroflexi. While members of this phylum are comm found in caves, this is the first identification of members of the Class Ktedonobacterales. An assessment of archaeal species demonstrates the dominance of phylotypes from the Thaumarchaeota Group I.1c (100%), which have previously been associated with acidic environments. While the Thaumarchaeota have been seen in numerous cave systems, the dominance of Group I.1c in RSC is unique and a departure from the traditional archaeal community structure. Geochemical analysis of the cave environment suggests that water entering the cave, rather than the nutrient-limited orthoquartzite rock, provides the carbon and energy necessary for microbial community growth and subsistence, while the poor buffering capacity of quartzite or the low pH of the environment may be selecting for this unusual community structure. Together these data suggest that pH, imparted by the geochemistry of the host rock, can play as important a role in niche-differentiation in caves as in other environmental systems.

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ORIGINALRESEARCHARTICLEpublished:26November2014 doi:10.3389/fmicb.2014.00615 MicrobialdiversityinaVenezuelanorthoquartzitecaveis dominatedbythe Chloro”exi (Class Ktedonobacterales ) and Thaumarchaeota GroupI.1cHazelA.Barton1*,JuanG.Giarrizzo2,PaulaSuarez3,CharlesE.Robertson4,MarkJ.Broering2, EricD.Banks2,ParagA.Vaishampayan5andKasthisuriVenkateswaran51DepartmentofBiologyandDepartmentofGeosciences,UniversityofAkron,Akron,OH,USA2DepartmentofBiologicalSciences,NorthernKentuckyUniversity,HighlandHeights,KY,USA3DepartamentodeBiologadeOrganismos,UniversidadSimnBolvar,Caracas,Venezuela4DepartmentofMolecular,CellularandDevelopmentalBiology,UniversityofColorado,Boulder,CO,USA5BiotechnologyandPlanetaryProtectionGroup,JetPropulsionLaboratory,CaliforniaInstituteofTechnology,Pasadena,CA,USA Editedby: DArcyReneeMeyer-Dombard, UniversityofIllinoisatChicago,USA Reviewedby: JohnR.Spear,ColoradoSchoolof Mines,USA JulieL.Meyer,UniversityofFlorida, USA *Correspondence: HazelA.Barton,Departmentof BiologyandDepartmentof Geosciences,UniversityofAkron, 185EastMillStreet,Akron, OH44325,USA e-mail:bartonh@uakron.eduThemajorityofcavesareformedwithinlimestonerockandhenceourunderstanding ofcavemicrobiologycomesfromcarbonate-bufferedsystems.Inthispaper,wedescribe themicrobialdiversityofRoraimaSurCave(RSC),anorthoquartzite(SiO4)cavewithin RoraimaTepui,Venezuela.Thecavecontainsahighlevelofmicrobialactivitywhen comparedwithothercavesystems,asdeterminedbyanATP-basedluminescence assayandcellcounting.Molecularphylogeneticanalysisofmicrobialdiversitywithin thecavedemonstratesthedominanceof Actinomycetales and Alphaproteobacteria in endolithicbacterialcommunitiesclosetotheentrance,whilecommunitiesfromdeeper inthecavearedominated(82%)byauniquecladeof Ktedonobacterales withinthe Chloro”exi.Whilemembersofthisphylumarecommonlyfoundincaves,thisisthe“rst identi“cationofmembersoftheClass Ktedonobacterales.Anassessmentofarchaeal speciesdemonstratesthedominanceofphylotypesfromthe Thaumarchaeota Group I.1c(100%),whichhavepreviouslybeenassociatedwithacidicenvironments.Whilethe Thaumarchaeota havebeenseeninnumerouscavesystems,thedominanceofGroup I.1cinRSCisuniqueandadeparturefromthetraditionalarchaealcommunitystructure. Geochemicalanalysisofthecaveenvironmentsuggeststhatwaterenteringthecave,rather thanthenutrient-limitedorthoquartziterock,providesthecarbonandenergynecessary formicrobialcommunitygrowthandsubsistence,whilethepoorbufferingcapacityof quartziteorthelowpHoftheenvironmentmaybeselectingforthisunusualcommunity structure.TogetherthesedatasuggestthatpH,impartedbythegeochemistryofthehost rock,canplayasimportantaroleinniche-differentiationincavesasinotherenvironmental systems.Keywords:orthoquartzite,cave, Ktedonobacterales, Thaumarchaeota,geomicrobiologyINTRODUCTIONThemajorityofcavesforminsolublerocksuchaslimestone,a sedimentaryrockmainlycomprisedofcalciumcarbonate( Klimchouketal.,2000 ).Classiccaveformation,orspeleogenesis, normallyoccursthroughthechemicaldissolutionofthisrock bywater,whichbecomesacidi“edviacarbonicacidwhenpassing throughCO2-richsoils.Occasionallymicrobiallyderivedacids, suchassulfuricacid,canalsoleadtothedissolutionofcaves (PalmerandPalmer,2000; Klimchouk,2007; Barton,2013).Once formed,cavesprovideauniqueportalintothedeepsubsurface (upto 2,000m)inwhichtostudygeomicrobialinteractions andprocessesunderrelativelystableconditions.Asmostcaves areformedinlimestonethemajorityofmicrobiologycarried outincaveshasbeendescribedinsuchsystems( Sarbuetal., 1996; Angertetal.,1998 ; Grothetal.,2001 ; Northupetal.,2003; CheliusandMoore,2004 ; Spearetal.,2007; Macaladyetal.,2008 ; Banksetal.,2010 ; Bhullaretal.,2012 ; Cuezvaetal.,2012; Barton, 2014).Suchstudiesdemonstratemicrobialspeciesoftenadapted tooligotrophy,withadominanceof Alpha -and Betaproteobacteria, presumablyinvolvedinnitrogen“xation,alongwithsigni“cantpopulationsof Firmicutes and Actinobacteria ,suggestingan importantroleforheterotrophicinteractionsandcarbonturnover (Barton,2014).Nonetheless,whendeep-sequencingtechnologies areusedtoexaminetheseenvironments,theresultssuggestthat thereismuchtolearnaboutthedepthandbreadthofmicrobial physiologyincaves( Tetuetal.,2013 ; Ortizetal.,2014). Theinsoluble,glass-likenatureoforthoquartzite(aquartzcementedsandstone)makesitresistanttotheweatheringprocesses thatroutinelyformcaves;sandstonecavesaretraditionallyshallow,near-surfacefeaturesformedviaaeolianortectonicprocesses (TurkingtonandParadise,2005 );however,tropicalsandstones demonstratekarst-likesolutionfeatures.Therecentexploration withintheTepuiMountainsofVenezuelahasidenti“edalarge numberofcaves,includingsomeofthelongestanddeepest www.frontiersin.orgNovember2014 | Volume5 | Article615 | 1

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Bartonetal. Unusualmicrobialdiversityinanorthoquartzitecave sandstonecavesintheworld( Wray,1997 ; Auler,2004 ; Aubrecht etal.,2008 , 2011 ; Smidaetal.,2008 ; PicciniandMecchia,2009 ). Theexactmechanismofthiscaveformationremainsunclear, althoughitappearsthatthewater-mediateddissolutionofthe quartzcementsplaysanimportantrole,asevidencedbytheformationofsandysedimentsonsurfaceswithinthecave( Piccini andMecchia,2009 ; Aubrechtetal.,2011 ).Asorthoquartziterock remainsrelativelyimpermeabletothemovementofwater,this dissolutionappearstooccurviasur facewaterpenetratingunconsilidatedlayerswithintherockmassif( PicciniandMecchia,2009 ; Aubrechtetal.,2011 ).Assuch,thesecaveswouldbeconsidered karstsystemsinthetraditionalsense,althoughtheirmorphology appearstobeuniquetotheTepuimountainsofVenezuelaand Brazil( Aubrechtetal.,2011 ). RoraimaTepui,a2,700mhighmassifconsistingofquartz (SiO2)cementedhorizontalandgentlydipping”uvialsandstones (quartzarenites),islocatedattheintersectionofVenezuela, GuyanaandBrazil( Figure1 ; Bricenoetal.,1990 ; Santosetal., 2003 ).ThesurfaceoftheTepuidemonstratesextensivemicrobialcolonizationthathasdramaticallychangedthelandscape, coveringexposedsurfaceswithathick(mm…cm)characteristicallyblackendolithiccommunitycomprisedof Cyanobacteria andfungi( Gorbushinaetal.,2001 ).Locatedwithinthismassifis RoraimaSurCave(RSC;akaCuevaOjosdeCristal; Figure1 ),one ofthelongestquartzitecavesyetdescribedatover16kminlength ( Galanetal.,2004 ; Smidaetal.,2008 ). Duetothelimitedweatheringandthepoornutrientavailabilityoforthoquartzite,theTepuimountainsareoftenbare FIGURE1 | (A) GeographiclocationofRoraimaTepui; (B) Microbialcolonies arepresentacrosstheceilingsinlocationswithinthecave;themicrobial coloniesareobviousaswhitemarkingsagainstthepink/redcolorofthe orthoquartzite; (C) MapofRoraimaSurCave,showingtheextentofthe 16kmcavesystem,includingthelocationofthethreesamplingsitesused. MapusedwithpermissionfromtheUniversityofOxfordSpeleological SocietyandtheSociedadVenezolanadeEspeleología. orcoveredonlywithathinsoil( Maguire,1970 ; Allenand Hajek,1989 ; Michelangeli,2000 ).Intheabsenceofsigni“cantsoils,surfaceecosystemsarenitrogenlimited,whichhas ledtoanabundanceofcarnivorousplantsinthelocal”ora ( Maguire,1970 ; Steyermark,1979 ).Duetothepoorbufferingcapacity(whencomparedtocarbonates)andthelimited nutrientavailabilityoforthoquartzite,weanticipatedthatany microbialactivitywithinRSCwouldbeminimal.Nonetheless, duringareconnaissancetripsigni“cantmicrobialactivitywas observedonexposedsurfaceswithinthecave( Figure1B )and appearedtobelinkedtoastream”owingthroughthecave. Examiningthesemicrobialcommunitiesusingmoleculartechniquesdemonstratedthatthecavecontainsanunusualmicrobial ecosystemdominatedbybothmembersofthe Chloro”exi (Class Ktedonobacterales )and Thaumarchaeota ,andisunlikeanypreviouscommunitydescribedincarbonatecaves( Northupetal., 2003 ; CheliusandMoore,2004 ; Spearetal.,2007 ; Tetuetal.,2013 ; Barton,2014 ; Ortizetal.,2014 ).Ourresultssuggestthatnitrogen andthepoorbufferingbyquartzmaydistinguishthemicrobialcommunitiesofsandstonecavesfromcomparativecarbonate systems.MATERIALSANDMETHODSSAMPLESITESANDATPANALYSESRoraimaSurCaveislocatedattheendofasurfacesinkholeon RoraimaTepuithattakesastreamdrainingthroughsurfacevegetationbeforeenteringthecave.Thiswater”owsintothecave at 0.5…2.0m3sŠ 1dependingonrainfall.Threesamplingsites wereusedwithinthecave,whichappearedtorepresentativeofan averagesurface(didnotcontainanyobviousmicrobialgrowth) andwere55,90,and300mfromthecaveentrance( Figure1 ). Thesethreesiteswere:CricketPool(CP),aceilingsite 2.5m aboveastillpoolinwhichforagingcricketshavebeenobserved (P.Sprouse,personalcommunication,2005);RedRiver(RR), aceilingsiteina1.5mhighpaleo-passagethatischaracterizedbythehighabundanceofironminerals;andLagoGrande (LG),locatedonawall 2mawayfromthelargestlakewithin thecave.Duetotheongoingspeleogenesisofthecave,allsampledsurfaceswerecoatedwithunconsolidated,sandysediments ( Aubrechtetal.,2011 ).Approximately10 g ofthesesediments collectedforanalysisateachsamplesitewithinthecaveinJanuary2007.Ceilingandwallsedimentswerecollectedusinga sterilescoop.Controlsampleswerecollectedfromoutsideofthe caveentrancefromareaswithoutobvious Cyanobacterial growth; however,thismaterialhadnotundergonethesameerosionalprocessesasthecavesamplesandremainedinitscemented,rock-like state.SamplesforDNAextractionwerestoredin70%ethanol andkeptat4Cuntilarrivalinthelab,whereuponsampleswere frozenat Š 80C.EachsitewasswabbedforATPbyswabbing an 2cm2areausingaportableluminescentChecklite-HSATP assay(KikkomanInternational,Noda,Japan; Venkateswaranetal., 2003 ).MICROSCOPYANDCELLCOUNTSUnlessotherwisestated,allchemicalswereobtainedfromSigma Chemical(St.Louis,MO,USA).Samplesforcellcountingwere “xedin4%paraformaldehyde/phosphatebufferedsaline(PBS) FrontiersinMicrobiology | ExtremeMicrobiologyNovember2014 | Volume5 | Article615 | 2

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Bartonetal. Unusualmicrobialdiversityinanorthoquartzitecave onsitefor4h,followedbywashingwithPBSandstorage in50%methanol/PBS.Sampleswerekeptat4Cuntilarrival inthelab,whereuponsampleswerefrozenat Š 20C.Forcell enumeration,1cm3ofsedimentwaswashedin1 × PBSand resuspendedin10mLSYBRGreenI/PBSfor15min.Tocount cells,10 µ lofsamplewasthenplaceonamicroscopeslideand examinedunder”uorescenceonaNikonEclipseE600microscope withaB-2E/Cband-passemissionFITC“lterandRemoteFocus Z-stagecontroller(Nikon,Melville,NY,USA).Anoculargridof 100 µ m2at1000 × magni“cationwithaverticalrangeof100 µ m allowedthenumberofcellswithinaknownvolumeofsediment (0.001mm3)tobecountedvisually.Thenumberofcellspercm3ofwallmaterialforanaverageofnineobservationswascalculatedas:averagenumberofvisualizedcells × [1/volumemeasured (0.001mm3)] × (1/dilutionfactor) × 1000].Forscanningelectron microscopy(SEM)analysis,paraformaldehyde-“xedsampleswere washedin70%ethanol/PBS,anddehydratedinanethanol/PBS seriesto100%.Samplesweredriedinacriticalpointdryerusing liquidCO2beforeexaminationundervacuumusingaFEIQuanta 200ESEM(Hillsboro,OR,USA).MOLECULARTECHNIQUESGenomicDNAwasobtainedfrom1.5 g ofcavesedimentby “rstblockingthequartzwith2 µ gofUV-irradiatedpolydI-dC ( Bartonetal.,2006 ),followedbythePowerSoilDNAKit(MO BIO,Carlsbad,CA,USA).Evenwithcrushing,wewereunable toobtainampli“ableDNAfromtherock-likesurfacecontrol samples.Toamplifythe16SribosomalRNAgenesequence,a 40 µ lPCRreactioncontaining10 µ l2X Taq MasterMix(New EnglandBiolabs,Ipswich,MA,USA;10mMTris-HCl,50mM KCl,1.5mMMgCl2,0.2mMdNTPs,5%Glycerol0.08%NP-40 0.05%Tween-20,0.5unitsof Taq DNAPolymerase) 100mM ofeachprimer,and50ngoftemplategDNAwassetupusing thebacterialprimers8F(5…AGAGTTTGATCMTGGCTC AG…3)and1391R(5…GACGGGCGGTGWGTRCA… 3; Spearetal.,2005 ).PCRampli“cationwascarriedoutwith ahot-startat94Cfor8min,followedby30sat94C,45sat 58Cand1minat72Cfor30cycles.Thiswasfollowedbya elongationcycleat72Cfor8min.ForArchaealsequencesthe Archaealprimers4Fa(5…TCCGGTTGATCCTGCCRG-3) and1100Ra(5…TGGGTCTCGCTCGTTG-3; Halesetal., 1996 ; Spearetal.,2005 )wereusedwitha62Cannealingtemperature.PCRproductswerepuri“edwithaZRDNAClean& Concentrator-25Kit(ZymoResearch,Orange,CA,USA)and clonedintoapTOPO-TAvectorandtransformedintocompetent Escherichiacoli accordingtomanufacturersprotocol(Invitrogen, Carlsbad,CA,USA).Cloneswerepickedandscreenedforunique phylotypesaspreviouslydescribed( Bartonetal.,2004 ).Sanger sequencingofthecloneswascarriedoutbyAgencourtBioscience, Beverly,MA,USAandassembledtogetherusingDNABaser software,obtainingminimallya3Xcoverageforeachexamined sequence(andinpracticeatleasta6Xcoverageforthemajorityofclones).Assembledsequenceswerealignedandchimeras removedusingtheGreengenesNASTalgorithm1.Allsequences 1http://greengenes.lbl.govweresubmittedtotheNCBIGenbankdatabaseunderaccessionnumbersGU205277…GU205318(bacterialsequences)and KM214004…KM214181(archaealsequences).CONSTRUCTIONOFPHYLOGENIESPhylogenetictreeswerebuiltusingbackbonesequencesfrom boththeRibosomalDatabaseProject(RDP; Coleetal.,2005 )and SILVA( Quastetal.,2013 )databasesandamendedwithadditional sequencesfromtheGenbankdatabase2asdescribed(seeFigure Legends).AllsequenceswerealignedusingtheARBsoftware packageversion5.1( Ludwigetal.,2004 )with“nescalealignmentgeneratedmanually.Gapswerecollapsedandthesequences weretrimmedinClustalW( Larkinetal.,2007 ).Thephylogenetic relationshipof1276(bacteria)and774(archaea)alignedbases ofsequencedataweredeterminedusingthemaximumlikelihood algorithmfor1000bootstrapreplicatesusingtheRAxMLBlackbox software( Stamatakisetal.,2008 )intheCIPRESgateway( Miller etal.,2010 ).Themodelusedandrelevantoutgroupsareshownin each“gure.FigTreeversion1.4.13wasusedtopreparethephylogenetictrees,whichwerepreparedforpublicationinwithAdobe IllustratorCS5.PHYSICALPARAMETERSANDGEOCHEMISTRYAsaroughestimateofavailableammonia,nitrateandnitriteand pHateachsamplesite,10cm3ofrockwasaddedto10mL deionizedwater(0mg/Lammonia,0mg/Lnitrite,0mg/Lnitrite, andpH6.9)andshakenbrie”y.Theparticulateswereallowed tosettleandthesupernatantwastestedfornitrogenouscompoundswitha“eld-availableassay(MardelLaboratories,Inc) withadetectionlimitof0.50mg/Lfornitrate,0.25mg/Lfor nitrite,and0.25mg/Lforammonia.Totaldissolvedsilicawas determinedusingaHachPortableColorimeterIIusingthesilicomolybdatemethod( Knudsonetal.,1940 ;Hach,Loveland,CO, USA),whilepHwasmeasuredusinganAccumetAP61portable pHmeter(FisherScienti“c,Pittsburg,PA,USA).Relativehumidity(RH)andtemperatureweremeasuredinthecaveusinga RH300Psychrometer(Extechinstruments,Waltham,MA,USA). Forelementalanalysis,sampleswerecrushedandexaminedvia X-ray”uorescenceinaBrukerGmbHS4Pioneer-4kWwavelengthdispersiveX-rayspectrometer(Billerica,MA,USA).The Mossbauerspectrumwasmeasuredusingaconventionalconstantacceleration-drivingunitfromHalder,GmbH(BadWaldsee, Germany),connectedtoa386personalcomputerbyaCanberra NuclearPHA/MCSinterfacecard(Meriden,CT,USA).Thespectrumwascollectedinmirror-imagemodeover1024channelsand foldedabouta0-pointofvelocit yde“nedbythespectrumofa thinfoilofmetallicironcollectedsimultaneouslywiththatof theunknownsample.Analysisofthespectrumwascarriedout usingaleast-squares“ttingroutinebasedonalorentzianpeak shapefortheabsorptionfeatures.RESULTSDespitethenutrient-limitednatureofRoraimaTepui,throughoutRSCtherewasevidenceofsigni“cantmicrobialactivityon 2http://www.ncbi.nlm.nih.gov/3http://tree.bio.ed.ac.uk/software/“gtree/ www.frontiersin.orgNovember2014 | Volume5 | Article615 | 3

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Bartonetal. Unusualmicrobialdiversityinanorthoquartzitecave therocksurfaces,primarilythroughthepresenceofobservable microbialcolonies( Figure1B ).Usingaluminescence-basedassay forATPtoserveasaproxyforthepresenceofmicroorganisms ( Venkateswaranetal.,2003 ),wemeasuredrelativeluminescent units(RLU)valuesashighas34,062fromsurfaceswithinthecave. Thesevaluessuggestahighamountofmicrobialactivitywhen comparedtoother(carbonate)cavesystems(suchcavesgenerally rangefrom80to1,400RLU;JohnstonandBarton,unpublished results).Toexplorethismicrobialactivity,weexaminedthreesites withinthecave:CP,RR,andLG,whichwereprogressivelyfurther fromtheentrance(CP 55m,RR 90m,andLG 300m,respectively; Figure1C ).Observablemicrobialcoloniesweregenerally associatedwithturbulentwaterinthecaveandbecamepatchier deeperintothecavesystem.Wethereforedecidedtocollectsampleswherespeci“cmicrobialgrowthwasnotobserved,whichwas morerepresentativeofthemajorityofsurfacesedimentswithin thecave.UsingtheATPassay,weobtainedarangeof4,025…15,352 RLUfromthesesurfacesites( Table1 ),whichprovidesanapproximationofsur“cialcellnumbers(assumingthatcellATPlevels average 1 × 10Š 18M)rangingfrom2.07to7.65 × 107cells/cm2( Table1 ; LaDucetal.,2007 ). TocorrelatetheobservedATPvalueswithtotalnumber ofmicrobialcellsateachsite,weattemptedbothdirectcell countingand”uorescent insitu hybridization(FISH).While cellcountingusingtheDNAstainSYBRGreenIwaspossible, theauto”uorescenceandDNAbindingpropertiesofthequartz grainsmeantthatFISHcouldnotbeeffectivelyusedtodistinguishcommunitycomposition.Nonetheless,cellcountsat LG( 0.52 × 108cells/cm3)andRR( 1.92 × 108cells/cm3) correlatedwellwiththesur“cialATPvalues(thecollectedCP samplewasdestroyedduringtransportandnocellnumbers wereobtained)andcon“rmedourobservationthatthecave containedahighmicrobialcellnumber( Venkateswaranetal., 2003 ; Bartonetal.,2005 , 2007 ).Whilewedidattemptcultivationstudiestodeterminethenumberofcolonyforming unitsateachsite,theculturedisolatessharednosimilaritywith thedominantphylotypesidenti“edbynon-cultivation(DNA) techniques,suggestingthatcolonycountingwasinnowayrepresentativeofthenumberofspeciesgrowing insitu (datanot shown).MICROBIALCOMMUNITYSTRUCTUREExtractionofDNAfromorthoquartziteisextremelydif“cultdue totheglass-likenatureoftherock,whichadsorbsDNAtoits surface( Maoetal.,1994 ).Nonetheless,byblockingthequartz withasyntheticnucleotidepolymerpriortoextraction,wewere abletoobtainsuf“cientDNAtogenerate16SribosomalRNA genesequencelibrariesforbacteriaandarchaea.Thebacterial clonelibrariescontained184,52,and43clonesforCP,RR,and LG;thisdiminishingnumberofclonesateachsitewasduetothe increasingdif“cultyinextractingDNAassamplesitesprogressed furtherintothecave.Atotalof87and91archaealphylotypeswere obtainedfortheCPandRRsites,respectively,whiletheLGsample wascompletelyconsumedinrepeatedattemptstoobtainsuf“cient DNAforPCRampli“cation.Theinabilitytoobtainarchaealclones fromtheLGsiteisthustheresultofDNAextractionproblems, ratherthantheabsenceoftheseorganisms. Inordertoobtainageneralideaofcommunitystructure ateachsitewecarriedoutaBLASTanalysis( Figure2 ).This analysisrevealedthatwithinthebacterialpopulationtherewere distinctcommunitydifferencesbetweenthenear-entrance(CP) anddeeper(RRandLG)samplesites.TheCPbacterialpopulation wasdominatedbythe Actinomycetales and Alphaproteobacteria , alongwithasigni“cantpopulationof Firmicutes and Acidobacteria ( Figure2 ).TheactinobacterialpopulationattheCPsitewasitself dominatedbymembersofthe Pseudonocardia ,whilethe Alphaproteobacteria wererepresentedbyanumberofthenitrogen-“xing Beijerinckiaceae and Methylocella ( Dedyshetal.,2004 ).ThecomparativeBLASToftheremainingsequencesrevealedthatmany shareidentitywithphylotypesalsoseeninmineralweathering horizonsorgeologicenvironments(caves,lavadeposits,mines, andiron-manganesenodules).Asinglecyanobacterialclonewas detectedintheCPclonelibrary;however,no Cyanobacteria were detectedatanyothersitewithinthecave. ThebacterialpopulationsatRRandLGsiteslookremarkably similar,withthedominanceofaphylotypeshowingalowlevel ofsequenceidentitytothe Chloro”exi (86%; Figure2 ).Despite thedominanceofthe Chloro”exi ,thesesitesdidcontainaminoritypopulationofboththe Actinobacteria (4%atRRand2%at LG)and Alphaproteobacteria (2%atRRand5%atLG).TheRR libraryalsocontainedmembersofthe Gemmatimonadetes and Planctomycetes ,whileLGcontainedanumberof Acidobacteria ( Figure2 );representativesofthesephylasharedahigherdegreeof identitytoorganismsexaminedinotherenvironmentsthanrepresentativesofthe Chloro”exi ( Figure2 ).Giventheuniqueness ofthe Chloro”exi ,wewantedtodeterminewhethertheyshared anyhomologywithother Chloro”exi previouslyidenti“edincaves ( Barton,2014 ).Theresultantphylogeny( Figure3 )demonstrates Table1 | Physiochemicalparametersatthedifferentsamplingsites. SiteATP(RLU)ATPcalculatedcell number(/cm2) Cellcounts(/cm3)TemperatureCHumidityStreampHDissolvedSi (mg/L) Surface4520.45 × 107………6.8755.0 CP4,0252.01 × 107Nd11.898.2%5.5614.0 RR15,3527.65 × 1071.92 × 108(SD ± 10.4%)11.699.6%5.0976.0 LG8,9334.45 × 1070.52 × 108(SD ± 12.1%)12.599.9%4.96815.0 nd,notdone;SD,standarddeviation. FrontiersinMicrobiology | ExtremeMicrobiologyNovember2014 | Volume5 | Article615 | 4

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Bartonetal. Unusualmicrobialdiversityinanorthoquartzitecave FIGURE2 | SequenceanalysisofidentiÞedphylotypesat eachsamplesitewithinRoraimaSurCave. Thebacterialand archaealpopulationswereanalyzedseparatelyanddemonstrate theoveralldistributionofphylaidenti“edateachsite(bar charts).ThedistributionsofphylotypeswithBLASTanalysis identityscores(%)tosequencesintheGenbankdatabase areshown,alongwiththemajorcommunitycompositionor environmentalsource. thattheRSC Chloro”exi groupfallswithintheClass Ktedonobacterales ,whilethe Chloro”exi thathavebeenidenti“edinpastcave studiesassociatewiththeClasses Dehalococcoidetes and Anaerolineae. Itisinterestingtonotethatthereisadistinctlineagewithin the Chloro”exi fromRSC,withphylotypesfromdeeperinthecave (RRandLG)formingauniquecladewithclonesfromafumarolecaveonMountErebus,Antarctica( Figure3 ),while(apart fromasinglephylotypeidenti“edattheLG;RSC_LGG05)those foundneartheentranceatCPshareanevolutionaryhistorywith soil-associatedclades( Figure3 ). TheBLASTanalysisofthearchaeaatCPandRRindicated thatallthephylotypesbelongedtothe Crenarchaeota intwo distinctpopulations:themajorityofphylotypesfromtheCP sitesharedsequenceidentitywithanoligotrophicpeatclone (CASN36; Akiyamaetal.,2011 ),whiletheRRsitewasdominated bytwophylotypes,onewithsimilaritytothesamepeatstudy (CASN28)andtheotherfromanacidicdesertsoil(Arc_DS16; Yingetal.,2010 ; Akiyamaetal.,2011 ; Figure2 ).Aswiththe Chloro”exi ,wecarriedoutaphylogeneticanalysistorelatetheRSC archaealclonestoother Crenarchaeota populationspreviously identi“edincaves.Giventheambientconditionswithinthecave ( Table1 ),itwasunsurprisingthatalloftheidenti“edphylotypes clusteredwiththemesophilic Crenarchaeota ammonia-oxidizing Class Thaumarchaeota ;however,theRSCphylotypesformeda distinctclusterwithinthe Thaumarchaeota GroupI.1c( Figure4 ). WhenwesearchedtheRDP,SILVA,andGenbankdatabasesfor additionalsequenceswithsharedhomology,theRSCarchaeal clonesclusteredwithinasubgroupdesignatedNRP-JbyDeSantis andcolleagues( McDonaldetal.,2012 ),whichhasvariouslybeen classi“edastheMBG-Aaf“liated,FSC,andFFSBGroup( Jurgens etal.,1997 ; Vetrianietal.,1999 ; Takaietal.,2001 ).Duetothis uncertaintyinthephylogenyofthe Thaumarchaeota ,weusedthe morerobustframeworkof DurbinandTeske(2012) todeterminethephylogeneticplacementofourclones.Theresultant phylogeny( Figure5 )demonstratedthattheRSCarchaealphylotypesclusteredwithintheFSC/NRP-Jgroupinfourdistinct clades,threeofwhichcorrelatedwellwiththepeaksobservedin ourinitialBLASTanalyses( Figure2 ).Allofthesequencesusedto determinethephylogenyoftheFSC/NRP-JGrouphavebeenidenti“edinacidicenvironments,includingacidic(Arc_DS16)and humicsoils(FRA27),oligotrophicpeat(CASN28andCASN36) andmines(HSM050P-A-8; Jurgensetal.,1997 ; Olineetal.,2006 ; Yingetal.,2010 ; Akiyamaetal.,2011 ),suggestingthatpHplays amajorroleinthearchaealcommunitystructurewithinthe cave.GEOCHEMICALANALYSISGiventhedramaticdifferenceindiversitybetweenCPandthe deepersiteswithinthecaveandthepotentialroleofpH(RRand LG),weexaminedwhetherthegeochemistryinRSCplayedarole indrivingcommunitystructure.Thephysiochemicalconditions inthecavewererelativestable,withatemperatureof11.8Cat CPincreasingto12.5CatLG,astheRHreachednear-saturation at99.9%( Table1 ).Giventheinsolubilityoftheorthoquartzite, thestreamprovidedtheonlyentryofallochthonousnutrients intothecave,whilepresumablybeingresponsiblefortheobserved www.frontiersin.orgNovember2014 | Volume5 | Article615 | 5

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Bartonetal. Unusualmicrobialdiversityinanorthoquartzitecave FIGURE3 | Phylogeneticanalysisof16SrRNAgenesequencesforthe majorClasses(Sub-phyla)withinthe Chloro”exi, alongwith representativephylotypesidentiÞedinothercaveenvironments(blue) andthisstudy(brown). Cultured Chloro”exi isolatesareshowninbold.The treetopologyisbasedonamaximumlikelihoodanalysisusingRAxMLand theevolutionarymodelCTR+G.Thelowestscoringtreeisshown,with branchsupport(percentage)of1,000bootstrapreplicatesshown.Thescale barrepresentstheestimatednumberofreplacementsateachsite.The bacterial16Ssequencesfor Aquifexpyrophilus (M83548)and Hydrogenobacterthermophilus (Z30214)wereusedasanoutgroup. FrontiersinMicrobiology | ExtremeMicrobiologyNovember2014 | Volume5 | Article615 | 6

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Bartonetal. Unusualmicrobialdiversityinanorthoquartzitecave FIGURE4 | Phylogeneticanalysisofthe Thaumarchaeota GroupI (asdeÞnedby DeLong,1992 )16SrRNAgenesequences,including representativephylotypesidentiÞedfromother(carbonate)cave environments(blue)andthisstudy(red). Cultured Thaumarchaeota isolatesareshowninbold.Thecladeindicatedwiththe(*) representstherelativelocationofthe Crenarchaeota identi“edfrom LechuguillaCave( Northupetal.,2003 ).Thetreetopologyisbasedon amaximumlikelihoodanalysisusingRAxMLandtheevolutionary modelCTR+G.Thelowestscoringtreeisshown,withbranch support(percentage)of1,000bootstrapreplicatesshown.Thescale barrepresentstheestimatednumberofreplacementsateachsite. Thearchaeal16Ssequencesfor Methanobacteriumaarhusense H2-LR (AY386124)and Ferroplasmaacidiphilum MT1(AF513710)wereused asanoutgroup. humidity.ThemeasuredpHofthestreamdidvary,droppingfrom 5.561atCPto4.968atLG( Table1 ).Grossexaminationofgeologic hand-samplesdemonstratedthattheorthoquartziteateachlocationhadlostitsrock-likestructureandwasturningintoasandy sediment,presumablythroughthedissolutionofthesilicacement ( Martini,2003 ).SEManalysisofthesedimentscon“rmedthis analysis,andrevealedthepresenceoftriangularetchpitswithinthe quartzgrainssuggestingthatchemicalweatheringofthemineral surfacewasoccurring( Figure6A ; TurkingtonandParadise,2005 ). Thedegradationofthehostrockintosandysedimentcorrelated www.frontiersin.orgNovember2014 | Volume5 | Article615 | 7

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Bartonetal. Unusualmicrobialdiversityinanorthoquartzitecave FIGURE5 | Phylogeneticanalysisofthe Thaumarchaeota GroupI.1c FSC/NRP-JGroup(asdeÞnedby DurbinandTeske,2012 ;inset)16SrRNA genesequences. Thesequencesfromthisstudyareshown(orange)aswell asBLASTidenti“edsequences(blue),theFSSB11sequenceofthe FSC/NRP-Jgroup(red),andsequencesintheFSC/NRP-Jgroupasidenti“ed fromtheRDP,SILVAandGenbankdatabases.Thetreetopologyisbasedona maximumlikelihoodanalysisusingRAxMLandtheevolutionarymodel CTR+G.Thelowestscoringtreeisshown,withbranchsupport(percentage) of1,000bootstraprepresentedbycircles(asshown).Thescalebar representstheestimatednumberofreplacementsateachsite,while backgroundcoloringwasusedtohighlighteachoftheputativeclades.The Thaumarchaeota GroupI.1a16Ssequencesfor Nitrosopumilusmaritimus SCM1(DQ085097)andtheunculturedcloneSAGMA-8(AB050238)were usedasanoutgroup. withanincreaseindissolvedsilicainthestream( Table1 ),which couldbecausedbytheproductionofaweaksilicicacid(H4SiO4). TotalelementalandX-raypowderdiffraction(XRD)analyses ofthesedimentscon“rmsthatSiO2representsthepredominant chemistryoftheorthoquartziteinitsmineralpolymorph -quartz ( Table1 ; Figure6B ).Otherpredominantelementswithinthe rockincludealuminum(1.96…3.00%),whichisenrichedinthe cavewhencomparedtotheTepuisurface,andiron(0.08… 0.12%),whichislikelyresponsibleforthepinkcolorofthe rock( Figure1 ; Table2 ).Weidenti“edtracephosphorous (0.02…0.05%),whichhasthepotentialtoserveasanutrient; potassium,strontium,calcium,barium,sodiumandmagnesiumwerebelowthe0.01%sensitivityoftheinstrument.In ordertoexaminewhethertheobservedironcouldcontributeto microbialmetabolism,eitherthroughautotrophicormixotrophic growth,weusedMossbauerspectroscopy.Theobtainedspectrumdemonstratesaverylowabsorptioneffectandpoorsignal/noiseratio( Figure6B )andcon“rmsboththelowlevel ofavailableironanditspresenceashematite[Fe(III)].Given thedominanceofammonia-oxidizingspecieswithinRSC,we alsotestedforthepresenceofnitrogenouscompoundsusinga “eld-availableassay.Whilewefoundnoreactivenitrogenouscompounds(NH+ 4,NOŠ 2,NOŠ 3)fromrocksonthesurfaceofthe Tepui,outsideofthecave( Table2 ),bothnitrateandammoniaweredetectedattracelevelsatallsamplesiteswithinthe cave.Togetherthesedatasuggestthatwhiletherearesomedifferencesbetweenthegeochemistryofthesurfaceandthecave sites,therearenosigni“cantgeochemistrydifferencestoaccount fortheobservedchangesinmicrobialcommunitystructurewithin thecave. FrontiersinMicrobiology | ExtremeMicrobiologyNovember2014 | Volume5 | Article615 | 8

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Bartonetal. Unusualmicrobialdiversityinanorthoquartzitecave FIGURE6 | GeochemicalanalysesofRoraimaSurCavesamples. (A) SEManalysisofthequartzgrainsrevealstheevidenceofmicrobial activityandtheetch-pits(indicatedbyarrows)characteristicofchemical dissolution. (B) XRDdiffractionpatternofhostrockmaterialfromoutsideof thecave(surface)andthesamplesites,CricketPool(CP),RedRiver(RR), andLargoGrande(LG).Qindicatesthepresenceofarecognized-quartz peak;( inset )MössbauerspectraoftheLGsite,withreferencepeaksfor hematite. DISCUSSIONTheTepuiMountainsofVenezuelaarearemarkableenvironment thatcontainssomeofthelongestanddeepestquartzitecavesin theworld( Martini,2003 ; Auler,2004 ; Galanetal.,2004 ; Aubrecht etal.,2008 , 2011 ).Theyarealsoamongthemostremoteand inaccessibleenvironmentsforresearch.Nonetheless,giventhehistoryofunusualfaunaand”orafoundonthesemountains( Im Thurn,1887 ; Maguire,1970 ; Steyermark,1979 ),itwasunsurprisingthatauniquemicrobialcommunitywasfoundwithinits caves;thisuniquenesswasevidentwhenwe“rstprocessedthe datafromthiscavein2008( Figure7 ).Atthetimethe Thaumarchaeota hadyettobedescribed,thecultivationof Nitrosopumilus maritimus stillappearedtobenovel,ourknowledgeofthecontributionbyammonia-oxidizingarchaea(AOA)intheglobal nitrogencyclewasstillinitsinfancy,andthe Ktedonobacterales had onlyrecentlybeendescribedfromthetypestrain Ktedonobacter racemifer ( Venteretal.,2004 ; Könnekeetal.,2005 ; Cavalettietal., 2006 ; Brochier-Armanetetal.,2008 ).Next-generationsequencingtechnologiesremainedlimitedandexpensive,andunableto amplifylow-biomasssamples( < 50ng)withoutsigni“cant(and Table2 | Geochemistryofsamplesites. SamplesiteChemicalParameter SiAlPFeTiNH+ 4NOŠ 2NOŠ 3 %mg/L Surfacerock99.400.450.020.070.07 bdlbdlbdl CP97.292.760.050.080.04 < 0.25 bdl < 0.5 RR95.993.000.040.120.07 < 0.25 bdl < 0.5 LG98.941.960.020.100.02 < 0.25 bdl < 0.5 bdl,belowdetectionlimit. FIGURE7 | Initial(2008)andsubsequentre-analysis(2014)ofRoraima SurCavephylotypesforthisvolume. Thedistributionsofphylotypes withBLASTanalysisidentityscores(%)tosequencesintheGenbank databaseareshown,alongwiththemajorcommunitycompositionor environmentalsource. biased)ampli“cation.Wewerethereforeconfrontedwitha16S rRNAclonelibrarythatcomprisedofsequenceswithverylittleidentitytocharacterizedspecieswithintheGenbankdatabase ( Figure7 ).EventhebacterialphylotypeswithintheCPsite,which demonstratedthebestidentitytoknownsequences,leftuswith whatappearedtobeasoil-likecommunity( Figure7 ).Thusthe www.frontiersin.orgNovember2014 | Volume5 | Article615 | 9

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Bartonetal. Unusualmicrobialdiversityinanorthoquartzitecave originalanalysisoftheRSCmicrobialpopulationsprovidedvery littlefunctionalinformation. Sincethattime,morethan6.1 × 107sequences(representingatotalof3.3 × 109bases)havebeenaddedtotheGenbank database(Genbankreleasenotes4),including16SrRNAsequences fromnewlyexploredgeochemicalenvironments( Connelland Staudigel,2013 ).Collectivelythisknowledgeincreaseproduceda noticeableshiftintheidentityofoursequencestothosewithin theGenbankdatabase( Figure7 )andhasimprovedourabilitytodetermineanenvironmentalphysiologyofthemicrobial communityinRSC.Forexample,attheCPsite,inaddition tosoil-associatedspecies,ourclonesnowdemonstratesequence identitytophylotypesfromcaves,mineralweatheringsurfaces, andendolithicenvironments( Figure7 ),includingheterotrophic, nitrogen-“xingspeciesidenti“edonthesurfacesofcarbonate cavesimpactedbytheintroductionoforganiccarbon( Stomeo etal.,2008 ).Thepresenceof Cyanobacteria atCPmaybeindicativeofthecloseproximityofthissamplesitetotheentranceand colonizationbysurfacespecies( Büdel,1999 );indeedthesiteis closeenoughtothecaveentrancethat,bypeeringaroundacorner,daylightcanbeseen( Figure1 ).Alternatively,thepresence ofsomanyheterotrophic,nitrogen-“xingspeciesatCPsuggests thatthemicroorganismsdependonsurface-derivedcarbonfor growth,whiletheabilityto“xnitrogenmayplayacriticalrole inmicrobialsubsistence.Takentogether,thesenewdatasuggestthattheCPcommunityexistswithinatransitionalzone, wherethemicrobialcommunityisstillin”uencedbysurfacecolonizationorallochthonouscarboninput,butdemonstratessome endolithic-likeadaptationtolifeonthesilicatemineralsurface. Asthesamplesitesextendedfurtherintothecave,therewasa dramaticshiftinbacterialcommunitystructure( Figure2 ).The impermeable,orthoquartziterockpreventsallochthonousin“ltration,andthusthesesamplesites(RRandLG)wouldhave todependonorganicinputdirectlyfromthecavestreamor autotrophicactivities.Insupportofthestreamhypothesis,the absenceofsurfacesoilsontheTepuismeansthatrainwaterrapidly accumulatesdissolvedorganicmatter(DOM)fromplantdetritus andhumicmaterialbefore”owingintothecave;theamountof humicmaterialinthewater”owingintothecaveandoffofthe Tepuiscanbesohighthatitgivestheriversinthisregiontheir famoustannicblackŽcolor( PicciniandMecchia,2009 ).While equipmentfailuremeantwewereunabletomeasureDOMatthe timeofsampling,thewateronTepuimountainshasanaverage dissolvedDOMcontentof 19mg/L( Gorbushinaetal.,2001 ), whichismuchhigherthantheaveragemeasuredDOMlevelsin carbonatecaves( < 0.5mg/L; Barton,2014 ).Thiscomparatively highDOM”owingintothecavecouldcertainlyserveasthesource ofenergythatdrivesthehighlevelsofmicrobialactivityobserved andindeed,themostvisiblemicrobialactivityseenwasassociated withturbulentwater”ow( Figure1 );however,ifthiswerethe case,itisunclearwhythecommunitydeeperintothecavecontainssuchadominantpopulation Ktedonobacterales ,ratherthan thephylaseenatthenear-entranceCPsitethataremorecommonlyassociatedwiththebreakdownofplantdetritus( Figures2 and 3 ; Hugetal.,2013 ). 4ftp.ncbi.nih.gov/genbank.gbrel.txtThe Chloro”exi representaremarkablydiversegroup,witha phylogeneticrangeasbroadasthatofthe Proteobacteria ( Ley etal.,2006 );yetoutofthe20,70216SrRNAsequencesinthe RDPdatabase,thereareonly187culturedrepresentatives(this compareswith96,507culturedisolatesforthe Proteobacteria ). Thismakesitverydif“culttoestimatethemetabolicfunction forunculturedspecieswithintheenvironment,particularlyin thisstudy,whentheclosestculturedrepresentative( Thermogemmatisporaonikobensis )onlyhas84%sequenceidentityatthe 16SrRNAgenelevel( Coleetal.,2005 ).Nonetheless,the Chloro”exi ,andinparticularthe Ktedonobacterales ,havearecognized roleasheterotrophicoligotrophsinsoils,includingtheabilitytosurviveonmorerecalcitrantplantpolymers( Yabeetal., 2010 , 2011 ; Hugetal.,2013 ; KingandKing,2014 ).Theirpresenceinoligotrophicenvironments,includingcaves,con“rms thisadaptationtogrowthundernutrientlimitation( Engeletal., 2010 ; Hugetal.,2013 ; Barton,2014 ).Thedifferencein Ktedonobacterales phylotypesbetweenthenear-entranceCPand deepercavesites,alongwiththeirdominanceatRRandLG, suggestthattherearespeci“cselectivepressuresdeeperwithin thecavefortheseorganisms.Theuniquecladeformedbythe RSCphylotypesandthosefromamesophilicfumarolecaveon MountErebus,Antarctica( Figure3 )hasthepotentialtotell usmoreaboutsuchpotentialselectivepressures( Yabeetal., 2011 ). TheRRandLGsitesaredominatedbyrepresentativesof the Ktedonobacterales growingonasilica-richsubstrateinthe formofquartz( Figure6 ).Thefumarolecaves,whichform atthecontactbetweentheicesheetanda”oorofphonolitic lava,similarlycontain Ktedonobacterales growinginalithosoil comprisedofsilica-richmontmorilloniteandkaolinite,witha pHof4.1…5.8( Ugolini,1965 ; HudsonandDaniel,1988 ; ConnellandStaudigel,2013 ).Incontrast,the Dehalococcoidetes and Anaerolineae dominatethe Chloro”exi inwell-buffered(pH8.0… 8.3)limestonecaves,aswellasandHawaiianlavatubecaves ( Figure4 ),wherethebasalticlava( < 20%quartz)ismuchless susceptibletochemicalweathering( Porderetal.,2007 ).Together, thesedatasuggestthattheselectionpressureforthe Ktedonobacterales mayberelatedtoeitherthehighlevelsofsilica,anda consequenceoftheselectivepressureoftheSi4 +ion,orthe resultofthepoorbufferingandthesurfaceacidityofquartzand phyllosilicates( Porderetal.,2007 ).Giventheobservationofthe Thaumarchaeota GroupI.1cinRSC,itislikelythatpHmaybethe predominantdriverofcommunitystructurewithinthemicrobial ecosystem. DeLong(1992) “rstidenti“edagroupofmesophilic,marine Crenarchaeota thatmetagenomicanalysessuggestedcontained anammoniamonooxygenase( amo A)geneandammoniaoxidizingactivity( Venteretal.,2004 ).Uponthediscoveryof relatedsequencesinthesoil,these Crenarchaeota weresubsequentlyreclassi“edintoGroupsI.1a,I.1b,andI.1c:Group I.1abeingassociatedwithmarineandfreshwaterenvironments,GroupI.1bwithsoilandsubsurfaceenvironments,and GroupI.1cwithforestsoils( Jurgensetal.,1997 ; Ochsenreiter etal.,2003 ).Thesigni“canceofthese Crenarchaeotal populationswithintheenvironmentwasdemonstratedbycultivationoftheammonia-oxidizing Nitrosopumilusmaritimus ,which FrontiersinMicrobiology | ExtremeMicrobiologyNovember2014 | Volume5 | Article615 | 10

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Bartonetal. Unusualmicrobialdiversityinanorthoquartzitecave con“rmedtheirammonia-oxidizingpotential( Könnekeetal., 2005 ).Sincethattime,thesemesophilicAOAhavebeenclassi“edintoanewphylum,the Thaumarchaeota ,whicharenow recognizedtobeimportant,ifnotthedominant,playersin globalnitri“cation( Brochier-Armanetetal.,2008 ; Monteiroetal., 2014 ). Asmoremembersofthe Thaumarchaeota wereidenti“ed withintheenvironment,thereseemedtobeanassociationof GroupI.1cwithacidicenvironments,eventhoughtheammoniaionisprotonatedtoitsunfavorableammoniumformatlow pH.Nonetheless,bothpHandnutrientconditionsappearto beimportantdriversofniche-differentiationwithinthe Thaumarchaeota ( Lehtovirtaetal.,2009 ; Martens-Habbenaetal.,2009 ; Gubry-Ranginetal.,2011 ; AuguetandCasamayor,2013 ).Thelack ofsuf“cientammoniaforammonia-oxidationunderlowpHwas solvedwiththecultivationoftheacidicAOA Nitrososphaeraviennensis ,whichusesureaforgrowth( Tournaetal.,2011 ).Thisurea, whichappearstoplayacriticalroleinammonia-oxidationunder acidicconditions,isdegradedbyintracellularureasestoreleasethe necessaryammoniaforgrowth( Luetal.,2012 ; LuandJia,2013 ). Thus,thelowpHwithinRSCmaythereforeexplainboththepresenceofthe Thaumarchaeota GroupI.1candthe Ktedonobacterales , whichencodeureasesandcanusenitriteandnitrate( Costello andSchmidt,2006 ; HanadaandPierson,2006 ; Wuetal.,2009 ; Sorokinetal.,2012 ).Ifammoniaand/orureaaredrivingcommunitystructurewithinRSC,thequestionremainsastoitssource. Theanswermayrequireustore-examinethestreamenteringthe cave. Cavemicrobiologyisarelativelyyoung“eld,althoughthere hasbeenadramaticincreaseinrecentyearsinboththenumberofresearchgroupsandresultantpublications( Leeetal., 2012 ).Yetmuchofthisworkhascenteredontraditionallimestone(karst)caves.Verylittlemicrobialexplorationhastaken placeinpseudokarst…cavesfoundinrockotherthanlimestone( Halliday,2007 ).Suchenvironmentsincludelavacaves, ice,glacierandfumarolecaves,taluscaves,ironcaves,littoralseacaves,andsandstonecaves( HudsonandDaniel,1988 ; Sooetal.,2009 ; Northupetal.,2011 ; ConnellandStaudigel, 2013 ; Hathawayetal.,2014 ).Yetsuchcavescontributesignificantlytoourunderstandingofthegeochemicalenvironments andsubsurfaceecosystemsthatcanbestudiedonEarth( Herboldetal.,2014 ).Thedif“cultyinaccessibility,unforgiving samplingenvironment,anddif“cultyinobtainingDNAfrom thesesamplesmeansthatthisinitialworkhasonlyallowedus asnapshotofthemicrobialdiversityfoundwithinthetepui sandstonecaves.Withadvancedtechnologiesinlow-biomass next-generationsequencing,metagenomicapproaches,andthe potentialtoculturethe Thaumarchaeota ,wehopethatfurther studywillallowustobetterunderstandboththeactivephysiologiesandthedriversofmicrobialselectionwithintheseunusual microbialecosystems.AUTHORCONTRIBUTIONSAllauthorscontributedtotheconceptionordesignofthework, “eldwork,dataanalysisandinterpretation.Themanuscriptwas preparedbyHazelA.Barton,withassistancefromJuanG. GiarrizzoandCharlesE.Robertson.ACKNOWLEDGMENTSThepresentedresearchwassupportedinpartbytheNSFCAREER award(NSF#0643462)toHazelA.Barton,alongwithan ExplorersClubGranttoEricD.Banks,andNorthernKentucky UniversitysupporttobothEricD.BanksandMarkJ.Broering. Theauthorsdeclarenoothercon”ictsofinterest.Theauthors wouldliketothankHenryFrancisforcarryingouttheelemental analysisandXRD,andFrankHugginsforcarryingouttheMossbauerspectroscopy.WewouldalsoliketothanAkananTraveland Toursforlogisticalsupportwithinthe“eldandcaversfromthe OxfordUniversityCavingClubfortheirusefuldiscussions.This researchwassupportedbysamplingpermits#I-111and#3953 providedbyTheVenezuelanEnvironmentalMinistryattheVice MinistryofEnvironmentalManagementandAdministration, Caracas,Venezuela.REFERENCESAkiyama,M.,Shimizu,S.,Sakai,T.,Ioka,S.,Ishijima,Y.,andNaganuma,T.(2011). SpatiotemporalvariationsintheabundancesoftheprokaryoticrRNAgenes, pmoA ,and mcrA inthedeeplayersofapeatboginSarobetsu-genyawetland, Japan. 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Barton,H.A.,Taylor,M.R.,andPace,N.R.(2004).Molecularphylogeneticanalysis ofabacterialcommunityinanoligotrophiccaveenvironment. J.Geomicrobiol. 21,11…20.doi:10.1080/01490450490253428 Barton,H.A.,Taylor,N.M.,Kreate,M.,Springer,A.J.,Oehrle,S.A.,andBertog, J.L.(2007).Theimpactofhostrockgeochemistryonbacterialcommunity structureinoligotrophiccaveenvironments. Int.J.Speleol. 36,93…104.doi: 10.5038/1827-806X.36.2.5 Barton,H.A.,Taylor,N.M.,Lubbers,B.R.,andPemberton,A.C.(2006).DNA extractionfromlow-biomasscarbonaterock:animprovedmethodwithreduced contaminationandthelow-biomasscontaminantdatabase. J.Microbiol.Methods 66,21…31.doi:10.1016/j.mimet.2005.10.005 Bhullar,K.,Waglechner,N.,Pawlowski,A.,Koteva,K.,Banks,E.D.,Johnston,M. D.,etal.(2012).Antibioticresistanceisprevalentinanisolatedcavemicrobiome. PLoSONE 7:e34953.doi:10.1371/journal.pone.0034953 www.frontiersin.orgNovember2014 | Volume5 | Article615 | 11

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Bartonetal. Unusualmicrobialdiversityinanorthoquartzitecave ofThermosporotrichaceaefam.nov.withintheclassKtedonobacteriaCavaletti etal.2007andemendeddescriptionoftheclassKtedonobacteria. Int.J.Syst.Evol. Microbiol. 60,1794…1801.doi:10.1099/ijs.0.018069-0 Yabe,S.,Aiba,Y.,Sakai,Y.,Hazaka,M.,andYokota,A.(2011). Thermogemmatisporaonikobensis gen.nov.,sp.nov.and Thermogemmatisporafoliorum sp.nov.,isolatedfromfallenleavesongeothermalsoils,anddescriptionof Thermogemmatisporaceaefam.nov.andThermogemmatisporalesord.nov. withintheclassKtedonobacteria. Int.J.Syst.Evol.Microbiol. 61,903…910.doi: 10.1099/ijs.0.024877-0 Ying,J.-Y.,Zhang,L.-M.,andHe,J.-Z.(2010).Putativeammonia-oxidizingbacteria andarchaeainanacidicredsoilwithdifferentlandutilizationpatterns. Environ. Microbiol.Rep. 2,304…312.doi:10.1111/j.1758-2229.2009.00130.x ConflictofInterestStatement: Theauthorsdeclarethattheresearchwasconducted intheabsenceofanycommercialor“nancialrelationshipsthatcouldbeconstrued asapotentialcon”ictofinterest. Received:01August2014;accepted:28October2014;publishedonline:26November 2014. Citation:BartonHA,GiarrizzoJG,SuarezP,RobertsonCE,BroeringMJ,BanksED, VaishampayanPAandVenkateswaranK(2014)MicrobialdiversityinaVenezuelan orthoquartzitecaveisdominatedbytheChloro”exi(ClassKtedonobacterales)and ThaumarchaeotaGroupI.1c.Front.Microbiol. 5 :615.doi:10.3389/fmicb.2014. 00615 ThisarticlewassubmittedtoExtremeMicrobiology,asectionofthejournalFrontiers inMicrobiology. Copyright©2014Barton,Giarrizzo,Suarez,Robertson,Broering,Banks, VaishampayanandVenkateswaran.Thisisanopen-accessarticledistributedunder thetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distribution orreproductioninotherforumsispermitted,providedtheoriginalauthor(s)orlicensor arecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewith acceptedacademicpractice.Nouse,distributionorreproductionispermittedwhich doesnotcomplywiththeseterms. FrontiersinMicrobiology | ExtremeMicrobiologyNovember2014 | Volume5 | Article615 | 14


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