Microbial Diversity in a Permanently Cold and Alkaline Environment in Greenland

22 Pages·2015·6.44 MB·English

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].
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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
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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:
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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.
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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
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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
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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.
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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
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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
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focusingonthecold-andalkaline-adaptedcommunity,whichisexpectedtoinhabittheinteri-
orofthecolumns.
Overalltaxonomicdistribution
Inordertoobtainamoreaccuraterepresentationoftheinterior/surfacecommunity,twosam-
pleswerereclassifiedbasedontheclusteranalysis(Fig3,seeabove).Oneinteriorsamplewas
groupedwiththesurfacesamples(sampleI38)andonesurfacesamplewasreclassifiedasan
interiorsample(sampleI24).Ananalysisofthedistributionofthemostabundantbacterial
phylarevealedthattheikaitecolumnsaredominatedbyProteobacteria,Firmicutes,Bacteroi-
detes,Actinobacteria,andCyanobacteria,whereasthemicrobialdiversityinseawaterandsedi-
mentsamplesweremainlyrestrictedtoProteobacteria,Bacteroidetes,andActinobacteria
(Fig4).Themostprominentdifferencesbetweencolumninteriorandsurfacewereobserved
fortheFirmicutes,at24.2%and4.7%,andtheCyanobacteria,at3.1%and36.4%,respectively.
ApartfromCyanobacteria,thesurfacewasalsoabundantinAlphaproteobacteria,orderRhodo-
bacterales(27.5%),whichincludenumerousphototrophicgenera(Table2).TheFirmicutes
werealmostexclusivelyoftheclassClostridia(23.7%),ordersNatranaerobiales(12.8%),Clos-
tridiales(7.7%),andThermoanaerobacterales(2.9%),mostofwhichgrowanaerobically[48].
Similarly,theBacteroidetesidentifiedinthecolumninterior,primarilyrepresentedbytheclass
Bacteroidia,orderBacteroidales(16.5%),aremostlyanaerobic.Theabundanceoftheseorders
suggeststhatthecolumninteriorisalargelyanoxicenvironment,muchlikethelowerlayers
andsedimentofsodalakes,whicharealsoabundantinanaerobicFirmicutesandBacteroidetes
[4,45,49,50].TheanaerobichaloalkaliphilicmembersoftheorderNatranaerobialeswerefirst
identifiedinsodalakesoftheWadiAnNatruninEgyptandtheabundantNatranaerobialesin
theikaitecolumnsmayrepresentlow-saline,cold-adaptedrelativesofthisnewlydescribed
order[48].Proteobacteriawereabundantinallsamples,althoughwithsignificantdifferences
inthedistributionbetweenclasses(Table2).Alpha-,Beta-andGammaproteobacteriawere
presentinbothcolumninteriorandatthesurface,whiletheDelta-andEpsilonproteobacteria
Fig4.Distributionofphylaandproteobacterialclassesineachsampletype.Thedatarepresentsthe
averageabundanceingroupsofsamplestakenfromcross-sections(interior)orsurfacesofikaitecolumnsor
fromseawaterorsedimentintheIkkaFjord.
doi:10.1371/journal.pone.0124863.g004
PLOSONE|DOI:10.1371/journal.pone.0124863 April27,2015 10/22