Phylogenetic Characterization of Marine Benthic Archaea in Organic-Poor Sediments of the

14 Pages·2016·1.22 MB·English

microorganisms
Article
Phylogenetic Characterization of Marine Benthic
Archaea in Organic-Poor Sediments of the Eastern
Equatorial Pacific Ocean (ODP Site 1225)
AntjeLauer1,2,KetilBerntSørensen1,3andAndreasTeske1,*
1 DepartmentofMarineSciences,UniversityofNorthCarolinaatChapelHill,ChapelHill,NC27599,USA;
[email protected](A.L.);[email protected](K.B.S.)
2 BiologyDepartment,CaliforniaStateUniversityBakersfield,Bakersfield,CA93311-1022,USA
3 Ramboll,CopenhagenDK-2300,Denmark
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
AcademicEditors:DonCowanandJohannesF.Imhoff
Received:20April2016;Accepted:22August2016;Published:6September2016
Abstract: Sequencing surveys of microbial communities in marine subsurface sediments have
focused on organic-rich, continental margins; the database for organic-lean deep-sea sediments
frommid-oceanregionsisunderdeveloped. Thearchaealcommunityinsubsurfacesedimentsof
ODPSite1225intheeasternequatorialPacific(3760mwaterdepth;1.1and7.8msedimentdepth)
was analyzed by PCR, cloning and sequencing, and by denaturant gradient gel electrophoresis
(DGGE)of16SrRNAgenes. Threeunculturedarchaeallineageswithdifferentdepthdistributions
werefound: MarineGroupI(MG-I)withintheThaumarchaeota,itssisterlineageMarineBenthic
Group A (MBG-A), the phylum-level archaeal lineage Marine Benthic Group B (also known as
Deep-SeaArchaealGrouporLokiarchaeota),andtheDeep-SeaEuryarchaeotalGroup3. TheMG-I
phylotypesincludedrepresentativesofsedimentclustersthataredistinctfromthepelagicmembers
of this phylum. On the scale from fully oxidized, extremely organic carbon-depleted sediments
(forexample,thosetheSouthPacificGyre)tofullyreduced,organiccarbon-richmarinesubsurface
sediments (such as those of the Peru Margin), Ocean Drilling Program (ODP) Site 1225 falls into
thenon-extremeorganiccarbon-leancategory,andharborsarchaealcommunitiesfrombothendsof
thespectrum.
Keywords: Marinearchaea;MarineGroupI;subsurface;marinesediment;OceanDrillingProgram
1. Introduction
Molecular surveys of microbial communities in marine subsurface sediments have detected
distinct archaeal populations in deep-sea sediments of the central oceanic basins, and near-shore
or continental margin locations; these differences most likely reflect the contrasting redox states
andorganiccarboncontentoforganic-lean,oxidizedopen-oceansedimentswithlowsedimentation
rates,andorganiccarbon-rich,reducedcoastalandcontinentalmarginsedimentsthatreceiveprimary
productionfromthewatercolumnandterrestrialinput[1,2].LowcellnumbersandpoorDNArecovery
havelimitedsequence-basedmicrobialcommunityanalysesoforganic-lean,open-oceansediments
torelativelyfewcasestudies[3–6]. Inconsequence,fundamentalaspectsofthesebenthicmicrobial
ecosystems—phylogenetic composition, functional gene repertoire, and metabolic activities—are
poorlyknown.
ThefirstOceanDrillingProgram(ODP)expeditiondedicatedtothestudyoflifedeepbeneath
the seafloor, ODP Leg 201, combined detailed geochemical analyses, cell counts, cultivations,
andmolecularscreeningofsubsurfacemicrobialcommunities[7],andsucceededinacomprehensive
Microorganisms2016,4,32;doi:10.3390/microorganisms4030032 www.mdpi.com/journal/microorganisms
Microorganisms2016,4,32 2of14
census of subsurface microbial life in the context of geochemical controls that shape microbial
communitycomposition,abundance,andactivity[8–14].Samplingsiteswereselectedthatrepresented
low-activity sediments from central oceanic basins as well as organic-rich coastal sediments from
upwelling areas (Figure 1). Subsurface sediment cores were obtained from open ocean sites with
organic-poorsedimentsintheeasternequatorialPacific(Sites1225and1226)andinthePeruBasin
(Site1231). Thesesiteshadcelldensitiesintherangeof105to5×106cells/cm3belowtheupperfew
metersofthesedimentcolumn,loworganicCcontent(1%–2%forODPSite1226;<0.4%forODPSites
1225and1231),near-linearorslowlychangingprofilesofsulfateandmethane,indicatinglittlesulfate
depletionormethaneaccumulation,andconsiderabledepthpenetrationofthehighlydynamicelectron
acceptornitrate[8]. Contrastingcoreswereobtainedfromorganic-richsedimentsonthePeruMargin
(Sites1227,1228,1229),andfromorganic-richdeep-seasedimentsinthePeruTrench(Site1230)[7].
Thesesiteswerecharacterizedbytotalorganiccarbon(TOC)intherangeof2.5%–5%,prokaryoticcell
countsmostlybetween5×106 and108 cells/cm3 inthesubsurfacesediment,andsteepporewater
sulfateandmethanegradientsindicatingdowncoresulfatedepletionandmethanebuildup[8].
ODP Site 1225 offered an opportunity to explore subsurface microbial communities in
organic-poor, oxidizedsedimentsthatcontrasttoorganic-rich, reducedsubsurfacesedimentsthat
havebeenthefocusofmost16SrRNA-basedmicrobialcommunityanalyses. Thearchaealcommunity
analysesfromSite1225havebeenabstractedanddiscussedincomparativebiogeographicalsurveys
andoverviewchaptersonsubsurfacemicrobiology[1,11,15–17],buthaveneverbeenpublishedin
adedicatedstudy. Herewereportthearchaeal16SrRNAgenesequencingsurveyofODPSite1225in
theeasternequatorialPacific,documentthevariableoutcomesofdifferentDNAextractionmethods,
andprovideadetailedphylogeneticanalysis. Wearealsore-examiningODPSite1225inthecontextof
morerecentlystudiedoligotrophicopen-oceansedimentsofthesubtropicalgyresintheSouthPacific
andtheNorthAtlanticOceans.
2. MaterialandMethods
Sitedescription. ODPSite1225(Figure1)islocatedintheeasternequatorialPacific(2◦36.25N,
110◦34.29W)at3760mwaterdepthnearthepresentdayboundaryoftheSouthEquatorialCurrent
andtheNorthEquatorialCountercurrent[18].
The clay-rich sediments of this site, consisting of carbonate and siliceous nannofossil oozes
andchalk,arerepresentativeforalargeportionofthecentralPacificOcean.Thesedimentlayerat
Site 1225 was 320 m thick and its age ranged from the Pleistocene (0–27 m below surface (mbsf),
1.8Ma)tothePliocene(28–98mbsf,5.2Ma)andtheMiocene(99–320mbsf,11.5Ma).Thewaterdepth
(3760 m) at Site 1225 reduces the input of photosynthetically derived organic material; shipboard
measurementsoftotalorganiccarbon(TOC)showedlowvalues(≤0.2%to0.42%)throughoutSite
1225. AcridineOrangecellcountsshowedthatcellswerepresentthroughoutthesedimentcolumn,
rangingbetweenapprox. 3×105and3×106cells/cm3. Thesenumberswereatthelowendofthecell
countsfortheorganic-richPeruMarginandPeruTrenchODPSites(approx. 5×106to108cells/cm3,
with some higher outliers [18]). Attenuated respiration rates in this organic-poor sediment were
reflected in deep penetration of electron acceptors; for example, porewater nitrate concentrations
decreasedfromca. 33µmtothedetectionlimitwithintheupper1.5mofthetopsedimentcore[18].
CharacteristicsforLeg201sitesaretabulatedinTableS1.
Sample collection. Deep-sea sediment was collected in February 2002 on ODP Leg 201 with
JOIDESResolutionatSite1225inthecentralPacificOceanat3760mwaterdepth. Sedimentcoreswere
retrievedbyadvancedpistoncoring(APC)andbroughttothesurfacewithin1–2h. Freshlyretrieved
cores(totallength9–9.5m)wereimmediatelydividedinto1.5msubcores,whichwereimmediately
slicedintheship’scoldroomintowholeroundcoresamplesof5–10cmlengthusinganN -flushed
2
corecuttingdevice[19]. Within2–4haftercoreretrieval,sedimentsampleswerefrozenandkeptat
−80◦Cuntilprocessinginthehomelaboratory.
Microorganisms2016,4,32 3of14
Microorganisms 2016, 4, 32 3 of 13

FFiigguurree1 1.. OODDPP SSiittee 11222255 iinn tthhee eeaasstteerrnn eeqquuaattoorriiaall PPaacciifificc,, aanndd ootthheerr ddrriilllliinngg ssiitteess ooff OODDPP LLeegg 220011 nneeaarr
GGaallaappaaggooss( 1(1222266),),o nonth tehPe ePruerMu aMrgairng(i1n2 2(172t2o71 t2o2 91)2,2in9)t,h ienP tehreu PTreernuc hTr(e1n2c3h0 ),(1a2n3d0)P, earnudB aPseinru( 1B2a3s1i)n.
M(1o2d3i1fi).e Mdfordoimfie[d7] f.rom [7].
DNA extraction from sediment samples. Two approaches were used for extracting DNA from
DNAextractionfromsedimentsamples. TwoapproacheswereusedforextractingDNAfrom
sediment samples. With direct lysis of microbial cells followed by DNA extraction from bulk
sedimentsamples. WithdirectlysisofmicrobialcellsfollowedbyDNAextractionfrombulksediment
sediment (Method 1), two parallel DNA extractions on a sediment sample from the uppermost 1.5 m
(Method1),twoparallelDNAextractionsonasedimentsamplefromtheuppermost1.5msubcore
subcore 1225C-1H1 (sample position 105–110 cm below surface, or cmbsf) yielded PCR-amplifiable
1225C-1H1 (sample position 105–110 cm below surface, or cmbsf) yielded PCR-amplifiable DNA;
DNA; extractions from other samples using this method remained unsuccessful. The sample
extractionsfromothersamplesusingthismethodremainedunsuccessful. Thesamplepositionwithin
position within the subcore represents a minimal depth estimate (1.05–1.10 mbsf), since the sediment
the subcore represents a minimal depth estimate (1.05–1.10 mbsf), since the sediment surface is
surface is heavily perturbed during drilling, and the immediate sediment surface is lost. As an
heavilyperturbedduringdrilling,andtheimmediatesedimentsurfaceislost. AsanalternativeDNA
alternative DNA extraction method, prokaryotic cells were separated from the sediment sample by
extractionmethod,prokaryoticcellswereseparatedfromthesedimentsamplebyshortsonication,
short sonication, followed by DNA extraction from isolated cells (Method 2). This method yielded
followedbyDNAextractionfromisolatedcells(Method2). ThismethodyieldedPCR-amplifiable
PCR-amplifiable DNA from sample 1225C-1H6, which had previously given negative results.
DNA from sample 1225C-1H6, which had previously given negative results. Subcore 1225C-1H6
Subcore 1225C-1H6 represents the sediment depth range from 7.5 to 9 mbsf; thus, the sample from
representsthesedimentdepthrangefrom7.5to9mbsf;thus,thesamplefromthe25–30cmintervalof
the 25–30 cm interval of this core segment originates from a depth of at least 7.75–7.8 mbsf.
thiscoresegmentoriginatesfromadepthofatleast7.75–7.8mbsf.
In Method 1, genomic DNA was extracted from 10 g of deep-sea sediment sample material as
InMethod1,genomicDNAwasextractedfrom10gofdeep-seasedimentsamplematerialas
follows. The sediment was suspended in 3 mL of DNA extraction buffer (200 mM NaCl, 200 mM
follows. Thesedimentwassuspendedin3mLofDNAextractionbuffer(200mMNaCl,200mMTris,
Tris, 2 mM sodium citrate, 10 mM CaCl2, 50 mM EDTA, pH 8) in a 50 mL Falcon tube and freeze–
2mMsodiumcitrate,10mMCaCl ,50mMEDTA,pH8)ina50mLFalcontubeandfreeze–thawed
2
thawed twice (−80 °C, 65 °C). Then, 100 µL of polyadenylic acid (polyA) (10 mg/mL), 100 µL of 6.7%
twice(−80◦C,65◦C).Then,100µLofpolyadenylicacid(polyA)(10mg/mL),100µLof6.7%sodium
sodium pyrophosphate, and 150 µL of lysozyme (100 mg/mL) were added in this order. The
pyrophosphate,and150µLoflysozyme(100mg/mL)wereaddedinthisorder. Thesedimentslurry
sediment slurry was briefly vortexed after the addition of each chemical and incubated for 40 min at
wasbrieflyvortexedaftertheadditionofeachchemicalandincubatedfor40minat37◦Cinaslowly
37 °C in a slowly shaking incubation chamber. Next, 50 µL of sodium dodecylsulfate (SDS) (20%)
shakingincubationchamber. Next,50µLofsodiumdodecylsulfate(SDS)(20%)and100µLproteinase
and 100 µL proteinase K (20 mg/mL) were added to the mixture followed by 3 h incubation at 55 °C
K(20mg/mL)wereaddedtothemixturefollowedby3hincubationat55◦Cwithgentleshaking.
with gentle shaking. The sample was extracted twice with an equal volume of phenol:chloroform:
Thesamplewasextractedtwicewithanequalvolumeofphenol:chloroform: isoamylalcohol25:24:1
isoamylalcohol 25:24:1 (pH 8). The aqueous phase was precipitated overnight with 0.1 vol of 5 M
(pH8). Theaqueousphasewasprecipitatedovernightwith0.1volof5Mice-coldNaClsolution,
ice-cold NaCl solution, 2.5 vol of ice-cold ethanol (96%), and 20 µg of glycogen per mL of solution
2.5volofice-coldethanol(96%),and20µgofglycogenpermLofsolution(20mg/mLstocksolution).
(20 mg/mL stock solution). The DNA was collected by centrifugation (16,000× g, corresponding to
TheDNAwascollectedbycentrifugation(16,000×g,correspondingto14,000rpminanEppendorf
14,000 rpm in an Eppendorf 5415C tabletop centrifuge), washed in ice-cold 70% ethanol, air dried,
5415Ctabletopcentrifuge),washedinice-cold70%ethanol,airdried,andresuspendedin50µLsterile
and resuspended in 50 µL sterile distilled water. DNA extracts were purified using the Wizard
distilledwater. DNAextractswerepurifiedusingtheWizardPlasmidPurificationKitbyPromega
Plasmid Purification Kit by Promega (Madison, WI, USA) and stored at −80 °C. A reagent blank was
also processed to assess the extent of laboratory or sample cross-contamination. Outer edges of
Microorganisms2016,4,32 4of14
(Madison,WI,USA)andstoredat−80◦C.Areagentblankwasalsoprocessedtoassesstheextentof
laboratoryorsamplecross-contamination. Outeredgesofsubcoreswerenotextracted,inorderto
avoidcontaminationintroducedduringcoringandpackaging.
InMethod2,extractionwasprecededbyashortsonicationstep. Sedimentsamples(15g)from
subcores1225C-1H6and1225C-1H1weresuspendedin4mLofphosphate-buffered3MKCl(pH7.5),
5mLofSDS(20%)and10mLofsterilewater,andsonicatedinawaterbathfor1min. Onlysample
1225C-1H6gaveapositiveresultinthenestedPCR.Longersonicationsteps(2.5and5min)were
alsotestedonbothsamples,withconsistentlynegativeresultsinallPCRexperiments. Thesediment
slurrieswerespundownfor1minat1000rpm,andthesupernatantwasextractedwithMethod1.
Amplificationof16SrRNAgenes. Theprimersusedinthisstudyandtheirreferencesarelisted
in Table 1. Primer acronyms followed by f refer to forward primers; r indicates reverse primers.
Foramplificationofarchaeal16SrRNAgenes, primersARC8fandARC1492r(Table1)wereused
initially, and the products were used as template in a subsequent nested PCR. In the nested PCR,
primersARC21fandARC915rwereusedforclonelibraryconstruction,whileprimersARC21f-GC
(primer ARC21F with GC-clamp 5(cid:48)-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA
CGG GGG G-3(cid:48) [24] attached at the 5(cid:48)-end) and ARC519r were used for denaturant gradient gel
electrophoresis(DGGE)analysis.
Table1.Archaeal16SrDNAprimers.
Primer Sequence AnnealingTemperature(◦C) Reference
ARC8f 5(cid:48)-TCCGGTTGATCCTGCC-3(cid:48) 55 [20]
ARC1492r 5(cid:48)-GGCTACCTTGTTACGACTT-3(cid:48) 55 [21]
ARC21f 5(cid:48)-TTCCGGTTGATCCYGCCGGA-3(cid:48) 65 [22]
ARC915r 5(cid:48)-GTGCTCCCCCGCCAATTCCT-3(cid:48) 65 [23]
ARC344f 5(cid:48)-AYGGGGYGCASCAGGSG-3(cid:48) 65 [24]
ARC519r 5(cid:48)-GGTDTTACCGCGGCKGCTG-3(cid:48) 65 [24]
PCRmixturescontained1µLof1:10dilutedDNAextract,2µLofeachprimer(10pmol/µL),
2µLof deoxynucleoside triphosphates (dNTPs) (10 mM each), 5 µL of 10× PCR buffer
(final concentration 1.5 mM MgCl ), 2 µL of bovine serum albumin (BSA, 10 mg/mL), 0.15 µL of
2
Taqpolymerase(5U/µL)(Promega,Madison,WI,USA)andsterilewatertoafinalvolumeof50µL.
The PCR amplification was performed using an iCycler (BioRad, Hercules, CA, USA). The PCR
mix was incubated at 94 ◦C for 4 min, followed by 35 cycles of denaturation at 94 ◦C for 1 min,
annealingatdifferenttemperatures(Table1)for1min,andextensionat72◦Cfor1min. ThePCR
amplificationendedwithasingle10minextensionstepat72◦C.ThePCRproductswereevaluatedby
gelelectrophoresison1.5%agarosegelsstainedwithethidiumbromide(0.5mg/L).SuccessfulPCR
amplificationsforarchaeawereobtainedwith1:10dilutedDNAextract;higherdilutionsundundiluted
DNAextractsdidnotconsistentlyproducePCRamplicons.
DGGEanalysis. DGGEwasperformedusingaDCodesystem(Bio-Rad,Hercules,CA,USA)
aspreviouslydescribed[24]. PCRsampleswereloadedonto6%(wt./vol)polyacrylamidegelsin
0.5×TAE (20 mM Tris, 10 mM acetate, 0.5 mM EDTA [pH 8]), cast with a gradient of 20%–70%
denaturing agent (100% denaturant contains 7 M urea and 40% formamide). Electrophoresis was
performedat60◦Cfor2hat250VonaBioradDCodesystem. Afterelectrophoresis,theDGGEgels
werestainedfor30mininanethidiumbromidebath(0.5mg/Lin0.5×TAEbuffer)anddestained
for at least 10 min in 0.5× TAE buffer. The gels were photographed on a UV transillumination
table(Spectroline®,BI-O-VISION,NewHaven,CT,USA)withadigitalcamera(KodakEDAS290).
ArchaealDGGEanalysishadtobelimitedtosample1225-1H1,duetopersistent,false-positiveDNA
amplicons in the negative controls of 1225-1H6. Archaeal DGGE bands of sample 1225-1H1 were
selectedforsequencing,punchedfromthegelwithsterilescalpelsandplacedinsterilevialscontaining
Microorganisms2016,4,32 5of14
100µLofsterilizeddeionizedwater. Afterovernightincubationat4◦C,2µLoftheeluatewasusedas
templateinaPCRwiththeDGGEprimers.
CloningofPCRproducts.ClonelibrarieswerepreparedbyusingtheTOPO®XLPCRcloningkit
(Invitrogen,Carlsbad,CA,USA).PCRproductswereligatedtopCR®-XL-TOPO®vectors,whichwere
transformed into E. coli JM 109 cells by electrophoresis. After streaking on Luria broth (LB)-agar
platesandincubationat37◦Caccordingtothemanufacturer’sinstructions,individualcloneswere
transferredtoliquidLB-mediumandgrownovernightonashakingtableat37◦C.Plasmidextraction
andpurificationwasperformedwiththeWizardPlasmidPurificationkit(Promega). Totestcloning
success,6–10randomlypickedcloneswerescreenedforinsertsbyPCRamplificationwithprimers
M13F(5’-GTAAAAACGACGGCCAG-3’)andM13R(5’-CAGGAAACAGCTATGAC-3’)[25]. ThePCR
contained1µLoftemplate,2.5µLofeachprimer(10pm/µL),2.5µLofdNTPs(10mM),2.5µLof
10×PCRbuffer(finalconcentration1.5mMMgCl ),0.15µLofTaqpolymerase(5U/µL),andsterile
2
watertoafinalvolumeof25µL.ThePCRreactionincludedaninitialdenaturationstepat94◦Cfor
2minfollowedby25cyclesofdenaturationat94◦Cfor30s,annealingat55◦Cfor30s,andextension
at72◦Cfor30s. Thestepat72◦Cwasextendedto5min.
Sequencing. Sangersequencingusingfluorescentdideoxyterminatorswasperformedatthe
GlaxoSequencingCenteroftheUniversityofNorthCarolinaatChapelHill. ReamplifiedDGGEbands
werepurifiedpriortocloningbyusingtheMOBIOPCRcleanupkit(MOBIO,SolanaBeach,CA,USA)
accordingtothemanufacturer’sprotocol. About50ngoftemplateand10pmolofprimersARC21f
andARC519rwereusedinthesequencingreaction. Clonelibrariesweresequencedusing10pmolof
primersM13FandM13Rinreactionswith0.7µgofplasmidtemplate.
Phylogenetic analysis. Sequence data were analyzed by BLAST against the entire
GenBank database [26]. Sequences were aligned in a first step with the program Multalign
(http://prodes.toulouse.inra.fr/multalin/), followed by manual alignment with SeqPup v0.6 [27].
Primersequenceswereexcludedfromalignmentsandphylogenies. Thesequenceswerecheckedfor
chimerasusingtheonlinedetectiontoolBellerophon[28],complementedbymanualinspectionof
the alignment and tree building using partial sequences. Phylogenetic analyses were performed
with PAUP4.0* [29] using maximum likelihood distances under minimum evolution optimality
criteria;empiricallyestimatedbasefrequenciesandtransition/transversionfrequencieswereused.
Bootstrapanalysesarebasedon200replicates.
Nucleotidesequence accessionnumbers. The archaeal16S rDNAsequences obtainedin this
study are available in GenBank under accession numbers AY800198 to AY800233 and DQ186521
(clone1CD3). The16SrDNAsequencesofthearchaealDGGEfragmentshaveGenBankaccession
numbersDQ137876toDQ137881.
3. Results
ExtractionofPCR-amplifiableDNA.DNAextractionwasattemptedfromnumeroussamples
throughoutthesedimentcolumn,usingMethods1and2asdescribedabove. WhentheDNAextracts
were evaluated by electrophoresis on 1% agarose gels, DNA concentrations were too low to be
visualizedbyethidiumbromidestaining.Nevertheless,archaealDNAwassuccessfullyamplifiedfrom
thetwoshallowestsamples(1225C-1H1and1225C-1H6). Method1workedintwoparallelextractions
andPCRamplificationsofarchaealDNAfromsample1225C-1H1,whichresultedin25and29archaeal
sequences,respectively. TheclonesobtainedfromparallelDNAextractionsandamplificationfrom
sample1225C-1H1differedinrelativeabundanceofphylogeneticgroups. Themajorityoftheclones
fromthefirstDNAextraction(clonenamesbeginningwith1C)areaffiliatedwithdifferentsubgroups
ofMarineGroup1(MG-I),whereasmostclonesfromthesecondDNAextraction(beginningwith1A)
weremembersofDeep-SeaArchaealGroup/MarineBenthicGroupB(DSAG/MBG-B)(Table2).
Since the same DNA extraction method was used, these variable results could reflect either
stochasticPCRbiasduetorandomamplificationfromasmallnumberofDNAtemplates[29]orspatial
heterogeneityinthesedimentsample. PCR-amplifiableDNAwasextractedusingMethod2from
Microorganisms2016,4,32 6of14
sample 1225C-1H6, and a total of 26 clones were sequenced. DNA extracted with Method 1 from
sample1225C-1H6gavenegativeresultsinthenestedPCR.
Table 2. Numbers of 16S rRNA gene clones affiliated with major archaeal groups retrieved after
DNAextractionsfromSite1225sedimentsamples.Thetworeplicateextractionsforsedimentsample
1225-1H1areseparatedintotwodatacolumns.
1225C-1H1 1225C-1H1 1225C-1H6
Affiliation Extraction1 Extraction2
Method1 Method1 Method2
MarineGroupI 17 3 4
MarineBenthicGroupA 5 3 22
DSAG/MarineBenthicGroupB 3 21 -
Euryarchaeota - 2 -
Sum 25 29 26
MarineGroupI.MostofthedetectedarchaealphylotypeswereaffiliatedwithMarineGroupI
(MG-I)withintheThaumarchaeota[30]. TheMG-IclonesfromSite1225couldnotbeaccommodated
solelybythepelagicMG-Isubgroupsα,β,andγ,definedoriginallybycomparativesequenceanalyses
ofMG-IArchaeafromdifferentoceansandwaterdepths[31]. SubgroupMG-Iαincludedclonesfrom
all water depths, but especially from the upper 200 m of the water column [31]; it also accounted
for almost half of the MG-I clones obtained from Site 1225 (Figure 2). Subgroup MG-Iγ harbored
mostlydeep-waterphylotypescollectedbetween450and3000mwaterdepth[31]. Followingthe
Greekalphabet,theadditionalclustersδ(delta),ε(epsilon),η(eta),andζ(zeta)weresubsequently
proposedtoaccommodatetheincreasingdiversityoftheMG-IArchaeafromhydrothermalventsand
subsurfacesediments[3,32]. ThreeadditionalclustersofsedimentMG-Iphylotypes(κ,kappa;ι,iota;
ν,upsilon)weredefinedtoaccommodatenovelMG-IphylotypesfromSite1225andothermarine
sedimentsandhabitats,includingcoastalandopenoceanwatersworldwide[22,31],LakeMichigan
sediment [33], deep-sea hydrothermal vents [32,34], subsurface water of a Japanese epithermal
goldmine[35],deep-seasubseafloorsediments[36],andfromwithinthewallsofanactivedeep-sea
sulfidechimneyontheJuandeFucaRidge[37]. ThesameMG-Isubgroupswereusedsubsequentlyfor
fine-scalephylogeneticplacementofMG-Iarchaeafromorganic-leansedimentsintheSouthPacific[4].
Interestingly, the MG-I phylotypes from Sites 1225, 1231 and the South Pacific showed very little
phylogeneticoverlapwiththeMG-Iclonesfromdeepsubsurfacesedimentsoforganic-richcontinental
margin sites (Peru Trench Site 1230 and Cascadia Margin Site 1251), which fell mostly into MG-I
groupγ[11].
Deep-Sea Archaeal group (DSAG). Altogether, 24 clones from sample 1225C-1H1 belonged
totheMarineBenthicGroupB(MBG-B)[38],synonymouswithDeep-SeaArchaealGroup[34,35],
andrecentlyrenamedasthedistinctphylumLokiarchaeota[39]. Thisunculturedarchaeallineage
has been found in a wide range of marine sediments and hydrothermal vent samples [1,6,40],
butappearstopreferorganic-rich,reducingsediments[1]. Whilemostdeeplybranchingmembers
ofthislineageweregenerallyfoundathydrothermalvents[20,34,41],atightlyclusteringbranchof
DSAG/MBG-BArchaeaincludespredominantlysequencesfromcoldmarinesediments,surficialas
wellassubsurface[3,35,36,38,42](Figure2). AllDSAG/MBG-BphylotypesfromSite1225fellintothis
clusterofmarinesedimentphylotypes,with100%bootstrapsupport(Figure2). Thesecondsediment
sample,1225C-1H6,didnotyieldDSAG/MBG-Bsequences.
Microorganisms2016,4,32 7of14

Nitrosopumilus maritimus (DQ085097)
Myojin Knoll pMC1A103 (AB019725)!
ODP1225-1H1-1CE1(AY800232)!
Mariana Trench sediment MARIANA15 (D87350) !
Methane seep sediment JTB167 (AB015275)!
88! ODP1225-1H1-1CB1(AY800233
 )!
Mud volcano HMMV47 (AJ579322)
100! ODP1225-1H1-1CB3 (AY800231)!

55! POaDciPfi1c2 d2e5e-1pH-s1e-1aC sBed2 i(mAYen8t0 0M2B2M5)P!A36 (AJ567658)

Pacific deep-sea sediment MBWPA100 (AJ870334)
92! Seawater SBAR5 (M88075)!
59! Cascadia Margin ODPB.A18 (AF121098)!
98! 100! MSeyaowjina tKern collol npeM WC2HAA1R (QAB (M01898702739))!!
 MG-I α !
ODP1230 Peru Trench A20.02 (AB177105)
99! Lake Michigan LMA299 (U87519)!
Lake Michigan LMA226 (U87520)!
66!86! N.Atlantic sediment APA4-0cm (AF119138) ! MG-I!
ODP1225-1H1-1CB5 (AY800224)!
Cenarchaeum symbiosum (U51469)!
57! 100! SSeeaawwaatteerr 4ABT-72 0(U03-79 6(A3F5)2!23113)!
 MG-I β!
100! ODNP1.A2t2la5n-1tHic1 s-1eCdiBm6e (n2t cAl.C) A(A1Y68-90c0m22 0(A)!F119
 144)MG-I κ!
64! 100! 95! ODUPra1n2i2a5 B-1aHs1in-1 sAeEd2im(AeYn8t0 10A22-512)! (AY627475)
  MG-I ϑ!
Pacific deep-sea sediment MBAA46 (AJ567635)
53! 100! 100! PJhaipliapnpeinsee SGeoald smubinsef .p pHPACuAA4-.A9 ((AABB007429702371))!!MG-I η!
Philippine Sea subsf. pPCA4.4 (AB049030)!
64! Japanese goldmine pHAub-30 (AB
 072725)!
84! 100! MuOdD vPo1l2c2a5n-o1 HH1M-1MCVA P6O(AGY3890 0(A21J587)!9320) MG-I ζ!
54! N. Atlantic sediment ACA3-0cm (AF119146)!
ODP1230 Peru Trench A.22.16 (AB177110)

90! 71! 69!UraNn.Aiat lBanastiicn sseeddiimmeenntt A 1CAA-3167 -(1A9Yc6m27 (4A6F01)1!9145)!
100! 80! 100! SOoDuPth12 A2f5r-ic1aHn1 -G1AolEd4m (i2n ec lS. /A 4G cMl.A) -(9A Y(A8B000251092)3!9)!MG-I ε!
67! N.Atlantic sediment CRA7-0cm (AF119125)!
100! PacOifiDc Ps1u2r2fi5c-i1aHl s1e-1dCmD. 6M(AWYP8A04042 1(A7)J!870956)

 MG-I ι!
100! SeawaUtrearn SiaB B95a.s5i7n (sUe7d8im19e9n)!t 1A-39 (AY627
 463)
94! 99! 88!KaOOirDDePPi 11V22e35n01t pPCCearIsRuc AaTd-rZeia1n cM(AhaB Ar0g.91in52 1.A23845. 2)(!A (BA1B7177079208)9)
  MG-I γ !
96! Iheya Basin pIVWA5 (AB019728)!
Kairei vent pCIRA104 (AB108844)!
100! ODP1230 Peru Trench A.10.04 (AB177086)!MG-I δ!
100! 80! Iheya Basin pIVWA1 (AB019727)!
Iheya Basin pIVWA108 (AB019726)!
N.Atlantic sediment ACA10-0cm (AF119143)!
88! 93! N.Atlantic sediment APA2-17cm (AF119135)!
96! 76! 100! ODPO1D22P51O-21D2HP561--124H2D150--111 CH(2F12-5 1 c(ClA.)AY (18A 0Y(208 2c01l0.3)2 )(1!A2Y)!800214)! 1!MBG-A! !
100! ODP1225-1H1-1AA2 (3 cl.) (AY800216)!2 !
ODP1225-1H1-1CC2 (2 cl.) (AY800215)!
Rocky Mountain soil FRD38 (AY016504)!
99! 100! 100! FFiinnnniisshh ffoorreesstt ssooiill FFFFSSBB12 ((XX9966668889))!!
Finnish forest soil FFSB11 (X96696)! FFSB!
99! 100! Finnish forest soil FFSB6 (X96693)!
Thaumarchaeota! 100! South AfricaFni nGnoilsdh M foinrees St AsoGiMl FAF-S2 A(A2B (0Y5008293835))!! SCG!
Wisconsin agricultural soil SCA11 (U62820)!
Lokiarchaeota! Japanese Goldmine pHAuA-5 (AB072723)!
and others! 72! 86!ODP1225-1H1-1AF5 (AY800209)!
74! NaOnDkPa1i 2T2ro5u-1gHh1 s-1uCbEsu2r (fAaYce8 0N0A2N08K)A!8 (AY
 436511)!
OODDPP11223300 PPeerruu TTrreenncchh A2H9.5014 ((DAQB310727013205))

92! ODP1227 Peru Margin 5H2-F22 (DQ30200
 3)
ODP1251 Cascadia Margin A1.17 (AB177262)
Okhotsk Sea subsurface OKHA2.14 (AB094531)!
66! 72! ODP1225-1H1-1AB3 (AY800206)!
52! ODP1O2D25P-112H215--11AHF14-1 (AAYE810 (0A2Y0830)0!207)! DSAG/MBG-B !
50! 60!73! ODP1O2D2P5-112H215--11AHD1-51 (A2Dcl1.) ((AAYY880000220045))!!
North Atlantic sediment CRA8-27cm (AF119128)!
100! Okhotsk Sea subsurface OKHA2.33 (AB094532) !
ODP1225-1H1-1CC3 (7 cl.) (AY800200)!
70! ODP1225-1H1-1AC2 (4 cl.) (AY800202)!
100! 95! O ODDPP11222255-1-1HH11-1-1AADE23 ( A(3Y 8cl0.)0 (1A9Y98)!00201)!
100! Nankai TrOoDuPg1h2 s2u5b-1sHu1rf-a1cAeA M4 A(A-AY18-030 1(A9Y80) 9 !3448
 ) !
91! ODP1227 Peru Margin 5H2-J01 (DQ302005)
100! North Atlantic sediment APA3-11cm (AF119137)!
99! Okhotsk Sea subsurface OKHA4.94 (AB094544)!
100! 79! 100! Japanese vJeunatn p dMeC F2uAc3a6 v (eAnBt 0fl1u9i7d2s0 3)!3-P92A98 (AF355807)

100! Mid-Atlantic vent VC2.1ARC31 (AF068822)!
Guaymas Basin C1R043 (AF419642)!
Myojin Knoll clone pMC2A308 (AB019721)!
74! Okhotsk Sea subsurface OKHA1.28 (AB0
 94525)!
94! 84! ODP1251 Hydrate Ridge A1.15 (AB177260)MHVG-3!
100! ODP1244 Hydrate Ridge A2.3 (AB177229)

Myojin Knoll clone pMC2A15 (AB019718)!
100! Thermococcus celer str. DSM 2474 (AY099174)

100! MethanPoaclaaledooccooccccuuss fjearnrnipahsicluhsii ssttrr.. DDMSJM (2A2B6011 9(M23599)1
 26)
  Cultured Euryarchaeota !
Mid-Atlantic vent sediment cl. pIR3AH01 (AY354110)!
78! 60! 100! 92! Mud OvSDoelPca1aw2na2ot5 ec-r1l ocHnl1oe-n1 KeA AMEZ6E3 -(A4A25Y708 _0(PA0Y92 15(19A)2F!024293)1D!5S0)E!G3!
ODP1225-1H1-1AD3 (AY800210)!
0.01 substitutions/site

Figure2.Phylogenetictree(minimumevolution)ofarchaeal16SrRNAgenes(E.colipositions25–914)
from clone libraries of sediment samples 1225C-1H1 and 1H6. Sequences in the phylogeny that
representmultipleclonesareannotatedwithclonenumbersinparentheses,betweenclonenameand
GenBanknumber. Theannotation“2cl/4cl”indicatesthatcloneODP1225-1H1-1AE4standsfor
2clonesobtainedfromsample1225-1H1and4clonesfromsample1225-1H6.Bootstrapnumbersare
basedon200resamplings. Euryarchaeota(culturedeuryarchaeotaandtheeuryarchaeotallineage
DSEG3)wereplacedastheoutgroup.
Microorganisms2016,4,32 8of14
Marine Benthic Group A (MBG-A). A single sequence from sample 1225C-1H1 (AY800213),
and 22 nearly identical clones from sample 1225C-1H6, represented by clone AY800212, grouped
with Marine Benthic Group A (MBG-A) (Figure 2). This uncultured archaeal cluster was initially
describedfromcoldcontinentalslopeanddeep-seasedimentsintheNorthAtlantic[38];itsmembers
constitutesubgroupMBG-A1[5]. Sevenclonesfromsample1225C-1H1(representedbyAY800214,
AY800215, and AY800216) formed two sister lineages to MBG-A (Figure 2). The latter two clones
belonged to subcluster MBG-A2, also consistently represented by phylotypes from oligotrophic
marinesediments[5].OtherrelativesofMBG-AaremembersoftheFinnishForestSoilBorealGroup
(FFSB) [43] found in boreal forests. These lineages and MBG-A share a common root with good
bootstrapsupportthatseparatesthemfromtheirclosestsistergroup,theSoilCrenarchaotalGroup
(SCG)fromagriculturalsoilsandfromgeothermalwaterinSouthAfricangoldmines[44]. Inturn,
MBG-A,FFSB,andSCGtogethersharebootstrapsupportof99%,andconstituteasisterlineageof
MG-IwithinthephylumThaumarchaeota(Figure2).
Euryarchaeota. Two sequences were members of the Euryarchaeota (Figure 2). Together with
marine sequences from Mediterranean mud volcano sediments (AY592049) and from metal-rich
sediments in the vicinity of hydrothermal vents (AY354110), clone 1AE6 formed a monophyletic
group, termed Deep-Sea Euryarchaeotal Group 3 [5]. Sequence 1AD3 could not be assigned to
aspecificlineage.
DGGEanalysis. TheDGGEprofileofsample1225C-1H1yieldedeightbands;afterexcisingand
elutingtheDGGEbands,sixofthesecouldbesuccessfullyreamplifiedandsequenced. Thesequences
of DGGE bands 1 and 2 were related to MBG-A clones, respectively, whereas the sequences of
bands4,5,7,and8fellwithintheMG-Iarchaea;bands3and6couldnotbeamplified(FigureS1).
Usingadifferentprimercombinationandmethodologythantheclonelibraries,theDGGEanalysis
demonstratedindependentlythatMG-IandMBG-Awereamongthedominantarchaealgroupsinthe
1225C-1H1sedimentsample.
Chimeras. Sevenarchaeal16SrRNAgenecloneswererecognizedaslikelychimeras,andwere
removed from the phylogeny in Figure 2. The last 85 positions of MG-I clone DQ186521 were
derivedfromaDSAG/Lokiarchaeotaphylotype. Thelast60positionsofcloneAY800222deviated
conspicuouslyfromthephylogeneticconsensusoftheMG-Iνgroup. CloneAY800223wasloosely
affiliatedwithMG-1α,butcouldnotbeplacedreliably. ThecloneAY800226combinedaphylotype
verycloselyrelatedtocloneAY800231andamoredistantlyrelatedphylotypewithintheMG-Iαgroup.
ThecloneAY800227combinedaphylotypeverycloselyrelatedtocloneAY800225andamoredistantly
relatedphylotypewithintheMG-Iαgroup. TheclonesAY800229andAY800230withintheMG-Iα
groupconsistedofuptoca. 60%ofadistinctphylotyperelatedtocloneAY800220intheMG-Iκgroup.
CloneAY800228,originallypositionedwithinMG-1α,wasidentifiedasachimerawithMG-Iεand
ζ phylotypes. Excluding these sequences from the phylogeny improved the bootstrap support of
especiallyMG-Iαconsiderably,fromnear50%to98%(Figure2). Wecallattentiontothesechimeras
anddocumentthemclonebyclone,sincetheyoccuralmostentirelyatthelevelofmutuallyclosely
relatedMG-Isubclustersthatareseparatedonlybymax. 3%–4%sequencedivergence,closetothe
thresholdfordefiningoperationaltaxonomicunits[4]. Afine-grained,andatthesametimerobust,
phylogeneticframeworkthatresolvestheactuallyexistingmicroclusterswithinaphylogeneticlineage
facilitatestheirdetection.
4. Discussion
Downcore trends in archaeal detection. Although deep sediment samples were repeatedly
extractedusingdifferentapproaches,onlytwosamplesfromtheuppersedimentcolumn,1225C-1H1
and1H6,yieldedPCR-amplifiableDNA.SimilardifficultiesofobtainingPCR-amplifiablearchaeal
DNAfromdeepersedimentlayersoccurredintheSouthPacificGyre,mostextremelyatSiteSPG11
whereonlytheupper30cmofthesedimentcolumnyieldedworkablematerial[4]. Quantifications
ofarchaealpopulationsinorganic-richsedimentcoresfromthePeruMarginandPeruTrench(ODP
Microorganisms2016,4,32 9of14
Sites1227and1230)using16SrDNA-targetedq-PCRshowedthatthehighestdensitiesofarchaeal
16S rRNA gene copies were found near the sediment surface, and decreased by several orders of
magnitudetowardsdeepersedimenthorizons[10]. Similarpatternsofrapidlydecreasingarchaeal
gene numbers were found by qPCR analysis of the oligotrophic sediment column at North Pond,
asedimentedbasinnearthemid-AtlanticRidge[45]. Thesetrendsarebroadlyconsistentwiththe
downcoreprofilesofdirectcellcountsinoligotrophicsedimentsintheSouthPacificGyre,withthe
caveatthatthemethoddoesnotdistinguishbetweenarchaeaandbacteria[2,46].
Range and diversity of MG-I archaea in marine sediments. Detecting members of the MG-I
ArchaeaatSite1225andinmanyotherbenthicmarinesediments[1,4,6]raisesthequestionwhether
theseArchaeacansurviveandgrowinmarinesediments, potentiallybynitratereductionorother
anaerobicmetabolisms,orwhethertheyrepresentsedimentationfrompelagic,aerobicpopulationsin
thewatercolumn.SinceMG-IArchaeaarehighlyabundantinoceanwater,andconstitutethedominant
cMoimcroporognaneinsmts o2f01b6,a 4c,t 3e2r ial and archaeal plankton in the mesopelagic water column [47], they c9o oufl 1d3
contribute to the archaeal populations innear-surface sediments. However, if theirrange extends
ienxttoenadnso xinictos uabnsouxrifca cseusbesduirmfaecne tss,edMimGe-Intasr,c hMaGea-Ia raercuhnaeliak ealyre tounrleipkreelyse ntot dreepproessietinotn dalepreomsintiaonntasl
frreommnathnetso fxryogme ntahtee doxwyagteenractoedlu mwnat,ear ncdolcuomnsnt,i taunted incodnigsteintuotues inpdopiguelnaotiuosn spoopfumlaatriionnes soefd immaernintse;
tsheidsiimnteenrtpsr; etthaitsi oinntweropureldtaatilosno bweocuolnds aisltseon btew ciothnstihsetednet twecittiho nthoef dspeteeccitfiiocns eodfi mspeenctifMic Gse-dIismubecnltu MsteGr-sI.
Asucbocmlupstielarsti.o An coofmcapsielastitound ioefs c(aisnec ls.tuthdeiesre (sinucltls. tohfet rheissuslttus doyf )thsihso swtusdtyh)a sthtohwesd tihstarti bthueti donistorfibMutGio-nI
aorfc hMaGea-Ii narmchaareian eins emdiamriennet sceodriemseanptp ceoarresst oapbpeelainrsk etod btoe tlhinekaevda tiloa bthileit yavoafiltahbeileitlyec otrfo tnhea cecleepcttroorns
oaxccyegpetnorasn doxnyitgreante ainndp onreitwraattee ri;nM pGo-rIeawrcahteare; aMcaGn-aI lsaorcehxateean dcabne loawlsot heexztoennde obfeploowre wthaete rznonitera toef
dpeoprelewtiaotneri fnnitirtraatete dreepmleatiinosna ivf aniiltarbaltee ardemsoaribnesd atvoatilhaebsleo laiddspohrbaseed (tFoi gthuer eso3l)i.d phase (Figure 3).

FFiigguurree 33.. BBaarr ddiiaaggrraamm sshhoowwiinngg ppeerrcceennttaaggeess ooff MMaarriinnee GGrroouupp II ((MMGG--II)) rreepprreesseennttaattiioonn iinn aarrcchhaaeeaall 1166SS
rrRRNNAA ggeenneec loclnoenleib lriabrriaersi,edse, ndaetunaratunrtagnrat dgireandtigeenlte lgeeclt reolpehctorroepsihso(rDeGsiGs E(D)aGnGdEp)y raonsdeq puyenrocsinegqupernoficilnegs
fporromfilaersi nfeosr edmimareinnet csoerdeimsaemnpt lecso,rceo lsoarm-cpodleesd, bcoyloelre-cctordoenda cbcye petolercatrvoanil aabciclietpy.toCro laovrasitlaribpielistyin. dCicoaloter
esvtriidpeensc iendfoicractoe eexvisidtienngcee lefocrtr oconeaxciscteinpgto ersleoctrropna tahcwceapytso,ros roarm pbaitghuwitayysi,n otrh eamdabtiag.uOityc eiann tDher idlliantag.
POrcoegarna mDr(iOlliDngP )Psriotgernamum (ObeDrsP)r espitree nseunmtbEeqrus arteoprrieaslePnat cEifiqcuaStioteria1l2 2P5ac[itfhicis Ssitteu d12y2],5 P[tehrius sMtuadrgyi]n, PSeirteu
1M22a7rg[i1n1 ]S,iPtee r1u22B7a s[i1n1]S,i tPee1r2u3 B1a[s3i]n, aSnidte P1e2r3u1 T[r3e]n, cahndS itPee1ru23 T0r[e1n1c]h. ASritceti c12O3c0e [a1n1]c.o ArersctGicC O12ceaannd cGoCre6s
fGroCm12s eadnidm GenCt6c forroems nseeadrimtheenht ycdorreost hneeramr athlley hayctdivroetGhearkmkaelllRy iadcgteivwe eGraekakneall yRzidedgeb wye1r6eS arnRaNlyAzegde nbey
p1y6Sro rsReqNuAe ngceinnge p[6y]r.oSsPeqGu1e1ncainndg S[6P]G. S1P2Gre1f1e rantod SSoPuGt1h2 Praefceifir ctoG Syoruethco PreacSifPicG G11yraen cdorSeo uSPthGP11a caifindc
cSooruethS PPGac1i2fic[ 4c,o5r].e DSPGGG1E2 [s4lu,5r]r. yDdGaGtaE rseluferrryt odaDtaG rGefEerr etos uDltGsGfrEo mressutlatbs lferocmar bstoanblies octaorpboenp irsoobtoinpge
epxrpoebriinmg eenxtpsewriimthensetsd iwmiethn tssefdriommenthtse fSroevme rtnheE sSteuvaerryn, UEKstu[a4r8y]., UnK.d .,[4n8o].t nd.edt.e, cnteodt ;dmet.edc.t,emd;i smsi.ndg.,
dmaitsas.inDga tdaautan.d Dearltyai ungndtherislybianrg dthiaigs rbaamr dairaegcroammp airleed coinmTpaibleled Sin1. Table S1.
In South Pacific sediments of core SPG12, MG-I archaea persisted at 1.53–1.63 mbsf but were no
longer detectable at 2.13–2.23 mbsf; porewater nitrate was entirely consumed near 2.5 mbsf [5]. In
sediments of the Peru Basin at ODP Site 1231, MG-I sequences were obtained only from a sample at
1.8 m depth in the near-surface core [3]; porewater nitrate at above-background concentrations was
detected within the upper 1 m [49]. At Site 1225, multiple MG-I subgroups were found in the upper
sediment sample at 1.05–1.10 mbsf where porewater nitrate remained available. Only MG-I
subgroup ε was detected in the deeper sample at 7.75–7.80 mbsf, below the penetration depth of
porewater nitrate. In a similar case from subsurface sediments of the Arctic Ocean, MG-I archaea
occurred in two gravity cores with a length of ca. 3 m each (GC6 and GC12 in Figure 3). These cores
were depleted in porewater nitrate below 30–40 cmbsf, but rich in Mn2+ and Fe2+; however, nitrate
remained available in the solid phase where it persisted, presumably due to absorption to mineral
particles [6]. Interestingly, MG-I archaea were found in DGGE analyses of stable carbon isotope
probing (SIP) incubations with 13C-CO2, inoculated with oxic and anoxic surficial intertidal
sediments from the Severn Estuary, UK [48]. When sulfate-reducing sediments below 20 cm depth
were used, MG-I archaea were no longer detected by DGGE but they appeared as a minority
population in a clone library analysis [48]. In a well-documented but so far isolated case, MG-I
archaea were detected consistently in clone library analyses of sulfate-reducing and methanogenic,
even hydrate-containing deep subsurface sediments of ODP Site 1230 in the Peru Trench [11], with
Microorganisms2016,4,32 10of14
In South Pacific sediments of core SPG12, MG-I archaea persisted at 1.53–1.63 mbsf but were
nolongerdetectableat2.13–2.23mbsf; porewaternitratewasentirelyconsumednear2.5mbsf[5].
InsedimentsofthePeruBasinatODPSite1231,MG-Isequenceswereobtainedonlyfromasample
at 1.8 m depth in the near-surface core [3]; porewater nitrate at above-background concentrations
wasdetectedwithintheupper1m[49]. AtSite1225,multipleMG-Isubgroupswerefoundinthe
uppersedimentsampleat1.05–1.10mbsfwhereporewaternitrateremainedavailable. OnlyMG-I
subgroup ε was detected in the deeper sample at 7.75–7.80 mbsf, below the penetration depth of
porewater nitrate. In a similar case from subsurface sediments of the Arctic Ocean, MG-I archaea
occurredintwogravitycoreswithalengthofca. 3meach(GC6andGC12inFigure3). Thesecores
weredepletedinporewaternitratebelow30–40cmbsf,butrichinMn2+ andFe2+;however,nitrate
remainedavailableinthesolidphasewhereitpersisted,presumablyduetoabsorptiontomineral
particles [6]. Interestingly, MG-I archaea were found in DGGE analyses of stable carbon isotope
probing(SIP)incubationswith13C-CO ,inoculatedwithoxicandanoxicsurficialintertidalsediments
2
fromtheSevernEstuary,UK[48]. Whensulfate-reducingsedimentsbelow20cmdepthwereused,
MG-IarchaeawerenolongerdetectedbyDGGEbuttheyappearedasaminoritypopulationinaclone
library analysis [48]. In a well-documented but so far isolated case, MG-I archaea were detected
consistentlyinclonelibraryanalysesofsulfate-reducingandmethanogenic,evenhydrate-containing
deepsubsurfacesedimentsofODPSite1230inthePeruTrench[11],withthecaveatthatMG-Iarchaea
couldnotbedetectedinanindependentarchaealcommunityanalysisofthesamesiteusingreverse
transcriptionofRNA[12].
Compiling the overall contribution of MG-I archaea to clone libraries, DGGE profiles,
andpyrosequencingsurveysofmarinesedimentsinrelationtooxic,nitrate-reducing,metal-reducing,
sulfate-reducing,ormethanogenicgeochemicalregimerevealsthepersistenceofMG-Iarchaeaunder
oxicandnitrate-reducingconditions,thelatteroverlappingwithmetal-reducingconditions;theyare
rarelyseenundersulfate-reducingconditions,andtheirabundantdetectioninmethanogenicsediments
ofODPSite1230standsoutasunique(Figure3,andTableS1).
ODPSite1225anditsarchaealcommunityincomparativeperspective. Extendingaprevious
comparative study [1], marine subsurface sediments can be placed into different categories of
organic carbon content and redox status in the subsurface sediment column (data tabulated in
TableS1). Extremelyorganic-depletedsediments,forexamplethoseoftheSouthPacificGyre[50],
arecharacterizedbyatotalorganiccarbon[TOC]contentbelow0.05wt. %andcontainporewater
oxygenandnitratethroughouttheirsedimentcolumndowntobasementbasalt;clonelibrariesfrom
thissedimenttypeareavailablefromSPG11[4]. Thenextcategory,organiccarbon-depletedsediments
withatotalorganiccarboncontentbelow0.5wt. %,arecharacterizedbyoxygenandnitratedepletion
withinafewmeters,followedbymetalreduction;yetporewatersulfateextendslargelyunchangedto
basementandsulfidedoesnotaccumulate. WithTOCcontentbetween0.2and0.4wt. %,ODPSite
1225representsthiscategory[18].SPG12ontheedgeoftheSouthPacificGyre[5]andODPsite1231[3]
alsoprovidegoodexamplesfororganiccarbon-depletedsediments. CoringsitesintheSouthChina
Seaappeartofallintothiscategoryaswell,withthecaveatthatnitrateconcentrationsarenotavailable
(TableS1). Thewell-documentedArcticBasincoresGC6andGC12[6]areattheorganic-richend
ofthiscategory;someTOCvaluesexceed>1%,duetotheshallowdepth(ca. 3m)ofthesegravity
cores(TableS1);however,theTOCcontentofsubsurfacesedimentsbelow3mbsfremainsunknown.
Forthefollowingcategory,intermediateorganic-depletedsediments,anupperTOCconcentration
thresholdof2wt. %wasselectedascutoffpoint,basedontheexamplesTableS1. Thesesediments
arecharacterizedbysurficialoxygenandnitratepenetration,andsignificantsulfatedepletionand
sulfide accumulation in the deep sediment column. ODP Site 1226 in the Equatorial Pacific near
GalapagosandIODPSite308inBrazosBasin,GulfofMexico,provideexamples(TableS1). Atlast,
organiccarbon-richsedimentswithTOCconcentrationsabove2%arecharacterizedbyoxygenand
nitratedepletionwithinafewcentimeters,andsulfatedepletionleadingtomethanogenesisinthe