Diversity of Methane-Cycling Archaea in Hydrothermal Sediment Investigated by General and ...

16 Pages·2015·2.27 MB·English

Diversity of Methane-Cycling Archaea in Hydrothermal Sediment
Investigated by General and Group-Specific PCR Primers
MarkA.Lever,a,b*AndreasP.Teskea
DepartmentofMarineSciences,UniversityofNorthCarolinaatChapelHill,ChapelHill,USAa;CenterforGeomicrobiology,DepartmentofBioscience,AarhusUniversity,
Aarhus,Denmarkb
Thezonationofanaerobicmethane-cyclingArchaeainhydrothermalsedimentofGuaymasBasinwasstudiedbygeneralprimer
pairs(mcrI,ME1/ME2,mcrIRD)targetingthealphasubunitofmethylcoenzymeMreductasegene(mcrA)andbynewgroup-
specificmcrAand16SrRNAgeneprimerpairs.ThemcrIRDprimerpairoutperformedtheothergeneralmcrAprimerpairsin
detectionsensitivityandphylogeneticcoverage.MethanotrophicANME-1Archaeaweretheonlygroupdetectedwithgroup-
D
specificprimersonly.Thedetectionof14mcrAlineagessurpassesthediversitypreviouslyfoundinthislocation.Mostphylo-
o
typeshavehighsequencesimilaritiestohydrogenotrophs,methylotrophs,andanaerobicmethanotrophspreviouslydetectedat w
n
GuaymasBasinorathydrothermalvents,coldseeps,andoilreservoirsworldwide.Additionally,fivemcrAphylotypesbelonging lo
tonewlydefinedlineagesaredetected.Twoofthesebelongtodeeplybranchingneworders,whiletheothersarenewspeciesor a
d
generaofMethanopyraceaeandMethermicoccaceae.Downcorediversitydecreasesfromallgroupsdetectedintheupper6cm e
((cid:2)2to40°C,sulfatemeasurableto4cm)toonlytwogroupsbelow6cm(>40°C).Despitethepresenceofhyperthermophilic d

genera(Methanopyrus,Methanocaldococcus)incoolersurfacestrata,nogenesweredetectedbelow10cm(>60°C).WhilemcrA- fr
o
basedand16SrRNAgene-basedcommunitycompositionsaregenerallycongruent,thedeeplybranchingmcrAcannotbeas- m
signedtospecific16SrRNAgenelineages.Ourstudyindicatesthatevenamongwell-studiedmetabolicgroupsandinpreviously h
t
characterizedmodelenvironments,majorevolutionarybranchesareoverlooked.Detectingthesegroupsbyimprovedmolecular tp
biologicalmethodsisacrucialfirststeptowardunderstandingtheirrolesinnature. :/
/
a
e
m
Hydrothermal surface sediments in Guaymas Basin support ThefactthatrevisedgeneralandnewANME-1-specificmcrA .a
exceptionallyhighmicrobialactivityandmicrobialdiversity primersdetectedawiderphylogeneticrangeofmcrAgenediver- s
m
(1–4).Thehighlyproductiveoverlyingwatercolumn,combined sitythanthatobtainedinpreviousstudiesonGuaymasBasinsed-
.
o
with terrestrial runoff, leads to sedimentation rates exceeding 1 iments (10, 11) raises several questions. (i) How do published r
mmyr(cid:3)1(5)andorganiccarboncontentsof2to4%byweight(6). generalmcrAprimerpairscompareindiversitycoverageandde- g/

Inaddition,theupwardflowofhydrothermalfluidssupplieshy- tection sensitivity? (ii) To what extent do these general mcrA o
n
drocarbons (methane, petroleum) and volatile fatty acids pro- primerpairscoverthediversityofmcrAgenespresentinGuaymas A
ducedbythermaldegradationofburiedorganicmatterindeeper Basinsediment?(iii)HowdodifferentsiteswithintheGuaymas p
r
sedimentlayerstosurfacesedimentsandtheirmicrobialcommu- Basin compare in terms of methanogen and anaerobic metha- il
nities(1,7–9). notrophdiversity?(iv)HowdoGuaymasBasinmethanogenicand 1
0
Methanogenesisandsulfate-dependentmethaneoxidationin anaerobic methanotrophic communities compare to methano- ,
2
GuaymasBasinarecarriedoutbydiversemicrobiallineages.16S genicandanaerobicmethanotrophiccommunitieselsewhereon 0
rRNAgene-basedsurveyshavedetectedknownmethane-cycling Earth? 1
9
groups(Methanococcoides,Methanocorpusculum,Methanoculleus, WeexaminethesequestionsinsurficialsedimentsoftheEver- b
Methermicoccus,ANME-1,ANME-2)(4,10(cid:3)12),anewlineageof est Mound area in the southern Guaymas vent field, home to y

ANME-1,ANME-1Guaymas(12),andunknowndeeplybranch- diverselineagesofmethanogensandmethanotrophs(4,10).We gu
ing euryarchaeotal groups within the phylogenetic vicinity of e
s
knownmethane-cyclingArchaea(4,10).Phylogeneticanalysesof t
mcrA,agenediagnosticofmethanogenicandanaerobicmetha- Received29October2014 Accepted10December2014
notrophicArchaea(13,14),indicateanevenhigherdiversityof Acceptedmanuscriptpostedonline19December2014
methane-cyclingArchaeathanthosedetectedby16SrRNAgene CitationLeverMA,TeskeAP.2015.Diversityofmethane-cyclingarchaeain
surveys (Methanococcoides, Methanohalophilus, Methanosaeta, hydrothermalsedimentinvestigatedbygeneralandgroup-specificPCRprimers.
ApplEnvironMicrobiol81:1426–1441.doi:10.1128/AEM.03588-14.
Methanoculleus, Methanocorpusculum, Methanocaldococcus, Me-
Editor:C.R.Lovell
thermicoccus,groupe,ANME-1,ANME-2[10,12])and,usinga
AddresscorrespondencetoMarkA.Lever,[email protected].
revisedgeneralmcrAprimerpair(15),anoveldeeplybranching
*Presentaddress:MarkA.Lever,InstituteforBiogeochemistryandPollutant
mcrA cluster (12). Perhaps surprisingly, the anaerobic metha-
Dynamics,DepartmentofEnvironmentalSystemsSciences,ETHZürich,Zürich,
notrophicANME-1cluster,adominantgroupinclonelibrariesof Switzerland.
archaeal16SrRNAgenes(4),wasinitiallyabsentfrommcrAclone Supplementalmaterialforthisarticlemaybefoundathttp://dx.doi.org/10.1128
libraries(10).Thisinconsistencyhassincebeenresolvedbyusing /AEM.03588-14.
anANME-1-specificmcrAprimerpair(15),withwhichhighdi- Copyright©2015,AmericanSocietyforMicrobiology.AllRightsReserved.
versityandwidespreadoccurrenceofANME-1inGuaymasBasin doi:10.1128/AEM.03588-14
sedimenthavebeenshown(12).
1426 aem.asm.org AppliedandEnvironmentalMicrobiology February2015 Volume81 Number4
Methane-CyclingArchaeainHydrothermalSediment
first compare the detection sensitivity and diversity coverage of Nucleotidesequenceaccessionnumbers.Nucleotidesequenceswere
three degenerate, general mcrA PCR primer pairs, designed by depositedtotheGenBankarchiveunderthefollowingaccessionnumbers:
Springeretal.(13),Halesetal.(16),andmorerecentlyLever(15). for mcrA, KM370762 to KM370786; for archaeal 16S rRNA genes,
Wethenevaluatedetectionsensitivityandphylogeneticbiasesby KM370787toKM370815).
comparing clone libraries based on these general mcrA primer
pairs to ones obtained using 27 nondegenerate, group-specific RESULTS
mcrAprimerpairs(15).Asafurthercheckforthephylogenetic Generalresults.DNAextractionsweresuccessfulto10cmbelow
rangeofgeneralandgroup-specificmcrAprimers,wecomparethe seafloor(cmbsf),atwhichthesedimenttemperaturewas(cid:2)60°C
total community detected based on mcrA sequences to that de- (Fig. 1A). Sulfate concentrations decreased steeply in the upper
tectedwith17newmethanogen-andanaerobicmethanotroph- cmbsf and were below the detection limit ((cid:2)0.1 mM) below a
targeted16SrRNAgeneprimerpairs.Weconcludewithananal- 4-cmdepth,indicatingactivesulfatereductionwithinthesurface
ysis of methane-cycling archaeal zonation within the steep layersandsulfatelimitationbelow(Fig.1B).Theabsenceofsulfate
thermalandsulfategradientsofthesite,investigatepossiblemet- inthedeepersedimentlayersindicatesminimalcoredisturbance
abolicpathwaysofphylotypesdetectedbasedonclosestrelatives andseawaterin-mixingduringsamplingandretrieval.
withknownmetabolisms,andcomparetheoverallphylogenetic ThecombinedmcrAandarchaeal16SrRNAgenesurveysiden- D
rangeandcommunitystructuretothosefoundinpreviousGuay- tifiedatotalof22phylotypesofmethane-cyclingArchaea.Here, ow
masBasinsedimentstudiesandotherhabitatsworldwide. distinctphylotypesweredefinedashaving(cid:5)97%sequencesimi- n
larity to other sequences detected in this study; the same 97% lo
a
MATERIALSANDMETHODS cutoffisusedconsistentlyformcrAand16SrRNAgenes.Simi- d
larly, all mcrA sequences from this and previous studies that e
Samplingandsitecharacteristics.Thesedimentcoreusedinthisstudy shared(cid:2)97%sequencesimilaritywereclassifiedasbelongingto d
wasobtainedduringAlvinDive3204fromthe“EverestMound”areain fr
thesamephylotype.Atotalof21phylotypesweredetectedamong o
thesouthernGuaymasTrench.Temperaturegradientsweredetermined m
themcrAsequences(Fig.2),andonlyoneadditionalphylotype,
insituusingAlvin’shigh-temperatureprobe.Theporewatersulfatecon-
belonging to the candidate order “Methanoplasmatales,” was h
centrationprofilewasdeterminedusingstandardionchromatographic t
t
methods(12).Anextendedsitedescriptionisprovidedinthesupplemen- found among the 16S rRNA gene sequences (see Fig. S4 in the p
:
talmaterial. supplementalmaterial).Severaladditional16SrRNAphylotypes //
a
DNAextractionandprimerdesign.DNAwasextractedaccordingto belongingtoeuryarchaeotalgroupswithoutknownphysiologies e
Leveretal.(17;seealsothesupplementalmaterial).Allprimerinforma- might, however, be involved in methane cycling. Of the phylo- m
tionisshowninthesupplementalmaterialandinTable1.Thegeneral typesthatcouldbelinkedtomethanecyclingbasedonmcrAde- .a
mcrIRDprimerpairandthegroup-specificmcrAprimerpairsaswellas tectionorbasedon16SrRNAgenesequencesthataremonophy- sm
allnewarchaeal16SrRNAgeneprimersweredesignedbasedonDNA
letic with known methane-cycling Archaea, 20 fall into groups .
o
sequence alignments in the ARB software (http://www.arb-home.de/
withknownenergysubstrates.Theremainingtwo mcrAphylo- r
[18]) and tested with the DNA extracts. Annealing temperatures were g
basedoncalculatedmeltingtemperatures.Foreachprimerpair,thelower types(termed“DeeplyBranchingmcrAgroupsIIandIII”)lack o/
close cultured relatives or in fact any environmental sequences n
ofthetwomeltingtemperatureswasusedasannealingtemperature.

withhighsequencesimilarity(Fig.2;Table1).Thenumbersof A
PCRprotocol.PCRassayswereperformedwiththeTaKaRaSpeed-
STARHSDNApolymerasekit(TaKaRaBioUSA,Madison,WI)usingthe phylotypeshadabimodaldistribution,withthehighestnumbers pr
manufacturer’srecommendedreactionmixture,exceptthatbovinese- incool,sulfate-containingsurfacesamples(0to2cm,(cid:2)2to12°C) il
1
rumalbuminwasaddedtoafinalconcentrationof1(cid:4)g(cid:4)l(cid:3)1.ThePCR andinawarm,sulfate-depletedlayer(5to6cm;(cid:2)20to30°C) 0
protocolwasasfollows:(i)one2-mindenaturation(98°C),(ii)40cycles (Fig.2;Tables2and3;seealsoFig.S4inthesupplementalmate- ,
2
of10-sdenaturation(98°C),30-sannealing(Table1fortemperatures), rial).Onlytwophylotypesweredetectedbelow6cmbsf. 0
and1-minextension(72°C),and(iii)one5-minextension(72°C).For Among the closest BLAST hits to the 21 mcrA phylotypes, 8 19
assayswithgeneralmcrAprimers,10(cid:4)lofDNAextractwasused.Forall werefromGuaymasBasinsediment,1wasfromaGuaymasBasin b
other assays, 1 (cid:4)l of extract was used. Where necessary, 1 (cid:4)l of PCR y
hydrothermalvent,and12werefromoutsideGuaymasBasin(Ta-
productfromthefirstPCRwastransferredtotubescontainingfreshPCR g
ble2).TheseclosestBLASThitsincludedthepure-cultureisolates u
reagentsandreamplifiedforasecondPCRof40cycles. e
MethanocaldococcusjannaschiiandMethanopyruskandleri,both
Cloningandsequencing.PCRproductswerepurifiedina2.5%low- s
ofwhichwerefirstisolatedfromahydrothermalventandahy- t
melting-point agarose gel using Tris acetate-EDTA (TAE) buffer. Gel
slicescontainingthecorrectPCRfragmentlengthwereexcisedandpuri- drothermalsedimentinGuaymasBasin,respectively(19,20).
fiedusingtheSN.A.P.minikit(Invitrogen,Carlsbad,USA)andcloned mcrAdiversity.Wedesignedasequencesimilaritymatrixfor
intoelectrocompetentEscherichiacoliusingtheTopoTAkit(Invitrogen, taxonomicclassificationofmcrAphylotypesthatisanalogousto
Carlsbad,CA,USA).Plasmidextraction,purification,andcyclesequenc- sequencesimilaritycutoffsthathavebeenemployedtoclassify16S
ingwereperformedattheJosephineBayPaulCenterattheMarineBio- rRNAgenesequencesfordecades(33,34).TodiscussmcrAdiver-
logicalLaboratory(WoodsHole,MA). sityinconsistentterms,weusetheterm“group”torefertose-
Phylogenetictrees.SequenceswereBLASTanalyzed(www.ncbi.nlm quencesclassifiedasformingtheirownfamily,order,orclass,the
.nih.gov/blast).Chimeraswereidentifiedbyvisualalignmentchecksin
term“cluster”torefertosequencesclassifiedasformingtheirown
ARBandusingtheonlinesoftwareDatabaseEnabledCodeforIdealProbe
genus,andtheterm“subcluster”torefertosequencesclassifiedas
Hybridization Employing R (DECIPHER; http://decipher.cee.wisc.edu
belonging to the same phylotype. In the following, we discuss
/index.html).PhylogenetictreeswerecreatedusingARBneighborjoining
mcrA diversity at Everest Mound based on this classification
with Jukes-Cantor correction and bootstrap analyses with 1,000 repli-
cates.ThetaxonomicidentificationandclassificationofnovelmcrAphy- scheme.Forunculturedclusters,thesedesignationsarenecessar-
lotypeswerecheckedandsubstantiatedwithnucleotidesequencesimilar- ilyqualified,forexample,byreferringto“genuslevellineages.”
itymatrixes,asspecifiedinthesupplementalmaterial. WithintheMethanosarcinales,wedetected3phylotypesinthe
February2015 Volume81 Number4 AppliedandEnvironmentalMicrobiology aem.asm.org 1427
LeverandTeske
rswith cSpecificity (cid:6) (cid:6) (cid:6) (cid:6) (cid:3) (cid:6) (cid:3) (cid:3) NA NA (cid:3) NA NA (cid:3) (cid:3) (cid:3) (cid:3)(cid:6)(/) (cid:3)(cid:6)(/) (cid:6) (cid:3) (cid:3)(cid:6)(/) (cid:3)(cid:6)/ (cid:3) (cid:6) (cid:6) (cid:3) (cid:6) (cid:3) (cid:3)
e
m
ri bon
genep mplificati
A A (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:3) (cid:3) (cid:6) (cid:3) (cid:3) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:3)
N
16SrR Tanneal(°C) 51 58 55 63 66 64 64 64 60 64 60 64 64 64 64 56 61 62 61 63 63 61 63 64 64 64 64 64 65
rchaeal Length(bp) (cid:2)490 (cid:2)750 (cid:2)490 (cid:2)480 425 155 305 385 525 480 480 560 510 445 550 335 310 290 355 515 365 490 315 385 410 255 395 330 955
a
mcrAand No.ofnucleotides 17;17 20;21 17;17 20;20 20;20 20;20 20;20 22/21;17 20;20 20;22 21;17 18;20 19;20 19;19 19;20 23;20 21;20 23;19 21;18 20;22 24;23 21;23 22;18 23;18 21;22 22;18 18;19 22;17 20;19 Dow
of n
ccess GGA loa
u T d
plifications ACACTGGTCC ed from
m C
a AC h
and CAC TG/C ttp
mperatures, CAGCAGCATA ACACTGGTCC ://aem.a
lengths,ampliconlengths,annealingte Primersequences =F,5-TAYGAYCARATHTGGYT=R,5-ACRTTCATNGCRTARTT=F,5-CMATGCARATHGGWATGTC=R,5-TCATKGCRTAGTTDGGRTAGT=F,5-TWYGACCARATMTGGYT=R,5-ACRTTCATBGCRTARTT=F,5-GACCAGTTGTGGTTCGGAAC=R,5-ATCTCGAATGGCATTCCCTC=F,5-GACCAGATCTGGCTCGGATC=R,5-TCGCCCTGGTAGGACAGAAC=F,5-GGATTCACGCAGTACGCAAC=R,5-CAAGAAGCGTTGGGTAGTCC=F,5-TACACCAACGATGTCCTGGA=R,5-CACTGATCCTGCAGGTCGTA=F,5-TATGCAACACCAGCATACACC/GTATGCCA=R,5-CACCGCACTGATCCTGC=F,5-GATATCATTCAGACAAGCCG=R,5-AGTTCAAGAGGCTCTCCTTC=F,5-CCTTGAGGTAGTCGGTGCAG=R,5-AGTTCAAGAGGCTCTCCTTCGT=F,5-GATATCATTCAGACAAGCCGT=R,5-CACCACACTGGTCCTGC=F,5-TACAAGATGTGCGCCGGT=R,5-CATGCTTCCTTGTGCAGGTA=F,5-AGCCAGGTGGCATCAAGTT=R,5-ACTGGTCCTGCAGGTCGTAG=F,5-AGCCAGGTGGCATCAAGTT=R,5-GACAGGTACCAGCCGTTCA=F,5-TGTCATCAACATGGCCCAC=R,5-TCGTAGCCGAAGAAACCAAG=F,5-GATGAGTTCACCTACTATGGTAT=R,5-CTGACAGAGAGTGAGTTGGT=F,5-CACCTACTACGGTATGGACTA=R,5-GAGTTTGCTGAACCACACTG=F,5-GGTATGGACTACATCAAGGACAA=R,5-ACTGGTCCTGGAGGTCGTA=F,5-CACCTACTACGGTATGGACTA=R,5-AGCTCTCCGAGCAGACCT=F,5-GCAAAACACGCAGAAGTTGT=R,5-GTCTGGAGTGCTGTTCTTTGTG=F,5-GGTTAGGTTCTTACATGTCTGGTG=-GCACCACATTGATCTTGTAAATC/TGCTCCR,5=F,5-AAGAAGAGCAAGAGGTCCAAA=R,5-TCGTATCCGTAGAATCCTAATCT=F,5-AGCCTACACAGACAACATCCTC=R,5-CACCACACTGGTCCTGGA=F,5-CTAGGATCCTACATGTCAGGAGG=R,5-CCTCACGCTCAGCGAGTT=F,5-GGGAGTAGGATTCACGCAGTA=R,5-GATAGTTTGGACCACGCAGTTC=F,5-ACGACTTCTGCTACTACGGTGC=R,5-CCTGCCCATCTCCTCCTT=F,5-GCAGTATGCAACCGCTGTT=R,5-GTCTGCACCTCTGAGCTCAAG=F,5-GTGTACACGGACAACATCCTGG=R,5-ACGCTCAGCGAGTTGGC =F,5-TGCTGGAATGCTTTATGCGT=R,5-CCGGAGGACATGCTGGTAA on April 10, 2019sm.org/
rgetgroups,nucleotidesequences,primeraEverestMound Targetgroups General General GeneralexceptANME-1 ANME-1,ANME-1-relatedgroup Methanosarcina ANME-2 Methanosaeta UnidentifiedRiceFieldSoilMcrA/Zoigecluster ANME-3 ANME-3 ANME-3,Methanococcoides,Methanomethylovorans,Methanolobus,MethanohalophilusRiceClusterI,Fencluster Fencluster Fencluster Methanocorpusculum Methanospirillum Methanomicrobiales Methanoculleusandcloserelatives GuaymasBasinMethanomicrobialesmcrAcluster Methanobacteriumaarhusensegroup MethanobacterialesexceptM.aarhusenseandMethanothermobacterMethanococcaceae Methanothermobacter Methanopyruskandleri DeeplyBranchingmcrAgroupII Clonemlas,cloneDEBITSandrelatives DeeplyBranchingmcrAgroupIII MethanopyrusmcrAsubclustersIandII Methanosarcina by guest
TABLE1Names,taDNAextractsfrom Name mcrAgeneprimersmcrI ME1/ME2 mcrIRD ANME-1-mcrI mcrMS mcrANME-2 mcrMsaeta mcrURFS mcrAM-3I mcrAM-3II mcrAM-3etal mcrRCI/FC mcrFCI mcrFCII mcrMcorp mcrMspir mcrMmicrob mcrMcul mcrGMcul mcrMbacA mcrMbac mcrMcoc mcrMtb mcrMpK mcrDBGrII mcrMlas mcrDBGrIII mcrMpyralesI&II Archaeal16SrRNAgeneprimersMS183F/MS1138R
1428 aem.asm.org AppliedandEnvironmentalMicrobiology February2015 Volume81 Number4
Methane-CyclingArchaeainHydrothermalSediment
(cid:3) (cid:3)(cid:6)/ (cid:3)(cid:6)(/) NA (cid:3)(cid:6)(/) (cid:3)(cid:6)(/) (cid:6) (cid:6) (cid:3)(cid:6)/)( NA NA NA NA NA (cid:3)(cid:6)(/) (cid:3)(cid:6)(/) plicable.
p
a
ot
n
A,
N
d.
fie
pli
m
(cid:6) (cid:6) (cid:6) (cid:3) (cid:6) (cid:6) (cid:6) (cid:6) (cid:6) (cid:3) (cid:3) (cid:3) (cid:3) (cid:3) (cid:6) (cid:6) werea
nes
ge
A
mcr
62 64 64 60 62 65 65 60 65 64 62 64 64 65 64 64 n-
o
n D
y
nl o
o w
(cid:3),
n
825 934 465 592 659 356 1123 856 1003 852 737 736 563 644 786 748 mplified; load
a e
were d
19;20 19;21 22;20 20;20 20;21 20;21 20;19 23;18 21;19 21;19 20;19 18;19 20;19 19;22 19;20 19;20 omLever(15). ntargetgroups httpfrom
fr no :
=F,5-GCTGGAATGCTTTATGCGT=R,5-TGGCCTACATATTGCTGTCG=F,5-CCTAAGGATGGATCTGCGG =R,5-CCACAGAGTACCCATCATCCC=F,5-TGGTGGCCGATATTATTGAGTC=R,5-TCAGCCTGGCCTTCATACAA=F,5-TCAGGTTGTAGTGGGTGTAA =R,5-CTGACACATAGCGAGCATCG=F,5-CCTACTAGCCTACGACGGGT=R,5-CCCGCCAATTCCTTTAAGTTT=F,5-TAAAGGGTCTGTAGCCGGCC=R,5-CCCGCCAATTCCTTTAAGTTT=F,5-TTAAGCCATGCGAGTCGAGA=R,5-TTTAGCAGAGGCGGTCCCA=F,5-GAGTTCGATTAAGCCATGTTAGT=R,5-CGACCGTACTCCCCAGAT=F,5-GCTATCAGCGTCCGACTAAGC =R,5-TAATCCGGCAGGGTCTTCA=F,5-CGGATAGGCCTCTGATACCTG =R,5-TAATCCGGCAGGGTCTTCA=F,5-CCTTAGGACTGGGATAACCC=R,5-TTAACAGCTTCCCTTCGGC=F,5-CTTGGGACCGGGATAACC=R,5-TTAACAGCTTCCCTTCGGC=F,5-CGGAGATGGAACCTGAGACA=R,5-TTAACAGCTTCCCTTCGGC=F,5-GCCCACCAAGCCTACGATC=R,5-TTTCAGTCTTGCGACCGTACTC=F,5-TGCGGCCGATTAGGTAGTT=R,5-AAGGTCATCAACCTGGCCAT=F,5-TGCGGCCGATTAGGTAGTT=R,5-TAAGGTTTCCGGCGTTGAAT ySpringeretal.(13)andHalesetal.(16),allprimersare (cid:3)(cid:6)groupandotherswereamplified;(/),mcrAgenesof on April 10, 20//aem.asm.org/
nedb arget 19
Methanosarcina Methanococcoides,Methanolobales,Methanohalophilus RiceClusterI ANME-2 Methanosaeta Methanosaeta Methanomicrobiales ANME-1 DeeplyBranchingANME-1 DeeplyBranchingANME-1 Methanobacteriales Methanobacteriales Methanobacteriales Methanococcales MethanopyralesandMethanothermales Methanopyrales mcrIandME1/ME2primers,whichweredesig(cid:3)urred;,noPCRamplificationoccurred.(cid:3)(cid:6)ouponlywereamplified;/,mcrAgenesoft by guest
MS184F/MS1009R MCC221F/MCC&Mlob1155R RCI549F/RCI1014R ANME-2244F/ANME-2836R Msta268F/Msta927R Msta571F/Msta927R MM48F/MM1171R ANME-142F/ANME-1898RANME-1Deep35F/ANME-1Deep1038R ANME-1Deep176F/ANME-1Deep1038R MB136F/MB873R MB137F/MB873R MB310F/MB873R MC266F/MC910R MP&MT235F/MT1021RMP&MT235F/MP983R Withtheexceptionofthe(cid:6),PCRamplificationocc(cid:6),mcrAgenesoftargetgr
a b c
February2015 Volume81 Number4 AppliedandEnvironmentalMicrobiology aem.asm.org 1429
LeverandTeske
supplementalmaterial).Oneofthesephylotypesclusterswithse-
quences from cold deep sea and mud volcano sediments. The
otherisnearlyidenticaltothermophilicANME-1previouslyen-
richedbyHolleretal.(11).AllANME-1archaeadetectedherefall
intothesamegenuslevellineage.TheJF937800phylotype,which
isthelikelymcrAequivalentoftheANME-1Guaymas16SrRNA
phylotype(12),formsaseparategenuslevellineagetogetherwith
a phylotype from hydrothermal fluid of the Endeavor Segment
(HQ635748;24).Wenamedthislineage“HydrothermalANME-1
cluster.”
Within the Methanopyrales, we detected three different
branches, all identified as members of the genus Methanopyrus.
BesidesaphylotypethatisnearlyidenticaltoMethanopyruskan-
dleri,wedetectedanewphylotype,whichwecalled“Methanopyrus
FIG1(A)Measuredtemperaturedataforthesedimentintervalexaminedby
mcrAsubclusterI,”andonephylotypethatclusterswithsequences D
Weber&Jørgensen(5to30cmbsf).Thedashedredlineindicatesthebest-fit
line,assumingatemperatureof2°Catthesedimentsurface.Theslopeofthis from hydrothermal vents, which we termed “Methanopyrus mcrA ow
linewasusedtomodeltemperaturesathigherdepthresolutionthroughoutthe subclusterII”(Fig.2). n
sedimentintervalexaminedinthisstudy.(B)Sulfateconcentrationprofile. Inadditiontothepreviouslyknowngroups,wedetectednovel lo
Sulfatecouldnotbedetectedbelow4cmbsf. a
deepbranchesonthemcrAphylogenetictree,whicheachconsist d
ofonephylotype.Wecallthese“DeeplyBranchingmcrAgroupsII e
d
and III.” Interestingly, these two groups appear equidistant to
f
r
ANME-2 family (Fig. 2), of which one is nearly identical to a each other and to Deeply Branching mcrA group I: Deeply o
m
previouslydetectedsequencefromGuaymasBasin.Threeother Branching mcrA group II has sequence similarities of 67.5 and

phylotypes are monophyletic with Methermicoccus shengliensis 70.6%,andDeeplyBranchingmcrAgroupIIIhassequencesimi- h
t
t
and related environmental sequences. One of these phylotypes laritiesof63.0and68.8%tothetwomembersofDeeplyBranch- p
:
belongstothesamegenusasM.shengliensis.Theothertwophy- ing mcrA group I (see Table S4 in the supplemental material). //
a
lotypesareconsiderablydivergentandlikelybelongtotwoaddi- SequencesimilaritiesofgroupsIIandIIItoeachotherarealsolow e
tionalgenerawithinthefamilyMethermicoccaceae(seeTableS4in (65.7%). Based on our taxonomic classifier, Deeply Branching m
.
thesupplementalmaterial).PhylotypesofMethanohalophilusand mcrAgroupsI,II,andIIIrepresentthreedistinctorders,withthe a
s
group e are virtually identical to previously detected sequences two phylotypes in group I being equivalent to separate families m
fromGuaymasBasinsedimentandahydrothermalvent,respec- within the same order. Our classification thus suggests that the .
o
tively. Group e forms a sister family to the family Unidentified methane-cycling Archaea consist of at least 10 rather than the r
g
Rice Field Soil mcrA group/Zoige cluster I (represented by the currentlyrecognized7orders(36). /
o
sequencewithaccessionnumberGU182109),whichinturnforms Inferred energy substrates of mcrA phylotypes. Based on n

asisterfamilytoitsneighboringbranch(representedbythese- publishedinformationontheclosestrelativeswithintheMetha- A
quencewiththeaccessionnumberAY354030). nomicrobiales,Methanococcales,andMethanopyrales,whichcon- p
r
WithintheMethanomicrobiales,aMethanoplanusphylotypeis sist almost exclusively of hydrogenotrophic methanogens il
1
nearlyidenticaltoapreviouslydetectedsequencefromaGuaymas (19(cid:3)20,35,37(cid:3)42),H and/orformateisalikelyenergysourceof 0
2
,
Basin hydrothermal vent and closely related to Methanoplanus 9of21mcrA phylotypesdetected.Inaddition,wedetectedone
2
petrolearius,whichwasisolatedfromanoilfield(35).Sequences phylotypeofthegenusMethanohalophilusandthreeofthefamily 0
1
ofthegenuslevelMethanomicrobialesseepmcrAclusterappearfor Methermicoccaceae,lineagesthatcatabolizemethanolandmeth- 9
thefirsttimeinGuaymasBasin.WealsodetectmcrAsequencesof ylamines (43, 44). Aceticlastic groups (Methanosaeta, Methano- b
thesamephylotypeasasequencepreviouslydetectedinGuaymas sarcina,ZoigeclusterI)(45(cid:3)46)werenotdetected.ANME-1and y
g
Basin sediment (AY837767); this phylotype clusters separately ANME-2Archaeahavebeenlinkedtoanaerobicmethaneoxida- u
e
fromothergeneraofMethanomicrobialesandhasonlylowDNA tion(47–50).Nothingisknownaboutthesubstratesofgroupeor
s
sequence similarity to the closest genera, Methanoculleus and thetwodeeplybranchinggroups. t
Methanofollis (83.0% (cid:7) 2.3% and 84.1%, respectively; Fig. 2 ComparisonofgeneralmcrAprimers.Thedetectionsensitiv-
[Methanofollisnotshown];seealsoTableS4inthesupplemental ityanddetectednumberofmcrAclustersvariedconsiderablybe-
material).Weclassifythisphylotype,whichsofarlacksmcrAse- tweenthethreegeneralprimerpairs(Fig.3).WiththeME1/ME2
quenceswithhighsequencesimilarityoutsideGuaymasBasin,as andmcrIprimerpairs,wedetectedmcrAgenesinfourhorizons,
amemberofthenewgenuslevel“GuaymasBasinMethanomicro- withonlyweakPCRamplificationandcloningsuccessinoneof
bialesmcrAcluster.” these horizons (for mcrI, 4 to 5 cmbsf; for ME1/ME2, 1 to 2
WithintheMethanococcales,onesequenceisnearlyidenticalto cmbsf). In contrast, the mcrIRD primer pair produced suitable
Methanocaldococcusjannaschii;asecondsequenceformsadistinct PCRproductsforcloningthroughouttheuppereighthorizons.
phylotypewithhighsequencesimilaritytootherMethanocaldo- ThenumberofmcrAclusterswasalsohigher;withthemcrIRD
coccusphylotypesfromhydrothermalvents,includingonefrom primerpair,12clustersweredetected,comparedto10and7clus-
GuaymasBasin(Fig.2). tersdetectedwiththemcrIandME1/ME2primerpairs,respec-
TwophylotypesfallintotheANME-1Archaea,whichformsits tively.
ownorderandevenaseparateclassalongwiththeneighboring Eachprimerpairproducedadifferentcommunityprofile.Not
orderlevelANME-1-relatedgroup(Fig.2;seealsoTableS4inthe surprisingly,profilesobtainedwiththemcrIRDandmcrIprimer
1430 aem.asm.org AppliedandEnvironmentalMicrobiology February2015 Volume81 Number4
Methane-CyclingArchaeainHydrothermalSediment
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FIG2mcrAgenephylogeny.Representativephylotypesfromthisstudyareshowninboldmagentatypefont.Thedepthintervaloforiginisshown,alongwith s
t
thenumberofclonesequencesinparentheses.PhylotypesfromotherstudiesontheGuaymasBasinhavethefollowingcolorcodes:boldblack,pureculture
isolates;blue,Dhillonetal.(10);yellow,Holleretal.(11);orange,Biddleetal.(12);green,Y.HeandF.Wang,unpublisheddata.Bootstrapvaluesof(cid:2)50%are
shownatbranchnodes.Basedonsequencesimilaritycalculations,wemadethefollowingphylogeneticdistinctions:sequenceslikelytobelongtothesamespecies
aremarkedbydottedlines,dashedlinesindicatemembersofthesamegenus,asterisks(*)indicategenuslevelmcrAbranchesthatalsorepresentfamilies,and
thicksolidlinesindicatesequencesthatbelongtothesameorder(formoreinformationonthesecalculations,seethesupplementalmaterial,aswellas
Discussion).
pairs, which target the same loci and are similar in primer se- primer pair; mcrIRD clone libraries were even dominated by
quence (Table 1), are more similar to one another than to the Deeply Branching group II in six of eight sediment horizons
communityprofileobtainedwiththeME1/ME2primerpair.The (Fig.3).
biggestdifferenceisthelackofdetectionofgroupe,bothdeeply Comparisonofgroup-specificmcrAprimers.Elevenofthe27
branching mcrA groups, and both Methanopyrales subclusters group-specificprimerpairsresultedinsuccessfulmcrAamplifica-
withtheME1/ME2primerpair.Threeofthesefivegroupswere tions.Fiveoftheseprimerpairsexclusivelyamplifiedtheirtarget
detected with the mcrI primer pair and four with the mcrIRD groups(Table2;seealsoTableS1Ainthesupplementalmaterial),
February2015 Volume81 Number4 AppliedandEnvironmentalMicrobiology aem.asm.org 1431
LeverandTeske
Reference(s) 21 22 23 Zhangetal.,unpublished24 2526,27 25Zhangetal.,unpublishedZhangetal.,unpublishedC.-X.SheandC.Tong,unpublished2826 26 23 29 30 30 26 26 31 31 31 32 32
milarity
%si 86 99 97 99 95 9694 8398 96 92 8799 95 92 92 72 72 74 75 94 89 89 89 90
a
ctedatEverestMound Origin SharkBayhypersalinemicrobialmatSeaofOhkotskcoldseeps Napolimudvolcano SeaofOhkotskcoldseeps OkinawaTroughhydrothermalsedimentHotAlaskanoilfieldMarianaArc,hydrothermalventfluidHotAlaskanoilfieldSeaofOhkotskcoldseep SeaofOhkotskcoldseep MinRiverestuary AgriculturalbiogasplantJuandeFucaRidgehydrothermalventfluidJuandeFucaRidgehydrothermalventfluidNapolimudvolcanohypersalinesedimentNapolimudvolcanohypersalinesedimentHuabeioilfieldwaterproductionHuabeioilfieldwaterproductionJuandeFucaRidgehydrothermalventfluidJuandeFucaRidgehydrothermalventfluidEPRdiffuseventcoveredbymatsEPRdiffuseventcoveredbymatsEPRdiffuseventcoveredbymatsRainbowultramafichydrothermalventfluidRainbowultramafichydrothermalventfluid Do
e
STsequencesimilaritytoonesdet ClosestBLASThitoutsideGuaymasBasin(accessionno.) Methanohalophilushalophilus(AB703633) (FJ403608)25H-0A-16 (HQ454478)C09b 25H-200A-3(FJ403654) p760_M_1.03(AB305373) PS23SGXP401(GU357470)FS448_53(HQ635543) PS23SGXP401(GU357470)40H-260A-4(FJ403643) 25H-270A-7(FJ403632) (JX430031)LW-6 ATB-EN-10393-M138(FJ226731)FS625_56(HQ635721) A4Sx12_84(HQ635491) (HM004884)NapMat-8_10-mcrE03 NapK-40_60-mcrH12(HM005070) M_mcrA-11(JQ406852) M_mcrA-11(JQ406852) FS625_63(HQ635729) FS625_63(HQ635729) MCR-A(AY354023) MCR-B(AY354018) MCR-B(AY354018) 1crD2T36(FN650318) 1crD2T36(FN650318) http://awnloaded from
A
L e
n,withhighestB Reference 11 Y.HeandF.Wang,unpublishedY.HeandF.Wang,unpublished12 11 1010 10Y.HeandF.Wang,unpublished Y.HeandF.Wang,unpublished 1019 Y.HeandF.Wang,unpublished 11 12 12 20 20 20 20 20 20 20 m.asm.org
masBasi milarity on A/
ay %si 99 98 93 99 93 8285 7782 99 9999 96 98 72 71 71 71 88 86 86 99 99 p
u r
doutsideG malsediment malvent malvent malsediment sedimentmal malsedimentmalsediment malsedimentmalvent malvent malsedimentmalsediment malsediment sedimentmal malsediment malsediment malsediment malsediment malsediment malsediment malsediment malsediment malsediment il 10, 20
Basin,an Habitat Hydrother Hydrother Hydrother Hydrother Hydrother HydrotherHydrother HydrotherHydrother Hydrother HydrotherHydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother 19 by
as g
ypesfromwithinGuaym ClosestBLASThitfromGuaymasBasin(accessionno.) Guaymas_37enr_mcrA28(FR682810)4559–4-2-MCR1(JX826408) 4559-3-R1-MCR65(JX826416) 4486_MCRird_191(JF937817) Guaymas_37enr_mcrA92FR682811)( A07(AY837773)A07(AY837773) A07(AY837773)4559-3-R1-MCR12(JX826415) None (JX826415)4559-3-R1-MCR12 AY837767)C01(Methanocaldococcusjannaschii(L77117) (JX826413)4559-3-R1-MCR2 None 63Guaymas_50enr_mcrA(FR682818)4483_MCRird_285(JF937797) 4483_MCRird_285(JF937797) kandleriMethanopyrus kandleriMethanopyrus kandleriMethanopyrus kandleriMethanopyrus kandleriMethanopyrus kandleriMethanopyrus kandleriMethanopyrus uest
Overviewofphylot Mhalo-mcrA GrpE-mcrA ANME-2-mcrA-2 ANME-2-mcrA-1 ANME-2-mcrA-3 Methermicoc-mcrA-2Methermicoc-mcrA-1 Methermicoc-mcrA-3Mmseep-mcrA-2 Mmseep-mcrA-1 Mplanus-mcrA Mcull-mcrAMcoc-mcrA-1 Mcoc-mcrA-2 ANME-1-mcrA-1 ANME-1-mcrA-2 DbrGrpII-mcrA-1 DbrGrpII-mcrA-2 DbrGrpIII-mcrA-1 DbrGrpIII-mcrA-2 MpyralesGrpI-mcrA MpyralesGrpII-mcrA-1 MpyralesGrpII-mcrA-2 Mpyrus-mcrA-2 Mpyrus-mcrA-1
TABLE2 OTUname mcrAgenesGB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd-GB-EvMd- GB-EvMd-GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd-GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd- GB-EvMd-
1432 aem.asm.org AppliedandEnvironmentalMicrobiology February2015 Volume81 Number4
Methane-CyclingArchaeainHydrothermalSediment
44 44 56,57 Tangetal.,unpublished58 Linetal.,unpublished59 Z.Fang,unpublished60 61 Twingetal.,unpublished Twingetal.,unpublished 27 27 62 6362 64 Yeungetal.,unpublishedJ.A.HuberandJ.A.Baross,unpublishedJ.Li,unpublished 65 66 67 68 followingpage)
n
o
d
ue
n
99 99 99 96 96 90 92 99 97 88 99 99 97 99 100 9895 99 99 97 94 98 96 99 99 onti
C
(
oilfield,ShengliproductionwaterShenglioilfield,productionwaterSaltonSea,hypersalinelakesedimentOilreservoir FreshwatersedimentenrichedwithhexadecaneKueishanIsshallowhydrothermalfieldOknawaTrough,hydrothermalventchimneyHigh-temperatureoilreservoirEPR,seafloorbasalticflanksAlmeria,Spain,seawater-processedactivatedsludgeGulfofMexicohydrocarbonseepsedimentGulfofMexicoseephydrocarbonsedimentEndeavorSegmentfluidhydrothermalEndeavorSegmentfluidhydrothermalKazanMudVolcano,sedimenthydrateHuabeioilfieldKazanMudVolcano,hydratesedimentVulcanosubmarinesolfataricfieldHiberniaoilfield,productionwaterAxialseamount,diffusehydrothermalvent Hydrothermalventchimney NorthSeachalkpetroleumreservoirCentralIndianRidgehydrothermalfieldOkinawaTrough,polychaetenest,hydroth.venthydrothermalventEPR D
o
w
Methermicoccusshengliensis(NR_043960)Methermicoccusshengliensis(NR_043960) SS_WC_06(FJ656258) QHYA-25(JF741950) cloneA3(AJ133793)Methanoculleussp. KSTwh-C1-7-A-028(JQ611039) (AB175594)IAN1-62 0DA-79(JQ772440) a87R29(DQ417481) ARCHDER07_1A1(FN598017) MC118_26C2(HM600925) MC118_26C2(HM600925) FS725(3)73(JQ740760) FS625(3)16(JQ740759) KZNMV-25-A37(FJ712390) Arch-Q12(JQ241421)KZNMV-25-A37(FJ712390) Thermococcusalcaliphilus(NR040870) HibPWCl-ARC3(JF789485) Thermococcussp.Ax00-27(AY559130) FR852947)(Q2-a15.seq PW15.7A(EU573156) pCIRA-L(AB095122) AB611429)HTM1039Pn-A31( AciduliprofundumisolateEPR07-39FR865186)( http://anloaded from
e
HeandF.Wang,unpublishedHeandF.Wang,unpublished LongneckerandA.-L.Reysenbach,unpublished LongneckerandReysenbach,A.-L.unpublishedLongneckerandReysenbach,A.-L.unpublished m.asm.o
10 10 51 10 10 Y. Y. 10 11 12 27 4 12 1210 52 53 53 K. 54 55 K. K. rg
/

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0 0 A
7 0 9 9 0 8 8 9 9 9 9 9 9 68 9 9 8 5 3 9 8 8
9 1 9 9 1 9 9 9 9 9 9 9 9 99 9 9 9 9 9 9 9 9 p
r
malsediment malsediment sedimentmal malsediment malsediment malvent malvent malsediment sedimentmal malsediment malventfluid malsediment malsediment malsedimentmalsediment sedimentmal malsediment sedimentmal mney malsediment malsediment mney mney il 10, 20
Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother Hydrother HydrotherHydrother Hydrother Hydrother Hydrother Sulfidechi Hydrother Hydrother Sulfidechi Sulfidechi 19 by

g
u
e
4A08(AY835419) (AY835419)4A08 GUAY_37enr_Arch72(FR682485)(AY835412)7H12 (AY835412)7H12 (JX507259)4559-4-C2-82 4559-4-C2-82(JX507259) (AY835423)7C08 None None GUAY_50enr_Arch41(FR682490) JF937751)Arch4483_112( JQ740749)BG410(1)9( GBa2r032(AF419632) Arch4486_075(JF937738) Arch4486_075(JF937738)4E09(AY835427) GB18Thermococcussp.(FJ862790)mexicalis(AY099181)T. T.coccusguaymasensisJQ346762)( AF356631)G26_C48( Archaeoglobusprofundus(AF297529)(FR692155)90-PY-10 G26_C56(AF356635) G26_C56(AF356635) st
16SrRNAgenesMd-Methermicoc-A Md-Methermicoc-C Md-Halo-C Md-Mcull-A Md-Mcull-B Md-MBGD-A Md-MBGD-B Md-Mplasmatales Md-DHVE5-A Md-DHVE5-B Md-ANME-1a-B Md-ANME-1a-C Md-ANME-1b-A Md-ANME-1b-C Md-GBEury-G Md-GBEury-EMd-GBEury-K Md-ThCoc-D Md-ThCoc-E Md-ThCoc-F Md-ThCoc-G Md-Archaeoglobales Md-DHVEG Md-AcidProf-A Md-AcidProf-B
ArchaealGB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-EvGB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev GB-Ev
February2015 Volume81 Number4 AppliedandEnvironmentalMicrobiology aem.asm.org 1433
LeverandTeske
d which are ANME-1 Archaea, ANME-2 Archaea, Methanopyrus
Reference(s) 68 Amendetal.,unpublishe69 67 D.R.getal.,W.C. dpkaerintmeIdncleteercrsdoi,(noaTtnnraladbysltDoente3oee)p.onMlueywroBrrrpeeahosnuyvcllehotrsti,nywcpgeiertmht(aAcitrnhNAepMghgrEyeonl-uo1ept)rysauplIseImisnawcngrdegArrIeopIIudr.iepmt-esecprtese,cdiwfiinec
%similarity 96 99 98 96 Burcea,andTeske;Yeun d2pertipomt3hecrimnpta,eiarrnsv,daelsm.g,.ow,sMhteserttrehiaktnhinoegpylyyhr,uaDdsekenaponltdylbBeerrieanantcd1hetitonegc2tgecrdmou,wApitNIhIMIg,ewEn-he2ircaahtl
C.A. wasshowntobepresentfrom0to6cmbsfratherthanonlyfrom
Origin hydrothermalventEPR Alkalinehotspring LogatchevultramafichydrothermalventfieldOkinawaTrough,polychaetenest,hydrothermalvent ndetal.,J.P.Amend,L.C.Martin,K.Lloyd,and 2pgnpprorlrtioiiobfimmuae3pceedctrrsem,srnpeib(oaa.gmsilner.f,tc.ssamr,ItMrnshgcttraieeblMttrlaecwdsms)aitediecr(nrteTnaAgalodfybotec,lrslwdeuiMgeis3tntthee)eetcrtdhotshartetnhaonnloerootsseaaentarae-dggtbmayerlotecoudreprAxpermtcesege.vccpeirItonMnteiuesdobtssnelaywa(comdiufft,ocAhntrrdhNMMgeeMetyseenactEaeehtmt-reaa1ad)--l, Do
%ClosestBLASThitoutsideGuaymasBasinHabitatsimilarityReference(accessionno.) Sulfidechimney95K.LongneckerandAciduliprofundumisolateEPR07-39FR865186)Reysenbach,A.-L.(unpublished(JF935159)PNG_Kap4_A49Hydrothermalvent99Y.HeandF.Wang,unpublished(FN554070)A257-49Hydrothermalvent97Y.HeandF.Wang,unpublished Hydrothermalvent100Y.HeandF.Wang,HTM1036Pn-A14(AB611436)unpublished uencesimilaritytotheoperationaltaxonomicunits(OTU)detectedinthisstudy.Unpublisheddata:AmeW.Cheng;Tangetal.,Y.-Q.Tang,Y.Guang,Y.Li,M.Cai,andX.-L.Wu;Twingetal.,K.Twing,J.Biddle,Greer;Zhangetal.,L.Zhang,H.Dang,andX.Luan. ((sgsMttwGc2tdpmTccMarp((hhauuiu7TMMlhne;irrhsiiulcsee4neessspiintataoerpmmleacehttAe)mebiepgArlGhhrtAeryn,rowmrlh.lMreesoaamceeeuatNccteTurrarisnnplhmoaaluhtdssaiAumnrsahMoohlpngoe1,.ylvuoasboeempteerAgirsemiTeeucDnnccEoE.cstFulraryga)aatiihlrrtoteugiab-boicue..leslaoegyoe1m,rsuederetcmansdl.ibs1oMhnMyplloptelBaeSmoaubm6eeaaoecl-it4wiaesdnmirnSosr2otSipfnaa,tessatncecigriehMrteiilttn(aehtlrcedMgenneaihhIerahtDmunRmosarosstgenaeagsHidae,hMevNHHtI(nab)oekpetonchtteet,alhyhorepneAtnaVh2hert)adnshsdraalttse,.eooeny(uaEshaoedelnrrga7rlIw—monlpcoa2gaoxpIomoe1IDsoeu)nrp)tnitlcI(naef,Mdrhtaoyfsuipolemwr(ht7eM(mie,uipeu1mecla7ilzT2eMemler)isie4pbpide3etfieen)minraishur-eb)ttc,eileeen(TabhisShav—rarsntlaa1dnln.loesohhrceeaaneeln0teelnboawrseannoPaao(u)1dVa,srift2n-lirofhe,hiaH6gsmri)memted;t-ptotwatoylteShhhncyeeecyhlnolslsncmaahiysoraeoe.ttdercrenoaotsencpiloReAtmMTrleEdctncultyiuhcoirNhktohdmtupdacncnailsitaehgecialra)leerhleAggcdunrs2oeAlele,llytsiuyaeoe)steck9nag(nwatri.hsgclcirhrecfhhnera,tempFiuTriecytodlcus)enyyteuohinBhslehauhAiarvddonddmrst,nyae(beealperorrttgooMlendeelrecotoMosVereewnarraohotr:mofggqutlMeglher)totiaptyieSeeutuanrghy,tnaie1tpeuhhnn1hyecrpheileateot6baevtrgiaAoohtae-gsghcgSmsyanEersttrgagrlggracwltrr(pomouonmieeeorerrhoonTonbmpesnRernuooetuippaor:acyeayhlcetpuuNcpldepttcihhtrlboayrrfihhehppouyoyaerDcaiilAAls5eeecccr-------ssssfl, on April 10, 2019 byhttp://aem.asm.org/wnloaded from
seqT.-W. (DHVE5)(73),theDeep-SeaHydrothermalVentEuryarchaeotal g
ClosestBLASThitfromGuaymasBasin(accessionno.) G26_C56(AF356635) 4559-4-C2-11(JX507250) JX507244)4559-4-C2-9-5( (JX507245)4559-4-C2-61 mentalsequenceswiththehighestH.Lin,W.-X.Tu,W.-Y.Tsai,andng,J.Bugden,L.G.Whyte,andC. ngatiipnmemraeaoiotelrpAuheunslspelsi.tlwfi/ssT1RCeueo6hdirprcrSeeepepnhardlCehRaryemcryldsNuhclireohgsaAontntaeetyteaeghrapoldeelVe1tnrmsat6meo(lSDaolgattifrHanerlRrorcevVgiNruaaeeeEgplnAtn.eGtgas(sg/rrMewRcoonhCufeCearpt-GeelhVsion)ep)oteraG(f(ael76gEvu66Meiua,,osry7Cu7yam5a7rsG)elr)a,yc.sfahaudTnBnaredhedattohosettihecTuanrtegh,e,dhMetdwhirsmipiiecsntyruchoiesspmaelstrllhedsoaedoer--- uest
TABLE2(Continued) OTUname GB-EvMd-AcidProf-F GB-EvMd-MCG-A GB-EvMd-MCG-B GB-EvMd-Thermoproteales-1 aBoldfaceindicatestheenvironMeyer-Dombard;Linetal.,L.-Yeung,K.Lee,S.Cobanli,T.Ki vGttpepmhhxohlueaeeciymatrrelGesmypo,lemuyttnaciyaoolatopyasnlnsemdleseBodsa-m(afehis1smetaaBi5hpntle)afees,nsr,(hDiitma1nasanH4ld,a)id)ii.nVnnthmhEclOrayel5uoudfufcdd(rtltgTioohvhnhamsoeegleblyscclhpethlaeoyhnyB2qdsydo;euLrlresoosAaotseltt.StehyBhpTpeaLerrelrAohmssmopiStaodTasFlrleifvthtgrvieeoo.ienctmnSnstte4sstfd.sroai,,onTnuomdhattisphelihwdperyoereisdttosuhhtrexhpoieriner----
1434 aem.asm.org AppliedandEnvironmentalMicrobiology February2015 Volume81 Number4
Methane-CyclingArchaeainHydrothermalSediment
b a mmmmmmmmmmmmmM P T DISCUSSION
Tosimplify,aOnlydatafor crDBGrIIIcrDBGrIIcrMpKcrMcoccrMbaccrGMculcrMculcrMmicrobcrFCIcrANME-2crANME-1crIRD0crI0E1/ME20 rimerM D ABLE3De Mvseedresitmihtyaennhetosct.yCscpoliolnltegcftaoirrvcemhlyae,ettahhlaelnimne-cecraAygcealsinn.dOg1ua6rrScshrtuRaedNayAidngeemGnouenadysatmtraaatisensBcalausddiine-
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crA the proportion of methylotrophs of the total methane-cycling
February2015 Volume81 Number4 AppliedandEnvironmentalMicrobiology aem.asm.org 1435