Diversity and Distribution of Archaea Community along a Stratigraphic Permafrost Profile from ...

12 Pages·2015·1.47 MB·English

Hindawi Publishing Corporation
Archaea
Volume 2014, Article ID 240817, 11 pages
http://dx.doi.org/10.1155/2014/240817
Research Article
Diversity and Distribution of Archaea
Community along a Stratigraphic Permafrost Profile
from Qinghai-Tibetan Plateau, China
ShipingWei,1HongpengCui,1HaoHe,1FeiHu,1XinSu,1andYouhaiZhu2
1SchoolofMarineSciences,ChinaUniversityofGeosciences,Beijing100083,China
2OilandGasSurvey,ChinaGeologicalSurvey,Beijing100029,China
CorrespondenceshouldbeaddressedtoShipingWei;[email protected];[email protected]
Received25August2014;Revised22October2014;Accepted3November2014;Published25November2014
AcademicEditor:ChuanlunZhang
Copyright©2014ShipingWeietal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Accompanying the thawing permafrost expected to result from the climate change, microbial decomposition of the massive
amountsoffrozenorganiccarbonstoredinpermafrostisapotentialemissionsourceofgreenhousegases,possiblyleadingto
positivefeedbackstothegreenhouseeffect.Inthisstudy,thecommunitycompositionofarchaeainstratigraphicsoilsfromanalpine
permafrostofQinghai-TibetanPlateauwasinvestigated.Phylogenicanalysisof16SrRNAsequencesrevealedthatthecommunity
waspredominantlyconstitutedbyCrenarchaeotaandEuryarchaeota.TheactivelayercontainedaproportionofCrenarchaeota
at 51.2%, with the proportion of Euryarchaeota at 48.8%, whereas the permafrost contained 41.2% Crenarchaeota and 58.8%
Euryarchaeota,basedon16SrRNAgenesequenceanalysis.OTU1andOTU11,affiliatedtoGroup1.3b/MCG-AwithinCrenarchaeota
and the unclassified group within Euryarchaeota, respectively, were widely distributed in all sediment layers. However, OTU5
affiliatedtoGroup1.3b/MCG-Awasprimarilydistributedintheactivelayers.SequenceanalysisoftheDGGEbandsfromthe16S
rRNAsofmethanogenicarchaeashowedthatthemajorityofmethanogensbelongedtoMethanosarcinalesandMethanomicrobiales
affiliatedtoEuryarchaeotaandtheunculturedZC-IclusteraffiliatedtoMethanosarcinalesdistributedinallthedepthsalongthe
permafrostprofile,whichindicatedadominantgroupofmethanogensoccurringinthecoldecosystems.
1.Introduction releaseofcarbonfrompermafrosttotheatmosphereoccurs
primarily through accelerated microbial decomposition of
Permafrostoccupiesapproximately25%ofEarth’sterrestrial organic matter [3]. Previous studies showed that a large
surface,occurringmostfrequentlyinhighlatitudesregions, varietyofmicroorganismsinhabitpermafrostenvironments
especiallythoseoftheNorthernHemisphere[1,2].However, [8, 9]. During the thawing, the organic matter becomes
alpine permafrost usually exists at low-temperature, high- more accessible to microbial degradation and results in
elevation sites in temperate latitudes. It is well known that greenhousegasemissions[4],whichisthoughttobeoneof
permafrost stores massive amounts of carbon; a recent themostsignificantfeedbacksfromterrestrialecosystemsto
estimateindicatesthat1672Pgoforganiccarbon,anamount theatmosphere,thuspotentiallyexacerbatingthegreenhouse
roughlyequivalenttothetotalcarboncontainedwithinland effectandfurtherriskingsignificantclimatechange[3].
plantsandtheatmosphere[3–5],mayexist inthenorthern Earlierstudiesshowedthatpermafrostharborsadiverse
permafrost region, which accounts for approximately 50% microbialcommunityincludingbacteriaandarchaea[10–12].
of the estimated global belowground organic carbon pool Most studies addressing permafrost microbial community
[6]. With global warming, the permafrost is starting to were limited to the sites in Siberian permafrost [13, 14]. A
thaw, with estimates of as much as 90% of the permafrost researchofSiberiantundrarevealedthatthetotalnumberof
being lost by 2100 [7], which raises the question about the bacterialcellsfromthetoptothebottomoftheactivelayer
fate of carbon in thawing permafrost. It is inferred that (thetoplayerofsoilthatthawsinthesummerandrefreezes
2 Archaea
in the winter) range from 2.3 × 109 − 1.2 × 108 cells per spring. The study area belongs to the natural ecosystem of
gramdrysoil[15].Archaeamayconstitutebetween1%and alpineswampmeadowswithalargequantityofsoilorganic
12%ofthetotalcellsinSiberianactivelayersoils[15,16].The carbonstorage.Theactivelayerofthepermafrostthawsevery
communityofarchaeawascomposedofEuryarchaeota(61%) summer,withthethicknessvaryingfrom20to60cm,while
andCrenarchaeota(39%)intheperenniallyfrozensediments thepermafrostlayeroccursthroughouttheareatoadepthof
[11].Methanogens,affiliatedtothephylumofEuryarchaeota, 600–700m.
areagroupofarchaeathatproducemethaneunderanaerobic SampleswerecollectedinJune,2013,andwereobtained
conditions, which are ubiquitous in the biosphere and are bydiggingtoadepthof65cm.Sampleswerethencollected
particularly found in a variety of ecosystems such as rice asepticallyfromtheuppermost3–5cmoftheactivelayerto
paddies,lakes,hydrothermalvents,andpermafrostsoiland 63–65cmofthepermafrostlayerat10cmintervals.Thesoil
sediments [17]. Some of them were proven to be viable wasplacedintosterilizedplasticbagsforchemicalanalyses
inhabitantsinthehighlatitudepermafrost[18,19].However, and50mLcentrifugetubesformicrobiologicalanalyses.All
the uncultured methanogen cluster, Zoige cluster I (ZC- the samples were kept frozen in insulated containers and
I),affiliatedtoMethanosarcinaleswithinEuryarchaeota,was transportedimmediatelytolaboratoryforfurtheranalyses.
found to be the dominant group in the alpine permafrost
wetland of Tibet plateau [20]. Phylogenetic analyses of the
2.2. Characteristics of the Permafrost Soil. The soil temper-
methanogenicArchaeacommunityrevealedagreatdiversity
ature was measured using the in situ method by inserting
of methanogens in the permafrost including families of
thepHprobeintoeachlayerofthesoil.ThepHofsoilwas
Methanobacteriaceae, Methanomicrobiaceae, Methanosarci-
determinedseparatelyoneachofthesoilsamplesforeachsite
naceae,Methanosaetaceae[16,17,19,20],methanogengroup
using a fresh soilto water ratio of1:5 (pH meter, Sartorius
Rice cluster I (RC-I) and uncultured Rice cluster II (RC-
PB-10). The water content was determined as the weight
II) within the phylogenetic radiation of Methanosarcinales ∘
loss of fresh soil dried at 105 C for 24h. Total soil carbon
and Methanomicrobiales [19, 21–23] and ZC-I affiliated to
contentforeachsoilsamplewasdeterminedbycombustion
Methanosarcinales [20], permafrost cluster I affiliated to ∘
for 16h at 375 C. For analysis of methane concentrations
Methanosarcinaceae [19], and permafrost cluster II and III
in the soil, 5g of each sample was taken with a cut-off
affiliated to Methanosarcinales [19]. The nonmethanogenic
syringeandinjectedintoa25mLbottlecontaining10mLof
archaea Group 1.1b/MCG affiliated to the uncultured Cre-
saturated NaCl solution. The bottles were sealed by a solid
narchaeota was mostly found in the High Arctic wetland
butylrubberseptumandshakenfor1hbeforetheywereused.
permafrost[24].Eventhoughwehavealreadyaccumulated
Theheadspacemethaneconcentrationwasdeterminedbygas
a modest amount of knowledge about the permafrost’s
chromatography using a Packard Model 438A fitted with a
methanogenicarchaea,theycontinuetoattracttheattention
flameionizationdetector.
of researchers due to their production of methane and the
implicationsfortheglobalwarming.
2.3. DNA Isolation. The DNA of the soil samples was
The Qinghai-Tibetan Plateau is the largest alpine per-
extracted using the Soil DNA Isolation Kit (MP) following
mafrost area on Earth. Earlier studies estimated the annual
themanufacturer’sinstruction.TheextractedDNAwasvisu-
methane emissions from cold wetlands in the Qinghai-
alizedona1%agarosegelusingethidiumbromidestaining
TibetanPlateauatabout0.7–0.9Tg[25].Withthepermafrost
andquantifiedbyNanodrop(ThermoScientiific,NanoDrop
thawing due to the greenhouse effect, the organic carbon
ND-2000).
stored in permafrost would become more accessible for
microbes, whose activities will dictate whether permafrost
environmentswillbeanetsourceorsinkofgreenhousegas 2.4. Denaturing Gradient Gel Electrophoresis (DGGE). A
[1].However,currentknowledgeofthemicrobialcommunity nested-PCR approach was used to amplify V3 region of
inthehigh-altitudepermafrostispoorlyunderstood.Inthis archaeal 16S rRNA. The full-length archaeal 16S rDNA
study, we described the diversity and vertical distribution gene was amplified from genomic DNA using the primer
󸀠 󸀠 󸀠
ofarchaealcommunityinthepermafrostprofile,attempting set 8f (5 -CGGTTGATCCTGCCGGA-3 ) and 1492r (5 -
󸀠
to elucidate the composition of archaeacolonizing both the GGCTACCTTGTTACGACTT-3 )[26].ThefirstroundPCR
active layer and permafrost and to further characterize the amplification was performed in 50𝜇L of reaction mixture
methanogencommunityinQinghai-TibetanPlateau. containing 0.25𝜇M of each primer, 0.2mM dNTP, 1.5mM
MgCl2,5𝜇LofTaqbuffer,5UTaqDNApolymerase(Invit-
2.MaterialandMethods rogen,USA),and10ngtemplateDNA.ThePCRconditions
∘ ∘
wereasfollows:95 Cfor5min,35cyclesof50sat95 C,50s
∘ ∘
2.1. Site Description and Sampling. The sampling site is at 45 C, and 1.5min at 72 C, followed by a final extension
∘ 󸀠 󸀠󸀠 ∘
locatedinQinghai-TibetanPlateau(QTP,N38 05 38.10 and for10minat72 C.ThePCRproductsweregel-purifiedand
∘ 󸀠 󸀠󸀠
E99 10 05.13 )withanelevationof4300mabovesealevel, used as a template to amplify the V3 region by using the
󸀠
wheresummersaremildandwintersarequitecold.Themean primer set 340f-GC (5 -CCCTACGGGGYGCASCAG) and
annual temperature is approximately −5.8∘C, with January 519r(TTACCGCGGCKGCTG)[26].ThesecondroundPCR
temperaturesrangingfrom−18to−7∘CandJulytemperatures conditionswereusedasfollows:95∘Cfor5min,35cyclesof
∘ ∘ ∘ ∘
ranging from 15 to 21 C. Significant rainfall occurs mainly 50sat95 C,50sat45 C,and1.5minat72 C,followedbya
∘
in summer, while precipitation is very low in winter and finalextensionfor10minat72 C.
Archaea 3
Table1:Physiochemicalpropertiesofsoilsamplesatdifferentdepths.
Soildepth Temperature Sand Silt Clay Averagegrain Standard Watercontent TOC CH
(cm) (∘C) pH (%) (%) (%) size(𝜇m) deviation(𝜎) (%) (%) (nmol4/g)
3–5 18.34 6.79 16.33 72.40 11.26 5.68 1.80 46.52 9.01 4.1
13–15 3.68 6.70 7.99 75.56 16.45 6.25 1.73 25.56 3.02 19.9
23–25 1.85 6.74 13.19 69.81 17.00 6.18 1.91 21.35 2.43 188.3
33–35 0.44 6.60 8.06 71.45 20.49 6.46 1.82 17.18 2.43 79.3
43–45 −0.12 6.44 10.85 69.79 19.36 6.30 1.90 19.23 1.76 6.7
53–55 −0.24 6.57 18.73 66.84 14.43 5.84 1.99 20.91 1.61 22.5
63–65 −0.45 6.43 15.00 68.96 16.05 6.06 1.94 25.54 1.96 16.1
DGGE was performed as described by Lazar et al. 2.7.StatisticalAnalysis. Clonelibrarycoveragewascalculated
[26] with modifications. PCR products were applied onto accordingtoGood[28].AcomputerprogramDOTURwas
8% (wt/vol) polyacrylamide gels in 1 × TAE buffer with usedtoassignsequencestoOTUs,andthentherarefaction
denaturantgradientfrom20to40%(100%denaturantcon- curveswereconstructedbyusingthepartial16SrRNAgene
sists of7M ureaand40%formamide).Electrophoresiswas sequences[29].AnothercomputerprogramMOTHURwas
∘
performedat60 Cand200Vfor6h.Afterelectrophoresis, usedtocalculatetheShannonandSimpsondiversityindices,
thegelswerestainedwithethidiumbromidefor30minand theabundance-basedcoverageestimator(ACE),andthebias-
photographedwithaUVimager(AlphaImagerMiniSystem). correctedChao1[30].
DGGEbandsweregelpurifiedandreamplifiedwithprimer
set 340f and 519r. The purified PCR products were cloned 3.Results
intovectorandtransformedintoEscherichiacoliTOP10for
sequencing. 3.1.PhysicalandChemicalCharacterizationofthePermafrost
Soil. The samples were collected at the end of June, 2013.
2.5. Clone Library Construction. Archaeal 16S rRNA genes The temperature along the soil profile showed a distinct
∘
were amplified from soil community DNA using the decreasing gradient from the top at 18.34 C to the bottom
primers 21f (5󸀠 TTCCGGTTGATCCYGCCGGA) and 958r at −0.45∘C. The pH was slightly acidic, ranging from 6.43
󸀠
(5 YCCGGCGTTGAMTCCAATT);PCRreactionsvolume to 6.79. Granulometric analysis revealed that the soils in
was the same with amplification of full length 16S rDNA the stratigraphic profile were composed of sand (7.99%–
of archaea, and PCR reactions were carried out according 18.73%), silt (66.84%–75.56%), and clay (11.26%–20.49%)
to DeLong [27] with modifications as follows: 1 cycle of with the average grain size ranging from 5.68 to 6.46𝜇m.
∘ ∘ ∘
95 C for 1min, 30 cycles of 94 C for 1min, 50 C for 1min, Sorting coefficients of the sample varied from 1.73 to 1.99,
∘ ∘
72 Cfor2min,andafinalextensionat72 Cfor10min.The which indicated that the sediments in the studied sites are
PCR products were purified as described above and cloned well-sorted (Table1). The water content was highest in the
followingthemanufacturer’sinstructionsusingthepMD18- uppermostlayerofthesoilbecauseofthefrequentrainfallin
T vector system (TaKaRa) together with competent E. coli thisareaduringsummermonthsandwaterloggingwasoften
JM109cells.Randomlyselectedclonesweresequenced. observedintheswamp.Theorganiccarbon(TOC)indried
sedimentdecreasedwithdepthfrom9.01%to1.76%,ofwhich
TOCinthetopmostlayersoilwassignificantlyhigherthan
2.6. Sequencing and Phylogenetic Analyses. The sequences
thatofothersamples,suggestinganabundantorganicmatter
weredeterminedonanABI3730automatedDNAsequencer
accumulationinthenearsurfacesoil.Themethanecontent
(AppliedBiosystems)usingtheuniversalprimerM13-47or
waslowinthetopmostfewcentimetersbutrapidlyincreased
archaeal 21f primer. Sequences of 16S rDNA from DGGE
to the maximum value at 188.3nmol/g in the other deeper
bands and clone libraries were analyzed by using NCBI
samples(Table1).
BLASTN search program to identify their putative closest
phylogenetic relatives. The sequences were aligned with
their relatives using Clustal W, and phylogenetic trees 3.2. Diversity of Archaeal 16S rRNA Gene Sequences in the
were constructed by the neighbor-joining method using Clone Libraries. The diversity and composition of archaea
the maximum-parsimony algorithm in MEG 4 software communitywas investigated by constructingclone libraries
with 1000 bootstrap replicates. Phylotypes or operational of archaeal 16S rRNA gene fragments. Seven clone libraries
taxonomicunits(OTUs)weredefinedassequencesshowing wereconstructedfromtheuppermostactivesoillayertothe
≥97% homology to each other. Nucleotide sequences have lowermostpermafrostlayerat10cmintervals.Ineachlibrary,
been deposited in the GenBank database and the accession thecoveragerangedfrom95.2%to97.7%(Table2).Thirtyto
numbersareasfollows:DGGEbands,KM251579-KM251601; fifty-fivesequencesineachlibrarywereretrieved,andatotal
archaealclones,KM251602-KM251632. of282sequenceswerecombinedforanalysisoftheirdiversity
4 Archaea
Table2:Sequencinginformationandstatisticalanalysesofarchaeal16SrRNAgeneclonelibraries.
Soildepth NC NO Coverage(%) ACE Chao1 Shannon Simpson
3–5cm 50 7 96.0 9.24(7.3–24.6) 7.5(7.0–15.3) 1.42(1.2–1.7) 0.30(0.21–0.40)
13–15cm 42 5 95.2 6.94(5.2–24.2) 6(5.1–18.5) 1.07(0.8–1.3) 0.43(0.29–0.56)
23–25cm 33 7 97.0 7.38(7.0–12.2) 7(0–7) 1.74(1.5–2.0) 0.18(0.11–0.25)
33–35cm 43 4 97.7 4.78(4.1–14.0) 4(0–4) 0.79(0.5–1.1) 0.58(0.41–0.74)
43–45cm 45 5 95.6 8(5.6–20.8) 5.5(5.0–13.3) 0.94(0.7–1.2) 0.48(0.35–0.60)
53–55cm 39 5 97.4 0(0-0) 8(5.4–29.4) 0.99(0.7–1.3) 0.42(0.33–0.51)
63–65cm 30 7 96.7 17(8.1–98.3) 10(7.4–30.0) 1.35(1.0–1.7) 0.31(0.21–0.41)
Notes:NCisthenumberofclonesineachlibrary.NOisthenumberofOperationalTaxonomicUnit(OTU)basedon97%nucleotideidentity.
and composition in the community. Fifteen operational the occurrence of OTU5 was reduced sharply with depths,
∘
taxonomicunits(OTUs) were identifiedat the species level especially when the temperature dropped below 0.4 C; we
(≥97% sequence similarity) through all the depths. Twelve inferred that OTU5 was probably controlled by the low
and ten of them were detected in the active layer and the temperatures.ThreeOTUs(OTU2,OTU3,andOTU4)were
permafrost layer, respectively, and 7OTUs were presented onlydetectedinthepermafrostlayerandwerenotfoundin
in both layers. Relatively more OTUs were observed in the the active layer. Conversely, distributions of OTU7, OTU8,
depths of 3–5cm (7OTUs), 23–25cm (7OTUs), and 63– OTU9,OTU12,andOTU14wereonlydetectedintheactive
65cm (OTUs). The Shannon and Simpson diversity indices layers, which suggested that the communities of archaea
varied from 0.79 to 1.74 and from 0.18 to 0.58, respectively, variedoverthedifferentdepths.
and the species richness estimators ACE and Chao 1 varied
from0to17andfrom4to10,respectively.TheShannonand 3.4. Denaturing Gradient Gel Electrophoresis Analysis of
Simpson diversity indices showed no significant difference, 16S rRNA of Methanogenic Archaea. Denaturing gradient
suggesting that diversity of the archaeal 16S rRNA gene gel electrophoresis (DGGE) fingerprinting was used to
librarieswassimilaramongthelayers(Table2). characterize the community composition of methanogenic
archaea. DGGE band patterns indicated that most DNA
3.3. Composition of Archaeal Communities. Phylogenetic bands were observed in the topmost layer of the profile
compositionofarchaeal16SrRNAgenesequencesrevealed (3–5cm), and similar band patterns were observed at all
that they were mainly affiliated to two phyla, Crenar- thedepthsbelow,whichindicatedtheoccurrenceofsimilar
chaeota and Euryarchaeota, which comprise 47.2%% and methanogenicarchaeacommunitiesinthelowerlayersofthe
52.8% of the total sequences, respectively. The retrieved profile. Major bands with strong intensity were commonly
crenarchaealsequenceswereclassifiedintoonlyonelineage, observedinalldepths,whichsuggestedthatdominantDGGE
Group 1.3b/MCG-A [31, 32], which accounted for 47.2% bands represented the abundant methanogenic archaea in
of archaeal clone sequences. The retrieved euryarchaeal the community. However, several DGGE bands detected
sequences could be classified into four lineages, Metha- in the different layers of soil were not present in other
nomicrobiales, Methanosarcinaceae, Methanosaetaceae, and layers, indicating depth-specific methanogenic populations.
unclassifiedEuryarchaeota,whichaccountedfor0.3%,1.1%, A total of 23 bands were excised and sequenced. Phylo-
1.4%, and 50.0% of archaeal clone sequences, respectively genetic analysis showed that twelve sequences fell within
(Figure1). the euryarchaeotal lineage, comprising three groups of ZC-
The relative abundance and distribution of different Icluster(12sequences),Methanosarcinales(1sequence),and
archaeal phylogenetic lineages with the depth were shown Methanomicrobiales(8sequences).Onlytwosequenceswere
in Figure2. Both active layer and permafrost consisted retrieved in the Miscellaneous Crenarchaeal Group (MCG)
predominantly of Crenarchaeota and Euryarchaeota. The under crenarchaeotal lineage. Sequences affiliated to ZC-
activelayercontainedaproportionofCrenarchaeotaat51.2% I cluster were distributed in all depths, whereas sequences
compared to 48.8% Euryarchaeota, whereas the permafrost associatedwithMethanomicrobialesweremainlydistributed
hadahigherproportionofEuryarchaeota(58.8%)thanCre- in the top and middle of the permafrost sediments, but
narchaeota(41.2%).VerticaldistributionofOTUsalongthe the sequences affiliated to MCG were only observed in the
sediment profile showed that OTU1 and OTU11, belonging topmost active layer of permafrost. It was very interesting
to Group 1.3b/MCG-A and the unclassified group within thatonlytwobandsaffiliatedtoZC-Iclusterwereobservedat
Euryarchaeota, respectively, were widely distributed in all thedepthof23–25cm,whichshowedthemaximummethane
sediment layers. The abundance of OTU1 was gradually concentration(Figures3and4andTable1).
increased with depths, whereas OTU11 gradually decreased
withdepthsexceptforthetopmostthreesoillayers.OTU5, 4.Discussion
another lineage belonging to Group 1.3b/MCG-A within
Crenarchaeota, was predominantly distributed only in the Permafrost is well-known for storing massive amounts of
top parts of the active layers. It was very interesting that organic carbon and is thought to be the most important
Archaea 5
DT5-60A-30-OTU2
50 Acidic fen soil clone A P2K30f (GU127872)
0.05
DT5-A-26-OTU8
Tidal freshwater wetland soil clone EP091-2.29 (JF789748)
Rich minerotrophic fen clone MH149212C (EU155994)
Sediment of Lake Monounclone MOAR9575m (JQ079952)
DT5-45A-31-OTU6
90 DT5-25A-2-OTU6
82
DT5-A-21-OTU6
DT5-35A-15-OTU6
88
DT5-50A-1-OTU4
Field soil clone UP1cl45 (KC784741)
DT5-50A-11-OTU1
54 Tidal freshwater wetland soil clone EC083-41 (JF789806) a
ot
Coal-rich sediment from freshly mined heaps of brown coal clone N07 (KJ424514) ae
7528 DDDTTT555---6A405-AA1---O1103T--UOO1TTUU11 Group 1.3b/MCG-A Crenarch
Fen soil microcosm clone 1813K140a (FR745081)
99 DT5-35A-12-OTU1
DT5-25A-10-OUT1
90
Peat soil clone 5AED11 (HG325678)
DT5-25A-1-OTU5
99 Permafrost soil samples from Kunlun Mountains Pass, Mt. Kunlun clone HWGA-11 (JQ684418)
DT5-15A-1-OTU5
62Glacier foreland clone GFS5-9500iii (AY601296)
Soil from Ruoergai plateau wetland clone 2PZ28.1 (FJ957963)
60 DT5-A-66-OUT9
Petroleum contaminated soil clone ASC27 (AB161330)
99
Kutch desert soil clone HKT-KArS2 (HE802749)
DT5-15A-13-OTU11
Anaerobic methane oxidation/denitrification reactor clone D-ARCH(DQ369741)
Petroleum contaminated soil clone ASN30 (AB161352)
Freshwater iron-rich microbial mat clone oytA083A (B722181)
DT5-35A-1-OTU11
DT5-50A-10-OTU11
Submarine permafrost clone PASLSS0.5m1(FJ982666)
Spring pit clone SSA0103-10 (JN397868)
61
DT5-60A-11-OTU11
85DT5-45A-10-OTU11 Unclassified
100 DT5-25A-16-OTU11
DT5-A-3-OUT10
Sediments containing oil near Cape Gorevoy Utes clone z3 (GU911421)
67
Iron-rich microbial mat clone hfmA004 (AB600438)
93 Groundwater clone arcFlank1063(KC604439) ota
DT5-A-13-OTU10 hae
Mixture of activated sludge and methanogenic sludge clone EAr23 (KC539785) arc
y
72 DT5-15A-35-OUT12 Eur
DT5-25A-12-OTU13
100
Antarctic sediment clone LH25 (AY177808) Methanosaetaceae
77 DT5-60A-24-OUT13
67
Chlorinated ethene-degrading culture clone KB-12 (AY780569)
74 59Neotropical forest clone Amr34G1(JN810750)
Richminerotrophic fen clone MHLsu4713B (EU155898)
100
61 DT5-50A-9-OTU15 Methanosarcinaceae
100
60 DT5-45A-24-OTU15
54 DT5-15A-8-OTU15
Deep subsurface groundwater from sedimentary rock milieu clone HDBW-WA05 (AB237738)
DT5-25A-11-OTU14
100
Athabasca oil sands formation water clone TS1A142 (JF789589) Methanomicrobiales
77
Richminerotrophic fen clone MH1100B1F (EU155974)
M83548
Figure1:Phylogenetictreeofarchaeal16SrRNAgenesequencesfromthestratigraphicsoilprofile.Thetreewasconstructedusingthe
neighbor-joiningmethodfromasimilaritymatrixbasedonthedistancedeterminedbyKimura’stwo-parametermodel.Bootstrapvalues(100
replications)generatedusingthemaximumparsimonymethod.Thephylotypeswerenamedaccordingtothesoildepthorigin.Numbersin
bracketswereGenBankaccessionnumbers.Thescalebarrepresents5%estimateddistance.
6 Archaea
OTU1
OTU11 14%
OT2U%10 24% 3–5cm
OTU9 OTU5
2% 48%
OTU8
6% OTU6
4%
OTU14 OTU15 OTU15
OTU13 0.6% 0.6% OTU12 2.4%
1.2% 2.4%
OTU2 OTU1
OTU12 0.0% 16.7%
0.6%
OTU1 O0T.0U%3 OTU11 O16T.7U%5 13–15cm
16.1% 61.9%
OTU4
0.0%
OTU11
45.2% OTU5 OTU13 OTU14
26.8% 6.1% 3.0%
OTU1
18.2%
OTU11
18.2% 23–25cm
OTU5
OTU6 OTU7 33.3%
OTU10 OTU7 4.2% 9.1%
0.6% OTU9 OTU8 1.8% OTU6
12.1%
0.6% 1.8%
OTU1 OTU5
16.3% 7.0%
OTU6 33–35cm
OTU11 2.3%
74.4%
Active layer
OTU15
2.2%
OTU1
26.7%
OTU13 O0T.0U%14 O1T.8U%15 O64T.U4%11 O4T.4U%5 43–45cm
1.8% OTU6
2.2%
OTU12
0.0%
OTU15
2.6%
OTU1
34.2%
OTU1
OTU11 39.5% 53–55cm
OTU11 52.6%
54.4% OTU2
0.9% OTU3 OTU5
0.9% OTU6 2.6%
OTU4 2.6%
0.9% OTU13
OTU5 6.5%
OTU6 2.6%
OTU10 1.8% OTU1
0.9% O0T.0U%9 O0T.0U%8 O0T.0U%7 O4T1.U9%11 38.7% 63–65cm
OTU2
Permafrost 3.2%
O3T.2U%10 OTU4 OTU3
3.2% 3.2%
Figure2:Frequencyofindividualtypes(OTU)andrelativedepth-dependentdistributionofgroupsofthearchaeafromthestratigraphic
permafrostprofilebasedonthegeneclonelibraries.GreensegmentsindicateOTUsbelongingtounclassifiedEuryarchaeota,bluesegments
indicateOTUsbelongingtoGroup1.3b/MCG-A,yellowsegmentsindicateOTUsbelongingtoMethanosaetaceae,redsegmentsindicateOTUs
belongingtoMethanosarcinaceae,andpinksegmentsindicateOTUsbelongingtoMethanomicrobiales.
Archaea 7
m m m m m m a lower archaeal diversity when compared with other envi-
m c c c c c c
5 5 5 5 5 5 ronments. Steven et al. [9] revealed 43OTUs of archaea
c 1 2 3 4 5 6
5 – – – – – –
– 3 3 3 3 3 3 in a permafrost/ground ice core from the Canadian High
3 1 2 3 4 5 6
Arctic. The lower diversity of archaea in QTP permafrost
soil may be related to the habitat environments, which did
notvarygreatlyasevidencedbythesimilarphysiochemical
1 variables shown in Table1. Frank-Fahle et al. [37] reported
2 thatmicrobialdiversitywashighestinthesurfacelayersand
3
4 decreased towards the permafrost layers. Our data showed
5
6 7 thatthediversityofthearchaeawassimilaramongthelayers
8
10 9 andloweroverallOTUnumbersineachlayerwereobserved.
11
13 12 Existence of overlapping OTUs of archaeal communities
14
from different depths suggested that the dominant archaeal
15
communitywassimilaroverthesamplingrange.
17 16 Thecommuntiycompositionrevealedby16SrRNAgene
18
19 clonelibrariesshowedthatCrenarchaeotaandEuryarchaeota
20 were the dominant phyla in both active layer and per-
mafrost.AsimilarabundanceofCrenarchaeota(51.2%)and
21
22
23 Euryarchaeota (48.8%) was observed in the active layer. In
contrast,alittlehigherabundanceofEuryarchaeota(58.8%)
and a little lower abundance of Crenarchaeota (41.2%) were
observed in the permafrost layer (Figure2). This result is
Figure3:DGGEofPCR-amplifiedarchaeal16SrRNAgenefrag-
mentsfromtotalcommunityDNAfromdifferentdepths(indicated quite similar with the report by Steven et al. [9] from the
above the lanes) in the soil profile. Marked DGGE bands were CanadianHighArctic,inwhichthe16SrRNAgenesequences
excisedandsequenced.ThenumbersofDGGEbandscorrespond belonging to the Crenarchaeota dominated the active layer
totheonesonthephylogenetictreeinFigure4. and permafrost table horizons, while Euryarchaeota were
predominantinthepermafrost.However,Wilhelmetal.[24]
reportedthatbothactivelayerandpermafrostconsistedpre-
natural methane emission source [3, 33]. Previous studies dominantly of Crenarchaeota at 71% and 95%, respectively;
estimated that a total of 33.52Pg of organic carbon and the active layer had a greater proportion of Euryarchaeota
0.7–0.9Tg of methane emission were emitted from the (22%) compared with permafrost (4%). The abundance of
Qinghai-Tibetan Plateau grassland soils [25, 34]. Recent
Crenarchaeota was inferred to be an increasing trend with
studies indicated a continuous increase in air temperatures
adecreasingtemperature,whichsuggestedthatmembersof
ontheQTPoverthelast40years,makingthemapotential
Crenarchaeotamaybeparticularlyadaptedtocoldconditions
source of considerable greenhouse gas emissions [35]. Our
[16].Nevertheless,ourdataindicatedthateventhoughboth
data showed that the organic carbon content ranged from
OTU1 and OTU5 belong to Group 1.3b/MCG-A within
1.61% to 3.02% at depths of 13–65cm, which is comparable
Crenarchaeota; 16.2% of OTU1 and 26.8% of OTU5 were
with1.6%inahighArcticpermafrostsoilfromSpitsbergen
distributed in the active layer; in comparison, 34.2% of
reported by Hansen et al. [8] and 1.2–3.0% in a permafrost
OTU1and3.5%ofOTU5weredistributedinthepermafrost
active layer soil from Lena Delta reported by Liebner and
(Figure2), suggesting that OTU1 may be more adapted to
Wagner [13]. However, the organic carbon content reached
the cold environment whereas OTU5 appears more cold
9.01% in the topmost soil (3–5cm) layers of our QTP sites.
sensitive.
Thehigherorganiccarbonwasinferredtoberelatedtothe
Basedon16SrRNAgenesequenceanalysis,bothCrenar-
decayedgrassrootsinthewetland.Themethanecontentwas
chaeota and Euryarchaeota showed a limited diversity with
much lower in both the top and bottom sediments along
only one group (Group 1.3b/MCG-A) underlying Crenar-
thepermafrostprofileexceptthesubsurfaceatdepthof23–
chaeotaandfourgroups(Methanomicrobiales,Methanosarci-
25cm,wherethemethanecontentcouldreachamaximum
naceae,Methanosaetaceae,andanunclassifiedgroup)under-
value at 188.3nmol/g. Patterns of methane content did not
lying Euryarchaeota. A total of 97.2% sequences belonged
exhibit any trends with depth nor correlate noticeably with
to both Group 1.3b/MCG-A (47.2%) and unclassified Eur-
thephysicalandchemicalpropertiesofthesediments,such
yarchaeota(50.0%),whichwerepredominantlycomprisedof
asorganiccarbonconcentration,sedimentaryproperties,or
OTU11 (48.9%) and OTU1 (23.4%), respectively (Figure2).
water content. This observation was consistent with that
Rivkina et al. [36] reported in the north-eastern Arctic Ochsenreiter et al. [31] reported that Group 1.3b, an non-
tundra. The higher methane content at depth of 23–25cm cultured group of Crenarchaeota, was widely distributed in
wasinferredtoberelatedwiththelowerredoxpotentialand different environments such as freshwater, wastewater, and
suitable substrates in this layer, which needs to be further soil.Sequencesofthe16SrRNAgeneaffiliatedtounclassified
proved. group within noncultured Euryarchaeota were frequently
A total of 15OTUs of archaea were observed from the present in freshwater environments. Our data showed that
Qinghai-Tibetan Plateau permafrost soil, which presented Group 1.3b/MCG-A and unclassified Euryarchaeota were
8 Archaea
DGGE-Band 12
Jing-Mei River clone SSA0101-9 (HM159365)
0.05 DGGE-Band 14
Freshwater iron-rich microbial mat clone oytA083 (AB722181)
Submarine permafrost clone SMPUFLSS70m1(FJ982704)
DGGE-Band 1
DGGE-Band 13
Sediments containing oil near Cape Gorevoy Utes clone 4.67 (HQ891194)
Iron-rich microbial mat clone hfmA004 (AB600438) ZC-I cluster
DGGE-Band 11
DGGE-Band 20
DGGE-Band 4
DGGE-Band 11
DGGE-Band 13
Cold wetland soil clone SD47 (EU283032)
DGGE-Band 5
63
55 Paddy soil clone FS-19 (KJ493699)
98 Sediments containing oil near Cape Gorevoy Utes clone 4.97 (HQ891149)
DGGE-Band 7
DGGE-Band 8
DGGE-Band 6
Anaerobic bioreactor clone AHPDArc1H03(KJ206655)
99 Methanogenic propionate-degradation clone SMS-T-Pro-5 (AB479409) Methanosarcinales
64 Sludge from full-scale anaerobic digester clone DH09 (KC676307)
Anaerobic granules clone 0A12 (KC502890)
DGGE-Band 10
72
Submarine permafrost clone PASLSS0.5m26(FJ982771)
DGGE-Band 16
94
DGGE-Band 9
99
Terrestrial permafrost clone PAS (FJ999541)
Cold wetland soil clone SD63 (EU283037)
DGGE-Band 21
Contaminated aquifer clone WCHA2-08 (AF050615) Methanomicrobiales
88
76 DGGE-Band 2
DGGE-Band 23
Mesophilic anaerobic wastewater sludge clone 105ZC05 (KF198716)
DGGE-Band 3
100
DGGE-Band 22
Anaerobic sludge clone ARC26 (AY693814)
Zoige wetland, Tibetan Plateau clone ZW-M-13 (KF360019)
DGGE-Band 18
Arctic peatland clone TCren8 (Dq188819)
MCG
100 DGGE-Band 19
Zoige wetland, Tibetan Plateau clone ZW-M-1 (KF360007)
M83548
Figure4:Phylogeneticrelationshipsofarchaeal16SrRNAgenesequencesobtainedfromDGGEbandsinthesoilprofile.Thescalebar
represents5%estimateddistance.
Archaea 9
predominant archaea distributed over all the depths. It was constitutedbyCrenarchaeotaandEuryarchaeota.Eachgroup
very interesting that OTU5 affiliated to Group 1.3b/MCG- ofthosetwophylahasauniquedistributionpatternbetween
A was overwhelmingly distributed in the active layers, and the active layers and the permafrost layers. The majority
sequences belonging to OTU1 tended to increase gradually of methanogens belonged to Methanosarcinales and Metha-
with depth, while sequences belonging to OTU11, affiliated nomicrobialesunderthephylaofEuryarchaeota.Thepresent
to unclassified Euryarchaeota, were primarily distributed studywillhelpimproveourunderstandingofthecommunity
in the layers with much lower tempertures (Figure2). To structureofarchaeainthepermafrostenvironment.
our knowledge, those findings have never been reported
previously. ConflictofInterests
Methanogenicarchaea,alsoknownasmethanogens,are
animportantgroupofEuryarchaeotathatproducemethane The authors declare that there is no conflict of interests
under anaerobic conditions and have proven to be viable regardingthepublicationofthispaper.
inhabitants of permafrost [36, 38]. Our DGGE sequences
analyses revealed that members of Methanosarcinales and Acknowledgments
Methanomicrobialesconstitutedthemajorityofmethanogens
inthesoils,andtheunculturedmethanogenofZC-Icluster The authors acknowledge James Hurley for making a crit-
affiliatedtoMethanosarcinalesshowedthestrongestintensity ical reading and revision of this paper. This research was
of bands in DGGE and was distributed in all the depths supported by the National Special Research Fund, China
along the permafrost profile, which indicated a dominant (GasHydrateResourceExplorationandProductionTesting
groupofmethanogensoccurringincoldecosystems.Similar Project).
observationwasreportedintheZoigewetlandoftheTibetan
Plateau [20]. Methanosarcinales are widespread in diverse References
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