Archaea Dominate the Ammonia-Oxidizing Community in Deep-Sea Sediments of the Eastern

16 Pages·2017·2.88 MB·English

by  Jun Sun

ORIGINALRESEARCH
published:16March2017
doi:10.3389/fmicb.2017.00415
Archaea Dominate the
Ammonia-Oxidizing Community in
Deep-Sea Sediments of the Eastern
Indian Ocean—from the Equator to
the Bay of Bengal
JingWang1,2,JinjunKan3,XiaodongZhang1,2,ZhiqiangXia1,XuechengZhang1,
GangQian1,2,YanyiMiao1,XiaoyunLeng1,2andJunSun1,2*
1CollegeofMarineandEnvironmentalSciences,TianjinUniversityofScienceandTechnology,Tianjin,China,2TianjinKey
LaboratoryofMarineResourcesandChemistry,TianjinUniversityofScienceandTechnology,Tianjin,China,3StroudWater
ResearchCenter,Avondale,PA,USA
Editedby:
MarkAlexanderLever, Ammonia-oxidizing Archaea (AOA) and ammonia-oxidizing Bacteria (AOB) oxidize
ETHZurich,Switzerland ammonia to nitrite, and therefore play essential roles in nitrification and global nitrogen
Reviewedby: cycling. To better understand the population structure and the distribution of AOA and
SteffenLethJørgensen,
AOBinthedeepEasternIndianOcean(EIO),ninesurfacesedimentsamples(>3,300m
UniversityofBergen,Norway
JamesA.Coker, depth)werecollectedduringtheinter-monsoonSpring2013.Onesedimentsamplefrom
UniversityofMarylandUniversity
theSouthChinaSea(SCS;2,510m)wasalsoincludedforcomparison.Thecommunity
College,USA
composition, speciesrichness,anddiversitywerecharacterizedbyclonelibraries(total
*Correspondence:
JunSun 1,238clones),andhigherdiversityofarchaealamoAgenesthanbacterialamoAgenes
[email protected] was observed in all analyzed samples. Real time qPCR analysis also demonstrated
higherabundances(genecopynumbers)ofarchaealamoAgenesthanbacterialamoA
Specialtysection:
Thisarticlewassubmittedto genes, and the ratios of AOA/AOB ranged from 1.42 to 8.49 among sites. In addition,
ExtremeMicrobiology, uniqueanddistinctcladeswerefoundinbothreconstructedAOAandAOBphylogeny,
asectionofthejournal
suggestingthepresenceofniche-specificammonia-oxidizingmicroorganismsintheEIO.
FrontiersinMicrobiology
The distribution pattern of both archaeal and bacterial amoA genes revealed by NMDS
Received:25October2016
Accepted:27February2017 (non-metric multidimensional scaling) showed a distinct geographic separation of the
Published:16March2017
samplefromtheSCSandmostofthesamplesfromtheEIOfollowingnitrogengradients.
Citation:
Higher abundance and diversity of archaeal amoA genes indicated that AOA may play
WangJ,KanJ,ZhangX,XiaZ,
ZhangX,QianG,MiaoY,LengXand a more important role than AOB in the deep Indian Ocean. Environmental parameters
SunJ(2017)ArchaeaDominatethe shapingthedistributionpatternofAOAweredifferentfromthatofAOB,indicatingdistinct
Ammonia-OxidizingCommunityin
metaboliccharacteristicsand/oradaptationmechanismsbetweenAOAandAOBinthe
Deep-SeaSedimentsoftheEastern
IndianOcean—fromtheEquatorto EIO,especiallyindeep-seaenvironments.
theBayofBengal.
Front.Microbiol.8:415. Keywords:EasternIndianOcean,deep-seasediment,ammonia-oxidizingarchaeaandbacteria(AOAandAOB),
doi:10.3389/fmicb.2017.00415 nitrogencycle,clonelibrary,qPCR
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Wangetal. AOADominatetheEIOSediments
INTRODUCTION very limited knowledge on these microorganisms in the Indian
Ocean.
Nitrification is an integrative component in the nitrogen cycle, TheIndianOceanisthethirdlargestoceanintheworld,which
whichincludesoxidizationofammoniatonitritebyammonia- is characterized by two semi-enclosed basins in the north, and
oxidizing Bacteria (AOB) and ammonia-oxidizing Archaea greatlyinfluencedbyseasonalmonsoon(Fineetal.,2008;Rixen
(AOA),andthennitriteisfurtheroxidizedtonitratebynitrite- etal.,2009).Strongstratificationinducedbymonsoonsuppresses
oxidizingBacteria(NOB).Asthefirstandalsotherate-limiting up welling and mixing of the deep waters, making the Eastern
step, ammonia oxidation to nitrite is mediated by both AOB EquatorialIndianOceanatypicaloligotrophicarea(Kumaretal.,
and AOA (Norton et al., 2002; Schleper and Nicol, 2010). 2009). The Arabian Sea (AS) and the Bay of Bengal (BOB) are
The chemolithotrophic AOB are placed taxonomically in the the two basins in the north part of the Indian Ocean and are
beta- and the gamma-subdivision of Proteobacteria, and their relatively less oligotrophic compared to the Equatorial region.
naturalpopulation/distributionhasbeenwidelyinvestigatedby ThefrequentdustinputtotheArabianSea,enhancedadvection,
molecular tools, such as using the 16S rRNA gene sequences and vertical eddy mixing bring small detritus and nutrients to
(Stephen et al., 1996, 1998; Kowalchuk et al., 2000) and the theCentral/EasternAS(McCrearyetal.,1993).Incontrast,lack
genes encoding ammonia monooxygenase (amoA; Bothe et al., of detritus transport limits organic matter accumulation in the
2000; Purkhold et al., 2000). In recent years, novel archaeal BOB(Kumaretal.,2004),meanwhile,riverrunoff,evaporation,
groups that function as ammonia oxidizers containing amoA seasonaladvection,andmixingresultinlowsurfacesalinityand
geneshavealsobeenobservedandtheymaycontributegreatlyto high surface stratification (George et al., 1994; Kumar et al.,
nitrificationprocessesaswell(Venteretal.,2004;Schleperetal., 2002).Theoceaniccirculationandheatstoragevariationcaused
2005).EnvironmentalAOAsequencesaremainlyaffiliatedwith by low surface salinity strengthens the vertical stratification
severalgroups,suchasmarinegroupI(MGI),putativelymarine (Nyadjroetal.,2013).Finally,theBOBisalsocharacterizedbyits
group pSL12, thermophilic AOA (ThAOA) and soil group I.1b lowproductivity(Madhupratapetal.,2003;Kumaretal.,2004)
(Durbin and Teske, 2011; Pester et al., 2011; Hatzenpichler, andhighnitrificationrate(Srinivasetal.,2011).Remineralization
2012). In order to better understand their roles in global that consumes oxygen in the BOB is also weaker, making
nitrogen cycling, it is critical to characterize the abundance, the BOB less hypoxic than AS, but still stronger than the
diversity, and distribution of AOA and AOB in natural Equator and other areas in the Eastern Indian Ocean (EIO;
environments. Kumaretal.,2002).
Nitrogenislikelythelimitingnutrientinmarineecosystems The Indian Ocean and sediments are highly oligotrophic,
(Gruber and Sarmiento, 1997), and therefore the nitrogen and most of the areas including the BOB are covered by
cycling in the ocean is of particular interest (Deutsch et al., volcanogenic sediments that are characterized by high illite
2007). AOA and AOB have been commonly found in marine and chlorite in the clay mineral assemblages (Venkatarathnam
sediments all over the world, including the East China Sea and Biscaye, 1973; Madhupratap et al., 2003). River borne
(Dang et al., 2008), the South China Sea (Jin et al., 2011), solids with terrestrial organic carbon (Goldberg and Griffin,
hydrothermal vents of the Pacific Ocean (Wang et al., 2009; 1970; Fontugne and Duplessy, 1986) also contribute to the
Nunoura et al., 2010), the western Pacific Ocean (Cao et al., surface sediments in the BOB, where bacterial oxidation and
2011a),thetropicalWestPacificContinentalMargin(Dangetal., respiration occurred (Peterson and Prell, 1985; Middelburg,
2009), the Northeastern Japan Sea (Nakagawa et al., 2007), the 1989;Walsh,2014).Oxidationandreductionoforganicmatter
SouthernNorthSea(Lipsewersetal.,2014),andalsothedeep- in the deep-sea sediments are active for resource and energy
seaenvironments(Nunouraetal.,2013;Xuetal.,2014;Lagostina flow, which provide ideal environments for microbial nitrogen
et al., 2015; Luo et al., 2015). Among these studies, ratios of transformations.However,nodirectmeasurementswerecarried
AOA/AOB gene copy numbers varied from 0.0003 to 232.3, out on the nitrification/denitrification processes in sediment of
and no consistent patterns have been observed. Obviously the theBOB.Thediversityanddistributionofbacterialcommunities
distributionandvariationsofAOAandAOBarerelevanttotheir inthewatercolumnoftheBOBandEquatorialareaoftheEIO
metabolic characteristics and their responses to environmental has been screened by a high throughput sequencing analysis
gradients as well. For instance, AOA has been reported to (Wangetal.,2016),butthedetailsofthecomposition,diversity
have a higher affinity to ammonium than AOB in oligotrophic anddistributionofnitrogentransformingmicrobesintheseareas
environment (Martens-Habbena et al., 2015). The dominant stillremainlargelyunknown.
groupanddistributionofAOBcouldbedrivenbysalinity,pH, In this study, deep-sea sediments (>3,300 m) were sampled
andnutrientavailability(Bernhardetal.,2007;Caoetal.,2012; to investigate the diversity and distribution of archaeal and
Li et al., 2015), while the distribution of AOA varied along bacterial amoA genes. Clone library and q-PCR analysis
water depth, redox variation, and concentration of ammonium were applied to characterize the community composition and
in water column or marine sediments (Wuchter et al., 2006; relative abundances of ammonia oxidizing genes (amoA).
Dang et al., 2009; Roussel et al., 2009; Flood et al., 2015). Based on the multivariate statistics and phylogenetic analysis,
Although a global distribution, diverse composition, and high diversity and distribution patterns of AOA and AOB in the
abundance of AOA and AOB have been reported (Rotthauwe EIO were compared and discussed. Environmental factors
et al., 1997; Nicol and Schleper, 2006; Francis et al., 2007; driving the distributions were also analyzed by redundancy
Cavicchioli et al., 2007), compared to other regions, we have analysis(RDA).
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Wangetal. AOADominatetheEIOSediments
METHODOLOGY Sedimental porewater was collected for measurement of
chemicalparameters.Totalcarbon(TC)andtotalnitrogen(TN)
Sample Collection and Environmental
contents were measured with a CHNSO elemental analyzer
Parameter Measurements
(Costech ECS 4010, Italy). The contents of nitrite, nitrate and
Sediment samples were collected during an open cruise ammoniuminsedimentsweremeasuredbyanAA3HRanalyzer
supportedbytheNationalNaturalScienceFoundationofChina (SEAL Analytical, USA). Detailed site descriptions including
in the EIO aboard the R/V “Shiyan 1” from March 22nd to time,waterdepth,locationetc.werelistedinTable1.
April 18th in 2013. Nine stations were selected and sediment
samples were collected along five main transections targeting
DNA Extraction and Clone Library
the Bay of Bengal (I1 and I2), the Equator area (I4 and I7),
Construction
and the transection (I5) paralle to the coastline of the Sumatra
(theEasternBoundaryoftheEIO;Figure1).Sedimentsamples DNAExtraction
were collected by a gravity sampler with a temperature probe Approximately 0.5g (wet weight) of sediment was transferred
attached. The surface of the sediment column was sealed with into a sterile 1.5 ml centrifuge tube. Total genomic DNA was
seawateruntilreleasefromthegravitysampler.Afterremoving extracted using the PowerSoil Isolation Kit (MoBio, Carlsbad,
the top 5 cm surface sediment that might be disturbed during CA,USA)accordingtomanufacturer’sinstructions.Dilutionwas
sampling, the sub-samples were taken from the 2 to 5 cm appliedwhennecessary.AllDNAextractswerestoredat−20◦C
of the column surface. Sub-samples were collected in sterile forfurtheranalyses.
plastic bags for microbial study and stored in liquid nitrogen
immediately. Sediment samples were transported on dry ice PCRAmplificationofamoAGenes
and stored in a −80◦C freezer until further analysis. For Primers Arch-amoAF, Arch-amoAR and amoA-1F, amoA-2R
comparisonpurpose,onesedimentsamplefromtheSouthChina were used to amplify archaeal and bacterial amoA genes,
Sea (SCS) was taken on March 20th 2013 following the same respectively (Rotthauwe et al., 1997; Francis et al., 2005).
protocol. PCR amplification was performed in a 25-µl reaction volume
FIGURE1|MapshowingthesamplingsitesintheEasternIndianOcean(EIO)andtheSouthChinaSea(SCS).
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Wangetal. AOADominatetheEIOSediments
TABLE1|Sitedescriptionandchemicalmeasurements.
SiteandSample I506 I503 I501A I412 I704 I207 I202 I105 I103 SCS
information
Stations
Samplingtime 2013/3/22 2013/3/24 2013/3/26 2013/3/27 2013/4/16 2013/4/27 2013/4/28 2013/4/29 2013/4/30 2013/3/20
Waterdepth(m) 5,725 4,810 4,527 4,532 4,426 3,515 3,386 3,437 3,614 2,510
Longtitude 97.76754667 95.317495 93.81465 90.95521667 82.02821667 86.49836667 88.72803333 89.12828333 89.44266667 112.8058167
Latitude −6.019785 −2.957325 −0.464666667 0.00823333 1.554216667 10.00263333 10.00371667 9.261833333 7.943716667 15.69273333
WaterContent(%) 77.30 78.26 60.81 59.36 64.94 60.65 64.68 61.43 62.00 55.28
TotalNitrogen(%) 0.1286** 0.0707 0.1568** 0.1626** 0.1519** 0.1574** 0.1413** 0.135** 0.133** 0.1901**
TotalCarbon(%) 0.6658 0.6304 5.4087* 8.8089** 2.3842 9.9288** 10.4103** 11.6196** 11.8085** 8.5872**
NO−(mg/L) 0.0001 0.0001 0.0003** 0.0003** 0.0003** 0.0003** 0.0002* 0.0004** 0.0003** 0.0003**
2
NO−(mg/L) 0.0024 0.0019 0.0034** 0.0037** 0.0017 0.0042** 0.003** 0.0034 0.0035** 0.0028*
3
NH+(mg/L) 0.0020 0.0019 0.0029** 0.0036** 0.0035** 0.0045** 0.0035** 0.0038** 0.0039** 0.0037**
4
DetectionlimitsforNO−andNO−are0.0001mg/L,forNH+is0.0004mg/L.
2 3 4
Differencesbetweendatasetofeachparameteraredeterminedbyt-test,significanceisrepresentedbyPvalue.*P<0.05;**P<0.01.
containing 0.25µM each primer, 0.5 U DNA polymerase examined and edited using BioEdit (Tom Hall, North Carolina
(Promega,Madison,WI,USA),2.5µl10×GoTag(cid:13)R FlexiBuffer, StateUniversity,Raleigh,NC,USA)andMEGA,version5.01.
50 mM MgCl solution, 500µM (PCR NucleotideMix, 10 mM
2
each)eachdeoxynucleoside triphosphate,2.5µl0.1%BSA,and Quantitative Real-Time PCR
25ngoftemplateDNAtoafinalvolumeof25µl.Amplification
The abundances of archaeal and bacterial amoA genes
was performed with the MJ Research PTC-200 Peltier Thermal
were determined in triplicate using an ABI Step One Plus
Cycler (Waltham, MA, USA). The thermal profile used for
Thermocycler. Quantification of each sample was based on the
amplification of the archaeal amoA gene followed Wang et al.
fluorescent dye SYBR Green I, which binded to the double-
(2014),whichincluded5minat94◦C,followedby40cyclesof
stranded DNA during PCR amplification. Reactions were
45sat94◦C,30sat57◦C,and1minat72◦C,andafinalextension performed ina 25µlvolumecontaining 1µlofDNA,0.15µM
of7minat72◦C.AmplificationofthebacterialamoAgenewas of each primer and 12.5µl of FastStart Universal SYBR Green
performed at 94◦C for 3 min, followed by 40 cycles of 30s at
Mastermix (Roche Diagnostic GmbH, Mannheim, Germany).
94◦C, 30s at 55◦C, and 45s at 72◦C, and a final extension of
Specificity of the amplification product was verified by melting
7 min at 72◦C. For quantitative polymerase chain reaction (q-
curveanalysisandvisualizedinagarosegels.PlasmidDNAwas
PCR) analysis, the same pairs of primer set were used with 45
extractedbyPlasmidKit(Promega,Madison,WI,USA)fromthe
cycles.
clones containing the correct insertion, and the concentration
was measured with an Eppendorf BioPhotometer (Eppendorf
CloneLibraryConstruction AG, Hamburg, Germany). The copy numbers of amoA genes
were then calculated based on the concentration of plasmid
Clone libraries of archaeal and bacterial amoA gene-amplified
DNAandampliconsize(Okanoetal.,2004;Heetal.,2007).The
products from all the samples were constructed following the
plasmids of the amoA gene clones with known concentrations
previously described protocol (Weidner et al., 1996). In brief,
were diluted to produce the standard curve over seven orders
after total DNA extraction and PCR amplification of the amoA
of magnitude (1.21 × 108 to 1.21 × 102 copies of template for
genes,PCRproductswereverifiedforthecorrectamplification
archaealamoAand1.14×108 to1.14×102 copiesoftemplate
by running a 1% agarose gel in 1 × TAE buffer at 90 V for
forbacterialamoA).
30 min. Gel slices containing the target PCR products were
excised with a sterilized scalpel and then purified using the
AxyPrep DNA Gel Extraction Kit (250-prep, Axygen, US). The Phylogenetic Analysis
sizeofpurifiedPCRproductswasconfirmedagainbyrunningon The partial archaeal and bacterial amoA sequences recovered
agarose gel before being ligated into pMD18-T vector (D101A, fromclonelibrariesoftheEIOandSCSwereblastedinGenBank
TaKaRa, Dalian, China) and then cloned into Escherichia coli using the BLAST (http://www.ncbi.nlm.nih.gov). The most
DH5-α cells according to the modified transformation method closelyrelatedsequencesandadditionalreferencesequenceswere
developedbyMandelandHiga(1970).Colonieswererandomly retrieved and subsequently aligned with representative clones
pickedfromeachclonelibraryandverifiedforcorrectinsertion in CLUSTALX (version 2.0.11). Phylogenetic neighbor-joining
of DNA fragment by PCR amplification with primer set treewereconstructedbyMEGA[version5.01].Thetreebranch
M13F (5′-GTTTCCCAGTCACGAC-3′) andM13R (5′-TCACA distances represented the substitution rate and the scale bar
CAGGAAACAGCTATGAC-3′).PCRproductsfromthepositive represented the expected number of changes per homologous
clones were sequenced by BGI, Beijing. DNA sequences were position. Cluster stability was assessed by bootstrap analyses
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Wangetal. AOADominatetheEIOSediments
based on 1,000 replicates, and the bootstrap values >50% were Nucleotide Sequence Accession Numbers
shownnearnodes. The archaeal and bacterial amoA gene sequences retrieved in
this study have been deposited in GenBank under accession
Data Analysis numbersKU595584toKU595656,andKU595657toKU595711,
respectively.
Similarity at 97% was used as cut-off value in defining OTUs
(operation taxonomy unit). The coverage of each clone library
was calculated as C = [1 − (n1/N)] · 100, where, n1 is RESULTS
the number of unique OTUs and N is the total number of
Environmental Parameters
clones in a library (Dang et al., 2009). Diversity indices of
The Environmental parameters measured in this study were
amoA gene (Shannon–Wiener H and Simpson D and Chao1)
summarizedinTable1.AllthesedimentsamplesfromtheEIO
were calculated based on the OTU data. Rarefaction analysis
were taken from 3,386 up to 5,725m depth, and the sample
and two non-parametric richness estimators, and the bias-
from the SCS was at a depth of 2,510 m. The sample from the
correctedChao1(S )werecalculatedusingDOTUR(Schloss
Chao1
SCSexhibitedhighestvalueintotalnitrogencontent,whilefour
and Handelsman, 2005). These diversity indices and richness
samplesfromtheBOB(I103,I105,I202,andI207)showedhigher
estimators are useful for comparing the relative complexity
content of total carbon than other samples. Samples from the
of communities and estimating the completeness of sampling
BOB also contained higher nitrate, nitrite and ammonium in
(Lozupone et al., 2007). Non-metric multidimensional scaling
general. The highest concentration of [NO− + NO−] reached
(NMDS) analysis was applied to characterize the distribution 2 3
0.0045 mg/L in sample I207, which was located at the BOB,
patterns of AOA and AOB. Pairwise similarities/distances
while the lowest concentration of [NO− + NO−] was 0.002
were calculated based on relative abundances of each OTU 2 3
mg/LinsamplesI704andI503,whichwasclosetotheequator
across all the samples analyzed, and NMDS was performed
and the Eastern Boundary of the EIO. Not surprisingly, the
with the multidimensional scaling (MDS) procedure of the
SAS System (SAS Institute Inc., 2008). Stress value <0.1 highest concentration of NH+4 was also detected at station
I207 (0.0045 mg/L), while the lowest one was at station I503
indicatedagoodordinationwithlittleriskofmisinterpretation
(0.0019mg/L).
of the results (Clarke and Ainsworth, 1993). Correlations
between amoA gene OTU distribution and environmental
Diversity of Archaeal and Bacterial amoA
factors were analyzed with a redundancy analysis (RDA)
using CANOCO 4.5 software (ver. 4.5, Microcomputer Power, Genes in the Eastern Indian Ocean
Ithaca, NY, USA) (Lepš and Šmilauer, 2003). The raw data Total 20 clone libraries were constructed for archaeal and
were processed by a log transformation before running RDA bacterial amoA genes. In 10 libraries of AOA, total 650 clones
(Lepš and Šmilauer, 2003). RDA was chosen to determine the were sequenced and 53 OTUs were identified at a cut-off value
relationships between AOA and AOB community structures of 97% nucleotide similarity; while for AOB, 588 clones were
andtheenvironmentalfactorsbecausethelongestgradientina randomly selected and sequenced, resulting in 22 OTUs with
detrendedcorrespondenceanalysiswasbetween3.0and4.0(Lepš the cut-off value of 97% nucleotide similarity as well (Table2).
andŠmilauer,2003). The coverage (C) ranged from 56 to 83% in AOA and 73
TABLE2|DiversityandpredictedrichnessofarchaealandbacterialamoAsequencesrecoveredfromtheEIOandSCS.
Sites I506 I503 I501A I412 I704 I207 I202 I105 I103 SCS
AOA No.ofclonesequenced 43 59 88 78 60 44 88 50 50 90
No.ofOTUs 17 26 27 32 10 18 28 19 14 19
Coverage(C%) 60.47 55.93 69.32 58.97 83.33 59.09 68.18 62.00 72.00 78.89
Shannon–Weiner 2.28** 2.88** 2.34** 2.54** 1.26 2.37** 2.73** 2.34** 2.32** 1.87*
1/D 6.74* 15.8** 5.15 5.15 2.18 7.65* 9.79** 6.92* 8.86** 3.15
Schao1 35.33 60.00** 69.75** 87.2** 13.33 57.00** 43.00* 45.00* 16.00 23.67
AOB No.ofclonesequenced 78 79 20 94 90 55 44 41 42 45
No.ofOTUs 11 9 4 13 6 14 10 11 5 7
Coverage(C%) 85.90 88.61 80.00 86.17 93.33 74.55 77.27 73.17 88.10 84.44
Shannon–Weiner 1.87** 1.08 0.71 1.73** 0.85 2.21** 2.24** 2.04** 0.94 1.48*
1/D 4.70* 1.88 1.56 3.26 1.73 7.00** 10.00** 7.07** 1.90 3.75
Schao1 11.25* 12.00* 4.50 13.00** 6.50 15.20** 14.00** 21.00** 5.00 7.33
UniqueOTUsoftheamoAsequencesweredeterminedusingtheDOTURprogram.Thecoverage(C),Shannon–Weiner(H),Simpson(D)andSchao1richnessestimatorswerecalculated
basedontheOTUdata.
Differencesbetweendatasetofeachindexaredeterminedbyt-test,significanceisrepresentedbyP-value.*P<0.05;**P<0.01.
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Wangetal. AOADominatetheEIOSediments
FIGURE2|Rarefactioncurvesforarchaeal(A)andbacterial(B)amoAgeneclonelibrariesgeneratedfromDOTURanalysis.
to 93% in AOB (Figure2). Rarefaction analysis indicated that Phylogeny of Archaeal and Bacterial amoA
AOA contained higher diversity than AOB (P < 0.01, t-test; Genes
Figure2). For AOA, sample I704 had the highest diversity The 53 representative archaeal amoA gene sequences shared
indices (H and 1/D) while the lowest indices occurred in 72.8–96.9% similarity among each other and 92–99% identities
sample I503; for AOB, the highest diversity indices occurred to the closely matched sequences from GenBank. The top hit
in sample I202 from the BOB and sample I501A contained sequences in GenBank were mainly retrieved from deep-sea
the lowest one. Together with the richness estimator Schao1, environments, including sediments from the SCS (Cao et al.,
higher diversity of AOA samples occurred in transections I4 2012),theWestPacificMarginandContinent(Luoetal.,2015),
and I5, while higher AOB diversity occurred in transections I1 the Ogasawara Trench hadopelagic (Nunoura et al., 2013), the
andI2. New Caledonia Basin deep subsea-floor (Roussel et al., 2009),
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Wangetal. AOADominatetheEIOSediments
hydrothermal vent at the Mid-Atlantic Ridge (Xu et al., 2014), identifiedinthephylogenetictree(Figure3).InClusterI,amoA
the South West Indian ridge, and the East China Sea (Dang sequences were mainly associated with deep-sea sediments. Six
et al., 2010). Sequences obtained from deep-sea water column sub-clusters were distinguished within cluster I, among which,
from the Mariana Trench (Nunoura et al., 2015), the Indian three of them included clones from the EIO exclusively; while
Ocean, coastal water, and sediment from the Arabian Sea were the rest of them contained clones from both the EIO and the
alsoincluded(Figure3).Noclonescorrelatedwithterrestrialor SCS together (Figure3). In Cluster II, three sub-clusters were
freshwater environment were detected, and most of the clones defined.Sub-cluster1containedclonesfromtheBOB(I103,I105,
wereoriginatedfromthemarinesedimentwithadepthranging and I207) and the Equator (I412-B48); sub-cluster 3 contained
from1,400to9,760m. clones from the BOB and the Eastern Boundary only. Two
All of the archaeal amoA sequences formed two big groups: clones from the SCS (SCS-al10) were grouped with a sequence
Marine sediment, and Marine/coastal water, and sediment. from the Northern SCS (JX537560) and formed sub-cluster 2.
Withinthetwogroups,fourclusterswithninesub-clusterswere In Cluster III, only clones from the Equatorial area (I412-BX8)
FIGURE3|Reconstructedphylogenetictreewithdistanceandneighbor-joiningmethodofpartialarchaealamoAsequencesrecoveredfromthe
sedimentsoftheEIOandtheSCS.Thetreebranchdistancesrepresentedthesubstitutionrateandthescalebarrepresentedtheexpectednumberofchanges
perhomologousposition.Bootstrapvalues>50%of1,000resamplingswereshownnearnodes.ThearchaealamoAsequencesobtainedinthisstudywereshown
withcoloredlabelsalongwiththesite,andclonenumbersforeachOTUwereincludedinbrackets.
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Wangetal. AOADominatetheEIOSediments
werepresented.WithinClusterIV,twoclonesfromtheSCS(SCS- sequencesfromGenBankwithasimilarityof97–99%(Figure4).
al21) and one clone from the Eastern Boundary (I503-BW40) The top hit sequences blasted in GenBank were from deep-sea
weregroupedtogether.OneuniqueclonefromsiteI202(I202- sediments, such as the Pacific Ocean deep-sea sediments (Luo
dp-4)formedasinglecladeandrepresentedastandaloneclade et al., 2015), the SCS Sediments (Cao et al., 2012), hadopelagic
from the BOB sediment. Among all the clusters, sub-cluster 4 sediments in the Ogasawara Trench (Nunoura et al., 2013),
andsub-cluster6withinClusterIcontainedmostoftheclones oligotrophicsurfacesedimentsoftheBenguelaupwellingsystem,
andaccountedfor23and25%ofthetotalsequences(Figure3). low-temperaturehydrothermalFe-Si-richprecipitates,anddeep-
In phylogenetic analysis of AOB, the 30 representative seahydrothermalsediments.AllbacterialamoAgenesobtained
bacterialamoAsequencesshared87.3–95.9%similaritiesamong inthisstudybelongedtobeta-proteobactetria(Nitrosospira);no
each other and quite high identities to the closest match clonesweregroupedwithNitrosomonasorNitrosococcus.Within
FIGURE4|Reconstructedphylogenetictreewithdistanceandneighbor-joiningmethodofpartialbacterialamoAsequencesrecoveredfromthe
sedimentsoftheEIOandtheSCS.Thetreebranchdistancesrepresentedthesubstitutionrateandthescalebarrepresentedtheexpectednumberofchanges
perhomologousposition.Bootstrapvalues>50%of1,000resamplingswereshownnearnodes.ThebacterialamoAsequencesobtainedinthisstudywereshown
withcoloredlabelsalongwiththesite,andclonenumbersforeachOTUwereincludedinbrackets.
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Wangetal. AOADominatetheEIOSediments
the big group of Nitrosospira, six sub-clusters were identified from the BOB, and also demonstrated the spatial differences
withtheircloselyalignedsequencesfromGenBank.Asshownin alongthetransect(I5).
Figure4,sub-cluster1andsub-cluster2werethetwolargestsub- Correlations of AOA/AOB community structures and the
clusters.Clonesretrievedfromthisstudywerepresentedinallsix associatedenvironmentalvariableswereanalyzedbymultivariate
sub-clusters with clone numbers in sub-clusters 1 and 2 higher analysisRDA(Figure7).Theenvironmentalvariablesinthefirst
than the other four sub-clusters. Clones in sub-cluster 3 were two RDA dimensions explained 94.3% of the total variance in
associatedwithsequencesfromtheEIOmainlywhilesub-cluster thearchaealamoAgenotypecompositionand61.5%ofthetotal
4 contained sequences from site I105, I506, and the SCS. Sub- variance in the bacterial amoA genotype composition. Results
cluster5andsub-cluster6containedsequencesfromtransections indicated that AOA community structure in the sediments
I5,I4,I1,andI2,whichwerealllocatedattheEIO. of EIO responded to water depth, ammonium and nitrate
concentration, and these three factors provided 33.4, 22.3,
Archaeal and Bacterial amoA Gene and 19.7% of the total RDA explanatory power. Ammonium
Abundance contributed significantly to archaeal amoA gene distributions
ThecopynumbersofAOAamoAgenesrangedfrom2.14×107 (P = 0.047, 533 Monte Carlo permutations). However, the
to3.14×107copies/gsediment(wetweight),whileAOBamoA community structure from SCS was more strongly correlated
gene copy numbers ranged from 3.27 × 106 to 2.08 × 107 withconcentrationoftotalnitrogenandnitrite(Figure7A).For
copies/g sediment (wet weight; Figure5). Ratios of AOA/AOB AOB, nitrateconcentration and water depth contributed to the
amoAgeneabundancerangedfrom1.42to8.49.Thegenecopy distributionsofbacterialamoAgenotype,whichprovided25and
numbersofAOAweresignificantlygreaterthanthatofAOBin 14.8% of the total RDA explanatory power, respectively. The
everysampleofthestudiedsites(t-test,P<0.01).Amongthe10 combination of other variables provided additionally 21.7% of
samples,AOBamoAgeneabundanceatsiteI412waslowerthan the total RDA explanatory power (Figure7B), but none of the
alltheothersites(t-test),andsitesI103,I105,I503andI506were parametersshowedsignificantcorrelations.
highercomparedtotherestsixsamples.Ingeneral,AOBamoA
genessharedbiggervariabilityamongthesamplesthanAOA. DISCUSSION
Distribution Patterns of amoA Genes and
Deep-seafloorsedimentconstitutesthelargestlandonearth.It
Potential Environmental Drivers ischaracterizedbyextremeconditionsincludinghighpressure,
DistributionpatternsofAOAandAOBweresimilarbasedonthe lowtemperature,lackoflightetc.,anditharborstaxonomically
NMDSresults:SCSandI704weredistinctfromalltheothers,and andmetabolicallydiversemicroorganisms(D’Hondtetal.,2004;
samplesfromtheBOB(I103,I105,I207,andI202)weregrouped Inagaki et al., 2006; Kallmeyer et al., 2012). Among those
together(Figure6).ThesamplesfromtheEquatorarea(I4)and microbes, bacteria dominated over archaea in the oxic surficial
the Eastern Boundary (I5) showed similarity with the samples sediments,andthedominantgroupsvariedwithenvironmental
FIGURE5|Relativeabundance(copiespergramofwetweightsediment)ofarchaealandbacterialamoAgenesinthedeep-seasedimentoftheEIO
andtheSCS.ErrorbarsrepresentedthestandarddeviationsoftheindependenttriplicateqPCRreactions.
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Wangetal. AOADominatetheEIOSediments
FIGURE6|NMDSordinationsofthearchaealamoA(A)(Stress=0.076)andbacterialamoA(B)(Stress=0.084)assemblagesfromthesedimentsoftheEIO
andtheSCS.Samplesfromthesametransectionswereindicatedbycolor.
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