Research Article Diversity and Distribution of …downloads.hindawi.com/journals/archaea/2015/968582.pdfResearch Article Diversity and Distribution of Archaea in the Mangrove Sediment

Research ArticleDiversity and Distribution of Archaea inthe Mangrove Sediment of Sundarbans

Anish Bhattacharyya1 Niladri Shekhar Majumder2 Pijush Basak1

Shayantan Mukherji3 Debojyoti Roy1 Sudip Nag1

Anwesha Haldar4 Dhrubajyoti Chattopadhyay1 Suparna Mitra5

Maitree Bhattacharyya1 and Abhrajyoti Ghosh3

1Department of Biochemistry University of Calcutta 35 Ballygunge Circular Road Kolkata West Bengal 700019 India2Roche Diagnostics India Pvt Ltd Block 4C Akash Tower Near Ruby Hospital 781 Anandapur Kolkata 700107 India3Department of Biochemistry Bose Institute P112 C I T Road Scheme VIIM Kolkata West Bengal 700054 India4Department of Geography University of Calcutta 35 Ballygunge Circular Road Kolkata West Bengal 700019 India5Norwich Medical School University of East Anglia and Institute of Food Research Norwich Research Park NorwichNorfolk NR4 7UA UK

Correspondence should be addressed to Dhrubajyoti Chattopadhyay dhrubajyoticgmailcomSuparna Mitra suparnamitrasmgmailcom Maitree Bhattacharyya bmaitreegmailcomand Abhrajyoti Ghosh aghosh78gmailcom

Received 30 March 2015 Revised 25 June 2015 Accepted 14 July 2015

Academic Editor William B Whitman

Copyright copy 2015 Anish Bhattacharyya et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Mangroves are among the most diverse and productive coastal ecosystems in the tropical and subtropical regions Environmentalconditions particular to this biome make mangroves hotspots for microbial diversity and the resident microbial communities playessential roles in maintenance of the ecosystem Recently there has been increasing interest to understand the composition andcontribution of microorganisms in mangroves In the present study we have analyzed the diversity and distribution of archaea inthe tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencingThe extraction of DNA from sedimentsamples and the direct application of 16S rRNA gene amplicon sequencing resulted in approximately 142Mb of data from threedistinct mangrove areas (Godkhali Bonnie camp and Dhulibhashani) The taxonomic analysis revealed the dominance of phylaEuryarchaeota andThaumarchaeota (Marine Group I) within our datasetThe distribution of different archaeal taxa and respectivestatistical analysis (SIMPERNMDS) revealed a clear community shift along the sampling stationsThe sampling stations (Godkhaliand Bonnie camp) with history of higher hydrocarbonoil pollution showed different archaeal community pattern (dominated byhaloarchaea) compared to station (Dhulibhashani) with nearly pristine environment (dominated by methanogens) It is indicatedthat sediment archaeal community patterns were influenced by environmental conditions

1 Introduction

Archaea representing the third domain of life were orig-inally anticipated to thrive under extreme environmentssuch as hydrothermal vents hot water springs salt brinesand extremely acidic and anoxic environments where theycontribute significantly towards the maintenance of thebiogeochemical cycles [1ndash7] However with the advent ofmolecular techniques it has become increasingly evident

that archaea are much more widespread and metabolicallydiverse than originally postulated A considerable portionof the microbial communities in a wide variety of ldquononex-tremerdquo environments for example soil ocean and lakes isconstituted by archaea [8ndash13] Despite the increasing interestto understand the ecophysiology of archaea the lack ofknowledge with respect to mesophilic and cold-adoptedarchaea is still enormous [9 14 15]

Hindawi Publishing CorporationArchaeaVolume 2015 Article ID 968582 14 pageshttpdxdoiorg1011552015968582

2 Archaea

Mangrovewetlands are typical example ofmesophilic andmoderately halophilic environmental niches They are com-monly situated at the intertidal zones along the tropical andsubtropical coasts and play a very important role in shapingthe coastal ecology [16] Mangrove forests are consideredto be highly productive niche that support detritus-basedfood web [16 17] Particularly in tropical mangroves thehigh turnover rates for organic matters and nutrient cyclingbetween the ocean and terrestrial habitats makes it the mostproductive ecosystem in the world [17] The high primaryproductivity of mangroves implies a high demand for nutri-ents essential for plant growth and this appears to be achievedby a highly efficient system of nutrient trapping uptake andrecycling in mangrove ecosystem [18] The diverse microbialcommunities residing in the mangroves play important rolein transformation of nutrients in the environment While theimportance of bacteria and fungi in mangrove biogeochem-ical cycles is well established our knowledge of archaea inmangrove habitats remains extremely limited [16]

Sundarbans is the worldrsquos largest tidal halophyticmangrove ecosystem covering 20400 square kilometers(7900 sqmi) of area and has been recognized as a UNESCOWorld Heritage site Situated in the delta of Ganges Meghnaand Brahmaputra rivers on the Bay of Bengal Sundarbansis shared between India and Bangladesh This mangroveecosystem is the home for diverse flora and fauna includingmangrove plant species like sundari (Heritiera fomes)goran (Ceriops decandra) geoa (Excoecaria agallocha)keora (Sonneratia apetala) and so forth and the worldrsquosfamous endangered royal Bengal tiger (Panthera tigris tigris)Microbial communities play an important role in generationof detritus in mangrove areas and Sundarbans is one suchecotype where microbes have been shown to be involvedin biogeochemical cycling of the nutrients [18ndash20] Despiteglobal advancement in understanding the microbial diversityand role of microbes in different mangrove environmentslittle has been performed in Sundarbans [21 22] In a veryrecent study a detailed description of the bacterial diversityhas been presented in the backdrop of seasonal changes [23]However no data are yet available regarding the abundanceand diversity of archaea in Sundarbans

Hitherto most of the research on mangrove environ-ments has focused on understanding the diversity andfunctions of bacteria and fungi [23ndash26] and very little isknown about archaeal assemblages in mangrove [16 27 28]In order to gain new insight into the archaeal communitypatterns and the influence of environmental conditions in themangrove sediments and to build foundational informationfor future research we have investigated the archaeal diversityin the sediments of Indian Sundarbans employing 454-pyrosequencing

2 Materials and Methods

21 Ethics Statement No specific permits were requiredfor the described field studies which complied with allrelevant regulation The studied locations are not privatelyowned Moreover the study did not involve endangered or

protected species Indeed the Indian Coastal Zone Manage-ment (ICZM) authority of the state ofWest Bengal India hasapproved this experimental exercise

22 Study Area and Sediment Sampling Samples werecollected in triplicate from surface (2 cm) and subsurface(32 cm) sediments of the Sundarbans mangrove wetlandwhich is located on the northeastern coast of India(Figure 1) The samples were collected from threedifferent stations for example Godkhali (station A22∘0610158403257010158401015840N 88∘4610158402222010158401015840E) Bonnie camp (station B21∘4910158405358110158401015840N88∘3610158404486010158401015840E) andDhulibhashani (stationC 21∘3710158404083710158401015840N 88∘3310158404776210158401015840E) spanning 90 km alongthe tidal gradient from the shoreline (Figure 1) In June 2013the sediment samples were collected in triplicate from eachstation A sediment corer (50 cm depth 6 cm diameter) wasused to collect the top 32 cm of sediments The uppermostsurface layer (0ndash2 cm) and deeper subsurface layer (30ndash32 cm)were sampled and immediately sieved by a 2mmmeshin the field The sieved fractions were stored in sealed sterilepolypropylene containers and brought to the laboratoryUpon arrival a portion of each sediment sample wasflash-frozen at minus80∘C for polyaromatic hydrocarbon (PAH)analysis and DNA extraction The remaining sediments werestored at 4∘C for other analytical procedures such as nutrientsand heavy metals estimation The individual collection fromeach sampling station was used for physicochemical analysisand in case of DNA extraction for sequencing analysisthree individual collections were mixed to homogeneity togenerate a representative composite sample

23 Sediment Analyses and Site Climate Microbiological andbiochemical analyses were performed with the field moistsediments Physical and chemical analyses were carried outwith air-dried sediment samples The sediment pH wasmeasured in 1 25 sediment-water suspensions and found tobe alkaline The total organic carbon (TOC) was measuredby the methods described previously [29 30] Briefly TOCin a sample was determined by combusting the air-driedsediment sample catalytically in oxygen atmosphere intoinstrument chamber at 500∘ndash900∘C and the resulting carbondioxide gas was detected by a nondispersive infrared (NDIR)detector in an Aurora TOC Analyzer that was calibrated todirectly display the detected carbon dioxide mass This masswas proportional to the TOC mass in the sediment sampleand calculated as total mass of carbon per unit of sedimentsample

Conductivity and salinity were measured in situ withHach Portable Meters (HQ40d) Measured salinity wasexpressed in parts per thousand (ppt) or gmKgminus1 asdescribed previously [31] Nutrients like inorganic nitrogen(ammonia nitrite and nitrate) soluble phosphate and reac-tive silicate were measured after quantitative extraction inrespective buffering conditions following standard method-ologies [32] Briefly nitrite was measured after complexingwith sulphanilamide followed by a coupling reactionwith n(1-napthyl)-ethylenediamene dihydrochloride which forms anazo dye upon coupling The resulting azo dye was measured

Archaea 3

Survey locationsReclaimed areas ofIndian SundarbansUnreclaimed areas ofIndian SundarbansTiger reserveforested area

Core area undertiger reserveSajnekhali wildlifesanctuaryInternational boundary

Godkhali

Bonniecamp

Dhulibhashani

MatlaEstuary

Bang

lade

sh

Saptamukhi

Estuary

Hugli E

stuar

yHariabhanga

21∘45

9984000998400998400N

22∘09984000998400998400N

22∘15

9984000998400998400N88

∘09984000998400998400E 88

∘15

9984000998400998400E 88

∘30

9984000998400998400E 88

∘45

9984000998400998400E 89

∘09984000998400998400E

88∘09984000998400998400E 88

∘15

9984000998400998400E 88

∘30

9984000998400998400E 88

∘45

9984000998400998400E 89

∘09984000998400998400E

0 5 10 20 30

(km)

21∘45

9984000998400998400N

22∘09984000998400998400N

22∘15

9984000998400998400N

WN

ES

Figure 1 Geographical location of the sampling stations (Jharkhali-A Sahidnagar-B and Godkhali-C) in Indian Sundarbans Coordinatesof the sampling points and description of the stations are presented in Section 2

spectrophotometrically at 543 nm The nitrate in contrarywas quantitatively reduced to nitrite using cadmium (Cd)granules prior to measurement The total nitrite was thenmeasured spectrophotometrically as described earlier andfurther subtraction of themeasured value of free nitrite in thesediment resulted in determination of nitrate in the sampleAmmonia was measured in a reaction with hypochloriteunder alkaline condition which results in formation ofmonochloramine In a successive reaction with phenol andnitroprusside monochloraminewas converted into indophe-nol blue which was measured spectrophotometrically at630 nmThe soluble phosphate was measured using acidifiedmolybdate reagent which yields phosphomolybdate com-plex upon reaction with soluble phosphate This complexwas further reduced into molybdenum blue and measuredspectrophotometrically at 880 nm The reactive silicate wasmeasured using the formation of yellow silicomolybdic acidin presence of molybdate under acidic condition

Organic pollutants (polyaromatic hydrocarbons PAH)were measured using a combined gas chromatography andmass spectrometry (GCMS) method described previously[33 34] Heavy metals in the sediment samples were assessedusing atomic absorption spectrophotometric technique (Agi-lent Technologies CA USA)

24 Sediment DNA Isolation For 454-pyrosequencing eachof the sediment samples from a station (total 119899 = 3) andaliquots of homogenized sediment of 05 g were subjected toDNA extraction using the MoBio PowerSoil DNA IsolationKit (MoBio Laboratories Carlsbad CA) After the extractionDNA from all three samples from each sampling station waspooled together (approximately 200 ng of DNA from eachextraction) and the pooledDNAwas concentrated in a speedvacuum centrifuge (2500 rpm 30min) to a final volumeof 25 120583L (approximately 24 ng 120583Lminus1) A NanoDrop (ThermoScientific Wilmington DE USA) spectrophotometer wasused to quantify the extracted pooled DNA and to measureother important parameters forDNAquality such as the ratioof absorbance at 260280 nm and 260230 nm

25 PCR Amplification of Archaeal 16S rRNA Gene Toanalyze archaeal diversity the V3ndashV5 region of archaeal16S rRNA gene was amplified by PCR The PCR reac-tion (25 120583L) contained 5 120583L of 5-fold Phusion GC buffer(Finnzymes Vantaa Finland) 200120583M of each of the fourdeoxynucleoside triphosphates 15mMMgCl

2 4 120583M of each

primer (see Table S1 of the Supplementary Material avail-able online at httpdxdoiorg1011552015968582) 25

4 Archaea

DMSO 1U of Phusion High Fidelity Hot Start DNA poly-merase (Finnzymes) and 25 ng of pooled sediment DNAThe following thermal cycling scheme was used on a VeritiThermal Cycler (Applied Biosystems USA) initial denatura-tion at 98∘C for 5min 25 cycles of denaturation at 98∘C for45 s and annealing at 68∘C for 45 s followed by extensionat 72∘C for 30 s The final extension was carried out at 72∘Cfor 5min Negative controls were performed by using thereaction mixture without template Primer sequences foramplification of theV3ndashV5 region [35] as well as 454 adaptorswith the unique MIDs for each sample are listed in Table S1

26 Library Preparation PCR amplicons were evaluated byelectrophoresis on a 15 agarose gel The amplicon librarywas purified with Agencourt AMPure XP beads (BeckmanCoulter Inc Canada) and quantified by fluorometry usingthe Quant-iT PicoGreen dsDNA Assay Kit (InvitrogenBurlington ON) according to the Roche 454 ldquoAmpliconLibrary Preparation Method Manualrdquo of the GS Junior Tita-nium Series (454 Life Sciences USA) Pooled amplicons werediluted as recommended and amplified by emulsion PCR onaThermal Cycler 9700 (Applied Biosystem) according to theRoche 454 ldquoemPCR Amplification Method Manual Lib-Lrdquo(454 Life Sciences USA)

27 Sequencing and Data Processing Pyrosequencing wasperformed for 200 cycles on a Roche 454 GS Junior sequenc-ing instrument according to the manufacturerrsquos protocol(454 Life Sciences USA) All reads were filtered using thestandard read rejecting filters of the GS Junior sequencernamely key pass filters dot and mixed filters signal intensityfilters and primer filters (454 Sequencing System SoftwareManual V 253 454 Life Sciences USA) Raw sequencingand processing data was carried out using a combination ofmothur (software version 1280) and Ribosomal DatabaseProject (RDP-II) The raw data were subjected to initialquality trimming using the mothur software and all readshaving an average quality value of lt20 were discarded Thenwe removed the number of chimeric sequences by usingUCHIME algorithm Then the chimeric free reads werefurther screened for the presence of ambiguous bases andany reads which have a length lt200 were discarded andforward primer sequence was removed from the final datasetThe obtained processed reads were then demultiplexed toseparate sample based on the 10 bp MID sequence and anyreads which do not match the MID were discarded Thehigh quality reads were then aligned to archaeal 16S rRNAsequences and clustering was performed at 97 similarityusing the RDP pipeline a representative sequence from eachcluster was selected based on abundance using the sequenceselection tool The representative sequences obtained fromeach cluster were then classified using the naıve BayesianClassifier (Ribosomal Data Project release 10) at a bootstrapconfidence of 80

28 Taxonomic Annotation After quality trimming all thesequence reads were aligned against the SILVA which is acurated ribosomal RNA sequence database using BLASTN

[36 37] The resulting output files of read sequences wereimported and analyzed using the paired-end protocol ofMEGAN [38] to obtain taxonomic profiles as describedearlier [39] When processing the BLAST output files byMEGAN we used parameter settings of ldquoMin Score = 35rdquoldquoTop Percent = 100rdquo ldquoMin Support = 5rdquo and ldquoMinimumSequence Complexity Threshold = 044rdquo Reads which didnot have any match to the respective database were placedunder ldquoNo hitrdquo node Any reads that were originally assignedto a taxon that did not meet our selected threshold criterionwere pushed back using the lowest common ancestor (LCA)algorithm to higher nodes where the threshold was metAfter importing the datasets in MEGAN we obtained a set ofMEGAN-proprietary ldquorma filesrdquo for each data mapped ontothe NCBI taxonomy based on our selected threshold for treevisualization purpose

All six RMA files were normalized to the smallest dataset size (without including reads classified as not having ataxonomic assignment) to allow intersample comparison oftaxonomic abundances and to obtain comparative tree viewfor all samples Comparative abundance of read counts atdifferent levels of NCBI taxonomy (class order family genusand species) was exported from all sample comparison-filefor later statistical analyses

29 Statistical Analyses Hierarchical cluster analyses havebeen performed using R 302 [40] with species abundancedata where Pearsonrsquos correlation is used for clustering theprobesgenes (rows) and Spearman rank correlation is usedfor clustering the sample datasets (cols) with completelinkage

Composition (presenceabsence) data were also analyzedto compare the community structure SIMPER analyses wereused to determine which taxafunction contributed mostto the observed differences Further data were analyzedusing common multivariate ordination techniques Non-metric Multidimensional Scaling (NMDS) using Bray Curtisdistance To understand the influence of physicochemicalparameters in different sampling stations Principal Compo-nent Analysis (PCA) was performed Additionally we haveperformed correspondence analyses on genomic data as wellas on PAH (polyaromatic hydrocarbons) data

We have also plotted the genomic data using Voronoidiagram which is a partitioning of a plane into regions basedon distance to points in a specific subset of the plane In thisplot we have highlighted 6 major phyla

210 Nucleotide Sequence Accession Numbers All 454-GSJunior sequence data from this study were submittedto the NCBI Sequence Read Archive (SRA) underaccession numbers SRR1632262 (Godkhali surface)SRR1632263 (Godkhali subsurface) SRR1632258 (Bonniecamp surface) SRR1632259 (Bonnie camp subsurface)SRR1632260 (Dhulibhashani surface) and SRR1632261(Dhulibhashani subsurface)

3 Results31 Environmental Parameters Physicochemical characteris-tics such as pH dissolved oxygen saturation total carbon

Archaea 5

Table 1 Environmental parameters of sampling sites analyzed in this study

Sample Site Latitude ∘N Longitude ∘E Depth (cm) 119879 (∘C)lowast Salinity (psu)lowast pHlowast DO (mgL)lowast

Surface Godkhali 22∘0610158403257010158401015840 88∘4610158402222010158401015840 2 3130 2100 788 732Subsurface Godkhali 22∘0610158403257010158401015840 88∘4610158402222010158401015840 32 3180 2080 785 724Surface Bonnie camp 21∘4910158405358110158401015840 88∘3610158404486010158401015840 2 3210 2160 798 715Subsurface Bonnie camp 21∘4910158405358110158401015840 88∘3610158404486010158401015840 32 3250 2140 797 705Surface Dhulibhashani 21∘3710158404083710158401015840 88∘3310158404776210158401015840 2 2970 2280 796 715Subsurface Dhulibhashani 21∘3710158404083710158401015840 88∘3310158404776210158401015840 32 3010 2210 795 702lowastAverage of three independent measurements 119899 = 3

total nitrogen and salinity were estimated for surface andsubsurface samples from all the three stations in the presentstudy (Table 1 and Table S2) The environmental parametersfor example salinity temperature dissolved oxygen pH andsaturation were measured in situ (Table 1) Temperature andsalinity ranged from 297 to 328∘C and from 21 to 228 psurespectivelyThe lowest temperature and highest salinity wererecorded in Dhulibhashani

Measurement of NO2minus NO3

minus and NH4+ in the surface

and subsurface samples from all the three sampling stationsrevealed a differential pattern for these nitrogen species(Table S2) Ammonia was found maximum in the sedimentsamples of Godkhali while it was minimum in the sedimentof Dhulibhashani (119875 lt 005) Nitrite was found negligiblein all the stations confirming very high rate of nitrificationresulting in nitrate formation which was evenly detectedin all three sampling stations (Table S1) Other parameterssuch as phosphate sulphate and silicate were found evenlydistributed in all the sampling stations possibly due to regularinundation

Heavy metals have been shown to play a pivotal anthro-pogenic role in the marine environment Previously it hasbeen shown that heavymetals like Fe Zn CuMnHg Pb NiCr and Cd are present in the Sundarbans estuaries [41ndash44]To ascertain the global ecological impact of different heavymetals in the present study concentrations of heavy metalslike Fe Cd Zn Cu Ni and Pd were measured in surface andsubsurface samples collected from all the three stations (TableS2) Heavy metal estimates for the Sundarbans sedimentfound in our study were fairly consistent with previouslyfound results [45]

32 Archaeal Community Structure Revealed by 16S rRNAGene Based Analysis We analyzed archaeal 16S rRNA gene(SSU) amplicons prepared from six sediment samples orig-inated from three sampling stations at two different depthsAfter quality filtering denoising and removal of potentialchimeric sequences of pyrosequencing dataset this resultedin recovery of 141816 sequences with an average lengthranges between 500 and 510 bases (506 bp average) (Table 2)Within our sequence dataset we were able to assign 139953sequences to the domain archaea and to classify all ofthese sequences below domain level using BLASTN 2225+in SILVA database and MEGAN (Table 2) The classifiedsequences were affiliated to two archaeal phyla and fivearchaeal classes or similar phylogenetic group (Figure 2 and

Table 2 Sample statistics

SamplesTotal number

of readssequenced

Assigned againstSILVA using

BLASTN 2225+and MEGAN

Godkhali surface 15939 15647Godkhali subsurface 23437 23091Bonnie camp surface 15371 15120Bonnie camp subsurface 15788 15654Dhulibhashani surface 33771 33395Dhulibhashani subsurface 37510 37046

Figure S1) Euryarchaeota often was the most abundantphylum (36ndash60) and Thermoplasmata was the predomi-nant class across all samples (39ndash62) (Figure S1) BesidesEuryarchaeotaThaumarchaeota (Marine group I) was foundto be highly abundant in all the samples (Table S3) Withineuryarchaeal sequences a number of members of the classHalobacteria for example Halosarcina Halorientalis Halo-lamina Halorhabdus Halogranum Haloferax HalomarinaHalorussus Haloplanus and Halarchaeum and of the classMethanomicrobia for example Methanosarcina Mether-micoccus Methanocella Methanococcoides MethanosalsumMethanolobus andMethanogeniumwere detected (Figure 2)Besides few sequences were also affiliated to classes Ther-moplasmata andMethanobacteria Unfortunately within ourdatasets we could not detect any representative of thearchaeal phyla Crenarchaeota Korarchaeota Nanoarchaeotaand Nanohaloarchaeota due to lower coverage of the degen-erate primers used in this study

33 Diversity and Species Richness of Archaeal CommunitiesA diversity index is a mathematical measure of speciesdiversity in a communityDiversity indices provide importantinformation about community composition rather than onlyspecies richness (ie the number of species present) andsimultaneously they also take the relative abundances ofdifferent species into account We evaluated the archaealdiversity and evenness using the normalized dataset Consid-ering all the sampling sites the Shannon-Weaver (H1015840) indexvaried from 067 to 108 and the Simpson diversity (1 minus 1205821015840)index varied from 029 to 052 (Table 3) Such variations ofdiversity indicated that the archaeal diversity is higher atthe surface samples both in Bonnie camp and Godkhali

6 Archaea

0 1 2Row Z-score

MethanosaetaHalosarcinaHalorientalisHalolaminaNitrosomonasHalorhabdusHalogranumHaloferaxHalomarinaHalorussusHaloplanusNatronoarchaeumHalarchaeumMethanosarcinaMethermicoccusMethanocellaMethanococcoidesMethanosalsumThaumarchaeotaMethanolobusMethanobacteriaceaeThermoplasmatalesMethanogenium

minus2 minus1

Dhu

libha

shan

i_su

bsur

face

God

khal

i_su

bsur

face

Bonn

ie ca

mp_

subs

urfa

ce

Bonn

ie ca

mp_

surfa

ce

Dhu

libha

shan

i_su

rface

God

khal

i_su

rface

Figure 2 Hierarchical clustering (complete-linkage) heat map generated from taxonomic abundance profile (using Spearmanrsquos rankcorrelation) reflecting spatial distribution of archaeal class in the sediment of Sundarbans (using Pearsonrsquos correlation)

However in Dhulibhashani archaeal diversity was foundto be almost similar both at the surface and at subsurfacelevel with a marginally higher diversity in subsurface sampleThe Pielou index (J1015840) was between 023 and 037 and theMargalef (d) index was between 152 and 369 (Table 3)Diversity and evenness are more informative for describingcommunity composition than simple phylotype richnesslevels Community diversity as reflected by the Shannonindex was highest in Bonnie camp subsurface and lowest inGodkhali subsurface and is by definition generally correlatedpositively with the number of unique phylotypes andor withgreater community evenness

34 Taxonomic Assignments and Statistics In addition tosurveying archaeal diversity at the surface and subsurfacesediments of three sites in Sundarbans pyrosequencingalso allowed assessment of the relative abundance of thetaxonomic levels of archaea detected A total of 23 genera(genusorderfamily) were commonly shared between six

samples (Figure 2 and Figure S1) Interestingly the dominantclassphylum showed some geographical characteristics Forexample Halobacteria were highly abundant in the sub-surface layers of the representative samples from Godkhaliand Bonnie camp In contrary Methanomicrobia were onlydominant in the subsurface sediment of Dhulibhashani

Cluster analysis showed that the archaeal communitydetected in the subsurface sediment ofDhulibhashaniwas themost dissimilar of all the sediment samples tested (Figure 2)Additionally among the subsurface sediments the mostdiverse archaeal community was detected in this sedimentAmong others there is a clear separation of two types ofsamples surface and subsurface The surface sediments haveshown an overall similarity among Godkhali and Bonniecamp while Dhulibhashani surface showed a different com-munity profile

Furtherwe have performedNonmetricMultidimensionalScaling (NMDS) to compare the community compositionusing presenceabsence data NMDS employs an iterative

Archaea 7

Table3Univaria

tediversity

indices

Sample

Total

species(119878)

Total

individu

als

(119873)

Speciesrichn

ess

(Margalef)

119889=(119878minus1)Lo

g(119873)

Pielo

ursquosevenness

119869

1015840

=119867

1015840

Lo

g(119878)

Shanno

n119867

1015840

=minusSU

M(119875

119894

lowast

Log(119875

119894

))

Simpson(1minusLambd

a1015840)=1minusSU

M(119873

119894

lowast(119873

119894

minus1)119873lowast(119873minus1))

God

khalisurface

11100

2171

03447

08264

05206

God

khali sub

surfa

ce17

100

3474

02384

06753

02998

Bonn

iecampsurfa

ce8

100

152

03597

0748

05104

Bonn

iecampsubsurface

18100

3692

03763

1088

04389

Dhu

libhashani surface

16100

3257

03454

09575

04761

Dhu

libhashani sub

surfa

ce13

100

260

60357

09157

04858

8 Archaea

DhulibhashaniGodkhaliBonnie camp

Transform presenceabsenceResemblance S17 Bray-Curtis similarity

Bonnie camp_surface

Godkhali_surface

Godkhali_subsurface

Bonnie camp_subsurface

Dhulibhashani_subsurface

Dhulibhashani_surface

2D stress 0

Similarity6080

Locations

Figure 3 Archaeal community compositional structure in thesediments of Sundarbans indicated byNonmetricMultidimensionalScaling (NMDS) using Bray-Curtis distance Gene abundance datawas transformed based on presenceabsence before creating Bray-Curtis resemblance matrix for NMDS analysis (showing similaritybased on community composition only)

algorithm that optimizes the position of samples into a low-dimensional space minimizing the difference between rank-order of multivariate ecological dissimilarity and distances inNMDS space (Figure 3) From the figure it is easily observedthat despite the presence of very few abundant taxa inmore than one sample surface and subsurface communitiesfrom different sampling stations or locations (Godkhali andBonnie camp) were more closely related to each other thanto samples from the same location Samples from Dhulib-hashani show similar results to cluster analyses showingmuch different community structure than the other locationsfor both surface and subsurface samples To this end webelieve that the presence or absence of certain taxonomicgroups in the sediment of Dhulibhashani is probably dueits proximity to the open ocean (Bay of Bengal) and thenutritional status of the sediment

Furthermore our PCA analysis revealed that the physic-ochemical parameters influence sampling stations differen-tially The first PCA axis showed high positive correlationswith heavy metals Zn Pb and Cu (Figure 4(a)) Samplesfrom Bonnie camp showed high positive correlations with allof these heavy metals In contrary samples from Godkhalishowed positive correlations with heavy metals Cd and Fe(Figure 4(a)) To this end PCA analysis revealed that both ofthese sampling stations at Godkhali and Bonnie camp wereat an elevated risk compared to Dhulibhashani regardingheavy metal pollution Notably these two sampling stationsalso showed positive correlations with pH DO silicatenitrite ammonia saturation and total nitrogen (Figure 4(a))

Dhulibhashani on the other hand showed positive correla-tions with TOC nitrate sulphate and salinity (Figure 4(a))

Further we have performed correspondence analysis(CA) to compare the influence of abundant taxonomic groupsand polyaromatic hydrocarbons (PAH) in these samplingsediments (Figures 4(b) and 4(c)) The correspondenceanalysis comparing abundant taxonomic groups in the sam-pling stations clearly indicated that the subsurface sedi-ments from Godkhali and Bonnie camp were heavily dom-inated by Halobacteriaceae (Figure 4(b)) whereas surfacearchaeal communities in these sampling stations were dom-inated by Methanomicrobiaceae Methermicoccaceae andMethanocellaceae (Figure 4(b)) Both the surface and sub-surface archaeal communities in Dhulibhashani were heavilydominated byMethanosarcinaceae andMethanobacteriaceae(Figure 4(b)) To summarize methanogens were commonlyencountered in the surface sediments of all three stationsHowever while Halobacteriaceae dominated the subsur-face archaeal population in Godkhali and Bonnie campmethanogenic Methanosarcinaceae and Methanobacteri-aceae dominated the subsurface sediment of Dhulibhashani

Correspondence analysis performed to understand theinfluence of PAHs in the sediments of the sampling stationsrevealed that both the surface and subsurface sediments ofGodkhali and Bonnie camp correlatedwith the detected PAHcongeners (Figure 4(c)) In contrary surface and subsurfacesediments of Dhulibhashani showed little correlation to anyof the identified PAHs To this end we believe that the patternof correlation of PAHs in the sediments of Godkhali andBonnie camp is in agreement with the detection of Halobac-teriaceae the most active hydrocarbon degrading archaealrepresentative in the subsurface sediments of these stationsIn general hydrocarbons by virtue of being hydrophobic innature are scarcely soluble inwater and tend to adsorb readilyonto organic matter particularly sediments In an envi-ronment like Sundarbans surface sediments are in generalmore dynamic due to regular inundationThe hydrocarbonstherefore remained mostly absorbed in the sediments andour data clearly shows that subsurface sediments containmore PAHs compared to surface sediments

Finally we have generated Voronoi plot (Figure 5) whichdescribes the proportionate taxonomic composition patternfor each sample as a partition of a plane

35 Multivariate SIMPER (Similarity Percentage) AnalysisTo reveal differences between three sampling stations God-khali Bonnie camp and Dhulibhashani and to identifymajor taxonomic groups that contributed to the similaritiesor dissimilarities SIMPER (Similarity Percentage) analysiswas performed (Tables 4(a) 4(b) and 4(c)) At the regionalscale there were differences observed at orderfamilygenuslevel between three sampling stations Overall distribu-tion of taxa was found similar in Godkhali and Bonniecamp with Thermoplasmatales being the major contributortowards the similarity (233) and with only exceptionof Methanosarcina the relative abundance of which washigher in Bonnie camp In general SIMPER analysis targetingrelative abundance of taxa in different stations revealedhigher relative abundance of class Halobacteria in Godkhali

Archaea 9

00 05

02

06

PC1

PC2

N

Cd

Cu

Pb

Zn

FeAmmonia

Silicate

Phosphate

NitriteNitrate

Sulphate

Temperature

pH

DO Saturation

Salinity

Total nitrogen

TOC



Bonnie camp_surface


Godkhali_surface

Godkhali_subsurfaceminus06

minus02

minus05

(a)

Dimension 1 (719)

Dim

ensio

n 2

(18

3)

00 05 10

00

05


Bonnie camp_surface

Dhulibhashani_subsurfaceGodkhali_surface

Bonnie camp_subsurfaceGodkhali_subsurface

Halobacteriaceae

Methanobacteriaceae

Methanocellaceae

Methanomicrobiaceae

Methanosaetaceae

Methanosarcinaceae

Methermicoccaceae

Thermoplasmatales Thaumarchaeota

minus10

minus05

minus15 minus10 minus05

(b)

Dimension 1 (771)

Dim

ensio

n 2

(19

7)

00 02 04 06

00

01

02

Dhulibhashani_surfaceDhulibhashani_subsurface

Bonnie camp_surface


Godkhali_surface

Godkhali_subsurfaceNaphthalene

Acenaphthylene

Fluorene

Phenanthrene

Fluoranthene

Acenaphthene

Pyrene

minus02

minus03

minus01

minus04 minus02

(c)

Figure 4 (a) Principal Component Analysis (PCA) of samples based on environmental parameters nutrient and heavy metal levels in thesediments samples (b) Correspondence analysis (CA) based on distribution of major taxonomic lineages generated analyzing genomic datain the sediment samples (c) Correspondence analysis (CA) based on distribution of polyaromatic hydrocarbons (PAH) in the sedimentsamples

and Bonnie camp while class Methanomicrobia was foundrelatively more abundant in Dhulibhashani (Tables 4(b)and 4(c)) Representatives of the class Methanomicrobiasuch as Methanolobus Methanococcoides Methermicoccusand Methanocella were either higher or only present inDhulibhashani In contrary representatives of class Halobac-teria such as Halogranum Halorientalis Haloferax andHalarchaeum were either higher or only present in God-khaliBonnie camp (Table 4(a)) Putative ammonia-oxidizingarchaea in the phylum Thaumarchaeota were highly abun-dant in all three sampling sites To our surprise the orderThermoplasmatales representing acidophilic Euryarchaeotawere highly abundant in all three sampling stations despitethe fact that the recorded pH fell between 785 and 798 inthese sites

4 Discussion

In marine environments microorganisms play an importantrole in sustainable maintenance of the ecosystem Theyare key mediators of global biogeochemical cycling of theessential elements in the marine environments Recentadvancement of molecular microbial ecology has emerged as

multiple studies on the diversity and abundance of microor-ganisms in these habitats and their role in shaping the sus-tainable ecosystem Most of these studies focused on marinebacteria and archaea whereas little is known on the diversityand ecology ofmangrove archaea [16 17 23 24] Interestinglyarchaea have been found to be ubiquitous and abundantmembers of the microbiome in diverse marine environmentsincluding coastal waters [46 47] marine sediments estuaries[48ndash50] stratified basins [7 51] mangrove sediments [52ndash54] and open ocean water columns [47 55] In marinehabitat archaea play crucial roles in nitrification [54 56 57]sulfur metabolism [58] methane oxidation (ANME) [59]and methanogenesis [60]

The present study focused on accessing the archaealcommunity structure and diversity in the worldrsquos largesttropical mangrove sediments of Sundarbans using 16S rRNAgene-based 454-amplicon sequencing To our knowledgethis is the first study on understanding archaeal diversity inSundarbans ecosystem The majority of sequences obtainedwere affiliated to the Euryarchaeota Previous studies by Sappet al on marine sediment and Wemheuer et al on GermanBight found high abundance of members of Euryarchaeota

10 Archaea


Godkhali_subsurfaceBonnie camp_subsurface

Bonnie camp_surfaceDhulibhashani_surface

Godkhali_surface

Figure 5 Voronoi diagramdepicting comparative taxonomic profile composition pattern for each sample as a partition of a plane highlighting6 major phyla

[35] We identified Thermoplasmatales as the most abun-dant euryarchaeal group in the investigated samples Mostsequences were affiliated to the MKCST-A3 CCA47 andVC21Arc6 groupsTheMKCST-A3 group was first identifiedin themangrove sediment of China andmost of themembersreported so far are uncultured archaeon [16] The CCA47group was originally identified by 16S rRNA gene analysis ofoxygen-depleted marine environment and anoxic subsalinesediments The number of sequences within this groupwas also affiliated to the Marine Group II (Euryarchaeota)Previous studies by DeLong and Karl have suggested thatthe members of Marine Group II are more abundant intemperate sea water than Marine Group I (Thaumarchaeota)[61] In our case however overall abundance of MarineGroup I (Thaumarchaeota) is higher compared to MarineGroup II (Euryarchaeota) possibly emphasizing geographicaldifferences between tropical and temperate marine sedimentmicrobial communityMoreover thismight also be due to thetidal current that influences the microbial community struc-ture within Sundarbans sediment The regular inundation ofthe subtidal zones within Sundarbans whirls up microbialcells to thewater columnAn in-depth analysis of the archaealdiversity and abundance in the water column might beuseful to know whether we could detect a large numberof sequences affiliated to Marine Group II Unfortunatelyonly limited studies have been performed targeting archaealcommunities in the water column in tropical mangroveDue to such knowledge gap the habitat preference of

Marine Group II cannot be addressed properly at this timeThe Thermoplasmatales cluster VC21Arc6 was originallydescribed within the microbial community obtained from insitu growth chamber placed on a deep-sea hydrothermal venton the Mid-Atlantic Ridge [62] Besides Thermoplasmatalesthe number of euryarchaeal sequences was affiliated toHalobacteria Members of this group can grow aerobicallyas well as anaerobically The majority of the halobacterialsequences analyzed in the present study were affiliated tothe Halogranum genus Besides a significant number ofsequences affiliated to Natronomonas Haloferax Halorhab-dus and Halolamina were identified Methanomicrobia andMethanobacteria were the other two abundant euryarchaealclasses in the investigated samples Methanomicrobia andMethanobacteria are known for their putative importancein sulfate reduction and methanogenesis in anoxic marinesediments such as mangroves The members of these classesare methanogens and are involved in carbon-cycle throughmethanogenesis

Beside euryarchaeota another archaeal group found in allsamples was the Marine Group I (MG1) or ThaumarchaeotaMarine Group I (MGI) Thaumarchaeota are one of the mostabundant and cosmopolitan chemoautotrophs and prokary-otic picoplankton within the global deep sea water Theywere originally identified by sequencing of environmental 16SrRNA genes derived from sea water All organisms of this lin-eage thus far identified are chemolithoautotrophic ammonia-oxidizers and may play important roles in biogeochemical

Archaea 11

Table 4 Results of SIMPER (Similarity Percentage) analyses indicating the contribution of specific taxa to observed differences in archaealcommunity structure among the stations (a) shows differences between Godkhali and Bonnie camp (b) shows differences betweenDhulibhashani and Godkhali and (c) shows differences between Dhulibhashani and Bonnie camp

(a) Godkhali versus Bonnie camp

Taxon1 Orderfamilygenus Contribution2 ()Average3 abundance inGodkhali

Average abundancein Bonnie camp

Averagedissimilarities

Thermoplasmata Thermoplasmatales 2330 485 464 523Halobacteria Halogranum 1172 150 164 263Thaumarchaeota Thaumarchaeota 1008 829 799 226Methanomicrobia Methanolobus 833 065 099 187Methanomicrobia Methanococcoides 646 040 082 145Halobacteria Haloferax 542 048 047 122Halobacteria Natronomonas 541 062 073 121Methanomicrobia Methanosarcina 500 055 103 112Halobacteria Halorhabdus 397 035 035 089Halobacteria Halolamina 349 038 030 078Methanomicrobia Methanosalsum 346 013 033 078Halobacteria Halarchaeum 317 008 030 071Halobacteria Halorientalis 217 019 016 049

(b) Dhulibhashani versus Godkhali

Taxon1 Orderfamilygenus Contribution2 ()Average3 abundance inDhulibhashani

Average abundancein Godkhali


Methanomicrobia Methanolobus 1980 234 065 428Thermoplasmata Thermoplasmatales 1875 459 485 405Methanomicrobia Methanococcoides 1214 143 040 262Thaumarchaeota Thaumarchaeota 964 829 829 208Halobacteria Halogranum 880 075 150 190Methanomicrobia Methermicoccus 402 034 000 087Methanomicrobia Methanosalsum 384 045 013 083Halobacteria Haloferax 358 018 048 077Halobacteria Natronomonas 349 042 062 075Methanomicrobia Methanocella 247 021 000 053Halobacteria Halorientalis 220 000 019 047Halobacteria Halolamina 190 024 038 041

(c) Dhulibhashani versus Bonnie camp

Taxon1 Orderfamilygenus Contribution2 ()Average3

abundance in stationA

Average abundancein station B


Thermoplasmata Thermoplasmatales 2076 459 464 512Methanomicrobia Methanolobus 1423 234 099 351Halobacteria Halogranum 1005 075 164 248Methanomicrobia Methanococcoides 865 143 082 213Thaumarchaeota Thaumarchaeota 672 829 799 166Halobacteria Natronomonas 473 042 073 117Halobacteria Haloferax 442 018 047 109Methanomicrobia Methanosarcina 396 070 103 098Methanomicrobia Methermicoccus 342 034 000 084Methanomicrobia Methanosalsum 340 045 033 084Halobacteria Halorhabdus 337 024 035 083Halobacteria Halolamina 292 024 030 072

12 Archaea

(c) Continued





Halobacteria Halarchaeum 270 000 030 067Methanomicrobia Methanocella 210 021 000 0521Phylum or class level for archaea2Contribution of each taxon to the overall dissimilarity between these two clusters3Average abundance of each taxon in the two clusters

cycles such as the nitrogen cycle and the carbon cycleWithinour sequence dataset the number of ammonia oxidizingThaumarchaeota was identified

We observed an increased abundance of Halobacteri-aceae in the investigated subsurface samples from Godkhaliand Bonnie camp In contrary the number of Methanosarci-naceae and Methanobacteriaceae was higher in the samplesderived from Dhulibhashani This might be correlated withthe high amounts of organic matter in resident algal bloomsin Godkhali and Bonnie camp (Figures 4(b) and 4(c))Furthermore correspondence analysis revealed that detectedPAHs were mostly correlated to the surface and subsurfacesediments of Godkhali and Bonnie camp Previous studiesfrom our group have shown that in Godkhali and Bonniecamp because of the presence of jetty for transportationlarge amount of hydrocarbon-derived chemicals and oils arereleased in water and in turn increase algal blooms in sur-rounding areas (Personal Communication) To this end webelieve that abundance of Halobacteriaceae in Godkhali andBonnie camp corroborates well to inflated detection PAHs inthe sediment In hypersaline environment Halobacteria arethemost active organisms capable of organicmatter degrada-tionThus the higher abundance of Halobacteria in Godkhaliand Bonnie camp might indicate an involvement in marineorganic matter degradation under high nutrient conditionsfound during algal blooms Similar results were reportedby Teeling et al where they demonstrated that bacterialcommunity structures were highly influenced by the presenceof an algal bloom [63] Furthermore Wemheuer et al haverecently demonstrated that archaeal diversity was influencedby algal blooms inGermanBight [35] Taken together presentobservation indicates that marinemicrobial communities areinfluenced by algal blooms or by environmental parameterscorrelated with bloom presence

5 Conclusions

In summary the spatial variations of the sedimentaryarchaeal diversity have been studied in the Sundarbansmangrove ecosystem for the first time by means of 16SrRNA454-pyrosequencingWe found highly diverse archaealcommunities in the sediment of Sundarbans Our analyseshave confirmed the influence of environment in shaping thearchaeal community diversity within the sediment Howeverdue to the lack of pure cultures and large comparativeinvestigations robust conclusions related to the involvementof identified archaeal communities in biogeochemical cycle

cannot be drawn Further research including analyses ofexpression of functional genes and determination of theactive archaeal populationwithin the sedimentmight unravelthe role of archaea in Sundarbans mangrove ecosystem

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to acknowledge the instrumentfacility provided by World Bank ICZM Project in theDepartment of Biochemistry University of Calcutta IndiaAnish Bhattacharyya Debojyoti Roy and Sudip Nag weresupportedwith Research Fellowships from the ICZMProjectWorld Bank Abhrajyoti Ghosh was supported by RamanujanFellowship from Department of Science and TechnologyIndia (SRS2RJN-1062012) and an extramural grant (no38(1391)14EMR-II) from the Council of Scientific amp Indus-trial Research (CSIR) Government of India

References

[1] S-V Albers P Forterre D Prangishvili and C Schleper ldquoThelegacy of Carl Woese and Wolfram Zillig from phylogeny tolandmark discoveriesrdquoNature Reviews Microbiology vol 11 no10 pp 713ndash719 2013

[2] K F Jarrell A D Walters C Bochiwal J M Borgia TDickinson and J P J Chong ldquoMajor players on the microbialstage why Archaea are importantrdquoMicrobiology vol 157 no 4pp 919ndash936 2011

[3] E F DeLong ldquoArchaea in coastal marine environmentsrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 89 no 12 pp 5685ndash5689 1992

[4] K Takai and K Horikoshi ldquoGenetic diversity of archaea indeep-sea hydrothermal vent environmentsrdquo Genetics vol 152no 4 pp 1285ndash1297 1999

[5] J L Macalady D S Jones and E H Lyon ldquoExtremely acidicpendulous cave wall biofilms from the Frasassi cave systemItalyrdquo Environmental Microbiology vol 9 no 6 pp 1402ndash14142007

[6] A Gittel K B Soslashrensen T L Skovhus K Ingvorsen andA Schramm ldquoProkaryotic community structure and sulfatereducer activity in water from high-temperature oil reservoirswith and without nitrate treatmentrdquoApplied and EnvironmentalMicrobiology vol 75 no 22 pp 7086ndash7096 2009

Archaea 13

[7] M J L Coolen B Abbas J van Bleijswijk et al ldquoPutativeammonia-oxidizing Crenarchaeota in suboxic waters of theBlack Sea a basin-wide ecological study using 16S ribosomaland functional genes and membrane lipidsrdquo EnvironmentalMicrobiology vol 9 no 4 pp 1001ndash1016 2007

[8] J Borneman and E W Triplett ldquoMolecular microbial diver-sity in soils from eastern Amazonia evidence for unusualmicroorganisms and microbial population shifts associatedwith deforestationrdquo Applied and Environmental Microbiologyvol 63 no 7 pp 2647ndash2653 1997

[9] D H Buckley J R Graber and T M Schmidt ldquoPhylogeneticanalysis of nonthermophilic members of the kingdom Crenar-chaeota and their diversity and abundance in soilsrdquoApplied andEnvironmentalMicrobiology vol 64 no 11 pp 4333ndash4339 1998

[10] G Jurgens K Lindstrom and A Saano ldquoNovel group withinthe kingdom Crenarchaeota from boreal forest soilrdquo Appliedand Environmental Microbiology vol 63 no 2 pp 803ndash8051997

[11] R Massana A E Murray C M Preston and E F DeLongldquoVertical distribution and phylogenetic characterization ofmarine planktonic Archaea in the Santa Barbara ChannelrdquoApplied and Environmental Microbiology vol 63 no 1 pp 50ndash56 1997

[12] C Schleper W Holben and H-P Klenk ldquoRecovery of cre-narchaeotal ribosomal DNA sequences from freshwater-lakesedimentsrdquo Applied and Environmental Microbiology vol 63no 1 pp 321ndash323 1997

[13] E F DeLong and N R Pace ldquoEnvironmental diversity ofbacteria and archaeardquo Systematic Biology vol 50 no 4 pp 470ndash478 2001

[14] K Knittel T Losekann A Boetius R Kort and R AmannldquoDiversity and distribution of methanotrophic archaea at coldseepsrdquo Applied and Environmental Microbiology vol 71 no 1pp 467ndash479 2005

[15] R Margesin and V Miteva ldquoDiversity and ecology of psy-chrophilic microorganismsrdquo Research in Microbiology vol 162no 3 pp 346ndash361 2011

[16] B Yan K Hong and Z-N Yu ldquoArchaeal communities inmangrove soil characterized by 16S rRNA gene clonesrdquo Journalof Microbiology vol 44 no 5 pp 566ndash571 2006

[17] T J Lyimo A Pol M S M Jetten and H J M Op den CampldquoDiversity of methanogenic archaea in a mangrove sedimentand isolation of a newMethanococcoides strain research LetterrdquoFEMS Microbiology Letters vol 291 no 2 pp 247ndash253 2009

[18] G Holguin P Vazquez and Y Bashan ldquoThe role of sedimentmicroorganisms in the productivity conservation and reha-bilitation of mangrove ecosystems an overviewrdquo Biology andFertility of Soils vol 33 no 4 pp 265ndash278 2001

[19] SDasMDe TK Jana andTKDe ldquoEnvironmental influenceon cultivable microbial community in the sediment of Sundar-ban mangrove forest Indiardquo African Journal of MicrobiologyResearch vol 7 no 38 pp 4655ndash4665 2013

[20] S Das P S Lyla and S A Khan ldquoMarine microbial diversityand ecology importance and future perspectivesrdquo CurrentScience vol 90 no 10 pp 1325ndash1335 2006

[21] A Ghosh N Dey A Bera et al ldquoCulture independent molecu-lar analysis of bacterial communities in the mangrove sedimentof Sundarban Indiardquo Saline Systems vol 6 no 1 article 1 2010

[22] A Chakraborty A Bera A Mukherjee et al ldquoChanging bacte-rial profile of Sundarbans the world heritage mangrove impactof anthropogenic interventionsrdquoWorld Journal of Microbiologyand Biotechnology vol 31 no 4 pp 593ndash610 2015

[23] P Basak N S Majumder S Nag et al ldquoSpatiotemporal analysisof bacterial diversity in sediments of Sundarbans using parallel16S rRNA gene tag sequencingrdquo Microbial Ecology vol 69 no3 pp 500ndash511 2014

[24] X-T Jiang X Peng G-H Deng et al ldquoIllumina Sequencingof 16S rRNA Tag Revealed Spatial Variations of BacterialCommunities in a Mangrove Wetlandrdquo Microbial Ecology vol66 no 1 pp 96ndash104 2013

[25] F L D D Sebastianes A S Romao-Dumaresq P T Lacavaet al ldquoSpecies diversity of culturable endophytic fungi fromBrazilian mangrove forestsrdquo Current Genetics vol 59 no 3 pp153ndash166 2013

[26] Y Arfi M Buee C Marchand A Levasseur and E RecordldquoMultiple markers pyrosequencing reveals highly diverse andhost-specific fungal communities on the mangrove trees Avi-cennia marinaand Rhizophora stylosardquo FEMS MicrobiologyEcology vol 79 no 2 pp 433ndash444 2012

[27] T J Lyimo A Pol M S M Jetten and H J M Op DenCamp ldquoDiversity of methanogenic archaea in a mangrovesediment and isolation of a newMethanococcoides strainrdquoFEMSMicrobiology Letters vol 291 no 2 pp 247ndash253 2009

[28] ACC PiresD F R Cleary AAlmeida et al ldquoDenaturing gra-dient gel electrophoresis and barcoded pyrosequencing revealunprecedented archaeal diversity in mangrove sediment andrhizosphere samplesrdquoApplied and Environmental Microbiologyvol 78 no 16 pp 5520ndash5528 2012

[29] D W Nelson and L E Somers Organic Carbon AcademicPress London UK 1975

[30] C A Black Methods of Soil Analysis American Society ofAgronomy Madison Wis USA 1965

[31] M Knudsen Hydrographical Tables GEC GAD CopenhagenDenmark 1901

[32] A Knap A Michaels A Close H Ducklow and A DicksonEds Protocols for the Joint Global Ocean Flux Study (JGOFS)Core Measurements JGOFS Report No 19 1996

[33] I Braganca A Placido P Paıga V F Domingues andC Delerue-Matos ldquoQuEChERS a new sample preparationapproach for the determination of ibuprofen and itsmetabolitesin soilsrdquo Science of the Total Environment vol 433 pp 281ndash2892012

[34] L Correia-Sa V C Fernandes M Carvalho C Calhau VM FDomingues and C Delerue-Matos ldquoOptimization of QuECh-ERS method for the analysis of organochlorine pesticides insoils with diverse organic matterrdquo Journal of Separation Sciencevol 35 no 12 pp 1521ndash1530 2012

[35] B Wemheuer F Wemheuer and R Daniel ldquoRNA-basedassessment of diversity and composition of active archaealcommunities in the German Bightrdquo Archaea vol 2012 ArticleID 695826 8 pages 2012

[36] E Pruesse C Quast K Knittel et al ldquoSILVA a comprehensiveonline resource for quality checked and aligned ribosomal RNAsequence data compatible with ARBrdquo Nucleic Acids Researchvol 35 no 21 pp 7188ndash7196 2007

[37] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[38] D H Huson S Mitra H-J Ruscheweyh N Weber and SC Schuster ldquoIntegrative analysis of environmental sequencesusingMEGAN4rdquoGenomeResearch vol 21 no 9 pp 1552ndash15602011

14 Archaea

[39] S Mitra M Stark and D H Huson ldquoAnalysis of 16S rRNAenvironmental sequences using MEGANrdquo BMC Genomics vol12 supplement 3 p S17 2011

[40] R Core Team R A Language and Environment for StatisticalComputing R Foundation for Statistical Computing ViennaAustria 2014

[41] S K Sarkar B Bhattacharya S Debnath G Bandopadhayaand S Giri ldquoHeavy metals in biota from Sundarban WetlandEcosystem India implications to monitoring and environmen-tal assessmentrdquo Aquatic Ecosystem Health amp Management vol5 no 4 pp 467ndash472 2002

[42] M T Rahman M S Rahman S B Quraishi J U Ahmad T RChoudhury and M A Mottaleb ldquoDistribution of heavy metalsin water and sediments in Passur River Sundarban MangroveForest Bangladeshrdquo Journal of International EnvironmentalApplication amp Science vol 6 no 4 pp 537ndash546 2011

[43] M Saha S K Sarkar and B Bhattacharya ldquoInterspecificvariation in heavy metal body concentrations in biota ofSunderban mangrove wetland northeast Indiardquo EnvironmentInternational vol 32 no 2 pp 203ndash207 2006

[44] K Banerjee B Senthilkumar R Purvaja and R Ramesh ldquoSed-imentation and trace metal distribution in selected locations ofSundarbans mangroves and Hooghly estuary Northeast coastof Indiardquo Environmental Geochemistry and Health vol 34 no1 pp 27ndash42 2012

[45] S Massolo A Bignasca S K Sarkar M Chatterjee B DBhattacharya andAAlam ldquoGeochemical fractionation of traceelements in sediments of Hugli River (Ganges) and Sundarbanwetland (West Bengal India)rdquo Environmental Monitoring andAssessment vol 184 no 12 pp 7561ndash7577 2012

[46] T J Mincer M J Church L T Taylor C Preston D M Karland E F DeLong ldquoQuantitative distribution of presumptivearchaeal and bacterial nitrifiers in Monterey Bay and the NorthPacific Subtropical Gyrerdquo Environmental Microbiology vol 9no 5 pp 1162ndash1175 2007

[47] J M Beman B N Popp and C A Francis ldquoMolecular andbiogeochemical evidence for ammonia oxidation by marineCrenarchaeota in the Gulf of CaliforniardquoThe ISME Journal vol2 no 4 pp 429ndash441 2008

[48] A C Mosier and C A Francis ldquoRelative abundance anddiversity of ammonia-oxidizing archaea and bacteria in the SanFrancisco Bay estuaryrdquo Environmental Microbiology vol 10 no11 pp 3002ndash3016 2008

[49] A E Bernhard Z C Landry A Blevins J R de la Torre AE Giblin and D A Stahl ldquoAbundance of ammonia-oxidizingarchaea and bacteria along an estuarine salinity gradient in rela-tion to potential nitrification ratesrdquo Applied and EnvironmentalMicrobiology vol 76 no 4 pp 1285ndash1289 2010

[50] H Urakawa W Martens-Habbena and D A Stahl ldquoHighabundance of ammonia-oxidizing archaea in coastal watersdetermined using a modified DNA extractionmethodrdquoAppliedand Environmental Microbiology vol 76 no 7 pp 2129ndash21352010

[51] P LamMM Jensen G Lavik et al ldquoLinking crenarchaeal andbacterial nitrification to anammox in the Black Seardquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 17 pp 7104ndash7109 2007

[52] Y-FWang Y-Y Feng XMa and J-D Gu ldquoSeasonal dynamicsof ammoniaammonium-oxidizing prokaryotes in oxic andanoxic wetland sediments of subtropical coastal mangroverdquoAppliedMicrobiology and Biotechnology vol 97 no 17 pp 7919ndash7934 2013

[53] M Li Y-G Hong H-L Cao and J-D Gu ldquoMangrove treesaffect the community structure and distribution of anammoxbacteria at an anthropogenic-polluted mangrove in the PearlRiver Delta reflected by 16S rRNA and hydrazine oxidoreduc-tase (HZO) encoding gene analysesrdquo Ecotoxicology vol 20 no8 pp 1780ndash1790 2011

[54] H Cao M Li Y Hong and J-D Gu ldquoDiversity and abundanceof ammonia-oxidizing archaea and bacteria in polluted man-grove sedimentrdquo Systematic and Applied Microbiology vol 34no 7 pp 513ndash523 2011

[55] M J Church BWai DMKarl and E FDeLong ldquoAbundancesof crenarchaeal amoA genes and transcripts in the PacificOceanrdquo Environmental Microbiology vol 12 no 3 pp 679ndash6882010

[56] D A Stahl and J R de la Torre ldquoPhysiology and diversity ofammonia-oxidizing archaeardquo Annual Review of Microbiologyvol 66 pp 83ndash101 2012

[57] H Cao J-C Auguet and J-D Gu ldquoGlobal ecological patternof ammonia-oxidizing archaeardquo PLoS ONE vol 8 no 2 ArticleID e52853 2013

[58] Y Zhang Z Zhao C-T A Chen K Tang J Su and N JiaoldquoSulfur metabolizing microbes dominate microbial commu-nities in Andesite-hosted shallow-sea hydrothermal systemsrdquoPLoS ONE vol 7 no 9 Article ID e44593 2012

[59] J Milucka T G Ferdelman L Polerecky et al ldquoZero-valentsulphur is a key intermediate in marine methane oxidationrdquoNature vol 491 no 7425 pp 541ndash546 2012

[60] K Knittel and A Boetius ldquoAnaerobic oxidation of methaneprogress with an unknown processrdquo Annual Review of Micro-biology vol 63 pp 311ndash334 2009

[61] E F DeLong and D M Karl ldquoGenomic perspectives inmicrobial oceanographyrdquoNature vol 437 no 7057 pp 336ndash3422005

[62] A-L Reysenbach K Longnecker and J Kirshtein ldquoNovelbacterial and archaeal lineages from an in situ growth chamberdeployed at a mid-atlantic ridge hydrothermal ventrdquo Appliedand Environmental Microbiology vol 66 no 9 pp 3798ndash38062000

[63] H Teeling B M Fuchs D Becher et al ldquoSubstrate-controlledsuccession of marine bacterioplankton populations induced bya phytoplankton bloomrdquo Science vol 336 no 6081 pp 608ndash6112012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology

2 Archaea

Mangrovewetlands are typical example ofmesophilic andmoderately halophilic environmental niches They are com-monly situated at the intertidal zones along the tropical andsubtropical coasts and play a very important role in shapingthe coastal ecology [16] Mangrove forests are consideredto be highly productive niche that support detritus-basedfood web [16 17] Particularly in tropical mangroves thehigh turnover rates for organic matters and nutrient cyclingbetween the ocean and terrestrial habitats makes it the mostproductive ecosystem in the world [17] The high primaryproductivity of mangroves implies a high demand for nutri-ents essential for plant growth and this appears to be achievedby a highly efficient system of nutrient trapping uptake andrecycling in mangrove ecosystem [18] The diverse microbialcommunities residing in the mangroves play important rolein transformation of nutrients in the environment While theimportance of bacteria and fungi in mangrove biogeochem-ical cycles is well established our knowledge of archaea inmangrove habitats remains extremely limited [16]

Sundarbans is the worldrsquos largest tidal halophyticmangrove ecosystem covering 20400 square kilometers(7900 sqmi) of area and has been recognized as a UNESCOWorld Heritage site Situated in the delta of Ganges Meghnaand Brahmaputra rivers on the Bay of Bengal Sundarbansis shared between India and Bangladesh This mangroveecosystem is the home for diverse flora and fauna includingmangrove plant species like sundari (Heritiera fomes)goran (Ceriops decandra) geoa (Excoecaria agallocha)keora (Sonneratia apetala) and so forth and the worldrsquosfamous endangered royal Bengal tiger (Panthera tigris tigris)Microbial communities play an important role in generationof detritus in mangrove areas and Sundarbans is one suchecotype where microbes have been shown to be involvedin biogeochemical cycling of the nutrients [18ndash20] Despiteglobal advancement in understanding the microbial diversityand role of microbes in different mangrove environmentslittle has been performed in Sundarbans [21 22] In a veryrecent study a detailed description of the bacterial diversityhas been presented in the backdrop of seasonal changes [23]However no data are yet available regarding the abundanceand diversity of archaea in Sundarbans

Hitherto most of the research on mangrove environ-ments has focused on understanding the diversity andfunctions of bacteria and fungi [23ndash26] and very little isknown about archaeal assemblages in mangrove [16 27 28]In order to gain new insight into the archaeal communitypatterns and the influence of environmental conditions in themangrove sediments and to build foundational informationfor future research we have investigated the archaeal diversityin the sediments of Indian Sundarbans employing 454-pyrosequencing

2 Materials and Methods

21 Ethics Statement No specific permits were requiredfor the described field studies which complied with allrelevant regulation The studied locations are not privatelyowned Moreover the study did not involve endangered or

protected species Indeed the Indian Coastal Zone Manage-ment (ICZM) authority of the state ofWest Bengal India hasapproved this experimental exercise

22 Study Area and Sediment Sampling Samples werecollected in triplicate from surface (2 cm) and subsurface(32 cm) sediments of the Sundarbans mangrove wetlandwhich is located on the northeastern coast of India(Figure 1) The samples were collected from threedifferent stations for example Godkhali (station A22∘0610158403257010158401015840N 88∘4610158402222010158401015840E) Bonnie camp (station B21∘4910158405358110158401015840N88∘3610158404486010158401015840E) andDhulibhashani (stationC 21∘3710158404083710158401015840N 88∘3310158404776210158401015840E) spanning 90 km alongthe tidal gradient from the shoreline (Figure 1) In June 2013the sediment samples were collected in triplicate from eachstation A sediment corer (50 cm depth 6 cm diameter) wasused to collect the top 32 cm of sediments The uppermostsurface layer (0ndash2 cm) and deeper subsurface layer (30ndash32 cm)were sampled and immediately sieved by a 2mmmeshin the field The sieved fractions were stored in sealed sterilepolypropylene containers and brought to the laboratoryUpon arrival a portion of each sediment sample wasflash-frozen at minus80∘C for polyaromatic hydrocarbon (PAH)analysis and DNA extraction The remaining sediments werestored at 4∘C for other analytical procedures such as nutrientsand heavy metals estimation The individual collection fromeach sampling station was used for physicochemical analysisand in case of DNA extraction for sequencing analysisthree individual collections were mixed to homogeneity togenerate a representative composite sample

23 Sediment Analyses and Site Climate Microbiological andbiochemical analyses were performed with the field moistsediments Physical and chemical analyses were carried outwith air-dried sediment samples The sediment pH wasmeasured in 1 25 sediment-water suspensions and found tobe alkaline The total organic carbon (TOC) was measuredby the methods described previously [29 30] Briefly TOCin a sample was determined by combusting the air-driedsediment sample catalytically in oxygen atmosphere intoinstrument chamber at 500∘ndash900∘C and the resulting carbondioxide gas was detected by a nondispersive infrared (NDIR)detector in an Aurora TOC Analyzer that was calibrated todirectly display the detected carbon dioxide mass This masswas proportional to the TOC mass in the sediment sampleand calculated as total mass of carbon per unit of sedimentsample

Conductivity and salinity were measured in situ withHach Portable Meters (HQ40d) Measured salinity wasexpressed in parts per thousand (ppt) or gmKgminus1 asdescribed previously [31] Nutrients like inorganic nitrogen(ammonia nitrite and nitrate) soluble phosphate and reac-tive silicate were measured after quantitative extraction inrespective buffering conditions following standard method-ologies [32] Briefly nitrite was measured after complexingwith sulphanilamide followed by a coupling reactionwith n(1-napthyl)-ethylenediamene dihydrochloride which forms anazo dye upon coupling The resulting azo dye was measured

Archaea 3



Godkhali

Bonniecamp

Dhulibhashani

MatlaEstuary

Bang

lade

sh

Saptamukhi

Estuary

Hugli E

stuar

yHariabhanga

21∘45

9984000998400998400N

22∘09984000998400998400N

22∘15

9984000998400998400N88

∘09984000998400998400E 88

∘15

9984000998400998400E 88

∘30

9984000998400998400E 88

∘45

9984000998400998400E 89

∘09984000998400998400E

88∘09984000998400998400E 88

∘15

9984000998400998400E 88

∘30

9984000998400998400E 88

∘45

9984000998400998400E 89

∘09984000998400998400E

0 5 10 20 30

(km)

21∘45

9984000998400998400N

22∘09984000998400998400N

22∘15

9984000998400998400N

WN

ES






2 4 120583M of each


4 Archaea












Archaea 5











SamplesTotal number

of readssequenced






6 Archaea

0 1 2Row Z-score


minus2 minus1

Dhu

libha

shan

i_su

bsur

face

God

khal

i_su

bsur

face

Bonn

ie ca

mp_

subs

urfa

ce

Bonn

ie ca

mp_

surfa

ce

Dhu

libha

shan

i_su

rface

God

khal

i_su

rface







Archaea 7

Table3Univaria

tediversity

indices

Sample

Total

species(119878)

Total

individu

als

(119873)

Speciesrichn

ess

(Margalef)

119889=(119878minus1)Lo

g(119873)

Pielo

ursquosevenness

119869

1015840

=119867

1015840

Lo

g(119878)

Shanno

n119867

1015840

=minusSU

M(119875

119894

lowast

Log(119875

119894

))

Simpson(1minusLambd

a1015840)=1minusSU

M(119873

119894

lowast(119873

119894


God

khalisurface

11100

2171

03447

08264

05206

God

khali sub

surfa

ce17

100

3474

02384

06753

02998

Bonn

iecampsurfa

ce8

100

152

03597

0748

05104

Bonn

iecampsubsurface

18100

3692

03763

1088

04389

Dhu

libhashani surface

16100

3257

03454

09575

04761

Dhu

libhashani sub

surfa

ce13

100

260

60357

09157

04858

8 Archaea



Bonnie camp_surface

Godkhali_surface

Godkhali_subsurface




2D stress 0

Similarity6080

Locations









Archaea 9

00 05

02

06

PC1

PC2

N

Cd

Cu

Pb

Zn

FeAmmonia

Silicate

Phosphate

NitriteNitrate

Sulphate

Temperature

pH

DO Saturation

Salinity

Total nitrogen

TOC



Bonnie camp_surface


Godkhali_surface


minus02

minus05

(a)

Dimension 1 (719)

Dim

ensio

n 2

(18

3)

00 05 10

00

05


Bonnie camp_surface



Halobacteriaceae

Methanobacteriaceae

Methanocellaceae

Methanomicrobiaceae

Methanosaetaceae

Methanosarcinaceae

Methermicoccaceae


minus10

minus05


(b)

Dimension 1 (771)

Dim

ensio

n 2

(19

7)

00 02 04 06

00

01

02


Bonnie camp_surface


Godkhali_surface


Acenaphthylene

Fluorene

Phenanthrene

Fluoranthene

Acenaphthene

Pyrene

minus02

minus03

minus01

minus04 minus02

(c)



4 Discussion




10 Archaea




Godkhali_surface





Archaea 11


















12 Archaea

(c) Continued








5 Conclusions





Acknowledgments


References







Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 3



Godkhali

Bonniecamp

Dhulibhashani

MatlaEstuary

Bang

lade

sh

Saptamukhi

Estuary

Hugli E

stuar

yHariabhanga

21∘45

9984000998400998400N

22∘09984000998400998400N

22∘15

9984000998400998400N88

∘09984000998400998400E 88

∘15

9984000998400998400E 88

∘30

9984000998400998400E 88

∘45

9984000998400998400E 89

∘09984000998400998400E

88∘09984000998400998400E 88

∘15

9984000998400998400E 88

∘30

9984000998400998400E 88

∘45

9984000998400998400E 89

∘09984000998400998400E

0 5 10 20 30

(km)

21∘45

9984000998400998400N

22∘09984000998400998400N

22∘15

9984000998400998400N

WN

ES






2 4 120583M of each


4 Archaea












Archaea 5











SamplesTotal number

of readssequenced






6 Archaea

0 1 2Row Z-score


minus2 minus1

Dhu

libha

shan

i_su

bsur

face

God

khal

i_su

bsur

face

Bonn

ie ca

mp_

subs

urfa

ce

Bonn

ie ca

mp_

surfa

ce

Dhu

libha

shan

i_su

rface

God

khal

i_su

rface







Archaea 7

Table3Univaria

tediversity

indices

Sample

Total

species(119878)

Total

individu

als

(119873)

Speciesrichn

ess

(Margalef)

119889=(119878minus1)Lo

g(119873)

Pielo

ursquosevenness

119869

1015840

=119867

1015840

Lo

g(119878)

Shanno

n119867

1015840

=minusSU

M(119875

119894

lowast

Log(119875

119894

))

Simpson(1minusLambd

a1015840)=1minusSU

M(119873

119894

lowast(119873

119894


God

khalisurface

11100

2171

03447

08264

05206

God

khali sub

surfa

ce17

100

3474

02384

06753

02998

Bonn

iecampsurfa

ce8

100

152

03597

0748

05104

Bonn

iecampsubsurface

18100

3692

03763

1088

04389

Dhu

libhashani surface

16100

3257

03454

09575

04761

Dhu

libhashani sub

surfa

ce13

100

260

60357

09157

04858

8 Archaea



Bonnie camp_surface

Godkhali_surface

Godkhali_subsurface




2D stress 0

Similarity6080

Locations









Archaea 9

00 05

02

06

PC1

PC2

N

Cd

Cu

Pb

Zn

FeAmmonia

Silicate

Phosphate

NitriteNitrate

Sulphate

Temperature

pH

DO Saturation

Salinity

Total nitrogen

TOC



Bonnie camp_surface


Godkhali_surface


minus02

minus05

(a)

Dimension 1 (719)

Dim

ensio

n 2

(18

3)

00 05 10

00

05


Bonnie camp_surface



Halobacteriaceae

Methanobacteriaceae

Methanocellaceae

Methanomicrobiaceae

Methanosaetaceae

Methanosarcinaceae

Methermicoccaceae


minus10

minus05


(b)

Dimension 1 (771)

Dim

ensio

n 2

(19

7)

00 02 04 06

00

01

02


Bonnie camp_surface


Godkhali_surface


Acenaphthylene

Fluorene

Phenanthrene

Fluoranthene

Acenaphthene

Pyrene

minus02

minus03

minus01

minus04 minus02

(c)



4 Discussion




10 Archaea




Godkhali_surface





Archaea 11


















12 Archaea

(c) Continued








5 Conclusions





Acknowledgments


References







Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Archaea












Archaea 5











SamplesTotal number

of readssequenced






6 Archaea

0 1 2Row Z-score


minus2 minus1

Dhu

libha

shan

i_su

bsur

face

God

khal

i_su

bsur

face

Bonn

ie ca

mp_

subs

urfa

ce

Bonn

ie ca

mp_

surfa

ce

Dhu

libha

shan

i_su

rface

God

khal

i_su

rface







Archaea 7

Table3Univaria

tediversity

indices

Sample

Total

species(119878)

Total

individu

als

(119873)

Speciesrichn

ess

(Margalef)

119889=(119878minus1)Lo

g(119873)

Pielo

ursquosevenness

119869

1015840

=119867

1015840

Lo

g(119878)

Shanno

n119867

1015840

=minusSU

M(119875

119894

lowast

Log(119875

119894

))

Simpson(1minusLambd

a1015840)=1minusSU

M(119873

119894

lowast(119873

119894


God

khalisurface

11100

2171

03447

08264

05206

God

khali sub

surfa

ce17

100

3474

02384

06753

02998

Bonn

iecampsurfa

ce8

100

152

03597

0748

05104

Bonn

iecampsubsurface

18100

3692

03763

1088

04389

Dhu

libhashani surface

16100

3257

03454

09575

04761

Dhu

libhashani sub

surfa

ce13

100

260

60357

09157

04858

8 Archaea



Bonnie camp_surface

Godkhali_surface

Godkhali_subsurface




2D stress 0

Similarity6080

Locations









Archaea 9

00 05

02

06

PC1

PC2

N

Cd

Cu

Pb

Zn

FeAmmonia

Silicate

Phosphate

NitriteNitrate

Sulphate

Temperature

pH

DO Saturation

Salinity

Total nitrogen

TOC



Bonnie camp_surface


Godkhali_surface


minus02

minus05

(a)

Dimension 1 (719)

Dim

ensio

n 2

(18

3)

00 05 10

00

05


Bonnie camp_surface



Halobacteriaceae

Methanobacteriaceae

Methanocellaceae

Methanomicrobiaceae

Methanosaetaceae

Methanosarcinaceae

Methermicoccaceae


minus10

minus05


(b)

Dimension 1 (771)

Dim

ensio

n 2

(19

7)

00 02 04 06

00

01

02


Bonnie camp_surface


Godkhali_surface


Acenaphthylene

Fluorene

Phenanthrene

Fluoranthene

Acenaphthene

Pyrene

minus02

minus03

minus01

minus04 minus02

(c)



4 Discussion




10 Archaea




Godkhali_surface





Archaea 11


















12 Archaea

(c) Continued








5 Conclusions





Acknowledgments


References







Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 5











SamplesTotal number

of readssequenced






6 Archaea

0 1 2Row Z-score


minus2 minus1

Dhu

libha

shan

i_su

bsur

face

God

khal

i_su

bsur

face

Bonn

ie ca

mp_

subs

urfa

ce

Bonn

ie ca

mp_

surfa

ce

Dhu

libha

shan

i_su

rface

God

khal

i_su

rface







Archaea 7

Table3Univaria

tediversity

indices

Sample

Total

species(119878)

Total

individu

als

(119873)

Speciesrichn

ess

(Margalef)

119889=(119878minus1)Lo

g(119873)

Pielo

ursquosevenness

119869

1015840

=119867

1015840

Lo

g(119878)

Shanno

n119867

1015840

=minusSU

M(119875

119894

lowast

Log(119875

119894

))

Simpson(1minusLambd

a1015840)=1minusSU

M(119873

119894

lowast(119873

119894


God

khalisurface

11100

2171

03447

08264

05206

God

khali sub

surfa

ce17

100

3474

02384

06753

02998

Bonn

iecampsurfa

ce8

100

152

03597

0748

05104

Bonn

iecampsubsurface

18100

3692

03763

1088

04389

Dhu

libhashani surface

16100

3257

03454

09575

04761

Dhu

libhashani sub

surfa

ce13

100

260

60357

09157

04858

8 Archaea



Bonnie camp_surface

Godkhali_surface

Godkhali_subsurface




2D stress 0

Similarity6080

Locations









Archaea 9

00 05

02

06

PC1

PC2

N

Cd

Cu

Pb

Zn

FeAmmonia

Silicate

Phosphate

NitriteNitrate

Sulphate

Temperature

pH

DO Saturation

Salinity

Total nitrogen

TOC



Bonnie camp_surface


Godkhali_surface


minus02

minus05

(a)

Dimension 1 (719)

Dim

ensio

n 2

(18

3)

00 05 10

00

05


Bonnie camp_surface



Halobacteriaceae

Methanobacteriaceae

Methanocellaceae

Methanomicrobiaceae

Methanosaetaceae

Methanosarcinaceae

Methermicoccaceae


minus10

minus05


(b)

Dimension 1 (771)

Dim

ensio

n 2

(19

7)

00 02 04 06

00

01

02


Bonnie camp_surface


Godkhali_surface


Acenaphthylene

Fluorene

Phenanthrene

Fluoranthene

Acenaphthene

Pyrene

minus02

minus03

minus01

minus04 minus02

(c)



4 Discussion




10 Archaea




Godkhali_surface





Archaea 11


















12 Archaea

(c) Continued








5 Conclusions





Acknowledgments


References







Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 Archaea

0 1 2Row Z-score


minus2 minus1

Dhu

libha

shan

i_su

bsur

face

God

khal

i_su

bsur

face

Bonn

ie ca

mp_

subs

urfa

ce

Bonn

ie ca

mp_

surfa

ce

Dhu

libha

shan

i_su

rface

God

khal

i_su

rface







Archaea 7

Table3Univaria

tediversity

indices

Sample

Total

species(119878)

Total

individu

als

(119873)

Speciesrichn

ess

(Margalef)

119889=(119878minus1)Lo

g(119873)

Pielo

ursquosevenness

119869

1015840

=119867

1015840

Lo

g(119878)

Shanno

n119867

1015840

=minusSU

M(119875

119894

lowast

Log(119875

119894

))

Simpson(1minusLambd

a1015840)=1minusSU

M(119873

119894

lowast(119873

119894


God

khalisurface

11100

2171

03447

08264

05206

God

khali sub

surfa

ce17

100

3474

02384

06753

02998

Bonn

iecampsurfa

ce8

100

152

03597

0748

05104

Bonn

iecampsubsurface

18100

3692

03763

1088

04389

Dhu

libhashani surface

16100

3257

03454

09575

04761

Dhu

libhashani sub

surfa

ce13

100

260

60357

09157

04858

8 Archaea



Bonnie camp_surface

Godkhali_surface

Godkhali_subsurface




2D stress 0

Similarity6080

Locations









Archaea 9

00 05

02

06

PC1

PC2

N

Cd

Cu

Pb

Zn

FeAmmonia

Silicate

Phosphate

NitriteNitrate

Sulphate

Temperature

pH

DO Saturation

Salinity

Total nitrogen

TOC



Bonnie camp_surface


Godkhali_surface


minus02

minus05

(a)

Dimension 1 (719)

Dim

ensio

n 2

(18

3)

00 05 10

00

05


Bonnie camp_surface



Halobacteriaceae

Methanobacteriaceae

Methanocellaceae

Methanomicrobiaceae

Methanosaetaceae

Methanosarcinaceae

Methermicoccaceae


minus10

minus05


(b)

Dimension 1 (771)

Dim

ensio

n 2

(19

7)

00 02 04 06

00

01

02


Bonnie camp_surface


Godkhali_surface


Acenaphthylene

Fluorene

Phenanthrene

Fluoranthene

Acenaphthene

Pyrene

minus02

minus03

minus01

minus04 minus02

(c)



4 Discussion




10 Archaea




Godkhali_surface





Archaea 11


















12 Archaea

(c) Continued








5 Conclusions





Acknowledgments


References







Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 7

Table3Univaria

tediversity

indices

Sample

Total

species(119878)

Total

individu

als

(119873)

Speciesrichn

ess

(Margalef)

119889=(119878minus1)Lo

g(119873)

Pielo

ursquosevenness

119869

1015840

=119867

1015840

Lo

g(119878)

Shanno

n119867

1015840

=minusSU

M(119875

119894

lowast

Log(119875

119894

))

Simpson(1minusLambd

a1015840)=1minusSU

M(119873

119894

lowast(119873

119894


God

khalisurface

11100

2171

03447

08264

05206

God

khali sub

surfa

ce17

100

3474

02384

06753

02998

Bonn

iecampsurfa

ce8

100

152

03597

0748

05104

Bonn

iecampsubsurface

18100

3692

03763

1088

04389

Dhu

libhashani surface

16100

3257

03454

09575

04761

Dhu

libhashani sub

surfa

ce13

100

260

60357

09157

04858

8 Archaea



Bonnie camp_surface

Godkhali_surface

Godkhali_subsurface




2D stress 0

Similarity6080

Locations









Archaea 9

00 05

02

06

PC1

PC2

N

Cd

Cu

Pb

Zn

FeAmmonia

Silicate

Phosphate

NitriteNitrate

Sulphate

Temperature

pH

DO Saturation

Salinity

Total nitrogen

TOC



Bonnie camp_surface


Godkhali_surface


minus02

minus05

(a)

Dimension 1 (719)

Dim

ensio

n 2

(18

3)

00 05 10

00

05


Bonnie camp_surface



Halobacteriaceae

Methanobacteriaceae

Methanocellaceae

Methanomicrobiaceae

Methanosaetaceae

Methanosarcinaceae

Methermicoccaceae


minus10

minus05


(b)

Dimension 1 (771)

Dim

ensio

n 2

(19

7)

00 02 04 06

00

01

02


Bonnie camp_surface


Godkhali_surface


Acenaphthylene

Fluorene

Phenanthrene

Fluoranthene

Acenaphthene

Pyrene

minus02

minus03

minus01

minus04 minus02

(c)



4 Discussion




10 Archaea




Godkhali_surface





Archaea 11


















12 Archaea

(c) Continued








5 Conclusions





Acknowledgments


References







Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

8 Archaea



Bonnie camp_surface

Godkhali_surface

Godkhali_subsurface




2D stress 0

Similarity6080

Locations









Archaea 9

00 05

02

06

PC1

PC2

N

Cd

Cu

Pb

Zn

FeAmmonia

Silicate

Phosphate

NitriteNitrate

Sulphate

Temperature

pH

DO Saturation

Salinity

Total nitrogen

TOC



Bonnie camp_surface


Godkhali_surface


minus02

minus05

(a)

Dimension 1 (719)

Dim

ensio

n 2

(18

3)

00 05 10

00

05


Bonnie camp_surface



Halobacteriaceae

Methanobacteriaceae

Methanocellaceae

Methanomicrobiaceae

Methanosaetaceae

Methanosarcinaceae

Methermicoccaceae


minus10

minus05


(b)

Dimension 1 (771)

Dim

ensio

n 2

(19

7)

00 02 04 06

00

01

02


Bonnie camp_surface


Godkhali_surface


Acenaphthylene

Fluorene

Phenanthrene

Fluoranthene

Acenaphthene

Pyrene

minus02

minus03

minus01

minus04 minus02

(c)



4 Discussion




10 Archaea




Godkhali_surface





Archaea 11


















12 Archaea

(c) Continued








5 Conclusions





Acknowledgments


References







Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 9

00 05

02

06

PC1

PC2

N

Cd

Cu

Pb

Zn

FeAmmonia

Silicate

Phosphate

NitriteNitrate

Sulphate

Temperature

pH

DO Saturation

Salinity

Total nitrogen

TOC



Bonnie camp_surface


Godkhali_surface


minus02

minus05

(a)

Dimension 1 (719)

Dim

ensio

n 2

(18

3)

00 05 10

00

05


Bonnie camp_surface



Halobacteriaceae

Methanobacteriaceae

Methanocellaceae

Methanomicrobiaceae

Methanosaetaceae

Methanosarcinaceae

Methermicoccaceae


minus10

minus05


(b)

Dimension 1 (771)

Dim

ensio

n 2

(19

7)

00 02 04 06

00

01

02


Bonnie camp_surface


Godkhali_surface


Acenaphthylene

Fluorene

Phenanthrene

Fluoranthene

Acenaphthene

Pyrene

minus02

minus03

minus01

minus04 minus02

(c)



4 Discussion




10 Archaea




Godkhali_surface





Archaea 11


















12 Archaea

(c) Continued








5 Conclusions





Acknowledgments


References







Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

10 Archaea




Godkhali_surface





Archaea 11


















12 Archaea

(c) Continued








5 Conclusions





Acknowledgments


References







Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 11


















12 Archaea

(c) Continued








5 Conclusions





Acknowledgments


References







Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

12 Archaea

(c) Continued








5 Conclusions





Acknowledgments


References







Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 13

































14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

14 Archaea

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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