research article seasonal effects in a lake sediment...

Research ArticleSeasonal Effects in a Lake Sediment Archaeal Community ofthe Brazilian Savanna

Thiago Rodrigues1 Elisa Catatildeo1 Mercedes M C Bustamante2 Betania F Quirino34

Ricardo H Kruger1 and Cynthia M Kyaw1

1 Department of Cell Biology Biological Sciences Institute University of Brasılia 70910-900 Brasılia DF Brazil2 Department of Ecology Biological Sciences Institute University of Brasılia 70910-900 Brasılia DF Brazil3 Department of Genomics Science and Biotechnology Catholic University of Brasılia 70790-160 Brasılia DF Brazil4 Embrapa-Agroenergy 70770-901 Brasılia DF Brazil

Correspondence should be addressed to Cynthia M Kyaw maltaunbbr

Received 23 April 2014 Revised 30 June 2014 Accepted 8 July 2014 Published 20 July 2014

Academic Editor William B Whitman

Copyright copy 2014 Thiago Rodrigues 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

The Cerrado is a biome that corresponds to 24 of Brazilrsquos territory Only recently microbial communities of this biome have beeninvestigated Herewe describe for the first time the diversity of archaeal communities from freshwater lake sediments of the Cerradoin the dry season and in the transition period between the dry and rainy seasons when the first rains occur Gene libraries wereconstructed using Archaea-specific primers for the 16S rRNA and amoA genes Analysis revealed marked differences between thearchaeal communities found in the two seasons I1a and I1c Thaumarchaeota were found in greater numbers in the transitionperiod while MCG Archaea was dominant on the dry season Methanogens were only found in the dry season Analysis of 16SrRNA sequences revealed lower diversity on the transition periodWe detected archaeal amoA sequences in both seasons but therewere more OTUs during the dry season These sequences were within the same cluster as Nitrosotalea devanaterrarsquos amoA geneThe principal coordinate analysis (PCoA) test revealed significant differences between samples from different seasonsThese resultsprovide information on archaeal diversity in freshwater lake sediments of the Cerrado and indicates that rain is likely a factor thatimpacts these communities

1 Introduction

The Brazilian savanna also known as the Cerrado corre-sponds to 24 of the countryrsquos territory [1] and is charac-terized by two defined seasons the dry season which occursfrom May to September and the rainy season which occursfrom October to April [2] Though there are some studies onthe diversity of Archaea in different Brazilian environments[3ndash7] little is known about archaeal communities in theCerrado Recently our group described the archaeal richnessin Cerrado soils [8] but there are currently no reports onthe diversity of Archaea in freshwater lake sediments in thisbiome

Lake sediments are environments with a high abundanceof microorganisms [9] which are subjected to changes innutrient composition associated with events of resuspension

and redeposition of the sediment surface caused bywater flow[10] Rain is one of many different environmental factors thatcause these events [11] Although there are many studies onthe diversity of Archaea in lake sediments [12ndash16] there arecurrently no reports that describe changes in the communitycaused by the occurrence of rain

Ammonia oxidation to nitrite has an important role inthe global biogeochemical nitrogen cycle [17] and it is knownthat Bacteria and Archaea are both capable of ammoniaoxidation [18 19] Many mesophilic Archaea found in soilswater and freshwater sediments [20] formerly classified asCrenarchaeota are now considered members of the recentlyproposedThaumarchaeota phylum [21] which includes all ofthe ammonia oxidizing Archaea (AOA) known so far [22]

Herewe describe for the first time the diversity of archaealcommunities from freshwater lake sediments of the Cerrado

Hindawi Publishing CorporationArchaeaVolume 2014 Article ID 957145 9 pageshttpdxdoiorg1011552014957145

2 Archaea

in the dry season and in the transition period between thedry and rainy seasons when the first rains occur Eight genelibraries were obtained and sequenced four using Archaea-specific primers for the 16S rRNA gene and the other fourwith amoA gene primers specific to ammonia-oxidizingArchaeaThe sequences obtained were analyzed to determinewhether there are differences between the communities inlake sediments of different seasons Our results show twovery different community profiles on each season indicatinga possible effect of seasonal change

2 Materials and Methods

21 Study Site and Sampling Sediments were obtainedfrom a lake in the ldquoParque Nacional das Sempre Vivasrdquoa National Park located in the Serra do Espinhaco in thestate of Minas Gerais Brazil within the Cerrado biomeThe sediments were sampled in the Baixo Inhancicabay near the margin and the distance between thesampling spots was 15m Samples will be referred hereas RIB-A (17∘461015840162107810158401015840S 43∘37101584035548110158401015840W) and RIB-B(17∘461015840164991810158401015840S 43∘371015840359463510158401015840W) and were consideredreplicates Samples were collected with PVC tubes 10 cm indiameter by introducing the tube into the sediments locatednear the lake shore Sediment samples were retrieved fromdepths between 0 and 10 cm and kept on ice until stored atminus20∘C

The dry season samples RIB-A and RIB-B were collectedon May 7 2010 and will therefore be referred to as RIB-A D and RIB-B D The samples from the transition periodbetween the dry and rainy seasons were RIB-A and RIB-B collected on September 13 2010 and will be referred toas RIB-A T and RIB-B T Using a thermometer the watertemperature of the sampling sites was obtained The pHof the sediments was measured with a pH electrode Totalnitrogen was quantified by the Nessler method [23] and totalphosphorus was quantified by the Murphy-Riley method[24]

22 16S rRNA and amoA Genes Libraries Construction Envi-ronmental DNA was extracted from 05 g of lake sedimentusing the PowerSoil DNA isolation kit (MO BIO Labora-tories Inc) according to the manufacturerrsquos instructionsPCR primers 21f and 958r [25] were used to amplify the 16SrRNAgene usingDNA from sediments samples as a templateFor the amoA gene the primers used were Arch amoAfand amoAr [26] PCR assay conditions were the same asthose originally describedThe amplifiedDNAwas visualizedon agarose gels stained with ethidium bromide (10mgmL)The PCR-amplified DNA fragments were purified using theWizard kit (SVGel and PCRClean-Up System Promega) andcloned into the pGEM T easy (Promega) plasmid accordingto the manufacturerrsquos instructions Recombinant plasmidswere inserted into Escherichia coli calcium chloride-treatedDH5120572 cells by heat shock treatment The presence of theinserts was verified by selection of clones in LB agar platessupplemented with ampicillin (150 120583gmL) IPTG (05mM)and Xgal (000625) Plasmid DNA was extracted with

Table 1 Comparison of physicochemical properties of freshwaterlake sediments from replicates of the dry season (RIB D) and of thetransition period between the dry and rainy seasons (RIB T)

RIB D RIB TSampling date May 2010 September 2010Water temperature (∘C) 2585 plusmn 035 2160 plusmn 014Depth (cm) 545 plusmn 007 470 plusmn 014pH 557 plusmn 009 578 plusmn 020P-total (120583gL) 900 plusmn 141 1850 plusmn 354N-total (120583gL) 1100 plusmn 141 2350 plusmn 212

phenol-chloroform-isoamyl alcohol at 25 24 1 (volvolvol)DNA was sequenced by the Sanger method at MacrogenInc (Korea) The sequences obtained were deposited in theGeneBank dataset under the accession numbers KF640285-KF640592 (16S rRNA gene) and KJ719076-KJ719244 (amoAgene)

23 Phylogenetic Analysis The sequences from the 16S rRNAgene were used for comparative analysis with the Greengenestaxonomical database [27] using the Mothur software [28]A threshold of 90 or higher identity with the database wasused Alignment of 16S rRNA and amoA gene sequences wasperformed with the ClustalX software [29]

Mothur was used to filter the gap columns generatedby the alignment and was also used to generate the AceChao Shannon and Simpson indexes The 16S rRNA genesequences were clustered with a sequence identity thresholdof 97 for species 95 for genus 90 for class and 80 forphylum [30] Phylogenetic analysis of the amoA sequenceswas OTU based and the identity threshold considered was90 [31] Rarefaction curves were constructed with theMothur software The principal coordinate analysis (PCoA)was performed with the Unifrac program [32] Sequencesof the rRNA 16S gene from isolates of Euryarchaeota Cre-narchaeota Thaumarchaeota and Korarchaeota as well assequences of uncultured Archaea found in the GenBankdataset were used to construct a phylogenetic tree withthe MEGA software [33] using the maximum-likelihoodmethod a bootstrap value of 1000 and the Tamura nucleotidesubstitution model

3 Results and Discussion

All the sediments analyzed in the present study were 6 cmdeep therefore they were part of the sediment layer mostrecently deposited [34] All the samples presented slightlyacidic pH values (Table 1) which may be due to hydro-gen ion liberation in the medium caused by microbialmetabolism and organic matter decomposition [10 35] Sed-iments obtained in the transition period (RIBT) had highervalues of total nitrogen and phosphorus when compared tothe samples obtained in the dry season (RIB D) (Table 1)As previously mentioned environmental factors such asthe occurrence of rain affect the nutrient content in lakesediments [11] which could explain the differences between

Archaea 3

Table 2 120572-Diversity analysis for Cerrado freshwater lake sediment rRNA 16S gene sequences

Season Similarity () Nseqs Sobs Chao Ace Shannon Simpson CoverageRIB Dlowast 97 142 85 28833 34085 421 001 5704RIB Tlowastlowast 97 163 25 3100 3436 228 017 9447lowastDry season lowastlowasttransition between dry and rainy seasons

the samples from each season For the Cerrado soils it hasalready been shown that the changes of seasons affect themicrobial community [36] and the present study suggests thatthese changes can also influence the microbial communitiesin freshwater lake sediments

Four 16S rRNA gene clone libraries were constructed tworeplicates for the dry season archaeal community (RIB-A Dand RIB-B D) and two for the transition period betweenthe dry and rainy seasons archaeal community (RIB-A Tand RIB-B T) This resulted in a total of 308 sequences(75 sequences from RIB-A D 70 sequences from RIB-B D75 sequences from RIB-A T and 88 sequences from RIB-B T) with high Phred quality (gt20) and size over 400 bpAdditionally four archaeal amoA libraries were obtainedfrom the same samples resulting in 169 sequences with highPhred quality (gt20) and size over 300 pb Sample amoA-A D(RIB-A D) contains 43 sequences amoA-B D (RIB-B D) 44sequences amoA-A T (RIB-A T) 42 sequences and amoA-BT (RIB-B T) 40 sequences

Using sequence similarity of 97or greater for the specieslevel [30] our results revealed a large difference in thenumber of OTUs in samples from different seasons (Table 2)We were able to detect 85 OTUs in the dry season (RIB D)and 25 OTUs in the transition period between the dry andrainy seasons (RIB T) suggesting a higher richness in the dryseason This is further observed with the richness indexesAce and Chao both of which estimated a higher number ofpredicted OTUs for the dry season Both diversity indexesShannon and Simpson also indicate a higher diversity forthe sediment archaeal community found in the dry seasonSimilarly the rarefaction curves (Figure 1) show a plateau forthe transition period samples even with a 97 16S rRNAgene sequence similarity level while no plateau is reachedfor the sediment samples from the dry season indicatinghigher richness and diversity in the dry season Changes inmicrobial diversity are usually related to changes in nutrientavailability caused by environmental factors including thepresence of nitrogen and phosphate fertilizers in soils andseasonal changes [37ndash44] Based on the increase in totalnitrogen detected in the transition period samples (Table 1)we can speculate that the reduced diversity observed in thesesamples was due to the higher nitrogen availability whichcould have favored selected groups of organisms and wasdetrimental to others as described in other studies [45]

The PCoA (Figure 2) was used to evaluate similaritiesbetween the communities ofArchaea in the samples analyzedThis analysis shows that the replicates from the same seasongrouped closer while samples retrieved in different seasonshad significant differences indicating a possible effect ofthe season change Similar results were also obtained withthe Libshuff test (data not shown) Most OTUs were shared

Table 3 Presence of the tenmost abundant OTUs from each seasonamong the lake sediments retrieved in the dry season (RIB-A D andRIB-B D) and the transition period (RIB-A T and RIB-B T)

OTU Rep RIB-A D RIB-B D RIB-A T RIB-B T

Dryseason

RIB-A D38 + minus minus minus

RIB-B D11 minus + minus minus

RIB-A D66 + + minus minus

RIB-B D50 + + minus minus






RIB-B D7 + + minus +

Transitionperiod

RIB-A T26 minus minus + minus

RIB-B T18 minus minus + +RIB-B T28 + minus minus +RIB-B T63 minus minus minus +RIB-B T74 minus minus minus +RIB-B T77 + + minus +RIB-B T85 minus minus + +RIB-B T89 minus minus + +RIB-B T91 minus minus + +RIB-B T87 minus minus + +

between samples of the same season while very few OTUswere shared between samples of different seasons (Table 3)further indicating two noticeably different community pro-files The overall decrease in diversity found in the transitionperiod differs from other studies A study from Cruz-Martınez et al [43] indicated that rain had little impact ona California soil microbial community However there is alack of microbial diversity studies on environments naturallycharacterized by dry and rainy periods and more studies areneeded to fully understand the ecological dynamics in thesekinds of environments

According to the Greengenes taxonomical database all16S rRNA gene sequences were from the Archaea domainOne aspect worth mentioning is that although this databaseconsiders Thaumarchaeota a class of the Crenarchaeotaphylum we classified Thaumarchaeota in the taxonomicallevel of phylum as suggested by Brochier-Armanet et al [21]The archaeal communities from the two seasons differedmarkedly in composition even at phyla level (Figure 3) Outof 142 sequences the dry season sample (RIB D) had pre-dominantly crenarchaeotal sequences (8028) According to

4 Archaea

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120 140 160

OTU

s

Sequences

Dry season

Unique9795

9080

(a)

0102030405060708090

0 50 100 150 200

OTU

s

Sequences

Transition period

Unique9795

9080

(b)

Figure 1 Rarefaction curves for archaeal 16S rRNA gene sequences from Cerrado freshwater lake sediments at different sequence identity ofsequences obtained in the dry season (a) and in the transition period (b) Curves were obtained using the Mothur software

RIB-A D

RIB-A T

RIB-B T

RIB-B D

PCA P1 versus P2

minus06 minus04 minus02 00 02 04 06

P2-p

erce

nt v

aria

tion

expl

aine

d2186

05

04

03

02

01

00

minus01

minus02

minus03

minus04

P1-percent variation explained 5645

Figure 2 Unifrac principal coordinates analysis of Cerrado fresh-water lake sediments archaeal 16S rRNA gene sequences obtained inthe dry season (RIB-A D and RIB-B D) and transition between dryand rainy season (RIB-A T and RIB-B T)

the database 8070 of the crenarchaeotal sequences wereof the pGrfC26 order This group is considered a part ofthe miscellaneous crenarchaeotic group (MCG) [46] MCGArchaea can be found in diverse habitats ranging fromterrestrial to marine environments [47 48] However thisgroup is interestingly considered one of the most abundantgroups in the sediment biosphere [49] It is also importantto note that the MCG group has no clear affiliation to any ofthe established archaeal phyla and has an unstable branchingorder in 16S rRNA trees [50] There are currently no cultured

0

10

20

30

40

50

60

70

80

90

RIB D RIB T

()

CrenarchaeotaThaumarchaeota

EuryarchaeotaUnclassified archaea

Figure 3 Histogram showing the taxonomic distribution of Cer-rado fresh water lake sediment archaeal sequences obtained inthe dry season (RIB D) and the transition between dry and rainyseason (RIB T) among different phyla according to the Greengenesdatabase

isolates of this group and little is known about the roles ofthese organisms in biogeochemical cycles [46] On the phy-logenetic tree constructed with sequences representative ofeach OTU (97) from both seasons (Figure 4) theMCG dryseason sequences clustered in a group with only unculturedsequences retrieved from other studies which was expectedIt was also possible to detect in the dry season sequencesclassified as Euryarchaeota (1760) and 9 of them wereaffiliated with methanogenic groups Methanogenic Archaeaare commonly described in freshwater lake sediments [51ndash54] which have limited oxygen and high amounts of organic

Archaea 5

RIB-B D22 (2)RIB-B D37 (2)

Groundwater from inner Mongolia clone JX1962121RIB-B D50 (4)Rice roots archaeon clone AM4953611

RIB-B D18 (5)Amazon river clone JX6699551

RIB-B D54 (4)RIB-B D55 (2)Confluence of the Ohio and Mississippi River clone GQ9065931

RIB-B D7 (9)RIB-B T83 (2)

RIB-A D66 (4)Tidal freshwater wetland soil clone JF7897441

RIB-B D6 (3)Mixing front of river and marine waters clone AB5385121

RIB-B T77 (13)RIB-B D35 (3)

Honghu Lake upper sediment clone HM2440531RIB-B D11 (3)

RIB-A D38 (3)RIB-B D10 (2)

Atlantic forest acidic peatland clone HQ6144361RIB-B D48 (6)

RIB-B D38 (4)El Zacaton water column clone FJ4854941

RIB-B D56 (4)RIB-B D58 (2)

RIB-B D19 (3)Coal rich sediment clone KJ4245111

RIB-B D25 (3)Lower Mississippi River clone GQ9066311

Thermoproteus neutrophilus (T) AB009618Thermocladium modestius (T) AB005296

Fervidicoccus fontis (T) EF552404Ignicoccus pacificus (T) AJ271794

Caldisphaera lagunensis (T) AB087499RIB-B T17 (3)Amazon river clone JX6700371RIB-B T89 (55)

Candidatus Nitrosotalea devanaterra JN2274881RIB-B T85 (9)

Deep south african gold mine water clone AB0502411RIB-B T87 (30)

Cenarchaeum symbiosum U51469Candidatus Nitrosopumilus maritimus DQ085097

Candidatus Giganthauma karukerense FN398074Candidatus Nitrososphaera gargensis EU281332

Candidatus Nitrososphaera EN76 FR773157Candidatus Nitrosocaldus yellowstonii EU239960

Freshwater meromictic lake sediment clone GU1354951RIB-B T42 (2)RIB-B T49 (2)

RIB-B T60 (2)Boreal oligotrophic peat wetland clone AB3649161

RIB-B T63 (4)Cerrado soil clone JX1251271RIB-A T26 (6)

Atlantic forest acidic peatland clone HQ6141051Cerrado soil clone JX1251791

RIB-B T18 (4)Oligotrophic alpine lake clone FN6916361RIB-B T21 (2)RIB-B T74 (3)Malaysian swamp forest clone EU4029861

RIB-B T91 (23)RIB-B T28 (3)

Candidatus Korarchaeum cryptofilum CP000968RIB-B D47 (2)

Methanosarcina acetivorans (T) NR 0447241Methanosaeta thermophila (T) NR 0742141RIB-B D13 (2)

Uncultured Acidobacterium sp JQ82522298

90

99

100

93

98

96

99

97

99

50

99

99

100

10053

77

94

93

58

84

96

59

78

88

93

71

88

96

83

99

96

95

62

80

63

98

82

86

96

8672

9864

75

98

98

94

78

96

94

60

66

60

83

01

MiscellaneousCrenarchaeoticgroup (MCG)

Crenarchaeota

I1a

HWCGIII

I1b

I1c

Thaumarchaeota

Euryarchaeota

Korarchaeota

Figure 4 Phylogenetic tree of archaeal 16S rRNA gene OTUs (97) obtained fromCerrado freshwater sediments in the dry season (RIB-ADand RIB-BD sequences) and in the dry to rainy season transition period (RIB-A T and RIB-B T sequences)The number of sequences that arerepresented by a specific node of the tree is indicated in parenthesis The phylogenetic tree was inferred by the maximum-likelihood methodusing theMEGA software bootstrap value of 1000 and Tamura-Nei nucleotide substitution modelThe percentage of bootstrap resamplingsthat supports each topological element is represented near the line Reference sequences from different phyla were used for comparison andthe bacterial sequence of the uncultured Acidobacterium sp JQ825222 was used as an outgroup Bootstrap values below 50 were not shownand singletons were not included

6 Archaea

Table 4 120572-Diversity analysis for the amoA gene sequences

Season Similarity () Nseqs SobsamoA Dlowast 90 87 7amoA Tlowastlowast 90 82 3lowastDry season lowastlowasttransition between dry and rainy seasons

matter [55] This can be also seen on the phylogenetic tree(Figure 4) where two dry season sequences (RIB-B D47 andRIB-B D13) aligned with methanogenic Archaea The factthat the sediment layer analyzed was recently deposited mayaccount for the low number of methanogenic Archaea foundin our samples although primer bias cannot be ruled out [5657] We were able to detect very few sequences classified asThaumarchaeota (141) or considered unclassified Archaea(071) in the dry season samples (Figure 3)

Interestingly among the samples of the transition periodbetween the dry and rainy seasons (RIB T) we were ableto detect predominantly sequences affiliated to the Thau-marchaeota phylum (6135) (Figure 3) All other sequenceswere affiliated to the Crenarchaeota phylum (3865) On thephylogenetic tree (Figure 4) the transition period sequencesmostly clustered with the Thaumarchaeota phylum theexception being sequences RIB-B T77 and RIB-B T83 whichclustered with the MCGThe sequences RIB-BT 89 and RIB-BT87 represent a significantly higher number of sequencesand were aligned within the I1a cluster This group iscomposed of organisms initially described in aquatic envi-ronments [58] All other transition period sequences werepart of the I1c group which contains organisms found inacidic soils [50] It is known that Cerrado soils have acidicpH [2] and our group recently reported the presence of I1cThaumarchaota in these environments [8] Given that onlytransition period sequenceswere foundwithin this group andthe fact that the sediments were retrieved near the margin itis plausible that the rain caused leaching of the soils near themargin into the lakeWewere not able to detect anymembersof the Euryarchaeota phylum in the transition period It hasbeen reported that ammonia oxidation is common amongmembers of theThaumarchaeota phylum [22 59] and in thissense the detection of a great number of sequences affiliatedto Thaumarchaeota might be correlated with the increase intotal nitrogen in the transition period between the dry andrainy seasons samples (Table 1 and Figures 3 and 4) On theother hand the absence of members of the Euryarchaeotaphylum in sediments of the transition period could also bea consequence of methanogenesis inhibition mediated bynitrogen compounds [60 61] It is also worth mentioningthat most uncultured sequences with high similarity to thosefound in the present study are from freshwater aquatic envi-ronments or soils with higher amounts of water (Figure 4)

Sequencing of amoA yielded 87 sequences from the dryseason samples and 82 sequences from the transition periodsamples 120572-Diversity analysis showed a higher number ofamoA geneOTUs (Sobs) in the dry season (Table 4)This datais consistent with the higher diversity found for the 16S rRNAgene for the dry season However the 16S rRNA gene clone

library failed to reveal this Thaumarchaeota diversity sinceonly 141 sequences from the dry season were consideredof theThaumarchaeota phylumThis result clearly highlightsthe importance of using more than one gene to describespecific microbial groups from natural environments [21]Another factor that may account for the much reducedphylogenetic diversity of the amoA sequences is the factthat while our 16S rRNA gene analysis showed overall highdiversity only four OTUs clustered in a confirmed ammonia-oxidizing group (I1a) [21 22] (Figure 4)

The amoA phylogenetic tree constructed (Figure 5) showsmost of our sequences in the same cluster of Nitroso-talea devanaterrarsquos amoA gene This is interesting as themost abundant rRNA 16S gene sequences also clusteredclose to this organism (Figure 4) N devanaterra was firstdescribed in acidic agricultural soils and was reported asa chemolithotrophic obligatory acidophilic soil ammoniaoxidizer [62] Similarly to our findings a study on AOAdiversity in high mountain lakes revealed sequences similartoN devanaterra in aquatic environments [63] and suggesteda phylogenetic conservation for the adaptive mechanisms tolow pH environments [64] However the ecological role ofArchaea in freshwater environments is still poorly under-stood [65ndash67] As with the 16S rRNA phylogenetic tree mostamoA uncultured sequences with high similarity to ours werefrom environments with higher water content

Although the number of samples analyzed imposes lim-itations this study describes for the first time the diversityof Archaea in freshwater lake sediments of the Cerrado andprovides evidence that rain can possibly affect archaeal diver-sity in these environments This can be concluded because ofthe reduced diversity in samples from the transition periodand markedly different archaeal community compositionsbetween seasons The events of resuspension and redepo-sition of the sediment surface that can be caused by rainalter nutritional composition (ie nitrogen and phosphorus)which in turn can influence microbial communities Futurestudies based on more sampling sites and use of quantitativemethods will be performed to further investigate these eventsand better understand the ecological roles ofArchaea in theseenvironments

4 Conclusions

We conclude that the occurrence of rain is likely a factor thatinfluences the archaeal communities in freshwater lake sedi-ments We were able to observe higher richness and diversityof organisms in the dry season with a few sequences beingfrommethanogenicmembersThe decrease of methanogenicArchaea in the transition period between the dry and rainyseasons could be due to an increase in nitrogen whichinhibitsmethanogenesisThe higher proportion of organismsfrom theThaumarchaeota phylum could also be explained bythis change in nutritional content While in both seasons thepresence of amoA genes was detected a higher number ofOTUs was observed in the dry season and in both cases thesesequences were more similar to Nitrosotalea devanaterrarsquosamoA gene

Archaea 7

Sediment of the Santa Fe River clone JN1798171Water of the Dongjiang River clone JQ3122531

amoA-A D48 (89)Candidatus Nitrosotalea devanaterra Nd1 amoA gene JN227489

amoA-A T7 (32)Wet sclerophyll forest soil clone JF5209891Wet tropical soil clone JX8853991

amoA-B T3 (48)Red soil clone HQ8960441

Candidatus Nitrosopumilus maritimus isolate SF amoA gene HM345611Candidatus Nitrosocaldus yellowstonii strain HL72 amoA gene EU239961

Candidatus Nitrososphaera gargensis clone GA15P03 amoA gene EU281321Candidatus Nitrososphaera viennensis strain EN76 amoA gene FR773159

Nitrosomonas europaea amoA gene AF037107

59

77

89

94

939595

57

97

7598

01

I1b

HWCGIII

I1a

Figure 5 Phylogenetic tree of archaeal amoA OTUs (90) obtained from Cerrado freshwater sediments in the dry season (amoA-A D andamoA-B D sequences) and in the dry to rainy season transition period (amoA-A T and amoA-B T sequences) The number of sequencesthat are represented by a specific node of the tree is indicated in parenthesis The phylogenetic tree was inferred by the maximum-likelihoodmethod using the MEGA software bootstrap value of 1000 and Tamura-Nei nucleotide substitution model The percentage of bootstrapresamplings that supports each topological element is represented near the line Reference sequences from different archaeal amoA geneswere used for comparison and the bacterial sequence Nitrosomonas europaea amoA gene AF037107 was used as an outgroup Bootstrapvalues below 50 were not shown and singletons were not included

Conflict of Interests

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

Acknowledgments

The authors would like to thank AdrianaMarinho Fernandesfor retrieving the lake sediment samples This research wassupported by Grants from CNPq FAPDF and DPP-UnB TRodrigues acknowledges a fellowship from CAPES

References

[1] BM TWalter AM de Carvalho and J F Ribeiro ldquoO conceitode savana e de seu componente Cerradordquo in Cerrado Ecologia eFloramdashVolume 1 SM Sano S P Almeida and JF Ribeiro Edscapıtulo 1 pp 21ndash45 Embrapa Cerrados Embrapa InformacaoTecnologica Brasılia Brazil 2008

[2] G Eiten ldquoThe cerrado vegetation of Brazilrdquo The BotanicalReview vol 38 no 2 pp 201ndash341 1972

[3] MM Clementino C C Fernandes R P Vieira AM CardosoC R Polycarpo and O B Martins ldquoArchaeal diversity innaturally occurring and impacted environments from a tropicalregionrdquo Journal of Applied Microbiology vol 103 no 1 pp 141ndash151 2007

[4] R P Vieira M M Clementino A M Cardoso et al ldquoArchaealcommunities in a tropical estuarine ecosystem guanabara bayBrazilrdquoMicrobial Ecology vol 54 no 3 pp 460ndash468 2007

[5] R G Taketani and S M Tsai ldquoThe influence of different landuses on the structure of archaeal communities in Amazoniananthrosols based on 16S rRNA and amoA genesrdquo MicrobialEcology vol 59 no 4 pp 734ndash743 2010

[6] D A Gracas P R Miranda R A Barauna et al ldquoMicrobialdiversity of an anoxic zone of a hydroelectric power stationreservoir in Brazilian AmazoniardquoMicrobial Ecology vol 62 no4 pp 853ndash861 2011

[7] T Bruce P M Meirelles G Garcia et al ldquoAbrolhos bank reefhealth evaluated by means of water quality microbial diversitybenthic cover and fish biomass datardquo PLoS ONE vol 7 no 6Article ID e36687 2012

[8] E Catao A P Castro C C Barreto R H Kruger and C MKyaw ldquoDiversity of archaea in Brazilian savanna soilsrdquo Archivesof Microbiology vol 195 no 7 pp 507ndash512 2013

[9] S Spring R Schulze J Overmann and K Schleifer ldquoIdentifica-tion and characterization of ecologically significant prokaryotesin the sediment of freshwater lakes molecular and cultivationstudiesrdquo FEMSMicrobiology Reviews vol 24 no 5 pp 573ndash5902000

[10] F A Esteves ldquoSedimentos Lımnicosrdquo in Fundamentos delimnologia pp 339ndash354 Interciencia Rio de Janeiro Brazil 3rdedition 2011

[11] R G Wetzel ldquoSediments and microflorardquo in Limnology Lakeand River Ecosystems pp 631ndash664 Elsevier San Diego CalifUSA 3rd edition 2001

[12] D R Lovley and M J Klug ldquoMethanogenesis from methanolandmethylamines and acetogenesis from hydrogen and carbondioxide in the sediments of a eutrophic lakerdquo Applied andEnvironmental Microbiology vol 45 no 4 pp 1310ndash1315 1983

[13] R Conrad T J Phelps and J G Zeikus ldquoGas metabolismevidence in support of the juxtaposition of hydrogen-producingand methanogenic bacteria in sewage sludge and lake sedi-mentsrdquo Applied and Environmental Microbiology vol 50 no 3pp 595ndash601 1985

[14] C Vetriani H W Jannasch B J Macgregor D A Stahl andA Reysenbach ldquoPopulation structure and phylogenetic char-acterization of marine benthic Archaea in deep-sea sedimentsrdquo

8 Archaea

Applied and Environmental Microbiology vol 65 no 10 pp4375ndash4384 1999

[15] C P Antony J Colin Murrell and Y S Shouche ldquoMoleculardiversity of methanogens and identification of Methanolobussp as active methylotrophic Archaea in Lonar crater lakesedimentsrdquo FEMS Microbiology Ecology vol 81 no 1 pp 43ndash51 2012

[16] D L Zhu C Sun andH He ldquoDetectionmethanogens in newlysettled sediments fromxuanwu lake inNanjing ChinardquoCurrentMicrobiology vol 64 no 6 pp 539ndash544 2012

[17] M Pester T Rattei S Flechl et al ldquoAmoA-based consensusphylogeny of ammonia-oxidizing archaea and deep sequencingof amoA genes from soils of four different geographic regionsrdquoEnvironmental Microbiology vol 14 no 2 pp 525ndash539 2012

[18] U Purkhold A Pommerening-Roser S Juretschko M CSchmid H-P Koops and M Wagner ldquoPhylogeny of all recog-nized species of ammonia oxidizers based on comparative 16SrRNA and amoA sequence analysis implications for moleculardiversity surveysrdquoApplied and EnvironmentalMicrobiology vol66 no 12 pp 5368ndash5382 2000

[19] M Konneke A E Bernhard J R de la Torre C B WalkerJ B Waterbury and D A Stahl ldquoIsolation of an autotrophicammonia-oxidizing marine archaeonrdquo Nature vol 437 no7058 pp 543ndash546 2005

[20] M Herrmann AM Saunders and A Schramm ldquoEffect of laketrophic status and rooted macrophytes on community com-position and abundance of ammonia-oxidizing prokaryotes infreshwater sedimentsrdquo Applied and Environmental Microbiol-ogy vol 75 no 10 pp 3127ndash3136 2009

[21] C Brochier-Armanet B Boussau S Gribaldo and P ForterreldquoMesophilic crenarchaeota proposal for a third archaeal phy-lum theThaumarchaeotardquoNature Reviews Microbiology vol 6no 3 pp 245ndash252 2008

[22] A Spang R Hatzenpichler C Brochier-Armanet et al ldquoDis-tinct gene set in two different lineages of ammonia-oxidizingarchaea supports the phylum Thaumarchaeotardquo Trends inMicrobiology vol 18 no 8 pp 331ndash340 2010

[23] S H Yuen and A G Pollard ldquoDetermination of nitrogenin agricultural materials by the nessler reagent IImdashMicro-determinations in Plant Tissue and in Soil Extractsrdquo Journal ofthe Science of Food and Agriculture vol 5 no 8 pp 364ndash3691954

[24] J Murphy and J P Riley ldquoA modified single solution methodfor the determination of phosphate in natural watersrdquoAnalyticaChimica Acta vol 27 pp 31ndash36 1962

[25] 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

[26] C A Francis K J Roberts J M Beman A E Santoro and B BOakley ldquoUbiquity and diversity of ammonia-oxidizing archaeain water columns and sediments of the oceanrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 102 no 41 pp 14683ndash14688 2005

[27] T Z DeSantis P Hugenholtz N Larsen et al ldquoGreengenesa chimera-checked 16S rRNA gene database and workbenchcompatible with ARBrdquo Applied and Environmental Microbiol-ogy vol 72 no 7 pp 5069ndash5072 2006

[28] P D Schloss S L Westcott T Ryabin et al ldquoIntroduc-ing mothur open-source platform-independent community-supported software for describing and comparing microbialcommunitiesrdquoApplied and EnvironmentalMicrobiology vol 75no 23 pp 7537ndash7541 2009

[29] M A Larkin G Blackshields N P Brown et al ldquoClustalW andclustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[30] P D Schloss and J Handelsman ldquoStatus of the microbialcensusrdquo Microbiology and Molecular Biology Reviews vol 68no 4 pp 686ndash691 2004

[31] Y Mao A C Yannarell and R I Mackie ldquoChanges in N-Transforming archaea and bacteria in soil during the establish-ment of bioenergy cropsrdquo PLoS ONE vol 6 no 9 Article IDe24750 2011

[32] C Lozupone M Hamady and R Knight ldquoUniFracmdashan onlinetool for comparing microbial community diversity in a phylo-genetic contextrdquo BMC Bioinformatics vol 7 article 371 2006

[33] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[34] S M Thomaz G Pereira and T A Pagioro ldquoMicrobialrespiration and chemical composition of different sedimentfractions in waterbodies of the upper Parana River floodplainBrazilrdquo Brazilian Journal of Biology vol 61 no 2 pp 277ndash2862001

[35] D C Zardo G C B Maas S Baia and O L S Weber ldquoCar-acterıstircas fısico-quımicas e microbiologicas do sedimentoda microbacia SamambaiamdashMT Brasilrdquo Revista Ciencias doAmbiente On-Line vol 7 no 2 pp 24ndash28 2011

[36] M R S S Silva Producao de serrapilheira biomassa e diver-sidade de comunidades bacterianas do solo em areas de Cer-rado sob diferentes usos e manejos [Dissertacao de mestrado]Departamento de Ecologia Instituto de Ciencias BiologicasUniversidade de Brasılia 2004

[37] 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

[38] V Torsvik and L Oslashvreas ldquoMicrobial diversity and function insoil From genes to ecosystemsrdquo Current Opinion in Microbiol-ogy vol 5 no 3 pp 240ndash245 2002

[39] T F Thingstad and R Lignell ldquoTheoretical models for thecontrol of bacterial growth rate abundance diversity andcarbon demandrdquo Aquatic Microbial Ecology vol 13 no 1 pp19ndash27 1997

[40] C Yang and D E Crowley ldquoRhizosphere microbial communitystructure in relation to root location and plant iron nutritionalstatusrdquo Applied and Environmental Microbiology vol 66 no 1pp 345ndash351 2000

[41] S U Sarathchandra A Ghani G W Yeates G Burch andN R Cox ldquoEffect of nitrogen and phosphate fertilisers onmicrobial and nematode diversity in pasture soilsrdquo Soil Biologyand Biochemistry vol 33 no 7-8 pp 953ndash964 2001

[42] J Pernthaler F Glockner S Unterholzner A Alfreider RPsenner and R Amann ldquoSeasonal community and populationdynamics of pelagic bacteria and archaea in a high mountainlakerdquo Applied and Environmental Microbiology vol 64 no 11pp 4299ndash4306 1998

[43] K Cruz-Martınez K B Suttle E L Brodie M E Power G LAndersen and J F Banfield ldquoDespite strong seasonal responsessoil microbial consortia are more resilient to long-term changesin rainfall than overlying grasslandrdquo The ISME Journal vol 3no 6 pp 738ndash744 2009

Archaea 9

[44] F Rasche D Knapp C Kaiser et al ldquoSeasonality and resourceavailability control bacterial and archaeal communities in soilsof a temperate beech forestrdquo ISME Journal vol 5 no 3 pp 389ndash402 2011

[45] S Ruppel V Torsvik F L Daae L Oslashvreas and J RuhlmannldquoNitrogen availability decreases prokaryotic diversity in sandysoilsrdquo Biology and Fertility of Soils vol 43 no 4 pp 449ndash4592007

[46] J Meng J Xu D Qin Y He X Xiao and FWang ldquoGenetic andfunctional properties of uncultivated MCG archaea assessed bymetagenome and gene expression analysesrdquoThe ISME Journalvol 8 no 3 pp 650ndash659 2014

[47] A Teske ldquoMicrobial communities of deep marine subsurfacesediments molecular and cultivation surveysrdquoGeomicrobiologyJournal vol 23 no 6 pp 357ndash368 2006

[48] K Kubo K G Lloyd J F Biddle R Amann A Teske and KKnittel ldquoArchaea of the miscellaneous crenarchaeotal group areabundant diverse and widespread in marine sedimentsrdquo ISMEJournal vol 6 no 10 pp 1949ndash1965 2012

[49] J C Fry R J Parkes B A Cragg A J Weightman andG Webster ldquoProkaryotic biodiversity and activity in the deepsubseafloor biosphererdquo FEMSMicrobiology Ecology vol 66 no2 pp 181ndash196 2008

[50] M Pester C Schleper and M Wagner ldquoThe Thaumarchaeotaan emerging view of their phylogeny and ecophysiologyrdquoCurrent Opinion in Microbiology vol 14 no 3 pp 300ndash3062011

[51] A Teske K Hinrichs V Edgcomb et al ldquoMicrobial diversityof hydrothermal sediments in the Guaymas Basin evidence foranaerobic methanotrophic communitiesrdquoApplied and Environ-mental Microbiology vol 68 no 4 pp 1994ndash2007 2002

[52] K Glissmann K-J Chin P Casper and R ConradldquoMethanogenic pathway and archaeal community structure inthe sediment of eutrophic Lake Dagow effect of temperaturerdquoMicrobial Ecology vol 48 no 3 pp 389ndash399 2004

[53] N Banning F Brock J C Fry R J Parkes E R C Horni-brook and A J Weightman ldquoInvestigation of the methanogenpopulation structure and activity in a brackish lake sedimentrdquoEnvironmental Microbiology vol 7 no 7 pp 947ndash960 2005

[54] C C On P Claus P Casper A Ulrich T Lueders and RConrad ldquoVertical distribution of structure and function of themethanogenic archaeal community in Lake Dagow sedimentrdquoEnvironmental Microbiology vol 7 no 8 pp 1139ndash1149 2005

[55] I C Torres K S Inglett and K R Reddy ldquoHeterotrophicmicrobial activity in lake sediments effects of organic electrondonorsrdquo Biogeochemistry vol 104 no 1ndash3 pp 165ndash181 2011

[56] G C Baker J J Smith and D A Cowan ldquoReview and re-analysis of domain-specific 16S primersrdquo Journal of Microbio-logical Methods vol 55 no 3 pp 541ndash555 2003

[57] S G Acinas R Sarma-Rupavtarm V Klepac-Ceraj and M FPolz ldquoPCR-induced sequence artifacts and bias Insights fromcomparison of two 16s rRNA clone libraries constructed fromthe same samplerdquoApplied and Environmental Microbiology vol71 no 12 pp 8966ndash8969 2005

[58] E F Delong ldquoEverything in moderation Archaea as lsquonon-extremophilesrsquordquo Current Opinion in Genetics and Developmentvol 8 no 6 pp 649ndash654 1998

[59] L A Sauder K Engel J C Stearns A P Masella R Pawliszynand J D Neufeld ldquoAquarium nitrification revisited thaumar-chaeota are the dominant ammonia oxidizers in freshwateraquarium biofiltersrdquo PLoS ONE vol 6 no 8 Article ID e232812011

[60] W L Balderston and W J Payne ldquoInhibition of methano-genesis in salt marsh sediments and whole-cell suspensionsof methanogenic bacteria by nitrogen oxidesrdquo Applied andEnvironmental Microbiology vol 32 no 2 pp 264ndash269 1976

[61] J Prochazka P Dolejs J Maca and M Dohanyos ldquoStabilityand inhibition of anaerobic processes caused by insufficiencyor excess of ammonia nitrogenrdquo Applied Microbiology andBiotechnology vol 93 no 1 pp 439ndash447 2012

[62] L E Lehtovirta-Morley K Stoecker A Vilcinskas J I Prosserand G W Nicol ldquoCultivation of an obligate acidophilic ammo-nia oxidizer from a nitrifying acid soilrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 38 pp 15892ndash15897 2011

[63] J C Auguet and E O Casamayor ldquoPartitioning of Thaumar-chaeota populations along environmental gradients in highmountain lakesrdquo FEMS Microbiology Ecology vol 84 no 1 pp154ndash164 2013

[64] C Gubry-Rangin B Hai C Quince et al ldquoNiche specializationof terrestrial archaeal ammonia oxidizersrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 52 pp 21206ndash21211 2011

[65] E O Casamayor G Muyzer and C Pedros-Alio ldquoCompo-sition and temporal dynamics of planktonic archaeal assem-blages from anaerobic sulfurous environments studied by 16SrDNA denaturing gradient gel electrophoresis and sequencingrdquoAquatic Microbial Ecology vol 25 no 3 pp 237ndash246 2001

[66] M Lliros E O Casamayor and C Borrego ldquoHigh archaealrichness in the water column of a freshwater sulfurous karsticlake along an interannual studyrdquo FEMS Microbiology Ecologyvol 66 no 2 pp 331ndash342 2008

[67] J-C Auguet N Nomokonova L Camarero and E OCasamayor ldquoSeasonal changes of freshwater ammonia-oxidizing archaeal assemblages and nitrogen species inoligotrophic alpine lakesrdquo Applied and EnvironmentalMicrobiology vol 77 no 6 pp 1937ndash1945 2011

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

in the dry season and in the transition period between thedry and rainy seasons when the first rains occur Eight genelibraries were obtained and sequenced four using Archaea-specific primers for the 16S rRNA gene and the other fourwith amoA gene primers specific to ammonia-oxidizingArchaeaThe sequences obtained were analyzed to determinewhether there are differences between the communities inlake sediments of different seasons Our results show twovery different community profiles on each season indicatinga possible effect of seasonal change

2 Materials and Methods

21 Study Site and Sampling Sediments were obtainedfrom a lake in the ldquoParque Nacional das Sempre Vivasrdquoa National Park located in the Serra do Espinhaco in thestate of Minas Gerais Brazil within the Cerrado biomeThe sediments were sampled in the Baixo Inhancicabay near the margin and the distance between thesampling spots was 15m Samples will be referred hereas RIB-A (17∘461015840162107810158401015840S 43∘37101584035548110158401015840W) and RIB-B(17∘461015840164991810158401015840S 43∘371015840359463510158401015840W) and were consideredreplicates Samples were collected with PVC tubes 10 cm indiameter by introducing the tube into the sediments locatednear the lake shore Sediment samples were retrieved fromdepths between 0 and 10 cm and kept on ice until stored atminus20∘C

The dry season samples RIB-A and RIB-B were collectedon May 7 2010 and will therefore be referred to as RIB-A D and RIB-B D The samples from the transition periodbetween the dry and rainy seasons were RIB-A and RIB-B collected on September 13 2010 and will be referred toas RIB-A T and RIB-B T Using a thermometer the watertemperature of the sampling sites was obtained The pHof the sediments was measured with a pH electrode Totalnitrogen was quantified by the Nessler method [23] and totalphosphorus was quantified by the Murphy-Riley method[24]

22 16S rRNA and amoA Genes Libraries Construction Envi-ronmental DNA was extracted from 05 g of lake sedimentusing the PowerSoil DNA isolation kit (MO BIO Labora-tories Inc) according to the manufacturerrsquos instructionsPCR primers 21f and 958r [25] were used to amplify the 16SrRNAgene usingDNA from sediments samples as a templateFor the amoA gene the primers used were Arch amoAfand amoAr [26] PCR assay conditions were the same asthose originally describedThe amplifiedDNAwas visualizedon agarose gels stained with ethidium bromide (10mgmL)The PCR-amplified DNA fragments were purified using theWizard kit (SVGel and PCRClean-Up System Promega) andcloned into the pGEM T easy (Promega) plasmid accordingto the manufacturerrsquos instructions Recombinant plasmidswere inserted into Escherichia coli calcium chloride-treatedDH5120572 cells by heat shock treatment The presence of theinserts was verified by selection of clones in LB agar platessupplemented with ampicillin (150 120583gmL) IPTG (05mM)and Xgal (000625) Plasmid DNA was extracted with

Table 1 Comparison of physicochemical properties of freshwaterlake sediments from replicates of the dry season (RIB D) and of thetransition period between the dry and rainy seasons (RIB T)

RIB D RIB TSampling date May 2010 September 2010Water temperature (∘C) 2585 plusmn 035 2160 plusmn 014Depth (cm) 545 plusmn 007 470 plusmn 014pH 557 plusmn 009 578 plusmn 020P-total (120583gL) 900 plusmn 141 1850 plusmn 354N-total (120583gL) 1100 plusmn 141 2350 plusmn 212

phenol-chloroform-isoamyl alcohol at 25 24 1 (volvolvol)DNA was sequenced by the Sanger method at MacrogenInc (Korea) The sequences obtained were deposited in theGeneBank dataset under the accession numbers KF640285-KF640592 (16S rRNA gene) and KJ719076-KJ719244 (amoAgene)

23 Phylogenetic Analysis The sequences from the 16S rRNAgene were used for comparative analysis with the Greengenestaxonomical database [27] using the Mothur software [28]A threshold of 90 or higher identity with the database wasused Alignment of 16S rRNA and amoA gene sequences wasperformed with the ClustalX software [29]

Mothur was used to filter the gap columns generatedby the alignment and was also used to generate the AceChao Shannon and Simpson indexes The 16S rRNA genesequences were clustered with a sequence identity thresholdof 97 for species 95 for genus 90 for class and 80 forphylum [30] Phylogenetic analysis of the amoA sequenceswas OTU based and the identity threshold considered was90 [31] Rarefaction curves were constructed with theMothur software The principal coordinate analysis (PCoA)was performed with the Unifrac program [32] Sequencesof the rRNA 16S gene from isolates of Euryarchaeota Cre-narchaeota Thaumarchaeota and Korarchaeota as well assequences of uncultured Archaea found in the GenBankdataset were used to construct a phylogenetic tree withthe MEGA software [33] using the maximum-likelihoodmethod a bootstrap value of 1000 and the Tamura nucleotidesubstitution model

3 Results and Discussion

All the sediments analyzed in the present study were 6 cmdeep therefore they were part of the sediment layer mostrecently deposited [34] All the samples presented slightlyacidic pH values (Table 1) which may be due to hydro-gen ion liberation in the medium caused by microbialmetabolism and organic matter decomposition [10 35] Sed-iments obtained in the transition period (RIBT) had highervalues of total nitrogen and phosphorus when compared tothe samples obtained in the dry season (RIB D) (Table 1)As previously mentioned environmental factors such asthe occurrence of rain affect the nutrient content in lakesediments [11] which could explain the differences between

Archaea 3









Dryseason











Transitionperiod





4 Archaea

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120 140 160

OTU

s

Sequences

Dry season

Unique9795

9080

(a)

0102030405060708090

0 50 100 150 200

OTU

s

Sequences

Transition period

Unique9795

9080

(b)


RIB-A D

RIB-A T

RIB-B T

RIB-B D

PCA P1 versus P2


P2-p

erce

nt v

aria

tion

expl

aine

d2186

05

04

03

02

01

00

minus01

minus02

minus03

minus04




0

10

20

30

40

50

60

70

80

90

RIB D RIB T

()





Archaea 5

RIB-B D22 (2)RIB-B D37 (2)







RIB-B T77 (13)RIB-B D35 (3)


RIB-A D38 (3)RIB-B D10 (2)



RIB-B D56 (4)RIB-B D58 (2)
















RIB-B T91 (23)RIB-B T28 (3)




90

99

100

93

98

96

99

97

99

50

99

99

100

10053

77

94

93

58

84

96

59

78

88

93

71

88

96

83

99

96

95

62

80

63

98

82

86

96

8672

9864

75

98

98

94

78

96

94

60

66

60

83

01


Crenarchaeota

I1a

HWCGIII

I1b

I1c

Thaumarchaeota

Euryarchaeota

Korarchaeota


6 Archaea









4 Conclusions


Archaea 7








59

77

89

94

939595

57

97

7598

01

I1b

HWCGIII

I1a




Acknowledgments


References















8 Archaea































Archaea 9
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 3









Dryseason











Transitionperiod





4 Archaea

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120 140 160

OTU

s

Sequences

Dry season

Unique9795

9080

(a)

0102030405060708090

0 50 100 150 200

OTU

s

Sequences

Transition period

Unique9795

9080

(b)


RIB-A D

RIB-A T

RIB-B T

RIB-B D

PCA P1 versus P2


P2-p

erce

nt v

aria

tion

expl

aine

d2186

05

04

03

02

01

00

minus01

minus02

minus03

minus04




0

10

20

30

40

50

60

70

80

90

RIB D RIB T

()





Archaea 5

RIB-B D22 (2)RIB-B D37 (2)







RIB-B T77 (13)RIB-B D35 (3)


RIB-A D38 (3)RIB-B D10 (2)



RIB-B D56 (4)RIB-B D58 (2)
















RIB-B T91 (23)RIB-B T28 (3)




90

99

100

93

98

96

99

97

99

50

99

99

100

10053

77

94

93

58

84

96

59

78

88

93

71

88

96

83

99

96

95

62

80

63

98

82

86

96

8672

9864

75

98

98

94

78

96

94

60

66

60

83

01


Crenarchaeota

I1a

HWCGIII

I1b

I1c

Thaumarchaeota

Euryarchaeota

Korarchaeota


6 Archaea









4 Conclusions


Archaea 7








59

77

89

94

939595

57

97

7598

01

I1b

HWCGIII

I1a




Acknowledgments


References















8 Archaea































Archaea 9
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Archaea

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120 140 160

OTU

s

Sequences

Dry season

Unique9795

9080

(a)

0102030405060708090

0 50 100 150 200

OTU

s

Sequences

Transition period

Unique9795

9080

(b)


RIB-A D

RIB-A T

RIB-B T

RIB-B D

PCA P1 versus P2


P2-p

erce

nt v

aria

tion

expl

aine

d2186

05

04

03

02

01

00

minus01

minus02

minus03

minus04




0

10

20

30

40

50

60

70

80

90

RIB D RIB T

()





Archaea 5

RIB-B D22 (2)RIB-B D37 (2)







RIB-B T77 (13)RIB-B D35 (3)


RIB-A D38 (3)RIB-B D10 (2)



RIB-B D56 (4)RIB-B D58 (2)
















RIB-B T91 (23)RIB-B T28 (3)




90

99

100

93

98

96

99

97

99

50

99

99

100

10053

77

94

93

58

84

96

59

78

88

93

71

88

96

83

99

96

95

62

80

63

98

82

86

96

8672

9864

75

98

98

94

78

96

94

60

66

60

83

01


Crenarchaeota

I1a

HWCGIII

I1b

I1c

Thaumarchaeota

Euryarchaeota

Korarchaeota


6 Archaea









4 Conclusions


Archaea 7








59

77

89

94

939595

57

97

7598

01

I1b

HWCGIII

I1a




Acknowledgments


References















8 Archaea































Archaea 9
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 5

RIB-B D22 (2)RIB-B D37 (2)







RIB-B T77 (13)RIB-B D35 (3)


RIB-A D38 (3)RIB-B D10 (2)



RIB-B D56 (4)RIB-B D58 (2)
















RIB-B T91 (23)RIB-B T28 (3)




90

99

100

93

98

96

99

97

99

50

99

99

100

10053

77

94

93

58

84

96

59

78

88

93

71

88

96

83

99

96

95

62

80

63

98

82

86

96

8672

9864

75

98

98

94

78

96

94

60

66

60

83

01


Crenarchaeota

I1a

HWCGIII

I1b

I1c

Thaumarchaeota

Euryarchaeota

Korarchaeota


6 Archaea









4 Conclusions


Archaea 7








59

77

89

94

939595

57

97

7598

01

I1b

HWCGIII

I1a




Acknowledgments


References















8 Archaea































Archaea 9
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 Archaea









4 Conclusions


Archaea 7








59

77

89

94

939595

57

97

7598

01

I1b

HWCGIII

I1a




Acknowledgments


References















8 Archaea































Archaea 9
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 7








59

77

89

94

939595

57

97

7598

01

I1b

HWCGIII

I1a




Acknowledgments


References















8 Archaea































Archaea 9
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

8 Archaea































Archaea 9
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 9
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article seasonal effects in a lake sediment...

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