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Hindawi Publishing CorporationArchaeaVolume 2013 Article ID 723871 11 pageshttpdxdoiorg1011552013723871

Research ArticleArchaeal Community Structures inthe Solfataric Acidic Hot Springs with DifferentTemperatures and Elemental Compositions

Tomoko Satoh Keiko Watanabe Hideo YamamotoShuichi Yamamoto and Norio Kurosawa

Division of Environmental Engineering for Symbiosis Graduate School of Engineering Soka University 1-236 Tangi-machiHachioji Tokyo 192-8577 Japan

Correspondence should be addressed to Tomoko Satoh stomoko76sokagrjp

Received 31 October 2012 Revised 20 January 2013 Accepted 24 March 2013

Academic Editor Yoshizumi Ishino

Copyright copy 2013 Tomoko Satoh et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Archaeal 16S rRNA gene compositions and environmental factors of four distinct solfataric acidic hot springs in Kirishima Japanwere comparedThe four ponds were selected by differences of temperature and total dissolved elemental concentration as follows(1) Pond-A 93∘C and 1679mg Lminus1 (2) Pond-B 66∘C and 2248mg Lminus1 (3) Pond-C 88∘C and 198mg Lminus1 and (4) Pond-D 67∘Cand 340mg Lminus1 In total 431 clones of 16S rRNA gene were classified into 26 phylotypes In Pond-B the archaeal diversity wasthe highest among the four and the members of the order Sulfolobales were dominant The Pond-D also showed relatively highdiversity and the most frequent group was uncultured thermoacidic spring clone group In contrast to Pond-B and Pond-D muchless diverse archaeal clones were detected in Pond-A and Pond-C showing higher temperatures However dominant groups in theseponds were also different from each other The members of the order Sulfolobales shared 89 of total clones in Pond-A and theuncultured crenarchaeal groups shared 99 of total Pond-C clones Therefore species compositions and biodiversity were clearlydifferent among the ponds showing different temperatures and dissolved elemental concentrations

1 Introduction

The extreme environments are unique places to study howorganisms interact with and adapt to the surroundingsSome of high temperature environments especially such asterrestrial hot springs and oceanic hydrothermal vents mayresemble volcanic habitats that are thought to have existed onearly Earth [1ndash3] Indeed some of the archaeal and bacteriallineages identified from hot springs appear to be related tolineages close to the root of the phylogenetic tree [4]

Hot spring microbial communities have been extensivelystudied in many areas such as Yellowstone National Park inthe United States [5ndash10] Kamchatka hot springs in Russia[11] the island of the Lesser Antilles [12 13] Icelandic hotsprings [11 14] Mt Unzen hot springs in Japan [15] Ohwaku-dani hot springs in Japan [16] Pisciarelli hot springs in Italy[17] Bor Khlueng hot springs in Thailand [18] Wai-o-tapugeothermal area in New Zealand [19] and Tengchong hotsprings in China [20]These pioneering works enabled better

appreciation of prokaryotic communities in the high temper-ature environments However despite decades of research westill understand relatively little about the relationship betweenthe environmental factors and hot spring prokaryotic com-munity It is important to reveal that which environmentalfactors affect prokaryotic community structures and diversityin individual hot spring habitats Temperature has perhapsreceived the most attention but other constraining factorsmay include pH oxidation redox potential elemental com-position and organic matter composition In this study wecompared the archaeal community structures and diversity offour distinct solfataric acidic hot springs in Kirishima Japan

2 Materials and Methods

21 Sample Collection and Analyses of Dissolved ElementalCompositions The investigated hot springs in this study arelocated in a field of one square kilometer in the Kirishima

2 Archaea

Table 1 Characteristics of sampling sites and ponds

Pond-A Pond-B Pond-C Pond-DTemp (∘C) 93 66 88 67pH 26 20 24 23Concentration (mg Lminus1)composition ()Fe 3889 23 1149 51 9630 5 2718 8S 6632 40 7028 31 5976 30 6190 18Al 4336 26 2879 13 1457 7 2021 1Mg 8674 5 4677 2 0001 0 4335 13Si 4788 3 4552 2 1039 53 1484 44Ca 5481 3 1088 0 7498 4 3926 12P 2850 0 4711 0 1265 1 1266 0Na 0001 0 0001 0 0001 0 8442 2K 0001 0 0001 0 0001 0 7384 2As 1079 0 1137 0 0879 0 0856 0Total 1679 100 2248 100 1975 100 3401 100Latitude (N) 31∘54101584037710158401015840 31∘54101584052410158401015840 31∘55101584005010158401015840 31∘55101584004510158401015840

Longitude (E) 130∘49101584000610158401015840 130∘48101584050310158401015840 130∘48101584041110158401015840 130∘48101584041010158401015840

Altitude (m) 759 842 884 885Color of sediments Light brown Light brown Gray GrayDetection limit is 0001mg Lminus1

Japan

N

Ishizaka River

500

Landslide

Spring

Pond-CPond-D

Pond-B

Pond-A

50131∘E

Kagoshima graben

Kirishima volcano32∘N

(Km)

(m)

Figure 1 Map of sampling site in Kirishima geothermal area Japan

geothermal area in Japan (KirishimaNational Park) (Figure 1Table 1) where the extensive volcanic activity occurred fromthe Pleistocene epoch to the present depositing a thick pileof volcanic rocks [21] Kirishima volcano one of the largestQuaternary volcanoes in Japan belongs to the northern partof the Kagoshima graben a volcano-tectonic depression [22]caused by the subduction of the Philippine Sea plate Thisvolcano occupies an area of about 20 kmtimes 30 km elongated inthe northwest to southeast direction and contains more than20 small volcanoes [23]

Sampling location within Kirishima geothermal area isa private land therefore people are usually not allowed totrespass on this area We got permission to take hot springwater soil and various other native samples in this area froman owner of the landThere are many hot springs and muddyponds showing a variety of temperatures and elementalcompositions

Muddy water sample was collected into sterile 100mLglass bottle at each pond Temperature and pH of thesamples were measured at each sampling site A part of eachsample was filtered using 022120583m membrane filter (AsahiGlass) and was subjected to analysis of dissolved elementalconcentrations using the inductively coupled plasma opticalemission spectroscopy (ICPS-7000 ver2 Shimadzu) In thepresent study we selected four ponds displaying a range oftemperatures and dissolved elemental compositions for thearchaeal community analysis

22 16119878 rRNA Gene Clone Libraries and Sequencing Theenvironmental DNA was extracted from 5 to 10 g of eachmuddy water sample using the UltraClean Soil DNA KitMega Prep (Mo Bio Laboratories) according to the manu-facturerrsquos instructions The precipitated DNA was purified

Archaea 3

using the GFX PCR DNA and Gel Band Purification Kit (GEHealthcare)

Purified DNA was used as the template for the ampli-fication of archaeal 16S rRNA gene by archaea-specificprimer A21F 51015840-TTCCGGTTGATCCYGCCGGA and uni-versal primer U1492R 51015840-GGYTACCTTGTTACGACTTThe PCR conditions included an initial denaturation step at94∘C for 3min followed by 35 cycles of denaturation at 94∘Cfor 30 sec annealing at 55∘C for 30 sec and extension at 72∘Cfor 2min using Ex Taq DNA polymerase (Takara Bio) Thiswas followed by a final extension at 72∘C for 10min

The PCR products were purified using the aforemen-tioned GFX Kit and were ligated into the pT7 Blue T-Vector(Novagen) E coli DH5120572 cells were transformed with theplasmid library and were plated onto LB plates including100 120583gmLminus1 ampicillin 40120583gmLminus1 X-gal and 05mM IPTGBluewhite selection was conducted by randomly pickingand subculturing individual white colonies in 100120583L of 2 timesYT medium containing 100120583gmLminus1 ampicillin in a 96-wellplate at 37∘C overnight The inserted 16S rRNA gene wasamplified using 1120583L of the culture as the template with thesame PCR procedure mentioned above About 800 bp of the51015840-region of each 16S rRNA gene clone was sequenced by theaforementioned archaea-specific primer A21F and used fortaxonomic and phylogenetic analysis

23 Identification of 16S rRNA Gene Clones and Phyloge-netic Analysis 16S rRNA gene sequences were edited usingthe MEGA5 (Molecular Evolutionary Genetics Analysishttpwwwmegasoftwarenet) [24] We also searched forchimera sequences by manually checking the sequencealignments using GENETYX ver 1003 software (Gene-tyx) Clones having 97 sequence similarity or higherwere treated as a phylotype The representative sequencesof each phylotype were compared with 16S rRNA genesequences published in the National Center for Biotech-nology Information DNA database using BLAST (BLASTNhttpwwwncbinlmnihgovBLAST) [25] to identify indi-vidual clonesThe representative sequences of each phylotypeand related sequences in the GenBank data base were alignedusing CLUSTALW ver 183 program [26] The maximumlikelihood tree including bootstrap probabilities (1000 sam-plings) was constructed using the MEGA5

24 Statistical Analyses Measurements of diversity ideallyinclude richness the number of different species or groupspresent and evenness the distribution of those groups [2728] The Shannon-Weaver index [29] 1198671015840 = minusΣ(119901119894)(ln119901119894)and Simpsonrsquos reciprocal index [30] 1D where 119863 = Σ(119901119894)2and pi is the proportion of phylotypes 119894 relative to the totalnumber of phylotypes both take richness and evenness intoaccount [13 28] The Shannon-Weaver index and Simpsonrsquosreciprocal index were calculated using ESTIMATES 80 [31]Evenness (1198691015840 = 1198671015840 ln 119878) was also calculated [32] ESTI-MATES 80 was also used to calculate Chao1 nonparamet-ric richness estimator [33] and abundance-based coverageestimator of species richness (ACE) [34] These coverageestimators determine the number of probable phylotypes in

the environment comparedwith the numbers observed in thesample The homologous coverage (biodiversity coverage) Cwas determined with the following equation 119862 = 1 minus (119873119899)where 119873 is the number of phylotypes sequences and 119899 isthe total number of analyzed clones [35 36] Additionallystatistical analyses including principal components analysisto determine the correlations among the archaeal diversitywith the environmental factors including temperature anddissolved elemental concentrations Canonical correlationanalysis was also performed to determine the correlationsbetween archaeal groups and temperature or dissolvedelemental concentrations by using the software XLSTAT(Addinsoft New York NY)

25 Nucleotide Sequence Accession Numbers The representa-tives of nucleotide sequences of the phylotypes are availablein the DDBJEMBLGenBank databases under the accessionnumbers AB753272-AB753298 and AB755799-AB755806

3 Results and Discussion

31 Water Chemistry The four ponds in Kirishima geother-mal area were selected based on the differences of tempera-tures and total dissolved elemental concentrations as follows(1) Pond-A 93∘C and 1679mg Lminus1 (2) Pond-B 66∘C and2248mg Lminus1 (3) Pond-C 88∘C and 198mg Lminus1 and (4) Pond-D 67∘C and 340mg Lminus1 The characteristics of sampling sitesand these ponds are shown in Table 1The range of pH valuesof the ponds was 20ndash26 In the ponds showing higher totaldissolved elemental concentration the concentrations andpercentages of Fe S and Al were especially higher than thosein other ponds There was no significant difference of theconcentrations of Mg Si Ca P Na K and as between theponds

32 16119878 rRNA Gene Clone Libraries 16S rRNA gene clonelibraries were successfully constructed using the environ-mental DNAs extracted from four muddy water samples Atotal of 432 clones of archaeal 16S rRNA gene were analyzedA chimerical sequence was detected during the analysis andwas not used for further study On the basis of the sequencesimilarity values a total of 431 clones (Pond-A 106 Pond-B 112 Pond-C 109 and Pond-D 104 clones) were classifiedinto 26 phylotypes consisting of 25 crenarchaeal phylotypesand a single euryarchaeal one (Table 2) The homologouscoverage values were 088 or above for all ponds indicatingthat approximately 90 of the 16S rRNA gene clones in theseponds could be considered in this study (Table 3)

The guanine-plus-cytosine (G + C) content in the 16SrRNA gene sequences detected from 26 phylotypes in thisstudy ranged from 566 to 690 with an overall averageof 624 plusmn 36 According to Kimura et al the growthtemperature of archaea are strongly correlated with the G +C content while the phylotypes containing this amount ofG + C were grouped as the moderately thermophilic andhyperthermophilic archaea [37] Therefore all phylotypesdetected in this study could possibly be related to moderatelythermophilic and hyperthermophilic archaea

4 Archaea

Table 2 Affiliation and closest published species or clones of 26 phylotypes

Phylotypes Affiliation Closest species or clones(accession number)

16S rRNA genesimilarity Number of clones detected from each site

() Pond-A Pond-B Pond-C Pond-DOrder Sulfolobales

ST2A1-3 Acidianus brierleyi Acidianus brierleyi (D26489) 989 3ST2A1-43 Acidianus sp Acidianus ambivalens (D85506) 955 1ST2A1-14 Metallosphaera sedula Metallosphaera sedula (D26491) 1000 7ST2A1-16 Sulfolobus solfataricus Sulfolobus solfataricus (D26490) 991 2

ST8A1-57 Sulfurisphaera sp Sulfurisphaera ohwakuensis(D85507) 951 1

ST2A1-5 Uncultured Sulfolobales Acidic hot spring cloneHO78W21A35 (AB600386) 807 39

ST2A1-32 Uncultured Sulfolobales Acidic sulfuric hot spring cloneLH2wa 90 (FJ797343) 983 6

ST8A1-12 Uncultured Sulfolobales Acidic sulfuric hot spring cloneHS3wa 52 (FJ797311) 966 92


Order Acidilobales

ST2A1-9 Caldisphaera lagunensis Caldisphaera lagunensis(AB087499) 985 10

ST2A1-27 Caldisphaera sp Caldisphaera draconis(EF057392) 954 2 21

(=ST15A1-7)Order Thermoproteales

ST8A1-8 Caldivirga maquilingensis Caldivirga maquilingensis(AB013926) 980 5 2

(=ST2A1-31)

ST8A1-40 Vulcanisaeta distributa Vulcanisaeta distributa(AB063630) 989 7

ST16A1-87 Thermocladium modestius Thermocladium modestius(AB005296) 994 1

Other crenarchaeal groups

ST2A1-8 UTSCG Acidic sulfuric hot spring cloneLH2wa 02 (FJ797332) 997 20 42 52

(=ST16A1-1ST15A1-1)

ST15A1-3 UTSCG Acidic hot spring cloneUzon4-5d (HQ395709) 961 2

ST16A1-6 UTSCG Acidic spring clone HO28S9A21(AB600335) 969 6

ST16A1-20 UTSCG Hot spring clone SK865(DQ834117) 969 13 6

(=ST15A1-6)

ST2A1-2 UTSCG II Hot spring clone BW303(DQ924843) 933 4

ST2A1-15 UTSCG II Hot spring clone SK993(DQ834245) 997 1

ST15A1-26 HWCG V Acidic spring clone HO28S9A51(AB600343) 963 1


Archaea 5

Table 2 Continued



() Pond-A Pond-B Pond-C Pond-D

ST2A1-25 HWCG VI Hot spring clone SK859(DQ834111) 958 13 46 2

(=ST16A1-2ST15A1-34)

ST2A1-52 HWCG VI Hot spring clone SK859(DQ834111) 960 2

ST15A1-32 HWCG VII Acidic sulfuric hot spring cloneHS4sa 15 (FJ797318) 912 1

Euryarchaeal groups

ST16A1-50 UnculturedEuryarchaeota

Thermal spring clone kmc048(HM150106) 992 1 17

(=ST15A1-8)Total 106 112 109 104

Table 3 Diversity index scores for clone libraries

Sample Shannon Simpson Rich Even 119878ACE 119878Chao1 Coverage Total clone numberPond-A 053 132 5 0332 704 600 095 106Pond-B 206 541 14 0780 154 142 088 112Pond-C 123 291 6 0687 101 700 094 109Pond-D 145 310 9 0659 109 925 091 104Temperatureapproximately 90∘C(Pond-A + Pond-C)

158 366 11 0659 160 170 095 215

Temperatureapproximately 70∘C(Pond-B + Pond-D)

220 582 20 0735 233 210 091 216

El conc gt 1600 ppm(Pond-A + Pond-B) 199 437 18 0689 210 195 092 218

El conc lt 350 ppm(Pond-C + Pond-D) 161 368 11 0673 155 133 095 213

Diversity index scoresmeasuredwere Shannon-Weaver index (Shannon) Simpsonrsquos reciprocal index (Simpson) Richness (Rich) Evenness (Even) the coverageestimators 119878ACE and 119878Chao1 and the homologous coverage El conc indicates total dissolved elemental concentration

33 Archaeal Community in Pond-A On the basis of 16SrRNA gene sequence similarities 106 clones derived fromPond-A which showed higher temperature and total dis-solved elemental concentration consisted of five phylotypesof Crenarchaeota (Table 2) The 5 and 7 sequencesof this pond were highly similar to those of culturedspecies (gt980) of the order Thermoproteales Caldivirgamaquilingensis and Vulcanisaeta distributa respectively Thetype strains of both species were hyperthermophilic archaeaoptimally growing at above 85∘C and they were originallyisolated from acidic hot springs in Philippines and Japanrespectively [38 39] On the other hand other 89 of Pond-Aclones did not show significant similarities with any culturedspecies Almost all the clones of them were assigned as aphylotype ST8A1-12 affiliated with the order SulfolobalesThis phylotype showed 95-96 sequence similarity withpublished environmental clones NAKO74-07 and HS3wa 52detected from Nakabusa hot spring Japan (DNA database

Accession no AB366602) [37] and Tatung Volcano hotspring Taiwan (DNA database Accession no FJ797311) Thediversity represented by the Shannon-Weaver index andSimpsonrsquos reciprocal index in the Pond-A was the lowestamong the four ponds (Table 3)

34 Archaeal Community in Pond-B In contrast to the Pond-A the largest number of phylotypes was detected in Pond-B which was characterized as lower temperature and highertotal dissolved elemental concentration resulting that thediversity indices and evenness value in this pondwere highestamong the four ponds (Table 3) A total of 112 clones consistedof 14 phylotypes that were classified into the following sixgroups the order Sulfolobales Acidilobales Thermopro-teales and three uncultured crenarchaeal groups (Table 2Figure 2) The 21 of the total clones were closely related toany of five cultured species (gt980) Sulfolobus solfataricus[40] Metallosphaera sedula [41] Acidianus brierleyi [42]

6 Archaea

Caldisphaera lagunensis [43] and Caldivirga maquilingensis[38] S solfataricus M sedula and A brierleyi are faculta-tively chemolithoautotrophic aerobes and require elementalsulfur or sulfidic ores These species and their close rela-tives have been isolated from acidic Solfatara fields aroundthe world [44] C lagunensis and C maquilingensis areheterotrophic anaerobes Their growths are stimulated orconstrained by the presence of sulfur as an electron acceptor

On the other hand nine phylotypes sharing 79 in totalof all Pond-B clones showed no significant similarity withany cultured species Nearly half of these uncultured cloneswere assigned as a phylotype ST2A1-5 This phylotype wasmost dominant (35) in Pond-B and was phylogeneticallydistant not only from any cultured species but also fromany published environmental clones This novel phylotypebelonged to a cluster in the order Sulfolobales (Figure 2)Thiscluster also harbored another Pond-B phylotype ST2A1-32which showed 98 16S rRNA gene sequence similarity withpublished environmental clone LH2wa 90 detected fromTaiwanese hot spring (DNA database Acc no FJ797343)

The phylotype ST2A1-8 belonging to the uncultured ther-moacidic spring clone group (UTSCG) [16] was secondarydominant in Pond-B and it shared 18 of the total Pond-B clones Interestingly phylotypes similar to ST2A1-8 werealso frequently detected in Pond-C and Pond-D suggestingthat this crenarchaeal species survive relatively wide range oftemperature and dissolved elemental composition in acidichot springs There might be unfavorable factors in Pond-Afor the presence of UTSCG The phylotypes ST2A1-2 andST2A1-15 were placed in the sister cluster of UTSCG withpublished clones detected from Yellowstone National Park(DNA database Acc no DQ834245) We call this cluster asUTSCG II in this study

The phylotype ST2A1-25 was thirdly dominant in Pond-B and was placed into the sister cluster of the hot water cre-narchaeotic group II (HWCG II) [15 45 46] with phylotypeST2A1-52 and published environmental clone SK859 detectedfrom acidic hot spring in Yellowstone National Park (DNAdatabase Acc No DQ834111) We call this cluster as HWCGVI in this study The phylotype ST2A1-25 was also dominantin Pond-C

35 Archaeal Community in Pond-C The other high temper-ature pond Pond-C showed relatively low value of speciesrichness as same as Pond-A (Table 3) A hundred andnine clones were classified into six phylotypes as followsThermocladium modestius of the order Thermoprotealesfour uncultured crenarchaeal phylotypes and unculturedeuryarchaeal phylotypes The type strain of T modestius wasoriginally isolated from solfataric mud at Noji-onsen Japanand is an anaerobic heterotroph growing optimally around75∘C pH 40 [47]

As mentioned in the previous section the unculturedphylotypes ST2A1-8 of UTSCG and ST2A1-25 of HWCG VIwere dominant in the clone library constructed for this pondsampleThese two phylotypes shared 81 in total of the Pond-C clones Three phylotypes sharing 56 of Pond-C cloneswere affiliated with uncultured thermoacidic spring clonegroup (UTSCG) [16]

36 Archaeal Community in Pond-D A hundred and fourclones derived from Pond-D which showed lower temper-ature and total dissolved elemental concentration consistedof nine phylotypes The diversity indices in Pond-D werelower than those in Pond-B but were higher than the valuesin Pond-A and Pond-C (Table 3) The phylotype sharing20 of the total clones was related to Caldisphaera draconiswith 95 sequence similarity of 16S rRNA gene This speciesis chemoorganotrophic anaerobe isolated from acidic hotspring in Yellowstone National Park [48]

Other phylotypes showed no significant similarity withany cultured species The most frequent phylotype wasST2A1-8 affiliated with UTSCG and it shared 50 of the totalclone in this pond In contrast to the archaeal communitiesin other three ponds the secondary dominant unculturedphylotype (ST16A1-50) was affiliated with Euryarchaeota andshowed 99 sequence similarity of 16S rRNA gene withthermal spring clone kmc048 detected from Kamchatka hotsprings in Russia (DNA database Acc no HM150106) Thisphylotype shared 16 of the total clones in this pond

The phylotype ST15A1-26 together with ST15A2-137 andST15A1-32 were barely detected in Pond-D and were placedinto the sister cluster of HWCG II We call these clusters asHWCG V and HWCG VII respectively in this study

37 Archaeal Diversity and Community Structures with Dif-ferent Temperatures and Total Dissolved Elemental Concentra-tions When the diversity was compared within ponds withdifferent temperatures (Temperature approximately 90∘CPond-A + Pond-C versus Temp approx 70∘C Pond-B +Pond-D) represented by the Shannon-Weaver index andSimpsonrsquos reciprocal index the lower temperature pondsshowed higher diversity (Table 3) On the other handwhen comparing within ponds with different total dissolvedelemental concentrations the diversity indices of highertotal dissolved elemental concentration ponds (El conc gt1600mg Lminus1 Pond-A + Pond-B) were higher than those ofPond-C + Pond-D (El conc lt 350mg Lminus1) As a result thearchaeal diversity was the highest in the pond characterizedas lower temperature and higher total dissolved elemen-tal concentration (Pond-B) In contrast the combinationof higher temperature and lower total dissolved elementalconcentration (Pond-A) caused the lowest diversity in thisstudy

When focusing on the species composition and distri-bution they were dissimilarity within ponds with differenttemperatures and total dissolved elemental concentrationsAs shown in Table 2 the phylotypes affiliated with the orderSulfolobales were only detected in the ponds showing highertotal dissolved elemental concentrations (Pond-A + Pond-B)The members of the order Sulfolobales are generally charac-terized as facultatively or obligately chemolithoautotrophicS0 metabolizers some members oxidize ferrous iron andsulfidic ores producing solublemetal sulfates [44]Thereforethe presence of Sulfolobales makes sense in these pondsincluding higher total dissolved elemental concentrationespecially sulfur as we would expect to detect microbes

Archaea 7

Acidic hot spring water clone pUWA36 (AB007308)

Deep gold mine clone SAGMA-W (AB050228)

Deep-sea hydrothermal vent clone pMC2A1 (AB019723) MG I

88100

100

100

99

100

98

100

68100

100

100

73

99

85

99

97

99

100

99

94

99

75

98

82

97

88

100

100

96

99

79

95

88

95

89

54

5299

90

94

99

10092

95

87

54

84

70

57

89

95

99

78

70

66

61

74 55

88

MHVG-I

Korarchaeota

Euryarchaeota

UTRCG

Thaumarchaeota

UTSCG

Thermoproteales

Acidilobales

ST2A1-8 (20) (AB753275)

ST16A1-6 (6) (AB753297)

ST15A1-6 (6) (AB753291)

ST2A1-2 (4) (AB753272)

ST8A1-12 (92) (AB753286)

ST8A1-52 (1) (AB753288)

ST8A1-57 (1) (AB753289)

ST2A1-16 (2) (AB753279)

ST2A1-3 (3) (AB753273)

ST2A1-14 (7) (AB753277)

ST2A1-43 (1) (AB753284)

ST2A1-32 (6) (AB753283)

ST2A1-5 (39) (AB753274)

ST2A1-27 (2) (AB753281)

ST2A1-9 (10) (AB753276)

ST2A1-31 (2) (AB753282)

ST16A1-87 (1) (AB753298)

ST15A2-137 (2) (AB753296)

ST15A1-26 (1) (AB753293)

ST2A1-52 (2) (AB753285)

ST15A1-32 (1) (AB753295)

ST16A1-50 (1) (AB755806)

ST8A1-40 (7) (AB753287)

ST2A1-15 (1) (AB753278)

ST15A1-7 (21) (AB755800)

ST8A1-8 (5) (AB755799)

ST16A1-20 (13) (AB755803)

ST15A1-3 (2) (AB753290)

ST15A1-1 (52) (AB755801)

ST16A1-1 (42) (AB755802)

ST2A1-25 (13) (AB753280)ST15A1-34 (2) (AB755804)

ST16A1-2 (46) (AB755805)

Thermal spring clone kmc048 (HM150106)

ST15A1-8 (17) (AB753292)

005

Hydrothermal vent water clone pOWA133 (AB007303) Hydrothermal sulfide structure clone Fhm1A36 (AB293205)

Korarchaeota SRI-306 (AF255604)

Acidic spring clone HO28S9A51 (AB600343)

Hot spring clone BY132 (EF156497)Hot spring clone SK859 (DQ834111)

Hot spring clone BY126 (EF156491)

Acidic spring clone HO28S21A56 (AB600373)Acidic red soil clone Arc-D93 (FJ174727)

Subterranean hot springs clone SUBT-14 (AF361211)Candidatus Nitrosocaldus yellowstonii (EU239960)

Hot spring clone BW303 (DQ924843)

Hot spring clone SK993 (DQ834245)


Acidic hot spring clone Uzon4-5d (HQ395709)

Hot spring clone SK295 (AY882844)Acidic spring clone HO28S9A21 (AB600335)

Acidic hot spring clone HO78W21A35 (AB600386) Acidic hot spring clone Hverd088Cs (DQ441530)

HWCG VII

UTSCG II

Thermal spring clone pSL12 (UCU63343)

Deep gold mine clone SAGMA-Z (AB050231)

Thermal spring clone pJP89 (L25305)Thermal spring clone pSL4 (UCU63341)

Deep-sea hydrothermal vent clone pMC2A36 (AB019720)Deep-sea hydrothermal vent clone pISA7 (AB019733) HWCG IV (UCII)

HWCG V

HWCG VI

HWCG IMCG

SAGMCGSCG

FSCG

HWCG II (UCIII)

Sulfolobus metallicus (D85519)

Sulfolobus solfataricus (D26490)Stygiolobus azoricus (D85520)

Sulfolobus acidocaldarius (D14876)Sulfurisphaera ohwakuensis (D85507)

Metallosphaera sedula (D26491)

Acidianus brierleyi (D26489)

Pyrodictium occultum (NR025933)Ignisphaera aggregans (NR043512)

Acidianus ambivalens (D85506)

Caldisphaera lagunensis (AB087499)Caldisphaera draconis (EF057392)

Acidic sulfuric hot spring clone LH2wa 62 (FJ797336)Acidilobus saccharovorans (AY350586)

Thermocladium modestius (AB005296)

Caldivirga maquilingensis (AB013926)

Vulcanisaeta distributa (AB063630)Thermofilum pendens (NR029214)

Acidic sulfuric hot spring clone LH2wa 02 (FJ797332)

DSAG (MBGB)

Acidic sulfuric hot spring clone HS4sa 15(FJ797318)

Thermoplasma acidophilum (M38637)Aciduliprofundum boonei (DQ451875)

Pyrococcus glycovorans (NR029053)

Candidatus Korarchaeum cryptofilum (CP000968)Aquifex pyrophilus (M83548)


Acidic sulfuric hot spring clone HS3wa 52 (FJ797311)


Sulfolobales

Figure 2 Phylogenetic tree of archaeal 16S rRNA gene sequences detected in Kirishima hot springs Bootstrap values (gt50) based on 1000replicates are indicated at nodesThe scale bar indicates the number of nucleotide substitutions per position Number in the parenthesis withphylotype name represents the number of clones of each phylotypeTheDNAdatabase accessionnumbers are also indicated in the parenthesisAquifex pyrophilus is used as an outgroup species The phylotype names derived from Pond-A Pond-B Pond-C and Pond-D shown in blueyellow red and green respectively UTSCG uncultured thermoacidic spring clone group [16] HWCG I hot water crenarchaeotic group I[6 46] HWCG II (known as UCIII) uncultured crenarchaeal group III [15 45 46] HWCG IV (also known as UCII) [45 49 50] DSAG(known as MBGB) deep-sea archaeal group (marine benthic group B) [49 51] MCG miscellaneous crenarchaeal group [5 50 52 53] MGI marine crenarchaeotic group I [49 54] SAGMCG South Africa gold mine group [52] SCG soil crenarchaeotic group [52] UTRCGuncultured Thaumarchaeota-related clone group [16] FSCG forest soil crenarchaeotic group [6 55] and MHVG-I marine hydrothermalvent group I [15 16 52]

8 Archaea

Table 4 Correlation matrix showing 119903 values for Pearsonrsquos correlation

Variables Shannon Temp Fe S Al Mg Si Ca P Na K As El ConcShannon 100 minus087 054 000 minus032 minus041 007 minus071 042 014 014 011 020Temp minus087 100 minus040 005 031 012 minus029 029 minus029 minus055 minus055 minus001 minus014Fe 054 minus040 100 084 062 034 minus076 minus026 099 minus046 minus046 090 092S 000 005 084 100 095 071 minus093 021 091 minus057 minus057 099 098Al minus032 031 062 095 100 082 minus090 044 072 minus057 minus057 090 087Mg minus041 012 034 071 082 100 minus049 082 044 minus002 minus002 061 064Si 007 minus029 minus076 minus093 minus090 minus049 100 000 minus083 084 084 minus094 minus089Ca minus071 029 minus026 021 044 082 000 100 minus015 033 033 007 008P 042 minus029 099 091 072 044 minus083 minus015 100 minus051 minus051 095 097Na 014 minus055 minus046 minus057 minus057 minus002 084 033 minus051 100 100 minus062 minus051K 014 minus055 minus046 minus057 minus057 minus002 084 033 minus051 100 100 minus062 minus051As 011 minus001 090 099 090 061 minus094 007 095 minus062 minus062 100 099El conc 020 minus014 092 098 087 064 minus089 008 097 minus051 minus051 099 100Values in bold are different from 0 with a significance level 120572 = 010 Diversity indices showed very similar correlations with each other and environmentalvariables only Shannon-Weaver index for archaeal clone libraries is shown Shannon Temp and El Conc indicate Shannon-Weaver index Temperature andtotal dissolved elemental concentration respectively

Shannon

Fe

S

AlMg

Si

Ca

P

As

minus1

minus075

minus05

minus025

0

025

05

075

1

minus1 minus075 minus05 minus025 0 025 05 075 1

Variables (axes F1 and F2 813)

F1 (5566)

Temperature

K Na

(a)

minus3

minus2

minus1

0

1

2

3

minus4 minus3 minus2 minus1 0 1 2 3 4 5

Observations (axes F1 and F2 813)

F1 (5566)

Pond-B

Pond-A

Pond-C Pond-D

(b)

Figure 3 Principal components analysis showing loadings on principal components 1 and 2 for environmental factors at sites and therelationship to each site Shannon Temp and El Conc indicate Shannon-Weaver index temperature and total dissolved elementalconcentration respectively

that metabolically depend on sulfur as an electron donorIt is also interesting that the species compositions withinthe order Sulfolobales between Pond-A and Pond-B wereclearly different from each other We detected sequencesclosely related to Sulfolobales species (989 similarity) inPond-B (66∘C) but most of the clones detected from Pond-A(93∘C) were affiliated with uncultured Sulfolobales forminga phylotype ST8A1-12 In addition the members of thegenus Caldisphaera of the order Acidilobales were frequentlydetected from lower temperature ponds (Pond-B + Pond-D)but not detected from higher temperature ponds (Pond-A +Pond-C)This may be due to the growth temperature limit ofthe members of the genus Caldisphaera which is 85∘C [56]

38 Geochemistry and Archaeal Diversity or Groups Correla-tions A matrix was created based on Pearsonrsquos correlation

coefficients (r) calculated from the Shannon-Weaver indextemperatures and dissolved elemental concentrations atthese four ponds (Table 4) Several dissolved elementalconcentrations were statistically correlated with each otherbut the archaeal diversity did not correlate with temperatureand any dissolved elemental concentrations (significancelevel 120572 = 010) Total dissolved elemental concentration wasstrongly correlated with S P and As (119875 value lt 005) andmoderately correlated with Fe (119875 lt 010) Al and S weremoderately correlated with each other Principal componentsanalysis showed that axes F1 and F2 accounted for 813 ofthe variation between sites S As and Si contributed equallyto PC1 while the archaeal diversity index contributed to PC2(Figure 3) Pond-A and Pond-B were higher in total dissolvedelemental concentration and variations at Pond-A and Pond-B appeared to be explained best by S and As of PC1 whereas

Archaea 9

Temperature

Fe

S

Al

Mg

SiCa

P

As


F1 (3333)


minus1

minus075

minus05

minus025

0

025

05

075

1

Acidilobales

UTSCG

HWCG

UnculturedEuryarchaeota

Sulfolobales

Thermoproteales K Na

Figure 4 Canonical correlation analysis showing correlative relationships between environmental factors and proportions of individualarchaeal groups Archaeal groups were shown in abbreviations and rhombus Environmental factors were shown in circle

the variations at Pond-A with higher temperature were bestexplained by PC2

Canonical correlation analysis showed that some archaealgroups strongly correlate with particular environmentalfactors such as the Sulfolobales with Al and unculturedEuryarchaeota with Na and K whereas UTSCG group wasnegatively correlated with Al (119875 lt 005) (Figure 4) Theorder Sulfolobales was also moderately correlated with S(119875 lt 010) Moreover the order Acidilobales UTSCG andHWCG groups were moderately negatively correlated withtemperature S and Mg respectively To date the elementrequirements in archaea were conducted on certain culturedspecies and little was known of the uncultured archaeaHowever the correlations between the uncultured archaealgroups and the dissolved elemental concentrations shown inthis study could givemore insights into how specific elementsaffect uncultured archaeal communities

4 Conclusion

In this study we have investigated the archaeal communitystructures of four distinct solfataric acidic hot springs inKirishima Japan The species compositions and biodiversitywere clearly different among the ponds showing differ-ent temperatures and dissolved elemental concentrationsAlthough other environmental factors also could have influ-enced on the archaeal community structures the presentstudy will be helpful in understanding the archaeal ecologyin the solfataric acidic hot springs

Conflict of Interests

The authors of this paper do not have a direct financialrelationwith the commercial identitiesmentioned herein thatmight lead to a conflict of interests

Acknowledgments

The authors thank Mr Fuchinoue and Mr Wada for theirsupport for field sampling They also thank Professor DrTaguchi andDr Kok for their valuable suggestions to improvethis paper

References

[1] N R Pace ldquoOrigin of lifemdashfacing up to the physical settingrdquoCell vol 65 no 4 pp 531ndash533 1991

[2] S L Miller and A Lazcano ldquoThe origin of lifemdashdid it occur athigh temperaturesrdquo Journal of Molecular Evolution vol 41 no6 pp 689ndash692 1995

[3] J A Baross ldquoDo the geological and geochemical records ofthe early earth support the prediction from global phylogeneticmodels of a thermophilic cenancestorrdquo in Thermophiles TheKeys to Molecular Evolution and the Origin of Life pp 3ndash18Taylor amp Francis Boca Raton Fla USA 1998

[4] N R Pace ldquoA molecular view of microbial diversity and thebiosphererdquo Science vol 276 no 5313 pp 734ndash740 1997

[5] S M Barns R E Fundyga M W Jeffries and N RPace ldquoRemarkable archaeal diversity detected in a YellowstoneNational Park hot spring environmentrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 91 no 5 pp 1609ndash1613 1994

[6] S M Barns C F Delwiche J D Palmer and N R PaceldquoPerspectives on archaeal diversity thermophily and mono-phyly from environmental rRNA sequencesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 93 no 17 pp 9188ndash9193 1996

[7] P Hugenholtz C Pitulle K L Hershberger and N R PaceldquoNovel division level bacterial diversity in a Yellowstone hotspringrdquo Journal of Bacteriology vol 180 no 2 pp 366ndash3761998

10 Archaea

[8] A L Reysenbach M Ehringer and K Hershberger ldquoMicrobialdiversity at 83∘C in Calcite Springs Yellowstone National Parkanother environment where the Aquificales and ldquoKorarchaeotardquocoexistrdquo Extremophiles vol 4 no 1 pp 61ndash67 2000

[9] C E Blank S L Cady and N R Pace ldquoMicrobial compositionof near-boiling silica-depositing thermal springs throughoutYellowstone National Parkrdquo Applied and Environmental Micro-biology vol 68 no 10 pp 5123ndash5135 2002

[10] D R Meyer-Dombard E L Shock and J P Amend ldquoArchaealand bacterial communities in geochemically diverse hot springsof YellowstoneNational Park USArdquoGeobiology vol 3 no 3 pp211ndash227 2005

[11] A A Perevalova T V Kolganova N K Birkeland C SchleperE A Bonch-Osmolovskaya and A V Lebedinsky ldquoDistribu-tion of Crenarchaeota representatives in terrestrial hot springsof Russia and IcelandrdquoApplied andEnvironmentalMicrobiologyvol 74 no 24 pp 7620ndash7628 2008

[12] N P Burton and P R Norris ldquoMicrobiology of acidic geother-mal springs of Montserrat environmental rDNA analysisrdquoExtremophiles vol 4 no 5 pp 315ndash320 2000

[13] L M Stout R E Blake J P Greenwood A M Martini and EC Rose ldquoMicrobial diversity of boron-rich volcanic hot springsof St Lucia Lesser Antillesrdquo FEMS Microbiology Ecology vol70 no 3 pp 402ndash412 2009

[14] T Kvist B K Ahring and PWestermann ldquoArchaeal diversity inIcelandic hot springsrdquo FEMS Microbiology Ecology vol 59 no1 pp 71ndash80 2007

[15] K Takai and Y Sako ldquoA molecular view of archaeal diversityin marine and terrestrial hot water environmentsrdquo FEMSMicrobiology Ecology vol 28 no 2 pp 177ndash188 1999

[16] S Kato T Itoh and A Yamagishi ldquoArchaeal diversity in aterrestrial acidic spring field revealed by a novel PCR primertargeting archaeal 16S rRNA genesrdquo FEMSMicrobiology Lettersvol 319 no 1 pp 34ndash43 2011

[17] T Kvist A Mengewein S Manzei B K Ahring and PWestermann ldquoDiversity of thermophilic and non-thermophiliccrenarchaeota at 80∘Crdquo FEMSMicrobiology Letters vol 244 no1 pp 61ndash68 2005

[18] P Kanokratana S Chanapan K Pootanakit and L Eurwilai-chitr ldquoDiversity and abundance of Bacteria and Archaea inthe Bor Khlueng Hot Spring in Thailandrdquo Journal of BasicMicrobiology vol 44 no 6 pp 430ndash444 2004

[19] A M Childs B W Mountain R OrsquoToole and M B StottldquoRelating microbial community and physicochemical parame-ters of a hot spring champagne pool wai-o-tapuNewZealandrdquoGeomicrobiology Journal vol 25 no 7-8 pp 441ndash453 2008

[20] Z Q Song J Q Chen H C Jiang et al ldquoDiversity ofCrenarchaeota in terrestrial hot springs in Tengchong ChinardquoExtremophiles vol 14 no 3 pp 287ndash296 2010

[21] K Goko ldquoStructure and hydrology of the Origi field WestKirishima geothermal area Kyushu Japanrdquo Geothermics vol29 no 2 pp 127ndash149 2000

[22] T Tsuyuki ldquoGeological study of hot springs in Kyushu Japan(5) Some hot springs in the Kagoshima graben with specialreferences to thermal water reservoir Reports of the Faculty ofScience Kagoshima Universityrdquo Earth Science and Biology vol2 pp 85ndash101 1969 (Japanese)

[23] R Imura T Kobayashi and C C S Senta Geological Map ofKirishimaVolcano (Japanese) Geological Survey of Japan 2001

[24] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysis

using maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 pp 2731ndash2739 2011

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

[26] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[27] S H Hurlbert ldquoThe nonconcept of species diversity a critiqueand alternative parametersrdquo Ecology vol 52 pp 577ndash586 1971

[28] G Stirling and B Wilsey ldquoEmpirical relationships betweenspecies richness evenness and proportional diversityrdquo Amer-ican Naturalist vol 158 no 3 pp 286ndash299 2001

[29] C E Shannon W Weaver R E Blahut and B Hajek TheMathematical Theory of Communication University of IllinoisPress Urbana Ill USA 1949

[30] E H Simpson ldquoMeasurement of diversityrdquoNature vol 163 no4148 article 688 1949

[31] R K Colwell 2006 EstimateS Statistical estimation ofspecies richness and shared species from samples Version 8httppurloclcorgestimates

[32] E C Pielou ldquoAssociation tests versus homogeneity tests theiruse in subdividing quadrats into groupsrdquo Vegetatio vol 18 no1ndash6 pp 4ndash18 1969

[33] AChao ldquoEstimating the population size for capturemdashrecapturedata with unequal catchabilityrdquo Biometrics vol 43 no 4 pp783ndash791 1987

[34] A Chao W H Hwang Y C Chen and C Y Kuo ldquoEstimatingthe number of shared species in two communitiesrdquo StatisticaSinica vol 10 no 1 pp 227ndash246 2000

[35] I J Good ldquoThe population frequencies of species and theestimation of population parametersrdquo Biometrika vol 40 pp237ndash264 1953

[36] D R Singleton M A Furlong S L Rathbun and WB Whitman ldquoQuantitative comparisons of 16S rRNA genesequence libraries from environmental samplesrdquo Applied andEnvironmental Microbiology vol 67 no 9 pp 4374ndash4376 2001

[37] H Kimura K Mori T Tashiro et al ldquoCulture-independentestimation of optimal and maximum growth temperatures ofarchaea in subsurface habitats based on the G plus C content in16S sequencesrdquo Geomicrobiology Journal vol 27 no 2 pp 114ndash122 2010

[38] T Itoh K I Suzuki P C Sanchez and T Nakase ldquoCaldivirgamaquilingensis gen nov sp nov a new genus of rod- shapedcrenarchaeote isolated from a hot spring in the PhilippinesrdquoInternational Journal of Systematic Bacteriology vol 49 no 3pp 1157ndash1163 1999

[39] T Itoh K I Suzuki and T Nakase ldquoVulcanisaeta distributagen nov sp nov and Vulcanisaeta souniana sp nov novelhyperthermophilic rod-shaped crenarchaeotes isolated fromhot springs in Japanrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 4 pp 1097ndash1104 2002

[40] W Zillig K O Stetter and S Wunderl ldquoThe Sulfolobus-ldquoCaldariellardquo group taxonomy on the basis of the structure ofDNA-dependent RNA polymerasesrdquo Archives of Microbiologyvol 125 no 3 pp 259ndash269 1980

[41] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus of

Archaea 11

aerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[42] A Segerer A Neuner J K Kristjansson and K O StetterldquoAcidianus infernus gen nov sp nov and Acidianus brierleyicomb nov facultatively aerobic extremely acidophilic ther-mophilic sulfur-metabolizing archaebacteriardquo InternationalJournal of Systematic Bacteriology vol 36 no 4 pp 559ndash5641986

[43] T Itoh K Suzuki P C Sanchez and T Nakase ldquoCaldisphaeralagunensis gen nov sp nov a novel thermoacidophilic cre-narchaeote isolated from a hot spring at Mt Maquiling Philip-pinesrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 53 no 4 pp 1149ndash1154 2003

[44] H Huber and K O Stetter ldquoOrder III Sulfolobalesrdquo in BergeyrsquosManual of Systematic Bacteriology J T Staley M P Bryant EN Pfenning and J G Holt Eds vol 1 pp 198ndash210 Williamsand Wilkins Baltimore Md USA 2nd edition 2001

[45] M O Schrenk D S Kelley J R Delaney and J A BarossldquoIncidence and diversity of microorganisms within the walls ofan active deep-sea sulfide chimneyrdquoApplied and EnvironmentalMicrobiology vol 69 no 6 pp 3580ndash3592 2003

[46] T Nunoura H Hirayama H Takami et al ldquoGenetic and func-tional properties of uncultivated thermophilic crenarchaeotesfrom a subsurface gold mine as revealed by analysis of genomefragmentsrdquo Environmental Microbiology vol 7 no 12 pp 1967ndash1984 2005

[47] T Itoh K I Suzuki and T Nakase ldquoThermocladium modestiusgen nov sp nov a new genus of rod-shaped extremelythermophilic crenarchaeoterdquo International Journal of SystematicBacteriology vol 48 no 3 pp 879ndash887 1998

[48] E S Boyd R A Jackson G Encarnacion et al ldquoIsolationcharacterization and ecology of sulfur-respiring Crenarchaeainhabiting acid-sulfate-chloride-containing geothermal springsin Yellowstone National Parkrdquo Applied and EnvironmentalMicrobiology vol 73 no 20 pp 6669ndash6677 2007

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

[50] F Inagaki K Takai H Hirayama Y Yamato K H Nealsonand K Horikoshi ldquoDistribution and phylogenetic diversity ofthe subsurface microbial community in a Japanese epithermalgold minerdquo Extremophiles vol 7 no 4 pp 307ndash317 2003

[51] C Vetriani H W Jannasch B J Macgregor D A Stahl and AL Reysenbach ldquoPopulation structure and phylogenetic charac-terization of marine benthic Archaea in deep-sea sedimentsrdquoApplied and Environmental Microbiology vol 65 no 10 pp4375ndash4384 1999

[52] K Takai D P Moser M DeFlaun T C Onstott and J KFredrickson ldquoArchaeal diversity in waters from deep SouthAfrican gold minesrdquo Applied and Environmental Microbiologyvol 67 no 12 pp 5750ndash5760 2001

[53] A Teske K U 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

[54] 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

[55] G Jurgens and A Saano ldquoDiversity of soil Archaea in borealforest before and after clear-cutting and prescribed burningrdquoFEMS Microbiology Ecology vol 29 no 2 pp 205ndash213 1999

[56] M I Prokofeva N A Kostrikina T V Kolganova et alldquoIsolation of the anaerobic thermoacidophilic crenarchaeoteAcidilobus saccharovorans sp nov and proposal of Acidilobalesord nov including Acidilobaceae fam nov and Caldisphaer-aceae fam novrdquo International Journal of Systematic and Evolu-tionary Microbiology vol 59 no 12 pp 3116ndash3122 2009

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

Table 1 Characteristics of sampling sites and ponds

Pond-A Pond-B Pond-C Pond-DTemp (∘C) 93 66 88 67pH 26 20 24 23Concentration (mg Lminus1)composition ()Fe 3889 23 1149 51 9630 5 2718 8S 6632 40 7028 31 5976 30 6190 18Al 4336 26 2879 13 1457 7 2021 1Mg 8674 5 4677 2 0001 0 4335 13Si 4788 3 4552 2 1039 53 1484 44Ca 5481 3 1088 0 7498 4 3926 12P 2850 0 4711 0 1265 1 1266 0Na 0001 0 0001 0 0001 0 8442 2K 0001 0 0001 0 0001 0 7384 2As 1079 0 1137 0 0879 0 0856 0Total 1679 100 2248 100 1975 100 3401 100Latitude (N) 31∘54101584037710158401015840 31∘54101584052410158401015840 31∘55101584005010158401015840 31∘55101584004510158401015840

Longitude (E) 130∘49101584000610158401015840 130∘48101584050310158401015840 130∘48101584041110158401015840 130∘48101584041010158401015840

Altitude (m) 759 842 884 885Color of sediments Light brown Light brown Gray GrayDetection limit is 0001mg Lminus1

Japan

N

Ishizaka River

500

Landslide

Spring

Pond-CPond-D

Pond-B

Pond-A

50131∘E

Kagoshima graben

Kirishima volcano32∘N

(Km)

(m)

Figure 1 Map of sampling site in Kirishima geothermal area Japan

geothermal area in Japan (KirishimaNational Park) (Figure 1Table 1) where the extensive volcanic activity occurred fromthe Pleistocene epoch to the present depositing a thick pileof volcanic rocks [21] Kirishima volcano one of the largestQuaternary volcanoes in Japan belongs to the northern partof the Kagoshima graben a volcano-tectonic depression [22]caused by the subduction of the Philippine Sea plate Thisvolcano occupies an area of about 20 kmtimes 30 km elongated inthe northwest to southeast direction and contains more than20 small volcanoes [23]

Sampling location within Kirishima geothermal area isa private land therefore people are usually not allowed totrespass on this area We got permission to take hot springwater soil and various other native samples in this area froman owner of the landThere are many hot springs and muddyponds showing a variety of temperatures and elementalcompositions

Muddy water sample was collected into sterile 100mLglass bottle at each pond Temperature and pH of thesamples were measured at each sampling site A part of eachsample was filtered using 022120583m membrane filter (AsahiGlass) and was subjected to analysis of dissolved elementalconcentrations using the inductively coupled plasma opticalemission spectroscopy (ICPS-7000 ver2 Shimadzu) In thepresent study we selected four ponds displaying a range oftemperatures and dissolved elemental compositions for thearchaeal community analysis

22 16119878 rRNA Gene Clone Libraries and Sequencing Theenvironmental DNA was extracted from 5 to 10 g of eachmuddy water sample using the UltraClean Soil DNA KitMega Prep (Mo Bio Laboratories) according to the manu-facturerrsquos instructions The precipitated DNA was purified

Archaea 3












4 Archaea











Order Acidilobales





(=ST2A1-31)





(=ST16A1-1ST15A1-1)




(=ST15A1-6)





Archaea 5

Table 2 Continued





(=ST16A1-2ST15A1-34)



Euryarchaeal groups



(=ST15A1-8)Total 106 112 109 104



158 366 11 0659 160 170 095 215


220 582 20 0735 233 210 091 216







6 Archaea












Archaea 7




88100

100

100

99

100

98

100

68100

100

100

73

99

85

99

97

99

100

99

94

99

75

98

82

97

88

100

100

96

99

79

95

88

95

89

54

5299

90

94

99

10092

95

87

54

84

70

57

89

95

99

78

70

66

61

74 55

88

MHVG-I

Korarchaeota

Euryarchaeota

UTRCG

Thaumarchaeota

UTSCG

Thermoproteales

Acidilobales

ST2A1-8 (20) (AB753275)

ST16A1-6 (6) (AB753297)

ST15A1-6 (6) (AB753291)

ST2A1-2 (4) (AB753272)

ST8A1-12 (92) (AB753286)

ST8A1-52 (1) (AB753288)

ST8A1-57 (1) (AB753289)

ST2A1-16 (2) (AB753279)

ST2A1-3 (3) (AB753273)

ST2A1-14 (7) (AB753277)

ST2A1-43 (1) (AB753284)

ST2A1-32 (6) (AB753283)

ST2A1-5 (39) (AB753274)

ST2A1-27 (2) (AB753281)

ST2A1-9 (10) (AB753276)

ST2A1-31 (2) (AB753282)

ST16A1-87 (1) (AB753298)

ST15A2-137 (2) (AB753296)

ST15A1-26 (1) (AB753293)

ST2A1-52 (2) (AB753285)

ST15A1-32 (1) (AB753295)

ST16A1-50 (1) (AB755806)

ST8A1-40 (7) (AB753287)

ST2A1-15 (1) (AB753278)

ST15A1-7 (21) (AB755800)

ST8A1-8 (5) (AB755799)

ST16A1-20 (13) (AB755803)

ST15A1-3 (2) (AB753290)

ST15A1-1 (52) (AB755801)

ST16A1-1 (42) (AB755802)

ST2A1-25 (13) (AB753280)ST15A1-34 (2) (AB755804)

ST16A1-2 (46) (AB755805)


ST15A1-8 (17) (AB753292)

005














HWCG VII

UTSCG II





HWCG V

HWCG VI

HWCG IMCG

SAGMCGSCG

FSCG

HWCG II (UCIII)














DSAG (MBGB)








Sulfolobales


8 Archaea



Shannon

Fe

S

AlMg

Si

Ca

P

As

minus1

minus075

minus05

minus025

0

025

05

075

1



F1 (5566)

Temperature

K Na

(a)

minus3

minus2

minus1

0

1

2

3



F1 (5566)

Pond-B

Pond-A

Pond-C Pond-D

(b)





Archaea 9

Temperature

Fe

S

Al

Mg

SiCa

P

As


F1 (3333)


minus1

minus075

minus05

minus025

0

025

05

075

1

Acidilobales

UTSCG

HWCG


Sulfolobales





4 Conclusion




Acknowledgments


References








10 Archaea




































Archaea 11
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 3












4 Archaea











Order Acidilobales





(=ST2A1-31)





(=ST16A1-1ST15A1-1)




(=ST15A1-6)





Archaea 5

Table 2 Continued





(=ST16A1-2ST15A1-34)



Euryarchaeal groups



(=ST15A1-8)Total 106 112 109 104



158 366 11 0659 160 170 095 215


220 582 20 0735 233 210 091 216







6 Archaea












Archaea 7




88100

100

100

99

100

98

100

68100

100

100

73

99

85

99

97

99

100

99

94

99

75

98

82

97

88

100

100

96

99

79

95

88

95

89

54

5299

90

94

99

10092

95

87

54

84

70

57

89

95

99

78

70

66

61

74 55

88

MHVG-I

Korarchaeota

Euryarchaeota

UTRCG

Thaumarchaeota

UTSCG

Thermoproteales

Acidilobales

ST2A1-8 (20) (AB753275)

ST16A1-6 (6) (AB753297)

ST15A1-6 (6) (AB753291)

ST2A1-2 (4) (AB753272)

ST8A1-12 (92) (AB753286)

ST8A1-52 (1) (AB753288)

ST8A1-57 (1) (AB753289)

ST2A1-16 (2) (AB753279)

ST2A1-3 (3) (AB753273)

ST2A1-14 (7) (AB753277)

ST2A1-43 (1) (AB753284)

ST2A1-32 (6) (AB753283)

ST2A1-5 (39) (AB753274)

ST2A1-27 (2) (AB753281)

ST2A1-9 (10) (AB753276)

ST2A1-31 (2) (AB753282)

ST16A1-87 (1) (AB753298)

ST15A2-137 (2) (AB753296)

ST15A1-26 (1) (AB753293)

ST2A1-52 (2) (AB753285)

ST15A1-32 (1) (AB753295)

ST16A1-50 (1) (AB755806)

ST8A1-40 (7) (AB753287)

ST2A1-15 (1) (AB753278)

ST15A1-7 (21) (AB755800)

ST8A1-8 (5) (AB755799)

ST16A1-20 (13) (AB755803)

ST15A1-3 (2) (AB753290)

ST15A1-1 (52) (AB755801)

ST16A1-1 (42) (AB755802)

ST2A1-25 (13) (AB753280)ST15A1-34 (2) (AB755804)

ST16A1-2 (46) (AB755805)


ST15A1-8 (17) (AB753292)

005














HWCG VII

UTSCG II





HWCG V

HWCG VI

HWCG IMCG

SAGMCGSCG

FSCG

HWCG II (UCIII)














DSAG (MBGB)








Sulfolobales


8 Archaea



Shannon

Fe

S

AlMg

Si

Ca

P

As

minus1

minus075

minus05

minus025

0

025

05

075

1



F1 (5566)

Temperature

K Na

(a)

minus3

minus2

minus1

0

1

2

3



F1 (5566)

Pond-B

Pond-A

Pond-C Pond-D

(b)





Archaea 9

Temperature

Fe

S

Al

Mg

SiCa

P

As


F1 (3333)


minus1

minus075

minus05

minus025

0

025

05

075

1

Acidilobales

UTSCG

HWCG


Sulfolobales





4 Conclusion




Acknowledgments


References








10 Archaea




































Archaea 11
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Archaea











Order Acidilobales





(=ST2A1-31)





(=ST16A1-1ST15A1-1)




(=ST15A1-6)





Archaea 5

Table 2 Continued





(=ST16A1-2ST15A1-34)



Euryarchaeal groups



(=ST15A1-8)Total 106 112 109 104



158 366 11 0659 160 170 095 215


220 582 20 0735 233 210 091 216







6 Archaea












Archaea 7




88100

100

100

99

100

98

100

68100

100

100

73

99

85

99

97

99

100

99

94

99

75

98

82

97

88

100

100

96

99

79

95

88

95

89

54

5299

90

94

99

10092

95

87

54

84

70

57

89

95

99

78

70

66

61

74 55

88

MHVG-I

Korarchaeota

Euryarchaeota

UTRCG

Thaumarchaeota

UTSCG

Thermoproteales

Acidilobales

ST2A1-8 (20) (AB753275)

ST16A1-6 (6) (AB753297)

ST15A1-6 (6) (AB753291)

ST2A1-2 (4) (AB753272)

ST8A1-12 (92) (AB753286)

ST8A1-52 (1) (AB753288)

ST8A1-57 (1) (AB753289)

ST2A1-16 (2) (AB753279)

ST2A1-3 (3) (AB753273)

ST2A1-14 (7) (AB753277)

ST2A1-43 (1) (AB753284)

ST2A1-32 (6) (AB753283)

ST2A1-5 (39) (AB753274)

ST2A1-27 (2) (AB753281)

ST2A1-9 (10) (AB753276)

ST2A1-31 (2) (AB753282)

ST16A1-87 (1) (AB753298)

ST15A2-137 (2) (AB753296)

ST15A1-26 (1) (AB753293)

ST2A1-52 (2) (AB753285)

ST15A1-32 (1) (AB753295)

ST16A1-50 (1) (AB755806)

ST8A1-40 (7) (AB753287)

ST2A1-15 (1) (AB753278)

ST15A1-7 (21) (AB755800)

ST8A1-8 (5) (AB755799)

ST16A1-20 (13) (AB755803)

ST15A1-3 (2) (AB753290)

ST15A1-1 (52) (AB755801)

ST16A1-1 (42) (AB755802)

ST2A1-25 (13) (AB753280)ST15A1-34 (2) (AB755804)

ST16A1-2 (46) (AB755805)


ST15A1-8 (17) (AB753292)

005














HWCG VII

UTSCG II





HWCG V

HWCG VI

HWCG IMCG

SAGMCGSCG

FSCG

HWCG II (UCIII)














DSAG (MBGB)








Sulfolobales


8 Archaea



Shannon

Fe

S

AlMg

Si

Ca

P

As

minus1

minus075

minus05

minus025

0

025

05

075

1



F1 (5566)

Temperature

K Na

(a)

minus3

minus2

minus1

0

1

2

3



F1 (5566)

Pond-B

Pond-A

Pond-C Pond-D

(b)





Archaea 9

Temperature

Fe

S

Al

Mg

SiCa

P

As


F1 (3333)


minus1

minus075

minus05

minus025

0

025

05

075

1

Acidilobales

UTSCG

HWCG


Sulfolobales





4 Conclusion




Acknowledgments


References








10 Archaea




































Archaea 11
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 5

Table 2 Continued





(=ST16A1-2ST15A1-34)



Euryarchaeal groups



(=ST15A1-8)Total 106 112 109 104



158 366 11 0659 160 170 095 215


220 582 20 0735 233 210 091 216







6 Archaea












Archaea 7




88100

100

100

99

100

98

100

68100

100

100

73

99

85

99

97

99

100

99

94

99

75

98

82

97

88

100

100

96

99

79

95

88

95

89

54

5299

90

94

99

10092

95

87

54

84

70

57

89

95

99

78

70

66

61

74 55

88

MHVG-I

Korarchaeota

Euryarchaeota

UTRCG

Thaumarchaeota

UTSCG

Thermoproteales

Acidilobales

ST2A1-8 (20) (AB753275)

ST16A1-6 (6) (AB753297)

ST15A1-6 (6) (AB753291)

ST2A1-2 (4) (AB753272)

ST8A1-12 (92) (AB753286)

ST8A1-52 (1) (AB753288)

ST8A1-57 (1) (AB753289)

ST2A1-16 (2) (AB753279)

ST2A1-3 (3) (AB753273)

ST2A1-14 (7) (AB753277)

ST2A1-43 (1) (AB753284)

ST2A1-32 (6) (AB753283)

ST2A1-5 (39) (AB753274)

ST2A1-27 (2) (AB753281)

ST2A1-9 (10) (AB753276)

ST2A1-31 (2) (AB753282)

ST16A1-87 (1) (AB753298)

ST15A2-137 (2) (AB753296)

ST15A1-26 (1) (AB753293)

ST2A1-52 (2) (AB753285)

ST15A1-32 (1) (AB753295)

ST16A1-50 (1) (AB755806)

ST8A1-40 (7) (AB753287)

ST2A1-15 (1) (AB753278)

ST15A1-7 (21) (AB755800)

ST8A1-8 (5) (AB755799)

ST16A1-20 (13) (AB755803)

ST15A1-3 (2) (AB753290)

ST15A1-1 (52) (AB755801)

ST16A1-1 (42) (AB755802)

ST2A1-25 (13) (AB753280)ST15A1-34 (2) (AB755804)

ST16A1-2 (46) (AB755805)


ST15A1-8 (17) (AB753292)

005














HWCG VII

UTSCG II





HWCG V

HWCG VI

HWCG IMCG

SAGMCGSCG

FSCG

HWCG II (UCIII)














DSAG (MBGB)








Sulfolobales


8 Archaea



Shannon

Fe

S

AlMg

Si

Ca

P

As

minus1

minus075

minus05

minus025

0

025

05

075

1



F1 (5566)

Temperature

K Na

(a)

minus3

minus2

minus1

0

1

2

3



F1 (5566)

Pond-B

Pond-A

Pond-C Pond-D

(b)





Archaea 9

Temperature

Fe

S

Al

Mg

SiCa

P

As


F1 (3333)


minus1

minus075

minus05

minus025

0

025

05

075

1

Acidilobales

UTSCG

HWCG


Sulfolobales





4 Conclusion




Acknowledgments


References








10 Archaea




































Archaea 11
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 Archaea












Archaea 7




88100

100

100

99

100

98

100

68100

100

100

73

99

85

99

97

99

100

99

94

99

75

98

82

97

88

100

100

96

99

79

95

88

95

89

54

5299

90

94

99

10092

95

87

54

84

70

57

89

95

99

78

70

66

61

74 55

88

MHVG-I

Korarchaeota

Euryarchaeota

UTRCG

Thaumarchaeota

UTSCG

Thermoproteales

Acidilobales

ST2A1-8 (20) (AB753275)

ST16A1-6 (6) (AB753297)

ST15A1-6 (6) (AB753291)

ST2A1-2 (4) (AB753272)

ST8A1-12 (92) (AB753286)

ST8A1-52 (1) (AB753288)

ST8A1-57 (1) (AB753289)

ST2A1-16 (2) (AB753279)

ST2A1-3 (3) (AB753273)

ST2A1-14 (7) (AB753277)

ST2A1-43 (1) (AB753284)

ST2A1-32 (6) (AB753283)

ST2A1-5 (39) (AB753274)

ST2A1-27 (2) (AB753281)

ST2A1-9 (10) (AB753276)

ST2A1-31 (2) (AB753282)

ST16A1-87 (1) (AB753298)

ST15A2-137 (2) (AB753296)

ST15A1-26 (1) (AB753293)

ST2A1-52 (2) (AB753285)

ST15A1-32 (1) (AB753295)

ST16A1-50 (1) (AB755806)

ST8A1-40 (7) (AB753287)

ST2A1-15 (1) (AB753278)

ST15A1-7 (21) (AB755800)

ST8A1-8 (5) (AB755799)

ST16A1-20 (13) (AB755803)

ST15A1-3 (2) (AB753290)

ST15A1-1 (52) (AB755801)

ST16A1-1 (42) (AB755802)

ST2A1-25 (13) (AB753280)ST15A1-34 (2) (AB755804)

ST16A1-2 (46) (AB755805)


ST15A1-8 (17) (AB753292)

005














HWCG VII

UTSCG II





HWCG V

HWCG VI

HWCG IMCG

SAGMCGSCG

FSCG

HWCG II (UCIII)














DSAG (MBGB)








Sulfolobales


8 Archaea



Shannon

Fe

S

AlMg

Si

Ca

P

As

minus1

minus075

minus05

minus025

0

025

05

075

1



F1 (5566)

Temperature

K Na

(a)

minus3

minus2

minus1

0

1

2

3



F1 (5566)

Pond-B

Pond-A

Pond-C Pond-D

(b)





Archaea 9

Temperature

Fe

S

Al

Mg

SiCa

P

As


F1 (3333)


minus1

minus075

minus05

minus025

0

025

05

075

1

Acidilobales

UTSCG

HWCG


Sulfolobales





4 Conclusion




Acknowledgments


References








10 Archaea




































Archaea 11
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 7




88100

100

100

99

100

98

100

68100

100

100

73

99

85

99

97

99

100

99

94

99

75

98

82

97

88

100

100

96

99

79

95

88

95

89

54

5299

90

94

99

10092

95

87

54

84

70

57

89

95

99

78

70

66

61

74 55

88

MHVG-I

Korarchaeota

Euryarchaeota

UTRCG

Thaumarchaeota

UTSCG

Thermoproteales

Acidilobales

ST2A1-8 (20) (AB753275)

ST16A1-6 (6) (AB753297)

ST15A1-6 (6) (AB753291)

ST2A1-2 (4) (AB753272)

ST8A1-12 (92) (AB753286)

ST8A1-52 (1) (AB753288)

ST8A1-57 (1) (AB753289)

ST2A1-16 (2) (AB753279)

ST2A1-3 (3) (AB753273)

ST2A1-14 (7) (AB753277)

ST2A1-43 (1) (AB753284)

ST2A1-32 (6) (AB753283)

ST2A1-5 (39) (AB753274)

ST2A1-27 (2) (AB753281)

ST2A1-9 (10) (AB753276)

ST2A1-31 (2) (AB753282)

ST16A1-87 (1) (AB753298)

ST15A2-137 (2) (AB753296)

ST15A1-26 (1) (AB753293)

ST2A1-52 (2) (AB753285)

ST15A1-32 (1) (AB753295)

ST16A1-50 (1) (AB755806)

ST8A1-40 (7) (AB753287)

ST2A1-15 (1) (AB753278)

ST15A1-7 (21) (AB755800)

ST8A1-8 (5) (AB755799)

ST16A1-20 (13) (AB755803)

ST15A1-3 (2) (AB753290)

ST15A1-1 (52) (AB755801)

ST16A1-1 (42) (AB755802)

ST2A1-25 (13) (AB753280)ST15A1-34 (2) (AB755804)

ST16A1-2 (46) (AB755805)


ST15A1-8 (17) (AB753292)

005














HWCG VII

UTSCG II





HWCG V

HWCG VI

HWCG IMCG

SAGMCGSCG

FSCG

HWCG II (UCIII)














DSAG (MBGB)








Sulfolobales


8 Archaea



Shannon

Fe

S

AlMg

Si

Ca

P

As

minus1

minus075

minus05

minus025

0

025

05

075

1



F1 (5566)

Temperature

K Na

(a)

minus3

minus2

minus1

0

1

2

3



F1 (5566)

Pond-B

Pond-A

Pond-C Pond-D

(b)





Archaea 9

Temperature

Fe

S

Al

Mg

SiCa

P

As


F1 (3333)


minus1

minus075

minus05

minus025

0

025

05

075

1

Acidilobales

UTSCG

HWCG


Sulfolobales





4 Conclusion




Acknowledgments


References








10 Archaea




































Archaea 11
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

8 Archaea



Shannon

Fe

S

AlMg

Si

Ca

P

As

minus1

minus075

minus05

minus025

0

025

05

075

1



F1 (5566)

Temperature

K Na

(a)

minus3

minus2

minus1

0

1

2

3



F1 (5566)

Pond-B

Pond-A

Pond-C Pond-D

(b)





Archaea 9

Temperature

Fe

S

Al

Mg

SiCa

P

As


F1 (3333)


minus1

minus075

minus05

minus025

0

025

05

075

1

Acidilobales

UTSCG

HWCG


Sulfolobales





4 Conclusion




Acknowledgments


References








10 Archaea




































Archaea 11
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 9

Temperature

Fe

S

Al

Mg

SiCa

P

As


F1 (3333)


minus1

minus075

minus05

minus025

0

025

05

075

1

Acidilobales

UTSCG

HWCG


Sulfolobales





4 Conclusion




Acknowledgments


References








10 Archaea




































Archaea 11
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

10 Archaea




































Archaea 11
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 11
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Archaeal Community Structures in the ...downloads.hindawi.com/journals/archaea/2013/723871.pdf · Acidic spring clone HOS A (AB ). Archaea T : Continued. Phylotypes