Cultureindependent characterization of a novel ... - Wiley Online Library

JOURNAL OF GEOPHYSICAL RESEARCH, VOL. 113, B08S06, doi:10.1029/2007JB005477, 2008

Culture-independent characterization of a novel microbial community at a hydrothermal vent at Brothers volcano, Kermadec arc, New Zealand M. B. Stott,1 J. A. Saito,2 M. A. Crowe,1 P. F. Dunfield,1 S. Hou,2 E. Nakasone,2 C. J. Daughney,1 A. V. Smirnova,1 B. W. Mountain,1 K. Takai,3 and M. Alam2 Received 31 October 2007; revised 22 April 2008; accepted 25 June 2008; published 7 August 2008.

[1] The bacterial and archaeal diversity of a hydrothermal vent microbial community at

Brothers volcano situated in the Kermadec arc, 400 km off the north coast of New Zealand, was examined using culture-independent molecular analysis. An unusual microbial community was detected with only 1% and 40% of the bacterial phylotypes exhibiting >92% small subunit (SSU) rRNA gene sequence similarity with cultivated and noncultivated microbes, respectively. Of the 29 bacterial representative phylotypes, over one third of the SSU rRNA gene sequences retrieved belonged to uncultivated candidate divisions including OP1, OP3, OP5, OP8, OD1, and OP11. All archaeal phylotypes belonged to the phylum Euryarchaeota in the uncultivated groups deep hydrothermal vent euryarchaeotal (DHVE) I and II or to the phylum Korarchaeota. Like the bacterial clone library, only a small proportion of archaeal SSU rRNA gene sequences (2% and 20%) displayed >92% sequence identity with any archaeal isolates or noncultivated microbes, respectively. Although the bacterial phylotypes detected were phylogenetically most similar to microbial communities detected in methane, hydrocarbon, and carbon dioxide-based hydrothermal and seep environments, no phylotypes directly associated with anaerobic methane oxidation and mcrA activity could be detected. The geochemical composition of the vent fluids at the Brothers-lower cone sample site is unusual and we suggest that it may play a prominent role in the species selection of this microbial community. Citation: Stott, M. B., et al. (2008), Culture-independent characterization of a novel microbial community at a hydrothermal vent at Brothers volcano, Kermadec arc, New Zealand, J. Geophys. Res., 113, B08S06, doi:10.1029/2007JB005477.

1. Introduction [2] Microbial communities inhabiting hydrothermal systems form the basis of food webs in deep-sea environments below the phototrophic zone. With the advent of cultureindependent techniques, the composition of microbial communities in hydrothermal vent environments have been explored in great detail and consequently our understanding of the diversity microbial ecosystems in hydrothermal environments has advanced [Emerson et al., 2007; Brazelton et al., 2006; Huber et al., 2006; Inagaki et al., 2006a; Nakagawa et al., 2005, 2006; Perner et al., 2007; Takai et al., 2001, 2004; Takai and Horikoshi, 1999; Teske et al., 2002]. [3] Our current knowledge of the microbial diversity of hydrothermal systems has been established primarily from 1

GNS Science, Extremophile Research Group, Taupo, New Zealand. Department of Microbiology, University of Hawaii, Honolulu, Hawaii, USA. 3 Subground Animalcule Retrieval Program, Japan Agency for MarineEarth Science and Technology, Yokosuka, Japan. 2

Copyright 2008 by the American Geophysical Union. 0148-0227/08/2007JB005477$09.00

studies of mid-ocean spreading ridges (MOR) [Huber et al., 2006; Reysenbach et al., 2000], back-arc basins (BAB) [Nakagawa et al., 2005; Takai et al., 2006], and hot spot seamounts [Emerson et al., 2007]. Microbial communities in these systems have been detected on rock surfaces, sediments, and chimney structures [Brazelton et al., 2006; Emerson et al., 2007; Takai et al., 2001] and as symbiont populations of vent macrofauna [Brazelton et al., 2006; Campbell et al., 2001; Goffredi et al., 2004]. Commonly detected species include Epsilonproteobacteria, Thermococcus, Archaeoglobus, Thiomicrospira, Aquificales, and methanogens such as Methanococcales and Methanosarcinales [Huber et al., 2003; Nakagawa et al., 2005; Reysenbach et al., 2000; Takai et al., 2006; Karl, 1995]. The diversity of these vent communities is strongly correlated with the physical and geochemical conditions associated with the vent fluids [Nakagawa et al., 2005] and result largely from subsurface interaction of seawater with magmatic basalt through a permeable crust. While the majority of microbial community analysis has been conducted on these types of hydrothermal environments, approximately 22,000 km of oceanic volcanism occurs where oceanic tectonic plates subduct, and is known as intraoceanic subducting arc (ISA) volcanism [de Ronde et al., 2003]. Vent

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STOTT ET AL.: MICROBIAL ECOLOGY OF BROTHERS SEAMOUNT

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Figure 1. Brothers cone sample site showing sulfur and biofilm encrusted dacite. Fluids emanating from the hydrothermal vent were measured at 67°C. fluid concentrations of dissolved CO2, H2S, and Fe at ISA submarine volcanoes can exceed those for MOR hydrothermal vents ecosystems by 5– 10 times or more [Massoth et al., 2003; Lupton et al., 2008] and presents the possibility of facilitating the generation of novel microbial communities. [4] The Kermadec arc is an ISA system that strikes a NE line from the North Island of New Zealand and contains as many as 82 major submarine volcanoes, of which 70% are actively venting [de Ronde et al., 2005]. One of the more active submarine volcanoes, Brothers volcano, is located approximately 400 km NE of New Zealand and was the subject of four manned dives with the submersible Shinkai 6500 in late 2004. Brothers volcano has two explored hydrothermally active zones, the geology and geochemistry of which is detailed elsewhere [de Ronde et al., 2005; C. E. J. de Ronde et al., Submarine hydrothermal activity and gold-rich mineralization at Brothers volcano, Kermadec arc, New Zealand, unpublished manuscript, 2008]. The first hydrothermally active area, known as Brothers NW (179.06°E/ 34.86°S) is perched midway up the caldera wall, has an extensive field of black smoking chimneys, and is the subject of a complementary microbial ecology investigation (K. Takai et al., Variability in microbial communities in black smoker chimneys at the NW caldera vent field of the Brothers volcano, Kermadec arc, unpublished manuscript, 2008). The second hydrothermally active area is on the flanks and summit of a cone structure situated within the caldera known as Brothers cone (179.07°E/ 34.87°S). At the time of sampling, Brothers cone contained a number of small diffuse venting systems of which one, designated B-LC (Brothers-lower cone) hosted a diverse microbial community on which this study is based.

2. Materials and Methods 2.1. Sample Collection [5] The hydrothermal vent material used in this study was collected on the second of two dives on the cone structure of Brothers volcano by the manned submersible Shinkai 6500

(Dive 854). A piece of dacite lava (sample 854– 2), covered in what appeared to be elemental sulfur and a white biofilm, was collected from the vent orifice (B-LC) on the lower flank of the cone structure at a depth of 1336 m (34 – 52.7232°S/179 – 4.3041°E) (Figure 1). The in situ temperature of the vent fluids was 67°C. Vent fluid was also collected from within the vent orifice for geochemical analysis (de Ronde et al., unpublished manuscript, 2008) (Table 1.) Upon surfacing, the elemental sulfur/white biofilm was aseptically scrapped off the dacite and immediately stored at 20°C. The sample was transported on dry ice to the laboratory and then stored at 80°C until processing. 2.2. DNA Extraction and PCR Amplification of SSU rRNA and Functional Genes [6] A total environmental DNA extraction was performed following the manufacturer’s protocol using the FastDNA extraction kit for soil (QBiogene) from approximately 0.3 g of sample. [7] All polymerase chain reactions (PCR), with the exception of the bacterial small subunit (SSU) rRNA gene reactions, were performed in a 50 mL volume containing 0.5 mM dNTP mix (Invitrogen), 0.5 mM of each primer, 5 mL of 10 PCR buffer (Bioline), 10 mL of PCR enhancer (Eppendorf), 1.5 mM MgCl2 (Bioline), 1 mL DNA template, and 1 U Taq polymerase (Bioline). The bacterial SSU rRNA gene PCR reactions were performed in a 50 mL volume containing 0.5 mM dNTP mix (Invitrogen), 0.5 mM of each primer, 1 PCR buffer (Stratagene), 10 mL of DMSO (Invitrogen), 1 mL DNA template and 1 U Pfu Turbo DNA polymerase (Stratagene). [8] The bacterial SSU rRNA genes were amplified from the DNA extract via PCR using the 27f/1492r primer set [Lane, 1991] according to the following conditions; 1 cycle of 10 min denaturation (94°C); 33 cycles of denaturation at 94°C for 2 min, annealing at 48°C for 2 min, extension for 72°C for 45 s; 1 final cycle of extension for 10 min for 72°C. Positive PCR products were cleaned with the Geneclean Spin kit (QBiogene) and eluted with 20 mL of sterile

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Fluids were collected from within the vent orifice prior to the microbial biofilm. Details of the chemical analysis used in this table are outlined by de Ronde et al. (unpublished manuscript, 2008). b All concentrations were measured from the liquid phase. BD, below detection limit.

a

25.6 103 33 7.8 10 5.6 10 1.2 10 3.0 10 3.1 1.8

Cl Vent/SW

BD 133 103 3.7 103 5.1 1336 854 – 2

67

H2 CO2 H2S Depth (m) Sample

Temp (°C)

pH

CH4

O2

C2

2

Alkanes

C3

2

Concentrationb (mmol/kg)

Table 1. Selected Physiochemical Data of the Vent Fluids Collected at Sample Site Brothers-Lower Conea

C2

2

Alkenes

C3

2

16

SO42 Fe NH4+

544/537


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HPLC water. The purified products were cloned into pCR4Blunt-TOPO vector (Invitrogen) and then were isolated using PerfectPrep Plasmid 96 Vac Direct Bind kit (Eppendorf) following the manufacturer’s protocols. [9] The environmental archaeal SSU rRNA gene set was amplified using the universal archaeal primer pair 109f/912r [Ramakrishnan et al., 2001] using the following conditions; 1 cycle of 5 min denaturation (94°C); 33 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension for 72°C for 90 s; 1 final cycle of extension for 6 min for 72°C. The presence of Korarchaeota was screened using the Korarchaeota-specific primer set Kb228f and Ua1406r [Brunk and Eis, 1998]. [10] PCR amplification was used to screen for a selection of functional genes including dsrA encoding the a-subunit of dissimilatory sulfite reductase (DSR1F/DSR4R [Wagner et al., 1998]), pmoA encoding a subunit of particulate methane monoxygenase (189f/682r [Holmes et al., 1996] and 189f/mb661 [Knief et al., 2003]), mcrA encoding methyl coenzyme M reductase (ME1/ME2 and AOM39_f/ AOM40_r [Hallam et al., 2003] and MCRf/MCRr [Lueders et al., 2001]) and coxL encoding the large subunit of aerobic carbon monoxide dehydrogenase (OMPf/O.Br [Dunfield and King, 2004]). These genes are an indication of sulfite/ sulfate reduction (dsrA), aerobic methanotrophy (pmoA), anaerobic methane oxidation (mcrA), methanogenesis (mcrA), and aerobic carbon monoxide oxidation (coxL), respectively. [11] Positive PCR products for all archaeal and functional gene amplifications were cloned using the TOPO TA cloning kit (Invitrogen) according to the manufacturer’s instructions. Transformed cells were picked and amplified directly using the M13F/M13R primer set using the same reaction mix as outlined above minus the PCR enhancer. The PCR amplification was conducted under the following conditions; 1 cycle of 10 min denaturation (94°C); 20 cycles of denaturation at 94°C for 2 min, annealing at 64°C for 2 min (decreasing temperature by 0.5°C per cycle), extension for 72°C for 45 s; 15 cycles of denaturation at 94°C for 2 min, annealing at 54°C for 2 min, extension for 72°C for 45 s; 1 final cycle of extension for 10 min for 72°C. Positive PCR products were cleaned using a Purelink PCR cleanup kit (Invitrogen) following the manufacturer’s protocol. 2.3. Sequencing [12] All clones were amplified using the BigDye Terminator v3.1 cycle sequencing kit with the T3 and T7 primers for the bacterial clones and M13F/M13R primers for the archaeal and functional gene clones. The clones were sequenced on an Applied Biosystems 3730 l DNA Analyzer. 2.4. Sequence Assembly and Interpretation [ 13 ] Sequences were assembled using Seqman 7.0 (DNAstar) and then checked for chimeras using the Bellerophon chimera check [Huber et al., 2004] linked with the Greengenes Web site [DeSantis et al., 2006], as well as examining the secondary structure. Determination of related sequences was conducted using discontiguous Mega BLAST search programs [Altschul et al., 1990] (http:// www.ncbi.nlm.nih.gov/blast/). Nonchimeric sequences were aligned in ARB [Ludwig et al., 2004] against the 3 of 9

4 of 9 AM712343 AM712345 AM712346

2 7 21 1 2 1 1 1 1 1 1 1 1

OP1 OP3 OP5 OP8 OP11

SB1

Syntrophobacterales Desulfobulbaceae Syntrophobacterales Desulfurella-Hippea

Camplyobacteriales

Uncultivated group

1 3 1 1 2

JS1/OPB46-affiliated OP8-affiliated

AF431240 - Alvinella symbiont clone (84%) AY280428 - Paralvinella symbionts clone (92%) AF419677 - Guaymas Basin clone (82%) AB252430 - Okinawa Trough CO2 lake clone (93%) AY197377 - Guaymas Basin clone (85%) AY197422 - Guaymas Basin clone (97%) AY197422 - Guaymas Basin clone (81%) DQ256300 - Kalahari subsurface clone (79%) AF507880 - Mono Lake clone (79%)

Thermodesulfobacteria AE013109 - Thermoanaerobacter tengcongensis (86%) U25627 - Thermodesulforhabdus norvegicus (83%) Thermotogae AB260048 - Thermotogales sp. Ag70 (82%) Undetermined Affiliation AY725424 - Desulfonatronum cooperativum (83%) AY725424 Desulfonatronum cooperativum (79%) AE013109 Thermoanaerobacter tengcongensis (78%) DQ095862 Thermolithobacter carboxydivorans (80%) AJ271450 Natronoanaerobium salstagnum (79%)

b

DQ071275 - hydrothermal sulfur mat clone (98%) AF449235 - Riftia symbionts clone (98%) AY327877 - hydrothermal gastropod clone (97%) AF299121 - E. Pacific Rise clone (95%) AF357197- Alvinella symbiont clone (92%)

AF420341 - Guaymas Basin clone (96%) AB252432 - Okinawa Trough clone (96%) AC150251 - Zodletone Spring clone (90%) AJ431226 - Alvinella sybiont clone (91%)

Spirochaetes AJ698859 - Spirochaeta bajacaliforniensis (84%)

Proteobacteria - Epsilonproteobacteria AB091292 - Sulfurovum lithotrophicum (95%) AB091292 - Sulfurovum lithotrophicum (98%) AM157656 - Proteobacterium AN-BI3A (98%) AB175498 - Thioreductor micantisoli (92%) AJ535664 - Caminibacter sp. CR (92%)

CP000252 - Syntrophus aciditrophicus (87%) AF230531 - Desulfomonile limimaris (87%) AJ237603 - Desulfobacterium cetonicum (85%) Y18292 - Hippea maritima (91%)

AB099985 - dead hydrothermal chimney clone (90%)

AF507706 - Arizona soil clone (82%) AB177219 - methane hydrate clone (87%) DQ521806 - Gulf of Mexico clone (92%) AY344402 - Hawaiian Archipelago clone (84%) AF419685 - Guaymas Basin clone (91%) AF419671 - Guaymas Basin clone (93%) AF419659 - Guaymas Basin clone (82%)

Candidate Divisions AJ431234 - Cytophaga sp. Dex80-43 (72%) AB235322 - Bacterium 55YT-8-4 (77%) Y18292 - Hippea maritima (81%) AF418169 - Thermodesulfobacterium commune (77%) DQ225186 - Geobacillus toebii (78%) Z70248 - Thermosipho melanesiensis (83%) AJ872269 - Thermotoga petrophila (82%) Chlorobi CP000108 - Chlorobium chlorochromatii (81%) Proteobacteria - Deltaproteobacteria

AM040098 - intertidal sediment clone (92%)

Bacteriodetes AJ431254 - Cytophaga sp. BHI60-95B (92%)

Identities to the closest cultured and noncultured species are based on a discontiguous megablast search (NCBI). Many of the Brothers-lower cone phylotypes do not group within the same phyla/division as the closest cultivated spp.

a

AM712348 AM712334 AM712333 AM712349 AM712347

AM712344

AM712340 AM712341 AM712342 AM712353 AM712354

AM712338 AM712339 AM712336 AM712337

AM712335

AM712328 AM712350 AM712329 AM712351 AM712331 AM712332 AM712330

AB099990 - dead hydrothermal chimney clone (97%) AJ535239 - Cascadia Margin clone (94%) AM086150 - Lake Kinneret clone (86%)

Closest Noncultivated Sequenceb (SSU rRNA Similarity)

Acidobacteria AF498753 - Acidobacterium Ellin371 (89%) L04711 - Bacterium OS- K (87%) AJ277897 - Desulfomicrobium norvegicum (83%)

Closest Isolateb (SSU rRNA Similarity)


Unaffiliated 1 Unaffiliated 2

3

Uncultivated group

Nautiliales

1 1 13 1 12 3 2

OD1

AM712327

1

Sphingobacteriales

AM712324 AM712325 AM712326

Accession Number

1 1 12

Percent of Clone Library

Group 3 Group 10 Novel group

Class, Order, or Group

Table 2. Phylogenetic Placement of Bacterial Operational Taxonomic Units Detected in Small Subunit rRNA Gene Clone Libraries Constructed From DNA Extracts of the Brothers-Lower Cone Sample Sitea

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Figure 2. Proportions of bacterial phyla and candidate divisions detected at the Brothers-lower cone sample site. Pie wedges are colorized according to their possible metabolic function.

SILVA (release 89) ribosomal RNA database (http:// www.arb-silva.de/). Distance matrices used for operational taxonomic unit (OTU) determination were generated via a neighbor-joining (NJ) algorithm (using an Olsen correction) with ARB. Operational taxonomic units were constructed by pair-wise identity (%ID) using the furthest-neighbor algorithm (precision 0.01) in DOTUR [Schloss and Handelsman, 2005]. Rarefaction and collectors curves were also constructed using data generated with DOTUR. Phylogenetic trees were constructed using NJ, maximumlikelihood (ML) and TREE-PUZZLE [Schmidt et al., 2002] treeing methods. The robustness of the inferred topologies were tested with Bootstrap resampling and TREE-PUZZLE quartet-support values (>10,000 steps). 2.5. Nucleotide Accession Numbers [14] The bacterial and archaeal SSU rRNA gene sequences determined in this study have been deposited in the EMBL/DDBJ/GenBank databases, under the accession numbers AM712324– 354 and AM749964 – 992.

3. Results and Discussion [15] Twenty-nine bacterial and 17 archaeal OTUs were identified using a 3% sequence dissimilarity value determined via the furthest-neighbor algorithm in DOTUR (Table S1 in the auxiliary material1) [Schloss and Handelsman, 2005]. The Chao1 estimator indicated that the bacterial and archaeal species richness (3% sequence dissimilarity) could be expected to be a minimum of 32 species (with a 95% confidence interval between 30 and 47) and 20 species (with a 95% confidence interval between 18 and 36), respectively. Similar values were also predicted using the ACE estimator. Rarefraction curves (figures not included), and Chao1 and ACE richness estimators indicate a low

1 Auxiliary materials are available in the HTML. doi:10.1029/ 2007JB005477.

frequency of further novel OTU discovery with continued sampling. [16] The bacterial community at the B-LC sample site exhibited a high phylogenetic diversity and contained a substantial proportion of uncultivated bacterial lineages (Table 2). Less than 1% and 40% of the bacterial library clones show >92% SSU rRNA gene sequence similarity with cultivated and uncultivated microorganisms respectively. The bacterial clone library was dominated by microorganisms belonging to the phyla Acidobacteria and Deltaproteobacteria, and candidate divisions OP1 and OP5 (Figure 2). In addition, clones grouping within candidate divisions OP3, OP8, OD1, and OP11 and the deep-branching phyla Thermodesulfobacteria and Thermotogae were also detected. [17] Approximately one-sixth of the bacterial community belonged to the phylum Acidobacteria (Figure 2). Acidobacteria are considered ubiquitous soil bacteria [Hugenholtz et al., 1998a], that have also been detected in hydrothermal vent ecosystems [Ludwig et al., 1997; Sievert et al., 2000; Teske et al., 2002]. It is difficult to infer the ecological function of the acidobacterial phylotypes due to the minimal number of cultivated Acidobacteria spp. and absence of any hydrothermal-associated acidobacterial isolates. However, in several studies where substantial acidobacterial communities have been detected, such as Lower Kane Cave [Meisinger et al., 2007] or the shallow hydrothermal vents at Milos [Sievert et al., 2000], it was suggested that Acidobacteria were involved in the oxidation of allochthonous organics, possibly in combination with dissimilatory sulfur reduction under microaerophilic conditions. The similarity in the geochemical fluid composition to these in sulfidic environments and B-LC suggests that the B-LC acidobacterial community may also operate in a similar way. Modest concentrations of short chain hydrocarbons that could serve as an energy source for organotrophic growth were detected in the vent fluids (Table 1), although complex carbohydrates known to support some acidobacterial isolates [Davis et al., 2005] were not analyzed.

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[18] Proteobacteria made up approximately one third of bacteria detected at B-LC (Figure 2) of which Deltaproteobacteria were the majority and included species grouping within classes containing sulfur or sulfate-reducers such as Desulfobulbaceae, Syntrophobacteriaceae or DesulfurellaHippea (Figure S1 in the auxiliary material). With the exception of the single Desulfurella-Hippea sp., all deltaproteobacterial clones grouped within families dominated by primarily SRB (sulfate-reducing bacteria). The presence of SRB activity was supported by the detection of the marker gene for bacterial dissimilatory sulfite reduction, dsrA. Comparative sequence analysis of inferred amino acid sequence of the only DsrA OTU detected (n = 24) in this study suggests it that belonged to either a Syntrophobacteriales or Thermodesulfobacteria spp. (data not shown), although a definitive phylogenetic inference could not be made as dsrA can be laterally transferred between microbial species [Klein et al., 2001]. Several of the deltaproteobacterial clones, grouped with other phylotypes (Table 2 and Figure S1 in the auxiliary material) detected in various methane and hydrocarbon-rich environments, including Guaymas Basin [Dhillon et al., 2003; Teske et al., 2002], Okinawa Trough [Inagaki et al., 2006a], and the Gulf of Mexico [Lloyd et al., 2006]. These environments are known hot spots of anaerobic methane oxidation (AOM) where sulfate-reducing Deltaproteobacteria grow symbiotically with anaerobic methane-oxidizing archaea. Epsilonproteobacteria made up only a minor component of the bacterial clone library (Figure 2), but their detection at the B-LC sample site further demonstrates the ubiquity of Epsilonproteobacteria across hydrothermal vent environments [Campbell et al., 2006; Longnecker and Reysenbach, 2001]. All Epsilonproteobacteria clones were phylogenetically related to species previously detected in hydrothermal, sulfur-dominated environments [Longnecker and Reysenbach, 2001; Moussard et al., 2006a] and are speculated to be involved in sulfur oxidation or reduction (Table 2). In particular, the phylogenetic similarity of two clones to sulfur-oxidizing chemolithotroph, Sulfurovum lithotrophicum suggests that they could have an oxidative sulfur/sulfide metabolism. Other minor phylotypes detected at BL-C include Bacteriodetes, Chlorobi, and Spirochaetes as well as deeply branching Thermodesulfobacteria and Thermotogae spp. (Figure 2). [19] The remaining bacterial species grouped within divisions containing no cultivated representatives (Figure S1 in the auxiliary material). These represented almost one third of the total SSU rRNA genes detected (Figure 2). The majority grouped in six candidate divisions including OP1, 3, 5, 8, and 11 [Hugenholtz et al., 1998b] and OD1 [Harris et al., 2004]. These divisions are comprised of phylotypes that have been detected across a geochemically diverse range of environments including hotsprings [Hugenholtz et al., 1998b], methane seeps [Dhillon et al., 2003; Inagaki et al., 2006a], bioreactors [Chouari et al., 2005], hypersaline mats [Ley et al., 2006], contaminated aquifers [Dojka et al., 1998], and hydrothermal vents [Alain et al., 2006; Brazelton et al., 2006; Perner et al., 2007]. As with other bacterial phylotypes detected at B-LC, the majority of the candidate division clones were most closely related to phylotypes detected in high methane, CO2 and hydrocarbon-rich

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environments, in particular Guaymas Basin [Dhillon et al., 2003; Inagaki et al., 2006a, 2006b; Teske et al., 2002]. [20] Only euryarchaeotal SSU rRNA gene sequences were detected using the archaeal clone library (Table S2 in the auxiliary material). Of the 105 clones sequenced, 17 OTUs (>3% sequence dissimilarity) were detected and belonged to only two major groups within the Euryarchaeota, the DHVE (Deep Hydrothermal Vent Euryarchaeotic) groups I (DHVE1, 2 and Marine Benthic Group D) and II (DHVE5 and 6) [Takai et al., 2001]. Both groups are ubiquitous in hydrothermal vent environments, although very little is understood about their physiology [Moussard et al., 2006b; Nakagawa and Takai, 2006; Nakagawa et al., 2005; Reysenbach et al., 2006; Takai et al., 2001]. To date, only one representative from the above archaeal groups, Candidatus ‘‘Aciduliprofundum boonei’’ has been isolated [Reysenbach et al., 2006]. Candidatus ‘‘A. boonei’’ groups in DHVE2 (DHVE group I) and is a thermoacidophilic Sand Fe-reducing heterotroph commonly detected at vent environments similar to B-LC. Both S and Fe were available in elevated concentrations at B-LC, so it seems likely that at least the DHVE2 clones (which share sequence similarities of 92 – 95% with Candidatus ‘‘A. boonei’’) participated in S cycling with the Deltaproteobacteria and Thermodesulfobacteria. All other clones detected at B-LC shared low sequence similarity with Candidatus ‘‘A. boonei,’’ and grouped with clones detected at other hydrothermal vent environments [Hoek et al., 2003; Dhillon et al., 2005; Nakagawa et al., 2005, 2006; Takai et al., 2001]. A separate clone library was also constructed using korarchaeotal-specific primers Kb228F and Ua1406R [Brunk and Eis, 1998]. Only one Korarchaeota phylotype was detected in the clone library (n = 24) and was most closely related to korarchaeotal clones (pOWA19, pOWA54) detected in shallow submarine hydrothermal vents off the coast of Japan [Takai and Sako, 1999] and also a (C1R025) detected in Guaymas Basin sediments [Teske et al., 2002]. Korarchaeota are a rarely detected candidate division of the Archaea with no cultivated representatives and generally found in hyperthermophilic (+80°C) environments. While the temperature measured at B-LC cannot be considered hyperthermophilic, the presence of Korarchaeota may suggest the input of microbial species from a subsurface environments [Takai et al., 2004] or that the temperature of the vent fluids at B-LC may vary. [21] Of the functional genes screened, only dsrA was detected at the sample site (discussed above). All other functional genes tested, including mcrA (methanogenesis and AOM activity), pmoA, and coxL, were negative. Although these results do not preclude the anaerobic and aerobic methanotrophy, methanogenesis, or carboxydotrophy, they do corroborate the SSU rRNA-based phylogenetic data which failed to detect the presence of known microbial species with these activities. [22] A comparison of the microbial communities from other hydrothermal features within the Brothers volcano caldera (K. Takai et al., unpublished manuscript, 2008) or other MOR, BAB, or hot spot hydrothermal systems do not assist in the interpretation of ecological function nor were there any similarities in microbial community makeup to that of B-LC. Within other hydrothermal systems, including Brothers NW, the microbial species such as Epsilonproteo-

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bacteria, Archaeoglobus, Aquificales, Thermococcus, and Methanococcales are commonly detected [Huber et al., 2003; Karl, 1995; Nakagawa et al., 2005; Reysenbach et al., 2000; Takai et al., 2006]. Yet, with the exception of Epsilonproteobacteria, none of the ‘‘commonly’’ detected hydrothermal vent species were discovered. In fact, a notable feature of the microbial community at B-LC was the substantial population of phylotypes not grouping within recognized phyla, in particular, the high proportion of clones grouping in bacterial candidate divisions and uncultivated euryarchaeotal ‘‘deep hydrothermal vent’’ communities. While microbial communities in marine hydrothermal settings containing multiple candidate divisions are not uncommon [Dhillon et al., 2003; Inagaki et al., 2006a, 2006b; Perner et al., 2007], to our knowledge, there are few studied ecosystems, most notably Obsidian Pool [Hugenholtz et al., 1998b], that contain high proportions of uncultivated bacterial divisions. Over one third of the bacterial community detected at B-LC belonged to at least six candidate divisions (Figure 2), making it a difficult community to infer ecological function. From a phylogenetic perspective, the most similar microbial communities to the B-LC sample site appear to be those in marine environments containing elevated concentrations of methane, CO2, and/or hydrocarbons, in particular, the methane and CO2-rich hydrothermal settings of the Okinawa Trough [Inagaki et al., 2006a], Gulf of Mexico [Lloyd et al., 2006], and Guaymas Basin [Dhillon et al., 2005]. Studies of these communities show that the microbial community centers on AOM with the concomitant microbial reduction of sulfate. Yet, neither the key members of the AOM communities, the anaerobic methane-oxidizing Euryarchaeota (ANME Groups 1–3) from the classes Methanosarcinales and Methanococcoides, nor the deltaproteobacterial SRBs from the Desulfosarcina or the Desulfococcus branch (DSS) normally associated with ANME [Orphan et al., 2002] were detected at B-LC. This observation was further supported by low methane concentrations (Table 1) and not detecting of AOM activity using specific mcrA primers. These results indicate that although phylogenetically similar clones were detected at B-LC and ecosystems with high AOM activity, it was unlikely that AOM is a primary driver behind the B-LC microbial community. Undoubtedly, the geochemistry of the B-LC vent fluids must play an important part in determining the microbial diversity. The hydrothermal vent fluids at Brothers volcano derive from arc-like lavas and are enriched in acid volatiles that are thought to aggressively dissolve the host rock generating extremes in vent fluid composition relative to most MOR hydrothermal systems [Massoth et al., 2003]. This is evident with the elevated concentrations at B-LC of Fe, H2S, and in particular CO2, relative to most MOR systems (de Ronde et al., unpublished manuscript, 2008) and yet low concentrations of H2 and CH4. The vent fluids also contained moderate concentrations of short chain hydrocarbons (Table 1). The predominance of alkenes over alkanes is typical of an oxidized vent fluid and is also consistent with the low O2, CH4, and H2 concentrations. [23] It is difficult to pinpoint the common geochemical or physical attribute that mediates the similar phylotypes detected at B-LC and similar CO2/hydrocarbon-rich communities. For example, it appears that low to moderate temperature ranges (4 – 74°C) and elevated concentrations of CO2, H2S, SO24 , and hydrocarbons feature at all sites

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[Dhillon et al., 2003; Inagaki et al., 2006a, 2006b; Lloyd et al., 2006; Teske et al., 2002]. Yet, other than dissimilatory sulfate reduction, none of the other common metabolic genes indicative of aerobic carbon monoxide oxidation, aerobic and anaerobic methanotrophy, or methanogenesis, were detected at the sample site. It was particularly interesting that no known methanogenic phylotypes or mcrA were detected despite elevated concentrations of CO2. While this does not preclude the methanogens, this observation was not necessarily surprising as low H2 concentrations and competition with SRB for available energy sources would restrict methanogenesis, and uncomplexed sulfide has been demonstrated to be toxic to methanotrophic and other vent archaea [Lloyd et al., 2005].

4. Conclusion [24] Insights into microbial ecology and function can be inferred to some extent using a three-pronged approach of collating the SSU rRNA gene phylogeny, surveying genes for particular microbial functional types, and geochemical analysis. We used this approach to assess the microbial diversity and biogeochemical processes at vent community at Brothers volcano and identified a microbial community, dissimilar in phylogenetic or functional gene makeup to vent systems previously reported. The only major microbial metabolism detected at B-LC was dissimilatory sulfite/ sulfate reduction, which was presumably part of a sulfur cycle with sulfur-oxidizing aerobes such as Epsilonproteobacteria. Allochthonous organic compounds may provide heterotrophic growth for SRB, Thermotogae, and Acidobacteria, although further work needs to be conducted to confirm these data. Despite the difficulties inferring microbial function at B-LC, the diversity of the microbial community and geochemistry of this hydrothermal ecosystem warrant further in-depth investigations to understand the ecology of this environment, possibly using a metagenomic approach. [25] Acknowledgments. The authors with to acknowledge the Captain and crew of the R/V Yokosuka and the Shinkai 6500 for their assistance obtaining samples on the SWEEP VENT 2004 expedition. We would also like to thank JAMSTEC as the funding agency for the SWEEP VENTS expedition to the Kermadec arc in late 2004 and all of the SWEEP VENTS scientific party for their assistance during and after the expedition. The geochemical data was supplied courtesy of J. Lupton, D. Butterfield (NOAA), J. Ishibashi, T. Yamanaka (Kyushu University), G. Massoth, and B. Christenson (GNS Science). The authors wish to thank C. de Ronde and G. Massoth for their useful discussions on Kermadec arc volcanology and the anonymous reviewers for their helpful suggestions. This work was partially funded by the Foundation for Research, Science and Technology grant C05X03030 (MS and CD) and by Intramural funding from UH Administration (MA).

References Alain, K., T. Holler, F. Musat, M. Elvert, T. Treude, and M. Kru¨ger (2006), Microbiological investigation of methane- and hydrocarbon-discharging mud volcanoes in the Carpathian Mountains, Romania, Environ. Microbiol., 8, 574 – 590, doi:10.1111/j.1462-2920.2005.00922.x. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman (1990), Basic local alignment search tool, J. Mol. Biol., 215, 403 – 410. Brazelton, W. J., M. O. Schrenk, D. S. Kelley, and J. A. Baross (2006), Methane- and sulfur-metabolizing microbial communities dominate the lost city hydrothermal field ecosystem, Appl. Environ. Microbiol., 72, 6257 – 6270, doi:10.1128/AEM.00574-06. Brunk, C. F., and N. Eis (1998), Quantitative measure of small-subunit rRNA gene sequences of the kingdom Korarchaeota, Appl. Environ. Microbiol., 64, 5064 – 5066.

7 of 9

B08S06


Campbell, B. J., C. Jeanthon, J. E. Kostka, G. W. Luther III, and S. C. Cary (2001), Growth and phylogenetic properties of novel Bacteria belonging to the Epsilon subdivision of the Proteobacteria enriched from Alvinella pompejana and deep-sea hydrothermal vents, Appl. Environ. Microbiol., 67, 4566 – 4572, doi:10.1128/AEM.67.10.4566-4572.2001. Campbell, B. J., A. S. Engel, M. L. Porter, and K. Takai (2006), The versatile Epsilon-proteobacteria: Key players in sulphidic habitats, Nat. Rev. Microbiol., 4, 458 – 468, doi:10.1038/nrmicro1414. Chouari, R., D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach, and A. Sghir (2005), Novel predominant archaeal and bacterial groups revealed by molecular analysis of an anaerobic sludge digester, Environ. Microbiol., 7, 1104 – 1115, doi:10.1111/j.1462-2920.2005.00795.x. Davis, K. E. R., S. J. Joseph, and P. H. Janssen (2005), Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria, Appl. Environ. Microbiol., 71, 826 – 834, doi:10.1128/ AEM.71.2.826-834.2005. de Ronde, C. E. J., G. J. Massoth, E. T. Baker, and J. E. Lupton (2003), Submarine hydrothermal venting related to volcanic arcs, in Volcanic, Geothermal, and Ore-forming Fluids: Rulers and Witnesses of Processes Within the Earth, edited by S. F. Simmons and I. Graham, Spec. Publ. Soc. Econ. Geol., 10, 91 – 110. de Ronde, C. E. J., et al. (2005), Evolution of a submarine magmatichydrothermal system: Brothers volcano, southern Kermadec arc, New Zealand, Econ. Geol., 100, 1097 – 1133, doi:10.2113/100.6.1097. DeSantis, T. Z., et al. (2006), Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB, Appl. Environ. Microbiol., 72, 5069 – 5072, doi:10.1128/AEM.03006-05. Dhillon, A., A. Teske, J. Dillon, D. A. Stahl, and M. L. Sogin (2003), Molecular characterization of sulfate-reducing bacteria in the Guaymas basin, Appl. Environ. Microbiol., 69, 2765 – 2772, doi:10.1128/ AEM.69.5.2765-2772.2003. Dhillon, A., M. Lever, K. G. Lloyd, D. B. Albert, M. L. Sogin, and A. Teske (2005), Methanogen diversity evidenced by molecular characterization of methyl coenzyme M reductase A (mcrA) genes in hydrothermal sediments of the Guaymas Basin, Appl. Environ. Microbiol., 71, 4592 – 4601, doi:10.1128/AEM.71.8.4592-4601.2005. Dojka, M. A., P. Hugenholtz, S. K. Haack, and N. R. Pace (1998), Microbial diversity in a hydrocarbon- and chlorinated-solvent- contaminated aquifer undergoing intrinsic bioremediation, Appl. Environ. Microbiol., 64, 3869 – 3877. Dunfield, K. E., and G. M. King (2004), Molecular analysis of carbon monoxide-oxidizing bacteria associated with recent Hawaiian volcanic deposits, Appl. Environ. Microbiol., 70, 4242 – 4248, doi:10.1128/ AEM.70.7.4242-4248.2004. Emerson, D., J. A. Rentz, T. G. Lilburn, R. E. Davis, H. Aldrich, C. Chan, and C. L. Moyer (2007), A novel lineage of Proteobacteria involved in formation of marine Fe-oxidizing microbial mat communities, PLoS One, 2, e667, doi:10.1371/journal.pone.0000667. Goffredi, S. K., A. Wareń, V. J. Orphan, C. L. Van Dover, and R. C. Vrijenhoek (2004), Novel forms of structural integration between microbes and a hydrothermal vent gastropod from the Indian Ocean, Appl. Environ. Microbiol., 70, 3082 – 3090, doi:10.1128/AEM.70.5.30823090.2004. Hallam, S. J., P. R. Girguis, C. M. Preston, P. M. Richardson, and E. F. DeLong (2003), Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing Archaea, Appl. Environ. Microbiol., 69, 5483 – 5491, doi:10.1128/AEM.69.9.5483-5491.2003. Harris, J. K., S. T. Kelley, and N. R. Pace (2004), New perspective on uncultured bacterial phylogenetic division OP11, Appl. Environ. Microbiol., 70, 845 – 849, doi:10.1128/AEM.70.2.845-849.2004. Hoek, J., A. B. Banta, R. F. Hubler, and A.-L. Reysenbach (2003), Microbial diversity of a deep-sea hydrothermal vent biofilm from Edmond vent field on the Central Indian Ridge, Geobiology, 1, 119 – 127, doi:10.1046/ j.1472-4669.2003.00015.x. Holmes, A. J., N. J. P. Owens, and J. C. Murrell (1996), Molecular analysis of enrichment cultures of marine methane oxidising bacteria, J. Exp. Mar. Biol. Ecol., 203, 27 – 38, doi:10.1016/0022-0981(96)02567-1. Huber, J. A., D. A. Butterfield, and J. A. Baross (2003), Bacterial diversity in a subseafloor habitat following a deep-sea volcanic eruption, FEMS Microbiol. Ecol., 43, 393 – 409, doi:10.1111/j.1574-6941.2003. tb01080.x. Huber, T., G. Faulkner, and P. Hugenholtz (2004), Bellerophon: A program to detect chimeric sequences in multiple sequence alignments, Bioinformatics, 20, 2317 – 2319, doi:10.1093/bioinformatics/bth226. Huber, J. A., H. P. Johnson, D. A. Butterfield, and J. A. Baross (2006), Microbial life in ridge flank crustal fluids, Environ. Microbiol., 8, 88 – 99, doi:10.1111/j.1462-2920.2005.00872.x. Hugenholtz, P., B. M. Goebel, and N. R. Pace (1998a), Impact of cultureindependent studies on the emerging phylogenetic view of bacterial diversity, J. Bacteriol., 180, 4765 – 4777.

B08S06

Hugenholtz, P., C. Pitulle, K. L. Hershberger, and N. R. Pace (1998b), Novel division level bacterial diversity in a Yellowstone hot spring, J. Bacteriol., 180, 366 – 376. Inagaki, F., et al. (2006a), Microbial community in a sediment-hosted CO2 lake of the southern Okinawa Trough hydrothermal system, Proc. Natl. Acad. Sci. U. S. A., 103, 14,164 – 14,169, doi:10.1073/pnas.0606083103. Inagaki, F., et al. (2006b), Biogeographical distribution and diversity of microbes in methane hydrate-bearing deep marine sediments on the Pacific Ocean Margin, Proc. Natl. Acad. Sci. U. S. A., 103, 2815 – 2820, doi:10.1073/pnas.0511033103. Karl, D. M. (1995), Ecology of free-living, hydrothermal vent microbial communities, in The Microbiology of Deep-Sea Hydrothermal Vents, edited by D. M. Karl, pp. 35 – 125, Chem. Rubber, Boca Raton, Fla. Klein, M., M. Friedrich, A. J. Roger, P. Hugenholtz, S. Fishbain, H. Abicht, L. L. Blackall, D. A. Stahl, and M. Wagner (2001), Multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes, J. Bacteriol., 183, 6028 – 6035, doi:10.1128/JB.183.20.6028-6035.2001. Knief, C., A. Lipski, and P. F. Dunfield (2003), Diversity and activity of methanotrophic Bacteria in different upland soils, Appl. Environ. Microbiol., 69, 6703 – 6714, doi:10.1128/AEM.69.11.6703-6714.2003. Lane, D. J. (1991), 16S/23S rRNA Sequencing, pp. 115 – 174, John Wiley, London. Ley, R. E., et al. (2006), Unexpected diversity and complexity of the Guerrero Negro hypersaline microbial mat, Appl. Environ. Microbiol., 72, 3685 – 3695, doi:10.1128/AEM.72.5.3685-3695.2006. Lloyd, K. G., V. P. Edgcomb, S. J. Molyneaux, S. Boër, C. O. Wirsen, M. S. Atkins, and A. Teske (2005), Effects of dissolved sulfide, pH, and temperature on growth and survival of marine hyperthermophilic archaea, A p p l . E n v i ro n . M i c ro b i o l . , 7 1 , 6 3 8 3 – 6 3 8 5 , d o i : 1 0 . 11 2 8 / AEM.71.10.6383-6387.2005. Lloyd, K. G., L. Lapham, and A. Teske (2006), An anaerobic methaneoxidizing community of ANME-1b archaea in hypersaline gulf of Mexico sediments, Appl. Environ. Microbiol., 72, 7218 – 7230, doi:10.1128/ AEM.00886-06. Longnecker, K., and A. L. Reysenbach (2001), Expansion of the geographic distribution of a novel lineage of e-Proteobacteria to a hydrothermal vent site on the Southern East Pacific Rise, FEMS Microbiol. Ecol., 35, 287 – 293. Ludwig, W., et al. (1997), Detection and in situ identification of representatives of a widely distributed new bacterial phylum, FEMS Microbiol. Lett., 153, 181 – 190, doi:10.1111/j.1574-6968.1997.tb10480.x. Ludwig, W., et al. (2004), ARB: A software environment for sequence data, Nucleic Acids Res., 32, 1363 – 1371, doi:10.1093/nar/gkh293. Lueders, T., K. J. Chin, R. Conrad, and M. Friedrich (2001), Molecular analyses of methyl-coenzyme M reductase a-subunit (mcrA) genes in rice field soil and enrichment cultures reveal the methanogenic phenotype of a novel archaeal lineage, Environ. Microbiol., 3, 194 – 204, doi:10.1046/ j.1462-2920.2001.00179.x. Lupton, J. E., M. Lilley, D. Butterfield, L. Evans, R. W. Embley, G. J. Massoth, B. Christenson, K.-I. Nakamura, and M. Schmidt (2008), Venting of a separate CO2-rich gas phase from submarine arc volcanoes: Examples from the Mariana and Tonga-Kermadec arcs, J. Geophys. Res., 113, B08S12, doi:10.1029/2007JB005467. Massoth, G. J., C. E. J. de Ronde, J. E. Lupton, R. A. Feely, E. T. Baker, G. T. Lebon, and S. M. Maenner (2003), Chemically rich and diverse submarine hydrothermal plumes of the southern Kermadec volcanic arc (New Zealand), in Intra-Oceanic Subduction Systems: Tectonic and Magmatic Processes, edited by R. D. Larder and P. D. Leat, Geol. Soc. London Spec. Publ., 219, 119 – 139. Meisinger, D. B., J. Zimmermann, W. Ludwig, K. H. Schleifer, G. Wanner, M. Schmid, P. C. Bennett, A. S. Engel, and N. M. Lee (2007), In situ detection of novel Acidobacteria in microbial mats from a chemolithoautotrophically based cave ecosystem (Lower Kane Cave, WY, USA), Environ. Microbiol., 9, 1523 – 1534, doi:10.1111/j.1462-2920.2007. 01271.x. Moussard, H., E. Corre, M. A. Cambon-Bonavita, Y. Fouquet, and C. Jeanthon (2006a), Novel uncultured Epsilonproteobacteria dominate a filamentous sulphur mat from the 13°N hydrothermal vent field, East Pacific Rise, FEMS Microbiol. Ecol., 58, 449 – 463, doi:10.1111/j.15746941.2006.00192.x. Moussard, H., D. Moreira, M. A. Cambon-Bonavita, P. Lo´pez-Garcıá, and C. Jeanthon (2006b), Uncultured Archaea in a hydrothermal microbial assemblage: Phylogenetic diversity and characterization of a genome fragment from a euryarchaeote, FEMS Microbiol. Ecol., 57, 452 – 469, doi:10.1111/j.1574-6941.2006.00128.x. Nakagawa, S., and K. Takai (2006), The isolation of themophiles from deep-sea hydrothermal environments, Methods Microbiol., 35, 55 – 91, doi:10.1016/S0580-9517(08)70006-0.

8 of 9

B08S06


Nakagawa, S., et al. (2005), Variability in microbial community and venting chemistry in a sediment-hosted backarc hydrothermal system: Impacts of subseafloor phase-separation, FEMS Microbiol. Ecol., 54, 141 – 155, doi:10.1016/j.femsec.2005.03.007. Nakagawa, T., K. Takai, Y. Suzuki, H. Hirayama, U. Konno, U. Tsunogai, and K. Horikoshi (2006), Geomicrobiological exploration and characterization of a novel deep-sea hydrothermal system at the TOTO caldera in the Mariana Volcanic Arc, Environ. Microbiol., 8, 37 – 49, doi:10.1111/ j.1462-2920.2005.00884.x. Orphan, V. J., C. H. House, K. U. Hinrichs, K. D. McKeegan, and E. F. DeLong (2002), Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments, Proc. Natl. Acad. Sci. U. S. A., 99, 7663 – 7668, doi:10.1073/pnas.072210299. Perner, M., R. Seifert, S. Weber, A. Koschinsky, K. Schmidt, H. Strauss, M. Peters, K. Haase, and J. F. Imhoff (2007), Microbial CO2 fixation and sulfur cycling associated with low-temperature emissions at the Lilliput hydrothermal field, southern Mid-Atlantic Ridge (9°S), Environ. Microbiol., 9, 1186 – 1201, doi:10.1111/j.1462-2920.2007.01241.x. Ramakrishnan, B., T. Lueders, P. F. Dunfield, R. Conrad, and M. W. Friedrich (2001), Archaeal community structures in rice soils from different geographical regions before and after initiation of methane production, FEMS Microbiol. Ecol., 37, 175 – 186, doi:10.1111/j.15746941.2001.tb00865.x. Reysenbach, A.-L., K. Longnecker, and J. Kirshtein (2000), Novel bacterial and archaeal lineages from an in situ growth chamber deployed at a midAtlantic ridge hydrothermal vent, Appl. Environ. Microbiol., 66, 3798 – 3806, doi:10.1128/AEM.66.9.3798-3806.2000. Reysenbach, A.-L., Y. Liu, A. B. Banta, T. J. Beveridge, J. D. Kirshtein, S. Schouten, M. K. Tivey, K. L. Von Damm, and M. A. Voytek (2006), A ubiquitous thermoacidophilic archaeon from deep-sea hydrothermal vents, Nature, 442, 444 – 447, doi:10.1038/nature04921. Schloss, P. D., and J. Handelsman (2005), Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness, Appl. Environ. Microbiol., 71, 1501 – 1506, doi:10.1128/ AEM.71.3.1501-1506.2005. Schmidt, H. A., K. Strimmer, M. Vingron, and A. von Haeseler (2002), TREE-PUZZLE: Maximum likelihood phylogenetic analysis using quartets and parallel computing, Bioinformatics, 18, 502 – 504, doi:10.1093/ bioinformatics/18.3.502. Sievert, S. M., J. Kuever, and G. Muyzer (2000), Identification of SSU ribosomal DNA-defined bacterial populations at a shallow submarine

B08S06

hydrothermal vent near Milos Island (Greece), Appl. Environ. Microbiol., 66, 3102 – 3109, doi:10.1128/AEM.66.7.3102-3109.2000. Takai, K., and K. Horikoshi (1999), Genetic diversity of Archaea in deepsea hydrothermal vent environments, Genetics, 152, 1285 – 1297. Takai, K., and Y. Sako (1999), A molecular view of archaeal diversity in marine and terrestrial hot water environments, FEMS Microbiol. Ecol., 28, 177 – 188, doi:10.1111/j.1574-6941.1999.tb00573.x. Takai, K., T. Komatsu, F. Inagaki, and K. Horikoshi (2001), Distribution of Archaea in a black smoker chimney structure, Appl. Environ. Microbiol., 67, 3618 – 3629, doi:10.1128/AEM.67.8.3618-3629.2001. Takai, K., T. Gamo, U. Tsunogai, N. Nakayama, H. Hirayama, K. H. Nealson, and K. Horikoshi (2004), Geochemical and microbiological evidence for a hydrogen-based, hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) beneath an active deep-sea hydrothermal field, Extremophiles, 8, 269 – 282, doi:10.1007/s00792004-0386-3. Takai, K., S. Nakagawa, A.-L. Reysenbach, and J. Hoek (2006), Microbial ecology of mid-ocean ridges and back-arc basins, in Back-Arc Spreading Systems: Geological, Biological, Chemical and Physical Interactions, Geophys. Monogr. Ser., vol. 166, edited by D. M. Christie et al., pp. 185 – 213, AGU, Washington, D.C. Teske, A., K. U. Hinrichs, V. Edgcomb, A. De Vera Gomez, D. Kysela, S. P. Sylva, M. L. Sogin, and H. W. Jannasch (2002), Microbial diversity of hydrothermal sediments in the Guaymas Basin: Evidence for anaerobic methanotrophic communities, Appl. Environ. Microbiol., 68, 1994 – 2007, doi:10.1128/AEM.68.4.1994-2007.2002. Wagner, M., A. J. Roger, J. L. Flax, G. A. Brusseau, and D. A. Stahl (1998), Phylogeny of dissimilatory sulfite reductases supports an early origin of sulfate respiration, J. Bacteriol., 180, 2975 – 2982. M. Alam, S. Hou, E. Nakasone, and J. A. Saito, Department of Microbiology, University of Hawaii, Snyder Hall 207, 2538 The Mall, Honolulu, HI 96822, USA. M. A. Crowe, C. J. Daughney, P. F. Dunfield, B. W. Mountain, A. V. Smirnova, and M. B. Stott, GNS Science, Extremophile Research Group, Private Bag 2000, Taupo, New Zealand. ([email protected]) K. Takai, Subground Animalcule Retrieval Program, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan.

9 of 9