Abstract

The bacterial diversity in a diffuse flow hydrothermal vent habitat at Axial Volcano, Juan de Fuca Ridge was examined shortly after an eruptive event in 1998 and again in 1999 and 2000 using PCR-amplified 16S rRNA gene sequence analyses. While considerable overlap with deep-sea background seawater was found within the α- and γ-proteobacteria, unique subseafloor phylotypes were distinguishable. These included diverse members of the ε-proteobacteria, high temperature groups such as Desulfurobacterium, Gram-positive bacteria, and members of novel candidate divisions WS6 and ABY1. Phylotype richness was highest in the particle-attached populations from all three sampling periods, and diversity appeared to increase over that time, particularly among the ε-proteobacteria. A preliminary model of the subseafloor is presented that relates microbial diversity to temperature, chemical characteristics of diffuse flow fluids and the degree of mixing with seawater.

Introduction

The subseafloor at oceanic ridges consists of complex gradients in porosity, temperature, and chemical composition. Imposed on these gradients are episodic geophysical processes including earthquakes and catastrophic magma intrusion events that result in seafloor volcanic eruptions [1,2]. Seafloor volcanic eruptions frequently start with the rapid heating of fluids in the upper porous portion of the crust (approximately 500 m) followed by ejection of these heated fluids into the water column. Subsequently, the sites of magma intrusion events can develop into chronic vents that either emit hot hydrothermal fluids or a mixture of hot fluid and entrained seawater called diffuse flow vents. These new eruption events provide an excellent opportunity to sample subseafloor microorganisms. Hyperthermophilic archaea have been isolated from cold event plume water indicating that these organisms may already be present in the subseafloor crust prior to the eruption [3–5]. Also, a morphologically and physiologically diverse group of microorganisms have been identified from these fluids and include iron oxidizers and bacteria that oxidize hydrogen sulfide to elemental sulfur [6,7]. Sulfide oxidation activity can be so extensive that it results in the frequently observed ‘snow blower’ vents at new eruption sites [8,9].

There are few reports on the phylogenetic diversity of diffuse flow fluids and the inferred subseafloor microbial communities. Deep ocean sediment cores show decreasing numbers of microorganisms with depth to levels that are at the lower limit for detection by microscopic and molecular techniques [10–12]. In contrast, high bacterial and archaeal diversity have been reported in hydrothermally affected sediment cores from Guaymas Basin in the Gulf of California [13]. Included in this study was the identification of anaerobic methane oxidizing consortia [14]. Hyperthermophilic microorganisms have been isolated directly from diffuse flow fluids associated with chronic diffuse flow vents or enriched in situ on mineral surfaces [15–18]. Subseafloor crustal environments are difficult to sample, and there are no reports of microbial diversity with depth within cores from oceanic basalt although there are numerous reports providing evidence for microorganisms associated with cracks in the basalt [19]. Diffuse flow vents at active hydrothermal sites have temperatures usually below 50°C. They harbor approximately 105 microorganisms ml−1 that include culturable mesophilic sulfur and metal oxidizers and hyperthermophilic methanogens and heterotrophs, regarded as indicators of high temperature biotopes in the subseafloor [5,16,17,20]. The diffuse flow vents associated with new deep-sea volcanic eruptions are frequently short-lived and during the first 3–5 years after the eruption show decreasing flow rates, decreasing concentrations of hydrothermally produced energy sources and greater entrainment of seawater [1,8,21,22]. These changes are also reflected in the phylogenetic diversity of archaeal communities based on one study [17]. However, nothing is known about the temporal changes in the bacterial communities associated with subseafloor habitats fueled by new volcanic eruptions.

In this study, we used 16S rRNA gene sequence analysis to follow changes in bacterial diversity in subseafloor fluids from a single diffuse flow vent shortly after a volcanic eruption created the site in 1998 and again in 1999 and 2000. This study was done in parallel with the archaeal diversity studies already published in which we found a higher diversity associated with particles than in the free-living fraction [17]. Over this time period, these diffuse flow fluids showed marked variation in temperature, flow rates, chemical characteristics and extent of mixing of hydrothermal fluids and seawater. Our results show that bacterial diversity is high in diffuse fluids, and that it changes with the post-eruptive evolution of vent fluid chemistry and temperature. The data also show increases in species richness with time within the ε-proteobacteria, the dominant phylotype found to be unique to the subseafloor environment. A preliminary model is presented that attempts to relate bacterial and archaeal diversity to chemical characteristics of diffuse flow fluids and the degree of mixing of seawater.

Materials and methods

Sampling

Axial Seamount (46°55′N; 130°00′W) is located 300 miles off the coast of Oregon on the Juan de Fuca Ridge at a depth of 1520 m (Fig. 1). Positioned between two rift zones to the north and south and bordered on three sides by a boundary fault, it is an active submarine volcano with a caldera (3×8 km) that rises 700 m above the mean ridge level [23]. Water column and seafloor observations of the southeast portion of the caldera after a week-long series of earthquakes at Axial in January 1998 found temperature and chemical anomalies, extensive new seafloor lava flows, large ‘snowblower’ type vents, and other characteristics commonly associated with diking-eruptive events [2,24–26].

1

Map showing the location of Axial Volcano (inset) and Marker 33 vent in Axial's caldera.

1

Map showing the location of Axial Volcano (inset) and Marker 33 vent in Axial's caldera.

Using the ROV ROPOS, diffuse fluids were collected from Marker 33 vent (Fig. 1) on dive R473 (September 1998), dive R485 (July 1999) and dive R551 (July 2000). Filtered and unfiltered fluids were sampled at the vent after finding a steady temperature on the intake probe and pumping fluid at a known rate using the hydrothermal fluid and particle sampler (HFPS). Fluid was pumped through a 47-mm Millipore (3-μm pore size) mixed cellulose ester filter, followed by a Sterivex-GP (0.22-μm pore size) filter. Temperature and volume of fluid collected were monitored throughout the 10–15-min sampling time required to obtain approximately 1 l of fluid. On shipboard, the filters were placed in 50-ml sterile Falcon tubes, frozen in liquid nitrogen, and stored at −20°C. Chemical analyses, such as sulfide, magnesium, and silica, as well as epifluorescent counts and culturing were performed on unfiltered and filtered fluid samples. Because the HFPS records temperature throughout the sampling procedure, the average temperature of the water collected can be calculated and compared with the average temperature of the water passed through the filters used for DNA analysis. It is sometimes difficult to achieve a steady temperature during diffuse fluid sampling due to extreme micro-gradients in temperature and fluid flow, and repositioning the intake probe by 1 cm or less can result in significant changes in sampled fluid temperature. Whenever possible, we collected multiple fluid and particle samples without changing position.

An 18-ml aliquot of each fluid sample was preserved in formaldehyde (3.7% final concentration) in duplicate, stored at 2°C, and enumerated using epifluorescence microscopy with DAPI (4′,6-diamidino-2-phenylindole; Sigma) [27]. A background (no detectable hydrothermal plume) seawater sample from 1275 m depth and approximately 700 m SE of the active vent site was collected using a 10-l Niskin bottle mounted on a CTD (conductivity, temperature, depth) and filtered through a sterile 47-mm 0.22-μm pore size filter shipboard and processed for further molecular analyses.

Geochemical analysis

Analytical methods are described in Butterfield et al. [1]. Fluids collected with HFPS were analyzed shipboard for H2S, pH, and dissolved silica. On shore, fluids were analyzed for major, minor, and trace elements. The precision (±1 S.D.) of the reported chemical analyses is: pH, 0.05 units; H2S 4%; Mg 1%; Cl 0.5%; silica 0.5%; Fe 4%.

DNA extraction and purification

DNA was extracted exactly as described by Huber et al. [17]. DNA was purified using the Qiaquick PCR purification columns (Qiagen) according to the manufacturer's instructions, and the DNA stored at −20°C.

Clone library construction

PCR was performed on environmental DNA with universal bacterial specific primers 8f (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492r (5′-GGT TAC CTT GTT ACG ACT T-3′). Each PCR reaction (20 μl) contained 3.0 mM MgCl2, 0.8 mM deoxynucleoside triphosphates, 0.25 μM (each) primer, 1×PCR buffer (Promega), and 1 U of Taq DNA polymerase (Promega). An initial denaturation step of 5 min at 94°C was followed by 22–24 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 2 min. The final extension step was 72°C for 10 min. PCR cycles were stopped while product concentration was still in the exponential phase, as visualized and quantified on 1% (w/v) agarose gels stained with SYBR green (Molecular Probes) at 15, 20, 25, and 30 cycles. To minimize PCR drift [28], 6–10 replicate amplifications were pooled, then concentrated and purified with Qiaquick PCR purification columns (Qiagen) in accordance with the manufacturers’ instructions.

Consolidated and cleaned PCR products were cloned with a TA cloning vector kit according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). A total of 100–150 white colonies for each library were selected and stored on agar plates. After sequencing 20 randomly chosen clones from each library, restriction fragment length polymorphism (RFLP) was then used to insure we had sequenced a representative community from each library. For RFLP analysis, clones were inoculated into 100 μl of Luria–Bertani broth and incubated with shaking at 220 rpm for 1 h at 37°C. The plasmid inserts were PCR-amplified with M13F (5′-GTA AAA CGA CGG CCA G-3′) and M13R (5′-CAG GAA ACA GCT ATG AC-3′) in a 20-μl reaction volume with 1 μl clone culture, 3 mM MgCl2, 0.8 mM deoxynucleoside triphosphates, 1 ng of each primer ml−1, 2.5 U of Taq DNA polymerase (Promega), and 1×PCR buffer (Promega). PCR amplification began with a 1-min denaturation at 94°C followed by 30 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1.5 min, and ended with a 5-min extension at 72°C. PCR products were digested with MspI (New England Biolabs) and RsaI (Gibco BRL) according to the manufacturer's instructions and visualized on 2.5% agarose gels (w/v) stained with SYBR green. Different banding patterns were noted. RFLP was not performed on the Marker 33 2000 or CTD background sample.

Clones with unique RFLP patterns as well as the randomly selected clones were used in sequencing. Each clone was grown in 100 μl of Luria–Bertani broth shaking at 220 rpm for 1 h at 37°C, and PCR-amplified with M13F and M13R. Each 50-μl reaction contained 5 μl of clone culture, 1 mM MgCl2, 0.8 mM deoxynucleoside triphosphates, 1 μM (each) primer, 1×PCR buffer (Promega), and 5 U of Taq DNA polymerase (Promega). The PCR conditions were as follows: two cycles of 1.5 min at 94°C, 45 s at 56°C, and 1.5 min at 72°C followed by 22 cycles of 30 s at 90°C, 30 s at 56°C, and 1 min at 72°C. The final step consisted of a 10-min extension at 72°C. PCR products were visualized on 1% (w/v) agarose gels stained with SYBR green.

Sequencing

PCR products were cleaned and 200 ng sequenced bidirectionally with primers 8f and 519r (5′-GWA TTA CCG CGG CKG CTG-3′). Either the Thermosequenase II Dye Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech Inc.) with analysis on a 373 A DNA Sequencer (Applied Biosystems) or the DYEnamic ET Dye Terminator kit (Amersham Pharmacia Biotech Inc.) with subsequent analysis on a MegaBACE 1000 (Molecular Dynamics) was used.

Phylogenetic analysis

The Sequencher program (Gene Codes Corporation) was used to assemble sequences, and they were subsequently checked for chimeras using the CHIMERA_CHECK program of the Ribosomal Database project website [29], as well as by examining secondary structure. Sequences found to be non-chimeric were submitted for alignment to the RDP Sequence Alignment program, with common gaps conserved, and manually manipulated in the BioEdit v4.7.8 Program Sequences [30]. To find closely related sequences for phylogenetic analysis, sequences were submitted to the Advanced BLAST search program (available through the National Center for Biotechnology Information). Approximately 500 nucleotide bases were used in phylogenetic analyses, with only homologous positions included in the comparisons. The Phylip 3.5 package (obtained from J. Felsenstein, University of Washington, Seattle, WA, USA) was used to construct maximum likelihood trees (DNAML). Bootstrap analysis (SEQBOOT) was used to provide confidence estimates for tree topologies. Negative branch lengths were prohibited.

Shannon–Wiener biodiversity index, species observed, rarefaction analysis, Chao1, and abundance-based coverage estimator (ACE) were computed based on 97% similarity phylotypes using EstimateS (version 6.9b1, R.K. Colwell, http://viceroy.eeb.uconn.edu/estimates). For each sample, 50 randomizations were performed; for ACE, Srare was set at 4 [31]. Coverage was calculated using the method of Good [32] with the equation C=(1−(n1/N))×100, where n1 is the number of single-occurrence phylotypes within a library and N is the number of clones examined.

Nucleotide sequence accession numbers

The GenBank nucleotide sequence accession numbers for the sequences determined in this study are as follows: ε-proteobacteria AF468696–AF468786; γ-proteobacteria AF469210–AF469324; α-proteobacteria AF469325–AF469358; all remaining clones AF469363–AF469409.

Results

Study site description

Marker 33 is located on an eruptive fissure and is a long crack in basaltic sheet flows with a width of 30 cm and a length of several meters. Some chemical characteristics of Marker 33 are shown in Fig. 2. For a more detailed analysis and interpretation of the vent fluid geochemistry, as well as microscopic counts and culturing results, refer to Huber et al. [17]. Briefly, the ratio of hydrogen sulfide to other hydrothermal components decreased, while the end-member chloride concentration [1,21,33] increased, which is consistent with a decreasing vapor component over time in the hot source fluids [1,21,33]. Also important to consider is the degree of mixing between the end-member source fluid and crustal seawater that occurs below the point of venting, which is indicated by the temperature and simple chemical properties. In 1999, we sampled less dilute hydrothermal fluids, as indicated by the maximum temperature of 78°C.

2

Chemical and thermal characteristics of Marker 33 from 1998–2000.

2

Chemical and thermal characteristics of Marker 33 from 1998–2000.

Clone libraries

Six bacterial 16S rRNA gene clone libraries were prepared from fractionated filtered vent fluids, as well as a bacterial clone library from 1275 m, representing background deep-sea water. A negative control of sterile frozen filters was extracted and showed no bacterial amplification products. The first 500 bp were sequenced in both directions from 290 clones, and three were found to be chimeric and eliminated from subsequent analysis. Table 1 lists all the non-chimeric sequenced clones organized by sample and grouped within a library based on ≥97% sequence similarity, and includes the phylogenetic group to which each belongs, the closest match organism or clone based on BLAST searches, and the calculated percent similarity. Table 2 shows those exact clones (≥97% similar) that appeared in more than one library, with a representative clone, and the distribution of that phylotype in the seven libraries. A summary of the phylogenetic data for the three sampling years, divided into particle-attached and free-living fractions, based on the 17 taxonomic groups found in this study, is shown in Fig. 3. A sub-set of clones representing diverse bacterial phyla is presented on one of four phylogenetic trees, shown in Fig. 4a–d. Phylogenetic relationships of the six taxonomic groups represented by five or more clones are described below, as well as the new candidate divisions, Gram-positive bacteria (low and high G+C), and the high temperature bacteria belonging to Thermodesulfobacterium and Desulfurobacterium. The remaining groups (Planctomycetales, Cyanobacteria/Chloroplast, Fibrobacter, Fusobacteria, Green sulfur bacteria) are listed in Table 1 and in Fig. 3.

1

Bacterial clones from each library listed with phylogenetic group, closest match, and percent similarity

Sample Clone Phylogenetic group Closest match organism or clone namea % Similarityb 
Free-living, 1998, 40 clones 33-FL2, 5, 9, 12, 15 17, 25, 35, 44, 46, 47, 51, 52, 58, 63, 64, 66, 68, 70, 75, 81, 84, 88 B98 γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 33-FL38, 48, 100 B98 γ-Proteobacteria Calyptogena symbiont (AF035724) 95.5–95.9 
 33-FL95B98 γ-Proteobacteria KTc1113 (AF235115) 95.9 
 33-FL1, 21, 41 B98 γ-Proteobacteria Pseudomonas spp. (AY040872) 99.1–99.8 
 33-FL111B98 γ-Proteobacteria JTB254 (AB015253) 94.2 
 33-FL49B98 ε-Proteobacteria S17sBac16 (AF299121) 95.6 
 33-FL69, 76 B98 ε-Proteobacteria S17sBac16 (AF299121) 96.7 
 33-FL33B98 ε-Proteobacteria PVB OTU 6 (U15106) 93.7 
 33-FL40B98 ε-Proteobacteria Thiomicrospira spp. (L40808) 89.1 
 33-FL14B98 ε-Proteobacteria Thiomicrospira spp. (L40808) 92.5 
 33-FL128B98 α-Proteobacteria ZD0409 (AJ400350) 99.5 
 33-FL82B98 α-Proteobacteria SAR220 (U75257) 96.7 
 33-FL60B98 Fusobacteria BSO19 (AF366265) 98.1 
Free-living, 1999, 39 clones 33-FL2, 15, 21, 36, 40, 47, 51, 53, 64, 79, 82, 91 B99 γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 33-FL4, 61, 89, 132 B99 γ-Proteobacteria TW-7 (AY028202) 99.3–100 
 33-FL1, 141 B99 γ-Proteobacteria ZD0405 (AJ400348) 98.9–99.1 
 33-FL10B99 γ-Proteobacteria OM23 (U70694) 98.7 
 33-FL26B99 γ-Proteobacteria Alteromonas spp. (Y18230) 96.8 
 33-FL131B99 γ-Proteobacteria Acinetobacter spp. (Z93438) 99.3 
 33-FL8, 92, 124 B99 ε-Proteobacteria S17sBac16 (AF299121) 92.3–92.5 
 33-FL37B99 ε-Proteobacteria S17sBac16 (AF299121) 98.3 
 33-FL35B99 ε-Proteobacteria S17sBac16 (AF299121) 93.6 
 33-FL80B99 ε-Proteobacteria VC2.1 Bac32 (AF068806) 95 
 33-FL71, 127 B99 ε-Proteobacteria Alvinella isolate (AJ309654) 95–95.3 
 33-FL49B99 ε-Proteobacteria UPB C3 (AF111864) 94.3 
 33-FL98B99 ε-Proteobacteria VC1.2-c151 97.5 
 33-FL11, 85, 97 B99 α-Proteobacteria ZD0409 (AJ400350) 98.6–99.7 
 33-FL9B99 α-Proteobacteria Sphingomonas spp. (AB033944) 99.8 
 33-FL34B99 β-Proteobacteria TAF-B73 (AY038714) 100 
 33-FL54B99 β-Proteobacteria TM24 (X97093) 99.1 
 33-FL67B99 Planctomycetales HstpL83 (AF159642) 98.3 
 33-FL95B99 Cyanobacteria 5×15 (AJ289785) 99.5 
Free-living, 2000, 40 clones 33-FL1, 12, 22, 40, 47, 62, 66, 82, 83, 86 89, 91, 97, 102, 103 B00 γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 33-FL33, 87 B00 γ-Proteobacteria ZD0405 (AJ400348) 98.8–98.9 
 33-FL84B00 γ-Proteobacteria DHB-2 (AF257292) 98.6 
 33-FL92B00 γ-Proteobacteria ZD0417 (AJ400353) 93.3 
 33-FL4, 11, 94 B00 ε-Proteobacteria JTB360 (AB015259) 90.3–91.3 
 33-FL80B00 ε-Proteobacteria 1014 (AB030587) 86.4 
 33-FL101B00 ε-Proteobacteria VC2.1 Bac4 (AF068786) 89.1 
 33-FL98B00 ε-Proteobacteria VC2.1 Bac32 (AF068806) 91.6 
 33-FL99B00 ε-Proteobacteria VC2.1 Bac32 (AF068806) 92 
 33-FL58B00 ε-Proteobacteria Vent worm symbiont (D83061) 90.8 
 33-FL74B00 ε-Proteobacteria Vent worm symbiont (D83061) 96.4 
 33-FL88B00 ε-Proteobacteria Vent worm symbiont (D83061) 89.9 
 33-FL70B00 ε-Proteobacteria CHA3-437 (AJ132728) 90.2 
 33-FL39B00 ε-Proteobacteria PVB OTU 6 (U15106) 95.6 
 33-FL51B00 ε-Proteobacteria Thiomicrospira spp. (L40808) 91.5 
 33-FL65B00 ε-Proteobacteria KTc1160 (AF235116) 84.6 
 33-FL48B00 ε-Proteobacteria VC2.1-c102 (AF367482) 96.1 
 33-FL34B00 ε-Proteobacteria BD1-29 (AB015529) 90.3 
 33-FL76B00 ε-Proteobacteria Arcobacter spp. (AJ271654) 93.9 
 33-FL60B00 α-Proteobacteria ZD0409 (AJ400350) 98.2 
 33-FL85B00 α-Proteobacteria ZD0418 (AJ400354) 99.3 
 33-FL50B00 Cand division ABY1 CL500-94 (AF316799) 81.4 
 33-FL71B00 Fibrobacter WCHB1-77 (AF050590) 80 
Particle-attached, 1998, 46 clones 33-PA3, 47, 60, 68, 89, 93, 102, 132 B98 γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 33-PA121, 129 B98 γ-Proteobacteria Bathymodiolus symbiont (AB036711) 93.4 
 33-PA143B98 γ-Proteobacteria Oceanospirillum spp. (AB006767) 95.2 
 33-PA80B98 γ-Proteobacteria Calyptogena symbiont (AF035724) 95.7 
 33-PA76B98 γ-Proteobacteria Alteromonas spp. (AJ294360) 97.1 
 33-PA4, 17, 64, 73 B98 ε-Proteobacteria JTB315 (AB015258) 88.5–90.4 
 33-PA72B98 ε-Proteobacteria JTB315 (AB015258) 93.8 
 33-PA2, 12, 14, 59, 71, 74, 79, 86 B98 ε-Proteobacteria S17sBac16 (AF299121) 92–92.5 
 33-PA42B98 ε-Proteobacteria S17sBac16 (AF299121) 95.6 
 33-PA70B98 ε-Proteobacteria S17sBac16 (AF299121) 92.8 
 33-PA84B98 ε-Proteobacteria Vent worm symbiont (D83061) 94.5 
 33-PA38, 65, 78 B98 ε-Proteobacteria PVB OTU 6 (U15106) 93.3–93.9 
 33-PA61B98 ε-Proteobacteria PVB OTU 6 (U15106) 92.3 
 33-PA75B98 ε-Proteobacteria Thiomicrospira spp. (L40808) 92.8 
 33-PA62B98 ε-Proteobacteria KTc1160 (AF235116) 93.4 
 33-PA26B98 ε-Proteobacteria 525 (AJ404370) 93.5 
 33-PA16B98 ε-Proteobacteria S17sBac17 (AF299122) 91.4 
 33-PA85B98 ε-Proteobacteria VC1.2-c115 (AF367502) 85.9 
 33-PA124B98 α-Proteobacteria MB11E07 (AY033306) 94.6 
 33-PA19B98 α-Proteobacteria MB13E08 (AY033327) 97 
 33-PA63B98 CFBc CD2E4 (AY038476) 97.1 
 33-PA27B98 β-Proteobacteria SAP II (AF052387) 99.7 
 33-PA45B98 β-Proteobacteria Nitrospira spp. (X84661) 87.5 
 33-PA119B98 δ-Proteobacteria NAC60-5 (AF245646) 93.7 
 33-PA95B98 δ-Proteobacteria Desulfuromusa spp. (X79412) 96.1 
 33-PA105B98 Thermodesulfobacterium Thermodesulfotobacterium spp. (AF255595) 85 
Particle-attached, 1999, 38 clones 33-PA38, 56, 61, 64, 84, 100, 105 B99 γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 33-PA5, 22, 37, 49 B99 γ-Proteobacteria TW-7 (AY028202) 98.6–99.5 
 33-PA57B99 γ-Proteobacteria Alteromonas spp. (AB049730) 99 
 33-PA4B99 ε-Proteobacteria JTB360 (AB015259) 91 
 33-PA132B99 ε-Proteobacteria S17sBac16 (AF299121) 91.4 
 33-PA25, 63 B99 ε-Proteobacteria S17sBac16 (AF299121) 95.8–96.1 
 33-PA99B99 ε-Proteobacteria S17sBac16 (AF299121) 92.5 
 33-PA52, 111 B99 ε-Proteobacteria Alvinella isolate (AJ309654) 95–95.5 
 33-PA54B99 ε-Proteobacteria NKB8 (AB013260) 97.5 
 33-PA39B99 α-Proteobacteria Sphingomonas spp. (AB033944) 99.6 
 33-PA93B99 α-Proteobacteria BHA-19 (AF090535) 94.3 
 33-PA14B99 α-Proteobacteria SAR 11 group (U75649) 97.4 
 33-PA17B99 α-Proteobacteria Sphingomonas spp. (X97776) 99.5 
 33-PA44B99 CFB JTB250 (AB015264) 88.7 
 33-PA87, 90, 91 B99 CFB O6 (AF361205) 91.2 
 33-PA7B99 β-Proteobacteria TM24 (X97093) 98.9 
 33-PA114B99 Cand division ABY1 CL500-13 (AF316798) 81.4 
 33-PA66B99 δ-Proteobacteria 2-400 C2.4 A/S6/S10/S15 (Z77531) 89.7 
 33-PA59B99 Gram-positive, high G+C CD3F7 (AY038453) 91.9 
 33-PA134B99 Gram-positive, high G+C OCS155 (AF001652) 90.4 
 33-PA109B99 Gram-positive, low G+C Streptococcus spp. (AF385523) 98.3 
 33-PA26B99 Fibrobacter OCS307 (U41450) 89.4 
 33-PA65B99 Planctomycetales HstpL8 (AF159639) 86.2 
 33-PA48B99 Cyanobacteria Cyanothece spp. (AF132771) 78 
 33-PA42B99 Desulfurobacterium VC2.1 Bac3 (AF068785) 94 
Particle-attached, 2000, 43 clones 33-PA70B00 γ-Proteobacteria Maorithyas symbiont (AB042413) 96.1 
 33-PA60B00 γ-Proteobacteria Pseudoalteromonas spp. (AJ391202) 99.7 
 33-PA34B00 γ-Proteobacteria Codakia spp. symbiont (X84979) 87.5 
 33-PA57B00 γ-Proteobacteria Moraxella spp. (X95304) 98.8 
 33-PA31, 43 B00 ε-Proteobacteria S17sBac16 (AF299121) 92 
 33-PA14B00 ε-Proteobacteria BD2-5 (AB015535) 86.4 
 33-PA61B00 ε-Proteobacteria BD2-5 (AB015535) 89.2 
 33-PA18, 79 B00 ε-Proteobacteria 1014 (AB030587) 86.3–87.1 
 33-PA3B00 ε-Proteobacteria VC2.1 Bac4 (AF068786) 90.6 
 33-PA46B00 ε-Proteobacteria PVB OTU 6 (U15106) 95 
 33-PA6, 33 B00 ε-Proteobacteria CHA3-437 (AJ132728) 95.5 
 33-PA74B00 ε-Proteobacteria CHA3-437 (AJ132728) 95 
 33-PA4B00 ε-Proteobacteria VC2.1-c102 (AF367482) 97.6 
 33-PA16B00 ε-Proteobacteria 525 (AJ404370) 91.2 
 33-PA8B00 ε-Proteobacteria NKB11 (AB013263) 92 
 33-PA11B00 ε-Proteobacteria JTB 146 (AB015257) 95.1 
 33-PA80B00 ε-Proteobacteria BD1-5 (AB015518) 84.5 
 33-PA55B00 ε-Proteobacteria SB-17 (AF029044) 94.2 
 33-PA19B00 ε-Proteobacteria ODPB-B3 (AF121088) 92.3 
 33-PA54B00 ε-Proteobacteria GCA014 (AF154101) 98.7 
 33-PA52B00 ε-Proteobacteria NKB13 (AB013265) 94.7 
 33-PA36B00 ε-Proteobacteria Wolinella spp. (AF273252) 85.2 
 33-PA28B00 ε-Proteobacteria Arcobacter spp. (AJ271654) 93.6 
 33-PA5B00 ε-Proteobacteria Arcobacter spp. (AJ271653) 91.5 
 33-PA27B00 ε-Proteobacteria Helicobacter spp. (M88137) 85.2 
 33-PA62B00 ε-Proteobacteria R. exoculata symbiont (U29081) 85.5 
 33-PA23B00 ε-Proteobacteria Campylobacter spp. (Y11763) 73.3 
 33-PA49, 53 B00 α-Proteobacteria ZD0409 (AJ400350) 98.9–99.5 
 33-PA75B00 CFB Flexibacter spp. (M58786) 82.5 
 33-PA1B00 CFB OM273 (U70709) 84.5 
 33-PA7B00 β-Proteobacteria TAF-B73 (AY038714) 99.7 
 33-PA77B00 Cand division ABY1 BD7-1 (AB015577) 85.3 
 33-PA24B00 Cand division ABY1 CL500-94 (AF316799) 80.3 
 33-PA51B00 Cand division WS6 BMS_29 (AF172926) 85 
 33-PA76B00 Gram-positive, high G+C Rhodococcus spp. (X85240) 78.3 
 33-PA12B00 Gram-positive, low G+C Ammoniphilus spp. (Y14580) 76.3 
 33-PA2B00 Fibrobacter Lactosphaera spp. (AF394926) 81.8 
 33-PA67B00 Chloroplast Chloroplast clone OM21 (U32671) 98.3 
 33-PA44B00 Green sulfur bacteria BSV40 (AJ229196) 89.1 
Background CTD, 41 clones CTD 8, 16, 21 B γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 CTD4B γ-Proteobacteria KTc1113 (AF235115) 96.1 
 CTD12B γ-Proteobacteria KTc1113 (AF235115) 90.6 
 CTD60B γ-Proteobacteria ZD0405 (AJ400348) 98.9 
 CTD27B γ-Proteobacteria Marinomonas spp. (AF063027) 88.9 
 CTD70B γ-Proteobacteria Alteromonas spp. (AF173965) 95 
 CTD42B γ-Proteobacteria NCE312 (AF295032) 99.4 
 CTD33, 41B γ-Proteobacteria Alteromonas spp. (AJ294360) 99.1–99.3 
 CTD10B γ-Proteobacteria Marinobacter spp. (AJ000647) 98.1 
 CTD53B γ-Proteobacteria CHAB-IV-19 (AJ240914) 97.8 
 CTD18B γ-Proteobacteria ZD0433 (AJ400356) 98.6 
 CTD47B γ-Proteobacteria Mussel symbiont (U05595) 93.4 
 CTD7B α-Proteobacteria ZD0409 (AJ400350) 98.9 
 CTD 3, 6, 31 B α-Proteobacteria SAR220 (U75257) 96.2–97.4 
 CTD44B α-Proteobacteria BHA-19 (AF090535) 94.8 
 CTD 2, 20, 67 B α-Proteobacteria SAR 11 group (U75649) 97.6–98.2 
 CTD 1, 36, 81 B α-Proteobacteria MB12A07 (AY033312) 97.1–97.9 
 CTD 23, 80 B α-Proteobacteria Erythrobacter spp. (AF025560) 99.3 
 CTD56B α-Proteobacteria QSSC9-5 (AF170750) 96.1 
 CTD30B α-Proteobacteria M3C203B-B (AF395031) 73.7 
 CTD14B α-Proteobacteria OM65 (U70682) 98.9 
 CTD49B α-Proteobacteria CD3E2 (AY038455) 93 
 CTD26B α-Proteobacteria SAR241 (U75258) 95.9 
 CTD 9, 37B CFB BIjiii32 (AJ318154) 86 
 CTD28B CFB R7695 (AJ278780) 99.1 
 CTD5B CFB ZD0255 (AJ400343) 96.5 
 CTD40B CFB OM188 (U70687) 94.5 
 CTD73B δ-Proteobacteria BD4-10 (AB015560) 90 
 CTD38B δ-Proteobacteria UPB A1 (AF111866) 84.2 
 CTD29B Planctomycetales Pirellula spp. (AJ231180) 86.9 
Sample Clone Phylogenetic group Closest match organism or clone namea % Similarityb 
Free-living, 1998, 40 clones 33-FL2, 5, 9, 12, 15 17, 25, 35, 44, 46, 47, 51, 52, 58, 63, 64, 66, 68, 70, 75, 81, 84, 88 B98 γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 33-FL38, 48, 100 B98 γ-Proteobacteria Calyptogena symbiont (AF035724) 95.5–95.9 
 33-FL95B98 γ-Proteobacteria KTc1113 (AF235115) 95.9 
 33-FL1, 21, 41 B98 γ-Proteobacteria Pseudomonas spp. (AY040872) 99.1–99.8 
 33-FL111B98 γ-Proteobacteria JTB254 (AB015253) 94.2 
 33-FL49B98 ε-Proteobacteria S17sBac16 (AF299121) 95.6 
 33-FL69, 76 B98 ε-Proteobacteria S17sBac16 (AF299121) 96.7 
 33-FL33B98 ε-Proteobacteria PVB OTU 6 (U15106) 93.7 
 33-FL40B98 ε-Proteobacteria Thiomicrospira spp. (L40808) 89.1 
 33-FL14B98 ε-Proteobacteria Thiomicrospira spp. (L40808) 92.5 
 33-FL128B98 α-Proteobacteria ZD0409 (AJ400350) 99.5 
 33-FL82B98 α-Proteobacteria SAR220 (U75257) 96.7 
 33-FL60B98 Fusobacteria BSO19 (AF366265) 98.1 
Free-living, 1999, 39 clones 33-FL2, 15, 21, 36, 40, 47, 51, 53, 64, 79, 82, 91 B99 γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 33-FL4, 61, 89, 132 B99 γ-Proteobacteria TW-7 (AY028202) 99.3–100 
 33-FL1, 141 B99 γ-Proteobacteria ZD0405 (AJ400348) 98.9–99.1 
 33-FL10B99 γ-Proteobacteria OM23 (U70694) 98.7 
 33-FL26B99 γ-Proteobacteria Alteromonas spp. (Y18230) 96.8 
 33-FL131B99 γ-Proteobacteria Acinetobacter spp. (Z93438) 99.3 
 33-FL8, 92, 124 B99 ε-Proteobacteria S17sBac16 (AF299121) 92.3–92.5 
 33-FL37B99 ε-Proteobacteria S17sBac16 (AF299121) 98.3 
 33-FL35B99 ε-Proteobacteria S17sBac16 (AF299121) 93.6 
 33-FL80B99 ε-Proteobacteria VC2.1 Bac32 (AF068806) 95 
 33-FL71, 127 B99 ε-Proteobacteria Alvinella isolate (AJ309654) 95–95.3 
 33-FL49B99 ε-Proteobacteria UPB C3 (AF111864) 94.3 
 33-FL98B99 ε-Proteobacteria VC1.2-c151 97.5 
 33-FL11, 85, 97 B99 α-Proteobacteria ZD0409 (AJ400350) 98.6–99.7 
 33-FL9B99 α-Proteobacteria Sphingomonas spp. (AB033944) 99.8 
 33-FL34B99 β-Proteobacteria TAF-B73 (AY038714) 100 
 33-FL54B99 β-Proteobacteria TM24 (X97093) 99.1 
 33-FL67B99 Planctomycetales HstpL83 (AF159642) 98.3 
 33-FL95B99 Cyanobacteria 5×15 (AJ289785) 99.5 
Free-living, 2000, 40 clones 33-FL1, 12, 22, 40, 47, 62, 66, 82, 83, 86 89, 91, 97, 102, 103 B00 γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 33-FL33, 87 B00 γ-Proteobacteria ZD0405 (AJ400348) 98.8–98.9 
 33-FL84B00 γ-Proteobacteria DHB-2 (AF257292) 98.6 
 33-FL92B00 γ-Proteobacteria ZD0417 (AJ400353) 93.3 
 33-FL4, 11, 94 B00 ε-Proteobacteria JTB360 (AB015259) 90.3–91.3 
 33-FL80B00 ε-Proteobacteria 1014 (AB030587) 86.4 
 33-FL101B00 ε-Proteobacteria VC2.1 Bac4 (AF068786) 89.1 
 33-FL98B00 ε-Proteobacteria VC2.1 Bac32 (AF068806) 91.6 
 33-FL99B00 ε-Proteobacteria VC2.1 Bac32 (AF068806) 92 
 33-FL58B00 ε-Proteobacteria Vent worm symbiont (D83061) 90.8 
 33-FL74B00 ε-Proteobacteria Vent worm symbiont (D83061) 96.4 
 33-FL88B00 ε-Proteobacteria Vent worm symbiont (D83061) 89.9 
 33-FL70B00 ε-Proteobacteria CHA3-437 (AJ132728) 90.2 
 33-FL39B00 ε-Proteobacteria PVB OTU 6 (U15106) 95.6 
 33-FL51B00 ε-Proteobacteria Thiomicrospira spp. (L40808) 91.5 
 33-FL65B00 ε-Proteobacteria KTc1160 (AF235116) 84.6 
 33-FL48B00 ε-Proteobacteria VC2.1-c102 (AF367482) 96.1 
 33-FL34B00 ε-Proteobacteria BD1-29 (AB015529) 90.3 
 33-FL76B00 ε-Proteobacteria Arcobacter spp. (AJ271654) 93.9 
 33-FL60B00 α-Proteobacteria ZD0409 (AJ400350) 98.2 
 33-FL85B00 α-Proteobacteria ZD0418 (AJ400354) 99.3 
 33-FL50B00 Cand division ABY1 CL500-94 (AF316799) 81.4 
 33-FL71B00 Fibrobacter WCHB1-77 (AF050590) 80 
Particle-attached, 1998, 46 clones 33-PA3, 47, 60, 68, 89, 93, 102, 132 B98 γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 33-PA121, 129 B98 γ-Proteobacteria Bathymodiolus symbiont (AB036711) 93.4 
 33-PA143B98 γ-Proteobacteria Oceanospirillum spp. (AB006767) 95.2 
 33-PA80B98 γ-Proteobacteria Calyptogena symbiont (AF035724) 95.7 
 33-PA76B98 γ-Proteobacteria Alteromonas spp. (AJ294360) 97.1 
 33-PA4, 17, 64, 73 B98 ε-Proteobacteria JTB315 (AB015258) 88.5–90.4 
 33-PA72B98 ε-Proteobacteria JTB315 (AB015258) 93.8 
 33-PA2, 12, 14, 59, 71, 74, 79, 86 B98 ε-Proteobacteria S17sBac16 (AF299121) 92–92.5 
 33-PA42B98 ε-Proteobacteria S17sBac16 (AF299121) 95.6 
 33-PA70B98 ε-Proteobacteria S17sBac16 (AF299121) 92.8 
 33-PA84B98 ε-Proteobacteria Vent worm symbiont (D83061) 94.5 
 33-PA38, 65, 78 B98 ε-Proteobacteria PVB OTU 6 (U15106) 93.3–93.9 
 33-PA61B98 ε-Proteobacteria PVB OTU 6 (U15106) 92.3 
 33-PA75B98 ε-Proteobacteria Thiomicrospira spp. (L40808) 92.8 
 33-PA62B98 ε-Proteobacteria KTc1160 (AF235116) 93.4 
 33-PA26B98 ε-Proteobacteria 525 (AJ404370) 93.5 
 33-PA16B98 ε-Proteobacteria S17sBac17 (AF299122) 91.4 
 33-PA85B98 ε-Proteobacteria VC1.2-c115 (AF367502) 85.9 
 33-PA124B98 α-Proteobacteria MB11E07 (AY033306) 94.6 
 33-PA19B98 α-Proteobacteria MB13E08 (AY033327) 97 
 33-PA63B98 CFBc CD2E4 (AY038476) 97.1 
 33-PA27B98 β-Proteobacteria SAP II (AF052387) 99.7 
 33-PA45B98 β-Proteobacteria Nitrospira spp. (X84661) 87.5 
 33-PA119B98 δ-Proteobacteria NAC60-5 (AF245646) 93.7 
 33-PA95B98 δ-Proteobacteria Desulfuromusa spp. (X79412) 96.1 
 33-PA105B98 Thermodesulfobacterium Thermodesulfotobacterium spp. (AF255595) 85 
Particle-attached, 1999, 38 clones 33-PA38, 56, 61, 64, 84, 100, 105 B99 γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 33-PA5, 22, 37, 49 B99 γ-Proteobacteria TW-7 (AY028202) 98.6–99.5 
 33-PA57B99 γ-Proteobacteria Alteromonas spp. (AB049730) 99 
 33-PA4B99 ε-Proteobacteria JTB360 (AB015259) 91 
 33-PA132B99 ε-Proteobacteria S17sBac16 (AF299121) 91.4 
 33-PA25, 63 B99 ε-Proteobacteria S17sBac16 (AF299121) 95.8–96.1 
 33-PA99B99 ε-Proteobacteria S17sBac16 (AF299121) 92.5 
 33-PA52, 111 B99 ε-Proteobacteria Alvinella isolate (AJ309654) 95–95.5 
 33-PA54B99 ε-Proteobacteria NKB8 (AB013260) 97.5 
 33-PA39B99 α-Proteobacteria Sphingomonas spp. (AB033944) 99.6 
 33-PA93B99 α-Proteobacteria BHA-19 (AF090535) 94.3 
 33-PA14B99 α-Proteobacteria SAR 11 group (U75649) 97.4 
 33-PA17B99 α-Proteobacteria Sphingomonas spp. (X97776) 99.5 
 33-PA44B99 CFB JTB250 (AB015264) 88.7 
 33-PA87, 90, 91 B99 CFB O6 (AF361205) 91.2 
 33-PA7B99 β-Proteobacteria TM24 (X97093) 98.9 
 33-PA114B99 Cand division ABY1 CL500-13 (AF316798) 81.4 
 33-PA66B99 δ-Proteobacteria 2-400 C2.4 A/S6/S10/S15 (Z77531) 89.7 
 33-PA59B99 Gram-positive, high G+C CD3F7 (AY038453) 91.9 
 33-PA134B99 Gram-positive, high G+C OCS155 (AF001652) 90.4 
 33-PA109B99 Gram-positive, low G+C Streptococcus spp. (AF385523) 98.3 
 33-PA26B99 Fibrobacter OCS307 (U41450) 89.4 
 33-PA65B99 Planctomycetales HstpL8 (AF159639) 86.2 
 33-PA48B99 Cyanobacteria Cyanothece spp. (AF132771) 78 
 33-PA42B99 Desulfurobacterium VC2.1 Bac3 (AF068785) 94 
Particle-attached, 2000, 43 clones 33-PA70B00 γ-Proteobacteria Maorithyas symbiont (AB042413) 96.1 
 33-PA60B00 γ-Proteobacteria Pseudoalteromonas spp. (AJ391202) 99.7 
 33-PA34B00 γ-Proteobacteria Codakia spp. symbiont (X84979) 87.5 
 33-PA57B00 γ-Proteobacteria Moraxella spp. (X95304) 98.8 
 33-PA31, 43 B00 ε-Proteobacteria S17sBac16 (AF299121) 92 
 33-PA14B00 ε-Proteobacteria BD2-5 (AB015535) 86.4 
 33-PA61B00 ε-Proteobacteria BD2-5 (AB015535) 89.2 
 33-PA18, 79 B00 ε-Proteobacteria 1014 (AB030587) 86.3–87.1 
 33-PA3B00 ε-Proteobacteria VC2.1 Bac4 (AF068786) 90.6 
 33-PA46B00 ε-Proteobacteria PVB OTU 6 (U15106) 95 
 33-PA6, 33 B00 ε-Proteobacteria CHA3-437 (AJ132728) 95.5 
 33-PA74B00 ε-Proteobacteria CHA3-437 (AJ132728) 95 
 33-PA4B00 ε-Proteobacteria VC2.1-c102 (AF367482) 97.6 
 33-PA16B00 ε-Proteobacteria 525 (AJ404370) 91.2 
 33-PA8B00 ε-Proteobacteria NKB11 (AB013263) 92 
 33-PA11B00 ε-Proteobacteria JTB 146 (AB015257) 95.1 
 33-PA80B00 ε-Proteobacteria BD1-5 (AB015518) 84.5 
 33-PA55B00 ε-Proteobacteria SB-17 (AF029044) 94.2 
 33-PA19B00 ε-Proteobacteria ODPB-B3 (AF121088) 92.3 
 33-PA54B00 ε-Proteobacteria GCA014 (AF154101) 98.7 
 33-PA52B00 ε-Proteobacteria NKB13 (AB013265) 94.7 
 33-PA36B00 ε-Proteobacteria Wolinella spp. (AF273252) 85.2 
 33-PA28B00 ε-Proteobacteria Arcobacter spp. (AJ271654) 93.6 
 33-PA5B00 ε-Proteobacteria Arcobacter spp. (AJ271653) 91.5 
 33-PA27B00 ε-Proteobacteria Helicobacter spp. (M88137) 85.2 
 33-PA62B00 ε-Proteobacteria R. exoculata symbiont (U29081) 85.5 
 33-PA23B00 ε-Proteobacteria Campylobacter spp. (Y11763) 73.3 
 33-PA49, 53 B00 α-Proteobacteria ZD0409 (AJ400350) 98.9–99.5 
 33-PA75B00 CFB Flexibacter spp. (M58786) 82.5 
 33-PA1B00 CFB OM273 (U70709) 84.5 
 33-PA7B00 β-Proteobacteria TAF-B73 (AY038714) 99.7 
 33-PA77B00 Cand division ABY1 BD7-1 (AB015577) 85.3 
 33-PA24B00 Cand division ABY1 CL500-94 (AF316799) 80.3 
 33-PA51B00 Cand division WS6 BMS_29 (AF172926) 85 
 33-PA76B00 Gram-positive, high G+C Rhodococcus spp. (X85240) 78.3 
 33-PA12B00 Gram-positive, low G+C Ammoniphilus spp. (Y14580) 76.3 
 33-PA2B00 Fibrobacter Lactosphaera spp. (AF394926) 81.8 
 33-PA67B00 Chloroplast Chloroplast clone OM21 (U32671) 98.3 
 33-PA44B00 Green sulfur bacteria BSV40 (AJ229196) 89.1 
Background CTD, 41 clones CTD 8, 16, 21 B γ-Proteobacteria Maorithyas symbiont (AB042413) 95.7–96.5 
 CTD4B γ-Proteobacteria KTc1113 (AF235115) 96.1 
 CTD12B γ-Proteobacteria KTc1113 (AF235115) 90.6 
 CTD60B γ-Proteobacteria ZD0405 (AJ400348) 98.9 
 CTD27B γ-Proteobacteria Marinomonas spp. (AF063027) 88.9 
 CTD70B γ-Proteobacteria Alteromonas spp. (AF173965) 95 
 CTD42B γ-Proteobacteria NCE312 (AF295032) 99.4 
 CTD33, 41B γ-Proteobacteria Alteromonas spp. (AJ294360) 99.1–99.3 
 CTD10B γ-Proteobacteria Marinobacter spp. (AJ000647) 98.1 
 CTD53B γ-Proteobacteria CHAB-IV-19 (AJ240914) 97.8 
 CTD18B γ-Proteobacteria ZD0433 (AJ400356) 98.6 
 CTD47B γ-Proteobacteria Mussel symbiont (U05595) 93.4 
 CTD7B α-Proteobacteria ZD0409 (AJ400350) 98.9 
 CTD 3, 6, 31 B α-Proteobacteria SAR220 (U75257) 96.2–97.4 
 CTD44B α-Proteobacteria BHA-19 (AF090535) 94.8 
 CTD 2, 20, 67 B α-Proteobacteria SAR 11 group (U75649) 97.6–98.2 
 CTD 1, 36, 81 B α-Proteobacteria MB12A07 (AY033312) 97.1–97.9 
 CTD 23, 80 B α-Proteobacteria Erythrobacter spp. (AF025560) 99.3 
 CTD56B α-Proteobacteria QSSC9-5 (AF170750) 96.1 
 CTD30B α-Proteobacteria M3C203B-B (AF395031) 73.7 
 CTD14B α-Proteobacteria OM65 (U70682) 98.9 
 CTD49B α-Proteobacteria CD3E2 (AY038455) 93 
 CTD26B α-Proteobacteria SAR241 (U75258) 95.9 
 CTD 9, 37B CFB BIjiii32 (AJ318154) 86 
 CTD28B CFB R7695 (AJ278780) 99.1 
 CTD5B CFB ZD0255 (AJ400343) 96.5 
 CTD40B CFB OM188 (U70687) 94.5 
 CTD73B δ-Proteobacteria BD4-10 (AB015560) 90 
 CTD38B δ-Proteobacteria UPB A1 (AF111866) 84.2 
 CTD29B Planctomycetales Pirellula spp. (AJ231180) 86.9 

aBased on BLAST search. GenBank accession numbers are in parentheses.

bBased on alignable base pairs.

cCFB, Cytophaga–Flexibacter–Bacteroides.

2

Distribution of identical bacterial clones (≥97% similarity) that appeared in more than one clone library

Phylogenetic group Representative clone Clone librarya 
  PA98 PA99 PA00 FL98 FL99 FL00 CTD 
γ-Proteobacteria 33-FL97B00 23 12 15 
 CTD33B      
 33-FL87B00     
 33-FL132B99      
 33-FL95B98      
 33-PA80B98      
β-Proteobacteria 33-FL34B99      
 33-FL54B99      
α-Proteobacteria CTD2B      
 CTD44B      
 CTD31B      
 33-FL9B99      
 33-FL60B00   
ε-Proteobacteria 33-PA28B00      
 33-FL48B00      
 33-FL127B99      
 33-PA75B98      
 33-PA46B00      
 33-FL74B00      
 33-PA79B00      
 33-FL76B98     
 33-PA42B98      
 33-PA70B98     
 33-PA86B98     
 33-FL4B00      
Phylogenetic group Representative clone Clone librarya 
  PA98 PA99 PA00 FL98 FL99 FL00 CTD 
γ-Proteobacteria 33-FL97B00 23 12 15 
 CTD33B      
 33-FL87B00     
 33-FL132B99      
 33-FL95B98      
 33-PA80B98      
β-Proteobacteria 33-FL34B99      
 33-FL54B99      
α-Proteobacteria CTD2B      
 CTD44B      
 CTD31B      
 33-FL9B99      
 33-FL60B00   
ε-Proteobacteria 33-PA28B00      
 33-FL48B00      
 33-FL127B99      
 33-PA75B98      
 33-PA46B00      
 33-FL74B00      
 33-PA79B00      
 33-FL76B98     
 33-PA42B98      
 33-PA70B98     
 33-PA86B98     
 33-FL4B00      

aPA, particle-attached; FL, free-living; CTD is the background seawater sample. The number is the last two digits of the year of sampling.

3

Composition of the Marker 33 particle-attached and free-living bacterial clone libraries in 1998, 1999, and 2000 based on taxonomic groupings of 16S rRNA sequences. The background sample is shown as well.

3

Composition of the Marker 33 particle-attached and free-living bacterial clone libraries in 1998, 1999, and 2000 based on taxonomic groupings of 16S rRNA sequences. The background sample is shown as well.

4

Phylogenetic trees as determined by maximum likelihood analyses of bacterial 16S rRNA clones for (a) α-, δ-, γ-proteobacteria, planctomyces, and Cytophaga–Flexibacter–Bacteroides, (b) Gram-positives, green sulfur bacteria, Fibrobacter, and β-proteobacteria, (c) ε-proteobacteria, and (d) high temperature bacteria and novel candidate divisions. Clones from this study are indicated in bold large font and labeled with PA (particle-attached), FL (free-living), CTD (background seawater), and the appropriate year (98, 99, or 00). Accession numbers for GenBank are provided for clones not from this study. The percentage of bootstrap resamplings above 50% is indicated. The scale bar represents the expected number of changes per nucleotide position.

4

Phylogenetic trees as determined by maximum likelihood analyses of bacterial 16S rRNA clones for (a) α-, δ-, γ-proteobacteria, planctomyces, and Cytophaga–Flexibacter–Bacteroides, (b) Gram-positives, green sulfur bacteria, Fibrobacter, and β-proteobacteria, (c) ε-proteobacteria, and (d) high temperature bacteria and novel candidate divisions. Clones from this study are indicated in bold large font and labeled with PA (particle-attached), FL (free-living), CTD (background seawater), and the appropriate year (98, 99, or 00). Accession numbers for GenBank are provided for clones not from this study. The percentage of bootstrap resamplings above 50% is indicated. The scale bar represents the expected number of changes per nucleotide position.

The γ-proteobacteria group

The largest number of clones found in all Marker 33 fluid samples and the deep-sea background seawater belonged to the γ-proteobacteria (115 clones, Fig. 4a). Sixty-nine of these clones were ≥97% identical to one another and most closely related (96–97%) to the endosymbionts of the cold seep, deep-sea bivalve Maorithyas hadalis[34]. Other clones in the γ-proteobacteria include those related to endosymbionts of hydrothermal vent mussels [35], deep-sea clams [36], and shallow tropical clams [37]. The remaining γ-proteobacteria were related to both cultured marine bacteria such as Marinomonas[38] and Alteromonas[39], as well as many uncultivated γ-proteobacteria from a variety of marine environments, including deep-sea sediments [40], North Atlantic coastal waters [41], surface waters in the North Sea [39], and deep-sea waters near Antarctica [42].

The ε-proteobacteria group

Clones belonging to the ε-proteobacteria (Fig. 4c) were found in every Marker 33 sample (92 clones) and were absent from the deep-sea background seawater. Twenty-four of these clones were most closely related (92–98%) to an uncultured bacterium, clone S17sBac16, from a hydrothermal vent microbial mat on the Southern East Pacific Rise [43]. Other clones in the ε-proteobacteria from Marker 33 were related to uncultivated marine bacterium, including clones from a deep-sea cold seep in the Japan Trench [40], a hydrothermal microbial mat [44], deep-sea sediments [45,46], a growth chamber associated with warm hydrothermal fluids [18,47], an ectosymbiont of the hydrothermal vent shrimp Rimicaris[48], and methane seep tubeworm endosymbionts [49]. Some of the clones from Marker 33 were also related to cultured ε-proteobacteria, such as Thiomicrospira denitrificans[50], Arcobacter spp. [51], and Campylobacter spp. [52]. There is also evidence for an increase in the species diversity of ε-proteobacteria from 1998 to 2000 (Table 3).

3

Bacterial clone library coverage, diversity, and species richness estimates

Samplea Coverage Species observed Shannon–Wiener ACEb Chao1 Epsilon phylotypes 
FL1998 77.50 13 1.69 32.47 40 
FL1999 66.67 19 2.52 45.35 52.8 
FL2000 50.00 23 2.56 157.66 156.33 15 
PA1998 54.35 26 2.88 140.73 173 13 
PA1999 47.37 25 2.97 102.45 105 
PA2000 18.60 39 3.63 209.62 175.11 23 
Total 1998 ND 35 2.77 113.69 104.44 16 
Total 1999 ND 37 3.09 124.87 141.14 10 
Total 2000 ND 57 3.69 240.86 266.45 35 
CTD 43.90 30 3.31 81.43 105.57 
Samplea Coverage Species observed Shannon–Wiener ACEb Chao1 Epsilon phylotypes 
FL1998 77.50 13 1.69 32.47 40 
FL1999 66.67 19 2.52 45.35 52.8 
FL2000 50.00 23 2.56 157.66 156.33 15 
PA1998 54.35 26 2.88 140.73 173 13 
PA1999 47.37 25 2.97 102.45 105 
PA2000 18.60 39 3.63 209.62 175.11 23 
Total 1998 ND 35 2.77 113.69 104.44 16 
Total 1999 ND 37 3.09 124.87 141.14 10 
Total 2000 ND 57 3.69 240.86 266.45 35 
CTD 43.90 30 3.31 81.43 105.57 

Total represents consolidated PA and FL libraries for that sample year.

CTD is background deep-sea water sample.

aPA is particle-attached, FL is free-living, followed by the year sampled.

bAbundance-based coverage estimator.

The α-proteobacteria group

Thirty-seven α-proteobacteria clones were found in the libraries, 18 of which were in the background seawater sample, and the remainder distributed throughout every Marker 33 vent sample (Fig. 4a). The majority of the clones were most closely related to uncultivated marine bacterioplankton, including the SAR11 cluster from coastal California waters [53], the Sargasso Sea [54], an ammonia biofilter [55], and the North Sea (unpublished). Additionally, a number of clones from this study were closely related to the cultured marine bacterium Sphingomonas spp. [56,57] and Erythrobacter spp. [58].

The Cytophaga–Flexiobacter–Bacteroides group

Clones belonging to the CFB group were found only in the particle-attached Marker 33 samples for all three years, as well as in the deep-sea background sample (12 clones, Fig. 4a). These clones were related to uncultivated marine CFB from a deep-sea cold seep [40], a mesoeutrophic reservoir [59], coastal waters off North Carolina [41], and the Sargasso Sea [54].

The β-proteobacteria group

Six β-proteobacteria clones were found in the Marker 33 samples, including all the particle-attached populations and the 1999 free-living population, and none was detected in background seawater (Fig. 4b). Clones were related to cultivated strains of Nitrospira spp. [60] as well as uncultured β-proteobacteria clones.

The δ-proteobacteria group

The five δ-proteobacteria clones from this study were found only in the particle-attached vent samples in two years (1998 and 2000), as well as in the background seawater sample (Fig. 4a). They were most closely related to uncultured δ-proteobacteria from the North Atlantic [61], deep-sea sediments [45], and New Jersey coastal waters [62].

Gram-positive bacteria, high G+C and low G+C

Five Gram-positive clones were found in the particle-attached Marker 33 populations, in both 1999 and 2000 (Fig. 4b). This included clones distantly related (<80%) to Rhodococcus spp. [63] and Ammoniphilus spp. [64], as well as those sequences from Marker 33 related to uncultured environmental clones.

Candidate divisions ABY1 and WS6

Four clones belonging to the candidate division ABY1 were found in the 1999 and 2000 particle-attached Marker 33 populations, as well as the 2000 free-living Marker 33 population (Fig. 4d). Three of these clones were most closely related to ABY1 environmental clones from Crater Lake [65], and the remaining clone to uncultivated bacteria from deep-sea sediments [45]. Only one clone from the 2000 particle-attached clone library, 33-PA51B00, fell into the candidate division WS6, and it was most closely related (85%) to an environmental clone from coastal marine sediments [66].

Thermodesulfobacterium/Desulfurobacterium

A single clone (33-PA105B98) belonging to the Thermodesulfobacterium was found in the 1998 particle-attached Marker 33 sample, and it was most closely related to an environmental clone from a hot spring sulfur mat in Iceland [67]. In the 1999 particle-attached Marker 33 sample, a single clone (33-PA42B99) was found that fell into the Desulfurobacterium group and was most closely related to a clone from the in situ growth chamber in warm hydrothermal fluid [18]. Both groups are shown in Fig. 4d.

Distribution of taxonomic groups and phylotypes in different clone libraries

Among the seven bacterial clone libraries, representation of the taxonomic groups and phylotypes differed greatly. Exact clones appeared in more than one library, as shown with a representative clone in Table 2; only α- and γ-proteobacteria were found in all seven libraries. Of the 17 taxonomic groups identified in this study, five were found in both the background library and the Marker 33 vent samples, and these included the α-, γ-, and δ-proteobacteria, Planctomycetales, and the Cytophaga–Flexibacter–Bacteroides (Fig. 4a). The background seawater sample was dominated by α- and γ-proteobacteria, compromising 80% of the clones from that library. The γ- and ε-proteobacteria made up >50% of sequenced clones from each Marker 33 library. The ε-proteobacteria was the only group found in all the Marker 33 libraries and not in the background seawater sample (Fig. 4c).

Diversity indices, species observed, and coverage

In each year, compared to the free-living Marker 33 populations, the particle-attached clone library from the same year always showed more species observed and a higher Shannon–Wiener index, ACE, and Chao1 estimator (Table 3). Additionally, the particle-attached populations for all three years had greater diversity of taxonomic groups than the free-living populations (Fig. 3). To compare the Marker 33 samples to the background sample, unique phylotypes in the particle-attached and free-living libraries for each year were consolidated to represent both fractions in one population, as they were in the background deep-sea sample. Based on species observed, the vent samples had more phylotypes than background seawater for all three years (Table 3). Vent samples also had more taxonomic groups than the background sample. The Shannon–Wiener index, though, is higher for the background sample compared to 1998 and 1999; the Marker 33 2000 sample has the highest biodiversity index, with a value of 3.69 (Table 3). ACE estimates show the background sample having the lowest richness; Chao1 estimates a similar value for 1998 and the background sample, with 1999 and 2000 having higher values. The Shannon–Wiener index, species observed, ACE and Chao1 estimators all increase over time at Marker 33 from 1998 to 2000 for the consolidated populations (Table 3). Rarefaction analysis (data not shown) showed the same result. The Marker 33 particle-attached 2000 library had the lowest coverage (19%), while the Marker 33 free-living 1998 library had the highest coverage (78%), indicating that between 19% and 78% of the diversity in all seven clone libraries was detected using this approach (Table 3).

Discussion

This study is part of an ongoing research program to describe the microbial diversity in the subseafloor at an active vent site (Marker 33, Axial Seamount) with the goals to identify microbial groups that are unique to these environments, gain an understanding of their physiology, and to determine if there is a correlation between species diversity and geochemical and physical characteristics of different subseafloor environments. We have already reported on the archaeal diversity at Marker 33, which showed the indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic Methanococcales, as well as many uncultured Euryarchaeota [17]. It was also shown that the particle-attached fraction showed greater archaeal diversity than the free-living fraction indicating that the ability to attach to mineral surfaces and form biofilms may be a hallmark characteristic of subseafloor microbes. Most probable number (MPN) technique for estimating the number of viable organisms showed that Marker 33, like other subseafloor samples from new eruption sites, supports a hyperthermophilic community, despite a large seawater intrusion into these environments as indicated by the abundance of clones belonging to the putative seawater Marine Groups I and II archaea. In this report we show a high diversity of bacteria in Marker 33 vent fluid samples analyzed in parallel for archaeal diversity. More than 100 phylotypes from 17 different groups were identified over the sampling period. These included phylotypes such as the ε-proteobacteria and thermophilic anaerobes that were found to be indigenous to the subseafloor. Like the results from the archaeal study, we found greater bacterial diversity associated with particles than in the free-living fraction and evidence for the presence of anaerobes and aerobes, and different thermal groups of bacteria.

One of the most interesting results from this study was the marked increase in diversity in the ε-proteobacteria over the sampling period of this study. This increase in diversity follows changes in the geochemical properties of the vent environment that is reflected mostly by the extent of seawater mixing with hydrothermal fluids and resulting temperature changes. For example in 1998, the first year of this study and a few months after the eruption, we found a total of 16 ε-proteobacteria phylotypes whereas in the year 2000, we detected 35. The lowest number of ε-proteobacteria phylotypes was from the 1999 samples that were also the hottest fluid sampled during this study period. We hypothesize that the ε-proteobacteria are favored by conditions caused by greater seawater mixing with hydrothermal fluids in the crust that resulted in decreasing temperatures and increasing levels of seawater electron acceptors. These conditions could lead to an increase in habitat area for organisms having an oxidative metabolism that could also then lead to an increase in species diversity. Similarly, we see a decrease in diversity in the ε-proteobacteria between 1998 and 1999 when the temperature of the vent increased while the amount of seawater mixing decreased. Moreover, seawater microorganisms can serve as indicators of mixing to track this trend as well. For example, in the Marine Group I archaea, a likely deep-sea water group, there were eight unique phylotypes detected in 1998 at Marker 33, only three in 1999, and 10 in 2000 [17]. At Marker 33, variations in geochemical characteristics of the subsurface habitat due to dilution of seawater with hydrothermal fluid have the greatest influence on microbial community diversity. These include short-term small-scale variations, such as the amount of mixing with crustal seawater that occurs in the shallow subseafloor, as well as long-term larger-scale changes, including changes in the end-member hydrothermal fluid over time following the eruption [17]. Previous studies of animal diversity at hydrothermal vents show increasing diversity with increasing habitat area, heterogeneity, and age, as well as increasing diversity correlated with a disturbance until a plateau is reached [68]. When or if a diversity plateau will be reached for the shallow subseafloor at Marker 33 is unknown, although based on our observations at this vent site in 2001 (D.A. Butterfield, M.D. Lilley, J.A. Huber, K.K. Roe, R.W. Embley, and G.J. Massoth, unpublished data), the vent appears to be stabilizing at similar temperatures to 2000, suggesting our samples from that same year may represent the high end of measured diversity at Marker 33 within the ε-proteobacteria.

Molecular studies show the ε-proteobacteria to be widespread at deep-sea hydrothermal vents associated with microbial mats [43,44,69], the vent polychete Alvinella pompejana[70,71], the vent worm Riftia pachyptila[72], and an in situ growth chamber with warm fluids [47,73]. Recently, the first representatives of this group were cultured and found to exhibit both heterotrophic and autotrophic sulfur reduction, moderate thermophily, and the ability to use hydrogen as an electron donor [74,75]. The ability of the ε-proteobacteria to oxidize or reduce sulfur, use a variety of electron acceptors such as oxygen and nitrate, and grow autotrophically makes them ideal candidates for the complex and constantly changing environmental conditions that are likely characteristic of the shallow subseafloor at hydrothermal vents.

Other phylogenetic groups that appear to be indigenous to the subseafloor at Marker 33 include clones related to Thermodesulfobacterium and Desulfurobacterium, Gram-positive bacteria (high and low G+C), novel candidate divisions WS6 and ABY1, as well as the ε-proteobacteria. The presence of clones related to thermophilic anaerobic bacteria is consistent with the detection of methanogens and Thermococcus spp. reported previously from Marker 33 [17] and is further support for the presence of a hot anaerobic habitat within the seafloor. This is the first report of the diverse WS6 division in marine hydrothermal vents. The candidate division WS6 is an uncultured phylogenetic group that has been found mainly in anaerobic environments, such as hot springs and marine sediments [66]. Such a distribution suggests these organisms are anaerobic and possibly thermophilic. Additionally, the candidate division ABY1 has been reported predominantly at deep hydrothermally active sites such as Crater Lake and deep-sea sediments [65], indicating that this uncultured division may also be indigenous to the subseafloor.

To determine the relative percentages of bacteria and archaea in these fluids, fluorescence in situ hybridization was applied to Marker 33 samples using oligonucleotide universal probes EUB338 and ARCH915 [76]. Due to extremely low hybridization percentages, precise numbers could not be obtained, but preliminary results show that bacteria dominate the fluids, compromising over 80% of the total DAPI-stained cells (data not shown). These difficulties in successful hybridization also indicate that the majority of bacteria and archaea in Marker 33 fluids may have a low ribosome content, similar to other natural marine microbial assemblages, and additional efforts will be necessary before they can be accurately quantified [77].

The microbial diversity in the background seawater sample from 1520 m, dominated by α- and γ-proteobacteria, is similar to studies from the deep Pacific, Atlantic, and Southern oceans [42,78]. One phylotype belonging to the γ-proteobacteria was found in all seven clone libraries and is most closely related (96–97%) to the endosymbionts of the cold seep, deep-sea bivalve M. hadalis[34]. Additional clones related to putative sulfide and methane oxidizing cold seep or vent symbionts were found in the Marker 33 clone libraries, suggesting that these volatile energy sources in vent fluids may be utilized not only by subsurface microorganisms but also by benthic seawater and sediment microbes as well [79].

We consistently found the highest diversity of bacteria in the particle-attached fraction at Marker 33 (Table 3), similar to our findings for the archaea at this vent [17]. While there was considerable overlap between the particle-attached and free-living populations, there were a number of groups found only in the larger fraction, including the CFB, β-proteobacteria, high temperature bacteria, and the Gram-positives (high and low G+C). Due to potentially high fluid flow rates, low levels of organics and possibly other nutrients in the subseafloor, we hypothesize that particle attachment with biofilm formation is a common mode of existence for resident subseafloor microbes. We believe that our sampling method captures both free-living microbes and sloughed off pieces of biofilm or mineral pieces. Cultured hyperthermophiles have been found to attach to mineral surfaces and form exopolysaccharides [20,80]. Additionally, we have isolated an autotrophic thermophilic sulfur reducing bacterium from Axial Seamount belonging to the Desulfurobacterium that produces copious amounts of extracellular material that may be involved in attachment to mineral surfaces and biofilm formation. A clone (33-PA42B99) within this group was also found in the particle-attached fraction of Marker 33 in 1999 (Fig. 4d).

There are still many questions about the subseafloor biosphere including its vertical and horizontal dimensions, fluid flow patterns, temporal changes in physical and geochemical properties, and sources and kinds of nutrients for microbes. However, the phylogenetic and inferred physiological diversity of the microbial communities along with changes in geochemistry can help constrain models for the subseafloor.

A schematic of the physiological diversity of the microbial community in the subsurface associated with the diking-eruptive event at Marker 33 is shown in Fig. 5. The geological, chemical and microbiological foundations of this model have been developed in a number of works [1,2,4–6,8,16,17,79,81]. Fig. 5 reflects the microbial population and chemistry found at Marker 33. The upper extrusive layer of the oceanic crust is divided into three different compartments based on a two-component system of hydrothermal fluid and seawater mixing, the temperature, possible microbial metabolisms and expected genera. In this top 500 m of the extrusive basaltic layer of the oceanic crust, extremely high porosities (>30%) exist [82]. Such high porosity can provide ample space for mixing of hydrothermal fluids with entrained seawater, water/rock chemical reactions as well as habitat for microbial communities including those that form biofilms. The shallow zone of the crust is likely dominated by seawater with little hydrothermal fluid component. This may make it possible for oligotrophic seawater microbes mixing through the subsurface to attach to surfaces and live in this zone provided there are sufficient carbon and energy sources and seawater electron acceptors. Additionally, low temperature sulfide or methane oxidizers, such as those in the γ-proteobacteria, could also occupy this zone. The deepest zones would be anaerobic, have the highest temperatures (>50°C) and minimal mixing of hot hydrothermal fluid with seawater. These conditions favor the growth of thermophilic and hyperthermophilic sulfur reducers, such as the Desulfurobacterium and Thermococcales, as well as methanogens. The intermediate zone would be the most variable, representing gradients of conditions of the zones above and below. This habitat may have gradients that include both anaerobic and aerobic conditions and mesophilic to moderately thermophilic temperatures. The presence of mesophilic methanogens and the inferred physiology of members of the novel candidate division ABY1 and WS6 support the existence of such an intermediate zone. The intermediate zone may also receive sufficient oxygen from seawater to form aerobic or microaerophilic gradients that could support sulfur, iron, and methane oxidizing bacteria represented in the γ- and ε-proteobacteria detected in this study.

5

Schematic of the subseafloor at Marker 33 based on inferred physiological characteristics of bacteria and archaea detected in clone libraries and in culture. Inset shows structure, rock type, and porosity of oceanic crust. Arrows indicate seawater mixing and hydrothermal fluid, the resultant thermal gradients and possible microbial processes and groups in these zones.

5

Schematic of the subseafloor at Marker 33 based on inferred physiological characteristics of bacteria and archaea detected in clone libraries and in culture. Inset shows structure, rock type, and porosity of oceanic crust. Arrows indicate seawater mixing and hydrothermal fluid, the resultant thermal gradients and possible microbial processes and groups in these zones.

Imposed on the above model are the temporal changes in physical and geochemical properties that are characteristic of diking-eruptive environments. The subseafloor associated with hydrothermal systems is therefore a dynamic environment in which habitat characteristics change over time, especially with disturbances such as volcanic eruptions and earthquakes [2,83]. In this study, we build on our previously reported archaeal work at Axial Seamount by identifying bacteria that are unique to the subseafloor at Marker 33. Following the deep-sea eruption, we see an increase both in overall bacterial diversity in Marker 33 fluids and in the microbes that are indigenous to the subseafloor, such as the ε-proteobacteria. This is the first report showing increases in bacterial species diversity over time at vent environments and implies that the dynamic nature of the subseafloor may create increasing habitat conditions that favor changes in bacterial diversity on short time scales. Further efforts to quantify and track these unique microbial assemblages as well as to identify their physiological characteristics will be necessary to fully understand the complex environment of the subseafloor at mid-ocean ridges.

Acknowledgements

We thank Bob Embley for his cooperation with sample collection, Kevin Roe for analyzing vent fluids, and the crews of the R/V Thompson, R/V Brown, and ROV ROPOS for their assistance. This work was supported by Washington Sea Grant (NA76RG0119), National Science Foundation (OCE 9816491), NSF IGERT (DGE-9870713), NASA Astrobiology Institute through the Carnegie Geophysical Institute, the NOAA/PMEL Vents Program (PMEL contribution #2530), NOAA West Coast and Polar Undersea Research Center, a NSF predoctoral fellowship to J.A.H., and by the Joint Institute for the Study of the Atmosphere and Ocean (JISAO contribution #952) under NOAA Cooperative Agreement No. NA117RJ1232.

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