Basalt panels were deployed and recovered by DSV Alvin at TicaVent on the EPR (9◦50.4 N, 104◦17.5 W: 2513-m depth) during R/V Atlantis research cruises AT11-07 (2004/02/03 –2004/02/19), AT11-10 (2004/04/07–2004/04/24), AT11-20 (2004/11/11–2004/11/24). Basalt panels were deployed either at a diffuse-flow vent site (Experiment site1: ∼15◦C) or a nearby, non-vent site (Control site: ∼1.8◦C). Experiment site1 was located within a thriving patch of tubeworms (Riftia pachyptila) and mussels (Bathymodiolus thermophilus). The Control site was located 2.5- m from the experimental site, ∼1/2-m from mussels marking the edge of the colonized vent. These panels are considered ‘pre-eruption’ samples because they were collected 19–28 months before the 2005/2006 EPR eruption. For the purpose of comparison with our experimental results, one post-eruption native basalt biofilm sample (hereafter referred to as ‘native Basalt’) was collected from Tamtown (9◦50.3 N, 104◦17.4 W: 2503-m depth; ∼0.58 km from TicaVent), during the RESET06 cruise (AT15-06, 2006/06/25-2006/07/01). [official dates: AT15-06 (2006/06/18-2006/07/07)]
Basalt panels (10.2 x 10.2 x 2.5 cm) were constructed of basalt collected in the area of 9oN on the EPR (Supplementary Figure 1D). Before deployment, all panels were sealed in aluminum foil and autoclaved. Each panel was deployed from and recovered into an ethanol-wiped biobox mounted on Alvin’s basket, filled prior to the dive with either 0.2-µm filtered seawater or double-distilled water to prevent contamination with surface seawater (as were the Basalt and Trap samples). All subsequent handling of the basalt panels was with sterilized gloves or tools. Control and experimental basalt panels were exposed for 5 time intervals: 4-days, 9-days, 13-days, 76-days, and 9-mos [283-days (control) and 293-days (experimental)]. One panel was lost (76-day, control), leaving a total of 9 panels for analysis, in addition to the native Basalt and Trap samples.
Environmental DNA was extracted from the basalt panel surfaces using a large-volume CTAB (hexadecyltrimethylammonium bromide) extraction. Individual basalt panels were placed into warmed (55oC) DNA extraction buffer, composed of 100 mM TRIS-HCl (pH 8.0), 20 mM EDTA (pH 8.0), 1.4 M NaCl and 2% CTAB, with mercaptoethanol added to 0.2% via syringe filter. Filter-sterilized Proteinase K solution and sodium dodecyl sulfate (SDS) solution were added to final concentrations of 0.1 mg/mL and 0.65% respectively. Covered by this extraction solution, each block was agitated on a shaker table for 2 hrs at 55oC. Biofilm removal was confirmed by inspection of the extracted blocks under a dissecting microscope. DNA was extracted from this solution with an equivalent volume of phenol:chloroform:isoamyl alcohol (25:24:1, pH 8.0), followed by an equivalent volume of chloroform:isoamyl alcohol (24:1). To precipitate the DNA, 0.1 volume of 3M sodium acetate and 2.5 volumes of cold 100% ethanol were added, then placed into -20oC for 3 hours before centrifuging at 10,000 x g for 15 min. After decanting the supernatant, the pellets were covered with cold 70% ethanol and centrifuged at 16,000 x g for 5 min. The ethanol was pipetted off and the pellets were dried. Isolated DNA was resuspended in 50-200 µl sterile water and kept at -80oC until use. Environmental DNA from Trap and native Basalt samples was extracted with using the Ultraclean Soil DNA extraction kit (MoBio laboratories).
The 16S rRNA region of environmental DNA was amplified (27F, 1492R primers) and replicates of 10 PCR amplifications (15 cycles each) were combined, precipitated using a QIAquick PCR purification kit (Qiagen), resuspended in 35 µl of sterile water and purified using the QIAquick gel extraction kit (Qiagen). Replicate PCR reactions were combined in order to minimize PCR bias (Polz and Cavanaugh, 1998) and only 15 cycles were used to decrease the formation of chimeric sequences and Taq error. The combined products were then reamplified with five additional PCR cycles to minimize the formation of heteroduplex molecules (Thompson et al., 2002) and purified using a QIAquick gel extraction kit (Qiagen).
Purified PCR products of the 16S rRNA gene were cloned with the Strataclone PCR cloning kit (Stratagene) for sequencing. Nearly complete, double-stranded sequences of 16S rRNA genes (~1,475 bp) were sequenced on a 96-capillary 3730xl DNA analyzer (Applied Biosystems) with primers M13F and M13R. For all libraries, single strand sequences were grouped by 97% similarity with the program Sequencher (version 4.1.2, Applied Biosystems) and one representative of each group was selected to sequence both forward and reverse strands. These groupings were designated operational taxonomic units (OTUs). Sequences were tested for the presence of chimeras with the program Mallard (Ashelford et al., 2006) and with the Bellerophon server (Huber et al., 2004). The ARB software package was used to analyze sequence data and construct trees (version 2.5b; O. Strunk and W. Ludwig, Technische Universitat Munchen, Munich, Germany).
Primers 517F and 806R were used to target the V4 region of the 16S rRNA gene (Caporaso et al., 2010). For multiplex sequencing, 8 forward primers were synthesized (Eurofin), each with a different tag. PCR reactions (50 µl volume) contained 250 nM of each of forward and reverse primers, 5 ng of template DNA and were performed using Picomaxx taq polymerase (Invitrogen, Carlsbad, CA) using the thermal profile 95°C for 2 min followed by 25 cycles of denaturation at 94°C for 15 s, primer annealing at 61°C and extension at 72°C for 45 s, with final extension of 72°C for 3 min. Amplicons were sequenced at EnGenCore (University of South Carolina) using 454 FLX chemistry. Raw sequences were processed with QIIME 1.8.0 (Caporaso et al., 2010). Sequences were excluded from analysis if they had a mean quality score < 25, were either < 200 or > 1000 bp in length, contained ambiguous nucleotides or had any mismatches in the forward and reverse primers. The sequences were assigned to individual samples by their adapter tags and the 16S rRNA primers were removed prior to analysis. The resulting data sets contained 254 bp of the bacterial V4 region. Potential chimeras were identified and removed using ChimeraSlayer. Trimmed sequences were then classified with RDP classifier. Operational taxonomic units (OTUs) were clustered at 97% similarity based on a distance matrix generated with the program QIIME.
Phylogenetic trees were constructed from aligned clone sequences and closely related environmental clones and cultures using the ARB software package (version 5.5; O. Strunk and W. Ludwig, Technische Universität Munchen, Munich, Germany). Tag sequences found at high abundances (>50 tags detected per sample), as well as those that were identical to a clone sequence or those that represent a unique lineage were inserted into bootstrapped trees using parsimony insertion tool. Alpha and beta diversity estimates were calculated using QIIME. After trimming each sample to an equal number of tags, bacterial diversity was estimated with N obs (observed richness), Chao1 (nonparametric richness estimator), nonparametic (np) Shannon diversity index, Simpson Evenness and Equitability. Structure of the pyrosequenced microbial communities was compared with two different methods. Principal coordinate analysis (PCoA) maps the samples on a set of orthogonal axes in order to explain the maximum amount of variation by the first coordinate and the second largest amount of variation by the second coordinate based on weighted UniFrac values (Lozupone et al., 2010). The UPGMA (unweighted pair group method with arithmetic mean) distance tree is based on a hierarchical clustering in which topological relationships are identified in order of similarity. The robustness of the UPGMA clusters was tested with jackknife analysis, based on 100 randomized subsamples.