phylogenetically diverse group whose range of functions in the environment have not yet been fully explored. For the archaea, taxonomic associations of the most commonly retrieved groups for the V6-tags generally supported the findings of the clone libraries. Deep samples within the mat and outside it were mostly composed of ANME-1 and ANME-2, with contribu- tions from common benthic groups MBG-D and other members of the Thermoplasmatales (Fig. S4b). As with the bacterial V6-tag dataset, some common benthic groups were represented by the archaeal V6-tags that did not appear in the RT-PCR clone libraries: MBG-B/DSAG, Miscellaneous Crenarcheotal Group, and a few others in lower abundance (Fig. S4b). The DHVE8 group appeared in RT-PCR clone libraries, but not in the V6-tags, although the V6-tag sequences may not have been phylogeneti- cally informative enough to distinguish this group. Messenger RNA for mcrA was found in all samples except for the top 3cm outside the mat, where no amplification was observed, even when nested RT-PCR and multiple primer sets were employed (Fig. 4b; Fig. S7) [61,62]. In the surficial sediment outside the mat, the lack of mcrA transcripts agrees with the absence of 16S rRNA transcripts from methane-cycling ANME archaea. Beneath the mat, transcripts of mcrA describe a similar population to that seen with 16S rRNA transcripts, containing multiple ANME-2 archaeal groups as well as group e, which has been found in similar methane seeps [44,63,64]. Although ANME-1 sequences were present in the 16S rRNA libraries, they were absent from the mcrA libraries, most likely because the primers used for mcrA are biased against ANME-1 (Table S2). Diversity Analysis Chao1 diversity estimates, based on 98% 16S rRNA similarity, ranged from 8 to 60 OTUs for archaea, and 9 to 232 OTUs for bacteria (Fig. 5). Chao1 diversity estimates based on V6-tag sequences at 97% OTU groupings, while higher, support the spatial trends predicted by the clone libraries. Sample sizes for clone libraries are in the range of 40 to 85 (Figs. 3a and 4a), whereas sample sizes for tags were 15,000 to 18,000 sequences. In sediments under the mat, bacterial and archaeal Chao1 diversity estimates based on 16S rRNA were in similar ranges to each other (9–63 for bacteria, 8–42 for archaea). Outside the mat bacterial community richness was higher than that of archaea; it peaked in surface sediments outside the mat and decreased with Patterns of CH4-Seep RNA depth outside the mat. All samples from under the mat had lower bacterial diversity than those from outside the mat. Archaeal community richness peaked at 12–15 cmbsf outside the mat. No consistent trends were seen for archaea relative to depth or presence of overlying mat. Discussion Correlation between Microbial Activity and Seeping Fluids Good correlation was observed in surface sediments between the composition of the active microbial community and geochem- ical processes. The abrupt decrease in sulfate flux and sulfate reduction rates just outside the mat was accompanied by a drop in the percentage of putative sulfate reducing groups in bacterial 16S rRNA-based clone libraries, as well as undetectable dsrAB transcripts (Fig. 6A). Correlation was also observed for methane flux, percentage of putative methane cyclers (ANME groups) in archaeal 16S rRNA transcript libraries, and mcrA in mRNA transcript clone libraries (Fig. 6A). Outside the mat, the bacterial community consists of phylotypes closely related to diverse aerobic, microaerophilic or nitrate-reducing bacteria, and the archaeal community is mostly composed of Marine Group I, whose cultured members are aerobic ammonia oxidizers and are commonly found in oxygenated seawater and sediments [65,66]. Also, transcripts of dsrAB and mcrA were below detection limits with the primers used, which correlates well with the low sulfate flux and sulfate reduction rates. These trends show tight spatial coupling between subsurface processes, the active microbial community, and the presence of bacterial mat on the seafloor. Deeper in the sediments underneath the mat, the tight spatial coupling between active microbial community and measured geochemical processes is absent (Fig. 6B). The transcript compositions of all three deep samples are similar to those of in- mat surficial sediments, but are not accompanied by high rates of sulfate reduction and methane oxidation (Figs. 2, 3, and 4). The phylotypes of sulfate reducers do not change after sulfate depletion at 12–15 cm under the mat and edge cores, suggesting that the dominant community composition does not change, even if their activity levels change in response to substrate limitation. Consistent retrieval of dsrAB genes in the deep sediments of all three cores is surprising since the sulfate reduction rates are so low Figure 5. Diversity estimates of bacteria and archaea. Chao1 diversity estimates for rRNA clone libraries (rRNA) and where available for tag sequencing (V6 Tag) are listed for (A) bacteria and (B) archaea at different depths in the mat, at its edge, and just outside the mat. Transparency of each block corresponds to diversity relative to the highest diversity sample. Clone library sample sizes are the same as those in Figs. 3a and 4a. doi:10.1371/journal.pone.0008738.g005 PLoS ONE | www.plosone.org 6 January 2010 | Volume 5 | Issue 1 | e8738