taken just below the bacterial mat, no sequences for Beggiatoa spp. were found, consistent with the frequently observed difficulty to amplify full-length Beggiatoa spp. 16S rRNA sequences from mixed environmental samples [47]. Similarly to the RT-PCR clone libraries, the V6-tag sequences (available from 12–15 cmbsf inside and outside the mat) were dominated by members of the Deltaproteobacteria (Fig. S4a). Deep samples within and outside the mat were similar to each other, and also had contributions from some groups that did not appear in RT-PCR clone libraries: Japan Sea Group 1 (JS1), Epsilonproteobacteria, Spirochaetes, Deferribacteres, Lenti- sphaera, OP8, and Actinobacteria, and a few others. V6-tag sequences are too short to allow further reliable phylogenetic identification [48]. Underneath the mat and at its edge dsrAB transcripts were recovered from all depths and the majority of them were related to the uncultivated Cluster B group [49] that is basal to the Desulfobacteraceae (Fig. 3b; Fig. S5). Other dsrAB transcripts are found only in the surface under the mat and include uncultured Group IV [50] and Group V [51], and Desulfobacterium anilini. No dsrAB transcripts of the Desulfobulbaceae were detected, even though they were present in the 16S rRNA transcript libraries. Primer bias most likely explains this result; one of the internal dsrAB primers used in this study had between three and four mismatches to cultured members of the Desulfobulbaceae (Table S2). No dsrAB transcripts were detected from surface sediments outside the mat, despite nested amplification with multiple primer sets. Deeper sediments outside the mat yielded dsrAB sequences Patterns of CH4-Seep RNA that were similar to those found under the mat, grouping with Cluster B (Fig. 3b). Archaeal 16S rRNA and mcrA Transcripts At all depths underneath the mat and at the mat’s edge, the majority of 16S rRNA sequences fall within the ANME-1b and ANME-2a and 2c groups, which are commonly thought to mediate sulfate-dependent anaerobic methane oxidation and are also found in net methane-producing sediments [52,53,54] (Fig. 4a and Fig. S6). The second most abundant sequence type, in all except the deep samples from the mat edge, are in the Deep Sea Hydrothermal Vent Euryarchaeota Group 8 (DHVE8) [55] within the DHVE II [56]. These sediment layers also contain 16S rRNA sequences from the Thermoplasmatales, Marine Benthic Group D (also called Marine Group III), and Marine Benthic Group B (also called the Deep Sea Archaeal Group). These uncultured archaea are commonly found in shallow and deep marine sediments [57]. Just outside the mat, the composition of the active archaeal community shifts to nearly exclusively Marine Group I at the sediment surface, and ANME-2a at 12–15 cmbsf (Fig. 4a). Marine Group I Crenarchaeota are the most abundant prokaryotic plankton in deep ocean water [58]. Genomics and physiology of a few species within Marine Group I have been studied using the naturally enriched candidate species Cenarchaeum symbiosum, an ammonia-oxidizing sponge symbiont, and the pure culture strain Nitrosopumilus marinus, an aquarium isolate capable of aerobic ammonium oxidation to nitrate chemolithoautotrophically [59]. A distantly related thermophilic representative has been cultured from a Yellowstone hot spring [60]. However, Marine Group I is a Figure 4. Archaeal community structure and stratification. Phylogenetic affiliations of (A) archaeal 16S rRNA and (B) mcrA mRNA transcripts at different depths across the mat transect. In A), shades of red and orange denote putative methane-oxidizing or methane-producing groups. Numbers in parentheses are the total number of clones analyzed including full length and short reads. ‘‘Not determined’’ means that no amplification was tried; ‘‘mcrA detected’’ means that mcrA was amplified but not sequenced. doi:10.1371/journal.pone.0008738.g004 PLoS ONE | www.plosone.org 5 January 2010 | Volume 5 | Issue 1 | e8738