underneath the mat and outside the mat. Archaeal diversity peaked at 13.5 cmbsf outside the mat, suggesting an additive effect of overlapping surface and seep archaeal communities; 16S rRNA clone libraries show that pelagic Marine Group I archaea and anaerobic ANME archaea were both present. Under the mat, the active archaeal and bacterial communities showed similar diversity (Chao1 indices of 9 to 63 for bacteria vs. 8 to 42 for archaea), in contrast to the oxic sediments outside the mat that strongly favored bacterial diversity. In fact, three out of the five clone libraries under the mat and at its edge had higher archaeal than bacterial diversity. To our knowledge, this is the first documentation of higher archaeal than bacterial diversity in marine sediments. Bacterial diversity has generally been found to be much higher than archaeal diversity in a given environment [48,68], although the discrepancy is much smaller in petroleum and natural gas seeps [68]. More research at a greater sampling depth is necessary to substantiate the higher archaeal than bacterial diversity, but these observations are consistent with the working hypothesis that archaea are low-energy specialists, and are widely adapted to highly reduced environments [69]. Other environmental studies substantiate this trend, with higher bacterial than archaeal diversity in microoxic mats [15] and seawater-mixed vent fluids [48]. RNA Transcripts as Indicators of the Active Microbial Community Our molecular analysis of bacterial and archaeal community structure and stratification has focused on the level of gene expression via RNA, not gene presence via DNA. Environmental microbial communities are most often studied by extracting, amplifying, and sequencing bulk DNA [70]. However, DNA- based clone libraries may not necessarily represent living microbes since extracellular DNA is preserved in cold anoxic environments [17], and eludes hydrolysis through adherence to mineral surfaces [71] from which it can nevertheless be amplified with PCR [72]. RNA, however, is an inherently less stable molecule than DNA, since it is mostly single-stranded and is susceptible to peptide backbone hydrolysis due to its extra 29 hydroxyl group which stabilizes the transition state. For this reason, it has been used as an indicator of the potentially active microbial population [27,44,73,74]. The short intracellular lifetime of messenger RNA (mRNA), and its direct link to metabolic processes makes it an even more promising indicator of microbial activity in environ- mental samples [33]. Indeed, we found that mRNA was more sensitive to environmental conditions than rRNA. In surface sediments outside the mat a few 16S rRNA transcripts from SRB were present, but these were not accompanied by dsrAB mRNA, as detectable by our primers. Unless these particular SRB have special adaptations for post-translational control of DSR protein, it is likely that they were not actively reducing sulfate. Given that the intracellular lifetime of mcrA mRNA molecules is on the scale of minutes and that of dsrAB is on the scale of days in laboratory cultures [31,33,34], and extracellular degradation is highly favorable, these molecules are likely indicators of active commu- nities. Extraction of RNA directly from marine sediments is difficult given the often low activity (and therefore low mRNA copy number) of microbes in anoxic environments and the susceptibility of RNA to RNases during the extraction process [26]. Therefore, microbes that are in low abundance or those with low cellular RNA content were likely missed by our analysis. The differences in the compositions of 16S rRNA RT-PCR clone libraries and V6-tag sequences may be due to far greater sampling depth in the V6-tags, or differences in primer bias, so it is difficult Patterns of CH4-Seep RNA to use these comparisons to infer differences in RNA- vs. DNA- based analyses. However, RNA-based studies allow access to the likely active members of the population and removes much of the uncertainty about the extent to which culture-independent methods describe functional populations. Relationship between Diversity Changes and Detection Limits Apparent changes in diversity might be impacted by detection issues. For example, rare groups that are still detected in sediments with high biomass may fall below detection level in sediments that have a lower biomass, resulting in erroneously low diversity indices [75]. However, some of our archaeal results show the opposite trend, arguing that archaeal diversity trends are not an artifact of total sample size bias. The amount of archaeal 16S rRNA, estimated by dilution PCR [76] was highest in the surface outside the mat, a sample with low archaeal diversity. Even if we assume equal detection sensitivity for different bacterial and archaeal groups, clone library representation can be read only as a relative, not as an absolute measure of their abundance. For example, what appears to be an increased contribution of sulfate reducing bacteria and ANME archaea at 12–15 cmbsf compared to the surface layer outside the mat, may instead reflect decreased contributions of surface-layer bacteria and archaea at depth, leaving SRB and ANMEs to comprise a larger percentage of the 12–15 cmbsf community. Finally, the 16S rRNA primers for bacteria and archaea are in principle subject to primer bias and mismatch problems [57]; however, 16S rRNA the primers used in this study were checked against phylum-level alignments of complete 16S rRNA genes, and they each detected a large number of lineages, including novel phylum-level bacterial lineages [47]. Spatial Scales of Mat-Associated Biogeochemical Activity One of the most interesting implications of this mat study is the extremely uneven spatial distribution of mat-associated microbial processes in surficial sediments at seep sites. Microbial mats cover only a small fraction of the total sediment surface area at methane and hydrocarbon seep sites. At MC118, Beggiatoa spp. mats are occasionally observed and recorded on JSL 2006 dive tapes, but an extensive video survey suggests that they cover only a small proportion of the seafloor, mostly in the northwestern crater of MC118, and to a lesser extent in the southeastern area [77]. At the same time they are hot spots of near-surface microbial sulfate- reducing and methane-oxidizing activity. The mat in our current study appears to be anomalously large (,10 m diameter) for this site, as no others of this size have been documented. A photomosaic survey of a limited area near the sampling site (Fig. 1c) indicates that only ,1% of the sediment surface is covered with microbial mats. Randomly taken gravity cores from the wider MC118 area have yielded only a few cores (4 out of 30) with steep sulfate and methane gradients, although they were not covered by bacterial mat and had much deeper sulfate depletion depths (50–100 cmbsf) [3]. In addition to the rarity of mat-covered, active sediments, the measured rates for sulfate reduction and methane oxidation in surficial sediments within and outside of mats diverge by an order of magnitude. Depth- integrated sulfate reduction rates (6 standard deviation) under- neath the mat are 12.366.2 mmol m22 d21; they drop to 2.160.8 outside the mat (Fig. 2, Table S1). These values generally agree with those averaged from 3 different Gulf of Mexico white Beggiatoa spp. mats with no tubeworms (26.9625.9 mmol m22 d21) [5]. Although mat-covered sediment accounts for only ,1% of sediment area at MC118, depth-integrated sulfate reduction rates in mat-associated surficial sediments (upper 20–25 cm) are an PLoS ONE | www.plosone.org 8 January 2010 | Volume 5 | Issue 1 | e8738