order of magnitude higher than in surrounding sediments. The abrupt changes in methane concentrations by two orders of magnitude indicate similar variability in methane oxidation rates (in mat, 2.561.2 mmol m22 d21) and methanogenesis rates as well (Fig. 2). Thus, mat-covered sediments at MC118 have a disproportionately large contribution to microbial processes relative to their small areal coverage. Conclusions High radiotracer rates of sulfate reduction, and methane oxidation, as well as steep methane and sulfate gradients in the center and edge of the Beggiatoa spp. mat suggest that the boundaries of rising methane-and hydrocarbon-rich fluids are delineated by overlying mat cover. Rates at the center and edge of the mat are nearly identical, and then drop sharply less than a meter outside the mat. These clear geochemical boundaries are reflected in the compositions of the active surface microbial community, with consistent community compositions of active sulfate reducers and methane- cycling microorganisms in the center and edge of the mat, but a large drop in their RNA expression levels immediately outside the mat. The deeper microbial communities outside the mat, however, look more similar to those under the mat. Therefore visually undistinguished sediments without conspicuous mat cover (and no porewater evidence for hydrocarbon seepage) can still harbor anaerobic methane- and sulfur-cycling communities that express genes for metabolic activities, but remain below detection limit in the geochemical measurements. High levels of sulfate reduction and methane oxidation in these sediments could resume quickly at the onset or reintroduction of active seeping, resulting in sulfide production and the rapid development of Beggiatoa spp. mats. As a result, microbial mat formation and the establishment of a sulfur- and methane-cycling, mat-associated microbial community in surficial sediments would be rapid and accessible to continuous in- situ observation over days and weeks [14]. These results validate that the often-observed patchiness and small-scale spatial architecture of microbial mats and methane seeps correspond to a profound reorganization of microbial community composition, activity patterns and geochemical imprint on spatial scales of tens of centimeters both vertically and horizontally in the sediments. Microbial mats play an important role as indicators of subsurface microbial heterogeneity and activity, a role proposed previously for seafloor fauna [78]. Systematic recording and documentation of visible seafloor heterogeneity and microbial mats over small spatial scales is therefore an essential component of microbial habitat studies and of foremost importance for sampling designs that capture the fundamental characteristic of microbial habitat patchiness. Methods Site Description and Sampling Mississippi Canyon Block 118 (MC118) in the Gulf of Mexico is characterized by seafloor-breaching methane hydrate deposits and thermogenic hydrocarbon-rich fluids pushed upwards through fractures in the sediments by salt domes [79]. It is located offshore of Louisiana in ,890 m of water at 5.5uC bottom water temperature (28u51.47, 88u29.52) (Fig. 1b). In September 2006 using the Johnson-Sea-Link submersible, four push-cores were taken across a wide (,10 m) white seafloor microbial mat: two near the center of the mat less than a meter away from each other, one at the edge of the mat (,10 cm from uncovered sediment, and ,50 cm outside the mat. The two cores taken from the center of the mat were underlain by a hard surface. Gas bubbles were fizzing from cores taken from the mat and edge of the mat upon Patterns of CH4-Seep RNA arrival at the ship, but the core from outside the mat was undisturbed. In a shipboard 4uC room, the cores were sub- sectioned into 3 cm intervals, and microbiological samples were taken in sterile 30 ml cut-off syringes and frozen immediately in liquid nitrogen. From each interval, subsamples were taken for porewater geochemistry and radiotracer rates. Only the mat and margin mat cores, not the outside mat core, smelled sulfidic. The mosaic was generated from a self-contained digital still stereo camera package developed at the Australian Centre for Field Robotics. The camera system was mounted on the Johnson- Sea-Link II and used to acquire 12 bit, 1.4 Mpixel imagery at 1 Hz from an altitude of 3 to 4 m from the seafloor. The speed of the submersible was such that high overlap (over 75%) was typical. The imagery was assembled into a composite view using the approach described in [80]. Porewater Geochemical Analysis For sulfate measurements, plastic 15 ml tubes filled completely with sediment were centrifuged and the resulting porewater was filtered at 0.2 mm, acidified with 10% HCl, and measured shipboard using a 2010i Dionex ion chromatograph (Sunnyvale, CA), as previously described [81]. For methane measurements, 4 ml sediments were added to 60 ml serum vials containing 1 ml 0.1 M KOH, and were stoppered and crimp-sealed. A 5 mL headspace aliquot was analyzed on a Shimadzu Mini II gas chromatograph (Kyoto, Japan) equipped with flame ionization detector. Carbon stable isotope ratios for dissolved methane were obtained using a pre-concentrating system on-line with a continuous flow 5890 Hewlett-Packard gas chromatograph (Palo Alto, CA), capillary combustion, and isotope ratio mass spectrom- etry as described in Rice et al. [82]. Results are reported using the standard ‘‘del’’ notation, d13C (%) = [R(sample)/R(PDB standard)– 1]*1000, where R is the ratio of the heavy to light isotope relative to the Pee Dee Belemnite standard [83]. The precision for replicate measurements of single samples was 63 percent for sulfate, chloride, and methane concentrations. Sulfate reduction rate and methane oxidation rate measurements were made as previously described [84]. These rate methods measure only methane oxidation and not methanogenesis; the direction of the net reaction can only be gleaned from trends of d13C of methane with depth. A 1-D, inverse, reaction-transport model was used to compare concentration profiles to radiotracer rate measurements based on the following equation [41,85]: 2 2DO L CPW z Lx2 3 DO L Lx { v? ? LCPW za(COW {CPW ) Lx zSRPW ~0 where Q is porosity, DO is molecular diffusivity, CPW is the concentration of the solute in sediment porewater, x is the depth interval in the sediment, v is the sedimentation rate, a is the bioirrigation coefficient, CPW is the concentration of the solute in the overlying water, and RPW is the reaction rate of the porewater constituent. The first term in the equation accounts for molecular diffusion, the second for sedimentation and compaction, the third for bioirrigation, and the fourth for reaction rate. RNA Extraction, Amplification, Cloning and Sequencing Total RNA was extracted following previously described methods [74,86], from the following samples: Mat-B (0–3 cmbsf), Mat-B (12–15 cmbsf), Edge (0–3 cmbsf,) Edge (12–15 cmbsf,) Edge (21–24 cmbsf), Outside (0–3 cmbsf), and Outside (12–15 cmbsf). Briefly, ,4 ml sediment was mixed with 5 ml phenol (pH 5), 5 ml PLoS ONE | www.plosone.org 9 January 2010 | Volume 5 | Issue 1 | e8738