Archaea of the Miscellaneous Crenarchaeotal Group are abundant, diverse and widespread in marine sediments

Abstract

Members of the highly diverse Miscellaneous Crenarchaeotal Group (MCG) are globally distributed in various marine and continental habitats. In this study, we applied a polyphasic approach (rRNA slot blot hybridization, quantitative PCR (qPCR) and catalyzed reporter deposition FISH) using newly developed probes and primers for the in situ detection and quantification of MCG crenarchaeota in diverse types of marine sediments and microbial mats. In general, abundance of MCG (cocci, 0.4 μm) relative to other archaea was highest (12-100%) in anoxic, low-energy environments characterized by deeper sulfate depletion and lower microbial respiration rates (P=0.06 for slot blot and P=0.05 for qPCR). When studied in high depth resolution in the White Oak River estuary and Hydrate Ridge methane seeps, changes in MCG abundance relative to total archaea and MCG phylogenetic composition did not correlate with changes in sulfate reduction or methane oxidation with depth. In addition, MCG abundance did not vary significantly (P>0.1) between seep sites (with high rates of methanotrophy) and non-seep sites (with low rates of methanotrophy). This suggests that MCG are likely not methanotrophs. MCG crenarchaeota are highly diverse and contain 17 subgroups, with a range of intragroup similarity of 82 to 94%. This high diversity and widespread distribution in subsurface sediments indicates that this group is globally important in sedimentary processes.

Figure 1

RaxML phylogenetic tree based on 16S rRNA genes showing MCG crenarchaeotal subgroups MCG-1–MCG-17. Subgroup designations follow those of Sørensen and Teske (2006) for MCG-1 through MCG-4, Nercessian et al. (2005) for MCG-16 (formerly pMARA-4), and Inagaki et al. (2006) for MCG-15 (formerly C3). MCG-13 through MCG-17 multifurcate because branching order was inconsistent between tree models. Sequences with no color were not assigned to a subgroup. Black bars indicate the total number of sequences from our MCG database within that subgroup, with the highest value of 1160 for MCG-6 and lowest value of 51 for MCG-5a. White Oak River estuary cDNA sequences (97% OTUs) are labeled with red or blue bars for methane oxidation or methane production zones, respectively. The first two numbers in the clone names are the depth of that sequence, followed by A for core July 05-1, B for July 05-2, E for December 06 and G for July 08-2. Additional membership in each OTU is listed after the initial clone name. Terrestrial Hot Springs Crenarchaeotal Group was used as outgroup and individual clone names are listed in Supplementary Tables S4 and S5. The web-based Interactive Tree of Life was used for tree and data set visualization (Letunic and Bork, 2011).

Figure 2

Relative abundance of MCG crenarchaeota compared with total archaea as determined for different marine habitats by rRNA slot blot hybridization (black bars), qPCR (white bars), and CARD-FISH (gray bars). Colors indicate samples from anoxic methanotrophic microbial mats (peach) or oxic surface sediments (pink) or sediments with sulfate depletion depths of <0.15 mbsf (yellow) or >0.15 mbsf (blue). Dots indicate samples with no MCG detected with slot blot (black) or qPCR (white); other samples with no visible data were not analyzed with that method. When a depth range is listed, values were averaged over adjacent depths. All individual measurements listed in Table 3.

Figure 3

Single cells of MCG crenarchaeota in subsurface sediments from the sulfate–methane transition zone of the White Oak River estuary (0.4 mbsf; a–f) and ODP site 1227 at Peru Margin (5H3, 37.38 mbsf; g–i), visualized by CARD-FISH. a and d show DAPI staining (blue); other panels show the corresponding FISH signals obtained by dual hybridization with the general archaeal probe ARCH915 (b, e, g; red) and MCG-specific probe MCG493 (c, f, h; green). i shows an overly of g and h. Arrows point to MCG cell signals. Scale bars, 5 μm.

Figure 4

qPCR of 16S rDNA for White Oak River estuary cores July 08-1 (a) and Jul 08-2 (b) and Hydrate Ridge core 19-2 (c). Primer sets used were for total archaea (ARCH915F-ARCH1059R, filled red squares, and ARCH806F-ARCH915R, open red squares), MCG (MCG528F-MCG732R, filled blue circles and MCG410F-MCG528R, open blue circles) or total bacteria (BAC340R-BAC515R, filled black diamonds). All replicate measurements at each depth are shown. In addition, rRNA concentrations of MCG (blue x's) and total archaea (red x's) measured with slot blot hybridization for Hydrate Ridge core 19-2 are shown in c. Sulfate (filled triangles) and methane (open squares or modeled line) concentrations for White Oak River estuary cores July 08-1 (d) and July 08-2 (e) and for Hydrate Ridge (f); (originally published in Lloyd et al. (2011), and in Treude et al., 2003). Dashed horizontal lines are the depth above which there is net anaerobic oxidation of methane (AOM) and below which there is net methane production (MP; Lloyd et al., 2011).

Figure 5

Phylogenetic groupings of all 16S rRNA cDNA clones and 16S rRNA gene V6 amplicon tag sequences for White Oak River estuary sediments that derived from (a) methane oxidation or (b) methane production zone. Numbers at the right side of bars are the total number of sequences in the library. Abbreviations and references for phylogenetic groups are listed in results section.

Figure 6

Slot blot-based determination of the ratio archaea:bacteria 16S rRNA in selected surface and subsurface sediments. The sum of detected archaeal and bacterial 16S rRNA was set as 100% of prokaryotic rRNA. The archaeal fraction is further resolved in the column that shows the proportion of MCG and other, yet unidentified archaea.