Ribosomal protein S3 was chosen as a single-copy phylogenetic marker, and the number of reads mapping to each rpS3-encoding scaffold was quantified to track organism abundance throughout the experiment ( Figure 1 B). Most groundwater-associated Archaea were detected prior to acetate amendment and decreased in relative abundance following acetate addition ( Figure 1 B). A few Archaea were detected near the onset of sulfate reduction, but none proliferated during the phases marked by increased ferrous iron and methane in the groundwater. Most likely, methanogenic Archaea had proliferated but were either nonplanktonic or removed by the 1.2-μm prefilter. The transcriptomic response of AR10 to acetate addition is discussed below.

A time series of anoxic groundwater samples were collected prior to (A) and during (B–E) an acetate biostimulation experiment, and after acetate addition ceased (F) in 2011 ( Figure 1 A; Table S1 ). Reduction of ferric iron was detected after ∼6 days, and sulfate reduction after ∼20 days. Methane monitoring began on day 79 ( Figure 1 A). For each groundwater sample, biomass was collected from 0.1- and 0.2-μm filters after groundwater was passed through a 1.2-μm prefilter ( Supplemental Experimental Procedures ). This strategy was designed specifically to target small cells, which were predicted to be abundant based on enrichment of organisms with small genomes in previous acetate amendment experiments []. Overall, ∼0.2 Tb of shotgun genomic sequence data was acquired for the groundwater samples. Assembly of this dataset using IDBA_UD [] yielded 3.08 Gb of genome sequence on contigs > 5 kb ( Supplemental Experimental Procedures; Table S1 ). The 0.2-μm filters were also used for RNA extraction and metatranscriptome sequencing ( Supplemental Experimental Procedures ).

(B) Relative abundance of subsurface Archaea. Abundance metrics were calculated utilizing total dataset size and coverage of ribosomal protein-containing scaffolds. The number of reads mapping to each scaffold was quantified to track abundance of Archaea throughout the acetate amendment experiment and to define the response to acetate addition. The rank abundance of archaeal genome bins (including AR1 to AR21) in sample A (0.2-μm filter) is shown in the line graph to the left of the heatmap.

(A) Groundwater samples were collected prior to (sample A), during (samples B–E), and after (sample F) acetate injection to the subsurface. Geochemical conditions during iron reduction, sulfate reduction, and methane production are indicated (methane monitoring began on day 79). Reduction of ferric iron was detected after ∼6 days and sulfate reduction after ∼20 days, as evidenced by increases in ferrous iron (Fe 2+ ) and decreases in sulfate at the sampling well, respectively.

This study was conducted in an aquifer adjacent to the Colorado River, near Rifle, CO, USA (39°31′44.69″ N, 107°46′19.71″ W; 1,617.5 m above sea level). DNA was extracted from sediment and filtered groundwater samples and was sequenced (paired 150-bp Illumina HiSeq 2000 reads). Drilling-recovered organic-rich sediments were obtained from 4, 5, and 6 m below ground surface in 2007 []. Sediments from 4 m depth experience strong variations in redox status due to seasonal variations in groundwater elevation. Assembly of the 163 Gbp of DNA sequence generated from the three sediment samples yielded ∼1.24 Gbp of sequence of contigs > 5 kbp ( Supplemental Experimental Procedures ). Previously, profiling of the 5-m sample indicated the presence of novel Archaea [].

Improved Resolution of the Archaeal Domain

21 Castelle C.J.

Hug L.A.

Wrighton K.C.

Thomas B.C.

Williams K.H.

Wu D.

Tringe S.G.

Singer S.W.

Eisen J.A.

Banfield J.F. Extraordinary phylogenetic diversity and metabolic versatility in aquifer sediment. 22 Hug L.A.

Castelle C.J.

Wrighton K.C.

Thomas B.C.

Sharon I.

Frischkorn K.R.

Williams K.H.

Tringe S.G.

Banfield J.F. Community genomic analyses constrain the distribution of metabolic traits across the Chloroflexi phylum and indicate roles in sediment carbon cycling. 25 Sorek R.

Zhu Y.

Creevey C.J.

Francino M.P.

Bork P.

Rubin E.M. Genome-wide experimental determination of barriers to horizontal gene transfer. 26 Wu D.

Hartman A.

Ward N.

Eisen J.A. An automated phylogenetic tree-based small subunit rRNA taxonomy and alignment pipeline (STAP). 21 Castelle C.J.

Hug L.A.

Wrighton K.C.

Thomas B.C.

Williams K.H.

Wu D.

Tringe S.G.

Singer S.W.

Eisen J.A.

Banfield J.F. Extraordinary phylogenetic diversity and metabolic versatility in aquifer sediment. 22 Hug L.A.

Castelle C.J.

Wrighton K.C.

Thomas B.C.

Sharon I.

Frischkorn K.R.

Williams K.H.

Tringe S.G.

Banfield J.F. Community genomic analyses constrain the distribution of metabolic traits across the Chloroflexi phylum and indicate roles in sediment carbon cycling. Figure 2 Phylogenetic Analyses Placing the 153 Genomically Sampled Subsurface Archaea Show full caption Maximum-likelihood phylogeny of the TACK and DPANN superphyla and Euryarchaeota phylum based on a 15-ribosomal-protein concatenated alignment. Black dots on nodes denote bootstrap support of 100%, while other support values > 50% are reported directly. Phyla are colored and named for clarity. Lineages reported in this study are marked with hexagons: blue for groundwater organisms and green for sediment-associated Archaea, with colored borders on green hexagons indicating the sediment depth from which the organism derives. Genome bins are further marked with stars, with the star color indicating genome completeness (see legend). See also Figure S1 for 16S rRNA analysis that clearly supports the definition of the two newly identified DPANN phyla in this study. To examine archaeal diversity and novelty, we used rpS3 genes to identify scaffolds with a conserved, syntenic block of ribosomal proteins []. Unlike other methods that use concatenation of proteins distributed across the genome for phylogenetic classification, no assignment of scaffolds to genome bins is required with this method []. Because these genes occur in single copy in genomes, each scaffold encoding the ribosomal protein block represents a distinct organism. The concatenated ribosomal protein-based phylogeny provided deep resolution of diverse archaeal lineages ( Figure 2 ), recapitulating the placement of most well-classified TACK superphylum Archaea with bootstrap support. In total, we identified 153 archaeal scaffolds that had at least 8 of the 15 ribosomal protein genes of interest and were thus included in the phylogenetic tree. All 153 archaeal genotypes identified from the Rifle sediment and groundwater are divergent from previously sequenced archaeal genomes and are distributed across the TACK and DPANN superphyla and the Euryarchaeota phylum. Notably, the sediment-associated Archaea studied here are evenly split between the Euryarchaeota phylum (17 genomes), the TACK superphylum (16 genomes), and the DPANN superphylum (14 genomes), while the groundwater-associated Archaea are associated only with the DPANN superphylum (106 genomes).

27 Holmes D.E.

Giloteaux L.

Orellana R.

Williams K.H.

Robbins M.J.

Lovley D.R. Methane production from protozoan endosymbionts following stimulation of microbial metabolism within subsurface sediments. 28 Dridi B.

Fardeau M.L.

Ollivier B.

Raoult D.

Drancourt M. Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces. 29 Borrel G.

Parisot N.

Harris H.M.B.

Peyretaillade E.

Gaci N.

Tottey W.

Bardot O.

Raymann K.

Gribaldo S.

Peyret P.

et al. Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine. 9 Lloyd K.G.

Schreiber L.

Petersen D.G.

Kjeldsen K.U.

Lever M.A.

Steen A.D.

Stepanauskas R.

Richter M.

Kleindienst S.

Lenk S.

et al. Predominant archaea in marine sediments degrade detrital proteins. 30 Iverson V.

Morris R.M.

Frazar C.D.

Berthiaume C.T.

Morales R.L.

Armbrust E.V. Untangling genomes from metagenomes: revealing an uncultured class of marine Euryarchaeota. Although evidence for their activity has been detected during in situ acetate amendment [], methanogenic Archaea did not appear to be abundant in the filtrate. From the sediment sample, we detected a novel species falling into the order of Methanosarcinales ( Figure 2 ). The additional sediment-associated euryarchaeotes form two clades, branching with, but distinct from, the Thermoplasmatales and Aciduliprofundum (DVHE2) orders ( Figure 2 ). One clade is defined by 14 genome sequences (green branches in Figure 2 ), which are most closely related to the recently isolated human-associated methanogen Methanomassiliicoccus luminyensis []. M. luminyensis, along with other recently identified methanogenic Archaea, belongs to a putative seventh order of methanogens, the Methanomassiliicoccales []. The second subgroup, represented by two organisms from the current study and two previously described organisms from the marine benthic group D (MBG-D) [] ( Figure 2 , purple branch), places as a deep branch basal to the putative Methanomassiliicoccales order and the uncultured marine group II []. Interestingly, the Methanomassiliicoccales order represents a monophyletic group independent of any previously known methanogenic order, sharing ancestry with the marine benthic group D, the marine group II, the DHVE2 group, and the Thermoplasmatales. To determine whether some sediment-associated Archaea are potentially methanogens, we searched for the metabolic mcrA gene from the 4-, 5-, and 6-m assemblies. However, only one full-length mcrA gene was retrieved from the entire dataset; thus, we cannot conclude whether the 14 organisms clustering with the human gut methanogen group are methanogens.

8 Meng J.

Xu J.

Qin D.

He Y.

Xiao X.

Wang F. Genetic and functional properties of uncultivated MCG archaea assessed by metagenome and gene expression analyses. 9 Lloyd K.G.

Schreiber L.

Petersen D.G.

Kjeldsen K.U.

Lever M.A.

Steen A.D.

Stepanauskas R.

Richter M.

Kleindienst S.

Lenk S.

et al. Predominant archaea in marine sediments degrade detrital proteins. 8 Meng J.

Xu J.

Qin D.

He Y.

Xiao X.

Wang F. Genetic and functional properties of uncultivated MCG archaea assessed by metagenome and gene expression analyses. 9 Lloyd K.G.

Schreiber L.

Petersen D.G.

Kjeldsen K.U.

Lever M.A.

Steen A.D.

Stepanauskas R.

Richter M.

Kleindienst S.

Lenk S.

et al. Predominant archaea in marine sediments degrade detrital proteins. 9 Lloyd K.G.

Schreiber L.

Petersen D.G.

Kjeldsen K.U.

Lever M.A.

Steen A.D.

Stepanauskas R.

Richter M.

Kleindienst S.

Lenk S.

et al. Predominant archaea in marine sediments degrade detrital proteins. Within the TACK superphylum, we recovered one genome from a member of the Thaumarchaeota and 15 draft genomes that associate with a recently proposed phylum sibling to the Thaumarchaeota and Aigarchaeota, the “Bathyarchaeota” [] ( Figure 2 ). This phylum is currently represented by the partial genome of MCGE09 [], sampled from cold anoxic marine sediment, and metagenome-derived sequences from estuarine sediment []; the estuarine sediment organisms could not be included in our concatenated ribosomal protein analysis because the sequences were incomplete. Metabolic analysis of MCGE09 and MBG-D (mentioned above) single-cell genomes revealed that these abundant marine Archaea might be capable of exogenous protein degradation in cold anoxic environments []. Detrital protein remineralization is mostly due to the action of extracellular protein-degrading enzymes such as gingipain and clostripain []. Here, we identified all of the archaeal scaffolds from the 4-, 5-, and 6-m assemblies; searched for such peptidases; and recovered more than 60 putative peptidases from the clostripain and gingipain families ( http://ggkbase.berkeley.edu/genome_summaries/434-Peptidases ). This expands the potential role of Archaea in protein remineralization to terrestrial anoxic sediment. In-depth metabolic analysis of sediment-associated Archaea will be discussed in a forthcoming manuscript.

14 Rinke C.

Schwientek P.

Sczyrba A.

Ivanova N.N.

Anderson I.J.

Cheng J.F.

Darling A.

Malfatti S.

Swan B.K.

Gies E.A.

et al. Insights into the phylogeny and coding potential of microbial dark matter. Euryarchaeota and TACK superphylum Archaea were not detected in the groundwater samples; however, retention on the prefilter or cell numbers below detection cannot be ruled out. From the DPANN superphylum, we recovered 106 genomes from groundwater and 14 from sediment ( Figure 2 ). Inclusion of these new sequences increases the sampling of this superphylum by a factor of 10. We resolved two new phylum-level lineages within this radiation based on 100 genomes. The first was defined with high bootstrap support (99%) based on 54 newly sampled archaeal genomes (50 from groundwater and 4 from sediment samples) and three previous single-cell sequences. Given that this level of genomic sampling greatly exceeds that used previously to name phyla and superphyla [], we propose the name Woesearchaeota for this phylum, recognizing the pioneering contribution to archaeal phylogeny of the late Professor Carl Woese.

The second highly supported (100%) monophyletic group closely related to the Woesearchaeota is represented by 39 groundwater-associated sequences, 7 sediment-associated sequences, and one previous single-cell sequence. We propose the name Pacearchaeota for this phylum, recognizing the contributions of Professor Norman Pace to archaeal phylogeny and cultivation-independent phylogenetic analyses.

The Aenigmarchaeota and Diapherotrites phyla (DPANN superphylum) are represented by seven and four Archaea sampled in this study, respectively ( Figure 2 ). A further four archaeal genomes improve the taxon representation of the Micrarcheota phyla. In our phylogenetic analyses, the Micrarchaeota and Parvarchaeota are now resolved as distinct lineages, with the Micrarchaeota well supported as a sibling lineage to the Diapherotrites phylum. The final five archaeal genomes (one from sediment and four from groundwater) form a novel, phylum-level clade ( Figure 2 , highlighted in light pink). Additional taxon sampling of this lineage is needed to confirm its placement as a distinct phylum within the DPANN superphylum.