Brockarchaeota, a novel archaeal lineage capable of methylotrophy

Single carbon (C1) compounds such as methanol, methylamines and formaldehyde are ubiquitous in nature and they are large components of the carbon cycle. In anoxic environments C1-utilizing microbes (methylotrophs) play an important role in controlling 25 global carbon degradation. Currently described anaerobic methylotrophs are limited to methanogenic archaea, acetogenic bacteria, and sulfate-reducing bacteria. Here, we report the first archaeal lineage outside of methanogenic taxa that are capable of anaerobic methylotrophy. Phylogenetic analyses suggest these archaea form a new phylum within the TACK superphylum, which we propose be named Brockarchaeota. A survey revealed 30 Brockarchaeota are globally distributed in geothermal springs. Metabolic inference from 15 metagenome-assembled genomes from hot springs and deep-sea sediments indicates that Brockarchaeota are strict anaerobes. They contain a variety C1 metabolisms including the methanol and trimethylamine methyltransferases system, the ribulose bisphosphate pathway coupled with the non-oxidative pentoses phosphate pathway, and reductive glycine 35 pathway. Brockarchaeota have an incomplete methyl-branch of the Wood-Ljungdahl pathway probably used for formaldehyde oxidation, since they lack several core genes involved in methanogenesis including methyl-CoM reductases. Brockarchaeota also appear to play an important role in the breakdown of plant-derived polysaccharides, especially cellulose, starch and xylan. Their broad distribution and their capacity to use both C1 compounds and complex polysaccharides via anaerobic metabolism suggest that the Brockarchaeota occupy previously overlooked roles in carbon cycling. (rGlyP). Enzymes: pyruvate ferredoxin oxidoreductase (PFO), and ligase (ADP-forming) (ACD), pyruvate formate lyase (PFL), tungsten-dependent aldehyde ferredoxin oxidoreductase (AFOR), tetrahydrofolate (THF) ligase methenyl-THF dehydrogenase


Introduction
Methylotrophs are organisms that are capable of using single-carbon (C1) compounds as a source of energy and carbon 1,2 . C1 compounds, such as methanol and methylamines, are derived from a variety of sources such as phytoplankton, plants, and the decay of organic matter [3][4][5] . As a result, they are ubiquitous in oceans and atmosphere and are important components of the global carbon and nitrogen cycles 4 . In oxic environments, C1 compounds are generally converted to settings, C1 compounds are used as substrate for methylotrophic-methanogenesis [6][7][8][9] and sulfate reduction 10 . Anaerobic methylotrophs utilize the methyltransferase system (MT) to break and transfer the methyl residue of C1 compounds to coenzyme M (in the case of methanogens) or tetrahydrofolate (in acetogens and sulfate reducers) [6][7][8][9][10] and couple this reaction to the Wood-

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Ljungdahl pathway (WLP) for energy conservation. Methylotrophic archaea include methanogenic orders (in Euryarchaeota): Methanosarcinales, Methanobacteriales, Methanomassiliicoccales and the recently discovered uncultured methylotrophic phylum, Verstraetearchaeota 6 . Methylotrophy has not been described in archaeal lineages outside of these methanogenic groups. Also, our understanding of C1-utilization is limited, as there is increasing 60 evidence that suggests C1 compounds serve as energetic substrates to fuel non-methanogenic heterotrophic communities in the deep subseafloor 11 and marine sediments 13 . However, little is known about the microorganisms or pathways mediating this process 4 . Here we characterize a new globally distributed archaeal lineages with metabolic pathways for C1 utilization from hot springs and the deep ocean.

Phylogeny and distribution in nature
Phylogenetic analyses of these MAGs based on a concatenated alignment of 37 conserved proteins (see Methods), revealed they form a distinct group within the TACK superphylum, basal to Aigarchaeota and Thaumarchaeota (Figure 1). A comparison of average amino acid identities (AAI) across 250 available TACK genomes (Supporting Table 2) including the recently 85 discovered phylum Marsarchaeota 13 , revealed that Brockarchaeota are distinct from neighboring phyla (Geoarchaeota, Aigarchaeota and Thaumarchaeota) and share up to 99% genome-wide nucleotide similarity to one another (Supporting Figure 1 and Supporting Table  3). The two GB MAGs (B48_G17 and B27_G9) are distinct from the hot springs at the AAI level (<50% similar to each other), and <45% AAI to members of Geoarchaeota, 90 Thaumarchaeota and Aigarchaeota, which is consistent with their phylogenetic placement. Phylogeny of 16S rRNA genes also indicated that Brockarchaeota do not fall within any described archaeal phyla (Figure 2A), with < 78% similarity to other TACK members. Together, these results support the classification of these MAGs as a new phylum. We propose that the phylum be named "Brockarchaeota", after Thomas Brock, an American microbiologist known for his 95 groundbreaking research in hot springs microbiology. Interestingly, only three 16S rRNA gene sequences with similarity (92-96%) to Brockarchaeota sequences have been described in PCR-based surveys, highlighting the inherent bias for primer choice in diversity studies. Therefore, we searched publicly available 120 metagenomic databases to examine the geographic distribution of this phylum. Notably, we almost exclusively found 16S rRNA gene sequences related to Brockarchaeota in sequence data generated from other hot springs from around the world (China, USA, South Africa; Figure 2A), revealing Brockarchaeota are globally distributed in hot springs ( Figure 2B). Three sequences, which cluster together, were recovered from lake sediments in Rwanda and the Gulf of Boni in Indonesia (28°C) (see Supporting Table 4), suggesting that some Brockarchaeota are mesophilic as well.

Utilization of C1 compounds and central carbon metabolism
To begin to understand the metabolism of the Brockarchaeota we compared the predicted proteins encoded by these genomes with a variety of functional databases (see Methods). This revealed a unique type of anaerobic methylotrophic metabolism. They contain the methyltransferase system (MT), that has been shown to be essential for anaerobic methylotrophy 16 and is composed of three components. The first is a methyltransferase involved in breaking the C-O bond of specific methylated compounds (MtaB for methanol, MtmB for monomethylamine, MtbB for dimethylamine, and MttB for trimethylamine). The second is the transferring methyl residue to the second component, a corrinoid protein ( Figure 3). The third is a methylated methylamine-150 specific corrinoid protein (MtaA for methanol, MtbA for methylamines), that is involved in transferring the methyl-group from the corrinoid protein to coenzyme M in methanogens, or tetrahydrofolate in acetogens. Brockarchaeota from hot springs encode proteins predicted to be methanol-CoM methyltransferases (MtaB) and trimethylamine-corrinoid protein methyltransferase (MttB) for the utilization of methanol and trimethylamine (TMA), respectively. Brockarchaeota also encode a putative B12-binding corrinoid protein, that once methylated, can act as a substrate for the third component of the MT system that is lacking in Brockarchaeota genomes. Another undescribed protein may be involved in the transfer of the methylated compound from the corrinoid protein to unknown methyl carrier (See details in Supporting Table 5). The distribution of methyltransferases among a current set of TACK 160 superphylum genomes indicate methanol-MT system is unique feature of Brockarchaeota within the TACK superphylum ( Figure 1). Prior to this, methanol-utilization is currently thought to be limited to Euryarchaeota and TACK (Verstraetaearchaeota 6 , Korarchaeota 17 ) archaea and some bacteria (Firmicutes and Deltaproteobacteria) 18 .
To our knowledge, no methylotrophic members of the archaea domain have been described outside methanogenic groups (Euryarchaeota and Verstraetearchaeota). Despite the presence of MT system, unlike phylogenetically related archaea such as Verstraetearchaeota 6 ; Brockarchaeota genomes do not possess the common core marker genes specific to methanogenesis including Methyl-coenzyme M reductase (MCR) (Figure 4 and Supporting Table 6). To ensure that the MCRs are not missing due to incomplete MAGs, we searched metagenomic datasets from each of 170 communities from which they were obtained for mcr genes belonging to Brockarchaeota and did not find any (See supporting Table 7 and Supporting Discussion). In addition to lacking MCR genes, they encode lack a complete Wood-Ljungdahl pathway essential for substrate utilization an energy conservation in methanogenic archaea. Furthermore, Brockarchaeota lack a methanol dehydrogenase (MDH) system that has been recently described in deep-sea sulfate reducing bacteria encoding methyltransferases 10 . The lack of previously described anaerobic methylotrophic pathways, raises the question of how C1 compounds are assimilated by Brockarchaeota, or if they are being used for energy conservation or biosynthetic purposes. Most of the Brockarchaeota encode all four enzymes of the non-oxidative pentoses phosphate pathway (NOPPP) and both key enzymes of the ribulose monophosphate pathway (RuMP) including 3-hexulose-6-phosphate synthases (HPS) and 6phospho-3-hexuloisomerases (PHI) (Supporting Figure 2). The presence of complete NOPPP, RuMP and reductive glycine pathway (rGlyP) suggests that these compounds are being assimilated into pyruvate, which can be converted to acetate for ATP formation at substrate-level phosphorylation.

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The RuMP pathway was originally described in methylotrophic bacteria, which use C1 compounds as a sole source of carbon and energy, however, it is now recognized as a widespread pathway for formaldehyde fixation and detoxification 19 . Formaldehyde was an essential building block to synthesize sugars on early Earth and is ubiquitous in nature, produced through the degradation of compounds containing methyl-or methoxyl-groups, e.g., lignin and pectin 20 . In geothermally environments autotrophic microbes are thought to produce formaldehyde from a variety of C1 compounds (i.e. CH4, CO2, CO) 21 . The RuMP pathway functions as an efficient system for trapping free formaldehyde at relatively low concentrations. The presence of HPS and PHI in Brockarchaeota suggests that formaldehyde can be fixed and detoxified via the RuMP pathway. Furthermore, formaldehyde can potentially be oxidized to CO2 or formate due to the presence of genes coding tungsten-dependent aldehyde ferredoxin oxidoreductase (AFOR). However, this enzyme can oxidize a wide range of different aldehydes derived from organic carbon degradation (e.g., of peptides) and may also be involved in electron transport reactions 22 . The presence of potential oxidative and assimilative formaldehyde pathways in Brockarchaeota may be an advantage for their survival in hot springs and hydrothermal deep-sea environments   Table 6 for specific details of the genomes used in this study and the specific marker genes named with m followed with a number in the figure). For comparison purposes the following known methanogenic archaea were included: Candidatus Methanoplasma termitum that lacks the entire pathway 230 for CO2 reduction to methyl coenzyme M and produces methane by hydrogen-dependent reduction of methanol or methylamine (Methanomassiliicoccales), Methanosphaera stadtmanae (Methanobacteriales) that can generate methane only by the reduction of methanol with H2 and is dependent on acetate as a carbon source, and Methanosarcina acetivorans (Methanosarcinales).
Some Brockarchaeota genomes code tetrahydrofolate (THF) ligase (fhs), methenyl-THF cyclohydrolase/methylene-THF dehydrogenase (folD), and methylene-THF reductase MTHFR (metF), which are key enzymes of the methyl-branch of the Wood-Ljungdahl pathway (Supporting Table 5). Nonetheless, given the absence of key carbonyl branch genes, Brockarchaeota appears to 240 be incapable of assimilating C1 compounds via the WL pathway used by other methylotrophic archaea. Yet, the presence of genes for the rGLYp pahtway (gcvP, glycine dehydrogenase; gcvT, aminomethyltransferase; gcvH, lipoate-binding protein; lpd, dihydrolipoyl dehydrogenase; glyA, serine hydroxymethyltransferase; and sdaA, serine deaminase) in Brockarchaeota genomes, offers another alternative route for assimilation of C1 compounds not yet described in methylotrophic archaea. Subsequent conversion of C1 compounds in Brockarchaeota can proceed via methylene-THF derivatives, either by MTHFR or FoldD (in the case of formaldehyde) to generate 5,10-CH2tetrahydrofolate which can be assimilated via glycine-cleavage system (GCS) 23 , followed by the assimilation into central metabolism with the conversion of serine into pyruvate (see figure 3).
A phylogenetic analysis of alcohol dehydrogenases from hot spring genomes revealed that 250 they encode a butanol dehydrogenase BDH (Supporting Figure 3) that catalyzes the reversible conversion of butyraldehyde to butanol. Brockarchaeota BDH's are homologues to sequences from obligately anaerobic, thermophilic bacteria that can degrade complex plant saccharides such as xylan (i.e Caldicoprobacter oshimai 24 and Hungateiclostridium thermocellum 25 ) or cellulose (Hungateiclostridium alkalicellulosi). To investigate if Brockarchaeota can oxidize or produce 255 butanol, we searched for genes involved in production of butanol in two model organisms; Clostridium acetobutylicum 26,27 which is one of the few organisms that produces butanol as a fermentation product, and Saccharomyces cerevisiae 28 involved in butanol and isopropanol production. We found that Brockarchaeota genomes lack the key enzymes involved in the fermentation of pyruvate to butanol (butanal dehydrogenase, butyryl-CoA dehydrogenase, enoyl-

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CoA dehydratase, 3-hydroxyacyl-CoA dehydrogenase). However, most of the genomes code a putative aldehyde dehydrogenase that could convert butyraldehyde to butyric acid. Also, we found a putative enoyl-CoA hydratase/isomerase protein that is coded by one bin (JZ-1.89), which could be involved in further converting butyric acid to acetyl-CoA. Our results suggest an alternative pathway for butanol oxidation that still remains unresolved (Supporting Figure 4).

Pathways for the utilization of extracellular organic carbon
In addition to anaerobic methylotrophy, Brockarchaeota may be able to degrade a variety of organic carbon compounds. They may utilize hexoses via Embden-Meyerhof-Parnas (EMP) pathway (Supporting Figure 5) and pentoses (xylose isomerase xylA and xylulose kinase xylB) via 270 the isomerase pathway (Supporting Figure 2). These enzymes were previously only found in bacterial thermophiles and halophilic archaea that ferment complex compounds and degrade xylose suggesting a similar physiology in Brockarchaeota 29 . Once assimilated into the cell, carbon complex compounds could enter the central metabolism and be converted to acetate and H2 via acetogenic fermentation. The ATP conserving step or either sugar or pyruvate fermentation to 275 acetate could be catalyzed by acetate-CoA ligase in the hot spring genomes. Acetate can also be assimilated to acetyl-CoA by acetyl-CoA synthetase (ACS), thus acetate might be a source of carbon and energy in the absence of other substrates in hot spring Brockarchaeota. The presence of pyruvate ferredoxin oxidoreductase (PFO) that couples pyruvate oxidation to H2 production, generating acetyl-CoA, could support fermentative metabolism via degradation of either acetate, 280 pyruvate, hexoses or pentoses. Brockarchaeota genomes appear to code a wide repertoire of ATPases such as the plasma-membrane proton-efflux P-type ATPase (only present in the hot spring genomes), Zn2+/Cd 2+exporting ATPase (present in DRTY7.37), and finally the V/A-type H+/Na+transporting ATPase (in most of the genomes). The existence of ATPase in Brockarchaeota suggests that members of these genotypes have the additional ability to couple acetogenic 285 fermentation to membrane potential generation of a transmembrane ion gradient across the membrane.
To complement their ability to degrade xylenes, Brockarchaeota also contain a relatively high number of carbohydrate-active enzymes (average of 27 CAZYmes per genome) which is 3 times what has been observed in other TACK archaea (Supporting Table 8 and Figure 5). Ten of 290 the 15 Brockarchaeota genomes have genes with similarity to α-L-fucosidase involved in the degradation of xyloglucan, which is the major component of hemicellulose in plant-cell walls 30 . All the hot spring genomes encode GH3 family proteins for detrital cellulose degradation, plant and bacterial cell wall remodeling, energy metabolism, and pathogen defense 31 . The hot spring genotypes contain a wider repertoire of CAZymes than the deep-sea GB genomes. Among these 295 are four predicted to be extracellular glycoside hydrolases, which are involved in the breakdown of high molecular-weight plant-derived polysaccharides, primarily xylanes, cellulose, and starch. Comparison of the CAZYmes across the TACK superphylum revealed that 17 extracellular enzymes, including enzymes for the degradation xylanes, are unique to Brockarchaeota (Supporting Table 8). The diversity and abundance of CAZYmes in members of the TACK superphylum 300 highlights that that despite the low number of sequenced Brockarchaeota genomes (15 described in this study) compared to Thaumarchaeota (89), the former encodes a wider arrange and in wider abundance that their phylogenetically related counterparts.

Hydrogen metabolism
Hydrogen is also abundant in hydrothermal systems due to volcanic processes 32 . Brockarchaeota might be able to use 3b [NiFe]-hydrogenases for H2 oxidation with NADP+ or NAD(P)+ as an electron acceptor 33 . The hot spring genomes also encode oxygen-tolerant group 3d [NiFe]hydrogenases, which may allow them to transfer electrons between NAD(P)H and H2 depending 320 on the availability of electron acceptors (Supporting Figure 6). Group 3d [NiFe]-hydrogenases are abundant in metagenomes from hot springs where microbial communities are relatively stable the production of H2 with NADPH or NAD(P)H as the electron donor. Therefore, Brockarchaeota might have the ability to reduce sulfur, using H2 or organic substrates as electron donors, which is common in hyperthermophilic archaea living in geothermally active environments 35

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Brockarchaeota gene contents suggests they are facultative or obligate anaerobic fermentative organisms that produce acetate, CO2, and H2 as byproducts (see Supporting Information for details). Brockarchaeota are also able to degrade complex carbon compounds such as xylenes and perform anaerobic methylotrophy. Xylenes are a major structural polysaccharide in plant cells, and is the 335 second most abundant polysaccharide in nature, accounting for approximately one-third of all renewable organic carbon on Earth after cellulose 37,38 . This suggests that Brockarchaeota are key players in organic matter degradation in hot springs and deep-sea sediments. Furthermore, Brockarchaeota have unique pathways for non-methanogenic methylotrophy which has not been described in the archaea domain prior to this and has not been attributed to any organism broadly 340 distributed in nature. This gives them a unique ecological position in nature, to degrade abundant methylamines in anoxic environments ( Figure 6).
The protein repertoire of GB and hot springs genomes have some important distinctions that likely reflects different anaerobic physiologies. GB genomes appear to be obligately fermenting organisms that rely mostly on substrate-level phosphorylation since they lack all the 345 complexes for the respiratory chain with exception of the ATPase. In contrast, hot spring genomes appear to have mechanisms to increase their ATP yield including the use of geothermally derived inorganic substrates as possible terminal electron acceptors such as mercury (Hg), arsenic (As) and hydrogen (H2). Deep-sea hydrothermal vents, hot springs, and fumaroles are natural sources of Hg 39 , H2 34 Arsenic 40 and sulfur 41 . Three hot springs Brockarchaeota genomes (DRTY735_44, DRTY-1.18 and DRTY.37) encode mercuric reductase (MerA), which detoxifies Hg (II) to Hg(0) 39 . Brockarchaeota also appear to have the genetic capacity for As-resistance including arsenate reductase (ArsC) for the reduction of arsenate to arsenite, which is present in most of the hot spring genomes, and arsenite efflux transporters (asrA and asrB) for cell export (Figure 3). The presence of this energy-dependent efflux process related detoxification proteins, could also indicate that Brockarchaeota in hot springs genomes could use arsenate as terminal electron acceptor, as seen in bacteria 42,43 The discovery of Brockarchaeota genomes from sediments around the world, overlooked by conventional rRNA gene diversity approaches, highlights the need for further exploration of subsurface microbial communities. The addition of these genomes to public databases, like other 360 recently described novel archaeal lineages [44][45][46] , will enhance their detection in future environmental studies. A lack of recognition of their existence prior to this, limited our ability to fully describe sediment community structure and function. Given their broad distribution, and versatile carbon metabolism, they are likely key players in global carbon cycling. However, this first description is limited to genomic characterization, thus culturing or in activity measurements are needed to confirm their physiological activities 47 . Overall, the description of this new phylum enhances our understanding of biodiversity of archaea and suggests they are mediating unique roles in anoxic carbon cycling.

Methods
Metagenomic assembly and binning. Two MAGs (B48_G17 and B27_G9) were obtained from Guaymas Basin sediments (Gulf of California; 27°N0.388, 111°W24.560) and were obtained as part of a larger study of these hydrothermal marine sediments 12 . Both samples were collected from the same location but G9 was sampled from 0-3 cm and G17 form 12-15 cm depth. The sediment cores from which these two MAGs were binned from were collected during Alvin dive 4571_4 in 2009 using polycarbonate cores (45-60 cm in length, 6.25 cm interior diameter), subsampled into cm layers under N2 gas in the ship's laboratory and immediately frozen at -80°C. Details on the sampling site and metagenomic sequencing effort is provided in Dombrowski et al., 2018. Briefly, total DNA from ≥10 g of sediment from each sample was extracted using the MoBio PowerMax soil kit using the manufacturer's instructions and adjusted to a final concentration of 10 ng/µl of each sample (using a total amount of 100 ng). Libraries for paired-end Illumina (HiSeq-2500 1TB) sequencing were prepared by the Joint Genome Institute (JGI).
Sequencing was performed on an Illumina HiSeq 2500 machine using the paired-end 2x125 bp run-type mode. All runs combined provided a total of ~280 gigabases of sequencing data. Quality control and sequence assembly was performed by JGI. For further binning, only scaffolds ≥ 2000 bps were included.
Metagenomic binning was performed on individual assemblies using the binning tools ESOM, Anvi'o (v2.2.2) 48 and Metabat (v1) 49 . For ESOM bins were extracted using getClassFasta.pl and the command -loyal 51. Anvi'o was run with default parameters and metabat was run using the following settings: in several hot springs. Sequencing was done on an Illumina HiSeq4000 (Beijing Novogene Bioinformatics Technology Co., Ltd). The Chinese MAGs were assembled using metaSPADES (version 3.10.1), with a k-mer set of "21, 33, 55, 77, 99, 127". For each sample only scaffolds larger than 2500 bp were binned using MetaBAT (v.1) with default parameters, considering both tetranucleotide frequencies (TNF) and scaffold coverage information. The scaffolds from the 420 obtained bins and the unbinned scaffolds were visualized using ESOM with a minimum length of 2500 bp and maximum length of 5000 bp as previously described 50 and the bins were modified by removing any out-of-range scaffolds (indicated by sequence points) or adding any unbinned scaffolds using ESOM related scripts 43 . Also, scaffolds ≥ 1000 bp from each sample were uploaded to ggKbase (http://ggkbase.berkeley.edu/), and the bins from ESOM analyses were evaluated and modified manually at ggKbase based on GC content, coverage and taxonomic information of scaffolds.

Phylogenetic analyses.
A phylogenetic tree was generated as recently described in ref 46 . Briefly, 36 conserved marker proteins were extracted using phylosift 51 , in a genomic dataset containing 430 3,549 archaeal genomes including Brockarchaeota, and 40 bacterial genomes. An alignment of the 36 individual proteins extracted from a total of 3,599 genomes was generated using MAFFT (algorithm autoselection) with a BLOSUM62 scoring and contains 4,962 characters after masking gaps present in at least 50% of the taxa. A tree was constructed withIQtree (v1.6.11) with a best fit LG+F+R10 model selected using the Bayesian Information Criterion (BIC) and bootstraps are 435 based on 1000 replicated trees. The bacterial genomes were used as an outgroup. The 16S rRNA sequences were extracted from Brockarchaeota genomes using Barrnap (https://github.com/Victorian-Bioinformatics-Consortium/barrnap) and used for a 16S rRNA gene phylogeny that included sequences derived from metagenomic surveys (NCBI accession EU924237, KX213943, and KX213897) and the IMG database. The rRNA phylogeny was generated using RAxML within the ARB software package (v. 2.5b). using default parameters.

Metabolic predictions.
Gene predictions for individual genomes were performed using Prodigal 52 (V2.6.2, default settings). Predicted genes of individual genomes were further characterized using a combination of several databases: KofamKOALA 53 , Interproscan v5.31.70 54 , HydDB 55 , 445 dbCAN2 56 , MEBS 57 and METABOLIC 58 . For KofamKOALA only hits above the predefined threshold for individual KOs were selected. Hydrogenases were extracted using the reference database described in ref 34,55 where there was conflict, the protein was manually reanalyzed using BLAST against non-redundant protein database, and genomic organization and annotation was confirmed using a web-based tool Operon Mapper 59 . The detected hydrogenases were used to generate a phylogenetic tree as previously described in ref 45 . Hits for key metabolic marker genes were verified across different databases KofamKOALA, PFAMv31 and TIGRFAMs and HydDB and were further verified using BLASTP using the NCBI web server tool. Genes encoding for carbohydrate degradation enzymes described in the Carbohydrate-Active enZYmes (CAZYmes) database 60 were identified by only retaining hits recovered by ≥ 2 tools. Protein localization of the selected CAZYmes was determined with the command line version of Psort (V3.0) 61 using the options -a and -terse for archaeal genomes in tabular format files. Finally, the presence of specific protein families was obtained with MEBS. The annotation was performed in a genomic dataset of 250 publicly available TACK genomes (Supporting Table 2) that were also used for the CAZYmes annotation.

Methyl coenzyme M reductase screening.
The mcrA gene was identified using GraftM v0.10.2 62 across metagenome assemblies where Brockarchaeota genomes from hot springs were detected 63 . The mcrA-containing scaffolds with sequence length < 2.5 Kbp were discarded since scaffolds with short length were not used during the 465 genome binning step. The taxonomic information of the corresponding bins which contain mcrA genes were determined using either GTDBtk v0.3.2 64 or phylogenetic placement (as reported in Supplementary Table 9). The mcrABGCD genes were identified in metagenome assemblies from deepsea assemblies previously described in ref 12 (Guay17 and Guay9; IMG genome ID 3300014887 and 3300013103 respectively).

Data availability
The final assembled and annotated genomic sequences of Brockarchaeota from deep sea sediments (B27_G9 and B48_G17) have been deposited in NCBI under BioProject ID PRJNA362212: BioSample id SAMN09215183 and SAMN09214986 respectively. Sequence data and sample 475 information of Brockarchaeota from hot springs are available at NCBI under Bio Project ID PRJNA544494.