Nutritional niches of endemic, facultatively anaerobic heterotrophs from an isolated Antarctic terrestrial hydrothermal refugium elucidated through metagenomics

Nutritional niches of endemic, facultatively anaerobic heterotrophs from an isolated Antarctic terrestrial hydrothermal refugium elucidated through metagenomics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Nutritional niches of endemic, facultatively anaerobic heterotrophs from an isolated Antarctic terrestrial hydrothermal refugium elucidated through metagenomics Craig W. Herbold, Stephen E. Noell, Charles K. Lee, Chelsea J. Vickers, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4805162/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Environmental Microbiome → Version 1 posted 10 You are reading this latest preprint version Abstract Background Tramway Ridge, a geothermal Antarctic Specially Protected Area ( 1 ) (elevation 3340 m) located near the summit of Mount Erebus, is home to a unique community composed of cosmopolitan surface-associated micro-organisms and abundant, poorly understood subsurface-associated microorganisms ( 2 – 5 ). Here, we use shotgun metagenomics to compare the functional capabilities of this community to those found elsewhere on Earth and to infer endemism and metabolic capabilities of abundant subsurface taxa. Results We found that the functional potential in this community is most similar to that found in terrestrial hydrothermal environments (hot springs, sediments) and that the two dominant organisms in the subsurface are primarily endemic. They were found to be facultative anaerobic heterotrophs that likely share a pool of nitrogenous organic compounds while specializing in different carbon compounds. Conclusions Metagenomic insights have provided a detailed understanding of the microbe-based ecosystem found in geothermally heated fumaroles at Tramway Ridge. This approach enabled us to compare Tramway Ridge with other microbial systems, identify endemic taxa and elucidate the key metabolic pathways that may enable specific organisms to dominate the ecosystem. endemic hydrothermal geothermal volcano fumarole metabolism sediments Figures Figure 1 Figure 2 Figure 3 Background Mt. Erebus, Victoria Land, Antarctica, is the highest, most southern, isolated geothermal feature on the planet ( 6 ). Since its original submarine eruption ~ 1.3Ma and through its multiple building phases ( 7 , 8 ), an ever-present Antarctic Circumpolar current has kept Mt. Erebus relatively isolated from volcanoes found elsewhere on Earth ( 9 ). Its biogeographical isolation is supported by previous research on the soil microbial communities at Tramway Ridge, a small geothermal feature on the summit plateau, where unique, deep-branching, and potentially endemic lineages of Bacteria and Archaea were found within highly thermally stratified fumaroles (Fig. 1 ) ( 2 – 4 , 10 , 11 ). The fumaroles of Tramway Ridge differ in many ways from other terrestrial hydrothermal features. They are characterized by hot (65°C) CO 2 -rich steam venting through slightly alkaline (pH 8) hydrothermally altered mineral soils ( 12 , 13 ). Oxygen levels within fumarolic sediments are approximately 1 mg L − 1 (roughly 25% saturated for the temperature) indicative of subsurface hypoxia ( 14 , 15 ). All Erebus hydrothermal features are driven by a phonolite magmatic source (Sims et al. 2021; Noell et al. 2022). Off-gasses tend to be composed primarily of steam, with low levels of sulfur and elevated concentrations of methane, hydrogen and CO ( 16 , 17 ). The high pH, moderate temperature, lack of standing water, and hypoxia distinguish them from other well-studied terrestrial hot springs and mud volcanoes and provide a unique geothermally-driven range of micro-environments for resident microbiota. Steam is vented through concentrated hotspots, generating steep temperature (-20 to 65°C) and pH (3.5–8) gradients over less than a meter that are major determinants of the composition of the thick cyanobacterial mats and associated microbial communities observed on the surface ( 3 , 4 ). Within fumaroles, the temperature is a relatively constant 65°C, even at > 5 cm depth, but can decrease suddenly to less than 20°C and stay low for 24 hours or more at a time ( 3 , 15 ). Beyond its unique physical characteristics, the hot fumarolic soils of Tramway Ridge also have an extremely low total C:N ratio (ranging from 1:3 to 3:1) ( 2 , 3 ), which suggests that the microbial community experiences continual carbon-limitation relative to nitrogen ( 18 ). The isolation of Tramway Ridge, its unique geochemical environment, and microbiota make it an exciting site for evaluating potential endemism and for identifying novel metabolic pathways. Driven by the novelty of the taxa encountered there and the lack of information regarding their metabolic potential, we launched a detailed metagenomic study of high-altitude Antarctic fumaroles. First, we used functional profiles to contextualize the functional repertoire of this particular community with respect to other types of microbial communities. Second, we improved on our previous effort to identify endemic taxa ( 2 ), where we relied on matching partial 16S rRNA gene amplicon sequences with database entries by developing a novel metric that is based on synonymous polymorphisms within reconstructed environmental populations to define/circumscribe endemic taxa. Finally, we sought to elucidate possible novel metabolic processes encoded by abundant and endemic taxa that are specifically localized to the fumarolic subsurface (depths > 2 cm). Methods Sample collection Soil samples were collected within the Tramway Ridge Antarctic Specially Protected Area (ASPA 130) in February 2009 from two sites (site A – 77° 31.103' S, 167° 6.682' S and site B – 77° 31.306' S, 167° 6.668' E). Sites were chosen based on measuring a surface temperature of 65°C with a stainless steel Checktemp1 temperature probe (Hanna Instruments, Rhode Island, USA) sterilized with 70% ethanol immediately prior to use. Temperature measurements were repeated for each layer sampled. Surface soil crusts were carefully set aside prior to collecting samples. Samples were collected by carefully removing 2 cm of soil in an approximately 5 cm x 5 cm square area using an autoclaved stainless steel spatula wiped with 70% ethanol just prior to sampling. Soil was placed into a fresh 50 mL Falcon tube and immediately frozen at -20°C. Sampling continued with the collection of a second layer (2–4 cm depth). DNA extraction, library preparation and sequencing DNA was extracted from samples using a modified CTAB (cetyltrimethylammonium bromide) bead-beating protocol ( 19 ) and quantified using the Quant-IT dsDNA BR Assay Kit (Invitrogen, Carlsbad, CA, USA). A portion of extracted metagenomic DNA was frozen and sent to sequencing facilities at the University of California-Los Angeles (USA) and the University of Waikato (Hamilton, New Zealand). At each location, samples were processed and sequenced using standard protocols for the 454-Ti platform (Roche 454 Life Sciences, Branford, CT, USA). Additional DNA fractions were sent to the sequencing facility at University of California, Davis, where paired-end libraries were prepared and sequenced using the Illumina Hi-Seq platform (Illumina, San Diego, CA, USA). Metagenome assembly and binning Assembly of metagenomic data was carried out using appropriate assemblers for the two different sequencing platforms. 454 reads were assembled from original sff files using Newbler (Roche). Reads from each dataset were assembled independently and were pooled for an additional assembly. Newbler assembly used default parameters except that minimum overlap was set to 100 nucleotides and overlap identity was set to 98% identity. Paired-end Illumina reads were pre-processed by removing any paired-end set for which identifying tags had at least one mismatch or for which the paired-end tags were not identical. Identifying tags were removed, and reads were end-trimmed at the first incidence of a quality score below 14. Any reads that were shorter than 20 nucleotides after tag removal and end-trim were removed from the dataset. 454 datasets were assembled in Newbler (-mi 98 -ml 100 -minlen 45 -a 500 -l 2000). Illumina assemblies were carried out using two sets of options (--careful –only-assembler; --cov-cutoff auto --careful -k 25,55,65,75 --only-assembler) in Spades ( 20 ), a single setting (--meta --only-assembler) in MetaSpades ( 21 ) and a single setting (--k-min 27 --k-max 127 --k-step 10 --min-contig-len 500 --prune-level 3 --no-mercy --min-count 3 --no-local) in Megahit ( 22 ). BBMap ( https://sourceforge.net/projects/bbmap/ ) was used to map all individual read sets (454 and Illumina) against all assemblies. Large (> 2 kb) contigs and scaffolds were clustered into Metagenome-assembled genomes by oligonucleotide frequency and read coverage using Maxbin 2 ( 23 ) and Metabat 2 ( 24 ). Redundant bins were subsequently dereplicated and evaluated using dRep ( 25 ) with a completeness cutoff of 40%, contamination cutoff of 10% and a minimum genome size of 200kb. Final MAGs (Table 1 ) were classified using GTDB-Tk v2.1 ( 26 ) with genome database release R214 ( 27 ) and annotated with the NCBI Prokaryotic Genome Annotation Pipeline ( 28 ). MAGs discussed in the text were additionally annotated with eggNOG mapper ( 29 ), Cytochrome c oxidases were Table 1 Species-level representatives of metagenome-assembled genomes (MAGs) from Tramway Ridge fumarolic soils. Lineage was assigned using GTDB-Tk v.2.1 ( 26 ) with Release R214 of the Genome Taxonomy Database ( 27 ). Completeness and contamination estimates were calculated in CheckM ( 50 ). Assembly quality follows the recommendations outlined previously ( 55 ). For a comprehensive list of genome quality attributes for all binned strains, see supplementary Table 3. Proposed name species group # of strains in species group Division assembly quality GC Genome size (bp) Blastocatellia bacterium ERB_27 27 2 Acidobacteria high 50.9 2698697 Pyrinomonas sp. ERB_32 32 1 Acidobacteria high 60 3512547 Acidimicrobiia bacterium ERB_23 23 3 Actinobacteria medium 55.1 1963147 Acidimicrobiia bacterium ERB_8 8 1 Actinobacteria medium 69.4 1352013 Thermoleophilia bacterium ERB_19 19 3 Actinobacteria high 69.5 2326210 Armatimonadetes bacterium ERB_24 24 1 Armatimonadetes medium 60.8 1563163 Armatimonadetes bacterium ERB_33 33 1 Armatimonadetes high 60.5 3260803 Armatimonadetes bacterium ERB_34 34 2 Armatimonadetes high 58 2190352 Armatimonadetes bacterium ERB_6 6 3 Armatimonadetes high 61.1 2058555 Chitinophagaceae sp. ERB_2 2 1 Bacteroidetes high 40.5 2908196 Chitinophagaceae sp. ERB_3 3 2 Bacteroidetes medium 39.3 2810122 Candidatus GAL15 bacterium ERB_18 18 2 candidate division GAL15 high 70.1 1986527 Candidatus Dadabacteria bacterium ERB_12 12 1 Candidatus Dadabacteria medium 44.2 3057813 Candidatus Fervidibacteria erebusii ERB_15 15 1 Candidatus Fervidibacteria high 56.3 2795547 Candidatus Lakebacteria bacterium ERB_C1 C1 3 Candidatus Lakebacteria medium 27.3 481934 Chloroflexi bacterium ERB_10 10 1 Chloroflexi medium 64.7 2882324 Chloroflexi bacterium ERB_11 11 1 Chloroflexi medium 68.5 2692019 Chloroflexi bacterium ERB_20 20 3 Chloroflexi high 63.6 3392850 Candidatus Nitrocaldera therma ERB_22 22 2 Chloroflexi high 65.1 2707038 Chloroflexi bacterium ERB_25 25 1 Chloroflexi Low-quality draft 64.8 1709950 Chloroflexi bacterium ERB_7 7 1 Chloroflexi medium 70.2 1489492 Chloroflexi bacterium ERB_9 9 2 Chloroflexi medium 62.8 3793921 Thermoflexus sp. ERB_21 21 3 Chloroflexi high 69.6 2384148 Leptolyngbya sp. ERB_1 1 2 Cyanobacteria medium 47.2 4634638 Mastigocladus sp. ERB_26 26 2 Cyanobacteria high 41.2 5965913 Meiothermus sp. ERB_29 29 2 Deinococcus-Thermus medium 66.3 2588060 Meiothermus sp. ERB_30 30 2 Deinococcus-Thermus medium 61.5 3765706 Meiothermus sp. ERB_31 31 1 Deinococcus-Thermus medium 68.5 2746960 Thermus sp. ERB_17 17 5 Deinococcus-Thermus high 65.2 1806162 Ignavibacteria bacterium ERB_28 28 1 Ignavibacteriae high 55.2 3309624 Nitrospiraceae bacterium ERB_14 14 2 Nitrospirae medium 56.2 3371732 Gemmataceae bacterium ERB_16 16 2 Planctomycetes high 60.1 3796795 Rhodanobacteraceae bacterium ERB_4 4 1 Proteobacteria medium 62.8 1332200 Candidatus Australlarchaeum erebusii ERB_5 5 1 Thaumarchaeota medium 63.6 1213694 Nitrososphaera sp. ERB_13 13 1 Thaumarchaeota medium 56.6 1122400 checked with the HCO classifier ( 30 ), hydrogenases were checked with HydDB ( 31 ), and CAZymes were checked with dbCAN3 ( 32 ). Endemicity Index Calculation Quality-trimmed Illumina reads were mapped to each MAG using BBmap and further filtered using a hard 97% identity cutoff where identity = number of matches/length of alignment, with the additional requirement that at least 50 nucleotides mapped. Raw diversity was compiled using Samtools mpileup using option: -d 1000000 ( 33 ) for each MAG and each dataset independently. SNPs were determined using Varscan2 pileup2snp (options: --min-var-freq 0.01 --p-value 0.05) ( 34 ) and further filtered using the Benjamini-Hochberg multiple testing correction FDR = 0.01. SnpEff ( 35 ) was used to classify SNPs as synonymous with gff files produced with Prodigal ( 36 ). The density of synonymous SNPs ( D SynSNP ) for a MAG was calculated as the number of Synonymous SNPs ( N SynSNP ) divided by MAG length in Mb ( L MAG ): D SynSNP = N SynSNP / L MAG . Because sensitivity increases as read depth increases, D SynSNP was corrected for read coverage of the MAG ( C MAG ) which was calculated as the number of reads mapped ( N M ) divided by MAG length ( L MAG ): C MAG = N M / L MAG . Endemicity Index ( EI ) was then the density of synonymous SNPs ( D SynSNP ) divided by read coverage ( C MAG ): EI = D SynSNP / C MAG . EI was calculated for each MAG/dataset combination only if N SynSNP ≥ 5 and reported EI values are the average of calculated values over four Illumina read sets (SRA accessions SRR6519253, SRR6519254,SRR6519255, SRR6519256). Functional profile comparisons Pfam ( 37 ) profiles were used to compare functional similarity between the metagenomic assembly from Tramway Ridge and publicly available metagenomic assemblies in the Integrated Microbial Genomes (IMG) database ( 38 ). A list of all assembled, published, and “unrestricted” environmental metagenomic datasets available through IMG was downloaded on 31 March, 2023. IMG-generated pfam profiles (counts of pfams present in metagenomic assembly) were downloaded for each available metagenome, resulting in 7652 total pfam profiles. The number of metagenomic datasets was reduced by removing datasets with total assembly length less 5x10 7 or greater than 1x10 9 bases. This subset was further reduced by removing metagenomic datasets with fewer than 3500 or greater than 7500 unique pfams. These filtering criteria resulted in a dataset of 4513 publicly sourced metagenomic datasets for comparative analysis. Profiles were analyzed in R 4.2.3. Jaccard dissimilarity was calculated using the vegdist() function from vegan 2.6-4 ( 39 ) based on presence/absence of pfams. Principle coordinate analysis (PCoA) was carried out using the pcoa() function from Version 5.7-1 of ape ( 40 ) and plotted in three dimensions using the plot3d() function from rgl 1.2.1 ( 41 ). t-distributed stochastic neighbor embedding (tSNE) was calculated using the Rtsne() function from Rtsne 0.16 ( 42 ) and plotted with ggplot2 3.4.3 ( 43 ). Rtsne settings were as follows: Rtsne(X, dims = 2,initial_dims = 5, perplexity = 300, theta = 0.5, check_duplicates = FALSE, pca = TRUE, partial_pca = FALSE, max_iter = 5000000, verbose = getOption("verbose",FALSE), is_distance = TRUE, Y_init = NULL, pca_center = TRUE, pca_scale = FALSE, normalize = TRUE, stop_lying_iter = 500000, mom_switch_iter = 500000, momentum = 0.5, final_momentum = 0.8, eta = 100, exaggeration_factor = 12). Phylogenomic analysis A collection of reference genomes for comparative phylogenomic analysis was assembled from representative species defined in release 214 (April, 2023) of the Genome Taxonomy Database ( 27 ). GTDB classifications of MAGs from the current study were used to select from GTDB species representatives for an informative tree. For instance, in the case where a MAG was classified into an order but not a family, one representative taxon from each family was included, the phylogeny was calculated, and if the MAG was associated with a particular family, the process was repeated using genus representatives from that family. These genomes were supplemented with additional genomes from thermophilic environments ( 44 , 45 ), thermophilic nitrifying enrichment cultures ( 46 , 47 ), additional Nitrospirota ( 48 ) and additional Nitrososphaeria ( 49 ). All genomes were downloaded and processed using CheckM ( 50 ) to generate concatenated alignments of 34 universal marker genes (43 marker HMMs). To be included in phylogenomic reconstruction, reference genomes were required to be at least 60% complete with less than 5% contamination and to have at least 4500 ungapped characters in the concatenated alignment (6988 total positions). All genomes used for phylogenetic analysis are listed in Supplementary Table 8. Phylogenetic reconstruction with IQ-TREE 2 ( 51 ) included model selection with ModelFinder ( 52 ) and calculation of bootstraps with UFboots ( 53 ). Results We found that the functional profiles from Tramway Ridge metagenomes resembled those from other thermally-influenced environments, in particular those from terrestrial hydrothermal systems. To learn this, we compared functional profiles of assembled Tramway Ridge metagenomes to 4513 publicly available and assembled environmental metagenomes broadly categorized as terrestrial hydrothermal / non-hydrothermal, freshwater, and marine hydrothermal / non-hydrothermal (Supplementary Table 1). Functional profiles for metagenomes were constructed based on the presence-absence of Pfam protein family domains ( 54 ), and dissimilarities were calculated using the Jaccard index. A post-hoc Tukey’s HSD test (Fig. 2 a) comparing Jaccard dissimilarity grouped according to the broad categories listed above showed that metagenomes from Tramway Ridge were most similar to terrestrial hydrothermal environments (Tukey category a) and least similar to metagenomes from marine non-hydrothermal and freshwater environments (Tukey category d). We continued with principal coordinate analysis (PCoA) and t-distributed Stochastic Neighbor Embedding (tSNE) visualizations to explore relationships that may not have been clear from grouped pairwise comparisons (Fig. 2bc). In both, Tramway Ridge microbial communities clustered loosely with microbial communities sourced from both terrestrial and marine hydrothermal environments to the exclusion of non-hydrothermal environments. T-SNE visualization (Fig. 2 c) recovered several distinct but associated clusters from hydrothermal systems, one of which was composed exclusively of profiles from Tramway Ridge. Metagenome-assembled genomes (MAGs) were constructed from DNA extracted from soils at two 65°C fumaroles at Tramway Ridge (supplementary Table 2). We recovered 63 MAGs at the strain level (99% average nucleotide identity, ANI), which clustered into 35 species-level representatives (> 96.5% average nucleotide identity) (Fig. 3 , Table 1 , supplementary Table 3). A total of 16 MAGs met the criteria for high-quality drafts, and 18 MAGs met the criteria for medium-level drafts ( 55 ); these include nearly complete (75–96%) genome bins for a novel order of Archaea within the Nitrososphaeria (Fig. 3 b) and novel lineages of Bacteria including Armatimonadota, Chloroflexota, Actinobacteriota, and Candidate division CSP1-3. Additional MAGs of interest include those belonging to the Candidatus Patescibacteria phylum (Fig. 3 c), Mastigocladus genus (Fig. 3 d) and Candidatus Fervidibacter genus (Fig. 3 e). MAGs were classified using GTDB-Tk v.2.1 ( 26 ) with Release R214 of the Genome Taxonomy Database ( 27 ), and novel taxa of note were given proposed names (Table 1 , supplementary Table 3). Tramway Ridge occupies a unique location on Earth that makes it ideal for the study of endemism due to its highly isolated nature and the geothermal enrichment that prevents intrusion of nearby psychrophilic and mesophilic microorganisms. Here, we quantified endemism for each of the MAGs we generated. We defined the degree of endemism of a given taxon as being proportional to the in situ diversity of that taxon, reasoning that endemic species have had an extended opportunity to diversify on-site, as opposed to recent arrivals that would be subject to founder effects. We developed a simple metric, which we called the endemicity index (EI) (Fig. 3 F) that measured the frequency of synonymous mutations in a MAG accounting for read depth. High values (e.g. 10 − 2 ) of EI indicate high diversity whereas low values (e.g. 10 − 6 ) indicate low diversity. The lowest median EI value (1 x 10 − 5 ) for a single species was calculated for ERB_26, (Fig. 3 f), which represented two individual strains of a cyanobacterium belonging to the Fischerella / Mastigocladus genus (supplementary Table 3) and dominated the near-surface (5–10% of near-surface reads). The highest median EI value (1 x 10 − 2 ) for a single species was calculated for ERB_C1 (Fig. 3 f), which represented three individual strains of Candidatus Patescibacteria (supplementary Table 3). Ca. Lakebacteria bacterium ERB_C1 classified into the HRBIN35 genus in the HR35 family (Fig. 3 c). Based on the percentage of reads recruited, it was slightly more abundant in the near-surface (0–2 cm depth) than in the subsurface (> 2 cm, Fig. 3 g). Due to this distribution pattern and the fact that an accelerated evolutionary process is a hallmark feature of members of this lineage ( 27 , 56 , 57 ), we did not pursue further analysis of this genome, which would be best accomplished in a comprehensive comparison with other Ca. Patescibacteria. Two MAGs, ERB_15_1 and ERB_5_1, with relatively high EI values (3 x 10 − 3 and 1 x 10 − 3 , respectively) dominated the subsurface community at depths greater than 2 cm (Fig. 3 f, Fig. 3 g). ERB_5_1 shared less than 40% average amino acid identity (AAI) with any other Nitrososphaeria ( formerly Thaumarchaeota) and formed a singular deep branch of the Nitrososphaeria in phylogenetic trees (FIgure 3 b). ERB_15_1 shared 69% AAI and 77% ANI with Candidatus Fervidibacteria sacchari, establishing it as a novel species of this genus (Fig. 3 c). Here we propose the names Candidatus Fervidibacteria erebusii (ERB_15_1) and Candidatus Australlarchaeum erebusii (ERB_5_1) to reflect their first observation and endemism at Mt. Erebus. We picked these two MAGs for further analyses based on their high EI values, subsurface abundance, and taxonomic novelty. Candidatus A. erebusii (ERB_5_1) branches near the base of the Nitrososphaeria (Fig. 3 b) and, like other deeply diverging lineages, it lacked marker genes for cobalamin biosynthesis ( cob/cbi / bluB ) and ammonia oxidation (archaeal amoABC, HAO ). Consistent with previous observations of deeply diverging Nitrososphaeria , the genome of Ca. A. erebusii encoded genes for the beta oxidation of fatty acids and peptide degradation and several genes that may be used for amino acid-based oxidation (Supplementary Table 5). Ca. A. erebusii encoded aminotransferases and glutamate dehydrogenase that could be used to produce NAD(P)H by converting glutamate to 2-oxoglutaric acid. A 2-oxoglutarate:ferredoxin oxidoreductase could then convert the 2-oxoglutaric acid to succinyl-CoA, which could then be converted to succinate with the production of ATP via ADP-forming succinate-CoA ligase (Supplementary Table 5). Unlike other deep-branching Nitrososphaeria, Ca. A. erebusii also additionally encoded two aa3 -type (low-affinity) cytochrome C-oxidases (Supplementary Table 5). We identified a putative aerobic carbon monoxide dehydrogenase (CODH) cluster of genes ( coxMLS ) that may confer the ability to use CO as an additional energy source. However, the large subunit of the CODH in Ca. A. erebusii contained a hallmark motif, AYXGAGR, of type II CODH, which oxidizes CO only very slowly and possibly incidentally ( 58 ). No predicted carbon fixation pathways were identified. Candidatus Fervidibacteria erebusii (ERB_15_1) shared 69% AAI and 77% ANI with Candidatus Fervidibacteria sacchari, establishing it as a novel species of this genus (Fig. 3 c). Based on conservative annotations, the Ca. F. erebusii genome encoded 144 CAZymes, including 69 glycosyl hydrolase (GH) domains spanning 32 GH families (Supplementary Table 6, Supplementary Table 7), and an impressive 15 variants of the unusual and poorly characterized GH109 family. Furthermore, when we group the Ca. F. erebusii CAZymes by substrate utilization capacity, the GH109s belong to the largest cohort (35.25%) which appear to be involved in depolymerisation cascades of N-acetylhexosamide glycans such as chitins and chitosan (Supplementary Table 7). Other major polysaccharide utilization cohorts include cellulose and hemicellulose (19.8%), pectin (14.3%) and ß-glucans (19.8%), with minor cohorts for ɑ-glucans, ɑ-mannans and ɑ-fucans. Ca. Fervidibacter genomes, including Ca. F. erebusii additionally encoded numerous aminotransferases, glutamate dehydrogenase, indole-pyruvate:ferrodoxin oxidoreductase and ADP-forming succinate-CoA ligase (Supplementary Table 6) which may explain the ability of Ca. Fervidibacter sacchari to grow solely on casamino acids ( 59 ). The Ca. F. erebusii genome also encoded Group 2a NiFe hydrogenases which are high-affinity hydrogenases that enable the survival of soil heterotrophs under carbon limitation ( 60 ) and enable hydrogenotrophic growth of autotrophic nitrite-oxidizing bacteria under nitrite limitations ( 61 ). The Ca. F. erebusii genome also encoded a cluster of putative CODH genes ( coxMLS ), which contained the hallmark motif, AYXGAGR, of type II CODH, indicating a low, incidental activity ( 58 ). No carbon fixation pathways were found. Discussion Metagenome Functional Profiles Mt. Erebus has evolved over time, starting with seafloor rifting and growing as a subaerial volcano into a modern-day stratovolcano ( 7 , 8 ). The current conditions at Tramway Ridge differ remarkably from other extant non-Antarctic terrestrial and marine hydrothermal systems, being primarily driven by the unique phonolite magmatic source resulting in alkaline fumaroles with low sulphidic content. It is unknown how long geothermal features such as the fumaroles at Tramway Ridge have been present on Mt. Erebus; however, volcanism was once widespread across the Ross Island massif ( 6 ). Extant geothermal features may represent a once widespread ecosystem of similar features. Given this complex history of Mt. Erebus, we questioned whether the Tramway Ridge community has retained a legacy signature of its origin on the seafloor or if the modern-day Tramway Ridge community better resembles other terrestrial hydrothermal sites. To answer this question, we compared functional profiles of assembled Tramway Ridge metagenomes to a large set of publicly available metagenomes. Metagenomes from Tramway Ridge were distinct from all others (Fig. 2 c) but showed the most similarity to terrestrial hydrothermal environments such as hot springs and associated sediments. This indicates that the legacy of a sea floor origin is less important than the extant conditions at Tramway ridge in defining the microbial community. This is also reflected in the prevalence of taxa at Tramway Ridge that are found in other terrestrial hydrothermal environments but not in seafloor hydrothermal systems, such as the genera Mastigocladus , Caldithermus , Meiothermus and phyla such as the Chloroflexota, Armatimonadota and Actinobacteriota. The unique nature of Tramway Ridge metagenomic profiles likely reflects the site’s relatively low diversity ( 2 ) and the type of functional profile analysis used here. First, pfams were used, which are very coarse functional units. Second, gene abundance was not available for most metagenomes, so we were forced to use presence-absence as data. Third, we found that the use of pfam presence-absence was sensitive to metagenome assembly size as well as the total number of unique pfams detected. In combination, these features had the potential to obfuscate any meaningful relationships. Our analysis was optimized to be as permissive as possible, allowing as many metagenomes into the analysis while still comparing like against like. Using this strategy, we were able to compare Tramway Ridge metagenomic datasets to 4513 out of 7652 publicly available and unrestricted metagenomes (see 10.5281/zenodo.10928974 ). Within continental Antarctica, only three known surface-expressed active geothermal areas exist (Mt. Rittmann, Mt. Melbourne and Mt Erebus), each of which is separated by vast ice fields ( 15 ). It is thought that intercontinental transport of microbe-bearing particulates into Antarctica occurs much less frequently than intracontinental transport ( 62 ), suggesting that the introduction of exogenous microbes is relatively infrequent. Recent studies have used database searches to identify endemic species ( 63 ), and a lack of sequence identity to database entries has been used in the past to suggest that novel sequences indicate endemism ( 2 ). However, defining endemism based on whether sequence matches exist within a database can be problematic as this definition is sensitive to database composition. Conclusions drawn may not withstand the inevitable growth in database size and the diversity it holds. For this study, we took an alternative approach and defined the degree of endemism of a given taxon as being proportional to the in situ diversity of that taxon. For these inferences, we assumed that rare colonization events have been limited to single clones due to the extreme isolation of Mt. Erebus. Therefore, populations arising from recently introduced taxa would be expected to exhibit relatively low levels of genetic polymorphism and endemic microbial populations would be expected to show high levels of genetic polymorphism. In particular, we focused on accumulations of synonymous mutations, which were assumed to be under reduced selection pressure. We developed the endemicity index (EI) (Fig. 3 F) to assess the diversity of a microbial population represented by a MAG. Similar calculations have been successfully applied to approximate effective population size and genomic fluidity ( 64 ). High values (e.g. 10 − 2 ) of EI indicate high diversity which we interpreted as reflecting a neutral evolutionary process occurring under minimal contemporary selection pressures. Low values (e.g. 10 − 6 ) indicate low diversity, which we interpreted as possible evidence of a relatively recent arrival, a local population bottleneck, or a recent selective sweep. A cyanobacterium belonging to either the Fischerella or Mastigocladus genus recovered the lowest median EI value (1 x 10 − 5 ) for a single species. This species dominated the near-surface. Although difficult to distinguish these two genera based on 16S rRNA gene sequence and GTDB classifies all members of the genera Fischerella and Mastigocladus as Fisherella , the distinction of Mastigocladus is recognized as a distinct genus by the List of Prokaryotic names with Standing in Nomenclature (LPSN). Therefore we classified this MAG as a member of the Mastigocladus genus to be consistent with the classification of specimens collected from Tramway Ridge in the past ( 65 ). The low endemicity index observed for this taxon was consistent with previous studies that showed that the global phylogeography of Mastigocladus reflects a geologically recent radiation from Yellowstone National Park, USA ( 66 ) and that the surface-associated microbial community at Tramway Ridge is likely dominated by aeolian-distributed cosmopolitan members of non-Antarctic temperate and terrestrial hydrothermal soil communities ( 2 ). We identified Candidatus Australlarchaeum erebusii (ERB_5_1) and Candidatus Fervidibacteria erebusii (ERB_15_1) as two MAGs with relatively high EI values and abundance in the subsurface at depths greater than 2 cm (Fig. 3 f, Fig. 3 g). In our earlier amplicon-based study, these two taxa were similarly identified as dominant, endemic, and associated with the subsurface, but were referred to as “Thaumarchaeota-like archaeon” and OCtSpA1-106 respectively ( 2 ). The most abundant endemic, subsurface-associated organism recovered was Candidatus A. erebusii (ERB_5_1), a member of the Nitrososphaeria (formerly Thaumarchaeota). This class of Archaea is a globally distributed group that is best known for the chemolithoautotrophic oxidation of ammonia ( 67 ) and for the apparent universal synthesis of cobalamin ( 68 ). However, several deeply divergent lineages of Nitrososphaeria identified through the construction of MAGs ( 45 , 69 , 70 ) and a single cultivated species, Candidatus Conexivisphaera calidus NAS-02 ( 71 ) have been shown to lack these hallmark attributes. These deeply diverging lineages have been predicted to be predominantly anaerobic heterotrophs ( 69 , 70 ) capable of beta oxidation of fatty acids and protein/peptide degradation ( 71 , 72 ). Candidatus A. erebusii (ERB_5_1) is one of the deepest-branching members of the Nitrososphaeria (Fig. 3 b) and like other deeply diverging lineages, it encodes genes for the beta oxidation of fatty acids and peptide degradation while lacking marker genes for cobalamin biosynthesis and ammonia oxidation (Supplementary Table 5). Candidatus A. erebusii may also employ amino acid-based oxidation, similar to a pathway used by Thermococcus kodakarensis ( 73 ) and a proposed alternative metabolism for Ca . Nitrosocaldus islandicus ( 74 ), a representative of thermophilic ammonia oxidizing archaea (AOA). In this proposed metabolism, glutamate could be utilized to generate both reducing power and ATP to power the cell. However, Ca. A. erebusii is also predicted to respire oxygen, a unique prediction among its closest, presumably anaerobic relatives. It encodes two aa3 -type (low-affinity) cytochrome C-oxidases which could presumably drive beta oxidation of fatty acids. However it is unclear whether aerobic respiration could be coupled with an amino acid degradation pathway, which is typically thought to be an anaerobic metabolism ( 73 , 74 ). It is unclear if these energy-generating pathways are mutually exclusive and operate under specific oxidative conditions. At least during summer, oxygen levels in the subsurface are around 30% saturation ( 2 ) and therefore, no organisms inhabiting the fumaroles are likely to be obligate anaerobes. However, it is also reasonable to assume that the wet, steamy subsurface experiences anoxia at least at small spatial scales. Therefore, we hypothesize that Ca. A. erebusii switches between metabolic pathways depending on the oxic environment, using beta-oxidation of fatty acids when oxygen levels are sufficiently high and peptide fermentation when oxygen levels are low. CO metabolism is likely to be used for maintenance during times of nutritional stress, as has previously been shown for Antarctic soil microbes ( 49 ). Another abundant and endemic subsurface MAG examined in detail belongs to the thermophilic genus Candidatus Fervidibacteria, which was first discovered in Octopus Spring, Yellowstone National Park (clone OctSpA1-106, ( 75 ). The genus is named after Ca. Fervidibacter sacchari, which encodes a large repertoire of carbohydrate-active enzymes (CAZymes) ( 76 ) and which has recently been isolated and described ( 59 ). Like other Ca. Fervidibacter, Ca. F. erebusii (ERB_15_1) encodes a significant number and diversity of CAZymes including a large, diverse cohort of the unusual and poorly characterized GH109 family. Previous findings suggest that these novel enzymes are involved in extracellular polysaccharide metabolism unique to thermophilic systems ( 59 , 77 , 78 ) with the diversity of Ca. F. erebusii GH109s suggesting a diverse and unique polysaccharide utilization profile typical of this genus. Interestingly, Ca. Fervidibacter genomes, including Ca. F. erebusii appear to also encode mechanisms that enable growth on casamino acids ( 59 ). The encoded hydrogenase and CODH are likely maintenance mechanisms to enable survival under carbon limitation with growth primarily supported through aerobic heterotrophic growth on saccharides and anaerobic growth on amino acids. Conclusion In the current study, we have used metagenomics to assess several aspects of the microbial community inhabiting the fumarolic soils of Tramway Ridge, Mt. Erebus, Antarctica. We observed a shared functional repertoire between Tramway Ridge and other geothermal systems, specifically terrestrial hydrothermal systems such as hot springs. We then assessed metagenome-assembled genomes (MAGs) using a novel endemicity measure to identify two highly endemic taxa that are more abundant in the Tramway Ridge subsurface (> 2 cm depth) than the near-surface (< 2 cm depth). We named these two taxa Candidatus Australlarchaeum erebusii and Candidatus Fervidibacter erebusii to reflect that they were first observed as dominant, unique and endemic to Mt. Erebus. A close examination of the metabolic repertoire of these taxa revealed that they are likely both facultative anaerobic heterotrophs that specialize in using different carbon sources under aerobic conditions, but that use similar organic compounds during anaerobic growth. Like other deep-branching non-AOA Nitrososphaeria, Ca. A. erebusii possesses a putative pathway for the beta-oxidation of fatty acids. Like other Candidatus Fervidibacter, Ca. F. erebusii is predicted to utilize sugars and scavenge hydrogen gas under aerobic conditions ( 76 , 79 ). Under oxygen-limited conditions, both may utilize similar peptides and amino acids for energy and carbon acquisition. We hypothesize that this pattern of metabolic utilization may reflect the extreme and carbon-limiting C:N ratios (1:3 to 3:1) encountered at Tramway Ridge. Dominant taxa may share nitrogen-rich compounds such as peptides and amino acids since the demand for such compounds may not be as high as for carbon-rich compounds. Instead, different taxa specialize in utilizing specific carbon compounds (sugars vs. fatty acids) through selective exclusion, allowing both to co-exist by carving out specific nutritional niches. Each is also equipped with form II aerobic carbon monoxide dehydrogenase genes that may provide maintenance energy during times of starvation. Together, these insights provide an unprecedented view into the dominant metabolic processes that may sustain life in this harsh, isolated environment. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and material All raw metagenomic sequence data and metagenome assembled genomes used in this manuscript are available under bioproject accession PRJNA431961 (https://www.ncbi.nlm.nih.gov/bioproject/431961) . Data and scripts necessary to reproduce comparative metagenomic work is available at 10.5281/zenodo.10928974 Competing interests Not applicable Funding Financial support was provided by grant UOW0802 from the New Zealand Marsden Fund to SCC and IRM and a CRE award from the National Geographic Society to S.C.C. Author contributions Planning and field sampling by CWH, CJV, SCC and IRM. Data analysis by CJV and CWH. All authors contributed to the interpretation of data and writing of the manuscript. Acknowledgements Antarctic logistic support for Event K-023 was provided by Antarctica New Zealand. References Parties of the Antarctic Treaty. 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Stott","email":"","orcid":"","institution":"Te Kura Pūtaiao Koiora - School of Biological Sciences, Te Whare Wānanga o Waitaha - University of Canterbury","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"B.","lastName":"Stott","suffix":""},{"id":341607873,"identity":"db935904-28de-4960-978d-4d1303ccddb5","order_by":5,"name":"Jonathan A. Eisen","email":"","orcid":"","institution":"University of California-Davis","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"A.","lastName":"Eisen","suffix":""},{"id":341607875,"identity":"2aea7893-9f0c-4a81-8373-8303412e9efd","order_by":6,"name":"Ian R. McDonald","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIiWNgGAWjYBACNgjFDMINEh8YGHgkSNDC2CA5A6yFmSjLIFqkeYAUQS187GcPMN2osZYzZz/YeNu2zU5Gsv38wQcMNXYM5vwHsDuMJy+BOedYurFlT2KzdW5bMo80TzKzAcOxZAbLBhxaGHIMmHMbDiduOJDYJp27jZlHjiGZTYKB7QCDwcEG7Fr430C1nH/YJm25rZ5Hjv8xUMs/oJbD2P3CJgGz5QbQFsZth3mkJYC2MLYBtRzDpeWNwWGQXwxuPGy27P13nEdyxmNjg8S+ZB6DM9i1yPfnGD7OAYaYwfnkgzd+nKm2lzif+PDBh292QBHs3gcBLDIJwDjFqX4UjIJRMApGAUEAAICFVadtV93BAAAAAElFTkSuQmCC","orcid":"","institution":"Te Aka Mātuatua - School of Science, Te Whare Wānanga o Waikato - University of Waikato","correspondingAuthor":true,"prefix":"","firstName":"Ian","middleName":"R.","lastName":"McDonald","suffix":""},{"id":341607878,"identity":"b5e90277-7132-4bee-b485-ec2aa43f262f","order_by":7,"name":"S. Craig Cary","email":"","orcid":"","institution":"Te Aka Mātuatua - School of Science, Te Whare Wānanga o Waikato - University of Waikato","correspondingAuthor":false,"prefix":"","firstName":"S.","middleName":"Craig","lastName":"Cary","suffix":""}],"badges":[],"createdAt":"2024-07-26 03:55:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4805162/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4805162/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40793-024-00655-5","type":"published","date":"2024-12-18T15:58:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63072607,"identity":"c47156c4-bde0-4391-8b3a-7b2983d84020","added_by":"auto","created_at":"2024-08-22 20:12:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":316815,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4805162/v1/e5adf0827dd55ab196ffd0c5.png"},{"id":63072637,"identity":"5ff7729e-52d5-4430-8dc8-6e1b593e990f","added_by":"auto","created_at":"2024-08-22 20:12:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116648,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4805162/v1/daae5766d1428cbb535968ca.png"},{"id":63072864,"identity":"bbef7970-1bf4-4902-9faa-e07b30a1a20a","added_by":"auto","created_at":"2024-08-22 20:13:06","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109883,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4805162/v1/1cbbcd9231feff8e272c0e1e.jpeg"},{"id":72201924,"identity":"0ec983b3-e254-43c8-b20f-cdcca16a67ae","added_by":"auto","created_at":"2024-12-23 16:12:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1330059,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4805162/v1/6d702470-5ddf-4f09-9222-33de87f4c5d2.pdf"},{"id":63072748,"identity":"bceeb885-97b0-4a8f-8e6b-86be89eee3fd","added_by":"auto","created_at":"2024-08-22 20:12:58","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1025222,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFig1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4805162/v1/a089457b78a50ad0c2b132b1.pdf"},{"id":63072457,"identity":"ca3daf97-3962-43f9-8d33-0111e5f079a8","added_by":"auto","created_at":"2024-08-22 20:12:21","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1320060,"visible":true,"origin":"","legend":"","description":"","filename":"SuppTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4805162/v1/855864d05d284baba68a4e55.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nutritional niches of endemic, facultatively anaerobic heterotrophs from an isolated Antarctic terrestrial hydrothermal refugium elucidated through metagenomics","fulltext":[{"header":"Background","content":"\u003cp\u003eMt. Erebus, Victoria Land, Antarctica, is the highest, most southern, isolated geothermal feature on the planet (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Since its original submarine eruption\u0026thinsp;~\u0026thinsp;1.3Ma and through its multiple building phases (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), an ever-present Antarctic Circumpolar current has kept Mt. Erebus relatively isolated from volcanoes found elsewhere on Earth (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Its biogeographical isolation is supported by previous research on the soil microbial communities at Tramway Ridge, a small geothermal feature on the summit plateau, where unique, deep-branching, and potentially endemic lineages of Bacteria and Archaea were found within highly thermally stratified fumaroles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe fumaroles of Tramway Ridge differ in many ways from other terrestrial hydrothermal features. They are characterized by hot (65\u0026deg;C) CO\u003csub\u003e2\u003c/sub\u003e-rich steam venting through slightly alkaline (pH 8) hydrothermally altered mineral soils (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Oxygen levels within fumarolic sediments are approximately 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (roughly 25% saturated for the temperature) indicative of subsurface hypoxia (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). All Erebus hydrothermal features are driven by a phonolite magmatic source (Sims et al. 2021; Noell et al. 2022). Off-gasses tend to be composed primarily of steam, with low levels of sulfur and elevated concentrations of methane, hydrogen and CO (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The high pH, moderate temperature, lack of standing water, and hypoxia distinguish them from other well-studied terrestrial hot springs and mud volcanoes and provide a unique geothermally-driven range of micro-environments for resident microbiota. Steam is vented through concentrated hotspots, generating steep temperature (-20 to 65\u0026deg;C) and pH (3.5\u0026ndash;8) gradients over less than a meter that are major determinants of the composition of the thick cyanobacterial mats and associated microbial communities observed on the surface (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Within fumaroles, the temperature is a relatively constant 65\u0026deg;C, even at \u0026gt;\u0026thinsp;5 cm depth, but can decrease suddenly to less than 20\u0026deg;C and stay low for 24 hours or more at a time (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Beyond its unique physical characteristics, the hot fumarolic soils of Tramway Ridge also have an extremely low total C:N ratio (ranging from 1:3 to 3:1) (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), which suggests that the microbial community experiences continual carbon-limitation relative to nitrogen (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe isolation of Tramway Ridge, its unique geochemical environment, and microbiota make it an exciting site for evaluating potential endemism and for identifying novel metabolic pathways. Driven by the novelty of the taxa encountered there and the lack of information regarding their metabolic potential, we launched a detailed metagenomic study of high-altitude Antarctic fumaroles. First, we used functional profiles to contextualize the functional repertoire of this particular community with respect to other types of microbial communities. Second, we improved on our previous effort to identify endemic taxa (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), where we relied on matching partial 16S rRNA gene amplicon sequences with database entries by developing a novel metric that is based on synonymous polymorphisms within reconstructed environmental populations to define/circumscribe endemic taxa. Finally, we sought to elucidate possible novel metabolic processes encoded by abundant and endemic taxa that are specifically localized to the fumarolic subsurface (depths\u0026thinsp;\u0026gt;\u0026thinsp;2 cm).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eSoil samples were collected within the Tramway Ridge Antarctic Specially Protected Area (ASPA 130) in February 2009 from two sites (site A \u0026ndash; 77\u0026deg; 31.103' S, 167\u0026deg; 6.682' S and site B \u0026ndash; 77\u0026deg; 31.306' S, 167\u0026deg; 6.668' E). Sites were chosen based on measuring a surface temperature of 65\u0026deg;C with a stainless steel Checktemp1 temperature probe (Hanna Instruments, Rhode Island, USA) sterilized with 70% ethanol immediately prior to use. Temperature measurements were repeated for each layer sampled. Surface soil crusts were carefully set aside prior to collecting samples. Samples were collected by carefully removing 2 cm of soil in an approximately 5 cm x 5 cm square area using an autoclaved stainless steel spatula wiped with 70% ethanol just prior to sampling. Soil was placed into a fresh 50 mL Falcon tube and immediately frozen at -20\u0026deg;C. Sampling continued with the collection of a second layer (2\u0026ndash;4 cm depth).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction, library preparation and sequencing\u003c/h2\u003e \u003cp\u003eDNA was extracted from samples using a modified CTAB (cetyltrimethylammonium bromide) bead-beating protocol (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) and quantified using the Quant-IT dsDNA BR Assay Kit (Invitrogen, Carlsbad, CA, USA). A portion of extracted metagenomic DNA was frozen and sent to sequencing facilities at the University of California-Los Angeles (USA) and the University of Waikato (Hamilton, New Zealand). At each location, samples were processed and sequenced using standard protocols for the 454-Ti platform (Roche 454 Life Sciences, Branford, CT, USA). Additional DNA fractions were sent to the sequencing facility at University of California, Davis, where paired-end libraries were prepared and sequenced using the Illumina Hi-Seq platform (Illumina, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMetagenome assembly and binning\u003c/h2\u003e \u003cp\u003eAssembly of metagenomic data was carried out using appropriate assemblers for the two different sequencing platforms. 454 reads were assembled from original sff files using Newbler (Roche). Reads from each dataset were assembled independently and were pooled for an additional assembly. Newbler assembly used default parameters except that minimum overlap was set to 100 nucleotides and overlap identity was set to 98% identity. Paired-end Illumina reads were pre-processed by removing any paired-end set for which identifying tags had at least one mismatch or for which the paired-end tags were not identical. Identifying tags were removed, and reads were end-trimmed at the first incidence of a quality score below 14. Any reads that were shorter than 20 nucleotides after tag removal and end-trim were removed from the dataset. 454 datasets were assembled in Newbler (-mi 98 -ml 100 -minlen 45 -a 500 -l 2000). Illumina assemblies were carried out using two sets of options (--careful \u0026ndash;only-assembler; --cov-cutoff auto --careful -k 25,55,65,75 --only-assembler) in Spades (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), a single setting (--meta --only-assembler) in MetaSpades (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and a single setting (--k-min 27 --k-max 127 --k-step 10 --min-contig-len 500 --prune-level 3 --no-mercy --min-count 3 --no-local) in Megahit (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). BBMap (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sourceforge.net/projects/bbmap/\u003c/span\u003e\u003cspan address=\"https://sourceforge.net/projects/bbmap/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to map all individual read sets (454 and Illumina) against all assemblies. Large (\u0026gt;\u0026thinsp;2 kb) contigs and scaffolds were clustered into Metagenome-assembled genomes by oligonucleotide frequency and read coverage using Maxbin 2 (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) and Metabat 2 (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Redundant bins were subsequently dereplicated and evaluated using dRep (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) with a completeness cutoff of 40%, contamination cutoff of 10% and a minimum genome size of 200kb. Final MAGs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were classified using GTDB-Tk v2.1 (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) with genome database release R214 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and annotated with the NCBI Prokaryotic Genome Annotation Pipeline (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). MAGs discussed in the text were additionally annotated with eggNOG mapper (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), Cytochrome c oxidases were\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpecies-level representatives of metagenome-assembled genomes (MAGs) from Tramway Ridge fumarolic soils. Lineage was assigned using GTDB-Tk v.2.1 (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) with Release R214 of the Genome Taxonomy Database (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Completeness and contamination estimates were calculated in CheckM (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Assembly quality follows the recommendations outlined previously (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). For a comprehensive list of genome quality attributes for all binned strains, see supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProposed name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003especies group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e# of strains in species group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDivision\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eassembly quality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGenome size (bp)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlastocatellia bacterium ERB_27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcidobacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2698697\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyrinomonas sp. ERB_32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcidobacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3512547\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcidimicrobiia bacterium ERB_23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eActinobacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e55.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1963147\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcidimicrobiia bacterium ERB_8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eActinobacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e69.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1352013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThermoleophilia bacterium ERB_19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eActinobacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e69.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2326210\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArmatimonadetes bacterium ERB_24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eArmatimonadetes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1563163\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArmatimonadetes bacterium ERB_33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eArmatimonadetes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3260803\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArmatimonadetes bacterium ERB_34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eArmatimonadetes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2190352\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArmatimonadetes bacterium ERB_6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eArmatimonadetes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e61.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2058555\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChitinophagaceae sp. ERB_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBacteroidetes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e40.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2908196\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChitinophagaceae sp. ERB_3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBacteroidetes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2810122\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCandidatus GAL15 bacterium ERB_18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecandidate division GAL15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e70.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1986527\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCandidatus Dadabacteria bacterium ERB_12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCandidatus Dadabacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e44.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3057813\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCandidatus Fervidibacteria erebusii ERB_15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCandidatus Fervidibacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e56.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2795547\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCandidatus Lakebacteria bacterium ERB_C1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCandidatus Lakebacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e481934\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloroflexi bacterium ERB_10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloroflexi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2882324\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloroflexi bacterium ERB_11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloroflexi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2692019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloroflexi bacterium ERB_20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloroflexi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3392850\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCandidatus Nitrocaldera therma ERB_22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloroflexi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2707038\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloroflexi bacterium ERB_25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloroflexi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLow-quality draft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1709950\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloroflexi bacterium ERB_7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloroflexi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e70.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1489492\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloroflexi bacterium ERB_9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloroflexi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e62.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3793921\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThermoflexus sp. ERB_21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloroflexi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e69.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2384148\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeptolyngbya sp. ERB_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCyanobacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4634638\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMastigocladus sp. ERB_26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCyanobacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e41.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5965913\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMeiothermus sp. ERB_29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeinococcus-Thermus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e66.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2588060\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMeiothermus sp. ERB_30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeinococcus-Thermus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e61.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3765706\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMeiothermus sp. ERB_31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeinococcus-Thermus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2746960\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThermus sp. ERB_17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeinococcus-Thermus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1806162\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgnavibacteria bacterium ERB_28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIgnavibacteriae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e55.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3309624\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrospiraceae bacterium ERB_14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNitrospirae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e56.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3371732\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGemmataceae bacterium ERB_16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePlanctomycetes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3796795\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRhodanobacteraceae bacterium ERB_4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProteobacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e62.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1332200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCandidatus Australlarchaeum erebusii ERB_5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThaumarchaeota\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1213694\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrososphaera sp. ERB_13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThaumarchaeota\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e56.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1122400\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003echecked with the HCO classifier (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), hydrogenases were checked with HydDB (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), and CAZymes were checked with dbCAN3 (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEndemicity Index Calculation\u003c/h2\u003e \u003cp\u003eQuality-trimmed Illumina reads were mapped to each MAG using BBmap and further filtered using a hard 97% identity cutoff where identity\u0026thinsp;=\u0026thinsp;number of matches/length of alignment, with the additional requirement that at least 50 nucleotides mapped. Raw diversity was compiled using Samtools mpileup using option: -d 1000000 (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) for each MAG and each dataset independently. SNPs were determined using Varscan2 pileup2snp (options: --min-var-freq 0.01 --p-value 0.05) (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) and further filtered using the Benjamini-Hochberg multiple testing correction FDR\u0026thinsp;=\u0026thinsp;0.01. SnpEff (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) was used to classify SNPs as synonymous with gff files produced with Prodigal (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The density of synonymous SNPs (\u003cb\u003eD\u003c/b\u003e\u003csub\u003e\u003cb\u003eSynSNP\u003c/b\u003e\u003c/sub\u003e) for a MAG was calculated as the number of Synonymous SNPs (\u003cb\u003eN\u003c/b\u003e\u003csub\u003e\u003cb\u003eSynSNP\u003c/b\u003e\u003c/sub\u003e) divided by MAG length in Mb (\u003cb\u003eL\u003c/b\u003e\u003csub\u003e\u003cb\u003eMAG\u003c/b\u003e\u003c/sub\u003e): \u003cb\u003eD\u003c/b\u003e\u003csub\u003e\u003cb\u003eSynSNP\u003c/b\u003e\u003c/sub\u003e = \u003cb\u003eN\u003c/b\u003e\u003csub\u003e\u003cb\u003eSynSNP\u003c/b\u003e\u003c/sub\u003e / \u003cb\u003eL\u003c/b\u003e\u003csub\u003e\u003cb\u003eMAG\u003c/b\u003e\u003c/sub\u003e. Because sensitivity increases as read depth increases, \u003cb\u003eD\u003c/b\u003e\u003csub\u003e\u003cb\u003eSynSNP\u003c/b\u003e\u003c/sub\u003e was corrected for read coverage of the MAG (\u003cb\u003eC\u003c/b\u003e\u003csub\u003e\u003cb\u003eMAG\u003c/b\u003e\u003c/sub\u003e) which was calculated as the number of reads mapped (\u003cb\u003eN\u003c/b\u003e\u003csub\u003e\u003cb\u003eM\u003c/b\u003e\u003c/sub\u003e) divided by MAG length (\u003cb\u003eL\u003c/b\u003e\u003csub\u003e\u003cb\u003eMAG\u003c/b\u003e\u003c/sub\u003e): \u003cb\u003eC\u003c/b\u003e\u003csub\u003e\u003cb\u003eMAG\u003c/b\u003e\u003c/sub\u003e = \u003cb\u003eN\u003c/b\u003e\u003csub\u003e\u003cb\u003eM\u003c/b\u003e\u003c/sub\u003e / \u003cb\u003eL\u003c/b\u003e\u003csub\u003e\u003cb\u003eMAG\u003c/b\u003e\u003c/sub\u003e. Endemicity Index (\u003cb\u003eEI\u003c/b\u003e) was then the density of synonymous SNPs (\u003cb\u003eD\u003c/b\u003e\u003csub\u003e\u003cb\u003eSynSNP\u003c/b\u003e\u003c/sub\u003e) divided by read coverage (\u003cb\u003eC\u003c/b\u003e\u003csub\u003e\u003cb\u003eMAG\u003c/b\u003e\u003c/sub\u003e): \u003cb\u003eEI\u003c/b\u003e\u0026thinsp;=\u0026thinsp;\u003cb\u003eD\u003c/b\u003e\u003csub\u003e\u003cb\u003eSynSNP\u003c/b\u003e\u003c/sub\u003e / \u003cb\u003eC\u003c/b\u003e\u003csub\u003e\u003cb\u003eMAG\u003c/b\u003e\u003c/sub\u003e. EI was calculated for each MAG/dataset combination only if \u003cb\u003eN\u003c/b\u003e\u003csub\u003e\u003cb\u003eSynSNP\u003c/b\u003e\u003c/sub\u003e \u0026ge; 5 and reported EI values are the average of calculated values over four Illumina read sets (SRA accessions SRR6519253, SRR6519254,SRR6519255, SRR6519256).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFunctional profile comparisons\u003c/h2\u003e \u003cp\u003ePfam (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) profiles were used to compare functional similarity between the metagenomic assembly from Tramway Ridge and publicly available metagenomic assemblies in the Integrated Microbial Genomes (IMG) database (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). A list of all assembled, published, and \u0026ldquo;unrestricted\u0026rdquo; environmental metagenomic datasets available through IMG was downloaded on 31 March, 2023. IMG-generated pfam profiles (counts of pfams present in metagenomic assembly) were downloaded for each available metagenome, resulting in 7652 total pfam profiles. The number of metagenomic datasets was reduced by removing datasets with total assembly length less 5x10\u003csup\u003e7\u003c/sup\u003e or greater than 1x10\u003csup\u003e9\u003c/sup\u003e bases. This subset was further reduced by removing metagenomic datasets with fewer than 3500 or greater than 7500 unique pfams. These filtering criteria resulted in a dataset of 4513 publicly sourced metagenomic datasets for comparative analysis. Profiles were analyzed in R 4.2.3. Jaccard dissimilarity was calculated using the vegdist() function from vegan 2.6-4 (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) based on presence/absence of pfams. Principle coordinate analysis (PCoA) was carried out using the pcoa() function from Version 5.7-1 of ape (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) and plotted in three dimensions using the plot3d() function from rgl 1.2.1 (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). t-distributed stochastic neighbor embedding (tSNE) was calculated using the Rtsne() function from Rtsne 0.16 (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) and plotted with ggplot2 3.4.3 (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Rtsne settings were as follows: Rtsne(X, dims\u0026thinsp;=\u0026thinsp;2,initial_dims\u0026thinsp;=\u0026thinsp;5, perplexity\u0026thinsp;=\u0026thinsp;300, theta\u0026thinsp;=\u0026thinsp;0.5, check_duplicates\u0026thinsp;=\u0026thinsp;FALSE, pca\u0026thinsp;=\u0026thinsp;TRUE, partial_pca\u0026thinsp;=\u0026thinsp;FALSE, max_iter\u0026thinsp;=\u0026thinsp;5000000, verbose\u0026thinsp;=\u0026thinsp;getOption(\"verbose\",FALSE), is_distance\u0026thinsp;=\u0026thinsp;TRUE, Y_init\u0026thinsp;=\u0026thinsp;NULL, pca_center\u0026thinsp;=\u0026thinsp;TRUE, pca_scale\u0026thinsp;=\u0026thinsp;FALSE, normalize\u0026thinsp;=\u0026thinsp;TRUE, stop_lying_iter\u0026thinsp;=\u0026thinsp;500000, mom_switch_iter\u0026thinsp;=\u0026thinsp;500000, momentum\u0026thinsp;=\u0026thinsp;0.5, final_momentum\u0026thinsp;=\u0026thinsp;0.8, eta\u0026thinsp;=\u0026thinsp;100, exaggeration_factor\u0026thinsp;=\u0026thinsp;12).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenomic analysis\u003c/h2\u003e \u003cp\u003eA collection of reference genomes for comparative phylogenomic analysis was assembled from representative species defined in release 214 (April, 2023) of the Genome Taxonomy Database (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). GTDB classifications of MAGs from the current study were used to select from GTDB species representatives for an informative tree. For instance, in the case where a MAG was classified into an order but not a family, one representative taxon from each family was included, the phylogeny was calculated, and if the MAG was associated with a particular family, the process was repeated using genus representatives from that family. These genomes were supplemented with additional genomes from thermophilic environments (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), thermophilic nitrifying enrichment cultures (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), additional \u003cem\u003eNitrospirota\u003c/em\u003e (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) and additional \u003cem\u003eNitrososphaeria\u003c/em\u003e (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). All genomes were downloaded and processed using CheckM (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) to generate concatenated alignments of 34 universal marker genes (43 marker HMMs). To be included in phylogenomic reconstruction, reference genomes were required to be at least 60% complete with less than 5% contamination and to have at least 4500 ungapped characters in the concatenated alignment (6988 total positions). All genomes used for phylogenetic analysis are listed in Supplementary Table\u0026nbsp;8. Phylogenetic reconstruction with IQ-TREE 2 (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) included model selection with ModelFinder (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) and calculation of bootstraps with UFboots (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eWe found that the functional profiles from Tramway Ridge metagenomes resembled those from other thermally-influenced environments, in particular those from terrestrial hydrothermal systems. To learn this, we compared functional profiles of assembled Tramway Ridge metagenomes to 4513 publicly available and assembled environmental metagenomes broadly categorized as terrestrial hydrothermal / non-hydrothermal, freshwater, and marine hydrothermal / non-hydrothermal (Supplementary Table\u0026nbsp;1). Functional profiles for metagenomes were constructed based on the presence-absence of Pfam protein family domains (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), and dissimilarities were calculated using the Jaccard index. A \u003cem\u003epost-hoc\u003c/em\u003e Tukey\u0026rsquo;s HSD test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) comparing Jaccard dissimilarity grouped according to the broad categories listed above showed that metagenomes from Tramway Ridge were most similar to terrestrial hydrothermal environments (Tukey category a) and least similar to metagenomes from marine non-hydrothermal and freshwater environments (Tukey category d). We continued with principal coordinate analysis (PCoA) and t-distributed Stochastic Neighbor Embedding (tSNE) visualizations to explore relationships that may not have been clear from grouped pairwise comparisons (Fig.\u0026nbsp;2bc). In both, Tramway Ridge microbial communities clustered loosely with microbial communities sourced from both terrestrial and marine hydrothermal environments to the exclusion of non-hydrothermal environments. T-SNE visualization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) recovered several distinct but associated clusters from hydrothermal systems, one of which was composed exclusively of profiles from Tramway Ridge.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMetagenome-assembled genomes (MAGs) were constructed from DNA extracted from soils at two 65\u0026deg;C fumaroles at Tramway Ridge (supplementary Table\u0026nbsp;2). We recovered 63 MAGs at the strain level (99% average nucleotide identity, ANI), which clustered into 35 species-level representatives (\u0026gt;\u0026thinsp;96.5% average nucleotide identity) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, supplementary Table\u0026nbsp;3). A total of 16 MAGs met the criteria for high-quality drafts, and 18 MAGs met the criteria for medium-level drafts (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e); these include nearly complete (75\u0026ndash;96%) genome bins for a novel order of Archaea within the \u003cem\u003eNitrososphaeria\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and novel lineages of Bacteria including Armatimonadota, Chloroflexota, Actinobacteriota, and Candidate division CSP1-3. Additional MAGs of interest include those belonging to the \u003cem\u003eCandidatus\u003c/em\u003e Patescibacteria phylum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), \u003cem\u003eMastigocladus\u003c/em\u003e genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) and \u003cem\u003eCandidatus\u003c/em\u003e Fervidibacter genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). MAGs were classified using GTDB-Tk v.2.1 (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) with Release R214 of the Genome Taxonomy Database (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), and novel taxa of note were given proposed names (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTramway Ridge occupies a unique location on Earth that makes it ideal for the study of endemism due to its highly isolated nature and the geothermal enrichment that prevents intrusion of nearby psychrophilic and mesophilic microorganisms. Here, we quantified endemism for each of the MAGs we generated. We defined the degree of endemism of a given taxon as being proportional to the \u003cem\u003ein situ\u003c/em\u003e diversity of that taxon, reasoning that endemic species have had an extended opportunity to diversify on-site, as opposed to recent arrivals that would be subject to founder effects. We developed a simple metric, which we called the \u003cem\u003eendemicity index\u003c/em\u003e (EI) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) that measured the frequency of synonymous mutations in a MAG accounting for read depth. High values (e.g. 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) of EI indicate high diversity whereas low values (e.g. 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e) indicate low diversity.\u003c/p\u003e \u003cp\u003eThe lowest median EI value (1 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e) for a single species was calculated for ERB_26, (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), which represented two individual strains of a cyanobacterium belonging to the \u003cem\u003eFischerella\u003c/em\u003e / \u003cem\u003eMastigocladus\u003c/em\u003e genus (supplementary Table\u0026nbsp;3) and dominated the near-surface (5\u0026ndash;10% of near-surface reads). The highest median EI value (1 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for a single species was calculated for ERB_C1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), which represented three individual strains of \u003cem\u003eCandidatus\u003c/em\u003e Patescibacteria (supplementary Table\u0026nbsp;3). \u003cem\u003eCa.\u003c/em\u003e Lakebacteria bacterium ERB_C1 classified into the HRBIN35 genus in the HR35 family (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Based on the percentage of reads recruited, it was slightly more abundant in the near-surface (0\u0026ndash;2 cm depth) than in the subsurface (\u0026gt;\u0026thinsp;2 cm, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Due to this distribution pattern and the fact that an accelerated evolutionary process is a hallmark feature of members of this lineage (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), we did not pursue further analysis of this genome, which would be best accomplished in a comprehensive comparison with other \u003cem\u003eCa.\u003c/em\u003e Patescibacteria.\u003c/p\u003e \u003cp\u003eTwo MAGs, ERB_15_1 and ERB_5_1, with relatively high EI values (3 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e and 1 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, respectively) dominated the subsurface community at depths greater than 2 cm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). ERB_5_1 shared less than 40% average amino acid identity (AAI) with any other \u003cem\u003eNitrososphaeria (\u003c/em\u003eformerly Thaumarchaeota) and formed a singular deep branch of the \u003cem\u003eNitrososphaeria\u003c/em\u003e in phylogenetic trees (FIgure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). ERB_15_1 shared 69% AAI and 77% ANI with \u003cem\u003eCandidatus\u003c/em\u003e Fervidibacteria sacchari, establishing it as a novel species of this genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Here we propose the names \u003cem\u003eCandidatus\u003c/em\u003e Fervidibacteria erebusii (ERB_15_1) and \u003cem\u003eCandidatus\u003c/em\u003e Australlarchaeum erebusii (ERB_5_1) to reflect their first observation and endemism at Mt. Erebus. We picked these two MAGs for further analyses based on their high EI values, subsurface abundance, and taxonomic novelty.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCandidatus\u003c/em\u003e A. erebusii (ERB_5_1) branches near the base of the \u003cem\u003eNitrososphaeria\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and, like other deeply diverging lineages, it lacked marker genes for cobalamin biosynthesis (\u003cem\u003ecob/cbi\u003c/em\u003e/\u003cem\u003ebluB\u003c/em\u003e) and ammonia oxidation (archaeal \u003cem\u003eamoABC, HAO\u003c/em\u003e). Consistent with previous observations of deeply diverging \u003cem\u003eNitrososphaeria\u003c/em\u003e, the genome of \u003cem\u003eCa.\u003c/em\u003e A. erebusii encoded genes for the beta oxidation of fatty acids and peptide degradation and several genes that may be used for amino acid-based oxidation (Supplementary Table\u0026nbsp;5). \u003cem\u003eCa.\u003c/em\u003e A. erebusii encoded aminotransferases and glutamate dehydrogenase that could be used to produce NAD(P)H by converting glutamate to 2-oxoglutaric acid. A 2-oxoglutarate:ferredoxin oxidoreductase could then convert the 2-oxoglutaric acid to succinyl-CoA, which could then be converted to succinate with the production of ATP via ADP-forming succinate-CoA ligase (Supplementary Table\u0026nbsp;5). Unlike other deep-branching Nitrososphaeria, \u003cem\u003eCa.\u003c/em\u003e A. erebusii also additionally encoded two \u003cem\u003eaa3\u003c/em\u003e-type (low-affinity) cytochrome C-oxidases (Supplementary Table\u0026nbsp;5). We identified a putative aerobic carbon monoxide dehydrogenase (CODH) cluster of genes (\u003cem\u003ecoxMLS\u003c/em\u003e) that may confer the ability to use CO as an additional energy source. However, the large subunit of the CODH in \u003cem\u003eCa.\u003c/em\u003e A. erebusii contained a hallmark motif, AYXGAGR, of type II CODH, which oxidizes CO only very slowly and possibly incidentally (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). No predicted carbon fixation pathways were identified.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCandidatus\u003c/em\u003e Fervidibacteria erebusii (ERB_15_1) shared 69% AAI and 77% ANI with \u003cem\u003eCandidatus\u003c/em\u003e Fervidibacteria sacchari, establishing it as a novel species of this genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Based on conservative annotations, the \u003cem\u003eCa.\u003c/em\u003e F. erebusii genome encoded 144 CAZymes, including 69 glycosyl hydrolase (GH) domains spanning 32 GH families (Supplementary Table\u0026nbsp;6, Supplementary Table\u0026nbsp;7), and an impressive 15 variants of the unusual and poorly characterized GH109 family. Furthermore, when we group the \u003cem\u003eCa.\u003c/em\u003e F. erebusii CAZymes by substrate utilization capacity, the GH109s belong to the largest cohort (35.25%) which appear to be involved in depolymerisation cascades of N-acetylhexosamide glycans such as chitins and chitosan (Supplementary Table\u0026nbsp;7). Other major polysaccharide utilization cohorts include cellulose and hemicellulose (19.8%), pectin (14.3%) and \u0026szlig;-glucans (19.8%), with minor cohorts for ɑ-glucans, ɑ-mannans and ɑ-fucans. \u003cem\u003eCa.\u003c/em\u003e Fervidibacter genomes, including \u003cem\u003eCa.\u003c/em\u003e F. erebusii additionally encoded numerous aminotransferases, glutamate dehydrogenase, indole-pyruvate:ferrodoxin oxidoreductase and ADP-forming succinate-CoA ligase (Supplementary Table\u0026nbsp;6) which may explain the ability of \u003cem\u003eCa.\u003c/em\u003e Fervidibacter sacchari to grow solely on casamino acids (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). The \u003cem\u003eCa.\u003c/em\u003e F. erebusii genome also encoded Group 2a NiFe hydrogenases which are high-affinity hydrogenases that enable the survival of soil heterotrophs under carbon limitation (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) and enable hydrogenotrophic growth of autotrophic nitrite-oxidizing bacteria under nitrite limitations (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). The \u003cem\u003eCa.\u003c/em\u003e F. erebusii genome also encoded a cluster of putative CODH genes (\u003cem\u003ecoxMLS\u003c/em\u003e), which contained the hallmark motif, AYXGAGR, of type II CODH, indicating a low, incidental activity (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). No carbon fixation pathways were found.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMetagenome Functional Profiles\u003c/h2\u003e \u003cp\u003eMt. Erebus has evolved over time, starting with seafloor rifting and growing as a subaerial volcano into a modern-day stratovolcano (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The current conditions at Tramway Ridge differ remarkably from other extant non-Antarctic terrestrial and marine hydrothermal systems, being primarily driven by the unique phonolite magmatic source resulting in alkaline fumaroles with low sulphidic content. It is unknown how long geothermal features such as the fumaroles at Tramway Ridge have been present on Mt. Erebus; however, volcanism was once widespread across the Ross Island massif (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Extant geothermal features may represent a once widespread ecosystem of similar features. Given this complex history of Mt. Erebus, we questioned whether the Tramway Ridge community has retained a legacy signature of its origin on the seafloor or if the modern-day Tramway Ridge community better resembles other terrestrial hydrothermal sites.\u003c/p\u003e \u003cp\u003eTo answer this question, we compared functional profiles of assembled Tramway Ridge metagenomes to a large set of publicly available metagenomes. Metagenomes from Tramway Ridge were distinct from all others (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) but showed the most similarity to terrestrial hydrothermal environments such as hot springs and associated sediments. This indicates that the legacy of a sea floor origin is less important than the extant conditions at Tramway ridge in defining the microbial community. This is also reflected in the prevalence of taxa at Tramway Ridge that are found in other terrestrial hydrothermal environments but not in seafloor hydrothermal systems, such as the genera \u003cem\u003eMastigocladus\u003c/em\u003e, \u003cem\u003eCaldithermus\u003c/em\u003e, \u003cem\u003eMeiothermus\u003c/em\u003e and phyla such as the Chloroflexota, Armatimonadota and Actinobacteriota.\u003c/p\u003e \u003cp\u003eThe unique nature of Tramway Ridge metagenomic profiles likely reflects the site\u0026rsquo;s relatively low diversity (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) and the type of functional profile analysis used here. First, pfams were used, which are very coarse functional units. Second, gene abundance was not available for most metagenomes, so we were forced to use presence-absence as data. Third, we found that the use of pfam presence-absence was sensitive to metagenome assembly size as well as the total number of unique pfams detected. In combination, these features had the potential to obfuscate any meaningful relationships. Our analysis was optimized to be as permissive as possible, allowing as many metagenomes into the analysis while still comparing like against like. Using this strategy, we were able to compare Tramway Ridge metagenomic datasets to 4513 out of 7652 publicly available and unrestricted metagenomes (see \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5281/zenodo.10928974\u003c/span\u003e\u003cspan address=\"10.5281/zenodo.10928974\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWithin continental Antarctica, only three known surface-expressed active geothermal areas exist (Mt. Rittmann, Mt. Melbourne and Mt Erebus), each of which is separated by vast ice fields (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). It is thought that intercontinental transport of microbe-bearing particulates into Antarctica occurs much less frequently than intracontinental transport (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e), suggesting that the introduction of exogenous microbes is relatively infrequent. Recent studies have used database searches to identify endemic species (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e), and a lack of sequence identity to database entries has been used in the past to suggest that novel sequences indicate endemism (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). However, defining endemism based on whether sequence matches exist within a database can be problematic as this definition is sensitive to database composition. Conclusions drawn may not withstand the inevitable growth in database size and the diversity it holds. For this study, we took an alternative approach and defined the degree of endemism of a given taxon as being proportional to the \u003cem\u003ein situ\u003c/em\u003e diversity of that taxon. For these inferences, we assumed that rare colonization events have been limited to single clones due to the extreme isolation of Mt. Erebus. Therefore, populations arising from recently introduced taxa would be expected to exhibit relatively low levels of genetic polymorphism and endemic microbial populations would be expected to show high levels of genetic polymorphism. In particular, we focused on accumulations of synonymous mutations, which were assumed to be under reduced selection pressure.\u003c/p\u003e \u003cp\u003eWe developed the \u003cem\u003eendemicity index\u003c/em\u003e (EI) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) to assess the diversity of a microbial population represented by a MAG. Similar calculations have been successfully applied to approximate effective population size and genomic fluidity (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). High values (e.g. 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) of EI indicate high diversity which we interpreted as reflecting a neutral evolutionary process occurring under minimal contemporary selection pressures. Low values (e.g. 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e) indicate low diversity, which we interpreted as possible evidence of a relatively recent arrival, a local population bottleneck, or a recent selective sweep.\u003c/p\u003e \u003cp\u003eA cyanobacterium belonging to either the \u003cem\u003eFischerella\u003c/em\u003e or \u003cem\u003eMastigocladus\u003c/em\u003e genus recovered the lowest median EI value (1 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e) for a single species. This species dominated the near-surface. Although difficult to distinguish these two genera based on 16S rRNA gene sequence and GTDB classifies all members of the genera \u003cem\u003eFischerella\u003c/em\u003e and \u003cem\u003eMastigocladus\u003c/em\u003e as \u003cem\u003eFisherella\u003c/em\u003e, the distinction of \u003cem\u003eMastigocladus\u003c/em\u003e is recognized as a distinct genus by the List of Prokaryotic names with Standing in Nomenclature (LPSN). Therefore we classified this MAG as a member of the \u003cem\u003eMastigocladus\u003c/em\u003e genus to be consistent with the classification of specimens collected from Tramway Ridge in the past (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). The low endemicity index observed for this taxon was consistent with previous studies that showed that the global phylogeography of \u003cem\u003eMastigocladus\u003c/em\u003e reflects a geologically recent radiation from Yellowstone National Park, USA (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e) and that the surface-associated microbial community at Tramway Ridge is likely dominated by aeolian-distributed cosmopolitan members of non-Antarctic temperate and terrestrial hydrothermal soil communities (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe identified \u003cem\u003eCandidatus\u003c/em\u003e Australlarchaeum erebusii (ERB_5_1) and \u003cem\u003eCandidatus\u003c/em\u003e Fervidibacteria erebusii (ERB_15_1) as two MAGs with relatively high EI values and abundance in the subsurface at depths greater than 2 cm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). In our earlier amplicon-based study, these two taxa were similarly identified as dominant, endemic, and associated with the subsurface, but were referred to as \u0026ldquo;Thaumarchaeota-like archaeon\u0026rdquo; and OCtSpA1-106 respectively (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe most abundant endemic, subsurface-associated organism recovered was \u003cem\u003eCandidatus\u003c/em\u003e A. erebusii (ERB_5_1), a member of the \u003cem\u003eNitrososphaeria\u003c/em\u003e (formerly Thaumarchaeota). This class of Archaea is a globally distributed group that is best known for the chemolithoautotrophic oxidation of ammonia (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e) and for the apparent universal synthesis of cobalamin (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). However, several deeply divergent lineages of \u003cem\u003eNitrososphaeria\u003c/em\u003e identified through the construction of MAGs (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e) and a single cultivated species, \u003cem\u003eCandidatus\u003c/em\u003e Conexivisphaera calidus NAS-02 (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e) have been shown to lack these hallmark attributes. These deeply diverging lineages have been predicted to be predominantly anaerobic heterotrophs (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e) capable of beta oxidation of fatty acids and protein/peptide degradation (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e). \u003cem\u003eCandidatus\u003c/em\u003e A. erebusii (ERB_5_1) is one of the deepest-branching members of the \u003cem\u003eNitrososphaeria\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and like other deeply diverging lineages, it encodes genes for the beta oxidation of fatty acids and peptide degradation while lacking marker genes for cobalamin biosynthesis and ammonia oxidation (Supplementary Table\u0026nbsp;5). \u003cem\u003eCandidatus\u003c/em\u003e A. erebusii may also employ amino acid-based oxidation, similar to a pathway used by \u003cem\u003eThermococcus kodakarensis\u003c/em\u003e (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e) and a proposed alternative metabolism for \u003cem\u003eCa\u003c/em\u003e. Nitrosocaldus islandicus (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e), a representative of thermophilic ammonia oxidizing archaea (AOA). In this proposed metabolism, glutamate could be utilized to generate both reducing power and ATP to power the cell.\u003c/p\u003e \u003cp\u003eHowever, \u003cem\u003eCa.\u003c/em\u003e A. erebusii is also predicted to respire oxygen, a unique prediction among its closest, presumably anaerobic relatives. It encodes two \u003cem\u003eaa3\u003c/em\u003e-type (low-affinity) cytochrome C-oxidases which could presumably drive beta oxidation of fatty acids. However it is unclear whether aerobic respiration could be coupled with an amino acid degradation pathway, which is typically thought to be an anaerobic metabolism (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e). It is unclear if these energy-generating pathways are mutually exclusive and operate under specific oxidative conditions. At least during summer, oxygen levels in the subsurface are around 30% saturation (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) and therefore, no organisms inhabiting the fumaroles are likely to be obligate anaerobes. However, it is also reasonable to assume that the wet, steamy subsurface experiences anoxia at least at small spatial scales. Therefore, we hypothesize that \u003cem\u003eCa.\u003c/em\u003e A. erebusii switches between metabolic pathways depending on the oxic environment, using beta-oxidation of fatty acids when oxygen levels are sufficiently high and peptide fermentation when oxygen levels are low. CO metabolism is likely to be used for maintenance during times of nutritional stress, as has previously been shown for Antarctic soil microbes (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother abundant and endemic subsurface MAG examined in detail belongs to the thermophilic genus \u003cem\u003eCandidatus\u003c/em\u003e Fervidibacteria, which was first discovered in Octopus Spring, Yellowstone National Park (clone OctSpA1-106, (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). The genus is named after \u003cem\u003eCa.\u003c/em\u003e Fervidibacter sacchari, which encodes a large repertoire of carbohydrate-active enzymes (CAZymes) (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e) and which has recently been isolated and described (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Like other \u003cem\u003eCa.\u003c/em\u003e Fervidibacter, \u003cem\u003eCa.\u003c/em\u003e F. erebusii (ERB_15_1) encodes a significant number and diversity of CAZymes including a large, diverse cohort of the unusual and poorly characterized GH109 family. Previous findings suggest that these novel enzymes are involved in extracellular polysaccharide metabolism unique to thermophilic systems (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e) with the diversity of \u003cem\u003eCa.\u003c/em\u003e F. erebusii GH109s suggesting a diverse and unique polysaccharide utilization profile typical of this genus. Interestingly, \u003cem\u003eCa.\u003c/em\u003e Fervidibacter genomes, including \u003cem\u003eCa.\u003c/em\u003e F. erebusii appear to also encode mechanisms that enable growth on casamino acids (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). The encoded hydrogenase and CODH are likely maintenance mechanisms to enable survival under carbon limitation with growth primarily supported through aerobic heterotrophic growth on saccharides and anaerobic growth on amino acids.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the current study, we have used metagenomics to assess several aspects of the microbial community inhabiting the fumarolic soils of Tramway Ridge, Mt. Erebus, Antarctica. We observed a shared functional repertoire between Tramway Ridge and other geothermal systems, specifically terrestrial hydrothermal systems such as hot springs. We then assessed metagenome-assembled genomes (MAGs) using a novel endemicity measure to identify two highly endemic taxa that are more abundant in the Tramway Ridge subsurface (\u0026gt;\u0026thinsp;2 cm depth) than the near-surface (\u0026lt;\u0026thinsp;2 cm depth). We named these two taxa \u003cem\u003eCandidatus\u003c/em\u003e Australlarchaeum erebusii and \u003cem\u003eCandidatus\u003c/em\u003e Fervidibacter erebusii to reflect that they were first observed as dominant, unique and endemic to Mt. Erebus. A close examination of the metabolic repertoire of these taxa revealed that they are likely both facultative anaerobic heterotrophs that specialize in using different carbon sources under aerobic conditions, but that use similar organic compounds during anaerobic growth. Like other deep-branching non-AOA Nitrososphaeria, \u003cem\u003eCa.\u003c/em\u003e A. erebusii possesses a putative pathway for the beta-oxidation of fatty acids. Like other \u003cem\u003eCandidatus\u003c/em\u003e Fervidibacter, \u003cem\u003eCa.\u003c/em\u003e F. erebusii is predicted to utilize sugars and scavenge hydrogen gas under aerobic conditions (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e). Under oxygen-limited conditions, both may utilize similar peptides and amino acids for energy and carbon acquisition. We hypothesize that this pattern of metabolic utilization may reflect the extreme and carbon-limiting C:N ratios (1:3 to 3:1) encountered at Tramway Ridge. Dominant taxa may share nitrogen-rich compounds such as peptides and amino acids since the demand for such compounds may not be as high as for carbon-rich compounds. Instead, different taxa specialize in utilizing specific carbon compounds (sugars vs. fatty acids) through selective exclusion, allowing both to co-exist by carving out specific nutritional niches. Each is also equipped with form II aerobic carbon monoxide dehydrogenase genes that may provide maintenance energy during times of starvation. Together, these insights provide an unprecedented view into the dominant metabolic processes that may sustain life in this harsh, isolated environment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw metagenomic sequence data and metagenome assembled genomes used in this manuscript are available under bioproject accession PRJNA431961 (https://www.ncbi.nlm.nih.gov/bioproject/431961) . Data and scripts necessary to reproduce comparative metagenomic work is available at 10.5281/zenodo.10928974\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinancial support was provided by grant UOW0802 from the New Zealand Marsden Fund to SCC and IRM and a CRE award from the National Geographic Society to S.C.C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlanning and field sampling by CWH, CJV, SCC and IRM. Data analysis by CJV and CWH. All authors contributed to the interpretation of data and writing of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntarctic logistic support for Event K-023 was provided by Antarctica New Zealand.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eParties of the Antarctic Treaty. Antarctic Treaty database [Internet]. 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Extremophiles. 2014;18(5):865\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"endemic, hydrothermal, geothermal, volcano, fumarole, metabolism, sediments","lastPublishedDoi":"10.21203/rs.3.rs-4805162/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4805162/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTramway Ridge, a geothermal Antarctic Specially Protected Area (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) (elevation 3340 m) located near the summit of Mount Erebus, is home to a unique community composed of cosmopolitan surface-associated micro-organisms and abundant, poorly understood subsurface-associated microorganisms (\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Here, we use shotgun metagenomics to compare the functional capabilities of this community to those found elsewhere on Earth and to infer endemism and metabolic capabilities of abundant subsurface taxa.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that the functional potential in this community is most similar to that found in terrestrial hydrothermal environments (hot springs, sediments) and that the two dominant organisms in the subsurface are primarily endemic. They were found to be facultative anaerobic heterotrophs that likely share a pool of nitrogenous organic compounds while specializing in different carbon compounds.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMetagenomic insights have provided a detailed understanding of the microbe-based ecosystem found in geothermally heated fumaroles at Tramway Ridge. This approach enabled us to compare Tramway Ridge with other microbial systems, identify endemic taxa and elucidate the key metabolic pathways that may enable specific organisms to dominate the ecosystem.\u003c/p\u003e","manuscriptTitle":"Nutritional niches of endemic, facultatively anaerobic heterotrophs from an isolated Antarctic terrestrial hydrothermal refugium elucidated through metagenomics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 19:54:06","doi":"10.21203/rs.3.rs-4805162/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-09T13:35:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-02T19:23:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-30T02:30:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176222804694270154924261648911013692841","date":"2024-08-29T09:23:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290623727464512726672717041935274360290","date":"2024-08-13T13:51:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28853234606080558728967886240261997784","date":"2024-08-07T16:13:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-07T05:34:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-01T13:58:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-27T18:48:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Microbiome","date":"2024-07-26T03:54:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c0154e5-bc6d-4a41-8287-cf4a959ac167","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:05:26+00:00","versionOfRecord":{"articleIdentity":"rs-4805162","link":"https://doi.org/10.1186/s40793-024-00655-5","journal":{"identity":"environmental-microbiome","isVorOnly":false,"title":"Environmental Microbiome"},"publishedOn":"2024-12-18 15:58:22","publishedOnDateReadable":"December 18th, 2024"},"versionCreatedAt":"2024-08-22 19:54:06","video":"","vorDoi":"10.1186/s40793-024-00655-5","vorDoiUrl":"https://doi.org/10.1186/s40793-024-00655-5","workflowStages":[]},"version":"v1","identity":"rs-4805162","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4805162","identity":"rs-4805162","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}