Geologic Legacy Spanning >90 Years Explains Unique Yellowstone Hot

Environmental Microbiology (2019) 21(11), 4180–4195 doi:10.1111/1462-2920.14775

Geologic legacy spanning >90 years explains unique Yellowstone hot spring geochemistry and biodiversity

Devon Payne,1† Eric C. Dunham,1† Elizabeth Mohr,2† Introduction Isaac Miller,1† Adrienne Arnold,1† Reece Erickson,1† Life in environments with temperatures exceeding the Elizabeth M. Fones,1† Melody R. Lindsay ,1 upper limit of photosynthesis (~73 C) is supported by Daniel R. Colman1 and Eric S. Boyd 1* chemical energy (Boyd et al., 2012). In volcanic hydro- 1Department of Microbiology and Immunology, Montana thermal ecosystems, reduced volatiles from magmatic State University, Bozeman, Montana 59717. degassing and solutes derived from subsurface water- 2Department of Land Resources and Environmental rock interaction mix with oxidized surface fluids to gener- Sciences, Montana State University, Bozeman, ate disequilibria in electron donor/acceptor pairs that Montana 59717. support chemosynthetic microbial metabolisms (Shock et al., 2010; Colman et al., 2019). Thus, the assembly of Summary microbial communities (i.e., taxonomic and functional Little is known about how the geological history of an diversity) in high temperature volcanic hydrothermal sys- environment shapes its physical and chemical proper- tems is controlled largely by the processes that source ties and how these, in turn, influence the assembly of them with chemical nutrients and that control their geo- communities. Evening primrose (EP), a moderately chemistry. This phenomenon has been particularly well acidic hot spring (pH 5.6, 77.4C) in Yellowstone documented across spatial chemical gradients in hot National Park (YNP), has undergone dramatic physico- springs in Yellowstone National Park (YNP), chemical change linked to seismic activity. Here, we U.S.A. (Amenabar et al., 2015) and those in the Taupo show that this legacy of geologic change led to the Volcanic Zone in New Zealand (Power et al., 2018). development of an unusual sulphur-rich, anoxic chemi- The current model for the development of chemical vari- cal environment that supports a unique archaeal- ation in continental hot springs begins with injection of dominated and anaerobic microbial community. magmatic gases [e.g., carbon dioxide (CO2), sulphur diox- Metagenomic sequencing and informatics analyses ide (SO2)] into a hydrothermal aquifer at depth (White reveal that >96% of this community is supported by dis- et al., 1971; Truesdell and Fournier, 1976; Truesdell et al., similatory reduction or disproportionation of inorganic 1977). Condensation of SO2 with water at high tempera- sulphur compounds, including a novel, deeply diverg- ture (>150C) leads to its disproportionation to form sul- 2− ing sulphate-reducing thaumarchaeote. When com- phate (SO4 ) and sulphide (H2S) (Nordstrom et al., pared to other YNP metagenomes, the inferred 2009). Hydrothermal fluids convect upward through a functions of EP populations were like those from series of successively shallower and cooler reservoirs, sulphur-rich acidic springs, suggesting that sulphur where they can undergo decompressional boiling that may overprint the predominant influence of pH on the leads to their separation into a liquid phase enriched in composition of hydrothermal communities. Together, non-volatile constituents [e.g., chloride (Cl−)] and a vapour these observations indicate that the dynamic geologi- phase enriched in volatile constituents [e.g., H2S] cal history of EP underpins its unique geochemistry (Fournier, 1989; Nordstrom et al., 2009; Lowenstern et al., and biodiversity, emphasizing the need to consider the 2012). Following this separation of phases, vapour can legacy of geologic change when describing processes migrate towards the surface where it can condense and that shape the assembly of communities. interact with oxygen (O2)-rich meteoric fluids. Near-surface 2− oxidation of H2SwithO2 to protons and SO4 leads to 2− springs that are often acidic and enriched in SO4 (Fournier, 1989; Nordstrom et al., 2005). In contrast, Received 10 June, 2019; revised 30 July, 2019; accepted 31 July, springs impacted by liquid-phase input tend to have lower 2019. *For correspondence. E-mail [email protected]; Tel. +1-406- 2− − 994-7046; Fax 1-406-994-4926. †These authors contributed equally to concentrations of SO4 are enriched in Cl and tend to this work. be circumneutral to alkaline. This results in a bimodal

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd. A Sulphur-Supported Hydrothermal Microbiome 4181 distribution of spring pH in hydrothermal fields, with vapour Seismic activity is concentrated in several locations in phase influenced springs having a pH <5.0 and liquid YNP, in particular along a west-northwest trending belt phase influenced springs having a pH >6.0 (Fournier, located in its northwest corner where several geyser 1989; Nordstrom et al., 2009). This bimodal distribution in basins exist (Lowenstern et al., 2005), including the Syl- the pH of hot springs is typical for those in YNP and TVZ, van Springs Geyser Basin (SSGB). Springs in SSGB among others, with distribution peaks centred at pH ~2–3 have undergone extreme changes due to seismic activity, and ~6–7 due to buffering by sulphuric acid and bicarbon- specifically those related to the 1959 Hebgen Lake earth- − ate (HCO3 ), respectively (Brock, 1971). quake (magnitude 7.3) and the 1975 Norris-Mary Moun- The types of chemical nutrients available to support tain earthquake (magnitude 6.1) (Hutchinson, 1978). In microbial communities vary markedly between acidic particular, historical observations of evening primrose springs and circumneutral-alkaline springs due to the (EP) spring in SSGB extending back to the early 20th influence of pH on the chemical speciation, thermody- century (Allen and Day, 1935; Hutchinson, 1978) show namic stability and kinetic stability of substrates (Colman that its hydrology (e.g., water level and temperature) and 2− − et al., In press; Amenabar and Boyd, 2019). As an exam- chemistry (e.g., pH and SO4 /Cl content) are closely ple, the product of incomplete H2S oxidation, thiosulfate tied to seismic activity (Fig. 1A). EP therefore provides a 2− (S2O3 ), is chemically unstable in aqueous solutions unique opportunity to examine how the historical legacy with pH <4.0 and rapidly disproportionates to form ele- of geological change shapes the chemical composition of 2− mental sulphur (S ) and sulphite (SO3 ) (Xu et al., a spring and to determine how this, in turn, influences the 2000), the latter of which is also unstable at low pH and taxonomic and functional diversity of microbial inhabitants 2− rapidly oxidizes abiotically to form SO4 (Nordstrom of a spring. Here, we examine historical accounts of the et al., 2005). S is chemically stable at temperatures physical and chemical characteristics of EP and integrate <100C (Nordstrom et al., 2005), a characteristic that can these with contemporary geochemical and metagenomic lead to its accumulation in acidic springs where it can data to provide new insight into the links between the leg- serve as an electron donor, acceptor, or both donor and acy of geological events that control hot spring geochem- acceptor (e.g., disproportionation) in microbial metabo- istry and the taxonomic and functional diversity of lism (Amenabar and Boyd, 2018). Moreover, acidic microbial communities that inhabit those hot springs. springs sourced by vapour phase gases are also likely to be enriched in volatiles such as H2S, hydrogen (H2) and methane (CH4) (Lindsay et al., 2019) and have higher total sulphur contents, including solid phase S and its Materials and methods oxidation product SO 2−, relative to alkaline springs 4 Sample collection, DNA extraction and sequencing (Nordstrom et al., 2009; Amenabar and Boyd, 2019). However, the chemical composition, pH and tempera- Sediment samples were collected from EP (GPS coordi- ture of hot springs can vary over time-scales that range nates: 44.699437, −110.767147) in the SSGB of YNP on from seconds to hours (e.g., earthquakes and geysing), July 3, 2015. Triplicate sediment samples (~250 mg) to days or seasons (e.g., diurnal cycling and precipita- were collected using aseptic techniques, immediately fro- tion), or even longer timescales (Hurwitz and Lowenstern, zen on dry ice and transported back to the laboratory. 2014). Variation in the chemical composition, pH, or tem- DNA was extracted from sediment samples using the perature of springs over these timescales is related to Fast DNA Spin Kit for Soil (MP Biomedicals, Irvine, CA) fluctuations in one or more of a thermal feature’s three following the manufacturer’s instructions. Equal volumes main components: water, heat and the flow path that of triplicate DNA extracts were then pooled for further delivers fluids to a spring (Heasler et al., 2009). Short metagenomics analyses. term and minor fluctuations in these components can be Spring temperature and pH were measured with a por- caused by changes in circulation of subsurface waters table pH meter and a temperature-compensated probe due to deposition of silica and subsequent sealing of the (WTW 3100; WTW, Weilheim, Germany). Spring water flow paths that source springs or to alteration of these conductivity was measured using a temperature- flow paths due to chemical and/or physical weathering compensated probe (YSI EC300; YSI Inc., Yellow (Hutchinson, 1978). Alternatively, large-scale changes in Springs, Ohio). Samples for determination of anion con- the three components of hot spring systems can occur centrations were filtered (0.22 μm) in the field and kept at 2− − instantaneously due to seismic activity (Hutchinson, 4 C until processing. SO4 and Cl concentrations were 1978). However, little is known of how the historical leg- quantified with Hach reagents (SulfaVer 4 sulphate acy of changes in these components that form hot reagent powder pillows and the model 8-P chloride low springs influence their contemporary chemical and bio- range test kit, respectively) and a field spectrometer logical compositions. (Hach Company, Loveland, CO).

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology, 21, 4180–4195 4182 D. Payne et al. Whole community shotgun metagenomic sequencing target downsampling average coverage set at 100×. was conducted on total genomic DNA from EP sediments MetaSPAdes (v.3.1.0) was used to assemble the down- (~21 ng total). Illumina library preparation and paired-end sampled reads over a range of k-mer lengths and using sequencing (2 × 150 bp) were conducted at the Genomics the default parameters. A final assembly was chosen for Core Facility at the University of Wisconsin-Madison using further analysis based on comparison of assembly statis- the Illumina NovaSeq platform. Reads were quality tics (e.g., N50 and contig length distributions) using the trimmed and cleaved of Illumina sequencing adapters metaquast function of quast v.4.3 (Mikheenko et al., 2018). using TrimGalore v.0.6.0 (https://github.com/FelixKrueger/ Finally, the non-downsampled, quality-filtered reads were TrimGalore) and Cutadapt v.1.18 (Martin, 2011). Prior to aligned and mapped to the final assembly contigs using assembly, the quality filtered reads were downsampled to Bowtie2 (Langmead and Salzberg, 2012). The assembled minimize data redundancy, mitigate uneven population metagenomic data are available under the IMG genome ID sequencing depth coverage and improve assembly. Down- 3300034404. sampling was conducted using the BBnorm script of the Contigs (>2.5 kbp) from the highest quality assembly (k- BBMap programme suite (https://sourceforge.net/projects/ mer size = 121) were binned into draft metagenome-assem- bbmap/), with a minimum depth specified at 5× and a bled-genomes (MAGs) using unsupervised binning in MetaBAT v.0.26.3 (Kang et al., 2015) based on tetranucleotide frequency distribution patterns and differential sequence coverage profiles with the ’verysensitive’ parameter settings. Draft genome bins were assessed for quality, contamination and completeness using CheckM v.1.0.5 (Parks et al., 2015). Manual curation of bins was conducted using several methods to improve the quality of bins that appeared to represent multiple populations based on marker gene ’contamination’ estimates. First, ’outlier’ contigs that were defined as outside of 95% of the distributions for tetranucleotide word frequency distance, G + C content, coding density or coverage of each genome bin were removed using RefineM (Parks et al., 2018). Second, differential recruitment of contigs to reference genomes was conducted when other tools were not capable of separating distinct populations (e.g., Acidilobus

Fig. 1. Historical geochemistry of EP hot spring. A. Timeline depicting major seismic events and inferred hydrological input throughout the history of scientific observation of EP. Selected dates of interest are connected by black lines to their temporal loca- tion on the timeline (grey line). Below timeline, black bars represent duration of inferred subsurface hydrologic regimes. Dotted black bars indicate periods of uncertain hydrology. B. Variation in the temperature and pH of EP hot spring waters as a function of time. Lines and arrows connect values measured in EP in specified years (red squares). The pH of EP spring waters in 1927 was set to 6.5 to represent neutrality at a temperature of 65C (Amend and Shock, 2001) as a conservative estimate of spring water pH. Prior Research Coordination Network data (www.rcn.mon tana.edu) collected from YNP springs between the years 2003 and 2013 (grey circles) are plotted for reference. A histogram plot of spring pH values used to construct this plot is also shown in Supple- mentary File 3, Fig. 1. 2− − C. Variation in sulphate (SO4 ) and chloride (Cl ) measurements in EP spring waters. Lines and arrows connect values measured for EP in specified years (red squares). Prior data collected from YNP springs between the years 2003 and 2013 (grey circles) is plotted for reference (Ball et al. (2006) and McCleskey et al. (2014)). The classi- fications of hot spring fluid types, as described previously (Nordstrom et al., 2009), are also plotted for reference: hydrothermal water only (HO), hydrothermal water with subsurface boiling (HB), meteoric water only (MO), meteoric water with hot gas discharge (MG) and hydrothermal water with subsurface boiling and hot gas discharge (HBG). Data correspond with that presented in Table 1.

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology, 21, 4180–4195 A Sulphur-Supported Hydrothermal Microbiome 4183 strains). Contig recruitment was conducted with the MG databases (IMG and National Center for Biotechnology Wrapser tool (https://github.com/dunfieldlab/mg_wrapser) Information) were downloaded based on homology using a 98% nucleotide identity level. The differential recruit- searches of RpoB proteins from each bin against each ment of the two Acidilobus bins to reference Acidilobus database. Only references with >~50% estimated com- genomes was sufficient to parse the two populations into two pleteness were included in the final trees. Each of the nearly complete, high-quality MAGs. Lastly, partially complete proteins was aligned individually with Clustal Omega MAGs were merged when they clearly belonged to the same v.1.2.0 (Sievers et al., 2011), and the protein alignments population but were initially binned into separate MAGs were concatenated into a super matrix for each domain. (i.e., partial Fervidicoccus and Thermofilum MAGs). Only The concatenated alignments for all major groups of medium-high quality draft genome bins (estimated >50% Archaea (n = 956; 66,060 informative amino acid posi- complete) are included in the analyses presented here with tions) and select groups of Bacteria (n = 57; 7,796 infor- the exception of a low abundance Thermocrinis MAG, Bin20, mative amino acid positions) were subjected to Maximum that was estimated to be 43% complete. Gene prediction and Likelihood phylogenetic analysis in RAxML v.8.2.9 annotations were generated using Prodigal v.2.6.3 (Hyatt (Stamatakis, 2014) specifying the LG protein substitution et al., 2010) as implemented in Prokka v.1.11 (Seemann, model and a Gamma-shape distribution. The robustness 2014) specifying default parameters. Relative abundances of each clade’s monophyly was assessed with 100 rapid were calculated for individual populations with the ’profile’ algorithm bootstraps. function within CheckM wherein relativized abundances are first calculated from the mapping of reads to individual MAGs Comparison of Metagenome functional composition and then normalized to estimated MAG sizes. Finally, relative abundances were relativized across all bins within the Fifty-one metagenomes from chemosynthetic communi- community. ties inhabiting YNP hot springs and associated environ- Following preliminary gene predictions and annotations mental measurements were compiled as a part of a for MAGs using Prodigal v.2.6.3, gene functions were further previous study (Colman et al., In press). Details regarding examined using the Kyoto Encyclopedia of Genes and the locations of hot spring metagenomes and compiled Genomes (KEGG) function database (Kanehisa and Goto, environmental metadata for each spring are provided in 2000) using the KEGG Automatic Annotation Server Supporting Information Appendix S2. To compare the (KAAS) (Moriya et al., 2007). The results from the KAAS- functional proteins encoded in each hot spring commu- based analyses were also cross-referenced against KEGG nity, including EP, the KEGG Orthology (KO) assign- annotations after annotation of the assembled metagenome ments for each assembled metagenome were collected via the Joint Genome Institute (JGI) Integrated Microbial from IMG-processed annotations on the IMG server. The Genomes (IMG) pipeline (Chen et al., 2019). MAGs were KO data were used to construct an abundance-weighted also queried via BLASTp for specific gene functions to table for each metagenome using custom scripts in screen for specified sulphur cycling functions, as guided by MATLAB. To account for unequal sequencing depths gene contents of closely related genomes. BLAST searches among metagenome assemblies, the KO abundances were conducted using bait sequences specific for active site were normalized to the total abundance of KOs for that subunits for each protein or protein complexes, in addition to metagenome using the normalize.rows function of the other requisite subunits. Positive matches within MAGs were vegetarian (v.1.2) R software package v.3.4.1. A Bray– considered as those with an E-value >1 × 10−6,>30% Curtis dissimilarity matrix was then constructed from the amino acid homology and > 60% of the length of the normalized dataset using the ‘vegdist’ function within the BLASTp bait sequence (Supporting Information Appendix vegan package (v.2.4-4) for R. Of the entire KO dataset, S1). BLAST hits were further screened based on the pres- only those database annotations associated with the ence of characteristic amino acid residues for select func- ’Metabolism’ category were used in further analyses to tional genes after alignment with Clustal Omega (Sievers include only data that were most pertinent in discerning et al., 2011), including for example conserved cysteinyl resi- the relationship between spring geochemistry and micro- dues demarcating [NiFe]-hydrogenases and membrane bial metabolism. bound oxidoreductases (Mbx) (Schut et al., 2016). To identify patterns in functional composition among metagenomes, the dissimilarity matrix was subjected to Principal Coordinates Analysis (PCO) using the ‘pco’ Phylogenetic analyses function in the labdsv v.1.8–0 package for R. The per- Curated genome bins were surveyed for the presence of centage variation that was explained by each axis of the 104 archaeal-specific or 31 bacterial-specific single copy ordination was calculated from the relative contributions phylogenetic marker genes with Amphora2 (Wu and of PCO Eigen values. The relative enrichment of KOs Scott, 2012). Publicly available references from genome involved in the ’Sulphur Metabolism’ KEGG pathway was

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology, 21, 4180–4195 4184 D. Payne et al. also determined for the 52 metagenomes by calculating metagenome to represent the relative fraction of the average of all ‘Sulphur Metabolism’ KO relative metagenome proteins attributable to each query popula- enrichment values per metagenome, inclusive of both tion. While these data lack population abundance infor- assimilatory and dissimilatory pathway modules. mation, they represent the percentage of each spring To estimate the distribution of populations represented community metagenome that is attributable to the query by the EP MAGs among other thermal spring environ- populations. The abundance-normalized data were then ments, the protein coding genes of each MAG were com- plotted by scaling individual points by the overall range pared against those of the other 51 metagenomes for each respective EP population. described above. The protein coding genes of the metagenomes were collated into a local BLAST database, and the individual EP MAG protein coding genes Results and discussion were searched against the database using BLASTp Historical geochemistry of Evening Primrose searches, considering positive matches as >95% amino acid identity over alignments of ≥100 amino acids in EP is in the SSGB of YNP, roughly 7.2 km southwest of length. The number of positive matches was then divided the Norris Geyser Basin. The spring ﬁlls a siliceous by the total number of protein coding genes in each sinter-lined crater that is approximately 5.8 m long (north

Table 1. Historical measurements of temperature, pH, chloride and various sulphur species in evening primrose hot spring.

− 2− Total Cl SO4 − 2− 2− 2− Temperature HS S Sx S2O3 SO3 Year (C) pH (mg L−1)a (mM)a (mg L−1)a (mM)a (μM) (mM) (μM) (μM) (μM) Reference

1927 64 6.5b 428 12.1 132 1.37 - - - - - Allen and Day (1935) 1973c 57–58 1.0 - - 7400 77.0 - - - - - Mosser et al. (1974) 1973c 56 1.1 ------Bohlool and Brock (1974) 1974 56 1.1 - - 6014 62.6 436.6 51 - - - Brock et al. (1976) 1975 45 1.1 38 1.08 6300 65.6 - - - - - Ball et al. (1998) 1976c 45 1.1 - - 5776 60.1 0.9 20 - - 18.7 Zinder and Brock (1977) 1976c 41 1.1 - - - 3.7 - - - bdd Zinder and Brock (1977) 1998 68 1.8 390 11.0 1700 17.7 12.8 - - - - Ball et al. (2001) 2000 82 2.3 ------RCNe 2004 83 5.4 - - - - 65.8 - 84–322 28 - Lorenson (2006) 2010 81 6.5 496 14.0 94.5 0.98 15.5 30.1 - 559 2.6 Kamyshny Jr. et al. (2014) 2012 78 5.1 - - - - bd(0.3)d - - - - Urschel et al. (2015) 2013 79 5.5 - - - - 7.4 - - - - Colman et al. (2016) 2015 77 5.6 552 15.6 130 1.35 - - - - - This study 2016 74 5.4 ------Dahlquist (2017)

a. Sulphate and chloride concentrations are reported in both mg L−1 and μM to facilitate comparisons among chemical species. b. pH was reported as ’neutral’ and is represented here as a conservative pH estimate for neutrality at a temperature of 65C. c. Sample year was not reported and is represented here as the year the published reference was received. d. Below detection, with detection limit in parentheses, if available. e. http://www.rcn.montana.edu/.

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology, 21, 4180–4195 A Sulphur-Supported Hydrothermal Microbiome 4185 to south) and 4.6 m wide (east to west), with a depth of constituents (Nordstrom et al., 2009). Allen and Day also 1.5 m (Hutchinson, 1978). In July 2015, spring waters in observed siliceous sinter lining the spring crater, which is EP had a pH of 5.6 and a temperature of 77.4 C (Fig. 1B; atypical of acidic springs and is more commonly Table 1). Springs with pH between ~4 and 6 are generally observed in circumneutral to alkaline features (Walter, rare among the YNP thermal system (Supporting Informa- 1976; Vitale et al., 2008). The siliceous sinter that lines tion Appendix S3, Fig. 1), and thermal fields globally the conduit and spring contours of EP are visible as of 2− −1 (Brock, 1971). Measurements of SO4 (130 mg L ) and 2016 (Supporting Information Appendix S3, Fig. 2K). − −1 Cl (552 mg L ) indicate that it is sourced primarily by As late as October, 1948, EP was reported to have a hydrothermal only (HO; Fig. 1C) waters with minimal input light overflow of water from the crater rim, which was of vapour phase gases. Despite being sourced by HO described as cadmium yellow (again, presumably due to waters, suspended and precipitated S is visually abun- precipitated S or orpiment) with the centre a dark green dant in both EP waters and sediments (Supporting Infor- colour due to refracted light from interaction of dissolved mation Appendix S3, Figs. 2B to 2K). Previous XRD silica and precipitated S (Watson and Condon, 1948). analyses conducted on sediments sampled in 2013 This suggests that the hydrology and chemistry of EP fi (pH 5.5, 78.6 C) con rm the presence of S in EP was similar to that observed in 1927, which is consistent (Colman et al., 2016) and HPLC analyses conducted on with an image of EP from 1950 that revealed similar char- waters collected in 2014 (pH 6.5, 81.0 C) reveal concen- acteristics [Supporting Information Appendix S3, Fig. 2A; trations of dissolved S as high as 3 mM dispersed in the (Hutchinson, 1978)]. However, shortly after the magni- water column (Kamyshny Jr. et al., 2014). Moreover, EP tude 7.1 Hebgen Lake earthquake on August 17, 1959, μ fi 2− waters contain up to 320 M polysul de (Sx ) (Lorenson, thermal activity in EP changed markedly. Field notes by 2006), which are soluble linear chains of S , and elevated Park Naturalists Al Mebane and Richard Frisbee report 2− μ 2− concentrations of sulphite (SO3 ;3 M) and S2O3 the water level at EP had dropped 0.6 m, and the temper- μ (560 M) have been detected in this spring (Kamyshny ature had increased to 87C by December 1959 Jr. et al., 2014). A comparison to other sulphur rich, acidic (M.A. Bellingham, personal communication). According ‘ ’ springs in YNP (e.g., Cinder Pool, Dragon Spring and to field notes by United States Geological Survey scien- Frying Pan Spring) indicates that the high amounts of tist Robert Fournier, the water level continued to 2− 2− suspended S , soluble Sx and S2O3 in EP is a unique decrease between 1960 and 1969 (Supporting Informa- feature in YNP (Kamyshny Jr. et al., 2014), particularly tion Appendix S3, Figs. 2B-2E; M.A. Bellingham, per- given its moderate acidity. sonal communication), concomitant with the To begin to understand how the unique geochemistry development of a 5 to 10 cm inch thick layer of pure S in EP may have developed, we examined historical sci- floating on its surface at a depth of 1.5 m below the crater fi fi fi enti c studies. The rst reported scienti c measurements rim. At this time, EP was the only spring in YNP with this of EP were made in August, 1927 by Allen and Day, who characteristic (Hutchinson, 1978). These observations determined the temperature to be 64 C and the acidity to signalled a change in the hydrology and chemistry of EP. be below detection via chemical titration (Allen and Day, Consistent with this interpretation, in summer of 1971, ‘ ’ 1935). Thus, the spring was reported as alkaline which, the pH of EP was 1.3 and the temperature was >90C without further information, is conservatively interpreted (Fig. 1B; Table 1; (Brock, 1978)). Sometime during the as neutral pH at this temperature (pH = 6.5 at 65 C winter of 1971 to 1972, the temperature and pH dropped (Amend and Shock, 2001); Fig. 1B). The spring was to ~60C and 0.9, respectively (Brock, 1978), which is named EP by Allen and Day for the veneer of yellow min- among the lowest pH measurements for any spring in eral lining the spring crater, which they assumed to be S YNP (Amenabar et al., 2015). From 1971 to 1977, the (Allen and Day, 1935). This colour could have also been temperature of EP remained moderate (45 to 60C) and attributable to an arsenic sulphide compound such as the pH hyperacidic, ranging from 1.0 to 1.1, while mea- orpiment, a mineral that has been detected in YNP 2− −1 surements of SO4 ranged from 1928 to 7400 mg L springs (Weed and Pirsson, 1891). Concentrations of (Fig. 1C; Table 1) (Mosser et al., 1974; Brock et al., 2− − −1 SO4 and Cl were 132 and 428 mg L , respectively, 1976; Zinder and Brock, 1977; Ball et al., 1998). These indicating that it was sourced by HO waters reflecting the 2− elevated concentrations of SO4 are consistent with sul- parent hydrothermal reservoir (Fig. 1C). More modern phuric acid buffering and indicate a significant input of estimates suggest that the parent hydrothermal reservoir vapour phase gas (Nordstrom et al., 2009). This is also 2− − ~ −1 has SO4 and Cl concentrations of 73 to 98 mg L consistent with reports that the spring hosted abundant − (Nordstrom et al., 2009) and 310 mg L 1 (McKenzie and microaerophilic Sulfolobus cells (Mosser et al., 1974; Truesdell, 1977), respectively. This suggests that the Brock et al., 1976; Zinder and Brock, 1977; Ball et al., waters sourcing EP in 1927 had undergone evaporative 1998), organisms that oxidize inorganic sulphur com- − 2− 2− concentration of Cl ,SO4 and likely other chemical pounds (e.g., S ) to generate SO4 and acidity (Huber

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology, 21, 4180–4195 4186 D. Payne et al. and Stetter, 2015), and reports of between 20 and reveal an obvious outflow channel. This suggests that 51 mM soluble total S at this time (Brock et al., 1976; since 1960, EP may have operated as a closed hydrolog- − Zinder and Brock, 1977). A single measurement of Cl ical system with only minimal fluid input and evaporative − (38.2 mg L 1) was made in 1975, and this is paired with losses. Evidence in potential support of this comes from 2− −1 18 2 aSO4 measurement of 6300 mg L (Ball et al., 1998). highly enriched δ O and δ H of spring waters (Dahlquist, Thus, in contrast to 1927, these observations indicate 2017) that indicate boiling and/or evaporative concentra- that EP in the 1960s and 1970s was sourced primarily by tion [Supporting Information Appendix S3, Fig. 3; meteoric water mixing with vapour phase gas (termed (Nordstrom et al., 2009)]. However, dilution of hyperacidic MG; Fig. 1C) and had a minimal input of deeply circulated waters in EP by input of HO waters alone cannot explain fl − HO uid (Nordstrom et al., 2009). The siliceous sinter the simultaneous increase in pH and Cl and the armouring the spring conduit, most likely deposited at a 2− decrease of SO4 observed between 1998 and 2004 time when the spring was circumneutral to alkaline and that characterize the spring in its present form. (Vitale et al., 2008), likely played a key role in resisting The following scenario is put forth to explain the shift in acid weathering of the spring crater during this period of the chemistry of EP. Input of deeply circulating HO hyperacidity. waters between 1998 and 2004 delivered alkalinity in the − The pH of EP increased to 1.8 (68 C) by September form of HCO that could consume remnant H+ and 3 1998 (Ball et al., 2001) and reached 2.3 (82 C) by August slowly raise the pH of the spring towards the pKa of 2000 (Fig. 1B; Table 1), changes that may be due to − HCO3 [~6.4 at 80 C (Amend and Shock, 2001)], while − increased seismic activity in YNP that began in 1994 also delivering Cl , allowing it to reach levels similar to 2− − (Waite and Smith, 2002). By 1998, SO4 had decreased the parent hydrothermal reservoir of ~310 mg L 1 −1 − −1 to 1700 mg L and Cl had increased to 390 mg L , (McKenzie and Truesdell, 1977). The increase in the pH indicating an increasing input of deeply circulating HO of EP also promoted the stability of compounds like fluid (Nordstrom et al., 2009). As of August 2004, the pH 2− 2− S2O3 and Sx , given that they are both thermodynami- of EP had increased to 5.4 and the temperature was cally unstable at more acidic pH (reviewed in Amenabar 82.8 C (Lorenson, 2006). Between 2012 and 2016, the and Boyd, 2019). The increase to moderately acidic pH pH of EP varied little (5.1 to 6.5), the temperature also promoted shifts in the microbial activities taking 2− remained high (74.4 to 81.0 C) (Kamyshny Jr. et al., place in the spring that may have included SO4 reduc- 2014; Urschel et al., 2015;Colman et al., 2016; Dahlquist, tion (SR), an activity that is generally favoured in more 2017), and the water level returned to those observed moderate (pH 5 to 9) conditions (Postgate, 1979). SR 2− prior to the 1959 earthquake (Supporting Information activity would decrease SO4 concentrations by conver- 2− − Appendix S3, Figs. 2H-2K). Based on SO4 and Cl ting it to sulphide that, in turn, either volatilized from the 2− numbers during this time interval, it was estimated that spring or reacted with remnant S to form soluble Sx , somewhere between 85% and 100% of the water in EP which have been measured at high concentrations in EP was from the parent HO water body (Kamyshny (Kamyshny Jr. et al., 2014). Importantly, dissolved O2 Jr. et al., 2014). concentrations in EP in 2013 and 2016 were 25.3 and Based on this historical account of EP, a temporal 12.6 μM (Colman et al., 2016; Dahlquist, 2017). While model is proposed to describe the development of the these dissolved O2 concentrations are low, they are likely unique, S -rich geochemical conditions of EP observed an overestimate of O2 availability due to our methodology today (Fig. 1A). Using the classification scheme of requiring spring water to be filtered (to remove Nordstrom et al., (2009), the waters sourcing EP from suspended S and sediment) before Winkler titration.

1927 to 1959 were HO and were likely circumneutral. Fol- Thus, some O2 may have diffused into the spring water lowing the Hebgen lake earthquake in 1959 and to at as it cooled during these ex situ assays. The low amount least 1977, waters sourcing EP were primarily MG of O2 infused from the atmosphere or delivered by HO (Nordstrom et al., 2009), allowing hyperacidic conditions waters would prevent extensive oxidation of reduced sul- to develop and a 5 to 10 cm thick layer of pure S to phur compounds, including remnant S that would other- accumulate on its surface, likely through the incomplete wise lead to the redevelopment of hyperacidic conditions. oxidation of vapour phase-sourced H2S (Nordstrom et al., In turn, this allowed the spring to maintain a moderately 2005). However, by 2004, the waters sourcing EP had acidic pH of 5.1 to 6.5 despite having an unusually high shifted to HO with minimal (if any) input of vapour phase concentration of precipitated and dissolved S gases. This shift was underway by 1998 (as evidenced (Kamyshny Jr. et al., 2014), the latter of which was likely − by rising pH, temperature and Cl concentration), per- deposited when the spring was hyperacidic. We suggest haps induced by the increasing seismic activity beginning that SR and other reductive sulphur cycling activities 2− in 1994 (Waite and Smith, 2002). As opposed to early such as S /Sx reduction and disproportionation are accounts of EP, photographs from 1960 onward do not enhanced in EP today given the abundance of oxidized

2− 2− 2− sulphur substrates [e.g., SO4 ,S2O3 ,S,Sx the EP sediment community forms a cluster with other (Kamyshny Jr. et al., 2014)] combined with the anoxic communities that are largely from acidic (pH <4.0) nature of deeply circulating HO waters that are now the springs. It is possible that this pattern of clustering occurs primary source of fluids to the spring (Fournier, 1989). because EP, much like the more acidic springs in YNP (Inskeep et al., 2013), is enriched in S. Indeed, sediment metagenomes from MV2 spring in the Mud Volcano area Taxonomic and functional composition of the EP (pH 3.8, 62 C) (Colman et al., 2016) and JC2E (pH 2.5, sediment community 75 C) in the Hot Springs Basin area (Kozubal et al., 2012), both of which are enriched in S, plot just to the fl ’ The potential in uence of EP s unusual sulphur rich and upper right of EP on the PCO ordination. Likewise, two anoxic geochemistry on taxonomic and functional biodi- metagenomes from Cistern Spring (pH 4.4, 79C, versity was examined using a metagenomics approach. labelled Cistern and YNP19.CIS) and two metagenomes Sequencing the EP sediment-associated community from Monarch Spring (pH 4.0, 79C, labelled Monarch metagenome yielded ~41.4 Gbp of reads, with assembly and YNP3.MG), which host sediments comprised of floc- generating a total of 43.6 Mbp of contigs (Supporting culant S (Inskeep et al., 2013), both plot to the lower left Information Appendix S1). The composition of proteins of EP on the ordination. This observation suggests that encoded by the EP sediment-associated metagenome the influence of sulphur geochemistry may overprint the was compared to those from 51 other chemosynthetic overarching and widely reported influence of pH on com- communities from hot springs across YNP using PCO munity diversity [e.g., (Inskeep et al., 2013; Colman analysis (Fig. 2). PCO axes 1 and 2 accounted for 46.3% et al., 2016)]. of the variation among proteins encoded in community To further examine whether the unusually high genomes. Communities formed clusters along PCO axis amounts of S in EP shape the functional composition of 1 and this pattern of clustering largely corresponded to the EP community, the relative enrichment of KO protein the pH of the hot spring from where they were sampled, families involved in ‘Sulphur Metabolism’ pathways were with acidic springs generally plotting on the left of PCO overlaid on the PCO ordination (Fig. 2). As expected, axis 1 and those from circumneutral to alkaline springs metagenomes from acidic hot spring environments generally plotting to the right of PCO axis 1. Despite hav- (i.e., plotting to the left of PCO axis 1) were enriched in ing a moderately acidic pH of 5.6 at the time of sampling, KOs predicted to be involved in sulphur metabolism relative to those from more circumneutral to alkaline hot springs (i.e., plotting to the right of PCO axis 2). Consis- tent with the elevated amounts of sulphur in EP, the sediment-associated community is also enriched in KOs predicted to be involved in sulphur metabolism, again suggesting that sulphur may overprint the overarching influence of pH on the functional composition of chemosynthetic communities (Colman et al., 2016; Power et al., 2018). To begin to define how the unusually high amounts of sulphur shape the taxonomic and functional composition of the EP community, as suggested by the PCO ordination and overlays of KO abundances on this ordination, assembled contigs were binned into metagenome assembled genomes (MAGs) based on tetranucleotide fi Fig. 2. Similarity in proteins putatively involved in energy metabolism frequencies and coverage pro les. A total of 17 draft among EP and 51 other published chemosynthetic community MAGs were compiled, 16 of which were estimated to be metagenomes from YNP. PCO shows the similarity in proteins that >50% complete and exhibited <7% contamination are putatively involved in energy metabolism pathways among metagenomes, where each point represents a metagenome. The (Fig. 3A, Supporting Information Appendix S1). Two distinct percentage of variation explained by each PCO axis is indicated. MAGs related to Acidilobus (30% and 21% genome-size Points are coloured based on the pH of spring waters, and the size corrected estimates of community relative abundance) domi- of each point corresponds to the mean relative enrichment of KEGG orthology (KO) protein families involved in ‘Sulphur Metabolism’ nated the sediment-associated community, followed in abun- pathways. The size scaling corresponds to relative enrichment dance by MAGs related to Thermogladius (11%), values for each KO within each metagenome, averaged across all Thermoproteus (7%) and Caldivirga (5%) (Fig. 3A; KOs involved in ‘Sulphur Metabolism.’ The data were further z-score transformed to facilitate the visualization of relative variation in Supporting Information Appendix S3, Figs. 4A to 4C). Addi- enrichment values. Spring labels are defined in the primary text. tional subdominant archaeal MAGs that were recovered

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology, 21, 4180–4195 4188 D. Payne et al. reduction or disproportionation of inorganic sulphur compounds. When the putative Acidilobus symbiont Nanopusillus, which, based on genomic data is inferred to be incapable of most biosynthetic functions and ATP formation (Supporting Information Appendix S1) (Wurch et al., 2016), is removed from consideration, this number increases to 99.7% of the community harbouring energy metabolisms that are dependent on inorganic sulphur. Importantly, it should be noted that Nanopusillus is likely to be dependent on its Acidilobus host for cellular growth, and thus is also likely to be indirectly dependent on inorganic sulphur respiration. Specifically, 89.4% of the total estimated EP community was represented by MAGs that are inferred to be anaerobic S reducers. These include two MAGs related to Acidilobus saccharovorans (each with 81% RpoB amino acid identity), a MAG related to Thermogladius calderae (87% ID), a MAG related to Thermoproteus uzoniensis (97% ID), a MAG related to Caldivirga maquilingensis (94% ID), a MAG related to Pyrobaculum aerophilum (92% ID), a MAG related to Thermofilum carboxydotrophus (100% ID), a MAG related to Vulcanisaeta distributa (93% ID), two MAGs related to Fervidicoccus fontis (71% and 79% ID), a MAG related to Thermocrinis ruber (98% ID) and a Thaumarchaeote-associated MAG (Supporting Informa- tion Appendix S1). Further, a divergent MAG that is dis- 2− Fig. 3. Rank-abundance plot of EP reconstructed population level tantly related to a S2O3 reducing Desulfurococcales bins. Each vertical bar represents a reconstructed genome bin that strain (e.g., 63% ID to Zestosphaera tikiterensis (St. John has an estimated completeness >50% (n = 16) as well as a lower et al., 2019)) was identified that exhibited the metabolic abundance Thermocrinis-affiliated bin with an estimated completeness of 43%. Genome bins are arranged by relative abundance potential for anaerobic S reduction. (as a percentage) in decreasing order, as determined by read map- To better assess the functional potential of MAGs, they ping. The taxonomy of genome bins was based on clustering with were screened for homologues of proteins in pathways that cultivated representatives with available genomes, as assessed by phylogenomic analyses (Supplementary File 3, Figs. 4A to 4G) and have been shown or suggested to enable S reduction BLASTp analyses with select housekeeping genes (Supplementary (Supporting Information Appendices S1 and S4). Homo- File 1). logues of a putative NAD(P)H sulphur oxidoreductase (NSR) complex and a ferredoxin:NAD(P)+ oxidoreductase, included those related to Pyrobaculum (4% estimated relative proposed to enable S reduction in A. saccharovorans abundance), Nanopusillus (4%), Thermofilum (3%), Vul- (Mardanov et al., 2010), were identified in both Acidilobus canisaeta (0.7%) and two distinct Fervidicoccus MAGs (0.2% MAGs as well as the Thermoproteus, Caldimicrobium, and 0.1%) (Supporting Information Appendix S3, Figs. 4B to Caldivirga, Vulcanisaeta, Desulfurococcales, Thermofilum, 4D). Several less abundant archaeal MAGs were distantly Thaumarchaeote Bin14 and both Fervidicoccus MAGs. In related to known archaeal lineages (<70% RpoB identities), addition, homologues of a membrane-bound oxidoreduc- including two that were associated with a deep-branching tase (Mbx), which is involved in reducing the persulfide 2− Thaumarchaeota clade, albeit in two different monophyletic bond in Sx in Pyrococcus furiousus (Wu et al., 2018), sub-groups (3% and 0.1% of total reads) and a MAG distantly were identified in the Thermogladius MAG. Based on (i) a related to characterized Desulfurococcales (2%) (Supporting lack of detected homologues for key enzymes involved in Information Appendix S3, Fig. 4E and B, respectively). The each of the four primary autotrophic pathways, (ii) the most abundant bacterial MAG was related to Caldimicrobium detection of homologues of for complete (or nearly com- (6% estimated relative abundance) with two additional MAGs plete) TCA and glycolytic pathways and (iii) the detection of affiliated with Thermocrinis (4% and 1%) (Supporting Informa- homologues of carbohydrate, polypeptide or amino acid tion Appendix S3, Fig. 4F and G, respectively). transporters, the Acidilobus, Thermogladius, Caldivirga, Analysis of MAGs based on the physiology of closely Vulcanisaeta, Desulfurococcales, Thaumarchaeote and related cultivars suggested that >96% of the sediment- Fervidicoccus-related MAGs are inferred to be heterotro- associated community is supported by dissimilatory phic. This is consistent with physiological characterizations

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology, 21, 4180–4195 A Sulphur-Supported Hydrothermal Microbiome 4189 of the most closely related type strains (Supporting Informa- enzyme pathways. The EP Caldimicrobium MAG tion Appendix S1 and S4). encodes homologues of all proteins required for autotro- In the case of the more abundant Thermocrinis MAG, phic growth via the Wood-Ljungdahl (reductive acetyl- homologues of a sulphur reductase (SreABC) system that CoA) pathway. 2− has been shown to reduce S or Sx to H2SinAquifex A number of EP MAGs are also inferred to be capable 2− aeolicus (Guiral et al., 2005), were identified. This MAG of generating energy via dissimilatory SO4 reduction, also encodes nearly all homologues of proteins required including those closely affiliated with Caldivirga for autotrophic growth via the rTCA cycle but does not maquilingensis (94% ID) and Vulcanisaeta distributa encode homologues of [NiFe]-hydrogenases, indicating it (90% ID) and a divergent MAG that branches at the base cannot use H2 as an electron donor. Intriguingly, however, of the Thaumarchaeota lineage (Supporting Information unlike other Thermocrinis strains, this EP Thermocrinis Appendix S3, Fig. 3). This physiological inference is MAG does not encode homologues for any subunits of a based on detection of homologues of Sat, Apr and cytochrome c oxidase, indicating it is unlikely to be able DsrABC in each of these genome bins and the phyloge- respire O2. In contrast, the less abundant and less com- netic placement of their DsrAB homologues (Supporting plete Thermocrinis MAG that was more closely related to Information Appendix S1 and S3, Fig. 5). DsrAB func- 2− those previously characterized in YNP springs [(Takacs- tions in both the forward (SO4 reduction) and reverse Vesbach et al., 2013); Supporting Information Appendix (S oxidation) directions and may also be potentially S3, Fig. 4G] encoded homologs of cytochrome c oxidase involved in S/polysulfide disproportionation (Müller et al. heme-Cu type oxidase subunits I (CoxA) and II (CoxB). 2014, Thorup et al. 2017). Importantly, catalytic direction- BLAST searches of the Aquificales-like CoxA or CoxB ality is generally consistent with phylogenetic placement subunits of Bin20 against the binned and unbinned EP of DsrAB (Müller et al. 2015). As such, all known metagenome did not yield any close homologues, crenarchaeote DsrAB from cultivated Vulcanisaeta spp., suggesting that the absence of these homologues in the Caldivirga spp., Pyrobaculum spp. and Thermoproteus 2− 2− more abundant Bin11 Thermocrinis MAG was not due to spp. are involved in either SO4 or SO3 reduction binning per se. Nevertheless, it is worth noting that the (Müller et al. 2015), implying a similar functionality for the more abundant Thermocrinis MAG was not complete Vulcanisaeta sp., Caldivirga sp. and the thaumarchaeote (75% estimated completeness; Supporting Information sp. that is further described below. Appendix S1), and the inferred lack of aerobic respiration Intriguingly, the less abundant thaumarchaeote MAG cannot be precluded in these populations and warrants from EP, which is related to MAGs from Obsidian Pool, further investigation. To this end, electron donors YNP (Berghuis et al., 2019) in addition to one from supporting chemolithotrophic growth of this population are ‘Beowulf Spring’, YNP (Beam et al., 2014), appears to 2− not known. have laterally acquired the ability to respire SO4 from The most abundant bacterial MAG (6% of the EP com- an ancestor of the crenarchaeal Vulcanisaeta lineage munity) is affiliated with Caldimicrobium thiodismutans (Supporting Information Appendix S3, Figs. 4E and 5). 2− (80% ID), a strain that has yet to be reported in a YNP The capacity for SO4 reduction has yet to be demon- hot spring system, let alone to be dominant among the strated or suggested in the Thaumarchaeota, although bacterial component of a YNP community. C. S oxidation via several different non-Dsr-based path- thiodismutans has been shown to couple autotrophic ways were proposed in other deeply branching 2− 2− growth to disproportionation of S ,S2O3 and SO3 but Thaumarchaeota, including those from ‘Beowulf Spring’ 2− not reduction of SO4 (Kojima et al., 2016). It has been (Beam et al., 2014). Further, S reduction was recently 2− suggested that during sulphur disproportionation, S2O3 demonstrated in a deep-branching thermophilic 2− and S are first converted to SO3 by thiosulfate reduc- thaumarchaeote (Kato et al., 2019). However, it was tase and a yet to be defined enzyme, respectively recently suggested that early thaumarchaeotes may have 2− 2− (Finster, 2008). SO3 is then proposed to be oxidized by lost the ability to reduce SO4 during their evolution into adenylyltransferase (Sat) and adenylylsulfate reductase aerobic ammonia oxidizers (Ren et al., 2019). This asser- (Apr) operating in reverse with the simultaneous reduction was partially based on the presence of some homo- 2− 2− tion of SO3 by dissimilatory sulphite reductase (Dsr). logues involved in SO4 reduction (e.g., adenylylsulfate Homologues of Sat, Apr and Dsr were identified in the reductase; AprAB) in the aforementioned MAG from EP MAG (Supporting Information Appendix S1), ‘Beowulf Spring’ that were originally proposed to be suggesting that this pathway may also facilitate dispro- involved in a novel, non-Dsr based S oxidation pathway portionation of inorganic sulphur compounds in this popu- (Beam et al., 2014). However, phylogenetic reconstruc- lation. While unlikely based on cultivation data from the tion of the early-evolving thaumarchaotes including Bin21 type strain (Kojima et al., 2016), it is possible that the EP (Supporting Information Appendix S3, Fig. 4E), in addi- 2− Caldimicrobium MAG could also reduce SO4 via these tion to a phylogenetic reconstruction of DsrAB

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology, 21, 4180–4195 4190 D. Payne et al. (Supporting Information Appendix S3, Fig. 5) rather suggests that the early-evolving clade including Bin21 and 2 the ’Beowulf Spring’ MAG acquired the capacity for SO4 − reduction relatively recently from a crenarchaeote, and likely a Vulcanisaeta strain. S oxidation via Dsr has not been physiologically demonstrated for archaeal organisms harbouring Dsr, which is consistent with the lack of accessory proteins (DsrEFH) necessary for bacterial S oxidation with Dsr (Dahl et al., 2005; Anantharaman et al., 2018) in any of the EP MAGs. The EP Caldivirga, Vulcanisaeta and divergent thaumarchaeote MAGs lacked homologues for key enzymes involved in autotrophic pathways but did encode homologues of complete TCA and glycolytic pathways as well as transporters for monosaccharides and polysaccharides and peptides, 2 suggesting that these populations are heterotrophic SO4 − Fig. 4. Proposed roles of members of the EP sediment-associated reducers. Consistent with this interpretation, the community in the sulphur cycle. Sulphur species are arranged with thaumarchaeote Bin21 MAG also lacked evidence for the respect to their oxidation states (y axis). Arrows indicate the predicted dissimilatory sulphur metabolism that supports specified ability to use H2 via [NiFe]-hydrogenases, CO via CO − MAGs. Line thickness corresponds to MAG relative abundance dehydrogenases (Cdh, Coo), or HCOO via formate (shown in parentheses); only those MAGs with a relative abundance dehydrogenases (Fdh) (Supporting Information Appen- greater than 0.5% are indicated by arrows. Blue arrows correspond dix S1). to MAGs with inferred strictly anaerobic metabolisms, red arrows correspond to MAGs with inferred facultative microaerophilic metabo- The majority of the EP community (89.9%) comprised lisms, and black arrows denote abiotic reactions. The putative role of archaeal MAGs and these are largely predicted to each MAG in dissimilatory sulphur cycling was inferred from proteins encode dissimilatory sulphur-based metabolisms. As indi- encoded by each MAG and physiological inference based on closest related cultivars. *The Thermocrinis-associated MAG from bin cated in Fig. 2, the enrichment of sulphur-based metabo- 11 could not be confirmed to be aerobic because cytochrome lisms is typical of communities from hyperacidic, sulphur- c oxidase homologues were not detected in that bin. Although the rich springs but is atypical for those inhabiting moderately lack of such proteins in an incomplete genome is not necessarily diagnostic, the MAG has been conservatively designated as a strict acidic or circumneutral springs such as EP (Colman anaerobe. et al., In press). It has been suggested that Archaea dominate the hottest and most acidic environments because previous analyses of a variety of environmental systems of adaptations that allow them to cope with chronic (Valentine, 2007): they are adapted to thrive under envi- energy stress (Valentine, 2007). Chief among these is ronmental conditions that impose chronic energy stress. the ability of cells to integrate O2 into their metabolism to We suggest that this phenomenon is driven either directly maximize energy yield, thereby coping with the high ener- or indirectly in both oxic acidic springs and anoxic moder- getic demand associated with synthesis and mainte- ately acidic springs by the presence of sulphur, since it is nance of biomolecules at high temperature and low pH responsible for the development of acidity in MO (Colman et al., 2018). However, unlike the aerobic influenced springs and for maintaining the anoxic nature thermoacidophilic Archaea that inhabit hyperacidic of HO influenced EP; both of these conditions impart springs, the Archaea that inhabit EP are inferred to be energy stress on cells (Valentine, 2007). anaerobes (Fig. 4). This is a likely consequence of EP Many of the microorganisms that dominate the EP being sourced by HO waters since ~2004 that deliver low sediment-associated community are related to cultivars

O2 (Fournier, 1989) together with the abundance of that have temperature optima, minima or maxima that reduced sulphur compounds that accumulated in the bracket the pH of EP spring waters at the time of sam- spring when it was hyperacidic (e.g., ~1959 to ~1998) pling (Supporting Information Appendix S3, Fig. 6B; and their tendency to react with O2. We suggest that this Supporting Information Appendix S4). However, these unique combination of characteristics limits the possible same cultivars are often not associated with springs with reactions capable of supporting microbial metabolism to moderately acidic pH. For example, Acidilobus is com- those that involve low energy yields, such as reduction or monly associated with acidic springs and the pH of EP at disproportionation of inorganic sulphur compounds the time of sampling (5.6) is near the maximum pH toler- (Amend and Shock, 2001; Finster, 2008). Thus, we ated by characterized Acidilobus cultivars (Supporting hypothesize that Archaea dominate the moderately Information Appendix S3, Fig. 6B; Supporting Information acidic EP and other hyperacidic springs for the Appendix S4). In contrast, other strains identiﬁed in EP same overarching reason and one that is consistent with are more commonly associated with higher pH springs.

© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology, 21, 4180–4195 A Sulphur-Supported Hydrothermal Microbiome 4191 For example, the minimum pH tolerated by Thermog- the prevalence of acidic (pH ~ 2 to 3) and circumneutral ladius, Caldimicrobium, Pyrobaculum, Thermocrinis and (pH 6 to 7) springs has biased cultivation studies toward Thermoﬁlum cultivars most closely related to EP MAGs is the characterization of strains that are adapted to these near or above the pH of EP at the time of sampling pH realms. It is also possible that sulphur rich, anoxic (Supporting Information Appendix S3, Fig. 6B; Supporting springs with moderately acidic pH are populated by cells Information Appendix S4). The only cultivars related to that are maladapted (albeit better adapted than other EP MAGs whose cardinal pH requirements closely match strains) to those conditions due to the uncommon geo- that of EP at the time of sampling are Thermoproteus, chemistry (sulphur rich, anoxic and moderately acidic pH) Desulfurococcus and Fervidicoccus. It is possible that and the ephemeral and dynamic nature of this spring type, which stymies long-term adaptation to these conditions. Alternatively, it is possible that the pH of the sediments is slightly more acidic than the water column thereby selecting for acidic populations. However, the abundance of reduced sulphur compounds and the low

abundance of dissolved O2, both of which are necessary to generate acidity in sediments (Colman et al., 2018), argues against this possibility. To gain further insight into whether EP populations are adapted to the pH and temperature regime of the spring, the relative abundance of proteins (>100 aa in length) encoded in 51 publicly available metagenomes from chemosynthetic YNP hot spring communities were mapped on EP MAGs at a > 95% sequence identity threshold. Like the analysis of the cardinal temperatures for cultivars that were most closely related to EP populations, protein sequences closely related to those identiﬁed in EP were found across YNP hot springs with similar temperatures to that measured at EP (Fig. 5B; Supporting Information Appendix S3, Fig. 7). This suggests that the realized niches of EP populations include the ~78C temperature condition measured at EP at the time of sampling. In contrast, sequences closely related to EP populations were rarely detected in springs with similar pH to that measured at EP (5.6) at the time of sampling (Fig. 5A). Rather, sequences closely related to EP MAGs were commonly detected in springs with more acidic pH, in particular for dominant MAGs putatively supported by S reduction (e.g., Acidilobus spp., Thermoproteus and Caldivirga). These observations are consistent with the cardinal pH for many cultivars from these genera (Supporting Information Appendix S3, Fig. 6B) and with the pattern of clustering in the PCO ordination with other more acidic springs (Figs. 2A & 2B). Together, these results suggest the unique chemical environment of EP, speciﬁcally the prevalence of intermediate oxidation state sulphur compounds, has selected for the dominance of archaeal strains that otherwise tend to inhabit more acidic sulphur-rich hot spring habitats. Fig. 5. Mapping of proteins from 51 YNP metagenomes to EP MAGs. Distribution of YNP metagenome encoded proteins (>100 aa in length) recruited to EP MAGs (>95% sequence identities) as a function of the temperature (A) and pH (B) of the springs. The size of symbols in both Conclusions plots is proportional to the relative abundance of total metagenome ORFs attributed to that MAG from each metagenome. Circle sizes are EP, a moderately acidic hot spring in YNP, hosts an unusu- scaled to the overall range of ’relative abundance’ for each respective ally high amount of intermediate oxidation state sulphur EP population among the 51 metagenomes. Shaded regions corre- 2− 2− compounds, including Sx ,S and S2O3 . Metagenomic spond to the temperature and pH of EP spring waters at time of sampling. An additional representation of the data presented in panels A sequencing and analyses indicate that EP hosts an and B in Supplementary File 3, Fig. 7. archaeal dominated community of which 96.3% (up to

99.7% pending inclusion of Nanopusillus) is predicted to be Head of the Department of Microbiology and Immunology at anaerobic and supported by dissimilatory reduction or dis- Montana State University, for supporting costs associated with proportionation of inorganic sulphur compounds. Several this Microbial Ecology and Evolution graduate class project. populations code for functionalities that are unique among their higher order taxonomic lineages, including a Thermocrinis bin that is inferred to be a strict anaerobe and Conflict of Interest reduce S . Similarly, a bin that branches at the base of the The authors declare that they have no conflicts of Thaumarchaeota codes for a complete dissimilatory sul- interest. phate reduction pathway. Surprisingly, despite its moderately acidic pH, the EP community is functionally more similar to communities recovered from hyperacidic hot References springs than circumneutral springs. We suggest this is due to the geological legacy of EP, specifically a shift toward Allen, E.T., and Day, A.L. (1935) Hot Springs of the Yellow- hyperacidic conditions due to an increased input of sulphur- stone National Park. Washington, DC: Carnegie institution rich vapour phase influenced waters following a well- of Washington. Amenabar, M.J., and Boyd, E.S. (2018) Mechanisms of min- documented seismic event, the Hebgen Lake earthquake eral substrate acquisition in a thermoacidophile. Appl in 1959. The increase in sulphur-rich vapour phase gases Environ Microbiol 84: e00334–e00318. to EP, combined with thermodynamic and kinetic instabil- Amenabar, M.J., and Boyd, E.S. (2019) A review of the ities of intermediate oxidation state sulphur compounds, mechanisms of mineral-based metabolism in early earth allowed S to accumulate during this time (~1959 to analog rock-hosted hydrothermal ecosystems. World J ~1998). Soon thereafter, for reasons that are not entirely Microbiol Biotechnol 35: 29. clear but that may be related to another swarm of seismic Amenabar, M.J., Urschel, M.R., and Boyd, E.S. (2015). In Metabolic and Taxonomic Diversification in Continental activity during the mid-1990s, the waters sourcing EP Magmatic Hydrothermal Systems, Bakermans, C. (ed). shifted back to anoxic liquid phase waters (HO). The deliv- Boston, MA: De Gruyter. ery of bicarbonate through HO waters likely neutralized the Amend, J.P., and Shock, E.L. (2001) Energetics of overall hyperacidic spring, resulting in the moderately acidic, metabolic reactions of thermophilic and hyperthermophilic anoxic spring, while maintaining the sulphur that was Archaea and bacteria. FEMS Microbiol Rev 25: 175–243. deposited during its hyperacidic phase. Anantharaman, K., Hausmann, B., Jungbluth, S.P., This dynamic geological legacy led to the development of Kantor, R.S., Lavy, A., Warren, L.A., et al. (2018) Expanded diversity of microbial groups that shape the dis- the unusual chemistry that selected for the unique archaeal similatory sulfur cycle. ISME J 12: 1715–1728. dominated, anaerobic, sulphur-dependent community that Ball, J.W., Nordstrom, D.K., Jenne, E.A., and Vivit, D.V. inhabits EP today. It is possible–if not likely–that repeated (1998) Chemical analyses of hot springs, pools, geysers, seismic episodes, such as those described herein, have and surface waters from Yellowstone National Park, Wyo- occurred at EP over its geological history and prior to scien- ming, and vicinity, 1974-1975. In United States Geological tific observation. Such episodes would be expected to also Survey Open File Report 98-182. have influenced the generation of unique chemical composi- Ball, J.W., Nordstrom, D.K., McCleskey, R.B., Schoonen, M. A.A., and Xu, Y. (2001) Water-chemistry and on-site sulfur tions that in turn could have influenced the assembly of EP speciation data for selected springs in Yellowstone communities and their unique biodiversity in ways similar to National Park, Wyoming, 1996–1998. In United States those described here. Considering that all environments are Geological Survey Open-File Report 01-49 geologically dynamic over varying timescales, their physical, Ball, J.W., McCleskey, R.B., Nordstrom, D.K., and chemical and biological properties should also change and Holloway, J.M. (2006) Water-chemistry data for selected create unique opportunities (open niches) that can promote springs, geysers, and streams in Yellowstone National diversification and influence the assembly of communities. Park, Wyoming, 2003-2005. In United States Geological Survey Open File Report 2006-1339. EP is an exceptionally dynamic system driven by seismic Beam, J.P., Jay, Z.J., Kozubal, M.A., and Inskeep, W.P. activity that allows for observation of these changes on a (2014) Niche specialization of novel Thaumarchaeota to timescale conducive to scientific study. The results reported oxic and hypoxic acidic geothermal springs of Yellowstone here implicate the need to consider the historical legacy of National Park. ISME J 8: 938–951. change in an environment when describing processes that Berghuis, B.A., Yu, F.B., Schulz, F., Blainey, P.C., shape the assembly of communities. Woyke, T., and Quake, S.R. (2019) Hydrogenotrophic methanogenesis in archaeal phylum Verstraetearchaeota reveals the shared ancestry of all methanogens. Proc Natl – Acknowledgements Acad Sci U S A 116: 5037 5044. Bohlool, B.B., and Brock, T.D. (1974) Population ecology of This work was supported by National Science Foundation Sulfolobus acidocaldarius. II. Immunoecolgical studies. grant EAR-1820658 to DRC and ESB. We thank Mark Jutila, Arch Microbiol 97: 181–194.

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