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