OPEN The ISME Journal (2018) 12, 225–236 www.nature.com/ismej ORIGINAL ARTICLE Geobiological feedbacks and the evolution of thermoacidophiles Daniel R Colman1, Saroj Poudel1, Trinity L Hamilton2, Jeff R Havig3, Matthew J Selensky1, Everett L Shock4,5,6 and Eric S Boyd1,6 1Department of Microbiology and Immunology, Montana State University, Bozeman, MT, USA; 2Department of Biological Sciences, University of Cincinnati, Cincinnati, OH, USA; 3Department of Geology, University of Cincinnati, Cincinnati, OH, USA; 4School of Molecular Sciences, Arizona State University, Tempe, AZ, USA; 5School of Earth and Space Exploration, Arizona State University, Tempe, AZ, USA and 6NASA Astrobiology Institute, Mountain View, CA, USA Oxygen-dependent microbial oxidation of sulfur compounds leads to the acidification of natural waters. How acidophiles and their acidic habitats evolved, however, is largely unknown. Using 16S rRNA gene abundance and composition data from 72 hot springs in Yellowstone National Park, Wyoming, we show that hyperacidic (pHo3.0) hydrothermal ecosystems are dominated by a limited number of archaeal lineages with an inferred ability to respire O2. Phylogenomic analyses of 584 existing archaeal genomes revealed that hyperacidophiles evolved independently multiple times within the Archaea, each coincident with the emergence of the ability to respire O2, and that these events likely occurred in the recent evolutionary past. Comparative genomic analyses indicated that archaeal thermoacidophiles from independent lineages are enriched in similar protein-coding genes, consistent with convergent evolution aided by horizontal gene transfer. Because the generation of acidic environments and their successful habitation characteristically require O2, these results suggest that thermoacidophilic Archaea and the acidity of their habitats co-evolved after the evolution of oxygenic photosynthesis. Moreover, it is likely that dissolved O2 concentrations in thermal waters likely did not reach levels capable of sustaining aerobic thermoacidophiles and their acidifying activity until ~ 0.8 Ga, when present day atmospheric levels were reached, a time period that is supported by our estimation of divergence times for archaeal thermoacidophilic clades. The ISME Journal (2018) 12, 225–236; doi:10.1038/ismej.2017.162; published online 13 October 2017 Introduction 2003). However, the events that led to the evolution o of acidophiles and their role in the generation of Hyperacidic environments (pH 3) are pervasive acidic habitats are underexplored. landscape features, albeit isolated, and are com- Two dominant processes are involved in the monly associated with volcanic systems or sulfide formation of hyperacidic natural waters: magmatic ore deposits. These environments define the lower degassing that contributes hydrochloric, hydrofluo- pH limit for life, are major sources of economically ric and sulfuric acid (from disproportionation of important metals and have the potential to contri- sulfur dioxide and subsequent oxidation of hydrogen bute to the pollution of natural waters (Johnson and sulfide or elemental sulfur) and the oxidation of Quatrini, 2016). As such, hyperacidic environments mineral sulfides that produces sulfuric acid are of substantial interest to microbiologists, geoche- (Nordstrom et al., 2000). Common to both processes mists, geologists and planetary scientists, and have are the oxidation of sulfur compounds, including been subject to extensive research to understand the free sulfide (H S), mineral sulfides (for example, processes that contribute to their formation and the 2 FeS ) or elemental sulfur (S0), and the concomitant adaptations that allow for their habitation. Consider- 2 able research has been conducted on the role of production of sulfate and protons. High-potential oxidants capable of driving sulfide/S0 oxidation microbial activity in the formation of hyperacidic − III waters (Johnson et al., 1993; Baker and Banfield, include nitrate (NO3), ferric iron ions (Fe ) and O2 (Falkowski et al., 2008). Mechanisms of generating − NO3 include lightning-catalyzed oxidation of atmo- Correspondence: ES Boyd, Department of Microbiology and spheric dinitrogen and precipitation of nitrogen Immunology, Montana State University, 109 Lewis Hall, PO Box − oxides as NO3, a process that has been decreasing 173520, Bozeman, MT 59717, USA. E-mail: [email protected] in importance since 3.8 Ga (Navarro-Gonzalez et al., Received 9 June 2017; revised 27 July 2017; accepted 28 August 2001), and biological oxidation of ammonia, which 2017; published online 13 October 2017 requires O2 (Godfrey and Falkowski, 2009). Evolution of thermoacidophiles DR Colman et al 226 Likewise, the generation of FeIII requires either springs in Yellowstone National Park (YNP), Wyom- abiological photo-oxidation of ferrous iron (FeII; ing, USA, to determine the abundance of taxonomic Braterman et al., 1983), which has been of negligible lineages found in thermal acidic springs. We then importance after the onset of an ozonated atmo- investigated the evolutionary history of thermoaci- sphere following the Great Oxidation Event (GOE) dophiles using phylogenomic analyses of publicly ~ 2.5 Ga (Kim et al., 2013), or one of several available genomes from cultured and uncultured biological oxidative processes (Emerson et al., taxa. To better understand the adaptations that led to II 2010). Biological oxidation of Fe requires O2 or increased acid tolerance, we used comparative − II NO3 as an oxidant (Emerson et al., 2010) or Fe - genomics to determine the protein complements oxidizing phototrophic organisms (Widdel et al., that differentiate thermoacidophiles from other 1993). The involvement of phototrophic organisms members of their higher-order lineages. Finally, we in FeIII generation in acidic environments is of present these results in context of changes in atmo- putative importance only in environments with spheric O2 over geologic time to evaluate the temperatures o56 °C—the upper temperature limit hypothesis that both thermoacidophiles and the for phototrophs in acidic (pHo4.0) environments habitats that they are putatively responsible for (Boyd et al., 2012). Thus, the remaining and most generating are recent evolutionary and geologic 0 likely oxidant capable of driving H2S/S oxidation innovations, respectively. and the development of hyperacidic hydrothermal ecosystems is atmospheric O2 rather than local O2, given temperature constraints on photosynthesis. Materials and methods Previous studies indicate that abiotic oxidation of H2S with O2 can be extremely slow in aqueous Spring temperature and pH were determined with a solutions with pH ⩽ 6 (Chen and Morris, 1972; Zhang portable pH meter and temperature-compensated and Millero, 1993; D’Imperio et al., 2008). Further, at probe (WTW 3300i or 3110; WTW, Weilheim, elevated pH (that is, in marine waters) where abiotic Germany) that was calibrated daily with standar- oxidation of sulfide is quicker, rates can be several dized buffer solutions. Hot spring sediments were orders of magnitude slower than biotic oxidation sampled from hot springs and DNA was extracted, (Luther et al., 2011), although biotic oxidation is not purified and quantified as previously described always necessary to explain observed rates (Millero, (Colman et al., 2016). Quantitative PCR quantifica- 2001). Further, the rate of abiotic oxidation of S0 with tion and bacterial/archaeal 16S rRNA gene amplifi- O2 is extremely slow (Nordstrom et al., 2005). cation and sequencing via the 454 Titanium platform Finally, the growth of several sulfur-oxidizing, were also performed as previously described aerobic thermoacidophiles results in the acidifica- (Hamilton et al., 2013). Raw untrimmed 454 tion of the cultivation medium (Shivvers and Brock, sequence data are deposited in the NCBI SRA 1973; Giaveno et al., 2013). These observations, database under SRA accession SRR3181942. Meta- coupled with evidence for the widespread distribu- data for aerobic respiration capability were inferred tion of aerobic organisms capable of oxidizing both from the nearest cultivated isolate (or genomic 0 H2S and S in acidic hot springs (Zillig et al., 1994; reference) and are provided in Supplementary Inskeep et al., 2013; Xie et al., 2014; Huber and Table S1. Statistical analyses of quantitative PCR Stetter, 2015) and the capability of sulfur-oxidizing data and community compositional differences in organisms to acidify their environments, strongly relation to geochemical parameters were conducted suggest that biological activity is involved in the in the base R package v. 3.2.0 (R Core Team, 2015). generation of many low-pH (o3.0) hydrothermal Available archaeal genomes were downloaded ecosystems (Nordstrom et al., 2005; Nordstrom et al., from the NCBI genome portal and cross-referenced 2009; Johnson and Quatrini, 2016). against the JGI Integrated Microbial Genomes data- Here, we hypothesize that O2-dependent biological base to ensure that representatives available in either oxidation of sulfur compounds in hydrothermal database were collected (Supplementary Table S2). ecosystems is dependent on a non-point source of Genome completeness of each downloaded genome O2 and that the widespread development of low-pH was estimated based on the presence of 104 single- (o3.0) hot spring ecosystems could only have copy archaeal-specific phylogenetic marker genes occurred after O2 began to accumulate in the atmo- using the Amphora2 software package (Wu and sphere to levels