Microbial communities and organic biomarkers in a Proterozoic-analog sinkhole The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Hamilton, T. L. et al “Microbial Communities and Organic Biomarkers in a Proterozoic-Analog Sinkhole.” Geobiology 15, 6 (October 2017): 784–797 © 2017 The Authors As Published http://dx.doi.org/10.1111/GBI.12252 Publisher Wiley Blackwell Version Final published version Citable link http://hdl.handle.net/1721.1/118593 Terms of Use Creative Commons Attribution 4.0 International License Detailed Terms http://creativecommons.org/licenses/by/4.0/ Received: 26 September 2016 | Accepted: 07 July 2017 DOI: 10.1111/gbi.12252 ORIGINAL ARTICLE Microbial communities and organic biomarkers in a Proterozoic- analog sinkhole T. L. Hamilton1,2 | P. V. Welander3 | H. L. Albrecht4 | J. M. Fulton5 | I. Schaperdoth4 | L. R. Bird4 | R. E. Summons6 | K. H. Freeman4 | J. L. Macalady4 1Department of Biological Sciences, University of Cincinnati, Cincinnati, OH, USA Abstract 2Department of Plant and Microbial Biology, Little Salt Spring (Sarasota County, FL, USA) is a sinkhole with groundwater vents at University of Minnesota, St. Paul, MN, USA ~77 m depth. The entire water column experiences sulfidic (~50 μM) conditions sea- 3Department of Earth System Science, sonally, resulting in a system poised between oxic and sulfidic conditions. Red pinnacle Stanford University, Stanford, CA, USA 4Department of Geosciences and the mats occupy the sediment–water interface in the sunlit upper basin of the sinkhole, Penn State Astrobiology Research Center and yielded 16S rRNA gene clones affiliated with Cyanobacteria, Chlorobi, and sulfate- (PSARC), The Pennsylvania State University, University Park, PA, USA reducing clades of Deltaproteobacteria. Nine bacteriochlorophyll e homologues and 5Department of Geosciences, Baylor isorenieratene indicate contributions from Chlorobi, and abundant chlorophyll a and University, Waco, TX, USA pheophytin a are consistent with the presence of Cyanobacteria. The red pinnacle mat 6 Department of Earth, Atmospheric and contains hopanoids, including 2- methyl structures that have been interpreted as bio- Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA markers for Cyanobacteria. A single sequence of hpnP, the gene required for methyla- tion of hopanoids at the C- 2 position, was recovered in both DNA and cDNA libraries Correspondence T. L. Hamilton, Department of Plant and from the red pinnacle mat. The hpnP sequence was most closely related to cyanobac- Microbial Biology, University of Minnesota, terial hpnP sequences, implying that Cyanobacteria are a source of 2- methyl hopa- St. Paul, MN, USA Email: [email protected] noids present in the mat. The mats are capable of light- dependent primary productivity and as evidenced by 13C- bicarbonate photoassimilation. We also observed 13C- bicarbonate J. L. Macalady, Department of Geosciences and the Penn State Astrobiology Research photoassimilation in the presence of DCMU, an inhibitor of electron transfer to Center (PSARC), The Pennsylvania State Photosystem II. Our results indicate that the mats carry out light- driven primary pro- University, University Park, PA, USA. Email: [email protected] duction in the absence of oxygen production—a mechanism that may have delayed the oxygenation of the Earth’s oceans and atmosphere during the Proterozoic Eon. Funding information National Science Foundation, Grant/ Furthermore, our observations of the production of 2- methyl hopanoids by Award Number: NSF EAR-0525503; NASA Cyanobacteria under conditions of low oxygen and low light are consistent with the Astrobiology Institute, Grant/Award Number: NNA04CC06A and NNA13AA90A recovery of these structures from ancient black shales as well as their paucity in mod- ern marine environments. 1 | INTRODUCTION present- day levels. The prevailing view suggests that during the Proterozoic, ~2–0.5 billion years ago, atmospheric oxygen levels were A growing body of evidence indicates a substantial increase in within 1–10% of present- day levels (Kump, 2008). Although recent atmospheric oxygen ~2.5 billion years ago. However, following the ini- research has revealed some complexity in the evolution of the global tial rapid rise of oxygen, a prolonged delay in the further rise of oxygen oceans during the Proterozoic, substantial evidence from marine sed- lasted until ~500 million years ago, when atmospheric oxygen reached imentary deposits indicate that oxygen- poor and sulfidic conditions This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2017 The Authors. Geobiology Published by John Wiley & Sons Ltd. 784 | wileyonlinelibrary.com/journal/gbi Geobiology. 2017;15:784–797. HAMILTON ET AL. | 785 have been an intermittent feature of the global oceans since the ini- in Earth’s evolution. The signal of 2- MeBHPs in the rock record may tial rise of oxygen (Canfield, 2005). Euxinic conditions have accompa- provide additional insights. nied remarkable events in Earth history including several Phanerozoic Little Salt Spring is a cover- collapse, karst- solution sinkhole lo- crises (Meyer & Kump, 2008). Sulfidic, anoxic environments are still cated 14 miles from the Gulf of Mexico in Sarasota, FL. This sinkhole widespread today although they tend to be confined to basins with has proven to be a valuable archaeological site because its anoxic bot- highly restricted circulation. They feature isotopic, mineralogical, or- tom waters preserve artifacts from some of North America’s earliest ganic, and morphological biosignatures that have the potential to be human populations (Alvarez Zarikian, Swart, Gifford, & Blackwelder, preserved in the geologic record, thereby enhancing our ability to in- 2005; Clausen, Brooks, & Weslowsky, 1975; Clausen, Cohen, Emiliani, terpret the presence or absence of the same biosignatures in earth’s Holman, & Stipp, 1979; Haynes, 2009; Peres & Simons, 2006). Data geologic record or on other planets. presented here and elsewhere (Alvarez Zarikian et al., 2005) indicate Hydrocarbon biomarkers derived from biologic lipid membranes the geochemical attributes of the sinkhole water are similar to those have historically played an important role in efforts to reconstruct thought to be common in the Proterozoic oceans, poised between events and environmental conditions in the deep past, including the oxic and sulfidic conditions. The microbial diversity of Little Salt Spring evolution of Cyanobacteria and oxygenic photosynthesis (Brocks, has not been investigated previously. Here, we report the discovery Buick, Summons, & Logan, 2003; Summons, Jahnke, Hope, & Logan, of conspicuous populations of microbial photoautotrophs that form a 1999). Hopanes, the diagenetic product of hopanoids (membrane lip- red pinnacle mat covering the sediment–water interface in the upper ids produced by numerous varieties of bacteria), are preserved in both basin. The primary populations in the mat are Cyanobacteria (oxygenic modern sediments and ancient sedimentary rocks, and are the most phototrophs) and green sulfur bacteria, Chlorobium spp. (anoxygenic abundant molecular fossils in the rock record (Brocks & Pearson, 2005; phototrophs). The mat contains multiple 2- methyl hopanoids and the Ourisson & Albrecht, 1992). Methylation of hopanoid A- rings is re- only hpnP gene recovered from the mat is affiliated with Cyanobacteria. tained upon diagenesis (Summons & Jahnke, 1992) and can have tax- Thus, Little Salt Spring provides an extraordinary opportunity to in- onomic relevance. For instance, C- 3 methylated hopanes are thought vestigate the factors affecting competition among oxygenic and an- to be diagnostic of microaerobic methanotrophs and acetic acid bac- oxygenic phototrophs and how these interactions may have affected teria (Zundel & Rohmer, 1985). Hopanoids modified by methylation at the oxygen budget throughout the Proterozoic Eon. Furthermore, the the C- 2 position, or 2- methyl hopanoids, have been recovered from recovery of 2- methyl hopanoids derived from Cyanobacteria in a low modern environments as well as from ancient sedimentary rocks and oxygen, sulfidic environment is consistent with both a cyanobacterial were long considered to be diagnostic of Cyanobacteria, and thus origin of these structures in ancient black shales and the paucity of a proxy for oxygenic photosynthesis in ancient sedimentary rocks 2- methyl hopanoids in modern marine sediments. (Summons et al., 1999). However, the discovery of their precursors, the 2- methylbacteriohopanepolyols (2- MeBHPs) in members of the 2 | MATERIALS AND METHODS Alphaproteobacteria (Bisseret, Zundel, & Rohmer, 1985; Bravo, Perzl, Hartner, Kannenberg, & Rohmer, 2001; Rashby, Sessions, Summons, & 2.1 | Site description, sample collection, and Newman, 2007; Renoux & Rohmer, 1985; Sahm, Rohmer, Bringermeyer, geochemistry Sprenger, & Welle, 1993) highlights the challenge associated with eval- uating the significance of 2- methylhopanes in sedimentary rocks. Little Salt Spring (Fig. S1) is a 78 meter diameter sinkhole lake located The function of 2- MeBHPs remains unknown, as well as the con- in Sarasota County, FL (lat. 27°04′30″N, long. 82°14′00″W). The ditions favoring the production of these lipids. These knowledge gaps geology and hydrology of the spring have been described previously