promoting access to White Rose research papers Universities of Leeds, Sheffield and York http://eprints.whiterose.ac.uk/ This is a copy of the final published version of a paper published via gold open access in Journal of Phycology. This open access article is distributed under the terms of the Creative Commons Attribution Licence (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/78621 Published paper Graham, L.E, Knack, J.J, Piotrowski, M.J, Wilcox, L.W, Cook, M.E, Wellman, C.H, Taylor, W, Lewis, L.A and Arancibia-Avila, P (2014) Lacustrine Nostoc (Nostocales) and associated microbiome generate a new type of modern clotted microbialite. Journal of Phycology. Doi: 10.1111/jpy.12152 White Rose Research Online [email protected] J. Phycol. 50, 280–291 (2014) © 2013 The Authors Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. DOI: 10.1111/jpy.12152 LACUSTRINE NOSTOC (NOSTOCALES) AND ASSOCIATED MICROBIOME GENERATE A NEW TYPE OF MODERN CLOTTED MICROBIALITE1 Linda E. Graham,2 Jennifer J. Knack, Michael J. Piotrowski, Lee W. Wilcox Department of Botany, University of Wisconsin-Madison, 430 Lincoln Drive, Madison, Wisconsin 53606, USA Martha E. Cook School of Biological Sciences, Illinois State University, Normal, Illinois 61790, USA Charles H. Wellman Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK Wilson Taylor Department of Biology, University of Wisconsin-Eau Claire, Eau Claire, Wisconsin 54702, USA Louise A. Lewis Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, Connecticut 06269, USA and Patricia Arancibia-Avila Department of Basic Sciences, University of Bio-Bio, Chillan, Chile Microbialites are mineral formations formed by Desulfomicrobium and Sulfospirillum and genes microbial communities that are often dominated encoding all known proteins specific to sulfate by cyanobacteria. Carbonate microbialites, known reduction, a process known to facilitate carbonate from Proterozoic times through the present, are deposition by increasing pH. Sequences indicating recognized for sequestering globally significant presence of nostocalean and other types of nifH, amounts of inorganic carbon. Recent ecological work nostocalean sulfide:ferredoxin oxidoreductase (indi- has focused on microbial communities dominated by cating anoxygenic photosynthesis), and biosynthetic cyanobacteria that produce microbial mats and pathways for the secondary products scytonemin, laminate microbialites (stromatolites). However, the mycosporine, and microviridin toxin were identified. taxonomic composition and functions of microbial These results allow comparisons with microbiota communities that generate distinctive clotted and microbiomes of other algae and illuminate microbialites (thrombolites) are less well understood. biogeochemical roles of ancient microbialites. Here, microscopy and deep shotgun sequencing were Key index words: microbialite; microbiome; Nostoc; used to characterize the microbiome (microbial taxa sulfate reduction; X-ray spectroscopy and their genomes) associated with a single cyanobacterial host linked by 16S sequences to Nostoc Abbreviations: EDS, energy-dispersive X-ray spectros- commune Vaucher ex Bornet & Flahault, which copy; ML, maximum likelihood; ORF, open reading dominates abundant littoral clotted microbialites frame in shallow, subpolar, freshwater Laguna Larga in southern Chile. Microscopy and energy-dispersive X-ray spectroscopy suggested the hypothesis that adherent hollow carbonate spheres typical of the The involvement of prokaryotic and eukaryotic clotted microbialite begin development on the rigid algae in the formation of diverse types of sedimen- curved outer surfaces of the Nostoc balls. A surface tary carbonates is important to the sequestration of biofilm included >50 nonoxygenic bacterial genera inorganic carbon for very long periods of time (taxa other than Nostoc) that indicate diverse (Graham et al. 2009). Cyanobacteria are thought to ecological functions. The Laguna Larga Nostoc have generated the earliest algal carbonate deposits microbiome included the sulfate reducers in the form of layered stromatolites, clotted thromb- olites, and other formations collectively known as 1Received 11 July 2013. Accepted 24 October 2013. microbially induced sedimentary structures or 2Author for correspondence: e-mail [email protected]. microbialites (Burne and Moore 1987). Most micro- Editorial Responsibility: P. Gabrielson (Associate Editor) 280 NOSTOC MICROBIALITE MICROBIOME 281 bialite formations are largely composed of carbon- et al. 2013) might explain the later appearance of ates, although other mineral types occur (Riding clotted microbialites in the fossil record. Modern 2011). clotted microbialites are known from marine and Cyanobacterial photosynthesis plays a role in freshwater settings, and understanding their micro- microbialite formation by increasing ambient pH bial ecology would inform our understanding of (Garcia-Pichel et al. 2004), thereby fostering carbon- modern biogeochemical function as well as the fos- ate precipitation, and extracellular polymeric sub- sil record. stances produced by cyanobacteria and associated Previous SSU rDNA analyses of clotted microbia- bacteria are thought to aid carbonate formation by lites (Airo 2010, Myshrall et al. 2010) indicated the binding Ca2+ (Van Lith et al. 2003, Braissant et al. presence of diverse cyanobacterial components. 2007). Sulfate reducing bacteria may also be crucial Although vertically oriented, tapered filaments of to the formation of microbialites, because sulfate heterocytous cyanobacteria identified as Dichothrix reduction fosters carbonate formation by increasing dominate button-size clotted microbialites in shallow local pH and carbonate alkalinity (Dupraz and marine systems (Highbourne Cay, Bahamas), 15 Visscher 2005, Baumgartner et al. 2006). The micro- additional diverse cyanobacterial OTUs also occur biomes, metagenomes, and other features of diverse (as do nonoxygenic Actinobacteria, Bacteroidetes, modern cyanobacteria-dominated mats and micro- Chloroflexi, Firmicutes, Planctomycetes, Alphaprote- bialites are being studied as model systems expected obacteria, Betaproteobacteria, Deltaproteobacteria, to yield insights into the biotic interactions and for- Gammaproteobacteria, Spirochaetes, and Verrucomi- mation of modern and ancient microbialites (Laval crobia; Myshrall et al. 2010). In a study of freshwater et al. 2000, Arp et al. 2001, Sheehan and Harris clotted microbialites, hemispherical colonies of verti- 2004, Breitbart et al. 2009, Couradeau et al. 2011, cally oriented, tapered, false-branching filaments Harris et al. 2012, Oliver and Rowland 2002). For having basal heterocytes, identified as the example, 16S amplicon sequencing of microbial cyanobacterial genus Rivularia, were reported as the mats in hypersaline salterns at Guerrero Negro prominent components of clotted microbialite com- (Mexico) revealed very high bacterial diversity (752 munities in Lago Sarmiento in southern Chile, species identifiable at the 97% level, in 42 phyla, 15 although more than 60 additional cyanobacterial of those new to science). In this model system the species were detected by SSU rDNA (in addition to filamentous, nonheterocytous (nonheterocystous) Proteobacteria, Firmicutes, Bacteroidetes, and Ver- genus Microcoleus (= Coleofasciculus; Siegesmund rucomicrobia; Airo 2010). The complexity of such cy- et al. 2008) was the dominant cyanobacterial com- anobacterial communities challenges efforts to ponent (Ley et al. 2006), as is the case for most understand how particular cyanobacterial species marine mats (Green and Jahnke 2010). might influence microbialite structure and func- Some modern freshwater lakes display large mi- tional interactions between the oxygenic photosyn- crobialite formations that are likewise regarded as thesizers and associated microbiota. While eukaryotic model systems for decoding the fossil record. 16S algae are known to provide diverse communities of clone library analysis revealed that mats whose epibiontic microbes with substratum, oxygen, and cyanobacterial components were mostly Pleurocapsa- organic exudates (Amin et al. 2012, Zulkifly et al. like coccoid forms promote carbonate precipitation 2012) and to receive growth-enhancing vitamins to form giant cone-shaped microbialites in Lake from bacterial associates (Croft et al. 2005), interac- Van, Turkey (Kempe et al. 1991, Lopez-Garcia et al. tions among bacterial epibionts and individual cy- 2005). Calothrix, a heterocytous cyanobacterial anobacterial host species are poorly understood. genus, and unidentified nonheterocytous filamen- Here, we report the results of correlative micros- tous cyanobacteria are associated with the formation copy, energy-dispersive X-ray spectros-copy (EDS), of structurally similar microbialites in freshwater and shotgun pyrosequencing analyses of a simpler Pavilion Lake, British Columbia, Canada (Schulze- microbialite system in which a single cyanobacterial