amoA‐based consensus phylogeny of ammonia‐oxidizing archaea and deep sequencing of amoA genes from soils of four different geographic regions
Identifieur interne : 000622 ( Istex/Corpus ); précédent : 000621; suivant : 000623amoA‐based consensus phylogeny of ammonia‐oxidizing archaea and deep sequencing of amoA genes from soils of four different geographic regions
Auteurs : Michael Pester ; Thomas Rattei ; Stefan Flechl ; Alexander Gröngröft ; Andreas Richter ; Jörg Overmann ; Barbara Reinhold-Hurek ; Alexander Loy ; Michael WagnerSource :
- Environmental Microbiology [ 1462-2912 ] ; 2012-02.
Abstract
Ammonia‐oxidizing archaea (AOA) play an important role in nitrification and many studies exploit their amoA genes as marker for their diversity and abundance. We present an archaeal amoA consensus phylogeny based on all publicly available sequences (status June 2010) and provide evidence for the diversification of AOA into four previously recognized clusters and one newly identified major cluster. These clusters, for which we suggest a new nomenclature, harboured 83 AOA species‐level OTU (using an inferred species threshold of 85% amoA identity). 454 pyrosequencing of amoA amplicons from 16 soils sampled in Austria, Costa Rica, Greenland and Namibia revealed that only 2% of retrieved sequences had no database representative on the species‐level and represented 30–37 additional species‐level OTUs. With the exception of an acidic soil from which mostly amoA amplicons of the Nitrosotalea cluster were retrieved, all soils were dominated by amoA amplicons from the Nitrososphaera cluster (also called group I.1b), indicating that the previously reported AOA from the Nitrosopumilus cluster (also called group I.1a) are absent or represent minor populations in soils. AOA richness estimates on the species level ranged from 8–83 co‐existing AOAs per soil. Presence/absence of amoA OTUs (97% identity level) correlated with geographic location, indicating that besides contemporary environmental conditions also dispersal limitation across different continents and/or historical environmental conditions might influence AOA biogeography in soils.
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DOI: 10.1111/j.1462-2920.2011.02666.x
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sort="Grongroft, Alexander" uniqKey="Grongroft A" first="Alexander" last="Gröngröft">Alexander Gröngröft</name><affiliation><mods:affiliation>Institute of Soil Science, University of Hamburg, Allende‐Platz 2, D‐20146 Hamburg, Germany.</mods:affiliation></affiliation></author><author><name sortKey="Richter, Andreas" sort="Richter, Andreas" uniqKey="Richter A" first="Andreas" last="Richter">Andreas Richter</name><affiliation><mods:affiliation>Chemical Ecology and Ecosystem Research, University of Vienna, Althanstrasse 14, A‐1090 Vienna, Austria.</mods:affiliation></affiliation></author><author><name sortKey="Overmann, Jorg" sort="Overmann, Jorg" uniqKey="Overmann J" first="Jörg" last="Overmann">Jörg Overmann</name><affiliation><mods:affiliation>Leibniz‐Institut DSMZ‐Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstraße 7B, D‐38124 Braunschweig, Germany.</mods:affiliation></affiliation></author><author><name sortKey="Reinhold Urek, Barbara" sort="Reinhold Urek, Barbara" 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Flechl</name><affiliation><mods:affiliation>Departments of Microbial Ecology,</mods:affiliation></affiliation></author><author><name sortKey="Grongroft, Alexander" sort="Grongroft, Alexander" uniqKey="Grongroft A" first="Alexander" last="Gröngröft">Alexander Gröngröft</name><affiliation><mods:affiliation>Institute of Soil Science, University of Hamburg, Allende‐Platz 2, D‐20146 Hamburg, Germany.</mods:affiliation></affiliation></author><author><name sortKey="Richter, Andreas" sort="Richter, Andreas" uniqKey="Richter A" first="Andreas" last="Richter">Andreas Richter</name><affiliation><mods:affiliation>Chemical Ecology and Ecosystem Research, University of Vienna, Althanstrasse 14, A‐1090 Vienna, Austria.</mods:affiliation></affiliation></author><author><name sortKey="Overmann, Jorg" sort="Overmann, Jorg" uniqKey="Overmann J" first="Jörg" last="Overmann">Jörg Overmann</name><affiliation><mods:affiliation>Leibniz‐Institut DSMZ‐Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstraße 7B, D‐38124 Braunschweig, Germany.</mods:affiliation></affiliation></author><author><name sortKey="Reinhold Urek, Barbara" sort="Reinhold Urek, Barbara" uniqKey="Reinhold Urek B" first="Barbara" last="Reinhold-Hurek">Barbara Reinhold-Hurek</name><affiliation><mods:affiliation>Department of Microbe‐Plant Interactions, University of Bremen, Postfach 330440, D‐28334 Bremen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Loy, Alexander" sort="Loy, Alexander" uniqKey="Loy A" first="Alexander" last="Loy">Alexander Loy</name><affiliation><mods:affiliation>Departments of Microbial Ecology,</mods:affiliation></affiliation></author><author><name sortKey="Wagner, Michael" sort="Wagner, Michael" uniqKey="Wagner M" first="Michael" last="Wagner">Michael Wagner</name><affiliation><mods:affiliation>Departments of Microbial Ecology,</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Environmental Microbiology</title><idno type="ISSN">1462-2912</idno><idno type="eISSN">1462-2920</idno><imprint><publisher>Blackwell Publishing Ltd</publisher><pubPlace>Oxford, UK</pubPlace><date type="published" when="2012-02">2012-02</date><biblScope unit="volume">14</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="525">525</biblScope><biblScope unit="page" to="539">539</biblScope></imprint><idno type="ISSN">1462-2912</idno></series><idno type="istex">071C3FCCD46A4C83B4AA567C8B28364AD9990C77</idno><idno type="DOI">10.1111/j.1462-2920.2011.02666.x</idno><idno type="ArticleID">EMI2666</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1462-2912</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Ammonia‐oxidizing archaea (AOA) play an important role in nitrification and many studies exploit their amoA genes as marker for their diversity and abundance. We present an archaeal amoA consensus phylogeny based on all publicly available sequences (status June 2010) and provide evidence for the diversification of AOA into four previously recognized clusters and one newly identified major cluster. These clusters, for which we suggest a new nomenclature, harboured 83 AOA species‐level OTU (using an inferred species threshold of 85% amoA identity). 454 pyrosequencing of amoA amplicons from 16 soils sampled in Austria, Costa Rica, Greenland and Namibia revealed that only 2% of retrieved sequences had no database representative on the species‐level and represented 30–37 additional species‐level OTUs. With the exception of an acidic soil from which mostly amoA amplicons of the Nitrosotalea cluster were retrieved, all soils were dominated by amoA amplicons from the Nitrososphaera cluster (also called group I.1b), indicating that the previously reported AOA from the Nitrosopumilus cluster (also called group I.1a) are absent or represent minor populations in soils. AOA richness estimates on the species level ranged from 8–83 co‐existing AOAs per soil. Presence/absence of amoA OTUs (97% identity level) correlated with geographic location, indicating that besides contemporary environmental conditions also dispersal limitation across different continents and/or historical environmental conditions might influence AOA biogeography in soils.</div></front></TEI><istex><corpusName>wiley</corpusName><author><json:item><name>Michael Pester</name><affiliations><json:string>Departments of Microbial Ecology,</json:string></affiliations></json:item><json:item><name>Thomas Rattei</name><affiliations><json:string>Computational Systems Biology</json:string></affiliations></json:item><json:item><name>Stefan Flechl</name><affiliations><json:string>Departments of Microbial Ecology,</json:string></affiliations></json:item><json:item><name>Alexander Gröngröft</name><affiliations><json:string>Institute of Soil Science, University of Hamburg, Allende‐Platz 2, D‐20146 Hamburg, Germany.</json:string></affiliations></json:item><json:item><name>Andreas Richter</name><affiliations><json:string>Chemical Ecology and Ecosystem Research, University of Vienna, Althanstrasse 14, A‐1090 Vienna, Austria.</json:string></affiliations></json:item><json:item><name>Jörg Overmann</name><affiliations><json:string>Leibniz‐Institut DSMZ‐Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstraße 7B, D‐38124 Braunschweig, Germany.</json:string></affiliations></json:item><json:item><name>Barbara Reinhold‐Hurek</name><affiliations><json:string>Department of Microbe‐Plant Interactions, University of Bremen, Postfach 330440, D‐28334 Bremen, Germany</json:string></affiliations></json:item><json:item><name>Alexander Loy</name><affiliations><json:string>Departments of Microbial Ecology,</json:string></affiliations></json:item><json:item><name>Michael Wagner</name><affiliations><json:string>Departments of Microbial Ecology,</json:string></affiliations></json:item></author><articleId><json:string>EMI2666</json:string></articleId><language><json:string>eng</json:string></language><originalGenre><json:string>article</json:string></originalGenre><abstract>Ammonia‐oxidizing archaea (AOA) play an important role in nitrification and many studies exploit their amoA genes as marker for their diversity and abundance. We present an archaeal amoA consensus phylogeny based on all publicly available sequences (status June 2010) and provide evidence for the diversification of AOA into four previously recognized clusters and one newly identified major cluster. These clusters, for which we suggest a new nomenclature, harboured 83 AOA species‐level OTU (using an inferred species threshold of 85% amoA identity). 454 pyrosequencing of amoA amplicons from 16 soils sampled in Austria, Costa Rica, Greenland and Namibia revealed that only 2% of retrieved sequences had no database representative on the species‐level and represented 30–37 additional species‐level OTUs. With the exception of an acidic soil from which mostly amoA amplicons of the Nitrosotalea cluster were retrieved, all soils were dominated by amoA amplicons from the Nitrososphaera cluster (also called group I.1b), indicating that the previously reported AOA from the Nitrosopumilus cluster (also called group I.1a) are absent or represent minor populations in soils. AOA richness estimates on the species level ranged from 8–83 co‐existing AOAs per soil. 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(+43) 1 4277 54390; Fax (+43) 1 4277 54389.</p></note><affiliation>Departments of Microbial Ecology,</affiliation></author></analytic><monogr><title level="j">Environmental Microbiology</title><idno type="pISSN">1462-2912</idno><idno type="eISSN">1462-2920</idno><idno type="DOI">10.1111/(ISSN)1462-2920</idno><imprint><publisher>Blackwell Publishing Ltd</publisher><pubPlace>Oxford, UK</pubPlace><date type="published" when="2012-02"></date><biblScope unit="volume">14</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="525">525</biblScope><biblScope unit="page" to="539">539</biblScope></imprint></monogr><idno type="istex">071C3FCCD46A4C83B4AA567C8B28364AD9990C77</idno><idno type="DOI">10.1111/j.1462-2920.2011.02666.x</idno><idno type="ArticleID">EMI2666</idno></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2012</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Ammonia‐oxidizing archaea (AOA) play an important role in nitrification and many studies exploit their amoA genes as marker for their diversity and abundance. We present an archaeal amoA consensus phylogeny based on all publicly available sequences (status June 2010) and provide evidence for the diversification of AOA into four previously recognized clusters and one newly identified major cluster. These clusters, for which we suggest a new nomenclature, harboured 83 AOA species‐level OTU (using an inferred species threshold of 85% amoA identity). 454 pyrosequencing of amoA amplicons from 16 soils sampled in Austria, Costa Rica, Greenland and Namibia revealed that only 2% of retrieved sequences had no database representative on the species‐level and represented 30–37 additional species‐level OTUs. With the exception of an acidic soil from which mostly amoA amplicons of the Nitrosotalea cluster were retrieved, all soils were dominated by amoA amplicons from the Nitrososphaera cluster (also called group I.1b), indicating that the previously reported AOA from the Nitrosopumilus cluster (also called group I.1a) are absent or represent minor populations in soils. AOA richness estimates on the species level ranged from 8–83 co‐existing AOAs per soil. Presence/absence of amoA OTUs (97% identity level) correlated with geographic location, indicating that besides contemporary environmental conditions also dispersal limitation across different continents and/or historical environmental conditions might influence AOA biogeography in soils.</p></abstract></profileDesc><revisionDesc><change when="2012-02">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><original>false</original><mimetype>text/plain</mimetype><extension>txt</extension><uri>https://api.istex.fr/document/071C3FCCD46A4C83B4AA567C8B28364AD9990C77/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Wiley, elements deleted: body"><istex:xmlDeclaration>version="1.0" encoding="UTF-8" standalone="yes"</istex:xmlDeclaration><istex:document><component version="2.0" type="serialArticle" xml:lang="en"><header><publicationMeta level="product"><publisherInfo><publisherName>Blackwell Publishing Ltd</publisherName><publisherLoc>Oxford, UK</publisherLoc></publisherInfo><doi origin="wiley" registered="yes">10.1111/(ISSN)1462-2920</doi><issn type="print">1462-2912</issn><issn type="electronic">1462-2920</issn><idGroup><id type="product" value="EMI"></id><id type="publisherDivision" value="ST"></id></idGroup><titleGroup><title type="main" sort="ENVIRONMENTAL MICROBIOLOGY">Environmental Microbiology</title></titleGroup></publicationMeta><publicationMeta level="part" position="02102"><doi origin="wiley">10.1111/emi.2012.14.issue-2</doi><titleGroup><title type="specialIssueTitle">Taxonomy and Biodiversity</title></titleGroup><numberingGroup><numbering type="journalVolume" number="14">14</numbering><numbering type="journalIssue">2</numbering></numberingGroup><coverDate startDate="2012-02">February 2012</coverDate></publicationMeta><publicationMeta level="unit" type="article" position="19" status="forIssue" accessType="open"><doi origin="wiley">10.1111/j.1462-2920.2011.02666.x</doi><idGroup><id type="unit" value="EMI2666"></id></idGroup><countGroup><count type="pageTotal" number="15"></count></countGroup><titleGroup><title type="tocHeading1">Research articles</title></titleGroup><copyright>© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd</copyright><eventGroup><event type="xmlConverted" agent="Converter:BPG_TO_WML3G version:3.1.3 standalone mode:FullText" date="2012-04-17"></event><event type="publishedOnlineEarlyUnpaginated" date="2011-12-05"></event><event type="publishedOnlineFinalForm" date="2012-02-03"></event><event type="firstOnline" date="2011-12-05"></event><event type="xmlConverted" agent="Converter:WILEY_ML3G_TO_WILEY_ML3GV2 version:3.8.8" date="2014-01-24"></event><event type="xmlConverted" agent="Converter:WML3G_To_WML3G version:4.3.4 mode:FullText" date="2015-02-25"></event></eventGroup><numberingGroup><numbering type="pageFirst" number="525">525</numbering><numbering type="pageLast" number="539">539</numbering></numberingGroup><correspondenceTo> E‐mail <email normalForm="wagner@microbial-ecology.net">wagner@microbial‐ecology.net</email>; Tel. (+43) 1 4277 54390; Fax (+43) 1 4277 54389. </correspondenceTo><linkGroup><link type="toTypesetVersion" href="file:EMI.EMI2666.pdf"></link></linkGroup></publicationMeta><contentMeta><unparsedEditorialHistory>Received 19 October, 2011; revised 7 November, 2011; accepted 7 November, 2011.</unparsedEditorialHistory><countGroup><count type="figureTotal" number="4"></count><count type="tableTotal" number="1"></count><count type="formulaTotal" number="0"></count><count type="referenceTotal" number="72"></count><count type="wordTotal" number="10087"></count><count type="linksPubMed" number="0"></count><count type="linksCrossRef" number="0"></count></countGroup><titleGroup><title type="main"><i>amoA</i>‐based consensus phylogeny of ammonia‐oxidizing archaea and deep sequencing of <i>amoA</i> genes from soils of four different geographic regions</title><title type="shortAuthors">M. Pester <i>et al</i>.</title><title type="short">Archaeal <i>amoA</i> phylogeny and diversity in soils</title></titleGroup><creators><creator creatorRole="author" xml:id="cr1" affiliationRef="#a1"><personName><givenNames>Michael</givenNames><familyName>Pester</familyName></personName></creator><creator creatorRole="author" xml:id="cr2" affiliationRef="#a2"><personName><givenNames>Thomas</givenNames><familyName>Rattei</familyName></personName></creator><creator creatorRole="author" xml:id="cr3" affiliationRef="#a1"><personName><givenNames>Stefan</givenNames><familyName>Flechl</familyName></personName></creator><creator creatorRole="author" xml:id="cr4" affiliationRef="#a4"><personName><givenNames>Alexander</givenNames><familyName>Gröngröft</familyName></personName></creator><creator creatorRole="author" xml:id="cr5" affiliationRef="#a3"><personName><givenNames>Andreas</givenNames><familyName>Richter</familyName></personName></creator><creator creatorRole="author" xml:id="cr6" affiliationRef="#a5"><personName><givenNames>Jörg</givenNames><familyName>Overmann</familyName></personName></creator><creator creatorRole="author" xml:id="cr7" affiliationRef="#a6"><personName><givenNames>Barbara</givenNames><familyName>Reinhold‐Hurek</familyName></personName></creator><creator creatorRole="author" xml:id="cr8" affiliationRef="#a1"><personName><givenNames>Alexander</givenNames><familyName>Loy</familyName></personName></creator><creator creatorRole="author" xml:id="cr9" affiliationRef="#a1" corresponding="yes"><personName><givenNames>Michael</givenNames><familyName>Wagner</familyName></personName></creator></creators><affiliationGroup><affiliation xml:id="a1"><unparsedAffiliation>Departments of Microbial Ecology,</unparsedAffiliation></affiliation><affiliation xml:id="a2"><unparsedAffiliation>Computational Systems Biology</unparsedAffiliation></affiliation><affiliation xml:id="a3" countryCode="AT"><unparsedAffiliation>Chemical Ecology and Ecosystem Research, University of Vienna, Althanstrasse 14, A‐1090 Vienna, Austria.</unparsedAffiliation></affiliation><affiliation xml:id="a4" countryCode="DE"><unparsedAffiliation>Institute of Soil Science, University of Hamburg, Allende‐Platz 2, D‐20146 Hamburg, Germany.</unparsedAffiliation></affiliation><affiliation xml:id="a5" countryCode="DE"><unparsedAffiliation>Leibniz‐Institut DSMZ‐Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstraße 7B, D‐38124 Braunschweig, Germany.</unparsedAffiliation></affiliation><affiliation xml:id="a6" countryCode="DE"><unparsedAffiliation>Department of Microbe‐Plant Interactions, University of Bremen, Postfach 330440, D‐28334 Bremen, Germany</unparsedAffiliation></affiliation></affiliationGroup><supportingInformation><p><b>Fig. S1.</b> Consensus tree illustrating the diversification of the <i>Nitrosopumilus</i> cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal <i>amoA</i> at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. <i>Nitrosopumilus</i> subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. <i>Nitrosopumilus</i> subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence.</p><p><b>Fig. S2.</b> Rarefaction analysis of forward and reverse sequenced <i>amoA</i> at the species level cut‐off of 85% <i>amoA</i> identity.</p><p><b>Fig. S3.</b> Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% <i>amoA</i> identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a <i>P</i>‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke <i>et al</i>., 2005; Hatzenpichler <i>et al</i>., 2008; de la Torre <i>et al</i>., 2008; Erguder <i>et al</i>., 2009; Martens‐Habbena <i>et al</i>., 2009; Di <i>et al</i>., 2010; Pratscher <i>et al</i>., 2011; Verhamme <i>et al</i>., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam <i>et al</i>., 2006; Jia and Conrad, 2009; Mußmann <i>et al</i>., 2011; Tourna <i>et al</i>., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber <i>et al</i>., 2009), with a maximum of species at slightly acidic pH (pH = 6).</p><p><b>Fig. S4.</b> Abundance plot showing sequence identities of soil archaeal <i>amoA</i> retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% <i>amoA</i> sequence identity is indicated by a dotted line.</p><p><b>Fig. S5.</b> <i>In silico</i> specificity analysis of the archaeal <i>amoA</i> primers used in this study against all archaeal <i>amoA</i> sequences covering the primer target regions. The primer regions of <i>Candidatus</i> Nitrosotalea devanaterra, <i>Candidatus</i> Nitrosocaldus yellowstonii and <i>Candidatus</i> Nitrosotenuis uzonensis (affiliated to <i>Nitrosopumilus</i> subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively.</p><p><b>Fig. S6.</b> Analysis of novel <i>amoA</i> that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% <i>amoA</i> identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin <i>et al</i>., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel <i>amoA</i> using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel <i>amoA</i> OTUs within the <i>amoA</i> consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig <i>et al</i>., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel <i>amoA</i> OTU representatives were added manually to the archaeal <i>amoA</i> consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences).</p><p><b>Fig. S7.</b> Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed <i>amoA</i> OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown).</p><p><b>Table S1.</b> Highest nucleic acid sequence identity between representing sequences of the major archaeal <i>amoA</i> clusters. Presented identities were not corrected by substitution models.</p><p><b>Table S2.</b> Soil samples and determined soil parameters.</p><p><b>Table S3.</b> Sequencing results and number of observed and estimated OTUs at the species level (85% <i>amoA</i> identity).</p><p><b>Table S4.</b> Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 <i>amoA</i> sequences per soil sample.</p><p><b>Table S5.</b> Number of indicator OTUs (97% <i>amoA</i> identity) in different <i>amoA</i> lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight).</p><p><b>Table S6.</b> Representing sequences and total retrieved sequences reads of novel archaeal <i>amoA</i> OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples.</p><p><b>Table S7.</b> Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils.</p><p><b>Table S8.</b> OTU classification of <i>amoA</i> sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given.</p><p><b>File S1.</b> Archaeal <i>amoA</i> ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal <i>amoA</i> genes by June 2010.</p><p><b>Appendix S1.</b> Supporting methods.</p><supportingInfoItem><mediaResource alt="supporting info item" href="urn-x:wiley:14622912:media:emi2666:EMI_2666_sm_archaeal-amoA-DB-for-publication"></mediaResource><caption>Supporting info item</caption></supportingInfoItem><supportingInfoItem><mediaResource alt="supporting info item" href="urn-x:wiley:14622912:media:emi2666:EMI_2666_sm_FigS1-7-TabS1-5-FileS1-AppS1"></mediaResource><caption>Supporting info item</caption></supportingInfoItem><supportingInfoItem><mediaResource alt="supporting info item" href="urn-x:wiley:14622912:media:emi2666:EMI_2666_sm_TabS6"></mediaResource><caption>Supporting info item</caption></supportingInfoItem><supportingInfoItem><mediaResource alt="supporting info item" href="urn-x:wiley:14622912:media:emi2666:EMI_2666_sm_TabS7"></mediaResource><caption>Supporting info item</caption></supportingInfoItem><supportingInfoItem><mediaResource alt="supporting info item" href="urn-x:wiley:14622912:media:emi2666:EMI_2666_sm_TabS8"></mediaResource><caption>Supporting info item</caption></supportingInfoItem></supportingInformation><abstractGroup><abstract type="main" xml:lang="en"><title type="main">Summary</title><p>Ammonia‐oxidizing archaea (AOA) play an important role in nitrification and many studies exploit their <i>amoA</i> genes as marker for their diversity and abundance. We present an archaeal <i>amoA</i> consensus phylogeny based on all publicly available sequences (status June 2010) and provide evidence for the diversification of AOA into four previously recognized clusters and one newly identified major cluster. These clusters, for which we suggest a new nomenclature, harboured 83 AOA species‐level OTU (using an inferred species threshold of 85% <i>amoA</i> identity). 454 pyrosequencing of <i>amoA</i> amplicons from 16 soils sampled in Austria, Costa Rica, Greenland and Namibia revealed that only 2% of retrieved sequences had no database representative on the species‐level and represented 30–37 additional species‐level OTUs. With the exception of an acidic soil from which mostly <i>amoA</i> amplicons of the <i>Nitrosotalea</i> cluster were retrieved, all soils were dominated by <i>amoA</i> amplicons from the <i>Nitrososphaera</i> cluster (also called group I.1b), indicating that the previously reported AOA from the <i>Nitrosopumilus</i> cluster (also called group I.1a) are absent or represent minor populations in soils. AOA richness estimates on the species level ranged from 8–83 co‐existing AOAs per soil. Presence/absence of <i>amoA</i> OTUs (97% identity level) correlated with geographic location, indicating that besides contemporary environmental conditions also dispersal limitation across different continents and/or historical environmental conditions might influence AOA biogeography in soils.</p></abstract></abstractGroup></contentMeta><noteGroup><note xml:id="fn1"><p>Re‐use of this article is permitted in accordance with the Terms and Conditions set out at <url href="http://wileyonlinelibrary.com/onlineopen#OnlineOpen_Terms">http://wileyonlinelibrary.com/onlineopen#OnlineOpen_Terms</url></p></note></noteGroup></header></component></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>amoA‐based consensus phylogeny of ammonia‐oxidizing archaea and deep sequencing of amoA genes from soils of four different geographic regions</title></titleInfo><titleInfo type="abbreviated" lang="en"><title>Archaeal amoA phylogeny and diversity in soils</title></titleInfo><titleInfo type="alternative" contentType="CDATA" lang="en"><title>amoA‐based consensus phylogeny of ammonia‐oxidizing archaea and deep sequencing of amoA genes from soils of four different geographic regions</title></titleInfo><name type="personal"><namePart type="given">Michael</namePart><namePart type="family">Pester</namePart><affiliation>Departments of Microbial Ecology,</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Thomas</namePart><namePart type="family">Rattei</namePart><affiliation>Computational Systems Biology</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Stefan</namePart><namePart type="family">Flechl</namePart><affiliation>Departments of Microbial Ecology,</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Alexander</namePart><namePart type="family">Gröngröft</namePart><affiliation>Institute of Soil Science, University of Hamburg, Allende‐Platz 2, D‐20146 Hamburg, Germany.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Andreas</namePart><namePart type="family">Richter</namePart><affiliation>Chemical Ecology and Ecosystem Research, University of Vienna, Althanstrasse 14, A‐1090 Vienna, Austria.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Jörg</namePart><namePart type="family">Overmann</namePart><affiliation>Leibniz‐Institut DSMZ‐Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstraße 7B, D‐38124 Braunschweig, Germany.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Barbara</namePart><namePart type="family">Reinhold‐Hurek</namePart><affiliation>Department of Microbe‐Plant Interactions, University of Bremen, Postfach 330440, D‐28334 Bremen, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Alexander</namePart><namePart type="family">Loy</namePart><affiliation>Departments of Microbial Ecology,</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Michael</namePart><namePart type="family">Wagner</namePart><affiliation>Departments of Microbial Ecology,</affiliation><description>Correspondence: E‐mail ; Tel. (+43) 1 4277 54390; Fax (+43) 1 4277 54389.</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="article" displayLabel="article"></genre><originInfo><publisher>Blackwell Publishing Ltd</publisher><place><placeTerm type="text">Oxford, UK</placeTerm></place><dateIssued encoding="w3cdtf">2012-02</dateIssued><edition>Received 19 October, 2011; revised 7 November, 2011; accepted 7 November, 2011.</edition><copyrightDate encoding="w3cdtf">2012</copyrightDate></originInfo><language><languageTerm type="code" authority="rfc3066">en</languageTerm><languageTerm type="code" authority="iso639-2b">eng</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType><extent unit="figures">4</extent><extent unit="tables">1</extent><extent unit="references">72</extent><extent unit="words">10087</extent></physicalDescription><abstract lang="en">Ammonia‐oxidizing archaea (AOA) play an important role in nitrification and many studies exploit their amoA genes as marker for their diversity and abundance. We present an archaeal amoA consensus phylogeny based on all publicly available sequences (status June 2010) and provide evidence for the diversification of AOA into four previously recognized clusters and one newly identified major cluster. These clusters, for which we suggest a new nomenclature, harboured 83 AOA species‐level OTU (using an inferred species threshold of 85% amoA identity). 454 pyrosequencing of amoA amplicons from 16 soils sampled in Austria, Costa Rica, Greenland and Namibia revealed that only 2% of retrieved sequences had no database representative on the species‐level and represented 30–37 additional species‐level OTUs. With the exception of an acidic soil from which mostly amoA amplicons of the Nitrosotalea cluster were retrieved, all soils were dominated by amoA amplicons from the Nitrososphaera cluster (also called group I.1b), indicating that the previously reported AOA from the Nitrosopumilus cluster (also called group I.1a) are absent or represent minor populations in soils. AOA richness estimates on the species level ranged from 8–83 co‐existing AOAs per soil. Presence/absence of amoA OTUs (97% identity level) correlated with geographic location, indicating that besides contemporary environmental conditions also dispersal limitation across different continents and/or historical environmental conditions might influence AOA biogeography in soils.</abstract><relatedItem type="host"><titleInfo><title>Environmental Microbiology</title></titleInfo><genre type="journal">journal</genre><note type="content"> Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods. Fig. S1. Consensus tree illustrating the diversification of the Nitrosopumilus cluster at the second and third phylogenetic level. The tree was determined using 592 unambiguously aligned positions of 735 nucleic acid sequences that evenly cover the known sequence space of archaeal amoA at a ≥ 97% sequence identity level. Numbers in circles represent the second phylogenetic level (e.g. Nitrosopumilus subcluster 1), whereas the third phylogenetic level is directly indicated at the tree branch (e.g. Nitrosopumilus subcluster 1.1); sequences that did not form stable sublineages of more than three representatives kept the affiliation of the higher phylogenetic level and are indicated by their NCBI accession number. The consensus tree and the source alignment of representing sequences can be found in File S1. The scale bar indicates 10% estimated sequence divergence. Fig. S2. Rarefaction analysis of forward and reverse sequenced amoA at the species level cut‐off of 85% amoA identity. Fig. S3. Correlation analysis of total nitrogen, organic carbon and soil pH to OTU richness at the species level cut‐off of 85% amoA identity when normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest was omitted from the analysis due to a sequence number of less than 1300 reads. The analysis of the reverse sequences is shown with all correlations having a P‐value < 0.05; analyses of forward sequences gave similar results (data not shown). Highest AOA species richness was observed at the lowest total nitrogen and organic carbon content, which agrees well with the cumulative recent findings that AOA are generally adapted to low ammonia concentrations and are inhibited by high loads of dissolved organic carbon (Könneke et al., 2005; Hatzenpichler et al., 2008; de la Torre et al., 2008; Erguder et al., 2009; Martens‐Habbena et al., 2009; Di et al., 2010; Pratscher et al., 2011; Verhamme et al., 2011). The few detected AOA species in soils with high loads of nitrogen and organic carbon indicate the existence of ecotypes adapted also to these conditions or represent AOA that perform a mixotrophic or heterotrophic lifestyle (Hallam et al., 2006; Jia and Conrad, 2009; Mußmann et al., 2011; Tourna et al., 2011). For soil pH, AOA species richness followed the general trend of microbial species richness observed in a large survey of soils (Lauber et al., 2009), with a maximum of species at slightly acidic pH (pH = 6). Fig. S4. Abundance plot showing sequence identities of soil archaeal amoA retrieved by 454 pyrosequencing to next relatives in public databases. The approximate species‐level threshold of 85% amoA sequence identity is indicated by a dotted line. Fig. S5. In silico specificity analysis of the archaeal amoA primers used in this study against all archaeal amoA sequences covering the primer target regions. The primer regions of Candidatus Nitrosotalea devanaterra, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrosotenuis uzonensis (affiliated to Nitrosopumilus subcluster 5.1) represent partly unpublished data and were kindly provided by Graeme Nicol (Institute of Biological and Environmental Sciences, University of Aberdeen), José de la Torre (Department of Biology, San Francisco State University) and Roland Hatzenpichler and Susanne Haider (Department. of Microbial Ecology, University of Vienna) respectively. Fig. S6. Analysis of novel amoA that shared less than 85% sequence identity to deposited sequences in public databases. A. Abundance of novel OTUs at the species level cut‐off of 85% amoA identity in the respective samples. Representing sequences of forward and reverse OTUs that overlapped by more than 260 nt and shared at least 97% sequence similarity (within the 454 sequencing error range, Kunin et al., 2009) were merged to represent one OTU. A detailed list of novel OTUs including their representing sequence is given in Table S6. B. Phylogenetic position of selected novel amoA using representing sequences of all merged OTUs. The phylogenetic position of representing sequences of novel amoA OTUs within the amoA consensus tree was deduced by two independent inference methods: (i) the interactive parsimony tool within the ARB software package (Ludwig et al., 2004) and (ii) and a distance matrix method (neighbour joining tree based on a Jukes‐Cantor corrected distance matrix). Thereafter, novel amoA OTU representatives were added manually to the archaeal amoA consensus tree (Fig. 1) without changing the overall tree topology (as indicated by the dotted branches of uniform length of the added sequences). Fig. S7. Principal component analysis based on OTU abundance (jackknifed weighted UniFrac) and separating soils according to their combined total nitrogen/organic carbon content or to soil pH. For this analysis, observed amoA OTUs at 97% sequence identity were used (representing the highest possible phylogenetic resolution) and normalized to 1300 reads per soil and sequencing direction. The Austrian spruce forest soil was omitted from the analysis due to a sequence number of less than 1300 reads. Analysis of the forward sequences is shown; analysis of reverse sequences gave similar results (data not shown). Table S1. Highest nucleic acid sequence identity between representing sequences of the major archaeal amoA clusters. Presented identities were not corrected by substitution models. Table S2. Soil samples and determined soil parameters. Table S3. Sequencing results and number of observed and estimated OTUs at the species level (85% amoA identity). Table S4. Correlation between beta diversity of sites (unweighted and weighted UniFrac) and measured soil characteristics as determined by Mantel's test. Determined parameters are averages based on 100 jackknifed OTU tables normalized to 1300 amoA sequences per soil sample. Table S5. Number of indicator OTUs (97% amoA identity) in different amoA lineages. Only OTUs with an indicator value of 1.000 (exclusively detected under a certain tested soil characteristic) were summarized. A detailed list of individual indicator OTUs with their next relatives in public databases is given in Table S7. dw: dry weight. N, total nitrogen (% dry weight); C, total org. carbon (% dry weight). Table S6. Representing sequences and total retrieved sequences reads of novel archaeal amoA OTUs at the species level cut‐off of 85% identity in the individual analysed soil samples. Table S7. Indicator OTU analysis for geographic location, pH, and combined total N and organic carbon content of soils. Table S8. OTU classification of amoA sequences at 97% identity. For each OTU the representing sequence, its affiliation, and the name and distance to the next relative in public databases is given. File S1. Archaeal amoA ARB database encompassing the consensus tree and the source alignment of sequences representing clusters at ≥ 97% sequence identity of all publicly available archaeal amoA genes by June 2010. Appendix S1. Supporting methods.Supporting Info Item: Supporting info item - Supporting info item - Supporting info item - Supporting info item - Supporting info item - </note><identifier type="ISSN">1462-2912</identifier><identifier type="eISSN">1462-2920</identifier><identifier type="DOI">10.1111/(ISSN)1462-2920</identifier><identifier type="PublisherID">EMI</identifier><part><date>2012</date><detail type="title"><title>Taxonomy and Biodiversity</title></detail><detail type="volume"><caption>vol.</caption><number>14</number></detail><detail type="issue"><caption>no.</caption><number>2</number></detail><extent unit="pages"><start>525</start><end>539</end><total>15</total></extent></part></relatedItem><identifier type="istex">071C3FCCD46A4C83B4AA567C8B28364AD9990C77</identifier><identifier type="DOI">10.1111/j.1462-2920.2011.02666.x</identifier><identifier type="ArticleID">EMI2666</identifier><accessCondition type="use and reproduction" contentType="copyright">© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd</accessCondition><recordInfo><recordContentSource>WILEY</recordContentSource><recordOrigin>Blackwell Publishing Ltd</recordOrigin></recordInfo></mods></metadata><enrichments><json:item><type>multicat</type><uri>https://api.istex.fr/document/071C3FCCD46A4C83B4AA567C8B28364AD9990C77/enrichments/multicat</uri></json:item></enrichments><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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