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ORIGINAL ARTICLE

Endospores of thermophilic bacteria as tracersof microbial dispersal by ocean currents

Albert Leopold Muller1,2, Julia Rosa de Rezende3,4, Casey RJ Hubert4, Kasper UrupKjeldsen3, Ilias Lagkouvardos1, David Berry1, Bo Barker Jørgensen3 and Alexander Loy1,2

1Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, Faculty of Life Sciences,University of Vienna, Vienna, Austria; 2Austrian Polar Research Institute, Vienna, Austria; 3Center forGeomicrobiology, Department of Bioscience, Aarhus University, Aarhus, Denmark and 4School of CivilEngineering and Geosciences, Newcastle University, Newcastle Upon Tyne, UK

Microbial biogeography is influenced by the combined effects of passive dispersal andenvironmental selection, but the contribution of either factor can be difficult to discern. Asthermophilic bacteria cannot grow in the cold seabed, their inactive spores are not subject toenvironmental selection. We therefore conducted a global experimental survey using thermophilicendospores that are passively deposited by sedimentation to the cold seafloor as tracers to studythe effect of dispersal by ocean currents on the biogeography of marine microorganisms. Ouranalysis of 81 different marine sediments from around the world identified 146 species-level 16SrRNA phylotypes of endospore-forming, thermophilic Firmicutes. Phylotypes showed variouspatterns of spatial distribution in the world oceans and were dispersal-limited to different degrees.Co-occurrence of several phylotypes in locations separated by great distances (west of Svalbard,the Baltic Sea and the Gulf of California) demonstrated a widespread but not ubiquitous distribution.In contrast, Arctic regions with water masses that are relatively isolated from global oceancirculation (Baffin Bay and east of Svalbard) were characterized by low phylotype richness anddifferent compositions of phylotypes. The observed distribution pattern of thermophilic endosporesin marine sediments suggests that the impact of passive dispersal on marine microbialbiogeography is controlled by the connectivity of local water masses to ocean circulation.The ISME Journal advance online publication, 19 December 2013; doi:10.1038/ismej.2013.225Subject Category: Microbial population and community ecologyKeywords: biogeography; endospores; marine microorganisms; ocean currents; thermophiles

Introduction

Microorganisms display distinct biogeographic pat-terns (reviewed in Foissner, 2006; Green andBohannan, 2006; Martiny et al., 2006; Ramette andTiedje, 2007; Lindstrom and Langenheder, 2012), yetthe mechanisms controlling their distribution in theenvironment are difficult to distinguish and thus notwell understood. Four fundamental processes—selection, drift, dispersal and mutation—have beenproposed for creating and maintaining microbialbiogeographic patterns (Hanson et al., 2012), updat-ing the classical concept of dispersal, speciation andextinction as the main factors determining biogeo-graphy. Regardless of theoretical framework,

dispersal of microbial cells has a central role in shapingthe spatial distribution of microbial biodiversity(Green and Bohannan, 2006; Fierer, 2008). In itsstrictest sense, microbial dispersal is defined as thephysical movement of cells between two locations,but an extended definition additionally includessuccessful establishment—that is, physiologicalactivity and growth of migrated cells—at the receiv-ing location (Hanson et al., 2012). The existence ofphysical dispersal barriers for microorganisms hastraditionally been questioned. Under the BaasBecking paradigm of microbial cosmopolitanism—‘everything is everywhere, but, the environmentselects’—it is hypothesized that microorganismspossess unlimited dispersal capabilities due to theirlarge population sizes and short generation times.In this paradigm, environmental factors are the soledeterminants of observed microbial distributionpatterns (Baas Becking, 1934; Finlay, 2002; Fencheland Finlay, 2004). More recently, multiple studieshave put forward evidence for dispersal limitationamong microorganisms (Papke et al., 2003; Whitakeret al., 2003; Green et al., 2004; Reche et al., 2005;Martiny et al., 2006; Ghiglione et al., 2012; Sul et al.,

Correspondence: CRJ Hubert, School of Civil Engineering andGeosciences, Newcastle University, Newcastle Upon Tyne, UK.E-mail: casey.hubert@newcastle.ac.ukor A Loy, Division of Microbial Ecology, Department ofMicrobiology and Ecosystem Science, University of Vienna,Althanstrasse 14, Wien 1090, Austria.E-mail: loy@microbial-ecology.netReceived 12 September 2013; revised 7 November 2013; accepted14 November 2013

The ISME Journal (2013), 1–13& 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13

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2013). Controversy remains, however, partly due todifferences in definitions of dispersal and theoperational taxonomic units used to classify micro-organisms (Hanson et al., 2012), but mostly becausethe biogeography of free-living microorganisms isthe result of a combination of various evolutionaryand ecological processes that make it difficult tounravel the relative influence of passive transport(Martiny et al., 2006; Ramette and Tiedje, 2007). Weuse the term dispersal here to describe physicalmovement by passive transport, but not colonizationof the new location.

The presence of dormant endospores of thermo-philic members of the bacterial phylum Firmicutesin cold marine sediments is the result of passivetransport from warm environments and has beenproposed as a natural model for selectively investi-gating the dispersal of microbial cells in the oceans(Bartholomew and Paik, 1966; Isaksen et al., 1994;Hubert et al., 2009; de Rezende et al., 2013). Theinactivity of these model organisms enables theirbiogeography to be investigated largely without anyconfounding influence of environmental selection.Therefore, any observed non-random spatial distri-bution patterns of these organisms should bedirectly attributable to the influence of dispersallimitation. Ocean currents and eventual sedimenta-tion have been invoked as dispersal vectors forsupplying thermophilic Firmicutes spores to Arcticfjord sediments off the coast of Svalbard at anestimated rate exceeding 108 spores per square meterper year (Hubert et al., 2009). Once deposited in thecold sediment, these spores lie dormant as membersof the rare biosphere and the ‘microbial seed bank’(Pedros-Alio, 2012; Gibbons et al., 2013), but theycan be induced to germinate rapidly during sedi-ment incubation experiments at high temperature(Hubert et al., 2009).

Enrichment of thermophilic anaerobes from dor-mant spores in high-temperature incubation experi-ments enables selective focus on a specific,culturable part of the rare biosphere that is ofexceptional interest for studying long-term andlong-distance dispersal. Bacterial endospore disper-sal represents an upper boundary for the dispersalcapabilities of vegetative cells, as bacterial endo-spores are metabolically inert, highly stress resistantand able to survive unfavorable conditions for longperiods (Nicholson et al., 2000). For example, a half-life of up to 440 years was estimated for endosporesof thermophilic sulfate-reducing bacteria depositedin Aarhus Bay in the Baltic Sea (de Rezende et al.,2013); these viable endospores decreased in abun-dance with depth but were still detected at 6.5 mbelow seafloor in 4500-year-old sediment, suggest-ing a life span that could allow the global dispersalof spores through thermohaline circulation thatfully connects the world oceans on a time scale ofB1000–2700 years (DeVries and Primeau, 2011).Diverse populations of dormant bacterial sporesbecome active when sediment is heated (to 50–60 1C).

Once germinated, the vegetative cells catalyze themineralization of organic matter via extracellularenzymatic hydrolysis, fermentation and sulfatereduction (Hubert et al., 2010). So far, the phyloge-netic composition of these dormant, thermophiliccommunities has only been studied in a fewlocations, that is, the sediments of West Svalbardfjords and Aarhus Bay. Even though these locationsare B3000 km apart, they share at least two thermo-philic Desulfotomaculum phylotypes with identical16S rRNA and dissimilatory (bi)sulfite reductase(dsrAB) gene sequences (de Rezende et al., 2013).Although this is the first intriguing evidence forlong-distance dispersal of these thermophilic endo-spores, their large-scale biogeography in the globalocean remains unexplored.

We thus analyzed the richness, phylogeny anddistribution of endospores of thermophilic bacteriain marine sediments on global and regional scales toaddress the following questions: are thermophilicspores randomly distributed, which would beindicative of unlimited dispersal, or does thebiogeography of thermophilic spores show signsof dispersal limitation? If these spores are notrandomly distributed, how do local hydrography,sedimentation and major ocean currents impacttheir distribution? To investigate the biogeographyof thermophilic spores, sediment samples from 81locations around the world ocean, including regio-nal Arctic samples from the Svalbard archipelagoand the Baffin Bay that connect differently to globalocean circulation, were amended with organicsubstrates, pasteurized and incubated at 50 1C underanoxic conditions. Pasteurization kills the vegeta-tive community of mesophilic or psychrophilicmicroorganisms. Shifts in microbial communityactivity and composition, owing to germinationand growth of thermophilic endospores during theincubation, were then monitored by measuringsulfate reduction rates and by using multiplexpyrosequencing of bacterial 16S rRNA gene amplicons.Subsequently, the phylogeny and biogeography ofenriched thermophilic phylotypes was analyzed.

Materials and methods

Marine sediment samplesThe sample set comprised marine sediments from81 locations around the world ocean, including tworegional Arctic sample sets from Svalbard fjords andthe Baffin Bay and samples from hydrothermallyinfluenced sediments in the Guaymas Basin(43–150 1C, Gulf of California) that comprisespotential source environments of endospore-formingthermophiles (Figure 1, Supplementary Table S1).Most sediment samples were collected from theseafloor surface (0–10 cm below seafloor) at coastalor deep sea, open ocean sites with in situ temperaturesranging from 0 to 30 1C. Samples were stored at 4 1Cor frozen at � 20 1C until germination experiments.

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Figure 1 Global and regional maps show the sediment-sampling sites and selected major ocean currents. Circle sizes represent richnessof thermophilic Firmicutes endospore phylotypes (crosses indicate a richness of 0). Symbol color indicates whether thermophilic sulfatereduction was detected during the high temperature incubation (green¼positive, red¼negative). Global map (top) shows the globalthermohaline circulation (warm/surface currents in red, cold/deep water currents in blue; adapted and simplified from Rahmstorf(2002)). Broad geographic regions studied are highlighted in lighter blue (WS, West Svalbard; ES, East Svalbard; BB, Baffin Bay; NE,Northern Europe; GB, Guaymas Basin; SA, South Asia; SP, South Pacific). Map of Baffin Bay (bottom left): local currents reproduced fromLloyd et al. (2005); BC, Baffin Island Current; WGC, West Greenland Current; LC, Labrador Current. Map of Svalbard (bottom right): localcurrents reproduced from Knies et al. (2007); WSC, West Spitsbergen Current; ESC, East Spitsbergen Current.

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Sediment incubation and sulfate reductionmeasurementsSediment was homogenized and mixed with sterileanoxic synthetic seawater medium at a 1:2 (w/w)ratio under constant flow of N2 gas. The sedimentslurries were amended with organic substrates:formate, lactate, acetate, succinate, propionate,butyrate, ethanol (each to a final concentration of0.5 mM) and/or with freeze-dried Spirulina cells(1.5 g l� 1). Two 12-ml aliquots of slurried sedimentwere transferred to Hungate tubes under constantflow of N2 and pasteurized for 20 min at 80 1C andincubated in parallel at 50 1C. Before the incubation,one aliquot received B720 kBq 35S-labeled carrier-free sulfate tracer for sulfate reduction measure-ment. The incubations were sub-sampled bysyringe-needle after 0, 56, 72 and/or 120 h. Aliquotsof 3 ml from the 35S-sulfate slurries were mixed with6 ml 20% zinc acetate solution and stored at � 20 1Cuntil sulfate reduction was determined using asingle-step cold chromium distillation method(Kallmeyer et al., 2004). From the non-radioactiveincubations, 2 ml subsamples were pelleted bycentrifugation and frozen at � 20 1C for subsequentDNA extraction.

Multiplex amplicon pyrosequencingDNA was extracted from sediment slurries using thePowerSoil DNA isolation kit (MO BIO Laboratories,Inc., Carlsbad, CA, USA). Bacterial 16S rRNA geneamplicons were obtained and supplied with bar-codes and pyrosequencing adaptors using a two-stepPCR approach with low cycle numbers (20þ 5) andpooling of triplicate PCR reactions to reducevariability associated with barcoded pyrosequen-cing primers (Berry et al., 2011). The PCR primerstargeting most bacteria (909F: 50-ACTCAAAKGAATWGACGG-30, 1492R: 50-NTACCTTGTTACGACT-30)and conditions were described previously (Berryet al., 2011). Final PCR products were purified usingthe Agencourt Ampure XP system (Beckman Coul-ter, Vienna, Austria) and the DNA concentrationwas determined using a Quant-iT PicoGreendsDNA Assay (Invitrogen, Darmstadt, Germany).For better coverage of samples with higherexpected bacterial diversity, amplicons were thenpooled at a 2:1 ratio of 0 h and 120 h time points,respectively. Sequencing was performed on a GSFLX or GS FLXþ instrument using Titaniumchemistry (Roche, Mannheim, Germany) by theNorwegian High-Throughput Sequencing Centre(Oslo, Norway) or by Eurofins MWG Operon(Ebersberg, Germany). In total, 1 493 577 reads withan average length of 567 nucleotides (nt) werereceived. Sequences were trimmed and erroneoussequencing reads removed using the PyroNoiseimplementation in mothur (Quince et al., 2009;Schloss et al., 2009) and sorted according tobarcode using QIIME (Caporaso et al., 2010),yielding a total of 1 196 847 usable reads with an

average length of 353 nt. A 97% identity thresholdwas used for clustering reads into phylotypes withUCLUST (Edgar, 2010). Representative sequenceswere aligned with mothur using the Needleman-Wunsch pairwise alignment method defaultsettings (Schloss et al., 2009). Chimeras weredetected using Chimera Slayer (Haas et al., 2011)and excluded from further analysis. Pyrosequen-cing data are archived at the NCBI Sequence ReadArchive under accession SRP028774.

Identification of putative thermophilic endosporephylotypesTwo criteria were used to identify putative thermo-philic endospore phylotypes. First, species-levelphylotypes had to be significantly enriched in atleast one sediment sample after incubation at hightemperature. This criterion is based on the assump-tion that thermophilic endospores will survive theinitial pasteurization and germinate and growduring the ensuing 50 1C incubation. Significantenrichment of phylotypes was determined using atwo-proportion T-test and P-values were correctedfor multiple comparisons using the false discoveryrate method in R (Benjamini and Hochberg, 1995) toaccount for uncertainty due to sequence samplingdepth. Corrected P-valuesp0.01 were consideredsignificant. Second, phylotypes had to be affiliatedwith the phylum Firmicutes, to which all knownendospore-forming bacteria belong (it was shownrecently that endosporulation likely evolved onlyonce at the base of the Firmicutes tree) (Abecasiset al., 2013). This approach does not survey allinactive cells in the sampled environments, buthones in on a physiological subset of the rarebiosphere (Lennon and Jones, 2011). The clearadvantage of this strategy is that taxa that wereactive and subject to environmental selection in situare excluded from the analysis, allowing passivedispersal to be evaluated in isolation. Representa-tive sequences of enriched Firmicutes phylotypeswere then automatically aligned using the web-based SINA aligner (Pruesse et al., 2012) andimported into the ARB-SILVA database SSU RefNR 111 (Quast et al., 2013) using the ARB softwarepackage (Ludwig et al., 2004). The alignment wasmanually curated and used to re-cluster the repre-sentative sequences into species-level phylotypes ofX97% sequence similarity using the average neigh-bor algorithm in mothur. A phylotype was calledpresent at a location if at least one sequence of thisphylotype was detected before and/or after the 120 hincubation of sediment from this location.

PhylogenyA maximum likelihood (RAxML) tree was calcu-lated with almost full-length 16S rRNA sequences(X1400 nt) of closely related reference bacteria orenvironmental clones based on 1222 alignment

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positions by using a 50% sequence conservationfilter for bacteria. Using the ARB ParsimonyInteractive tool, the short amplicon pyrose-quences were subsequently added to this treeone at a time by using the 50% sequenceconservation filter and alignment filters coveringthe individual length of each representativephylotype sequence, without changing the overalltree topology. Trees were visualized using iTOL(Letunic and Bork, 2006).

Network analysisTwo different kinds of networks were built basedon the phylotype presence/absence matrix. First, anetwork of phylotype co-occurrence was producedfor phylotypes present in at least five sites andwith a minimum Spearman correlation coefficientof 0.6 (and Po0.0001 based on permutationtesting) (Barberan et al., 2012). Focus on phylo-types that occurred at multiple sites reducednetwork complexity and facilitated identificationof the core thermophilic endospore community.Second, a network of sites based on phylotypediversity was produced based on Bray-Curtissimilarity of at least 0.6. Networks were plottedusing the ‘network’ package in R (Butts et al.,2012).

Distance-decay analysisJaccard similarities were calculated from thephylotype presence/absence matrix using the‘vegan’ package in R (Oksanen et al., 2012).Distances were calculated from latitude and long-itude coordinates of sampling sites using theVincenty inverse formula for ellipsoids and WorldGeodetic System (WGS) 84 ellipsoid parametersand calculated using R (R Development CoreTeam, 2008). A linear model was fit to the dataand the statistical significance of the resultingregression parameters was evaluated by analysis ofvariance in R. To determine which phylotypeswere dispersal-limited, a permutation-testingapproach was used. The geographical dispersalof each phylotype was calculated as the meandistance between sites at which it was found.A null distribution of mean distances was producedby randomly selecting from all sites an equallysized subset (e.g. if a phylotype was found at sevensites, then seven sites would be randomlyselected). The mean distance between the randomsubsets was calculated, and this process wasrepeated for 10 000 re-samplings. The probabilitythat the observed mean distance was due to chancewas calculated and corrected for multiple compa-risons and P-values p0.05 were consideredsignificant.

Geographical maps were drawn using GenericMapping Tools (Wessel and Smith, 1998).

Results and discussion

Non-uniform distribution of thermophilic Firmicutesendospores in marine sedimentsWe studied the biogeography of thermophilicendospores in Arctic and other permanently coldor temperate marine sediments, because longevityof endospores makes them ideal study objectsfor understanding the time-averaged impact ofdispersal on marine microbial biogeography.While there might be differences in endosporesurvival between different bacteria (Nicholsonet al., 2000; McKenney et al., 2013), results areinterpreted under the assumption that differencesin endospore distribution are dependent ondispersal and not on environmental selection.

Anoxic, high-temperature incubations of pasteur-ized marine sediment (mostly from the topfew centimeters) (Supplementary Results andDiscussion, Supplementary Figure S1) from 81different locations (Supplementary Table S1) ledgenerally to a reduction in bacterial alpha diversitydue to germination and growth of thermophilicendospores combined with the death and DNAdecay of vegetative cells (Supplementary Materialsand Methods, Supplementary Table S2). Principlecoordinate analysis of weighted UniFrac distancesconfirmed a shift in phylogenetic compositionof bacterial 16S rRNA genes after the incubation ofmost, but not all, sediment samples (SupplementaryFigure S2). In total, we identified 146 thermophilicendospore phylotypes (hereafter called ‘thermo-spore phylotypes’) across all high-temperatureincubations (Supplementary Table S3). Thermo-spore phylotypes were detected in samples fromalmost all of the investigated locations (n¼ 79/81)(Figure 1). Most of the 146 thermospore phylotypeswere affiliated with the orders Clostridiales(61.0%) and Bacillales (17.8%); the most repre-sented families were Clostridiaceae (32.2%),Peptococcaceae (17.8%) and Bacillaceae (14.4%)(Supplementary Figure S3, Supplementary Table S3).The phylogenetic identity and potential physiologyof thermospore phylotypes corroborate and greatlyexpand upon previous studies that investigatedthermophilic endospores in marine sediments(Bartholomew and Paik, 1966; Isaksen et al., 1994;Vandieken et al., 2006; Hubert et al., 2009, 2010; Jiet al., 2012; de Rezende et al., 2013) (SupplementaryResults and Discussion). A screening of environmentalamplicon databases showed that 16S rRNA genesequences of the identified thermospore phylotypesare very rarely detected by environmental surveys inthe marine environment (Supplementary Results andDiscussion, Supplementary Table S4), which is likelyconsistent with the widespread occurrence ofendospore taxa in low relative abundance in manyhabitats.

The different sampling locations showed con-siderable differences in thermospore phylotyperichness, despite an absence of strict endemism of

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phylotypes that occurred at multiple locations (thatis, exclusive presence in only one geographicregion). The number of detected thermosporephylotypes per site ranged from 0 to 51 (on average,13.3±11.6 (s.d.), n¼ 81) (Supplementary Table S1).Thermospore phylotype richness was generallyhigher in Northern Europe (36.4±10.2 per site,n¼ 5), the Guaymas Basin (28.7±18.6 per site,n¼ 3), West Svalbard (17.9±8.3 per site, n¼ 23)and South Asia (22.8±15.2 per site, n¼ 4), whereassites in the Baffin Bay (5.4±4.7 per site, n¼ 25), theSouth Pacific (9.5±6.5 per site, n¼ 4), East Sval-bard (10.3±6.2 per site, n¼ 4) and other regions(7.2±5.7 per site, n¼ 13) showed comparativelylower phylotype richness (Figure 1). These differ-ences in site occupancy indicate non-randomvariation in the distribution of thermosporephylotypes across the investigated sites. Out ofthe 146 thermospore phylotypes, 21 were detectedat more than 15 locations and were widelydistributed across different oceanic regions (‘cos-mopolitan phylotypes’, Figure 2, SupplementaryFigures S4 and 5). This cosmopolitan distribution

of thermospore phylotypes suggests that these taxa(i) are globally dispersed by ocean circulationand/or (ii) derive from multiple, globally wide-spread source environments that support growthof similar communities of thermophilic, endo-spore-forming bacteria. The cosmopolitan ther-mospore phylotypes were related to Bacillus(TSP005, TSP010, TSP013, TSP021), the Aeriba-cillus-Geobacillus lineage (TSP003, TSP007,TSP016), Desulfonispora (TSP014), Desulfotoma-culum (TSP004, TSP006, TSP015), Clostridium(TSP018, TSP019, TSP020), the Brassicibacter-Sporosalibacterium lineage (TSP002, TSP009,TSP012), or were only assigned at the family ororder level (Clostridiales Family XI. IncertaeSedis: TSP008; Christensenellaceae: TSP017;Bacillales: TSP001, TSP011) due to lack of close,cultivated relatives (Figure 2). In contrast, 82thermospore phylotypes were found only at fiveor fewer locations (Supplementary Figures S3 and4), indicating more restricted occurrence and/orlow abundance below the detection limit of ourapproach.

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Figure 2 Phylogeny and geographic distribution of cosmopolitan thermophilic endospores. 16S rRNA-based phylogenetic tree ofthermospore phylotypes (TSPs) that were detected at 15 or more sites. Scale bar indicates 1% sequence divergence as inferred fromRAxML. Phylogenetic affiliations are highlighted in different colors. Colored bars indicate broad geographic regions where thethermospore phylotypes were present. Numbers indicate the number of sites at which a thermospore phylotype was detected, whereasthe height of each bar color indicates the number of those phylotypes detected in each ocean region. TSP, thermospore phylotype. SeeSupplementary Figure S3 for an extended tree showing the phylogeny and geographic distribution of all 146 thermospore phylotypes.

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Global biogeography patterns of thermophilicendospores show the influence of dispersal limitationTo analyze the relationship between geography anddifferences in endospore community structure, weplotted similarity between thermophilic endosporecommunities vs geographic distance for eachlocation pair. This analysis revealed a significant,negative distance-decay curve (linear regression oflog10-transformed variables with slope¼ � 0.68177,intercept¼ � 0.09818, Po0.001) (Figure 3). Thisresult shows that thermophilic endospore commu-nities are non-randomly distributed, which suggeststhat dispersal limitation is influencing the beta-diversity of thermospore phylotypes. Alternatively,the shape of the distance-decay curve may beinfluenced by differences in the ability of endosporephylotypes to resist decay during dispersal (Nicholsonet al., 2000; McKenney et al., 2013). To account forthis, we investigated if the distribution of individualphylotypes, which should be less biased by within-phylotype variances in endospore survival, issignificantly different from a random geographicdistribution. Twelve of 146 thermospore phylotypes(TSP001, TSP007, TSP008, TSP009, TSP011, TSP015,TSP020, TSP027, TSP060, TSP061, TSP096, TSP111)were seemingly dispersal-limited because they hadsignificantly geographically clustered occurrence; thatis, the average geographic distance between thelocations of each phylotype was significantly lessthan would be expected from its random occurrence atthe same number of locations.

Correlation network analyses reveal a widely distributedcore of frequently co-occurring thermophilic endosporesNetwork analysis was recently incorporatedinto microbial biogeography research to explore

co-occurrence patterns of microbial taxa (Fuhrman,2009; Barberan et al., 2012). We used correlationnetwork analysis to better describe the biogeographypatterns of thermophilic endospores identified bythe distance-decay curve and investigated whichphylotypes tend to co-occur; that is, which phylo-types are found together at some sites and arecommonly absent from others. The co-occurrence ofthermospore phylotypes was calculated using non-parametric Spearman correlations of thermosporephylotype presence/absence across all samplingsites and, to explore groups of co-occurring phylo-types, network analysis was used for visualization.We identified eight small networks of differentcomplexity, that is, numbers of nodes and edges(connections) (Supplementary Figure S6A). Thelargest network was characterized by four highlyconnected, central phylotypes (X4 connections;TSP004, TSP008, TSP009, TSP015) and nine per-ipheral phylotypes that occurred at several locations(Figure 4a). Once we identified the most frequentlyco-occurring phylotypes, we explored similaritiesbetween thermospore phylotype communities byinferring association networks of sediment loca-tions. The largest network contained 18 locations, ofwhich many harbored several thermospore phylo-types (Figure 4b), whereas 10 other, much smallernetworks were composed of p4 locations(Supplementary Figure S6B). The 18-location net-work was composed of most West Svalbard samples,but also samples that were geographically verydistant from the Svalbard archipelago, namely twosamples from the Baltic Sea (Aarhus Bay, ArkonaBasin) and one sample from the Gulf of California(Guaymas Basin) (Figure 4b). This network containsa phylogenetically diverse core of co-occurringthermophilic endospores that are widely but notubiquitously distributed in the oceans (for example,one of the phylotypes, TSP001, was detected in 39sediments). This raises questions about whethercertain thermospore phylotypes share commonsource environments from where they are dissemi-nated in a non-random manner over long distancesand along similar, as yet unidentified travel routes.

Hydrothermal sediments of the Guaymas Basin aspotential source environments for endospore-formingthermophilesBased on the identity, physiology and sedimentationrates of thermophilic spores in Svalbard and AarhusBay, we have previously postulated biogeochemicaland geological characteristics of hypothetical sourceenvironments for these bacteria (Hubert et al., 2009,2010; de Rezende et al., 2013). First, such anoxicmarine environments must be warm enough to allowvegetative growth of the diverse community ofanaerobic, endospore-forming thermophiles. Second,sufficiently strong fluxes (e.g., fluid transport) fromthese environments into the water column mustphysically transport cells into circulating seawater.

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Consistent with this general description are, forexample, pressurized gas or oil reservoirs in seabedsediments, and parts of oceanic spreading centersincluding hydrothermal vents and the sedimentsoverlying them (Head et al., 2003; Cowen, 2004;Brazelton et al., 2006; Judd and Hovland, 2007;Hutnak et al., 2008; Hubert and Judd, 2010).Anaerobic thermophiles in these hot sediment andocean crust habitats are connected with theoverlying cold ocean water via advection of seabedfluids such as hydrocarbons and recirculating waterdischarged from the ocean crust (Judd and Hovland,2007; Hutnak et al., 2008).

The hydrothermal surface sediments and thegeneral geology and geography of Guaymas Basinsatisfy these criteria (Supplementary Results andDiscussion). Accordingly, a very large proportion

(44.5%) of all 146 thermospore phylotypes weredetected overall in the three Guaymas Basinsediments investigated. Although surveys withmuch finer genetic resolution than offered by the16S rRNA gene approach are required to prove thatidentical microorganisms co-occur at distant loca-tions, it is intriguing that the Guaymas Basinsediment community comprised 15 of 25 cosmopo-litan thermospore phylotypes and 8 of the 13co-occurring phylotypes (including all four highlyconnected phylotypes in the largest network)(Figure 4a). High thermospore phylotype richnessdespite restricted access due to the geographicisolation of the semi-enclosed Gulf of Californiasupports the hypothesis that environments in theGuaymas Basin are sources of thermophilic sporesto the overlying water column. Thermohaline water

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Figure 4 Network analysis of thermophilic endospore co-occurrence (a) and location (b). (a) Largest network of co-occurring thermosporephylotypes. Each node represents a thermospore phylotype (TSP). Presence of an edge between two nodes shows a strong correlationbetween these two phylotypes, which is indicative for co-occurrence. Circle size indicates site occupancy. Map shows locations wherephylotypes of the network are present. The circle color indicates how many of the 13 phylotypes comprising the network are present at thissite. (b) Largest location network. Each node represents a location. Presence of an edge between two nodes corresponds to a high Bray-Curtissimilarity (X0.6) between the endospore communities at these two locations. Circle size indicates thermospore phylotype richness. Mapsshow the global locations and correlations of these sites. The map on the right shows a magnification of Svalbard for enhanced resolution.

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circulation in the Gulf of California is primarilyinfluenced by wind, which alters directions andwater transport between the gulf and the PacificOcean seasonally (Bray, 1988). It is conceivable thatGuaymas Basin-derived spores embark on andsurvive long-distance travel to far away Svalbardand North Sea sediments via the global thermoha-line circulation (Rahmstorf, 2000) (Figure 1). How-ever, it may be more likely that these colddestinations are supplied with thermophilic sporesfrom nearby warm environments that were notinvestigated in this study, and that dispersal barriersmay prevent thermophilic endospores that escapethe hot anoxic sediments in the Guaymas Basin fromreaching far away water masses in the northAtlantic. Possible examples of potential sourceenvironments closer to Svalbard and the Baltic Seacould include systems such as the Lost Cityhydrothermal vent field associated with the mid-Atlantic ridge (Kelley et al., 2005; Brazelton et al.,2006) or the oil-bearing sediments in the North Sea(Gittel et al., 2009), which both support thermo-philic sulfate-reducing Desulfotomaculum speciesthat are closely related to the thermospore phylo-types identified in this study. It is conceivable thatthermophilic bacteria get distributed along the mid-oceanic ridges throughout the world oceans, asexemplified by hydrothermal vent animals and theirsymbionts (Petersen et al., 2010; Vrijenhoek, 2010;Teixeira et al., 2011), creating an extended series ofsource habitats from where thermophilic endo-spores may be further dispersed to the coldsediments tested in this study. The location networkshown in Figure 4b, thus likely represents a fractionof a much larger network of warm sources and warmor cold destinations for dispersing thermophilicendospores that are interconnected through oceancurrents.

Anthropogenic sources may also contribute tothe presence of anaerobic thermophiles in marinesediments. North Sea oil production systems havebeen proposed as possible sources for thermophilicDesulfotomaculum species in Aarhus Bay sediments(de Rezende et al., 2013) and close relatives to

thermospore phylotypes detected in the presentstudy were found in oil production water (Gittelet al., 2009). Close relatives were also detected inseveral anthropogenic, as well as natural terrestrialhabitats such as bioreactors, activated sludge, com-post and soil environments (Supplementary TablesS3 and 4). However, the phylotypes we haveidentified were enriched in artificial seawater,which is suggestive of a marine origin. Also, thepresence of a widely distributed thermophilic Desul-fotomaculum alkaliphilum phylotype (Figure 2) atdepths corresponding to B4500 years of sedimen-tation in Aarhus Bay (de Rezende et al., 2013)indicates that there is a natural long-term dispersalof thermophilic endospores occurring in the marineenvironment that is independent of human activity.

Differences in thermophilic spore richness andphylogeny in Arctic regions correlate with connectivityof major water massesThe variable compositions of thermophilic endo-spore communities in Arctic sediments from theBaffin Bay and the east and west sides of theSvalbard archipelago suggest that thermospore phy-lotypes are not equally dispersed across these Arcticregions. Sediments from the Baffin Bay contained asignificantly lower number of thermospore phylo-types than sediments from West Svalbard (Table 1).While sediments from only four locations on theEast coast of Svalbard were available, we detected asimilar yet not statistically significant trend of lowerrichness in East compared with West Svalbardsediments. Analysis of more samples from EastSvalbard is required to confirm this trend. Weadditionally revealed a contrasting geographicaldistribution in the potential for thermophilicsulfate reduction that was generally consistent withthe observed differences in thermospore phylotyperichness (Figure 1). Thermophilic sulfate reductionwas significantly more prevalent in West Svalbard thanin East Svalbard and Baffin Bay sediments (Table 1).

The differences in thermophilic spore richnessand metabolic potential were also reflected by the

Table 1 Comparison of thermospore phylotype richness, phylogenetic composition and prevalence of thermophilic sulfate reductionbetween Arctic regions

West Svalbard(n¼23)

East Svalbard(n¼ 4)

Baffin Bay(n¼ 25)

West Svalbard vsBaffin Baya,b

West Svalbard vsEast Svalbarda

East Svalbard vsBaffin Baya

Thermospore phylotype richnessc 17.9±8.3 10.3±6.2 5.4±4.7 Po0.001 Not significant Not significantClostridiaceae richness 6.0±2.8 2.0±1.8 1.5±2.1 Po0.001 P¼ 0.02 Not significantPeptococcaceae richness 3.4±2.9 2.0±1.6 0.6±0.9 Po0.001 Not significant Not significantBacillaceae richness 2.2±1.9 3.0±1.4 2.4±1.4 Not significant Not significant Not significantThermophilic sulfate reduction(% of sites tested positive)

87 25 24 Po0.001 P¼ 0.02 Not significant

aSignificant differences between Arctic regions were determined by pairwise comparisons using the Mann–Whitney U-test for richness andFisher’s exact test for thermophilic sulfate reduction. P-values p0.05 were considered significant.bAll significant P-values in this column are also significant (Po0.01) when only surface (0–10 cm, n¼ 13) or only deep (10–121 cm, n¼ 12)sediment samples are analyzed.cAll richness values are presented as mean±s.d.

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phylogenetic composition of thermophilic endo-spore communities in these Arctic regions (notwith-standing the aforementioned weak indications forstrict endemism for any individual phylotypes).Of the three major families, West Svalbard sedimentsexhibit significantly more thermospore phylotypesbelonging to the Clostridiaceae and the Peptococcaceaethan Baffin Bay sediments, whereas Bacillaceaephylotypes were not differentially distributed betweenthe Arctic regions (Table 1). Again, we observed asimilar pattern between West Svalbard and EastSvalbard, although differences were only significantfor Clostridiaceae phylotypes (Table 1). No significantdifferences between East Svalbard and Baffin Baysediments were found among these three taxonomicfamilies (Table 1). Furthermore, frequently co-occurringthermospore phylotypes (e.g. TSP004, TSP008,TSP009, TSP015), as identified by network analysis(Figure 4), were almost exclusively limited to WestSvalbard sediments. The observed distribution patternis not impacted by the fact that 12 of 25 Baffin Baysediment samples originate from a sediment depthbelow 10cm (Supplementary Table S1). Restricting ouranalyses to only the shallow and deeper Baffin Baysediments showed (i) no significant difference betweenthem (e.g., thermospore phylotype richness: surfacesamples¼ 4.8±4.7, deeper samples¼ 6.1±4.9) and(ii) that both had the same significant differencescompared with West Svalbard regarding richness,phylogenetic composition and prevalence of thermo-philic sulfate reduction (Table 1).

One dispersal-limiting factor that might beresponsible for the different thermophilic endosporecommunities in Svalbard and Baffin Bay sedimentsis the rate at which spores are deposited to theseafloor. Based on sediment thickness and age,sedimentation rates in the Baffin Bay and off thecoast of Svalbard are estimated to be in the sameorder of magnitude (5–25 cm ka� 1, Kallmeyer et al.(2012)). In contrast, reports for a few locations inthese regions suggest that sedimentation rates arelower in the Baffin Bay (6.5 cm ka� 1, Simon et al.(2012)) than in Svalbard fjords (180 cm ka�1, Haldet al. (2001); 190 cm ka� 1, Hubert et al. (2009)).However, it is currently unknown how efficientendospores in the water column are deposited to thesediment, that is, how the specific rate of sporesedimentation relates to the overall sedimentationrate. It is noteworthy that for most locations ourenrichment inoculum derives from homogenizedsediment from 0 to 10 cm depth and thereforeintegrates endospore communities that accumulatedover 50 to 1500 years of deposition. Given thelongevity of thermophilic spores (de Rezende et al.,2013), our approach may diminish the impact ofsedimentation rates on the recovered richness ofthermophilic spore communities.

Besides the impact of local hydrography, sedi-mentation, and the possibility that unknown, localsources contribute to biogeography of thermophilicendospores, we hypothesized that dispersal-driven

distribution also depends on how local currents inthe sampled regions are connected to global oceancirculation. The observed West–East difference inthermophilic endospore community structure inSvalbard appears to correlate with the regionalhydrography of the archipelago, although the differ-ence is not statistically significant in all parameters(Table 1). Two major ocean currents define thehydrography of Svalbard. The warm West Spitsber-gen Current, which is an extension of the GulfStream, flows from the Atlantic Ocean northwards,whereas the East Spitsbergen Current transportscold water from the Arctic Ocean southwards(Loeng, 1991) (Figure 1). Although the core of theWest Spitsbergen Current that flows along thecontinental slope is separated from the WestSpitsbergen shelf waters by the Arctic Front,extensive cross-front exchange takes place below50 m depth (Saloranta and Svendsen, 2001) and ithas been shown that the West Spitsbergen Currentextends its influence deep into the fjord system ofWest Svalbard (MacLachlan et al., 2007).

The formation of deep and bottom water masses inthe Baffin Bay are not fully understood (Tang et al.,2004), but they are generally influenced by coldArctic Ocean water via the Baffin Island Current inthe north and by the Atlantic Ocean via the WestGreenland Current (Figure 1). The low thermosporephylotype richness in the Arctic water-influencedsediments in the Baffin Bay suggests a lower influxof thermophiles derived from the Arctic Ocean.Reduced thermospore phylotype richness in theArctic Ocean is possibly due to limited thermophilesources and lower connectivity to water masses fromthe rest of the world oceans where thermophilesources may be far more abundant.

Recent studies of vegetative microbial commu-nities in the global ocean have suggested thathydrography and geographic isolation of the ArcticOcean represents an effective dispersal barrier formicroorganisms (Galand et al., 2010; Ghiglioneet al., 2012; Hamdan et al., 2013; Sul et al., 2013).By tracking thermophilic endospores whose Arcticbiogeography is only controlled by passive disper-sal, our results demonstrate this dispersal limitationto be true. Importantly, evidence presented heresuggests that this dispersal limitation even holdstrue for marine bacteria with enhanced survivalcapacities that are less prone to death during long-term and long-distance dispersal under unfavorableenvironmental conditions, that is, perhaps the mostlikely candidates to not be dispersal-limited. Differentphylotypes of globally widespread thermophilicendospores co-occur at multiple distantly separatedlocations and thus could be simultaneouslydispersed from common sources and/or alongsimilar paths according to non-random mechanisms.Global dispersal routes and frequencies of marinemicrobial taxa are largely dependent on the degree ofconnectivity between major oceanic water masses.Dispersal barriers of this kind exert greater control on

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the composition of marine microbial communities inwater masses that participate minimally in globalocean circulation such as the Arctic Ocean.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

We gratefully acknowledge the provision of sedimentsamples by Jan Amend, Gail Lee Arnold, Carol Arnosti,Elisa Bayraktarov, Antje Boetius, Gerhard Bohrmann,Ellen Damm, Steve D’Hondt, Tim Ferdelman, MoritzHoltappels, Lars Holmkvist, Sabine Kasten, Martin Kruger,Connie Lovejoy, Maren Nickel, Aude Picard, Roy Price,Nils Risgaard-Petersen, Alberto Robador, Søren Rysgaard,Joanna Sawicka, Verona Vandieken, and ThorstenWilhelm. We thank China Hanson for her valuablecomments on a draft version of this paper. This workwas financially supported by the Austrian Science Fund(FWF, P20185-B17 and P25111-B22 to AL).

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