Published in 1 162, issue 1, 14-32, 2011 which should be used for any reference to this work

Highly Diverse and Seasonally Dynamic Protist Community in a Pristine Peat Bog

Enrique Laraa,b,c,d,1,2, Edward A.D. Mitchellb,c, David Moreiraa, and Purificación López Garcíaa a Unité d’Ecologie, Systématique et Evolution, UMR CNRS 8079, Université Paris-Sud, bâtiment 360, 91405 Orsay Cedex, France b Swiss Federal Research Institute WSL, Wetlands Research Group, Lausanne, Switzerland c École Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Ecological Systems, Station 2, Lausanne, Switzerland d Institute of Biology, Laboratory of Soil Biology, CH-2009 Neuchâtel, Switzerland

Culture-independent molecular methods based on the amplification, cloning and sequencing of small- subunit ribosomal RNA genes (SSU rDNAs) are powerful tools to study the diversity of microorganisms. Despite so, the eukaryotic microbial diversity of many ecosystems, including peatlands has not yet received much attention. We analysed the eukaryotic diversity by molecular surveys in water from the centre of a pristine Sphagnum-dominated peatland in the Jura Mountains of Switzerland during a com- plete seasonal cycle. The clone libraries constructed from five different temporal samplings revealed a high diversity of with representatives of all major eukaryotic phyla. In addition, four sequence types could not be assigned to any known high-level eukaryotic taxon but branched together with a rather good statistic support, raising the possibility of a novel, deep branching eukaryotic clade. The analysis of seasonal patterns of phylotypes showed a clear change in the eukaryotic communities between the warm period (late spring and summer) and the cold period (autumn and winter). Chryso- phytes dominated the samples in the cold period while (Arcellinida and ) and a few other groups peaked in summer. A few phylotypes (such as a cryptomonad and a perkinsid) were abundant at given sampling times and then almost disappeared, suggesting bloom-like dynamics.

Key words: Environmental diversity; Sphagnum-dominated peatland; chrysophytes; new phyla; testate amoe- bae; seasonal patterns.

Introduction ganisms, both prokaryotic and eukaryotic, from environmental samples. In the past few years, Culture-independent molecular methods based on progress in the study of microbial eukaryotic the amplification, cloning and sequencing of small- phylogeny and diversity has been rapid. As a subunit ribosomal RNA genes (SSU rDNAs) are consequence, both old-established phylogenetic a powerful tool to study the diversity of microor- relationships among major eukaryotic lineages (Baldauf 2003) and previous estimates of eukary- otic diversity have been fundamentally questioned 1Corresponding author; fax +41327182103 2Current address: University of Neuchâtel, Institute of Biology, (Epstein and López-García 2008). However, only a Laboratory of Soil Biology, CH-2009 Neuchâtel, Switzerland fraction of the existing ecosystem types have been e-mail [email protected] (E. Lara). studied in this way and it is therefore possible that 2

some microbial groups remain to be discovered. Warner 1992; Lamentowicz and Mitchell 2005; Peatlands are among the ecosystems that have Mitchell et al. 2008; Tolonen et al. 1992) which received little attention until now with respect to make up a high fraction of the total biomass of eukaryotic microbial diversity. microorganisms, and also for metazoans (Gilbert Peatlands are globally widespread biomes, et al. 1998a,b; Mitchell et al. 2003). However, to which occupy up to 3% of the Earth’s terrestrial date, the diversity of microbial in the surface (Kivinen and Pakarinen 1981). Among the Sphagnum peat bog environment has never been different types of peatlands, Sphagnum-dominated investigated with molecular methods. These could peatlands (bogs and poor fens) are most common be useful to study the small colourless flagellates in high latitudes (and locally high altitudes), where and naked amoebae which are easily overlooked they can be a dominant feature of the landscape. in microscope-based studies but play a key role They are characterised by very low concentrations in nutrient cycling in many environments, as illus- of mineral nutrients (5 to 50 mg per litre in total), trated in the paradigm of the microbial loop (Azam low pH values (sometimes below 4.0) (Dedysh et al. et al. 1983; Clarholm 1985). In addition, parasitic 2006), and high concentration of humic acids which organisms, difficult to detect solely by observation, are known to have biocidal properties (Steinberg may also play a fundamental role in freshwater et al. 2006). Under such environmental conditions, ecosystems (Lefèvre et al. 2008). Moreover, it can which can be considered relatively extreme (mostly be speculated that strongly selective environments due to the lack of certain nutrients), the degrada- such as Sphagnum-dominated peatlands can host tion rate of organic matter is very low, resulting in an undetected diversity, perhaps with novel clades. an accumulation of organic carbon. At the global Microscopic observation of bog communities has scale, this process is of considerable significance: revealed the existence of organisms growing exclu- 30% of the world’s pool of organic carbon is stored sively in these environments (Foissner 1996; Heal in peat bogs (Gorham 1991). It has been found that 1961). In this work, we have analysed by molec- they act also as carbon sinks, sequestering 12% of ular methods the eukaryotic microbial diversity of all anthropogenic emissions of CO2 (Moore 2002). a pristine ombrotrophic, (i.e. receiving most of its Despite their importance in the global carbon water and nutrient supply from rain) peat bog in cycle, it is only recently that the microbial diversity of the Jura Mountains (Switzerland), the Praz Rodet peatlands has attracted the interest of the scientific bog, along a full seasonal cycle. The aims of our community, mainly because of their possible con- study were (1) to investigate the eukaryotic molec- tribution to greenhouse gas emissions and hence ular diversity in the peat bog environment and (2) global warming (Dedysh et al. 2006). To date, most to assess if this diversity varies seasonally. studies on microbial diversity in peatlands focussed on prokaryotes involved in CH4 cycling, leading to the discovery of a phylogenetically diverse and Results and Discussion mostly unknown prokaryotic community (Dedysh et al. 2006) and also of remarkable adaptations to In order to explore the protist diversity in the Praz- the harsh environmental conditions (Bräuer et al. Rodet peat bog and its temporal variability, we 2006). The taxonomic composition of these com- analysed the diversity of eukaryotic SSU rRNA munities is not fixed, but dynamic in time; increased genes in five water samples taken during a full sea- temperature (as in summer) is paralleled by a pro- sonal cycle between 2007 and 2008 (March 2007, portional increase of methanogenic archaea, which June 2007, November 2007, January 2008 and May in turn correlates with enhanced methane produc- 2008). In addition, original clones obtained from tion (Høj et al. 2008). peat-only samples (coded PRS) were also included In contrast to bacteria and archaea, the study in the phylogenetic trees as part of the overall peat of the diversity of eukaryotic micro-organisms in bog diversity. In the framework of this study we Sphagnum-dominated peatlands is currently lim- define an operational taxonomic unit (OTU) as the ited to morphological observations of the general taxonomic entity represented by a group of SSU protist community (Kreutz and Foissner 2006; rRNA gene sequences sharing >97% identity, and Strüder-Kypke and Schönborn 1999). Data on the it is hence equivalent to phylotypes (environmental ecology of certain species do exist for certain con- sequences) defined on the same criterion. This is a spicuous groups, such as fungi (Thormann and very conservative proxy for the eukaryotic species, Rice 2007 and references therein; Thormann et since many described eukaryotic species differ by al. 2007), diatoms (Bertrand et al. 2004; Delluomo less than 1% at this marker. However, since we are 1992) and testate amoebae (e.g. Charman and covering the whole eukaryotic diversity, including a 3

wide variety of phyla, we prefer to use this criterion 73% of the total 90 phylotypes from the bog water to facilitate the analyses, although this is likely an samples were retrieved using, respectively, the underestimation of the real specific diversity. primer combinations 1F/1498R and 82F/1498R. Of these, 28 phylotypes were retrieved using both Overall Eukaryotic Diversity in primer sets (31% of the total) (Supplementary Table S2). This illustrates the advantage of using more Praz-Rodet Peat Bog than one set of primers to screen environmen- Our five clone libraries from peat bog water and tal eukaryotic molecular diversity (López-García three from peat yielded a total of 1070 pos- et al. 2001; Von Wintzingerode et al. 1997). The itive clones (i.e. having incorporated an insert biases appear if the composition of the clone with the expected SSU rDNA fragment amplified), libraries is carefully examined; for instance the which corresponded to 132 different phylotypes clone PR3_4E_61 (an ), which is one of including sequences ranging between 1310 bp the most frequent of the June 2007 clone library (PR3_3E_134) and 2076 bp (PR3_3E_45). These was only amplified with forward primer EK 82F in length differences are due to length heterogeneities that sample, and never with primer EK 1F. Con- in the SSU rRNA gene. Clones derived from versely, euglyphid sequences were never obtained metazoans (15 phylotypes) or vascular (1 with primer EK 82F (Supplementary Table S2). For phylotype, closest BLAST hit Sphagnum palustre these reasons, an equivalent number of clones Y11370, similarity 99%) were not sequenced at were sequenced in each of the clone libraries built full length because the study of their respective for each sample. phylogenetic positions was beyond the scope of Full length sequences from each OTU were this paper (taxonomic affiliation of the metazoan then included into different alignments follow- sequences is given in Supplementary Table S1). ing their phylogenetic affiliation and having taken As the clone libraries derived from peat-only sam- into account the total number of sequences ples (directly associated with the decomposing retrieved for each group: (1) Opisthokonta, (2) Sphagnum biomass) were largely dominated by , (3) Alveolata, (4) Heterokonta (=Stra- metazoan sequences (up to more than 90% of menopiles) + , and (5) Chrysophyceae. all the clones), they were not further analysed, Unclassified sequences and other less represented although clones derived from eukaryotic microbes groups were included in a general eukaryotic align- which were exclusively found in these samples ment (6). All these alignments comprised both were fully sequenced and included in molecular environmental sequences in GenBank that were phylogenetic analyses (26 phylotypes). We recov- closest to our sequences and culture/isolated cell- ered a total of 745 positive clones corresponding derived sequences that served as reference and to 90 phylotypes (Supplementary Table S2) from covered a representative diversity of the respective our filtered water clone libraries. These libraries groups. Partial sequences shorter than 2/3 of the yielded respectively 206, 91, 176, 142 and 130 total SSUrRNA gene length were excluded from the positive clones and 33, 24, 29, 37 and 23 phylo- analysis. types, respectively, in the clone libraries derived The taxonomic distribution of our sequences was from the samples collected in March 2007, June large, since we identified sequences belonging to 2007, November 2007, January 2008 and May all major eukaryotic super-groups: Opisthokonta, 2008. Heterokonta, Rhizaria, Alveolata, , Rarefaction analysis was performed on all clone Viridiplantae (=Archaeplastida), and Amoebozoa libraries derived from peatbog water to estimate (Supplementary Fig. S2). Other groups of as yet to what extent the diversity of the samples could more ambiguous classification were also repre- be described with the number of clones analysed. sented, including the Centrohelida (Sakaguchi et Accumulation curves tended to flatten in all sam- al. 2007), and the Cryptophyta, the latter recently ples, indicating that the coverage of the diversity classified within the SAR eukaryotic supergroup was satisfactory (Supplementary Fig. S1) and that (Burki et al. 2008) and proposed to be member diversity of clone libraries could thus be compared. of a new super-group, the Hacrobia (Okamoto et Libraries built with two different primer sets at al. 2009). In addition, four OTUs (PR2_3E_18, the same sampling time were significantly different PR4_4E_41, PR3_3E_89 and PR3_3E_134), between each other with a probability of more than which represented five different sequences, could 95% except for January 2008, as indicated by LIB- not be assigned to any high-level eukaryotic taxon. SHUFF (Singleton et al. 2001). A total of 52 and These sequences branched robustly together in 66 phylotypes, representing respectively 58% and ML analysis (bootstrap value: 80%, see Fig 1); this 4

A. General eukaryotic tree Carpediemonas membranifera AY117416 Fornicata Dysnectes brevis AB263123 Streblomastix strix AY188885 1.00/92 1.00/87 1.00/98 Monocercomonoides sp. AY831435 Oxymonas sp. AB326383 1.00/97 0.96/68 Saccinobaculus sp. DQ525690 0.97/54 Preaxostyla Trimastix marina AF244905

1.00/82 Trimastix pyriformis AF244903 0.62/56 1.00/88 Trimastix pyriformis AF244904 Malawimonas jakobiformis AY117420 PR4_4E_41 0.99/65 Unidentified eukaryotic lineage PR2_3E_18 (+PR3_3E_134) 0.99/80 PR3_3E_89 Diphylleia rotans AF420478 anathema AF153206 0.82/99 0.77/61 ALVEOLATA 0.79/85 HETEROKONTA RHIZARIA HAPTOPHYTA Undertermined JJP-2003 AY268041 Heterophrys marina AF534710 Acanthocystis nichollsi AY749632 0.88/73 PR3_3E_45 Centrohelida 0.99/71 PR1_4E_11 0.86/59 Raphidiophrys ambigua AY305008 Pterocystis sp. JJP 2003 AY268043 Chlamydaster sterni AY268042 RHODOPHYTA B. Marine microhelizoan TCS2002 AF534711 100 Euglena gracilis AY029409 PRS2_3E_36 62 Phacus ranula AJ532484 Euglenea 0.05 0.92/73 Protoderma sarcinoidea Z47998 Peranema trichophorum AF386636 95 Astasia curvata AY004245 Volvox carteri X53904 1.00/80 100 Distigma sennii AF386644 Aphagea PR1_3E_30 88 Distigma proteus AF386638 0.99/80 PR3_4E_22 PRS2_4E_52 100 freshwater CH1_S2_19 AY821958 Scenedesmus obliquus AJ249515 100 PR3_3E_63 Spheno- 0.82/63 100 Petalomonas cantuscygni U84731 Oedogonium cylindrosporum DQ078297 Notosolenus ostium AF403159 monadales PRS2_4E_3 100 marine AT4-56 AF530520 Bodo saltans DQ207575 Diplonemida + 0.99/80 PRS2_4E_42 Diplonema ambulator AF380996 89 100 Kinetoplastida Schefferlia dubia X68484 marine LC22_5EP_32 DQ504349 Helianthus annuus AF107577 1.00/99 Psilotum nudum X81963 Viridiplantae Mesostigma viride AJ250108 Goniomonas truncata U03072 Cryptomonas ovata AF508270 PR4_4E_57 Cryptophyta Guillardia theta X57162 GLAUCOPHYTA AMOEBOZOA APUSOZOA

OPISTHOKONTA 0.08 Figure 1. A: Bayesian phylogenetic tree of a broad sampling of eukaryotes, illustrating the position of several unidentified eukaryotic sequences found in the Praz-Rodet peat bog, and also of some clones belonging to groups which presented lower diversity (Centrohelida, Cryptophyta and Viridiplantae).The tree was based upon an alignment of 108 sequences and 1090 characters. Numbers indicate respectively Bayesian posterior proba- bilities (PP) and maximum likelihood bootstrap values (BV). Only values above 0.75 and 50% are shown here, except for the nodes at the base of the novel eukaryotic group and of the Fornicata + Preaxostyla + Malawimonas group. The root is placed between unikonts and bikonts. B: ML phylogenetic tree representing sequences from euglenids encountered in this study. Alignment comprised 15 sequences and 1188 characters. Numbers at nodes represent bootstrap values; all values below 50% were omitted. Clones obtained in this study are shown in bold. 5

was confirmed by Bayesian analysis (PP = 0.99). isms considered as typical inhabitants of peat This clade branched within a group composed of bogs. The clone sequences PR3_4E_134 and Malawimonas + Fornicata + Preaxostyla (Excavata) PR4_4E_85 shared more than 99% similarity in the ML and the Bayesian analyses. However, with the sequences of, respectively, Hyalosphe- the statistical values which support this branching nia papilio (EU392153) and Nebela carinata are very weak (PP = 0.62; BV = 56); similar results (EU392144). These two species of arcellinid testate were obtained with or without the long-branch amoebae are considered as indicators for the most forming sequence PR3_3E_134. Its removal from oligotrophic conditions in peat bogs; when these the analysis did not change the topology of the conditions are met, they can be extremely common tree (Fig. 1). Thus, we consider this group as (Mitchell 2004; Mitchell et al. 2000). Microscopic an unidentified lineage, potentially belonging to observations confirmed the presence of these two excavates, which could also possibly be a novel, species in the samples. A third arcellinid clone deep branching eukaryotic clade. Further work sequence, PR3_4E_25, branched with Argynnia is needed to confirm these observations and to dentistoma. Its position in the arcellinid tree (Fig. 4) characterise this clade morphologically. The rest shows that, unlike PR3_4E_134 and PR4_4E_85, of the clone sequences branched robustly within it does not belong to the “core Nebelas”, the only described eukaryotic taxa; they are discussed here group of arcellinids which has been extensively in reference to their phylogenetic affiliation. characterised at the SSU rRNA gene level (Lara et al. 2008). Opisthokonta Two other amoebozoan clone sequences from peat (PRS2_3E_84 and PRS2_4E_19) branched The (excluding metazoans) appeared robustly with the flagellated form Phalansterium to be the most diverse eukaryotic high-level taxon, solitarium. Members of Phalansterium and with 33 different OTUs of which 28 were affiliated to the morphologically related Rhipidodendron are Fungi (Fig. 2 and Supplementary Table S2). Many also considered as characteristic inhabitants of clones derived from fungi were closely related to oligotrophic peat bogs (Kreutz and Foissner 2006; species typically isolated and cultured from peat Strüder-Kypke and Schönborn 1999). bogs such as Mortierella sp., Umbelopsis sp., Rhi- zophidium sp., Blittyomyces helicus, Cryptococcus sp., Candida sp., Mrakia sp., Penicillium purpuro- Alveolata genum, and Botrytis cinerea (Thormann and Rice were the most diverse group within 2007; Thormann et al. 2003). In addition, other the , with 16 OTUs, including rep- sequences were probably derived from symbiotic resentatives from numerous classes such as species, such as PR1_4E_47, which is closely , , and related to Symbiotaphrina spp., a genus of arthro- Spirotrichea (Fig. 5). Such a high diversity of pod symbionts of uncertain affiliation within the ciliates is an expected result in freshwater and Ascomycota (Noda and Kodama 1996). ˇ We also encountered sequences associated to soil environments (Lara et al. 2007b; Slapeta et a clade that may represent the deepest branch- al. 2005). Clone PR2_3E_66, closely related to ing group of fungi. This highly diverse clade, from Uroleptus pisces, could correspond to U. caudatus, which the only isolate-derived sequences belong described as one of the most characteristic species to the genus Rozella, has been tentatively named of acidic and oligotrophic peat bogs, reaching its “” (Lara et al. 2010) highest density in the submerged Sphagnum habi- In addition to Fungi, other sequences tat (Grolière 1977; Strüder-Kypke and Schönborn corresponded to free-living organisms such as 1999). As in the case of Rozella spp., the joint nucleariids and a choanoflagellate, and also organ- analysis of environmental and isolate-derived SSU isms that were most likely parasites, such as the rRNA gene sequences helped to give a taxonomic mesomycetozoans PR2_4E_07 and PR4_4E_73. identity to a clade represented only by environ- The latter, as a member of the Eccrinales, was likely mental sequences; the environmental LKM63 clade to be an arthropod parasite (Marshall et al. 2008). (Lara et al. 2007b; Van Hannen et al. 1999)was identified as corresponding to the Cyrtolophosida Amoebozoa as described in Dunthorn et al. (2008). Clone PR5_3E_39 branched within a clade that has been Seven OTUs belonged to the Amoebozoa (Fig. 3 erroneously claimed to correspond to freshwater and Supplementary Table S2). Here again, some representatives of the marine parasitic of our sequences were derived from organ- clade (Di Giuseppe and Dini 2004; 6

93 Bullera unica D78330 74 Cryptococcus aureus DQ437076 53 Cryptotrichosporon anacardii DQ242636 Basidiomycota 100 Cryptococcus humicola AB032637 66Cryptococcus skinneri AB032646 58 PR4_4E_66 Tremellales 70 Tremella foliacea L22262 100 60 Cryptococcus gastricus DQ645513 PR4_4E_90 Bullera taiwanensis AB072234 66 Cryptococcus aquaticus AB032621 Mrakia frigida DQ831017 68 Mrakia psychrophilia AJ223490 Cystofilobasidiales 99 PR1_4E_31 Itersonilia perplexans AB072228 Calvatia gigantea AF026622 68 Lycoperdon sp. AF026619 74 Lopharia mirabilis AY293141 53 PR1_4E_64 68 Agaricus aff. campestris DQ113914 PR3_4E_14 Psilocybe montana DQ465342 Agaricales 71 61 Armillaria mellea AY787217 PR2_3E_38 97 Xerula radicata AY654884 96 100 85 Flammulina velutipes AY665781 83Clitocybe nebularis DQ437681 PRS2_4E_20 Tricholoma myomyces DQ367422 78 Camptobasidium hydrophilum DQ198783 96 PR1_3E_31 62 Leucosporidium antarcticum DQ785788 Leucosporidiales 100 Rhodotorula yarrowii AB032658 + Microbotryales Microbotryum violaceum U77062 79 Friedmanniomyces endolithicus DQ066715 50 Hobsonia santessonii AF289658 Ascomycota 94 PRS2c_4E_01 Baudoinia compniacensis EF137357 Capnodiales 72 63 Hyperacidic freshwater RT3n2 AY082971 100 82 PR2_4E_93 97 Penicillium purpurogenum DQ365947 100 Aspergillus oryzae D63698 Eurotiales Coccidioides immitus X58571 77 Dothideomycete sp. G6-R56 AY190268 100 Leptosphaeria maculans DQ470993 Pleosporales 93 PR4_3E_67 Cenococcum geophilum L76616 Symbiotaphrina buchneri AY227716 100 60 98 Symbiotaphrina kochii DQ248314 PR1_4E_47 69Cryomyces minteri DQ066714

66 Ascocalyx abietina AF548076 F 65 PR3_4E_20

77 U Hyphozyma variabilis var. odora AJ496240 Helotiales 98 Heyderia abietis AY789295 99 Botrytis cinerea EF110887 N PR5_4E_15 74 Dipodascopsis anomala DQ519001 G 82 PR2_4E_28

98 Dipodascopsis tothii DQ519002 I 71 Dipodascopsis uninucleata U00969 Saccharomycetales 87 Myxozyma neotropica DQ519023 76 Candida sp. AB054525 PR5_3E_80 Trigonopsis vinaria AB018135 66 Rhizophydium sp. JEL223 DQ536492 62 Rhizophydium sp. JEL316 DQ536485 100 PR1_4E_45 Chytridiomycota 77 90 freshwater PFB11AU2004 DQ244014 84 Rhizophydium sp. JEL136 AY601710 Polychytrium aggregatum AY601711 Cladochytrium replicatum AY546683 65 97 Entophlyctis confervae-glomeratae EF014367 63 Spizellomycete sp. JEL371 DQ536490 Catenomyces sp. JEL342 AY635830 56 Rhizophlyctis rosea AY635829 66 100 Blyttiomyces helicus DQ536491 PR3_4E_79 88 freshwater P34.43 AY642701 90 Entophlyctis helioformis AY635826 PRS2c_4E_5 93 52 anoxic marine CCW64 AY180029 95 anoxic marine D1P02C04 EF100195 100 100 Chytriomyces angularis AF164253 PR1_4E_32 64 Chytridium polysiphoniae AY032608 100 freshwater PFA12SP2005 EU162643 95 freshwater PFG9SP2005 EU162638 100 soil Amb_18S_778 EF023558 Mortierella verticillata AF157145 74 PR5_4E_14 99 Mortierella parvispora AY129549 Echinosporangium transversale AF113424 83 PR3_4E_28 Mucorales 2 77 Spinochlamydosporium variabile EU688964 97 Endogone lactiflua DQ536471 Endogone pisiformis DQ322628 58 PRS2_4E_05 100 Umbelopsis ramanniana DQ322627 Mucorales 1 Umbelopsis isabellina AF157166 81 freshwater CV1_B2_34 AY821997 80 99 freshwater LKM11 AJ130849 100 freshwater Zeuk2 AY916571 “Rozellida” 57 PR2_4E_03 PRS2_4E_06 78 94 Rozella allomycis AY635838 Rozella sp. JEL347 AY601707 PR5_4E_71 92 100 anoxic marine CCW24 AY230211 83 PRS2_4E_31 66 84 anoxic saline NAMAKO_35 AB252775 soil WIM48 AM114806 anoxic marine D1P02G09 EF100212 84 Nuclearia delicatula AF349563 96 PR2_3E_125 100 Nuclearia moebiusi AF349565 72 Nuclearia pattersoni AY364635 87 Nuclearia simplex AF484687 PRS2_3E_65 78 Eccrinidus flexilis AY336700 PR4_4E_73 Mesomycetozoa 64 100 Enterobryus oxidi AY336710 Eccrinales 63 Palavascia patagonica AY682845 Ichthyophonus hoferi U25637 100 anoxic marine TAGIRI_27 AB191435 64 freshwater LKM51 AJ130859 100 PR2_4E_07 Anurofeca richardsi AF070445 89 62 Sphaeroforma arctica Y16260 70 soil Amb_18S_701 EF023370 100 Sphaeroeca volvox Z34900 59 PR5_4E_81 Choanomonada 89 98 Monosiga ovata AF271999 58 Salpingoeca amphoridium DQ059032 gracilis AY149897 100 Parazoanthus axinellae U42453 72 Tripedalia cystophora L10829 96 Leucosolenia sp. AF100945 Metazoa Plakortis sp. AF100948

0.03 Figure 2. ML phylogenetic tree representing the position of the opisthokont sequences (excluding Metazoa) from Praz-Rodet, based on an alignment of 139 taxa and 1195 characters. Root is placed between Meta- zoa + Choanomonada + Mesomycetozoa and Fungi + “Rozellida” + Nucleariida. Numbers at nodes represent bootstrap values; all values below 50% were omitted. Clones obtained in this study are shown in bold. Branches presenting a double barred symbol are reduced to half for clarity. 7

100 Physarum polycephalum X13160 100 Stemonitis flavogenita AF239229 89 Trichia persimilis AY643826 94 Acramoeba dendroida AF293897 soil isolate AND16 AY965863 FLABELLINEA CONOSEA 62 73 Filamoeba nolandi AF293896 72 hyperacidic freshwater RT5iin44 AY082989 64 sp. EU273440 Dictyostelium discoideum AY171066 64 100 Entamoeba equi DQ286371 75 Entamoeba histolytica X65163 83 Endolimax nana AF149916 Mastigamoeba simplex AF421218 99 83 87 Phreatamoeba balamuthi L23799 51 93 Mastigella commutans AF421219 Mastigamoeba sp. AF421220 Pelomyxa palustris AF320348 Multicilia marina AY268037 Dermamoeba algensis AY294148 61 Vannella placida AY294150 92 Vannella miroides AY183888 70 100 Vannella plurinucleolus AY121849 52 Vannella aberdonica AY121853 Vannellida 55 Vannella anglica AF099101 93 Vexillifera minutissima AY294149 71 Clydonnella sp. 50884 AY183892 Lingulamoeba leei AY183886 98 Korotnevella monacantholepis AY121854 72 88 Korotnevella stella AY183893 74 Korotnevella hemistylolepis AY121850 100 Neoparamoeba branchiphila AY714366 Dactylopodida 100 Neoparamoeba sp. SM53 AY193726 100 Neoparamoeba aestuarina AY121848 Vexillifera armata AY183891 73 proteus AJ314604 100 Chaos carolinense AJ314607 76 Amoeba leningradensis AJ314605 85 Hartmannella cantabrigiense AY294147 90 100 PR5_3E_71 Tubulinida 82 Glaeseria mira AY294146 Saccamoeba limax AS293902 anoxic marine BOLA868 AF372795 92 100 PR3_4E_134 (Hyalosphaenia papilio) 70 PR4_4E_85 (Nebela carinata) 93 Centropyxis laevigata AY848965 Arcellinida 98 92 PR3_4E_25 Heleopera sphagni AY848961 100 Paraflabellula hoguae AY277797 100 98 Paraflabellula reniformis AF293900 Leptomyxida AF293898 100 Echinamoeba exundans AF293895 Echinamoeba thermarum AJ489261 81 Vermamoeba vermiformis AY502959 58 Acanthamoeba castellanii M13435 96 Acanthamoeba lenticulata U94730 Acanthamoeba tubiashi AF019065 Acanthopodida 87 Balamuthia mandrillaris AF477019 63 Protacanthamoeba bohemica AY960120 100 freshwater LKM74 AJ130863 54 PR1_3E_80 79 Sappinia diploidea DQ122380 99 Thecamoeba similis AY294145 Thecamoebida Stenamoeba stenopodia AY294144 64 73 PRS2_3E_84 Phalansterium 98 Phalansterium solitarium AF280078 PRS2_4E_19 group 63 Mayorella sp. AY294143 68 Plakortis sp. AF100948 61 Trichoplax adhaerens L10828 64 Sphaeroforma arctica Y16260 Monosiga ovata AF271999 OPISTHOKONTA Nuclearia moebiusi AF349565 74 99 Saccharomyces cerevisiae EU011664 Schizosaccharomyces pombe X58056

0.06 Figure 3. ML phylogenetic tree representing the position of the amoebozoan sequences from Praz-Rodet. The tree was constructed using an alignment of 74 sequences and 939 positions and was rooted with 7 representative opisthokont sequences. Numbers at nodes represent bootstrap values; all values below 50% were omitted. Clones obtained in this study are shown in bold. Branches presenting a double barred symbol are reduced to half length for clarity. 8

Hyalosphenia papilio EU392153 PR3_4E_134 Arcellinida 96 Core Nebela carinata EU392144 nebelas 100 99 PR4_4E_85 Apodera vas EU392156 65 Bullinularia indica AY848970 68 Centropyxis laevigata AY848965 Heleopera rosea EU392157 75 Arcella hemisphaerica EU273445

73 90 PR3_4E_25 Argynnia dentistoma EU392158 Heleopera sphagni AY848964 Glaeseria mira AY294146 Tubulinida 99 Hartmannella cantabrigiensis AY294147

80 PR5_3E_71 Amoeba leningradensis AJ314605 100 Chaos carolinense AJ314607 Saccamoeba limax AF293902

0.05 Figure 4. ML phylogenetic tree representing the position of the arcellinid sequences from this study. The tree was derived from an alignment of partial SSU rRNA sequences (18 sequences, 668 positions) and was rooted with 6 representative Tubulinida sequences. Numbers at nodes represent bootstrap values; all values below 50% were omitted. Clones obtained in this study are shown in bold.

Lefèvre et al. 2008). Our analysis showed that (PR1_4E_51). are reg- these sequences are related to irri- ularly found in freshwater systems, where their tans, a prostomatean ectoparasite of marine flagellated free-living stage is part of the picoplank- fishes (Wright and Colorni 2002), in agreement ton assemblages (Lefèvre et al. 2008; Richards et with Guillou et al. (2008). Moreover, we showed al. 2005). One alveolate (PR2_3E_74) did not clus- here that the species Balanion masanensis (Kim ter with any previously known group (Fig. 5). et al. 2007), a marine free-living species is also included within this clade (Fig. 5). This illustrates Heterokonta and Rhizaria the wide ecological range of this ciliate group which includes both free-living and parasitic species, The largest diversity of was found in encountered from acidic and mineral-poor freshwa- the Chrysophyceae clade, with 13 different OTUs ter to marine environments. Despite the ecological widely distributed in the tree (Fig. 6), and there- width of the group, our clone was included in a very fore one of the most diversified taxa in our clone well supported clade of environmental sequences libraries. Such a result is in agreement with sim- deriving exclusively from freshwater environments ilar findings in an environmental DNA survey of (Fig. 5). another freshwater system, the oligotrophic, dim- We also identified a dinoflagellate clone that ictic Lake George (Richards et al. 2005). However, was closely related to the species Gloeodinium other less oligotrophic freshwater environments did montanum, a freshwater species of uncertain affil- not seem to host such great chrysophyte diversity iation, but which might be a representative of a (Berney et al. 2004; Lefèvre et al. 2008; Slapetaˇ larger undersampled freshwater clade (Logares et al. 2005). Chrysophyceae comprise both pho- et al. 2007). Clones affiliated to alveolate para- tosynthetic/mixotrophic and phagotrophic forms; it sitic taxa were also found in our survey, namely is difficult to predict the physiology of most of one Perkinsea OTU (PR2_3E_17), two Coccidi- the organisms from which our clone sequences asina (PR3_4E_33 and PR2_3E_116) and one derived, because the photosynthetic ability has 9

B. Gregarinasina

77 Parauronema longum AY212807 anoxic freshwater LEMD003 AF372797 77 Uronema marinum Z22881 Oligohymeno 61 anoxic freshwater LEMD154 AF372802 100 Anophyroides haemophila U51554 Metanophrys similis AY314803 99 PR1_4E_51 74 Mesanophrys carcini AY103189 -phorea 79 98 Gregarina niphandrodes AF129882 PR2_3E_53 87 freshwater CV1_B1_45 AY821939 soil Elev_18S_1214 EF024723 freshwater CV1_B2_52 AY821941 50 75 marine M3_18A05 DQ103857 Scuticociliatia Gregarina chortiocetes L31841 80 73 marine cLA12B08 EU446379 60 Gregarina caledia L31799 93 Cyclidium glaucoma Z22879 88 PRS2c_4E_17 100 Leidyana migrator AF457130 66 PR3_4E_59 Ophriocystis elektroscirrha AF129883 100 100 PR4_4E_42 PRS3_4E_78 freshwater CH1_S2_24 AY821921 98 Cyclidium plouneouri U27816 COCCIDIASINA 100 calkinsi AF100301 0.04 96 Paramecium multimicronucleatum AB252006 90 Apofrontonia dohrni AM072621 97 leucas AM072622 Peniculia 99 freshwater CV1_5A_22 AY821929 92 Lembadion bullinum AF255358 86 PR2_4E_22 83 freshwater PAB10AU2004 DQ244026 97 freshwater PAG3SP2005 EU162617 PR5_3E_39 100 freshwater cloneF AY829526 A. General alveolates 94 freshwater cloneE AY829525 Cryptocaryon 68 marine SCM27C43 AY665049 group C

97 100 Balanion masanensis AM412525 I 78 freshwater PAF4SP2005 EU162618 L I

Cryptocaryon irritans AF351579 O 95 Cyrtolophosis mucicola EU039899 freshwater LKM63 AJ130861 LKM63/ P

97 PR2_4E_94 Cyrtolophosida I Colpodea H 95 soil Amb_18S_1429 EF023962 O 99 cucullus EU039893

Colpodida R 97 Colpoda steinii DQ388599 98 100 Bryometopus atypicus EU039886 A 100 Bursaria truncatella U82204 Bursariomorphida Bryometopus pseudochilodon EU039887 +Bryometopida 82 PRS3_4E_35 88 soil Elev_18S_549 EF024200 100 PRS3_4E_60 Microthoracida Nassophorea 79 77 PRS2_3E_08 Pseudomicrothorax dubius X65151 +Synhymeniida Orthodonella apohamatus DQ232761 64 Colpidium caudatum EU264560 93 Obertrumia georgiana X65149 Nassulida Furgasonia blochmanni X65150 66 Oxytricha grandis AJ310486 Oxytricha sp. “Misty” AF508764 PR5_4E_23 Spirotrichea 84 100 Pattersoniella vitiphila AJ310495 lemnae AM233913 71 PR2_4E_33 86 Hemiurosoma terricola AY498651 Oxytricha granulifera X53486 92 freshwater CV1_2A_16 AY821922 Halteria grandinella AF164137 78 marine UI14B05 EU446357 PR2_3E_37 Stichotrichia freshwater PAC10AU2004 DQ244023 82 soil Elev_18S_1438 EF024903 97 PR2_4E_02 100 soil Amb_18S_5861 EF024076 Pseudourostyla cristata DQ019318 96 PR2_3E_66 99 Parauroleptus lepisma AF164132 100 Uroleptus pisces AF164131 100 Strombinium caudatum AY143573 Tintinnopsis tubulosoides AF399108 Choreotrichia 97 Aspidisca steini AF305625 Euplotidium arenarium Y19166 Hypotrichia 91 Gloeodinium montanum EF058238 PRS2c_4E_04 Thecadinium dragescoi AY238479 Pentapharsodinium tyrrhenicum AF022201 84 Dinoflagellata 74 Galeidinium rugatum AB195668 Prorocentrum micans AY585526 60 semilunatum 100 marine DH147-EKD18 AF290073 100 73 sp. AB295041 100 marine LC22_5EP_6 DQ504316 Marine alveolates marine DH144-EKD3 AF290064 Gr I D

79 I

100 Amoebophrya sp. AF472553 N 81 marine DH148-EKD27 AF290077 78 marine DH147-EKD3 AF290069 O 100 Syndiniales marine DH148_EKD14 AF290079 Z 88 =Marine alveolates

marine OLI11012 AJ402330 O 100 marine LC22_5EP_37 DQ504356 Gr II 100 sp. DQ146406 A 61 freshwater PAA8SP2005 EU162629 100 freshwater PAB5AU2004 DQ244038 62 Rana sphenocephala pathogen EF675616 93 PR2_3E_17 100 Perkinsea 82 71 freshwater PAD1SP2005 EU162628 100 marine SA2_2G9 EF527175 marine AT4_98 AF530536 andrewsi AY305326

82 pontica AY078092 A 92 77

Colpodella tetrahymenae AF330214 P 80 DQ174731 I freshwater LG32_03 AY919792 C freshwater PAF8AU2004 DQ244031 O 90 edax AY234843 100 marine anoxic BOLA566 AF372780 M 62

PR3_4E_33 P 64 PR2_3E_116 100 muris AF093497 L Coccidiasina E 85 91 Cryptosporidium parvum L16996 99 janae AY043206 X 88 U03070 A 99 Montastrea annularis symbiont AF238265 Adelina dimidiata DQ096835 PR2_3E_74 Unidentified alveolate HETEROKONTA 0.05 Figure 5. ML phylogenetic tree representing the position of A. general alveolate sequences from this study, based on an alignment of 130 taxa and 1233 characters, rooted with 13 heterokonts. B. the position of PR1_4E_51 within gregarines, based on an alignment of partial sequences (14 taxa, 798 characters) and rooted with 4 Coccidiasina. Clones obtained in this study are shown in bold. 10

82 soil Elev_18S_1490 EF024948 98 Spumella-like JBNA46 DQ388542 89 Poterioochromonas malhamensis AB023070 Ochromonadales 59 Poterioochromonas sp. AY699607 100 Ochromonas vasocystis EF165111 78 Spumella-like JBAS38 DQ388538 Ochromonas sp. CCMP2761 EF165126 57 Uroglena sp. EF165132 96 69 Ochromonas sp. AY520447 100 Ochromonas danica EF165108 Spumella-like JBC21 DQ388540 Ochromonas perlata EF165143 82 Dinobryon pediforme EU024993 Dinobryon sociale var. americana Ochromonas marina EF165138 69 Chrysoxys sp. AF123302 Ochromonas sp. aestuarii EF165124 73 Chrysolepidomonas dendrolepidota AF123297 88 Chrysonephele palustris U71196 83 Epipyxis aurea AF123301 Epipyxis pulchra AF123298 65 deep sea cold seep CYSGM7 AB275090 81Spumella danica AJ236861 Spumella elongata AJ236859 57 PR2_4E_39 Uroglena americana AF123290 93 66 PR4_4E_17 85 Spumella-like 194f DQ388551 61 Spumella-like 1036 DQ388565 Spumella-like1013 DQ388559 82 Spumella sp. AND30 AY965871 78 Spumella sp. SpiG AJ236862 92 Spumella obliqua AJ236860 PR2_4E_24 59 Chromulina chionophila M87332 57 Chrysosaccus sp. EF165121 100 Chromophyton cf. rosanoffii EF165106 Chrysosphaera parvula AF123299 55 Lagynion scherffelii AF123288 97 Chrysamoeba mikrokonta AF123287 Chrysamoeba pyrenoidifera AF123286 Chromulinales 92 Chrysamoeba tenera EF165102 PR3_4E_06 freshwater CV1_B1_76 AY821972 100 86 Oikomonas sp. SA2.2 AY520451 97 PR2_3E_07 71 Oikomonas mutabilis U42454 58 PR3_4E_15 Chromulina nebulosa AF123285 62 deep sea cold seep CYSGM8 AB275091 65 freshwater LG2105 AY919756 100 PR2_4E_09 Oikomonas sp. SA2.1 AY520450 92 Synurales 95 Chrysonebula flava EF165104 74 Chromulina sp. SAG17.97 EF165103 100 Hibberdia magna M87331 Hibberdiales 73 Chrysocapsa paludosa EF165145 57 98 Chrysocapsa vernalis AF123283 PRS2_3E_78 100 Ochromonas tuberculata AF123293 PR5_3E_64 57 PR2_3E_81 Ochromonadales 82 PR2_3E_45 85 Spumella-like JBM18 AY651092 54 PR2_3E_121 100 PR4_4E_14 Spumella-like JBM08 AY651098 99 Paraphysomonadales 99 Other heterokonts 0.03 Figure 6. ML phylogenetic tree representing the position of the chrysophyte clones from this study (86 taxa, 1261 characters), rooted with 6 other heterokonts. Clones obtained in this study are shown in bold. been lost independently several times in many lin- (1 OTU), bicosoecids (1 OTU), and labyrinthulids eages (Boenigk et al. 2005). Other photosynthetic (2 OTUs). The first group comprises sequences groups were represented by two eustig- from saprotrophic or parasitic organisms. They matophytes (PRS2_4E_40 and PRS2_3E_43) and have complex life cycles that include a motile one diatom (PR4_4E_05). The latter belonged to flagellated stage (zoospore), which is planktonic. genus Eunotia, a genus typical of acidic environ- MAST-12 is a clade of uncultured heterokonts ments (Flower 1986)(Fig. 7). whose sequences were first identified in marine Non-photosynthetic groups were represented by oxygen-depleted environments (Massana et al. (3 OTUs), uncultured MAST-12 clade 2004). These organisms were identified as small 11

Achlya bisexualis M32705 98 Leptolegnia caudata AJ238659 PR4_4E_25 91 Pythiopsis cymosa AJ238657 Oomycetes 75 Saprolegniales 91 Aphanomyces invadans DQ403202 78 PR3_3E_94 freshwater LG18_11 AY919745 87 Pseudoperonospora cubensis AY742760 64 97 Plasmopara viticola EF426540 Peronosporales Lagenidium giganteum X54266 83 76 marine BL010320.2 AY381206 Halocrusticida baliensis AB284578 100 anoxic marine BOLA515 AF372763 99 Haliphthoros sp. AB178865 PR3_4E_61 66 Eurychasma dicksonii AB368176 Haptoglossa sp. EU271966 74 soil Amb_18S_394 EF023114 51 MAST-12 KKTS_E5 EF219382 93 97 PRS2_4E_2 freshwater PSC4SP2005 EU162644 95 MAST-12 uncultured stramenopile clade MAST-12 KKTS_D3 EF219381 57 100 brackish anoxic BAQA72 AF372754 saline anoxic NAMAKO_29 AB252769 58 100 marine OLI51105 AF167414 100 Blastocystis cycluri AY266474 77 Blastocystis hominis AY244620 Opalinata Proteromonas lacertae U37108 Developayella elegans U37107 Hyphochytriales 99 53 Labyrinthuloides haliotidis U21338 66 Ulkenia visurgensis DQ100296 91 Ulkenia profunda AB022114 100 Thraustochytrium aureum AB022110 Thraustochytriales Labyrinthulida 87 Thraustochytrium striatum AB022112 HETEROKONTA 92 Quahog parasite QPX AF155209 Thraustochytrium pachydermum AB022113 54 73 84 freshwater LG03_03 AY919687 100 freshwater LG07_03 AY919701 Uncultured 81 freshwater LG08_03 AY919705 100 freshwater CV1_B1_3 AY821979 labyrithulid clade 100 PR2_3E_77 hyperacidic freshwater RT5iin25 AY082983 100 Labyrinthula sp. AN-1565 AB022105 88 100 Labyrinthula sp. L59 AB095092 80 Labyrinthulales Labyrinthuloides minuta L27634 marine HE001005.112 AY381171 100 Diplophrys sp. AF304465 PR3_4E_52 88 PR2_3E_73 98 52 freshwater PSA11SP2005 EU162646 50 Paramonas globosa AY520452 Pseudodendromonadales 100 Siluania monomastiga AF072883 freshwater CH1_2B_3 AY821965 100 89 Cafeteria roenbergensis AY827851 100 74 Bicosoeca petiolata AY520444 Bicosoeca vacillans AY520445 Anoecales and Bicoecales 100 Caecitellus parvulus AY642126 Caecitellus pseudoparvulus DQ220712 86 Chrysophyceae 91 Synchroma sp. grande DQ788730 Picophagus flagellatus AF185051 64 Eustigmatos magna U41051 76 Vischeria helvetica AF045051 99 Pseudocharaciopsis minuta U41052 Eustigmatophyceae PRS2_4E_40 59 Nannochloropsis gaditana AF133819 100 PRS2_3E_43 Monodopsis subterranea U41054 Pseudotetraedriella kamillae EF044311 89 Eunotia sp. AM501963 PR4_4E_05 71 Eunotia formica AM502040 Bacillariophyceae 66 freshwater CV1_B2_38 AY821975 85 Eunotia monodon var. asiatica AB085831 Bacillaria paxillifer M87325 Bacillariophytina Nitzschia frustulum AJ535164 90 Cylindrotheca closterium DQ082739 82 Licmophora juergensii AY633759 99 92 Tabularia fasciculata EF423417 Diatoma vulgare var. linearis EF465466 Biddulphia sp. EF585585 Coscinodiscophytina 71 80 Bolidophyceae 94 compressa EF456755 62 PR4_3E_18 Euglypha rotunda X77692 87 Euglypha cf . ciliata EF456754 Euglyphida Filosa 100 PR2_3E_98 97 Assulina seminulum EF456749 72 60 PR1_3E_44 chromatophora X81811 Cercomonas metabolicus DQ211597 66 freshwater LKM48 AJ130858 96 Pseudodifflugia cf. gracilis AJ418794 98 PR1_4E_55 Tectofilosida

73 PR3_4E_92 RHIZARIA 75 Bodomorpha minima AF411276 84 Proleptomonas faecicola AF411275 92 Heteromita globosa U42447 Glissomonadida 100 soil Amb_18S_1122 EF023523 69 PR2_3E_130 100 soil Amb_18S_469 EF023178 Gymnophrys cometa AJ514866 57 69 freshwater PCG6AU2004 DQ243994 96 89 hyperacidic freshwater RT5iin4 AY082993 Proteomyxida 91 freshwater PCH11AU2004 DQ243993 71 PRS2_4E_63 PR1_3E_70 Nuclearia-like filose amoeba N-Por AF289081 67 soil Amb_18S_7211 EF024097 100 Polymyxa betae AF310902 67 100 PR1_3E_26 Spongospora subterranea f. sp. nasturtii AF310901 79 99 anoxic saline NAMAKO_19 AB252759 Phagomyxa bellerocheae AF310903 100 PR4_3E_90 Vampyrellida Platyreta germanica AY941201 PRS2_3E_116 Unidentified Rhizarian 0.05 Figure 7. ML phylogenetic tree representing the position of the heterokont and rhizarian clones from this study (146 taxa, 1161 characters), with the root placed between these two groups. Clones obtained in this study are shown in bold. Branches presenting a double barred symbol are reduced to a quarter for clarity. 12

phagotrophic flagellates by combining fluores- Other Taxa cent in situ hybridisation and scanning electron microscopy (Kolodziej and Stoeck 2007). In the One cryptophyte OTU (PR4_4E_57), closely meanwhile, other related sequences have been related to Cryptomonas ovata, a species already found in freshwater (Lefèvre et al. 2008) and reported in peat bogs (Kreutz and Foissner 2006), soil, though being misidentified as a member appeared in our survey. Three identical sequences of the apicomplexan parasitic group of Cryptomonas nucleomorph were also obtained (Lesaulnier et al. 2008). In our analysis, all (not shown). Viridiplantae were represented in MAST-12 clones from freshwater and soil form particular by sequences related to the gen- together a robust clade to which our clone era Oedogonium (PRS2_4E_3 and PRS2_4E_42) belongs. It seems that there is only little over- and Scenedesmus (PRS2_4E_52), two ubiquitous lap between freshwater/soil and marine genotypes, freshwater genera. In addition, two centrohelid which suggests that the barrier imposed by salin- sequences related to genus Acanthocystis, and two ity has been crossed relatively rarely during the euglenids also appeared. All these clones, which evolution of this clade. A similar situation has belong to groups whose diversity was lower in our been observed for many other protists such study, are represented in Figure 1. as cryptophytes (Shalchian-Tabrizi et al. 2008), dinoflagellates (Logares et al. 2007) and euglyphid Relationship Between Protist testate amoebae (Heger et al. 2010). Bicosoe- Communities and Environmental cids are common members of the freshwater picoplankton; the low diversity observed in our Variables samples contrasts with observations in another The different physico-chemical variables mea- oligotrophic, though very different, freshwater sys- sured (Supplementary Table S3) were typical tem (Lake George), where a wide diversity was for ombrotrophic Sphagnum-dominated peatlands: found (Richards et al. 2005). Labyrinthulids were high DOC (>30 mg/l on average) and values for both represented by one OTU (PR2_3E_77) branching total nitrogen (tot-N) (<1.25 mg/l on average) and + within a clade constituted only by environmental NH4 (<0.13 mg/l on average) were comparable to sequences, commonly found in freshwater sys- those observed in other European ombrotrophic tems (Amaral Zettler et al. 2002; Richards et peat bogs (Hoosbeek et al. 2002). High DOC al. 2005; Slapetaˇ et al. 2005). The other one concentration in peatlands is mainly due to the clustered robustly with the enigmatic long branch- presence of recalcitrant humic acids while low N forming Diplophrys sp., confirming its position content is due to the fact that these ecosystems are within labyrinthulids as suggested by Cavalier- fed only by atmospheric deposition and are there- Smith and Chao (2006). fore nutrient-poor. Several OTU affiliated to Euglyphida were found Total nitrogen values were higher in the sum- in our study, in agreement with previous obser- mer and fall samples (late June 2007, November vations of peat bog environments based on light 2007 and May 2008) than in winter and early spring + microscopy (Kreutz and Foissner 2006; Strüder- samples. In contrast, NH4 values were highest in Kypke and Schönborn 1999). Clone PR4_3E_18 early spring at snowmelt (March 2007), remained branched robustly (bootstrap 94%) with the species low in June 2007, November 2007 and January Euglypha compressa, which belongs to a cluster 2008 and increased again in May 2008. In con- of moss-inhabiting testate amoebae (Lara et al. trast, total nitrogen was higher in the June 2007 2007a). Another clone (PRS2_4E_63) was related and November 2007 samples. Such trends are in to the newly described rhizarian family Limnofil- accordance with previous observation in other bog idae, a group of freshwater filamentous protists environments (Kilroy et al. 2008; Vitt et al. 1995). (Bass et al. 2009). In addition to Filosa, a sequence DOC has its lowest value in June 2007, and then of a Phytomyxea (a clade of obligate para- increases gradually to reach its highest value in sites) was retrieved, as well as a vampyrellid (a January 2008. March 2007 and May 2008 show group which contains some apparently exclusive intermediate values. TOC followed almost exactly predators). Moreover, one clone from these values. the peat (PRS2_3E_116) did not cluster with any As a measure of the biological variables that known rhizarian group. Its affiliation to Rhizaria could be associated to eukaryotic dynamics, we is nevertheless justified, as it branched robustly counted prokaryotic cells from all the water sam- with other rhizarians in the general eukaryotic tree ples. These counts showed lower values in January (BV = 100%; Fig. 1). 2008 and comparable values for the other four 13

samples; these numbers increased with higher while they represented only 10% and 4%, of the temperatures, being maximal in late spring and total number of sequences in May and June, summer (Supplementary Table S3). Our prokary- respectively (Supplementary Fig. S2). In the DCA otic counts were roughly one order of magnitude ordination, they appear linked to low tempera- below the results of Gilbert et al., in other peat- tures and low bacterial numbers (Fig. 8), which bog systems (1998a). However, the environment might be perceived as a contradiction with the they investigated (squeezed Sphagnum fallax)was bacterivorous/mixotrophic lifestyle of most of these a poor-fen, which is less oligotrophic than the Praz- organisms. A possible explanation could be the Rodet peat bog and thus would be expected to predation pressure during the most productive contain a higher density of bacteria. periods. Interestingly, the only two chrysomonad sequences which had a peak of abundance Seasonal Patterns of Microbial Eukaryotic in the summer samples were most likely pho- totrophic: PR3_4E_06 branched within the genus Communities Chrysamoeba, a very distinctive genus of amoe- We carried out a detrended correspondence anal- boid organisms which comprises only phototrophs ysis (DCA) in which the water chemistry variables (Preisig and Andersen 2002) and PR5_3E_64 and the prokaryotic cell counts were passively pro- sequence shared >99% identity to Ochromonas jected in the ordination space defined by the clone tuberculata, another photosynthetic chrysomonad data (abundance of phylotypes in each sample). In (Fig. 6). the DCA ordination (Fig. 8) samples which appear Fungal diversity (excluding “Rozellida”) was low- close together in the ordination space have simi- est in March and did not vary much the rest lar community structure while samples which are of the year. Clone PR1_4E_41 branched within distant have different community structure. The the Mrakia clade, a known group of psychrophilic environmental variables were then projected on this yeasts whose relatives have been found in glaciers multivariate space. The length of the vectors repre- and polar environments (De García et al. 2007). senting environmental variables indicates how well Interestingly, this clone was only found in the Jan- the variable fits the multivariate ordination space. uary sample. Sequences from multicellular fungi Thus Bacteria + Archaea counts and DOC are less can derive from various sources, such as spores or correlated to seasonal changes in community struc- mycelium fragments. Therefore, their relative abun- + ture than total N, NH4 and temperature. The fall dance in the different libraries did not necessarily and winter samples clustered together in the ordi- reflect their true abundance in the environment, and nation space (Fig. 8) while the May and June will not be discussed further. samples are quite different from this group and also Among the other groups, ciliate diversity showed from each other. Separate analyses on different a bimodal pattern with peaks in spring and fall subsets of the data showed that this pattern was (Supplementary Fig. S2). Some clone sequences mostly due to the 16 most abundant species, which were typically found in high numbers during win- each accounted for at least 5% of the total clones ter conditions, such as for instance PR2_3E_66 (data not shown). (Supplementary Fig. S3). This clone appeared Although a detailed assessment of the ecology associated with many chrysomonad OTUs in the and seasonal pattern of each taxonomic group DCA ordination (Fig. 8). Given that members is beyond the goals of this study, the follow- of genus Uroleptus are large protist predators ing observations can be made from the position (Foissner and Berger 1996) chrysophytes might of clones and samples in the DCA ordination. be their main prey. In contrast, PR3_4E_59, affil- Fungi and chrysophytes were the two most diverse iated to the genus Cyclidium (Fig. 5), a genus taxa within the clones selected for this analy- of small bacterivorous ciliates, appeared associ- sis (Supplementary Table 2) with respectively 14 ated with high bacterial numbers found during and 11 different OTUs. Therefore an analysis the warm months. This clone represented up of seasonal patterns of diversity may be perti- to 42% of the total number of sequences in nent for those two groups. Interestingly, for both May 2008 (Supplementary Fig. S3). Such a groups the highest diversity was not observed peak in abundance of small bacterivorous cili- during the summer period but in January. Chrys- ates during the summer has been observed in ophyte diversity then declined during the spring a eutrophic freshwater system in Finland, where and summer and increased again in November. they were shown to be the most important reg- The abundance of chrysophyte sequences repre- ulators of bacterial populations (Zingel et al. sented 62% of all sequences retrieved in January, 2007). 14

3.80

PR5_3E_39 PR5_3E_80 PR5_4E_71 PR5_3E_64 PR5_4E_14

PR2_4E_22

PR2_4E_07 PR2_3E_125 PR3_4E_59

2.55 May 2008 PR3_3E_94 PR3_4E_20

PR3_4E_28

PR4_4E_57 PR3_4E_15

PR1_4E_35 PR2_3E_45 N-NH4 PR1_3E_31 PR3_4E_52 1.29 PR1_3E_18 PR1_4E_47 PR1_3E_70 PR4_3E_90 PR4_4E_66 PR4_4E_25 March 2007 Bacteria + Archaea PR4_4E_90 PR4_4E_17 November 2007 C/N January 2008 PR2_3E_07 PR2_3E_121 T PR2_3E_81 PR4_4E_14 PR2_3E_66 PR2_4E_02PR2_4E_09 PR4_3E_67 PR2_4E_03 PR2_4E_28 PR2_3E_37 PR2_4E_33 PR2_3E_130 PR2-4E-24 June 2007 0.04 PR2_4E_111 PR2_3E_17 PR2_3E_98 DOC

PR3_4E_06 PR3_4E_92 PR3_3E_45 PR3_3E_63 PR3_4E_79 PR3_3E_89 PR4_3E_18 PR3_4E_134 PR3_4E_22 PR3_4E_25 PR3_4E_33 PR3_4E_61 PR3_3E_134 PR3_3E_16 PR3_4E_14

TotN -1.22 -0.95 0.29 1.52 2.76 4.00

Chrysophyta Various Ciliophora Centrohelida Fungi Parasitic alveolates Euglyphida Cryptophyta Arcellinida Undetermined labyrinthulids Oomycota “Rozellida” Euglenida Nucleariida Others Figure 8. Detrended correspondence analysis (DCA) analysis of Eukaryotic clones obtained from water sam- ples collected at the Praz-Rodet Bog (Swiss Jura). Environmental variables are projected passively in the ordination space. Only clones representing more than 0.97% of the total number of clones of the sample were included in the analysis. Metazoa were excluded. Symbols represent the taxonomic affiliation of the clones. The length of the vectors representing the environmental parameters has been multiplied five times for clarity. Symbols and clone codes figuring in boxes linked by a line to a point share the exact same coordinates in the ordination space. 15

The total amount of OTUs related to testate Methods amoebae (Arcellinida and Euglyphida) was higher in abundance and diversity in the warmer months Sampling: Water samples were taken at the Praz-Rodet peat ◦ ◦ (summer and fall), in agreement with direct obser- bog, located in Switzerland (46 33’N; 06 10’E; altitude 1041m) vations of Sphagnum mosses taken from the field at five different times: 08.03.2007 (ice melting, early spring), 25.06.2007 (summer conditions), 01.11.2007 (autumn condi- (Heal 1964), although such differences are not tions), 24.01.2008 (winter, under ca. 5 cm ice) and 08.05.2008 always clear (Warner et al. 2007). Likewise, sphe- (late spring). Water (1000 ml) was sampled at the same nomonad euglenids (PR3_3E_63) and centrohelid location, in a pool where vegetation was dominated by half followed the same trend (PR3_3E_45), submerged Sphagnum cuspidatum, with presence of Drosera as well as an unidentified stramenopile related to rotundifolia, Scheuchzeria palustris and Carex limosa. 500 ml of water were filtered through a 50 ␮m-pore mesh and then Diplophrys sp. (PR3_4E_52, Supplementary Fig. through a 0.2 ␮m-pore GTTP filter (Millipore). The 0.2 ␮m filter S3). was used for DNA extraction and the 0.2 ␮m filtrate for water The clone sequence PR4_4E_57, affiliated to chemistry analysis. Filtered water was conserved at -20 ◦Cfor cryptomonads, was found in great numbers in further chemical analyses. The remaining 500 ml of water was fixed with glutaraldehyde (4% final concentration) and stored the November sample, where it represented 19% at 4 ◦C for prokaryotic cell counts. All sequences derived from of all sequences. This percentage fell in Jan- these water samples are coded PR1 to PR5. Additionally peat uary and in May (respectively 4% and less than sediments were collected. Sequences derived from these peat 1%). Such a peak of abundance may suggest a sediment samples are coded PRS. bloom, which is typical for Cryptomonas species Water chemistry analyses: Temperature (T), total nitrogen population dynamics in peat bogs, although these (tot-N), total carbon (tot-C), dissolved organic carbon (DOC), and ammonium (NH4) were measured on the water samples. events generally occur in summer (Kreutz and Tot-N was measured using a Shimadzu TOC-V TNM1 analyser Foissner 2006). Another clone which might have by thermal decomposition (chemiluminescence method). Tot-C followed a “bloom-like” dynamic was PR2_3E_17 and DOC were measured by the company CAR (Illkirch, France) (Supplementary Fig. S3), a member of the para- by chemical oxidation of carbon and subsequent measuring of the released CO2.NH4 was determined by Colorimetric- sitic alveolate clade Perkinsea which represented Autoanalyzer Method by Dr. Luca Bragazza at the University up to 14% of all clones encountered in the March of Ferrara (Italy). The C/N ratio was calculated from the tot-C sample, and which did not appear elsewhere. Since and tot-N data. Perkinsea are parasites of a variety of animal and Prokaryotic cell counts: Prokaryotic cells were stained with ␮ protozoan hosts, it is likely that their population DAPI (4,6 diamino 2 phenylindol), filtered on 0.2 m black mem- brane filters, and examined by epifluoresence microscopy. The dynamics followed the variation of their host’s abun- image was recorded using a digital camera. Twenty to 30 ran- dance. dom fields were counted for each sample, with an average This study demonstrates that Sphagnum bogs of 9-14 cells per field. Counts were expressed as number of are home to a broad diversity of eukaryotic microor- prokaryotic cells per millilitre of water. DNA extraction, PCR amplification, cloning and ganisms. Testate amoebae are one of the best sequencing: DNA was extracted from the filters as described known groups of eukaryotic microorganisms living elsewhere (Lara et al. 2009). In addition, peat was also in peatlands. With a maximum identified diversity collected for DNA extraction, which was performed using a of eight phylotypes it is likely, though, that only a MoBio Power SoilTM DNA extraction kit (Carlsbad, CA USA) fraction of the total diversity of testate amoebae following the manufacturer’s instructions. In order to limit potential PCR biases, two forward eukaryotic -specific was recorded. Indeed, an average number of 22 primers and one reverse were used, resulting in two sets species has been reported in bog pool ecosystems of primers. These primers were, respectively, EK 1F (CTG- with Sphagnum cuspidatum in the Jura Moun- GTTGATCCTGCCAG), EK 82F (GAAACTGCGAATGGCTC) tains (Mitchell et al. 1999). In the case of the and EK 1498R (CACCTACGGAAACCTTGTTA). Consequently, arcellinids (three clone types), DNA extraction and two clone libraries per sample were built. PCR reactions were carried out in 25 ␮l of reaction buffer containing 1 ␮l DNA PCR biases might explain their low level of detec- template (∼1-5 ng), 1.5 mM MgCl2, dNTPs (10 nmol each), 20 tion, as is often the case with amoebozoans in pmol of each primer, and1UTaqDNApolymerase (Promega). general (Berney et al. 2004). Similar conclusions PCR reactions were performed under the following conditions: ◦ ◦ can be drawn for other groups as well which present 35 cycles (denaturation at 94 C for 15 s, annealing at 50 C for 30 s, extension at 72 ◦C for 2 min) preceded by 2 min long and/or divergent SSU rRNA genes, such as ◦ ◦ denaturation at 94 C, and followed by 8 min extension at 72 C. the other amoebozoans, the euglenids, the cen- Amplicons were cloned into pCR2.1 Topo TA cloning vector trohelids or members of the putative new clade (Invitrogen) and transformed into E. coli TOP10’ One Shot presented in this work. Group-specific amplifica- cells (Invitrogen) according to the manufacturer’s instructions. tion protocols would be required to assess the true Clone inserts were amplified with vector T7 and M13R primers, and inserts of the expected size were sequenced directly using diversity of individual taxa along temporal and spa- either specific or vector primers by Cogenics (Meylan, France). tial scales, which would improve considerably our Sequence and phylogenetic analyses: Initially, we understanding of peat bog microbial ecology. obtained partial SSU rRNA gene sequences, which were 16

trimmed for ambiguities and aligned using the software MUS- correspondence analysis (DCA) on this community data (phy- CLE v. 3.6 (Edgar 2004). Groups of similar sequences (greater lotype absolute frequency data) in which the water chemistry than 97% identity), which will be referred in this work as OTUs variables and the prokaryotic cell counts were passively pro- (Operational Taxonomic Units), were identified by building dis- jected in the ordination space defined by the clone’s data. The tance (neighbour joining) trees using the software Clustal X DCA was done using the software Canoco version 3.1 (Ter v.1.82 (Thompson et al. 1997). Representative sequences of Braak 1988-1992). the different OTUs were then selected and fully sequenced to obtain almost complete SSU rDNAs. Full-length sequences were checked for the presence of chimeras: the affiliation of each half of the suspicious sequences was determined using Acknowledgements BLAST (Altschul et al. 1997). These sequences were also anal- ysed with KeyDNAtools (Zimmerman et al., unpublished). Final phylogenetic trees were reconstructed using the maximum like- We thank Laure Guillou for fruitful discussion, lihood (ML) method with the software TREEFINDER (Jobb et Thierry J. Heger and Thierry Perruchoud for help al. 2004), under the GTR + + I model with four substitution rate with sampling, Andy Siegenthaler for advice with categories. Nonparametric bootstrap analyses were performed prokaryote counting, and Jean-David Teuscher with TREEFINDER using 1000 replicates. In parallel, the same (CEAL, ENAC, EPFL) and Luca Bragazza (Univer- datasets were analysed using RAxML (Stamatakis et al. 2008) to test if the general topology of the trees remained congru- sity of Ferrara, Italy) for water chemistry analyses. ent, which they did, when the RAxML algorithm was applied. This work was supported by an ATIP Plus grant Ambiguous positions in the alignment were discarded from phy- of the French Centre National de la Recherche logenetic analyses. When the placement of a sequence in a Scientifique (CNRS), section “Dynamique de la tree required confirmation, we performed a Bayesian analy- biodiversité” to P.L.G. E.L. was personally funded sis on the same dataset, using the software Mr Bayes v. 3.1.2 (Ronquist and Huelsenbeck 2003) with the GTR + + I model by an ATIP-associated CNRS research assis- of sequence evolution. They were repeatedly run from a ran- tant contract and by an SNFR (Swiss National dom starting tree and run well beyond convergence. We used Fund for Research) Ambizione fellowship (PZ00P2- four hidden Markov chains, and up to 5 000 000 generations. 122042). Funding to EM by Swiss NSF project no. The number of sequences and that of unambiguously-aligned positions used in the construction of the different trees were, 205321-109709/1 is kindly acknowledged. respectively: (1) 139 and 1195 for the Opisthokonta, the root being placed between Fungi + Nucleariida and Mesomyce- tozoa + Choanomonadea + Metazoa; (2) 74 and 939 for the Appendix A. Supplementary data Amoebozoa, rooted with some Opisthokonta; (3) 130 and 1233 for the Alveolata, rooted with the Heterokonta; (4) 146 and Supplementary data associated with this arti- 1161 for the Heterokonta plus Rhizaria, the root being placed between these two groups; (5) 86 and 1261 for the Chryso- cle can be found, in the online version, at phyta, rooted with some closely related heterokonts; and (6) doi:10.1016/j.protis.2010.05.003. 108 and 1090 for the general eukaryote alignment, with the root placed between unikonts and bikonts. In this last tree, the analysis was performed with and without long-branch forming sequence PR3_3E_134, to ensure that the topology of the tree References was not affected by long branch attraction. The eukaryotic taxa that formed too long branches were analysed separately; these Amaral Zettler LA, Gomez F, Zettler E, Keenan BG, Amils R, were the Gregarinasina (14 sequences, 798 positions, rooted Sogin ML (2002) Eukaryotic diversity in Spain’s River of Fire. with the Coccidiniasina) and the Euglenida (15 sequences, Nature 417:137 1188 positions, rooted with the Kinetoplastida + Diplonemida). Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, In addition, Arcellinida were treated separately due to the Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: fact that most sequences available in GenBank are signifi- a new generation of protein database search programs. Nucleic cantly shorter than the whole SSU rRNA gene sequence (19 Acids Res 25:3389–3402 sequences, 668 positions, rooted with some Tubulinea). Align- ments are available from the authors upon request. Sequences Azam F, Fenchel T, Field JG, Gray JS, Meyerreil LA, reported in this paper have been deposited in the GenBank Thingstad F (1983) The ecological role of the water column database under accession numbers GQ330569-GQ330643 microbes in the sea. Mar Ecol -Progr Ser 10:257–263 and FJ976648-FJ976650. Statistical analyses: Rarefaction calculations as a measure Baldauf SL (2003) The deep roots of eukaryotes. Science of coverage of the clone libraries were performed using the 300:1703–1706 software DOTUR (Schloss and Handelsman 2005). In addition, Bass D, Chao EEY, Nikolaev S, Yabuki A, Ishida K, the composition of clone libraries from the same sample and Berney C, Pakzad U, Wylezich C, Cavalier-Smith T (2009) different primer combinations were compared between them- Phylogeny of novel naked filose and reticulose Cercozoa: selves using the software LIBSHUFF v 0.96 (Singleton et al. Granofilosea cl. n. and revised. Protist 160: 2001). We assessed the seasonal patterns of eukaryotic micro- 75–109 bial communities and associated environmental variables by studying a reduced data set that included 59 phylotypes, which Berney C, Fahrni J, Pawlowski J (2004) How many novel appeared with a minimum percentage of 0.97% of all clones, eukaryotic kingdoms? Pitfalls and limitations of environmental thus removing 32 rare phylotypes. We performed a detrended DNA surveys. BMC Biol 2:1–13 17

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Supplementary Tables

Highly diverse and seasonally dynamic protist community in a pristine peat bog

Lara, E. a, b, c, d, Mitchell, E.A.D. b, c, d, Moreira, D. a, López García, P. a

a Unité d’Ecologie, Systématique et Evolution, UMR CNRS 8079, Université Paris- Sud, bâtiment 360, 91405 Orsay Cedex, France. b Swiss Federal Research Institute WSL, Wetlands Research Group, Lausanne, Switzerland c École Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Ecological Systems, Station 2, Lausanne, Switzerland d University of Neuchâtel, Institute of Biology, Laboratory of Soil Biology, CH-2009 Neuchâtel, Switzerland

correspondence: [email protected] 21

Table S1. Total numbers of metazoan clones per phylotype (de ned with a 3% threshold) encountered in the libraries derived from peat bog water in relation with closest BLAST hit. Numbers at the bottom of the table represent total clone numbers.

Number of sequences per date Metazoa taxa First BLAST hit (% sequence similarity) 3.2007 6.2007 11.2007 1.2008 5.2008 Uncultured Chaetonotus-like gastrotrich Gastrotrich 1 0 105 10 0 21 clone CH1_S1_38 AY821982 (97%) Uncultured Chaetonotus-like gastrotrich Gastrotrich 2 1 30 0 4 3 clone CH1_S1_38 AY821982 (97%)

Gastrotrich 3 0 1 0 0 0 Chaetonotus neptuni AM231774 (93%)

Stenostomum bryophilum isolate K04_50 Turbellaria 1 0 7 5 0 0 FJ384807 (99%)

Turbellaria 2 0 0 6 0 2 Eukaryote clone Elev_18S_5021 EF025005 (94%)

Prismatolaimus cf. dolichurus JH-2004 Nematod 1 36 3 0 0 0 isolate PriMDol1Z AY284727 (99%)

Nematod 2 0 0 1 0 0 Malaconothrus gracilis EF091424 (99%)

Uncultured nematode clone SType 5 Nematod 3 0 0 0 0 1 AJ875122 (98%)

Copepod 1 Harpacticoida 0 0 0 6 0 Attheyella crassa EU380307 (98%)

Copepod 2 Cyclopoida 0 1 0 0 0 Cyclops sp. UK-RJH-2004 AY626998 (98%)

Acari: Oribatid 0 0 1 1 0 Hydrozetes lacustris EU433987 (99%)

Rotifera: Bdelloid 0 5 0 0 0 Rotaria rotatoria 18S DQ089736 (99%)

Rotifera: Monogonont 1 0 11 0 0 0 Polyarthra remata DQ297716 (99%)

Uncultured metazoan clone Orly20 Rotifera: Monogonont 2 0 2 0 0 1 FJ577821 (97%)

Rotifera: Monogonont 3 0 9 3 2 6 Lepadella patella 18S AY218117 (99%) total clones 280 265 202 155 130 (unicell + metazoan) 22 Table S2. Total numbers of microbial eukaryote clones per phylotype (dened with a 3% threshold) encountered in the libraries derived from peat bog water. Clones that were considered for DCA analysis are indicated, as well as the primer set which successfully amplied them. In addition, taxonomic identication, sequence length and percentage of similarity with closest BLAST hit are indicated. Number of sequences Ampli ed with Ampli ed with Retained for Sequence Phylotype Taxonomic identi cation primers primers First BLAST hit (%sequence similarity) 3.2007 6.2007 11.2007 1.2008 5.2008 DCA analysis length 1F/1498R 82F/1498R

PR2_4E_24 Heterokonta; Chrysophyceae 8 1 40 30 0 yes yes yes 1446 Soil agellate AND30 AY965871 (98%)

PR2_3E_81 Heterokonta; Chrysophyceae 4 0 4 14 0 yes yes yes 1445 Chrysophyceae sp. CCMP2296 (96%) EU247834

PR2_3E_07 Heterokonta; Chrysophyceae 40 0 0 3 0 yes yes yes 1441 Oikomonas sp. SA-2.2 AY520451 (98%)

PR2_3E_45 Heterokonta; Chrysophyceae 11 1 13 22 4 yes yes yes 1444 Chrysophyceae sp. CCCM41 EF165134 (94%)

PR5_3E_64 Heterokonta; Chrysophyceae 0 0 0 0 3 yes yes yes 1440 Ochromonas tuberculata AF123293 (99%)

Uncultured eukaryote clone: CYSGM-8 AB275091 PR2_4E_09 Heterokonta; Chrysophyceae 2 0 2 0 0 yes no yes 1445 (97%) Chromophyton cf. rosanofi strain CCMP2751 PR2_3E_121 Heterokonta; Chrysophyceae 3 0 0 7 0 yes yes yes 1466 EF165106 (92%) Chrysamoeba tenera strain UTCC273 (96%) PR3_4E_06 Heterokonta; Chrysophyceae 0 2 5 1 0 yes yes yes 1436 EF165102

PR3_4E_15 Heterokonta; Chrysophyceae 20 2 0 1 6 yes yes yes 1436 Spumella-like agellate JBAF35 AY651071 (96%)

PR4_4E_17 Heterokonta; Chrysophyceae 0 0 10 9 0 yes yes yes 1448 Spumella-like agellate 194f DQ388551 (99%)

Uncultured marine eukaryote clone SA2_1F7 PR4_4E_14 Heterokonta; Chrysophyceae 0 0 3 1 0 yes no yes 1497 EF527128 (90%)

PR4_4E_05 Heterokonta; Bacillariophyceae; Eunotia 0 0 1 1 0 no no yes 1445 Eunotia sp. AT-73Gel02 AM501963 (98%)

Uncultured stramenopile clone BOLA320 AF372762 PR3_4E_61 Heterokonta; Oomycetes, Unidentied Oomycetes 1 20 0 2 0 yes no yes 1466 (91%)

PR4_4E_25 Heterokonta; Oomycetes, Saprolegniales 0 0 2 0 0 yes no yes 1461 Aphanomyces invadans DQ403202 (97%)

PR3_3E_94 Heterokonta; Oomycetes, Saprolegniales 0 1 0 1 6 yes yes yes 1453 Pythiopsis cymosa AJ238657 (98%)

PR4_4E_12 Heterokonta; bicosoecides; pseudodendromonadales 0 0 1 0 0 no no yes 525 Adriamonas peritocrescens AF243501 (95%)

Uncultured freshwater eukaryote clone LG33-04 PR2_4E_64 Heterokonta; bicosoecides; pseudodendromonadales 1 0 0 0 0 no no yes 207 AY919797 (94%) Uncultured stramenopile clone PSA11SP2005 PR2_3E_73 Heterokonta; bicosoecides; pseudodendromonadales 1 0 0 0 0 no yes no 1444 EU162646 (94%) Uncultured labyrinthulid clone CV1_B1_3 AY821979 PR2_3E_77 Heterokonta; labyrinthulida (environm. Group RT5iin25) 1 0 0 1 0 no yes no 1488 (86%)

PR3_4E_52 Heterokonta; labyrinthulida; undetermined labyrithulids 4 9 3 3 14 yes yes yes 1574 Uncultured eukaryote clone 528-O7 EF586082 (92%)

PR2_3E_66 Alveolata; Ciliophora; Intramacronucleata; Spirotrichea 44 0 4 8 0 yes yes yes 1435 Uroleptus pisces AF164131 (99%)

Environmental sample clone Elev_18S_1438 PR2_4E_02 Alveolata; Ciliophora; Intramacronucleata; Spirotrichea 3 0 1 0 0 yes yes yes 1438 EF024903 (99%)

PR2_3E_37 Alveolata; Ciliophora; Intramacronucleata; Spirotrichea 6 0 0 0 0 yes yes yes 1438 Halteria grandinella AF194410 (98%)

PR2_4E_33 Alveolata; Ciliophora; Intramacronucleata; Spirotrichea 4 0 0 0 0 yes yes yes 1437 Hemiurosoma terricola AY498651 (98%) 23 Table S2. Total numbers of microbial eukaryote clones per phylotype (de ned with a 3% threshold) encountered in the libraries derived from peat bog water. Clones that were considered for DCA analysis are indicated, as well as the primer set which successfully ampli ed them. In addition, taxonomic identi cation, sequence length and percentage of similarity with closest BLAST hit are indicated. Number of sequences Ampli ed with Ampli ed with Retained for Sequence Phylotype Taxonomic identi cation primers primers First BLAST hit (%sequence similarity) 3.2007 6.2007 11.2007 1.2008 5.2008 DCA analysis length 1F/1498R 82F/1498R

PR5_4E_23 Alveolata; Ciliophora; Intramacronucleata; Spirotrichea 0 0 0 0 1 no no yes 1434 Oxytricha sp. Misty AF508764 (97%)

PR3_4E_59 Alveolata;Ciliophora; Oligohymenophorea; Scuticociliatia 0 6 10 0 54 yes yes yes 1440 Cyclidium glaucoma EU032356 (91%)

Uncultured marine eukaryote clone NA2_2E2 PR2_3E_53 Alveolata;Ciliophora; Oligohymenophorea; Scuticociliatia 1 0 0 0 0 no yes no 1424 EF526761 (92%)

PR4_4E_42 Alveolata;Ciliophora; Oligohymenophorea; Scuticociliatia 0 0 0 1 0 no no yes 1447 Cyclidium glaucoma EU032356 (91%)

Uncultured marine eukaryote clone M2_18E08 PR2_4E_22 Alveolata; Ciliophora; Oligohymenophorida; Peniculida 1 0 0 0 2 yes no yes 1396 DQ103844 (88%) Environmental sample clone Amb_18S_1429 PR2_4E_94 Alveolata; Ciliophora; Colpodida; Cyrtolophosida 1 0 1 0 0 no no yes 1417 EF023962 (94%)

PR5_3E_39 Alveolata; Ciliophora; Prostomatea 0 0 0 0 8 yes yes yes 1434 "Uncultured Amoebophrya" clone F AY829526 (97%)

Uncultured eukaryote clone LEMD012 AF372799 PR1_4E_51 Alveolata; ; Gregarinida 0 0 0 1 0 no no yes 1417 (97%) Uncultured alveolate clone BOLA566AF372780 PR3_4E_33 Alveolata; Apicomplexa; 0 1 0 0 0 no no yes 1435 (96%) Uncultured marine alveolate clone PAF8AU2004 PR2_3E_1 16 Alveolata; Apicomplexa; 1 0 0 0 0 no yes no 1453 DQ244031 (89%)

PR2_3E_17 Alveolata; Perkinsea 28 0 0 0 0 yes yes yes 1447 Uncultured alveolate clone AT4-98AF530536 (92%)

PR2_3E_74 Alveolata 1 0 0 0 0 no yes no 1434 Ichthyophonida sp. LKM51 AJ130859 (84%)

PR2_3E_98 Rhizaria; Cercozoa; Filosa; Euglyphida; Euglyphidae 2 4 4 0 1 yes yes no 1471 Euglypha cf .ciliata EF456754 (98%)

PR4_3E_18 Rhizaria; Cercozoa; Filosa; Euglyphida; Euglyphidae 0 22 14 1 0 yes yes no 1469 Euglypha compressa EF456755 (99%)

PR1_3E_44 Rhizaria; Cercozoa; Filosa; Euglyphida; Assulinidae 0 0 0 1 1 no yes no 1469 Assulina seminulum EF456749 (99%)

Uncultured cercozoan isolate DB-2703-11 EU567236 PR1_3E_70 Rhizaria; Cercozoa; Filosa; Proteomyxidae 0 0 0 4 0 yes yes no 1913 (96%)

PR1_4E_55 Rhizaria; Cercozoa; Filosa; Tecto losida 0 0 0 1 0 no no yes 1454 Pseudodifugia cf. gracilis AJ418794 (97%)

PR3_4E_92 Rhizaria; Cercozoa; Filosa; Tecto losida 0 1 0 0 0 no no yes 1472 Protaspis obliqua isolate 2 FJ824122 (92%)

PR2_3E_130 Rhizaria; Cercozoa; Filosa; Glissomonadina 2 0 0 0 0 yes yes no 1467 Cercozoa clone Amb_18S_1122 EF023523.1 (97%)

Rhizaria; Cercozoa; Endomyxa; PR4_3E_90 0 0 2 0 0 no yes no 1457 Uncultured eukaryote clone LEMD004 (87%) Phytomyxea; Rhizaria; Cercozoa; Endomyxa; Phytomyxea; Uncultured plasmodiophorid clone D20 EU910610 PR1_3E_26 0 0 0 1 0 no yes no 1480 Plasmodiophorida (96%)

PR4_4E_73 Opisthokonta; Ichthyophonida; Eccrinales 0 0 1 0 0 no yes no 1465 Eccrinidus exilis isolate SP A11C45 AY336700 (90%)

PR2_4E_07 Opisthokonta; Ichthyophonida 3 0 0 0 1 no yes yes 1450 Ichthyophonida sp. LKM51 AJ130859 (97%)

PR5_4E_81 Opisthokonta; Choanoagellida; Codonosigidae 0 0 0 0 1 no no yes 1435 Sphaeroeca volvox Z34900 (97%) 24 Table S2. Total numbers of microbial eukaryote clones per phylotype (de ned with a 3% threshold) encountered in the libraries derived from peat bog water. Clones that were considered for DCA analysis are indicated, as well as the primer set which successfully ampli ed them. In addition, taxonomic identi cation, sequence length and percentage of similarity with closest BLAST hit are indicated. Number of sequences Ampli ed with Ampli ed with Retained for Sequence Phylotype Taxonomic identi cation primers primers First BLAST hit (%sequence similarity) 3.2007 6.2007 11.2007 1.2008 5.2008 DCA analysis length 1F/1498R 82F/1498R

PR2_3E_125 Opisthokonta; Nucleariidae 1 0 4 0 4 yes yes yes 1538 Nuclearia thermophila AB433328 (97%)

PR2_4E_03 Opisthokonta; "Rozellida" 4 0 0 0 0 yes yes yes 1415 Uncultured eukaryote clone Ivry06 FJ577810 (87%)

Rhizophlyctis rosea isolate AFTOL-ID 43 AY635829 PR5_4E_71 Opisthokonta; "Rozellida" 0 0 0 0 2 no no yes 1450 (90%) Lepidostroma akagerae voucher Ertz 7673 FJ171730 PR3_3E_16 Opisthokonta; Fungi; Basal fungal lineages 0 1 0 0 0 yes yes no 593 (85%) Uncultured clone T6_II_2b_09 EF629013 PR1_3E_18 Opisthokonta; Fungi; Zoopagomycotina 0 0 0 4 0 yes yes no 300 (91%)

PR5_4E_14 Opisthokonta; Fungi; Mucoromycotina; Mucorales 2 0 0 0 0 5 yes no yes 1448 Mortierella parvispora AY129549 (99%)

Basidiobolus ranarum isolate AFTOL-ID 301 PR3_4E_28 Opisthokonta; Fungi; Mucoromycotina; Mucorales 2 0 2 0 0 4 yes no yes 1478 AY635841 (87%) Chytriomyces angularis isolate AFTOL-ID 630 PR1_4E_32 Opisthokonta; Fungi; Chytridiomycota; Chytridiales 0 0 0 1 0 no no yes 1447 AF164253 (99%) Rhizophydium sp. JEL223 isolate AFTOL-ID 2007 PR1_4E_45 Opisthokonta; Fungi; Chytridiomycota; Rhizophydiales 0 0 0 1 0 no no yes 1450 DQ536492 (96%) Blyttiomyces helicus isolate AFTOL-ID 2006 PR3_4E_79 Opisthokonta; Fungi; Chytridiomycota 0 1 0 0 0 yes no yes 1450 DQ536491 (99%) Opisthokonta; Fungi; Ascomycota; pezizomycotina; Botryotinia fuckeliana strain DSM877 (=Botropsis PR5_4E_15 0 0 0 0 1 yes no yes 1452 Helotiales cinerea) EF1 10887 (100%) Opisthokonta; Fungi; Ascomycota; pezizomycotina; PR3_4E_20 0 1 1 1 7 yes no yes 1452 Hyphozyma variabilis var. odora AJ496240 (99%) Helotiales Opisthokonta; Fungi; Ascomycota; pezizomycotina; Penicillium purpurogenum strain HS-A82 DQ365947 PR2_4E_93 1 0 0 0 0 no no yes 1461 Eurotiales (99%) Opisthokonta; Fungi; Ascomycota; pezizomycotina; Phoma exigua var. exigua clone 26-7 EU342890 PR4_3E_67 0 0 2 1 0 yes yes yes 1452 dothideales (96%) Opisthokonta; Fungi; Ascomycota; pezizomycotina; Symbiotaphrina kochii isolate AFTOL-ID 1902 PR1_4E_47 0 0 0 4 0 yes yes yes 1452 leotideomycetes; dothideales FJ176833 (98%) Opisthokonta; Fungi; Ascomycota; Saccharomycotina; Dipodascopsis (=Babjevia) anomala strain NRRL Y- PR2_4E_28 2 0 0 0 0 yes no yes 1460 Saccharomycetes; Lipomycetales 7931 DQ519001 (99%) Opisthokonta; Fungi; Ascomycota; Saccharomycotina; PR5_3E_80 0 0 0 0 2 yes yes yes 1451 Candida sp. BG02-7-15-009-2-1 AY520194 (95%) Saccharomycetes; Lipomycetales Opisthokonta; Fungi; Basidiomycota; Pucciniomycotina; Leucosporidium antarcticum isolate AFTOL-ID 1550 PR1_3E_31 0 0 0 2 0 yes yes yes 1458 Microbotryomycetes; Leucosporidiales DQ785788 (99%) Opisthokonta; Fungi; Basidiomycota; PR1_4E_31 0 0 0 1 0 no no yes 1450 Mrakia frigida AFTOL-ID 1818 DQ831017 (99%) Agaricomycotina;Tremellomycetes; Cysto lobasidiales Opisthokonta; Fungi; Basidiomycota; PR4_4E_66 0 0 2 2 0 yes no yes 1452 Cryptococcus skinneri AB032646 (99%) Agaricomycotina;Tremellomycetes; Tremellales Opisthokonta; Fungi; Basidiomycota; PR4_4E_90 0 0 2 0 0 yes yes yes 1450 Bullera taiwanensis AB072234 (98%) Agaricomycotina;Tremellomycetes; Tremellales Opisthokonta; Fungi; Basidiomycota; Agaricomycotina; PR1_4E_64 0 0 0 1 0 no no yes 1455 Lopharia mirabilis AY293141 (99%) Agaricomycetes; Agaricales; Lycoperdaceae Opisthokonta; Fungi; Basidiomycota; Agaricomycotina; Psilocybe montana isolate AFTOL-ID 820 DQ465342 PR3_4E_14 0 1 0 0 0 yes no yes 1456 Agaricomycetes; Agaricales; Strophariaceae (99%) Opisthokonta; Fungi; Basidiomycota; Agaricomycotina; PR2_3E_38 1 0 0 0 0 no yes no 1878 Oudemansiella radicata AY654884 (93%) Agaricomycetes;Agaricales; Armillaria group 25 Table S2. Total numbers of microbial eukaryote clones per phylotype (de ned with a 3% threshold) encountered in the libraries derived from peat bog water. Clones that were considered for DCA analysis are indicated, as well as the primer set which successfully ampli ed them. In addition, taxonomic identi cation, sequence length and percentage of similarity with closest BLAST hit are indicated. Number of sequences Ampli ed with Ampli ed with Retained for Sequence Phylotype Taxonomic identi cation primers primers First BLAST hit (%sequence similarity) 3.2007 6.2007 11.2007 1.2008 5.2008 DCA analysis length 1F/1498R 82F/1498R

PR3_3E_45 Centrohelida; Acanthocystidae 0 5 0 0 1 yes yes yes 2076 Acanthocystis spinifera AY749630 (95%)

PR1_4E_1 1 Centrohelida; Acanthocystidae 0 0 0 1 0 no no yes 2069 Acanthocystis sp. Oxford8 AY749626 (87%)

PR3_4E_25 Amoebozoa; Tubulina; Arcellinida; unidenti ed Arcellinid 0 3 1 0 0 yes no yes 1541 Argynnia dentistoma EU392158 (91%)

Amoebozoa; Tubulina; Arcellinida; core Nebelas; PR3_4E_134 0 1 0 0 0 yes no yes 1958 Hyalosphenia papilio from Sweden EU392153 (99%) Hyalosphenia

PR4_4E_85 Amoebozoa; Tubulina; Arcellinida; core Nebelas; Nebela 0 0 1 0 0 no no yes 1989 Nebela carinata from Sweden EU392143 (99%)

PR5_3E_71 Amoebozoa; Tubulina; Euamoebida; Hartmanellidae 0 0 0 0 1 no yes no 1577 Hartmannella cantabrigiensis AY294147 (81%)

Environmental clone Elev_18S_1278 AF386643 PR2-3E-03 Amoebozoa; Phalansterium clade 1 0 0 0 0 no yes no 1442 EF024776 (94%) Environmental clone Elev_18S_1517 EF024971 PR1_3E_80 Amoebozoa; LKM74 environmental clade 0 0 0 1 0 no yes no 1581 (87%) Uncultured sphenomonad euglenozoan clone PR3_3E_63 Excavata; Euglenozoa; Euglenida; Sphenomonadales 0 2 7 0 0 yes yes no 1716 CH1_S2_19 AY821958 (96%)

PR3_4E_22 Excavata; Euglenozoa; Euglenida; Aphagea 0 1 0 0 0 no no yes 1814 Distigma elegans SAG 224.80 (79%)

Archaeplastida; Viridiplantae; ; PR1_4E_35 0 0 0 2 0 yes no yes 513 Trebouxia asymmetrica Z21553 (99%) Trebouxiophyceae; Microthamniales Archaeplastida; Viridiplantae; Chlorophyta; PR2_4E_1 11 2 0 0 0 0 yes no yes 513 Chloromonas sp. 047-99 AF514406 (99%) Trebouxiophyceae; Microthamniales Archaeplastida; Viridiplantae; Chlorophyta; Chlorophyceae; Chloromonas reticulata strain SAG 29.83 AJ410448 PR1_3E_30 0 0 0 1 0 no yes no 1448 Chlamydomonadales (97%)

PR4_4E_57 Cryptophyta; Cryptomonadina 0 0 34 5 1 yes yes yes 1432 Cryptomonas sp. M1634 AM901361 (99%)

PR4_4E_41 EK Incertae sedis 0 0 1 0 0 no no yes 1485 Cyanidium caldarium strain:55B AB091232 (76%)

PR3_3E_134 EK Incertae sedis 0 2 0 0 0 yes yes no 1310 Uncultured eukaryote clone R T5iin3 AY082996 (80%)

PR2_3E_18 EK Incertae sedis 1 0 0 0 0 no yes no 1386 Uncultured eukaryote clone R T5iin3 AY082996 (79%)

Environmental sample clone Elev_18S_792 PR3_3E_89 EK Incertae sedis 0 1 0 0 0 no yes no 1371 EF024493 (80%) total Unicell 206 91 176 142 130 26

Table S3. Values obtained for the di erent environmental parameters measured in peat bog water at the di erent sampling times. TN= total nitrogen, N-NH4= total ammonium, DOC =dissolved organic carbon, TOC =total organic carbon, Temp= water temperature, and Bact/ml = bacterial cells per ml water.

Date TN (mg/l) N-NH4 (mg/l) DOC (mg/l) TOC (mg/l) Temp °C Bact / ml March 2007 0.903 0.232 24.9 25.1 2.0 9.08E+04 June 2007 1.780 0.084 10.1 10.3 16.5 1.36E+05 November 2007 1.674 0.059 34.3 34.9 6.5 1.27E+05 January 2008 0.737 0.092 55.3 57.2 1.0 1.21E+05 May 2008 1.102 0.178 31.9 32.5 20.0 1.39E+05 27

Supplementary Figures

Highly diverse and seasonally dynamic protist community in a pristine peat bog

Lara, E. a, b, c, d, Mitchell, E.A.D. b, c, d, Moreira, D. a, López García, P. a

a Unité d’Ecologie, Systématique et Evolution, UMR CNRS 8079, Université Paris- Sud, bâtiment 360, 91405 Orsay Cedex, France. b Swiss Federal Research Institute WSL, Wetlands Research Group, Lausanne, Switzerland c École Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Ecological Systems, Station 2, Lausanne, Switzerland d University of Neuchâtel, Institute of Biology, Laboratory of Soil Biology, CH-2009 Neuchâtel, Switzerland

correspondence: [email protected]

28

Figure S1: Rarefaction analysis performed on the clone libraries derived from peat bog water 29

Figure S2: Taxonomic composition of the clone libraries of all peat bog water samples as percentages of total clone numbers. 30

Figure S3: Histograms representing the repartition of the most representative phylotypes in all peat bog water samples as percentage of total clone numbers. The phylotypes presented here either represented more than 5% of all clones, either represented more than 10% of all clones derived from a single library.