Complex Communities of Small Protists and Unexpected Occurrence of Typical Marine Lineages in Shallow Freshwater Systems

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Environmental Microbiology (2014) doi:10.1111/1462-2920.12591

Complex communities of small protists and unexpected occurrence of typical marine lineages in shallow freshwater systems

Marianne Simon, Ludwig Jardillier, Introduction Philippe Deschamps, David Moreira, Aquatic ecosystems occupy most of the Earth surface. Gwendal Restoux, Paola Bertolino, Oceans alone cover around 71% of that surface Purificación López-García* (Costanza, 1999). Lakes, ponds and reservoirs cover Unité d’Ecologie, Systématique et Evolution, CNRS more than 3% (nearly 4.5 million km2) of non-oceanic UMR 8079, Université Paris-Sud, 91405 Orsay, France. regions (Downing et al., 2006). Although comparatively smaller, these freshwater systems offer a large array and Summary diversity of ecological niches including various trophic levels, light accessibility, temperature and oxygen con- Although inland water bodies are more heterogene- centrations. This is especially true for small and shallow ous and sensitive to environmental variation than lentic inland ecosystems (sizes from 0.001 to 0.1 km2), oceans, the diversity of small protists in these eco- which are widespread, varied and numerous, correspond- systems is much less well known. Some molecular ing to more than 99% of the total number of lakes on Earth surveys of lakes exist, but little information is avail- (Downing et al., 2006). Microbial communities dominate able from smaller, shallower and often ephemeral aquatic ecosystems and their activity has profound impact freshwater systems, despite their global distribution at global scales, being largely implicated in carbon fixation and ecological importance. We carried out a compara- (Li, 1994; Jardillier et al., 2010) and climate regulation tive study based on massive pyrosequencing of (Simó, 2001). Within these communities, microbial amplified 18S rRNA gene fragments of protists in the eukaryotes play key roles in nutrient cycling acting as 0.2–5 μm size range in one brook and four shallow photosynthesizers, heterotrophs (predators, parasites) or ponds located in the Natural Regional Park of the mixotrophs (Caron, 1994; Zubkov and Tarran, 2008; Chevreuse Valley, France. Our study revealed a wide Jardillier et al., 2010; Massana, 2011). diversity of small protists, with 812 stringently Small eukaryotes (< 20 μm) have been known to consti- defined operational taxonomic units (OTUs) belong- tute a non-negligible part of aquatic microbial communities ing to the recognized eukaryotic supergroups (SAR − in both freshwater and marine systems for a long time Stramenopiles, Alveolata, Rhizaria − Archaeplastida, (Johnson and Sieburth, 1982; Corpe and Jensen, 1992). Excavata, Amoebozoa, Opisthokonta) and to groups Based on their cell size, small protists were initially classi- of unresolved phylogenetic position (Cryptophyta, fied in nanoeukaryotes (cells between 2 and 20 μm in Haptophyta, Centrohelida, Katablepharida, Telone- diameter) and picoeukaryotes (cells ≤ 2 μm). In the last mida, Apusozoa). Some OTUs represented deep- two centuries, many small eukaryotic species have been branching lineages (Cryptomycota, Aphelida, described, including phototrophs such as prasinophytes Colpodellida, Tremulida, clade-10 Cercozoa, HAP-1 and other chlorophytes (Knight-Jones and Walne, 1951; Haptophyta). We identified several lineages previ- Stockner, 1988; Guillou et al., 1999), which suggested a ously thought to be marine including, in addition to potentially significant role in primary production (Johnson MAST-2 and MAST-12, already detected in freshwater, and Sieburth, 1982), but also heterotrophs (Fenchel, 1982; MAST-3 and possibly MAST-6. Protist community Patterson and Larsen, 1991). Indeed, the ecological impor- structures were different in the five ecosystems. tance of heterotrophic nanoflagellates as predators has These differences did not correlate with geographical long been acknowledged (Fenchel, 1982; Wright and distances, but seemed to be influenced by environ- Coffin, 1984). However, being too small to show easily mental parameters. identifiable, unambiguous morphological differences, their true diversity remained largely inaccessible and their tax- Received 13 July, 2014; accepted 5 August, 2014. *For correspondence. E-mail [email protected]; Tel. +33 169 157 608; Fax onomy poorly studied or oversimplified (many of these tiny +33 169 154 697. protists were simply classed as incertae sedis).

In the past 15 years, the use of molecular methods current knowledge, to be specific to marine (e.g. based on 18S rRNA gene analysis has largely overcome Syndiniales; Guillou et al., 2008) or freshwater (e.g. that problem, allowing studying the phylogenetic diversity HAP-1 lineage of haptophytes; Šlapeta et al., 2005; and distribution of small protists at unprecedented scales. Shalchian-Tabrizi et al., 2011) environments. Even within Most such studies largely explored oceanic systems, lineages present both in marine and freshwater systems, including surface waters (Diez et al., 2001; Moon-van der 18S rRNA sequences obtained from freshwater samples Staay et al., 2001), the deep sea (López-García et al., often form phylogenetic clades distinct from those of 2001) and the coastal regions (Massana et al., 2004a; oceanic sequences, suggesting a limited number of, Romari and Vaulot, 2004; Cheung et al., 2010). They mostly ancient, freshwater colonization events followed revealed a huge protist diversity encompassing members by radiations (Logares et al., 2009). That scarcity of of all recognized eukaryotic supergroups (SAR − marine-freshwater transitions has been observed for Stramenopiles, Alveolata, Rhizaria − Archaeplastida, many small eukaryote groups such as the perkinsids Excavata, Amoebozoa and Opisthokonta) as well as lin- (Bråte et al., 2010), haptophytes (Shalchian-Tabrizi et al., eages of uncertain position in the eukaryotic tree, such as 2011; Simon et al., 2013) or MAST lineages (Massana Haptophyta, Cryptophyta or Picozoa (López-García and et al., 2014), and could be explained by the difficulty Moreira, 2008; Massana, 2011; Seenivasan et al., 2013; of crossing the salinity barrier (Logares et al., 2009). Moreira and López-García, 2014). Molecular surveys also However, freshwater aquatic systems remain largely allowed the discovery of lineages previously unknown in understudied and massive high-throughput sequencing spite of their abundance, such as new clades affiliated techniques have been applied to very few among them. to alveolates (Groups I and II), which turned out to be Therefore, it might be possible that failure to detect some members of the classical Syndiniales (Groisillier et al., of these lineages is due to undersampling. Furthermore, 2006; Harada et al., 2007; Guillou et al., 2008) and the vast majority of protist molecular diversity studies in stramenopiles (the MAST clades, for marine strameno- freshwater have been conducted in lakes (e.g. Richards, piles) (Diez et al., 2001; López-García et al., 2001; 2005, Lepère and Boucher, 2006; Lepère et al., 2008). Moon-van der Staay et al., 2001; Stoeck et al., 2003; However, smaller systems like ponds or brooks have Massana et al., 2004b). Thought for a long time to be been overlooked, despite their abundance, distribution exclusively marine, the MAST groups remain poorly and ecological importance. Yet, previous analyses known. In a few cases a correspondence has been found suggest that they host several lineages undetected in between particular organisms and their environmental marine environments or lakes (Šlapeta et al., 2005; sequences (e.g. the colonial protist Solenicola setigera Simon et al., 2013). In addition, because they are and the MAST-3; Gómez et al., 2011). However, many shallow and small, this kind of freshwater systems may other MAST lineages have not yet been linked to any display very different local physico-chemical conditions cultured or described organisms (Massana et al., 2014), and, hence, they constitute ideal models to test whether even though fluorescent in situ hybridization labelling has local environmental selection is more influential than provided some hints on their morphology, life style and geographic distance in determining community composi- ecology (Massana et al., 2002; 2006). tion, which remains controversial (Green et al., 2004; The molecular exploration of very small protists in Martiny et al., 2006). A recent study on lakes suggest freshwater began slightly later than in the oceans and that geographic distance might be important to explain also revealed a large diversity (Lefranc and Thénot, protist biogeography (Lepère et al., 2013), but lakes 2005; Richards, 2005; Šlapeta et al., 2005). In lakes, have much more buffering capacity than small shallow alveolates (especially ciliates and Perkinsozoa, by con- systems. trast to the Syndiniales-Duboscquellids-dinoflagellate In this work, we have investigated the diversity of small dominance in marine systems), stramenopiles, crypto- eukaryotes (essentially the 0.2–5 μm cell size fraction) in phytes and fungi were found to be abundant, but protists a set of shallow freshwater environments by amplification belonging to the Archaeplastida, Rhizaria and Cercozoa of 18S rRNA gene fragments and direct high-throughput or groups of uncertain affiliation were also diverse 454-pyrosequencing. We selected one brook and four (Lefranc and Thénot, 2005; Šlapeta et al., 2005; Lepère ponds located in the same geographic area (the Natural et al., 2008; Zhao et al., 2011; Mangot et al., 2012). In Regional Park of the Chevreuse Valley, France) but dif- some cases, a significant part of the detected diversity fering in size, depth and physico-chemical conditions. The was composed only of environmental sequences without objectives of our work were threefold: (i) describing protist known close relatives in databases (Lefèvre et al., 2008; diversity at unprecedented depth in this kind of habitats, Mangot et al., 2012; Triadó-Margarit and Casamayor, (ii) checking whether increasing sequence depth leads to 2012). Some groups were observed in both oceans and the discovery in freshwater systems of eukaryotes previ- freshwater systems whereas others seem, according to ously thought to be exclusively marine and (iii) testing

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Protist diversity in shallow freshwater systems 3 which parameters (geographical distance versus physico- ria to avoid sequence artefacts and chimeras. The reads chemical characteristics) determine eukaryotic microbial from the 12 different samples (the 0.2–5 μm cell fraction community structure. of five systems with replicas plus the two replicas of the 5–30 μm for the Etang des Vallées) were treated together in order to define operational taxonomic units (OTUs) that Results could be fully compared among samples. All those To study protist diversity in five small and shallow fresh- sequences grouped in 812 OTUs defined using a cut-off water ecosystems, we selected one brook and four value of 98% sequence identity. From these, 768 OTUs shallow ponds in the Natural Regional Park characterized (128 661 reads) were detected at least in the 0.2–5 μm by different physico-chemical parameters and trophic size fraction, the remaining 44 OTUs corresponding to status (Table 1; Fig. S1). The Mare Gabard is a small pond protists found only in the larger 5–30 μm at the Etang de located in the middle of the forest (Fig. S1). It smelled Vallées (Table 1). The different OTUs were then assigned strongly of H2S when sediments were stirred and had high to known taxonomic groups based on sequence similarity, phosphate concentrations (0.15 mg l−1) but the lowest pH, which revealed a wide phylogenetic diversity of protists in conductivity and TDS (total dissolved solids) values. Saint general and of small eukaryotes in particular. Our strin- Robert pond is the most anthropized system selected gently defined OTUs affiliated to the major recognized because of its location in a hamlet (Fig. S1). It contained eukaryotic supergroups SAR (Stramenopiles, Alveolata, very high ammonia (1.22 mg l−1) and, to a lesser extent, Rhizaria), Archaeplastida, Excavata, Amoebozoa and also nitrite concentrations (Table 1, Fig. 1), likely influ- Opisthokonta (López-García and Moreira, 2008; Adl enced by the permanent presence of a sizeable duck et al., 2012) and to several lineages of unresolved population. La Claye pond is part of a complex of several phylogenetic position such as Cryptophyta, Haptophyta ponds on ancient peat bog substrate, with a dense popu- and Apusozoa (Fig. 2). lation of eagle ferns in the surroundings. Lying on acidic To evaluate the reliability of the community composition and organic-rich soils, it was characterized by high determined for each ecosystem at this stage, we com- concentrations of dissolved organic carbon (DOC; pared the pairwise Bray–Curtis distances between the 36.3 mg l−1). The Etang des Vallées is a nearly 1.5 m deep two replicates for each ecosystem. These were very small pond and, contrary to other sampled systems, was super- (0.26 on average, min. 0.12, max. 0.36) as compared with saturated in oxygen (116.8%; Table 1). It also had high distances between libraries from distinct ecosystems temperature at the time of sampling (13.1°C), and high (0.84 on average, min. 0.44, max. 0.99). Replicates concentrations of nitrate (5.1 mg l−1) as compared with the grouped together in both non-metric multidimensional other systems. The Ru Sainte Anne is the brook and had scaling (NMDS; Fig. S2) and Correspondence (CoA) the highest amounts of total dissolved solids (746 mg l−1) analyses (Fig. 3), revealing their high similarity in OTU possibly due to sediment input in a very shallow (∼ 10 cm) composition. system of running waters (Table 1). Principal component The composition of protist communities from the small analysis (PCA) of the different physico-chemical param- size fraction samples (0.2–5 μm) differed greatly between eters measured showed that, despite their close proximity ecosystems (Fig. 2). First, richness and diversity indexes (distances between these systems varied from 2 to 9 km; were highly variable (Table 1). Samples from Ru Sainte Fig. S1), the ponds and brook were clearly distinct from Anne and Etang des Vallées were the richest and the each other (Fig. 1). This made of this set of shallow aquatic most diverse, whereas La Claye was highly dominated by ecosystems a good model to study whether geographic few abundant OTUs (evenness = 0.34–0.27 in replicates). proximity is more influential than physico-chemical param- La Claye and Saint Robert appeared relatively close in eters in determining community composition. NMDS plots (Fig. S2) and clustered close together in CoA plots (Fig. 3). These two systems displayed relatively similar physico-chemical parameters, notably oxygen Overall protist community composition content and conductivity, and low diversity as compared The composition of protist communities was estimated for with the other ecosystems (Table 1). The similarity the five selected shallow freshwater systems based on between La Claye and St Robert community composition 454-pyrosequenced 18S rDNA fragments amplified from was likely influenced by the fact that they were largely DNA of plankton of the 0.2–5 μm cell diameter fraction. In dominated by the same cryptophyte OTU (Fig. 2; see addition, protist diversity in the size fraction 5–30 μm was below). studied for the largest sampled ecosystem, the Etang des Second, the relative abundance of taxonomic groups Vallées. Replicate samples were included in all cases. We varied between ecosystems, even though OTUs affiliated described protist diversity in these systems from a total of to Cryptophyta, Stramenopiles (or Heterokonta) and 146 549 quality-filtered reads using highly stringent crite- Alveolata accounted for the majority of sequences in all

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology 4 .Simon M. Table 1. Characteristics of freshwater systems studied and diversity and richness estimates for the different samples.

Sampled ecosystem Gabard (MG) Saint Robert (SR) Etang des Vallées (EV) Sainte Anne (RSA) La Claye (LC) tal et Ecosystem type Forest pond Village pond Large pond Forest brook Pond on peaty soil

GPS coordinates 48°39′15.83″N 1°55′20.26″E 48°39′54.82″N 1°56′45.28″E 48°41′23,0″N 001°54′59,2″E 48°36′45.91″N 1°58′16.61″E 48°36′31.72″N 1°56′17.33″E . Approximate surface (m2) 850 (210 × 75 m) 495 (20 × 28 m) 12 880 (210 × 75 m) 1 m width at sampling point 265 (24 × 10 m) Approximate depth (cm) 50 50 150 20 60 Sampling Date April 5, 2012 April 5, 2012 March 30, 2012 April 6, 2012 April 6, 2012 Water temperature (°C) 9.0 10.7 13.1 7.1 7.8 pH 6.6 7.2 7.31 7.36 7 Oxygen (%) 78.9 58.9 116.8 72.8 61.2

04SceyfrApidMcoilg n onWly&Sn Ltd, Sons & Wiley John and Microbiology Applied for Society 2014 © Conductivity (μS cm−1) 81.4 531 288 746 520 TDS (mg l−1) 81 531 288 746 520 Chlorophyll (μgl−1) 74.9 61.9 44.7 2.6 10.7 − −1 NO3 (mg l ) 1.41 1.17 5.08 1.45 2.02 − −1 a NO2 (mg l ) ∼0 0.054 0.047 0.010 ∼0 −1 NH3 (mg ) 0.02 1.22 0.02 0.03 0.04 DOC (mg l−1) 19.0 15.7 9.8 17.7 36.3 3− −1 PO4 (mg l ) 0.15 0.03 0.03 0.03 0.03 Size fraction studied 0.2–5 μm 0.2–5 μm 5–30 μm 0.2–5 μm 0.2–5 μm 0.2–5 μm

Sample EV34 EV34b Nameb MG25 MG25b SR25 SR25b (5–30 µm) (5–30 µm) EV33 EV33c RSA25 RSA25b LC25 LC25b

Number of 15 020 5110 85 803 6724 28 418 14 168 22 314 7938 37 766 7140 21 283 14 215 reads before ﬁltering Number of 10 616 4197 42 034 4506 10 982 6906 17 670 3947 19 652 4243 11 191 10 605 quality- ﬁltered reads Observed 76 55 15 37 147 132 177 87 427 198 66 68 number of OTUs Richnessc 67.9 (2.37) 55.0 (0.15) 11.4 (1.06) 36.8 (0.44) 134.4 (2.94) 126.9 (2.07) 148.5 (3.99) 87.0 (0.00) 313.7 (7.52) 197.8 (0.44) 57.9 (2.34) 57.0 (2.67) (Standard error) Diversity 0.86 0.80 0.75 0.68 0.96 0.95 0.95 0.95 0.92 0.91 0.50 0.38 niomna Microbiology Environmental (Simpson Index) Evenness 0.58 0.56 0.61 0.45 0.75 0.72 0.69 0.78 0.64 0.7 0.34 0.27

a. Nitrite concentrations in Gabard and La Claye ponds were under the kit detection limit of 0.006 mg l−1 and were set as 0 in analysis. b. Samples correspond to 0.2–5 µm cell-size fractions except stated otherwise. Replicate samples for the same site are labeled “b”. c. Expected number of OTUs in random subsamples of the size of the smallest sequence library (3947 reads in EV33c). Replicate samples are indicated by a small case letter after the sample name. DOC, dissolved organic carbon; TDS, total dissolved solids. Protist diversity in shallow freshwater systems 5

libraries (Figs 2 and 4). Cryptophyte sequences were remarkably abundant, representing up to 49% and 53% of the total number of reads and reads of the 0.2–5 μm size fraction respectively. Cryptophytes were the dominant group in Saint Robert and La Claye (around 75% of reads in those samples). They were also the dominant group in the brook Sainte Anne (between 32–44% of reads) and were the second most abundant group in the two other systems (Fig. 2, Table S1). Most cryptophyte reads belonged to a unique OTU affiliated to Cryptomonas curvata (OTU_52; Figs 4 and S3). Stramenopiles constituted the second most abundant group in our systems and represented 25% of all reads and nearly 24% of the reads from the 0.2–5 μm size fraction samples (Fig. 2, Table S1). Within this supergroup, Chrysophyceae were abundant in all samples. However, the most abundant Fig. 1. Principal component analysis (PCA) plot of the measured stramenopile groups in Mare Gabard and Ru Sainte physico-chemical parameters. Sampled ecosystems appear in grey Anne were, respectively, Synurophyceae and Bacillario- (dots) and physico-chemical parameters in black. MG, Mare phyceae, two lineages producing silica skeletons or Gabard; EV, Etang des Vallees; LC, La Claye; RSA, Ru Sainte Anne; SR, Saint Robert. TDS, total dissolved solids; DOC, scales. Oomycetes were also relatively abundant in the dissolved organic carbon; OrthoP, orthophosphate. brook Sainte Anne. Alveolates constituted the third most abundant supergroup in our study. It was the dominant group in Etang des Vallées (32–41% of reads in replicates

Fig. 2. Histogram showing the relative proportion of 18S rRNA gene amplicon reads assigned to high-rank taxa in the ﬁve shallow ecosystems studied. Replicate samples are labelled as ‘b’ or ‘c’. In all cases, the distribution corresponds to protists in the 0.2–5 μm size range, except in the Etang des Vallées, where the 5–30 μm size fraction was additionally analysed.

pond (12–20%). However, they represented only a small proportion of the taxa detected in the brook Sainte Anne and La Claye pond (less than 5% of reads; Fig. 2, Table S1). Among alveolates, ciliates were the most represented in all samples; although OTUs affiliated to dinoflagellates and other alveolates had occasionally significant proportions. Along with these three abundant supergroups, members of the Opisthokonta, Amoebozoa, Excavata, Archaeplastida, Rhizaria and of groups of uncertain position in the eukaryotic tree were also detected. Because we purposefully used general eukaryotic primers biasing against Metazoa, opisthokonts were essentially represented by fungi, although choanoflagellates and ichthyosporeans were also detected. The highest abun- Fig. 3. Correspondence analysis (CoA) plot showing protist community composition similarities and differences among the five dance of fungi was observed in Mare Gabard and Ru ecosystems studied. MG25 and MG25b, Mare Gabard; LC25 and Sainte Anne, but they generally represented less than LC25b, La Claye; SR25 and SR25b, Saint Robert; RSA25 and 10% of reads. Rhizarian OTUs were retrieved in all RSA25b, Ru Saint Anne; EV33 and EV33c, Etang des Vallées (0.2–5 μm size samples). EV34 and 34b, Etang des Vallées ecosystems and were only represented by cercozoans. (5–30 μm size samples). They were not abundant except for the Ru Saint Anne, where they reached 6–8% of the reads. Katablepharid sequences represented as much as 8–11% of reads from of the 0.2–5 μm size fraction, and similar values for the the 0.2–5 μm fraction in the Etang des Vallées but were larger size fraction analysed) and one of the dominant less abundant elsewhere (0.03% on average in each groups in Mare Gabard (22–40% of reads in duplicate small size fraction sample). Archaeplastida (streptophytes samples). It was also a major component in Saint Robert and, mainly, chlorophytes) were detected in all ecosys-

Fig. 4. Rank abundance curve for the total 768 OTUs detected collectively in the 0.2–5 μm size fraction of the ﬁve shallow freshwater systems studied. Sequence data from all samples were pooled to deﬁne OTUs with high stringency. The relative abundance of protist OTUs representing more than 0.5% of the total number of reads are shown in the inset. The identity number of the respective OTUs and their taxonomic affiliation are shown below and above the histogram bars.

Fig. 5. Distribution of protist OTUs in the five shallow freshwater systems studied. Sequence data from replicates were pooled. A. Five-set Venn diagram showing the number of specific and shared OTUs in the different freshwater systems (0.2–5 μm fraction size). B. Venn diagram showing OTUs shared by the two fraction sizes analysed in Etang des Vallées. C. Clustering analysis of the five ecosystems based on the presence of shared OTUs, as shown by the heatmap. Only 0.2–5 μm size fractions are considered. Heatmaps show all OTUs and OTUs shared by two, three or four ecosystems. No OTU is shared by all five ecosystems. Each row represents an ecosystem and each vertical bar an OTU. Black: OTU present, Grey: OTU absent. The coloured sidebar indicates the taxonomic affiliation of the OTU represented by the bar below. Colour codes representing different phylogenetic affiliation are indicated in the box. CCTHK: Cryptophyta, Centroheliozoa, Telonemia, Haptophyta and Katablepharida. For presentation reasons, the width of OTU bars is not the same in all the heatmaps. RSA25d, Ru Sainte Anne; LC25d, La Claye; SR25d, Saint Robert; MG25d, Mare Gabard; EV33d, EV34d, Etang des Vallées.

tems. Haptophytes were also identified, although in low OTUs (Fig. 4). Remarkably, OTU_52, affiliated to proportions, and only in the Etang des Vallées and the Ru Cryptomonas curvata, was particularly abundant in St Sainte Anne. OTUs affiliated to Excavata, Labyrinthulida, Robert and La Claye samples where it represented as Xanthophyceae, Apicomplexa, Centroheliozoa, Telone- much as 43–52% and 69–78% of reads respectively. mida, Amoebozoa and Apusomonadida (Apusozoa) were However, OTU_52 dropped to 4–7% of reads in the detected only in the highly diverse Sainte Anne brook 0.2–5 μm fraction of the Etang des Vallées and was (Fig. 2, Table 1). Three additional apusozoan OTUs not detected in Mare Gabard. OTU_25, affiliated to were detected in Sainte Anne brook and the Etang des stramenopiles, was also shared by four systems, but rep- Vallées. resented only 0.13% of all reads in 0.2–5 μm fractions. Despite some similarity in the distribution of large The composition of protist communities within the two phylogenetic groups, with cryptophytes, stramenopiles different size fractions (0.2–5 and 5–30 μm) of the Etang and alveolates dominating the different shallow water des Vallées was very similar, as revealed by the NMDS systems, protist communities were very different at the and CoA analyses (Figs 3 and S2) as well as by low phylotype scale. Indeed, no OTU was shared by all the Bray–Curtis distances (0.35 on average, min: 0.29, max: systems and the vast majority of the 768 OTUs detected 0.43). Moreover, the community structure was similar in in the small size fraction was specific to the libraries of a both size fractions from the Etang des Vallées, which single ecosystem (Fig. 5A). Sixty-seven and twelve OTUs displayed the highest diversity and evenness recorded of were shared by two and three different ecosystems all samples (Table 1). The taxonomic composition was respectively (Fig. 5C). Only three OTUs were shared also similar at high-rank taxa level, although diatoms and by four systems (Fig. 5C), two of which affiliated to green algae were in slightly higher proportions in the cryptophytes and corresponded to the most abundant largest size fraction (Fig. 2). At a finer level of resolution,

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology 8 M. Simon et al. more OTUs were shared by both size fractions (131 OTUs related to sequences of putative freshwater OTUs) than specific to each of them (Fig. 4B). (Amaral-Zettler et al., 2008; Monchy et al., 2011) or marine (Behnke et al., 2010; Scheckenbach et al., 2010) perkinsid parasites. Interestingly, several OTUs grouped Phylogenetic diversity with Apicomplexa and Colpodellida and clustered into two In order to get more detailed information on the different distinct groups (Fig. S5). Eight OTUs clustered with OTUs identified, we carried out phylogenetic analyses Colpodella edax (Leander et al., 2003) and many environ- with partial 18S rRNA gene sequences representative of mental sequences from freshwater lakes or ponds the different OTUs. Although cryptophytes were the most (Richards, 2005; Lefèvre et al., 2007; Nakai et al., 2012; abundant group in our samples, they were not very Oikonomou et al., 2012). Three other OTUs clustered diverse. Only 27 OTUs were affiliated to that group (3.3% with sequences affiliated to apicomplexans (mostly of all OTUs). Most of these OTUs were closely related to Cryptosporidium) coming from more diverse environ- existing sequences, affiliating to photosynthetic genera, ments, e.g. peat bog (Lara et al., 2011), marine sediment especially Cryptomonas (containing the highly overrepre- (Dawson and Pace, 2002), humans (Yuan et al., 2012) or sented OTU_52 corresponding to C. curvata mentioned ostrich faecal samples (R. Martinez-Diaz, unpublished). above) and Chroomonas, but also to the phagocytic From the 131 OTUs affiliated with opisthokonts, the plastid-lacking Goniomonas (Fig. S2). However, several great majority (125 OTUs) related to fungi, mostly to OTUs appeared to be quite divergent; for instance, rep- chytrids but also to ascomycetes, basidiomycetes or to resentative sequences of OTUs 838 and 1812 shared no the basal Rozellida/Cryptomycota lineage (Fig. S7). In more than 93% of identity with their first BLAST hit in the addition, 11 OTUs were related to the aphelids, the sister NCBI database, the sequences HM135076 from freshwa- group to fungi, which so far mostly contains highly diver- ter (Luo et al., 2011) and AM901364 from the cultured gent 18S rRNA gene environmental sequences (Karpov Cryptomonas commutata strain M1975 respectively et al., 2014). Several OTUs clustered with ichtyosporeans (Fig. S3). and choanoflagellates (Fig. S7). A total of 268 OTUs (33% of all OTUs) were affiliated All rhizarian OTUs branched with cercozoan with stramenopiles, the second most abundant group sequences. Even though cercozoan OTUs were not very in our samples. Nearly half of them were related to abundant in our samples and represent only 1.2% of the Chrysophyta-Synurophyta (125 OTUs) whereas the total number of reads, they were composed of numerous remaining ones affiliated to Eustigmatophyta, Dictyo- phylotypes (106 OTUs) that represented altogether 13% chophyta, Bicosoecida, Oomycetes, Bacillariophyta, of all OTUs. The vast majority of these OTUs affiliated Xanthophyta and Labyrinthulida (Fig. S4). Surprisingly, to the Filosa (Fig. S8). Several OTUs branched with 14 OTUs seemed to belong to various MAST groups Endomyxa, although they shared only 93.6% identity on (Fig. 6), originally thought to be exclusively marine. Nine average with their first BLAST hits in GenBank (calculated OTUs affiliated to the group MAST-12, being related to on the 13 OTU representative sequences affiliated sequences previously detected in a wide variety of eco- to Endomyxa, min: 89% between OTU_12985 and systems, such as a suboxic Norwegian estuary (Kolodziej AB526843, max: 99% between OTU_1810 and and Stoeck, 2007), freshwater lakes (Lefèvre et al., 2008; EU910610). Furthermore, five OTUs clustered with Monchy et al., 2011) and a peat bog (Lara et al., 2011). sequences from the Novel Clade 10, and OTUs_2162 and OTU_116 was closely related (99% identity) to MAST-2 608 shared 92% and 91% identity respectively with sequences detected in a freshwater lake (Luo et al., 2011) sequence EU567287 from Novel Clade 11 – Tremulida, and the Mediterranean (Diez et al., 2001). OTUs_1850 two recently defined deep-branching cercozoan lineages and 3339 affiliated to MAST-3, which so far was known to (Bass et al., 2009; Howe et al., 2011). contain only sequences from marine environments. Sixty-five OTUs (8% of all OTUs) belonged to Finally, OTU_222 and OTU_3247 had similarity by BLAST Archaeplastida (Fig. S9). Five of them strongly affiliated to to MAST-6 sequences retrieved from the Mediterranean Embryophyta. Given the pre-filtration steps, these OTUs (AF363207, Diez et al., 2001) and other MAST-6 must correspond to pollen grains, free DNA or cells from sequences, with 94% and 91% identity respectively. dead leaves. Strikingly, they represented altogether only However, from the phylogenetic analysis, their affiliation 0.04% of all reads, in spite of the dense vegetation around to this clade is unclear and will require a more in-depth most sampled systems. Four additional OTUs most exploration of these groups. likely corresponded to unicellular Streptophyta, affiliated Alveolates were also found to be diverse (Figs S5 and to Clausteriaceae and Desmidiaceae. The remaining S6), but the vast majority of OTUs were assigned to archaeplastid OTUs affiliated to the chlorophyte classes ciliates (88 out of 125 alveolate OTUs). Dinoflagellates Mamiellophyceae, Trebouxiophyceae, Nephroselmido- were also represented in our samples, along with 17 phyceae and, mainly, Chlorophyceae. In addition, we

Fig. 6. Approximate maximum likelihood (ML) phylogenetic tree of partial 18S rRNA gene stramenopile sequences showing the presence of several MAST clades in shallow freshwater systems. Non-MAST stramenopile lineages have been collapsed. A total of 392 unambiguously aligned positions were used to reconstruct the tree. Two alveolate sequences were used to root the tree. Representative sequences of OTUs from this work are shown in bold. The name of samples where MAST OTUs were detected and the respective proportion of reads are shown within brackets. The scale bar represents the number of estimated substitutions per position for a unit branch length. RSA25/RSA25b, Ru Saint Anne; MG25/MG25b, Mare Gabard; EV33/ EV33b (0.2–5 μm) and EV34/EV34b (5–30 μm), Etang des Vallées. detected a few OTUs belonging to other, less abundant brackish pond (Simon et al., 2013). Sequences belonging eukaryotic groups. Thus, four, one and six OTUs affiliated to the basal haptophyte group HAP-1 (Šlapeta et al., to katablepharids, telonemids and haptophytes respec- 2005) were detected in the Ru Sainte Anne (OTU_3063; tively (Fig. S3). Most haptophyte OTUs were very close to Fig. S3). Six additional OTUs belonged to centro- other freshwater sequences, but the OTU_3295 had heliozoans (Fig. S3). Their closest relatives were a sequence nearly identical to Jomonlithus littoralis sequences from soil or freshwater sediment and were (AM490979; 99% identity), a coastal species and to two only retrieved in Sainte Anne brook, the most narrow and environmental sequences (JX680345, JX680344) from a less deep ecosystem (Table 1). That observation, along

(2.1 km away). However, this OTU was detected in all other systems, up to 9.5 km away. As mentioned above, each of our five ecosystems was characterized by a set of specific environmental variables, with the Mare Gabard and the Ru Saint Anne being, respectively, the less and most charged in TDS and Saint Robert being highly enriched in ammonia (Table 1; Fig. 1). All these differences in environmental parameters correlated to the structure of protist communities in these ecosystems as revealed by a Mantel test linking environmental and community pairwise distances between samples (r = 0.7323, P-value = 0.014). However, additional canonical correspondence analyses (CCA) did not reveal any clear relationship between any of the detected eukaryotic supergroups and one or several environmental variables, although some OTUs appeared to be associ- Fig. 7. Canonical correspondence analysis (CCA) plot. Only ated with particular samples according to their specific 0.2–5 μm size fractions are considered. Each dot represents an OTU. The colours indicate the taxonomic affiliation. Black triangles presence (Fig. 5) and distribution in CCA (Fig. 7). At the indicate samples. Duplicate samples appear superimposed. phylum or class level (Fig. S10), haptophytes, katable- CCTHK: Cryptophyta, Centroheliozoa, Telonemia, Haptophyta and pharids, choanoflagellates, cercozoans and cryptophytes Katablepharida. TDS: total dissolved solutes, DOC: dissolved organic carbon, OrthoP: orthophosphate. appeared to reach higher proportions where pH was the highest and phosphate concentration the lowest, the two latter variables being tightly correlated in PCA and CCA with the higher proportions and diversity in Sainte Anne (Figs 1 and 7). Streptophytes were associated with high brook of cercozoa, fungi and diatoms, known to be usually values of DOC whereas bacillariophytes and fungi were composed of large and/or benthic cells, could be more abundant where total dissolved solutes were the explained by a higher influence or contribution of benthic highest. These features could thus partially explain the communities in this system. One excavate OTU differences in composition of the small eukaryotes among (OTU_1857; 99% identical to Trimastix marina) and one our contrasting ecosystems. apusomonad OTU were also detected in Sainte Anne brook. Three amoebozoan OTUs from the brook and two ancyromonad OTUs could also be detected in our small Discussion freshwater systems (Fig. S9). Finally, 66 of the total 812 OTUs determined could not be affiliated with confidence Shallow freshwater systems, reservoirs of to any group. protist diversity Compared with oceans, molecular protist diversity surveys in freshwater ecosystems are still scarce. Para- Effects of physico-chemical parameters and distance on doxically, inland water bodies are collectively much more community structure and composition heterogeneous than oceans and much more sensitive to The five sampled ecosystems were separated by dis- environmental variation. Despite so, only a handful of tances between 2 and 9.5 km (Fig. S1). To see whether studies on freshwater protist communities exist, mainly the differences in community composition increased with from a variety of lakes, from temperate regions and moun- geographical distance, we pooled sequence data from tains to polar areas (e.g. Charvet et al., 2012; Lepère replicate samples for the 0.2–5 μm size fraction, calcu- et al., 2013; Taib et al., 2013). However, nearly nothing lated Bray–Curtis distances between all systems based is known from smaller, shallower and often ephemeral on the pooled data and performed a Mantel analysis freshwater systems, despite their global distribution and to test the correlation between Bray–Curtis and geo- ecological importance (Downing et al., 2006). Recent graphical distance matrices. The Mantel test showed no approaches based on massive sequence analysis, correlation between differences in protist community com- although prone to a variety of methodological errors and position and distance between ecosystems (r = 0.2151, biases, are called to facilitate comparative studies among P-value = 0.236). As an illustration, OTU_52 (Crypto- those ecologically relevant ecosystems. This is especially monas curvata) reached 44% of all reads (43–52% in true for tiny protists, because morphology-based explora- replicates) in the Saint Robert pond, but it could not be tion very often miss their phylogenetic diversity (Massana detected in the Gabard pond (Fig. 5), its nearest system et al., 2002; Moreira and López-García, 2002; Šlapeta

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Protist diversity in shallow freshwater systems 11 et al., 2006). To contribute to that task, we carried out a Although protist community composition was different study based on massive pyrosequencing of amplified 18S in the five analysed systems, the comparison of protist rRNA gene fragments of protists in the 0.2–5 μm size diversity in two cell fraction sizes (0.2–5 and 5–30 μm) in range in five shallow freshwater ecosystems from a the most diverse site, the Etang des Vallées, did not temperate area. Many of the high-rank taxa detected differ much in terms of high-rank taxa (Figs 2 and 3). occur, although with variable relative abundances, in Indeed, despite the presence of OTUs apparently con- other freshwater systems, such as lakes. Cryptophytes, fined to each cell-size fraction (21% and 25%, for the stramenopiles and alveolates were by far the most abun- smaller and larger size ranges, respectively), a consid- dant supergroups detected, although their internal diver- erable proportion of OTUs (53%) were shared between sity varied greatly among ecosystems (Fig. 2). Although the two cell fractions (Fig. 5B). Several explanations are less abundant in the open sea (Shi et al., 2009; Kirkham possible and non-mutually exclusive. One is that many et al., 2013), small cryptophytes seem to be common in small organisms may be retained in filters of larger pore- freshwater (Lefranc and Thénot, 2005; Šlapeta et al., diameter if the filtered biomass is high, because they 2005; Lepère et al., 2008; Mangot et al., 2012; Taib et al., might be entangled with larger organisms. On the oppo- 2013). Stramenopiles, as well as ciliates, which dominate site, flexible organisms of relatively bigger sizes than the alveolate diversity in our samples, are also common in those of filter pores may pass through them under the protist communities from freshwater lakes (Lefranc and filtration pressure applied. Finally, many organisms may Thénot, 2005; Lepère et al., 2008; Taib et al., 2013). encompass a size range that spans the 5 μm diameter Fungi, Ichthyosporea and choanoflagellates within the barrier imposed by our filters. This may be due to either Opisthokonta, cercozoans within the Rhizaria, and mem- differential sizes during their life cycle (e.g. gametes or bers of the Amoebozoa, Archaeplastida, Excavata as spores and vegetative forms) or to size variability under well as members of the phylogenetically unresolved a given life stage. Haptophyta, Centrohelida, Katablepharida, Telonemida- The broad protist diversity unveiled in the five freshwa- and Apusozoa were also detected. ter systems also reflects a wide ecological diversity of Surprisingly, fungi and their relatives, which are often functions. At a very general level, typical photosynthetic, detected in very large proportions in some lakes (Lefranc heterotrophic and parasitic groups were detected. Among and Thénot, 2005; Lepère et al., 2008), accounting for up photosynthesizers, cryptophytes followed by photosyn- to 94% of all the 454-pyrosequences in some of them thetic stramenopiles (diatoms, chrysophytes, synuro- (Taib et al., 2013), were in very low proportions (generally phytes, xantophytes) largely exceeded green algae. Many much less than 10%; Fig. 2) in our systems. This may of these lineages may also contribute to heterotrophic reveal a real difference between shallow ecosystems and activities, because many photosynthetic protists are deeper lakes, but it could also reflect varying population mixotrophs (Zubkov and Tarran, 2008; Massana, 2011; dynamics along the year (Nolte et al., 2010). Our samples Hartmann et al., 2012). Typical predators span most of the were collected in early spring and many of the fungal eukaryotic diversity identified, from choanoflagellates and OTUs detected correspond to potential parasitic fungi or amoeba to bicosoecids, ciliates, cercozoa, centrohelids, related lineages, such as chytrids, rozellids/cryptomycota katablepharids or apusozoa (Fig. 2). However, hetero- or aphelids (Fig. S7), which might experience fluctuation trophic activities also encompass the degrading activity of depending on host population dynamics along the year. osmotrophic taxa, including several fungi and labyrin- Temporal surveys would be needed to test such a hypoth- thulids and that of parasitic or parasitoid protists (many esis. Several OTUs in our freshwater systems branched chytrids, cryptomycota, aphelids, oomycetes, apicom- deeply within known eukaryotic supergroups. Thus, the plexa, perkinsids). Rozellida-Cryptomycota and the aphelids have been con- Altogether, our extensive 18S rDNA-based survey sidered the deepest lineages of fungi (Lara et al., 2010; of small protists suggests that shallow freshwater Jones et al., 2011), although recently, they have been systems are important reservoirs of eukaryotic diversity. re-classified as forming part of the superphylum Not only members of all supergroups are present, Opisthosporidia together with Microsporidia and would but also several members of uncertain, poorly known be the sister group to fungi (Karpov et al., 2014). or deep-branching lineages. Given the heterogeneity of Other detected deep-branching lineages include the this kind of systems on Earth, it can be advanced that Colpodellida (Leander et al., 2003) within the alveolates, further studies on shallow freshwater systems will the poorly known novel Clade-10 Cercozoa and uncover yet-to-characterize protists, the study of which Tremulida (Bass et al., 2009; Howe et al., 2011) or the will be of relevance to understand the ecology of these early diverging haptophyte lineage HAP-1, so far only ecosystems and, from an evolutionary perspective, detected in freshwater systems (Šlapeta et al., 2005; to reconstruct poorly resolved areas in the eukaryotic Shalchian-Tabrizi et al., 2011). tree.

Marine-freshwater barriers transgressed Most classical views posit that small free-living protists would find little barriers to dispersal and, hence, be widely One of the potential surprises that the study of protist distributed. Differences in protist community structure diversity in varied freshwater systems may bring is the would then be essentially explained by local environmen- increasing awareness that salinity barriers can be over- tal parameters. This ‘everything is everywhere, but the come more easily than previously thought. Although line- environment selects’ view seems supported by the occur- ages with members in both freshwater and marine rence of cosmopolitan protist species (Baas-Becking, systems exist, such as dinoflagellates, haptophytes, 1934; Finlay, 2002; Šlapeta et al., 2006). On the opposite perkinsids or stramenopiles (Logares et al., 2007; Bråte extreme, a variety of studies seem to suggest that et al., 2010; Shalchian-Tabrizi et al., 2011; Simon et al., endemic protists (Foissner, 2006) and taxa–area re- 2013), the marine-freshwater transition is thought to be lationships exist for microbial eukaryotes (Green et al., rare (Logares et al., 2009). However, this view may be 2004). Comparing community compositions sidesteps biased by current undersampling of freshwater systems. undersampling and the nearly impossible task of demon- Indeed, in our study, we have detected protist lineages strating true microbial endemisms (Martiny et al., 2006). A thought to be exclusively marine in the past, notably recent study suggested that the beta-diversity of the small several MAST lineages. Thus, we did not only identify eukaryotes between lakes was linked to the geographic members of MAST-2 and MAST-12 lineages, which have distances between ecosystems (Lepère et al., 2013). recently been detected in other freshwater systems However, it is extremely difficult to disentangle the effect (Massana et al., 2014), but also members of MAST line- of local environmental parameters from physical distance. ages never identified in freshwater systems before. This Furthermore, other factors, such as the temporal (Nolte was the case of MAST-3, for which we identified two bona et al., 2010) and the phylogenetic scale (Ragon et al., fide OTUs and, potentially, of MAST-6 and even MAST-1 2012) need to be taken into account. In our case, we lineages, although OTUs related to the latter are of much sampled the five systems at the same or correlative dates more uncertain affiliation (Fig. 6). to limit temporal effects and, although the distances Moreover, in addition to the occurrence of the emblem- involved were different from those in the study of Lepère atic MAST groups in these shallow freshwater systems, we and colleagues (2013) (10 versus 100 km scale), our also identified many OTUs widespread in the eukaryotic study clearly rejects geographic distance as a driver of tree that share 99% identity or more with sequences community composition. On the contrary, Mantel tests retrieved from marine environments. Examples are the show significant correlations between differences in com- haptophyte OTU_3295, practically identical to Jomonlithus munity structure and physico-chemical parameters. littoralis (Fig. S3), the stramenopile OTU_116 (Fig. S4), However, clear associations between high taxon levels the ciliate OTU_255 (Fig. S6) or the excavate OTU_1857 and environmental parameters were not identified (Fig. 7 affiliating to Trimastix marina (Fig. S9). But there are many and S10). Differences at finer, OTU scale might provide other OTUs whose closest relatives are sequences from hints about the role of environmental selection in deter- marine systems, even if similarity is slightly lower. mining community structure. At any rate, testing which In fact, even if salinity seems to be a relevant ecological factors more greatly influence the composition of these determinant structuring microbial communities (Lozupone communities would require the inclusion of biotic param- and Knight, 2007), the marine-freshwater barrier seems to eters, notably the diversity and relative abundance of be relatively easy to cross. On the one hand, several bacteria, archaea and viruses from the same systems. protists, notably cryptophytes, are osmotolerant and can cope with various salinity concentrations by the means of contractile vacuole regulation (Hoef-Emden, 2014). On Experimental procedures the other hand, many freshwater systems contain rela- Sampling and measurement of tively high levels of dissolved solutes (organic and/or inor- physico-chemical parameters ganic) requiring similar adaptations as those require for life in seawater salts. In this sense, it is interesting to note Samples were collected in spring 2012 from five small and that most of the ‘typically marine’ lineages detected in our shallow freshwater ecosystems at the Natural Regional Park of the Chevreuse Valley (France, South of Paris) (Table 1 freshwater systems were identified in the Ru Sainte Anne, and Fig. S1). The systems were chosen to represent which has the highest TDS content and seems greatly a variety of conditions at local scale, from forest ponds influenced by this parameter (Table 1; Fig. 7). rich in organic matter (Mare Gabard) to more urban and agricultural-influenced systems (Saint Robert). Surface Elements of protist biogeography water was collected using sterile plastic carboys and pro- cessed immediately back in the laboratory. Water samples There are two essentially opposed views with regard to were serially filtered through 30 μm pore-size nylon filters microbial and, more specifically, protist biogeography. (Millipore), and through 5 and 0.2 μm pore-size diameter

Nucleopore membranes (Whatman). Filters were stored nium technology from Roche (Beckman Coulter Genomics). frozen at −20°C until DNA extraction. The water tempera- Sequences have been deposited at NCBI under the ture, pH, the concentration of total dissolved solutes and the BioProject number PRJNA259710. level of dissolved oxygen were measured in situ using a multiparameter probe (Multi 350i, WTW). The concentrations 454 Pyrosequence analysis − − of dissolved nitrate (NO3 ), nitrite (NO2 ), ammonia (NH3), 3− orthophosphate (PO4 ) and DOC were measured in We obtained a total of 265 899 pyrosequences (Table 1). A water samples filtered through 0.2 μm pore-size diameter series of filters were applied in order to retain only high- Nucleopore membranes on the same day of sampling quality sequences. First, pyrosequences containing errors using manufactured colorimetric tests (Hach-Lange). in the primer region and positions with undetermined Chlorophyll a concentrations were determined after harvest- bases were eliminated using a local pipeline (Bachy et al., ing plankton biomass on glass microfiber filters (GF/F, 2013). The remaining sequences were analysed with Whatman) that were stored at −20°C until ethanol pigment AmpliconNoise (Quince et al., 2011) to further eliminate extraction. For chlorophyll extraction, filters were dried errors introduced during PCR reactions or 454 sequencing, then ground in 7 ml of absolute ethanol, and heated at and build OTUs. Filtered reads were clustered by pairwise 70°C for 20 min. After centrifugation, 1 ml of supernatant alignment and average linkage into OTUs with a 98% simi- was collected and optical densities at 665 and 750 nm larity cut-off using AMPLICONNOISE integrated in our local pipe- were measured (spectrophotometer DR5000 Hach- line (Bachy et al., 2013). Singletons, i.e. OTUs composed of Lange). Chlorophyll a concentration was determined only one read, were eliminated for precaution. The most on pigment extract by spectrophotometry, as follows: abundant sequence in each OTU was used as reference. OTU reference sequences were blasted against the Silva Chla 11. 9 ()OD665− OD 750 Ve with [Chla]: Chla []=∗ w ∗ Vf []= SSU111 database (Pruesse et al., 2007) and assigned to OD OD taxonomic groups based on sequence similarity. The 11. 9 ∗()665− 750 ∗Ve chlorophyll a concentration w Vf sequences in all OTUs were then attributed to the different −1 (μgl ), 11.95: Reciprocal specific absorbance coefficient of samples according to their MIDs. Chimerical OTUs were −1 chlorophyll a at 665 nm (μg cm ml ), OD665: optical density eliminated by a stringent procedure combining automated at 665 nm, OD750: optical density at 750 nm, w: width of the and manual steps. OTUs including sequences from at least spectroscopic cuvette (1 cm), Ve: volume of the pigment two different samples were considered to be real. OTUs extract (in ethanol, ml) and Vf: volume of water filtrated on composed of sequences from only one sample were checked the glass microfiber filter (l). The protocol was adapted from for chimeras using KEYDNATOOL (http://KeyDNAtools.com). Ritchie (2006). The sequences considered suspect by the software were double checked by comparing BLAST hits recovered from DNA extraction, amplification and sequencing of 18S independent sequence fragments (sequences were split in rRNA genes two and three fragments). Finally, OTUs whose representative sequence had a coverage of less than 90% with its first DNA was extracted from cells collected onto filters that were BLAST hit were eliminated if they were present in only one cut into pieces using the PowerSoil DNA extraction kit sample; they were kept but with their taxonomic affiliation (MoBio) according to the manufacturer’s instructions. DNA changed to ‘uncertain’ if they were present in at least two was eluted in 80 μl of 10 mM Tris, pH 8.0. 18S rRNA gene samples. OTUs affiliated to cryptophyte nucleomorphs were fragments of approximately 550 bp, encompassing the excluded from our analysis. After filtering, we kept 146 549 V4 hypervariable region, were amplified using the newly correct reads (Table 1). designed primer EK-565F (5′-GCAGTTAAAAAGCTCG TAGT) and primer 18s-EUK- 1134-R- UNonMet (5′-TTTAA Phylogenetic analyses GTTTCAGCCTTGCG) biased against Metazoa (Bower et al., 2004). Both forward and reverse primers were tagged with 20 Phylogenetic trees were built for each eukaryotic super-group different 10 bp molecular identifiers (MIDs) to allow pooling or for several high-rank taxonomic lineages if they com- and later differentiation of polymerase chain reaction (PCR) prised only a few OTUs. Analyses included representative amplification products from 20 distinct samples. PCR ampli- sequences of OTUs, their first BLAST hit and sequences from fications were conducted in a total reaction volume of 25 μl the closest cultured members. Sequences were aligned using 1.5 mM MgCl2, 0.2 mM of each deoxynucleotide using PROBCONS (Do et al., 2005). Positions retained to build (dNTP) mix (PCR Nucleotide Mix, Promega), 0.3 μM of each trees were selected from the multiple alignments using primer, 0.3–2 μl of DNA sample and 0.5 U HotStart Taq poly- GBLOCKS (Castresana, 2000) with the less stringent param- merase (Taq Platinum, Invitrogen). The amplification condi- eters. Phylogenetic reconstructions were then carried out by tions consisted of 25 cycles (94°C for 30 s, 58°C for 45 s and maximum likelihood approximation using FASTTREE (Price 72°C for 90 s), preceded by 3 min of denaturation at 94°C et al., 2010). Trees were visualized using FIGTREE (http:// and ending with a 10 min final extension step at 72°C. tree.bio.ed.ac.uk/software/figtree/). Sequences with particu- Amplicons from 5–7 independent PCR products for each larly interesting positions in trees were then blasted against sample were pooled together and then purified using the the NCBI database (http://blast.ncbi.nlm.nih.gov) to have an QIAquick PCR purification kit (Qiagen), according to the insight of their similarity with additional sequences in data- manufacturer’s instructions. The same amounts (around bases. The taxonomic indications given in trees are based on 200 ng) of purified amplicons from 20 samples were pooled. the taxonomic affiliation proposed in the PR2 database Amplicons were pyrosequenced using the 454 GS FLX Tita- (http://ssu-rrna.org/index.html).

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Statistical analyses were conducted using the R SOFTWARE Adl, S.M., Simpson, A.G.B., Lane, C.E., Lukeš, J., Bass, D., (http://cran.r-project.org) (R Development Core Team, 2013). Bowser, S.S., et al. (2012) The revised classification of To assess overall differences between microbial community eukaryotes. J Eukaryot Microbiol 59: 429–493. compositions, we calculated pair-wise Bray–Curtis distances Amaral-Zettler, L.A., Rocca, J.D., Lamontagne, M.G., between all samples on the basis of OTU proportions Dennett, M.R., and Gast, R.J. (2008) Changes in microbial (number of reads from each OTU for each sample normal- community structure in the wake of Hurricanes Katrina and ized to the total number of reads in the corresponding Rita. Environ Sci Technol 42: 9072–9078. sample) among replicates or sampling sites (Bray and Curtis, Baas-Becking, L. (1934) Geobiologie of inleiding tot de 1957). This would avoid heterogeneity due to different milieukunde. Hague, the Netherlands: W.P. van Stock & numbers of sequences generated per sample. NMDS ordi- Zoon. nation analyses were conducted based on Bray–Curtis Bachy, C., Dolan, J.R., López-García, P., Deschamps, P., and distances (after applying a Wisconsin standardization to Moreira, D. (2013) Accuracy of protist diversity assess- balance the influence of the most and least abundant OTUs) ments: morphology compared with cloning and direct using the ‘Vegan’ R PACKAGE (Oksanen et al., 2013). CoA on pyrosequencing of 18S rRNA genes and ITS regions using all OTU frequencies for all 12 samples were done using the the conspicuous tintinnid ciliates as a case study. ISME J ‘Ade4’ R PACKAGE (Dray and Dufour, 2007). Diversity and 7: 244–255. richness indices were determined using the ‘Vegan’ Bass, D., Chao, E.E.-Y., Nikolaev, S., Yabuki, A., Ishida, K., package. Richness was estimated by rarefaction analysis as Berney, C., et al. (2009) Phylogeny of novel naked Filose the estimated number of OTUs in a random subsample of and Reticulose Cercozoa: Granofilosea cl. n. and each sequence library (raw counts of OTUs), of the same Proteomyxidea revised. Protist 160: 75–109. size as the smallest one (Hurlbert, 1971). Simpson index Behnke, A., Barger, K.J., Bunge, J., and Stoeck, T. (2010) S Spatio-temporal variations in protistan communities along 2 is defined as Df′ =1−∑( i ) (Simpson, 1949) and evenness an O/HS gradient in the anoxic Framvaren Fjord (Norway). i =1 S FEMS Microbiol Ecol 72: 89–102. − ffiiln() Bolker, B., Lodewijk, B., Gentleman, R., Liaw, W.H.A., was calculated as e = ∑i =1 (Pielou, 1966) with ln()S Lumley, T., Maechler, M., et al. (2012) gplots: Various R

S being the observed number of OTUs and fi the frequency programming tools for plotting data., v2.12.1 [WWW docu- of each OTUi in the sample. Venn diagrams showing the ment]. URL http://cran.r-project.org/web/packages/gplots/ number of OTUs shared by, or exclusive to, the different index.html. samples, and heatmaps showing the presence/absence of Bower, S.M., Carnegie, R.B., Goh, B., Jones, S.R.M., Lowe, OTUs were built using the ‘GPLOTS’ PACKAGE (Bolker et al., G.J., and Mak, M.W. (2004) Preferential PCR ampliﬁcation 2012). To test whether community composition correlated of parasitic protistan Small subunit rDNA from metazoan with environmental parameters in the different samples, we tissues. J Eukaryot Microbiol 51: 325–332. constructed a matrix of Bray–Curtis dissimilarities based Bray, J.R., and Curtis, J.T. (1957) An ordination of the upland on OTU frequencies (sequences from replicate samples forest communities of southern Wisconsin. Ecol Monogr were pooled given that they clustered together in previous 27: 325–349. analyses) and a matrix of Euclidean distances based on Bråte, J., Logares, R., Berney, C., Ree, D.K., Klaveness, D., physico-chemical parameters for all ecosystems using the Jakobsen, K.S., and Shalchian-Tabrizi, K. (2010) ‘Vegan’ package. Both matrices were compared using a Freshwater Perkinsea and marine-freshwater coloniza- Mantel test. The Bray–Curtis matrix was also compared with tions revealed by pyrosequencing and phylogeny of envi- a matrix of geographical distances between ecosystems. ronmental rDNA. ISME J 4: 1144–1153. Geographical distances were estimated based on coordi- Caron, D.A. (1994) Inorganic nutrients, bacteria, and the nates (http://biodiversityinformatics.amnh.org/open_source/ microbial loop. Microb Ecol 28: 295–298. gdmg/), using the mean earth radius (Moritz, 2000) as Castresana, J. (2000) Selection of conserved blocks from spheroid. PCA and CCA were conducted on centred and multiple alignments for their use in phylogenetic analysis. scaled physico-chemical parameters and OTU frequencies Mol Biol Evol 17: 540–552. in replicate samples (0.2–5 μm size fractions) using the Charvet, S., Vincent, W.F., Comeau, A., and Lovejoy, C. ‘ADE4’ PACKAGE. (2012) Pyrosequencing analysis of the protist communities in a High Arctic meromictic lake: DNA preservation and change. Front Microbiol 3: 422. Acknowledgements Cheung, M.K., Au, C.H., Chu, K.H., Kwan, H.S., and Wong, C.K. (2010) Composition and genetic diversity of We are thankful to F. Hardy, the Parc Naturel Régional de la picoeukaryotes in subtropical coastal waters as revealed Haute Vallée de Chevreuse and the Office National des by 454 pyrosequencing. ISME J 4: 1053–1059. Forêts du Parc de Rambouillet. We also thank Giselle Walker Corpe, W.A., and Jensen, T.E. (1992) An electron micro- for critical reading of the manuscript. The research leading to scopic study of picoplanktonic organisms from a Small these results received funding from the CNRS EC2CO Lake. Microb Ecol 24: 181–197. program and the European Research Council under the Costanza, R. (1999) The ecological, economic, and European Union’s Seventh Framework Program ERC Grant social importance of the oceans. Ecol Econ 31: 199– Agreement 322669 ‘ProtistWorld’. 213.

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Shallow freshwater systems sampled in this study in ‘picobiliphytes. PLoS ONE 8: e59565. April 2011. Shalchian-Tabrizi, K., Reier-Røberg, K., Ree, D.K., A. Localization of the five ecosystems in the Parc Naturel Klaveness, D., and Bråte, J. (2011) Marine–freshwater Régional de la Haute Vallée de Chevreuse, SouthWest of colonizations of haptophytes inferred from phylogeny of Paris, France (http://www.parc-naturel-chevreuse.fr). environmental 18S rDNA sequences. J Eukaryot Microbiol B. Distances as the crow flies between the sampled ponds 58: 315–318. and brook (km). Photographs of the sites are shown on the Shi, X., Marie, D., Jardillier, L., Scanlan, D., and Vaulot, D. left. (2009) Groups without cultured representatives dominate Fig. S2. Non-metric multidimensional scaling (NMDS) plot eukaryotic picophytoplankton in the oligotrophic South showing protist community composition similarities and dif- East Pacific Ocean. PLoS ONE 4: e7657. ferences among the five ecosystems studied. MG25 and Simó, R. (2001) Production of atmospheric sulfur by oceanic MG25b: Mare Gabard, SR25 and SR25b: Saint Robert, EV33 plankton: biogeochemical, ecological and evolutionary and EV33c: Etang des Vallées (0.2–5 μm size sample), EV34 links. Trends Ecol Evol 16: 287–294. and EV34b: Etang des Vallées (5–30 μm size samples), Simon, M., López-García, P., Moreira, D., and Jardillier, L. RSA25 and RSA25b: Sainte Anne, LC25 and LC25b: La (2013) New haptophyte lineages and multiple independent Claye. colonizations of freshwater ecosystems. Environ Microbiol Fig. S3. Approximate maximum likelihood (ML) phylogenetic Rep 5: 322–332. tree of partial 18S rRNA gene sequences of cryptophytes, Simpson, E.H. (1949) Measurement of diversity. Nature 163: haptophytes, telonemids, katablepharids and centro- 688. heliozoans. A total of 354 unambiguously aligned positions Stockner, J.G. (1988) Phototrophic picoplankton: an over- were used to reconstruct the tree. Two chlorophyte view from marine and freshwater ecosystems. Limnol sequences were used as outgroup (not shown) to root the Oceanogr 33: 765–775. tree. Representative sequences of OTUs from this work are Stoeck, T., Taylor, G.T., and Epstein, S.S. (2003) Novel shown in bold green. For abundant OTUs (> 0.5% reads), eukaryotes from the permanently anoxic Cariaco Basin their proportions in term of total number of reads and reads (Caribbean Sea). Appl Environ Microbiol 69: 5656–5663. from the 0.2–5 μm size fraction are given within square Šlapeta, J., Moreira, D., and López-García, P. (2005) The brackets. Statistical local support values higher than 0.5 are extent of protist diversity: insights from molecular ecology shown at nodes. The scale bar represents the number of of freshwater eukaryotes. Proc Biol Sci 272: 2073–2081. estimated substitutions per position for a unit branch length. Šlapeta, J., López-García, P., and Moreira, D. (2006) Global Fig. S4. Approximate ML phylogenetic tree of partial 18S dispersal and ancient cryptic species in the smallest rDNA sequences of stramenopiles. A total of 336 unambigu- marine eukaryotes. Mol Biol Evol 23: 23–29. ously aligned positions were used to reconstruct the tree. Two Taib, N., Mangot, J.-F., Domaizon, I., Bronner, G., and alveolate sequences were used as outgroup to root the tree. Debroas, D. (2013) Phylogenetic affiliation of SSU rRNA Representative sequences of OTUs from this work are shown genes generated by massively parallel sequencing: new in bold green. Local support values greater than 0.5 are insights into the freshwater protist diversity. PLoS ONE 8: shown at nodes. The scale bar represents the number of e58950. estimated substitutions per position for a unit branch length. Triadó-Margarit, X., and Casamayor, E.O. (2012) Genetic Fig. S5. Approximate ML phylogenetic tree of partial 18S diversity of planktonic eukaryotes in high mountain lakes rRNA gene sequences of alveolates. Diloflagellate and ciliate (Central Pyrenees, Spain). Environ Microbiol 14: 2445– branches are shown collapsed. A total of 325 unambiguously 2456. aligned positions were used to reconstruct the tree. Four

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology 18 M. Simon et al. stramenopile sequences were used as outgroup to root the root the tree. Representative sequences of OTUs from this tree. Representative sequences of OTUs from this work are work are shown in bold green. Local support values greater shown in bold green. Local support values higher than 0.5 are than 0.5 are shown at nodes. Scale bar represents the shown at nodes. The scale bar represents the number number of estimated substitutions per position for a unit of estimated substitutions per position for a unit branch branch length. length. Fig. S9. Approximate ML phylogenetic tree of partial 18S Fig. S6. Approximate ML phylogenetic tree of partial 18S rDNA sequences of Archaeplastida. A total of 450 unambigu- rRNA sequences of alveolates. Branches leading to groups ously aligned positions were used to reconstruct the tree. Two other than dinoﬂagellates and ciliates (see Fig. S4) are haptophyte sequences were used as an outgroup to root the shown collapsed. A total of 325 unambiguously aligned posi- tree. Representative sequences of OTUs from this work are tions were used to reconstruct the tree. Four stramenopile shown in bold green. Local support values greater than 0.5 sequences were used as outgroup to root the tree. Repre- are shown at nodes. Scale bar represents the number of sentative sequences of OTUs from that work are shown in estimated substitutions per position for a unit branch length. bold green. Local support values greater than 0.5 are shown Fig. S10. Canonical correspondance analysis (CCA) plot. All at nodes. The scale bar represents the number of estimated plots are from the same analysis as in Fig. 6, with ellipses substitutions per position for a unit branch length. emphasizing the distribution of OTUs affiliated to (A) Fig. S7. Approximate ML phylogenetic tree of 18S rDNA alveolates, (B) rhizarians, archaeplastids and excavates, partial sequences of opisthokonts. A total of 342 unambigu- (C) cryptophytes, (D) centroheliozoans, telonemids, hapto- ously aligned positions were used to reconstruct the tree. Two phytes, katablepharids and apusozoans, stramenopiles and amoebozoan sequences were used as outgroup to root the (E) unikonts. Projected inertia on CCA1: 34.07% and on CA: tree. Representative OTU sequences from our work are 29.77%. Black dots correspond to OTUs. Grey triangles indi- shown in bold green. Local support values greater than 0.5 cate the samples. SR, Saint Robert; EV, Etang des Vallées; are shown at nodes. Scale bar represents the number of MG, Mare Gabard; LC, La Claye; RSA, Ru Sainte Anne. estimated substitutions per position for a unit of branch Replicate samples are superimposed. DOC, dissolved length. organic carbon; OrthoP, ortho phosphate; TDS, total dis- Fig. S8. Approximate ML phylogenetic tree of partial solved solids. 18S rDNA sequences of cercozoans. A total of 353 Table S1. Number and percentage of reads assigned to dif- unambiguously aligned positions were used to reconstruct ferent taxa in the ﬁve studied ecosystems. Replica samples the tree. Two ciliate sequences were used as an outgroup to are labelled ‘b’.

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Table S1. !%IGV`':JR']V`HVJ :$V'Q`'`V:R1':111$JVR' Q'R1cV`VJ ' :6:'1J' .V'0V'1 %R1VR'VHQ171 VI18':V]C1H:'1:I]CV1':`V'C:GVCVR':G;8

Sampled ecosystem Gabard pond Saint Robert pond Etang des Vallées Sainte Anne brook La Claye pond Size fraction 0.2 - 5 µm 0.2 - 5 µm 5 - 30 µm 0.2 - 5 µm 0.2 - 5 µm 0.2 - 5 µm Sample names MG25 MG25b SR25 SR25b EV34 EV34b EV33 EV33c RSA25 RSA25b LC25 LC25b 10616 4197 42034 4506 10982 6906 17670 3947 19652 4243 11191 10605

Fungi 2958 2.6 3.5 0.1 0.2 0.9 0.3 0.3 0.3 8.6 11.9 0.7 0.4 Opisthokonta Ichthyosporea 30 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.2 0.1 0.0 Choanoflagellida 16 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 Amoebozoa Discosea 5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Other Amoebozoa 4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Excavata Metamonada 2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 !`H.:V]C:)R: Chlorophyta 3081 4.7 7.0 0.0 3.1 5.6 3.0 2.1 2.1 3.2 2.9 0.6 0.6 Streptophyta 115 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.2 0.3 0.3 0.2 MAST 368 0.0 0.0 0.0 0.0 0.6 0.6 0.8 0.0 0.5 0.6 0.0 0.0 Bicosoecida 757 2.3 1.2 0.0 0.1 0.5 0.2 1.6 1.0 0.0 0.0 0.4 0.2 Bacillariophyceae 8473 0.0 0.0 0.0 0.0 13.1 3.5 4.4 3.1 27.1 13.3 0.0 0.0 Oomyceta 1471 0.0 0.1 0.0 0.0 0.1 0.0 0.1 0.4 6.1 4.8 0.0 0.0 Stramenopiles Chrysophyceae 11872 6.2 3.4 8.2 2.2 14.2 12.7 14.9 13.6 5.9 5.1 2.9 2.0 Synurophyceae 7964 20.1 32.0 4.3 2.1 7.3 5.7 3.7 4.4 1.4 0.9 1.2 0.8 Labyrinthulida 14 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 Xanthophyceae 56 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.3 0.1 0.0 0.0 Other Stram 5541 1.8 1.0 0.0 0.4 3.0 1.9 5.8 5.7 0.9 0.8 19.1 11.5 Ciliophora 23037 36.2 16.1 12.1 19.8 21.8 33.9 28.8 37.7 2.9 1.3 3.1 2.5 Dinoflagellata 730 3.7 4.6 0.0 0.0 0.6 0.3 0.3 0.0 0.0 0.0 0.0 0.0 Alveolata Apicomplexa 14 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 Perkinsea 362 0.5 0.8 0.0 0.0 0.3 0.2 0.3 0.3 0.3 0.3 0.3 0.4 Other Alveolata 1273 0.0 0.0 0.0 0.7 4.9 2.0 2.2 3.3 0.2 0.1 0.1 0.0 Rhizaria Cercozoa 1829 0.1 0.1 0.0 0.1 1.0 1.0 0.6 0.1 5.9 7.5 0.3 0.2 Haptophyta 24 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.1 0.0 0.0 0.0 Cryptophyta 71564 21.9 29.9 75.2 71.4 19.1 25.3 22.8 20.0 31.7 43.7 69.9 80.7 Centroheliozoa 38 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.1 0.0 0.0 Katablepharida 3630 0.0 0.0 0.0 0.1 6.7 9.2 10.9 7.8 0.1 0.0 0.0 0.0 Telonemida 18 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 Ancyromonadida 17 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0 Apusomonadidae 2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Uncertain 1284 0.0 0.2 0.0 0.0 0.1 0.1 0.2 0.1 4.2 5.6 1.1 0.2 Mare Gabard (MG) A France

Etang des Vallées (EV) EV SR MG

RSA LC

Ponds Brooks or rivers Stretch of water Open wetlands Forest wetlands Villages

La Claye (LC)

B Gabard Saint Robert Etang des Vallées Sainte Anne La Claye Gabard 02.11 3.95 5.87 5.2 Saint Robert 0 3.48 6.13 6.30 Etang des Vallées 0 9.46 9.14 Sainte Anne 02.48 La Claye 0 Saint Robert (SR)

Figure S1. Shallow freshwater systems sampled in this study in April 2011. A, localizaton of the ﬁve ecosystems in the Parc Naturel Régional de la Haute Vallée de Chevreuse, SouthWest of Paris, France (htp://www.parc- naturel-chevreuse.fr). B, distances ias the crow ﬂies between the sampled ponds and brook (km). Photographs of the sites are shown on the lef.

Ru Sainte Anne (RSA) z,5

z,µ EV33 EV33c EV34b µ,5 EV34

SR25 µ,µ SR25b LC25 LC25b RSA25 −µ,5 RSA25b Stress= µ,zµ2 Stress=

−z,µ MG25 MG25b −z,5

−2,µ −2 −zµ z 2

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637852494 ;8829V75618917129 862176!"#2$%;&'(&9 6)961&0126&6271929(8&91216 279 661&627761&3&470#5662&97968&20;19169 89@(65A7(9(&67289&7179&72(&21929&12(1 112049(2&1265987&'(&72(&26&9(129(81929911120B82&1617)&'(&9 CLE& 295161792F62 &977A922096#(88921)6(&261216G0@62&976192&0L&6A62282&1&1(5A29 &175612 &(A&171(179&8289&7179 926(71A2610 0,99 OTU_2804 HQ219425 Uncultured ichthyosporean clone AY2009D4 AY336698 Eccrinidus flexilis 0,96 Y19155 Amoebidium parasiticum Y16260 Sphaeroforma arctica EU124914 Creolimax fragrantissima Ichtyosporea OTU_2938 AB191435 Uncunltured eukaryote clone TAGIRI-27 0,75 OTU_1815 AJ130859 Ichthyophonida sp. LKM51 0,86 AF070445 Anurofeca richardsi OTU_1355

0,86 GU647190 Uncultured freshwater eukaryote clone ML2_dino18S-46 EU011929 Salpingoeca sp. ATCC 50153 0,88 AJ867611 Uncultured Sphaeroeca clone WS 3-Uni05 0,86 AF271999 Monosiga ovata 0,9 HM135088 Uncultured choanoflagellate clone STFeb_136 0,99 OTU_3259 0,92 AY821949 Uncultured choanoflagellate CV1_B2_17 0,84 OTU_597 Choanoflagellates 0,95 FJ848511 Uncultured eukaryote clone DLN-J-95 0,76 0,76 FJ848488 Uncultured eukaryote clone DLN-J-69 0,84 EF432541 Choanoflagellida sp. SL1363 clone D10 EF432540 Choanoflagellida sp. SL163 clone D3 EU011928 Savillea microspora 0,78 0,99 L10823 Acanthocoepsis unguiculata 0,52 GU647170 Uncultured freshwater eukaryote clone AP_dino18S-6 0,91 0,55 EU011930 Salpingoeca sp. ATCC 50929 0,78 OTU_2504 JN090879 Uncultured eukaryote clone KRL01E19 0,99 OTU_615 OTU_327 HQ191302 Uncultured eukaryote clone PA2009E13 0,94 0,95 0,97 JN090865 Uncultured eukaryote clone KRL01E5 GU067958 Uncultured fungus clone ESS270706.065 GU067917 Uncultured fungus clone ESS270706.024 OTU_548 0,92 0,78 JN090898 Uncultured eukaryote clone KRL01E38 1 JN090889 Uncultured eukaryote clone KRL01E29 GU067817 Uncultured fungus clone ESS220206.038

0,67 JN090896 Uncultured eukaryote clone KRL01E36 FJ157332 Uncultured eukaryote clone kor_110904_24 Aphelids 0,76 GQ995418 Uncultured Chrytridiomycota clone T6P2AeG07 0,97 OTU_1818 0,88 OTU_3090 OTU_3337 0,89 OTU_1846

0,86 0,91 OTU_1856 113926798_Uncultured_fungus_partial_18S OTU_3143 GQ995419 Uncultured Chytridiomycota clone T4P1AeE08 GQ995421 Uncultured Chytridiomycota clone T5P1AeH09 0,84 HM487005 Uncultured fungus clone GA089 OTU_3163 0,88 OTU_690 0,99 GU067982 Uncultured fungus clone ESS270706.089 AY635838 Rozella allomycis 0,89 OTU_2916 0,97 0,6 OTU_1286 AM114814 Uncultured fungus clone WIM27 OTU_2298 0,78 FM178229 Uncultured fungus clone hww3 0,93 0,92 DQ244011 Uncultured fungus clone PFA12AU2004 OTU_2669 1 OTU_3294 0,75 EF023360 Nucleariidae clone Amb_18S_686

1OTU_1826 0,99 AY821991Uncultured fungus clone CH1_S2_50 FR874575 Uncultured marine picoeukaryote isolate ws_101 clone 1807A08 0,81 0,93 OTU_961 0,85 JN054664 Uncultured fungus clone B26 JN054676 Uncultured fungus clone C10 HM487048 Uncultured fungus clone HA004 0,81 0,9 HM628685 Uncultured eukaryote clone E-H6_1 Rozellida / 0,88 0,94 OTU_35 AB695466 Uncultured eukaryote clone MPE1-23 Cryptomycota 1 0,87 HM486990 Uncultured fungus clone GA082 OTU_1827 OTU_329 JN054661 Uncultured fungus clone B5 0,81 GU070875 Invertebrate environmental sample clone F05 OTU_359 AY642700 Uncultured eukaryotic picoplankton clone P34.42 0,94 OTU_315 0,7 1 OTU_2566

0,92 OTU_725

0,94 AF372716 Uncultured fungus clone LEM108 0,93 AY916644 Uncultured eukaryote clone wweuk6 0,88 OTU_3186 0,97 JN054654 Uncultured fungus clone A17 OTU_273 OTU_434 EF024493 Uncultured eukaryote clone Elev_18S_792 0,88 EU091852 Uncultured Banisveld eukaryote clone P2-3m5 HQ865510 Uncultured eukaryote clone SGSF770 0,91 0,73 OTU_2503 HM487038 Uncultured fungus clone HA056 0,98 0,98 AY635844 Endochytrium sp. isolate AFTOL-ID AF164283 Nowakowskiella hemisphaerospora OTU_673 Chytridiomycetes 0,97 JN705500 Uncultured microeukaryote clone 226Jun21_01A AY635835 Nowakowskiella 0,79 sp. JEL127 isolate AFTOL-ID OTU_1297 0,79 OTU_15 DQ534690 Rhizopogon olivaceotinctus OTU_1084 OTU_217 0,98 EF025022 Uncultured eukaryote clone Elev_18S_6061 AY336769 Diplomitoporus crustulinus JF972577 Fomitopsis lilacinogilva 0,8 AY293134 Hydnochaete olivacea D85646 Rhizoctonia sp. strain AG-A 0,85 GU824528 Uncultured eukaryote clone AB3F14RJ9G07 1 DQ873602 Hyphodontia alutaria AY705967 Fomitopsis pinicola DQ873608 Hyphodontia barba-jovis 1 0,87 OTU_199 AY436631 Phellinus igniarius 0,75 OTU_202 EU091863 Uncultured Banisveld eukaryote clone P4-3m3 DQ645524 Cystofilobasidium infirmominiatum OTU_235 HM486973 Uncultured fungus clone GA114 Basidiomycetes DQ831017 Mrakia frigida 0,79 OTU_2130 AB072227 Udeniomyces pannonicus AB032636 Cryptococcus huempii GQ330612 Uncultured Tremellales clone PR4_4E_90 L22262 Tremella foliacea

1 AB032646 Cryptococcus skinneri 0,92 OTU_947

0,98 FJ153086 Cryptococcus sp. CFL-2008b strain SJ8L06 OTU_960 JN939405 Trichosporon porosum 0,64 0,9 EU647050 Uncultured Agaricomycotina clone D0735_50_M OTU_293 0,84 JN043557 Tremella indecorata 0,97 OTU_1552 OTU_271 1 0,81 AY211545 Bullera sakaeratica 0,76 AB032635 Cryptococcus heveanensis 0,97 OTU_243 AJ001983 Arthrobotrys conoides Anguillosporea rosea 0,88 AY357265 GQ154641 Phaeomoniella zymoides 0,9 GU250343 Davidiellaceae sp. CCFEE 5414 JN397376 Cladosporium bruhnei AB220204 Physalospora scirpi

0,86 0,93 OTU_1849 0,78 AF130976 Hyponectria buxi 0,88 OTU_2629 OTU_808 AY382452 Uncultured soil fungus clone S_Canopy_300_02_13 AY357283 Tricladium chaetocladium 0,89 JN938701 Phaeophleospora atkinsonii 0,89 GU823078 Uncultured eukaryote clone AI5F14RJ2C03 0,94 0,71 OTU_2679 OTU_195 DL137895 Ascomycota FJ237044 Phacidiopycnis washingtonensis 8 AY524846 Phialocephala fortinii AY789422 Mitrula paludosa AY541482 Farlowiella carmichaeliana 0,91 U45445 Neobulgaria premnophila Asomycetes 0,93 AY544712 Sarcosphaera crassa 0,96 U53373 Boudiera acanthospora OTU_1799 GY714768 Sequence 5 from patent US 8084237 OTU_1873 0,72 0,97 FJ176839 Pyxidiophora arvernensis OTU_3183

0,78 OTU_218 0,99 AY204599 Anguillospora longissima GU296134 Amniculicola parva 0,93 0,76 OTU_453 0,91 U43449 Mycosphaerella mycopappi U42485 Lophiostoma crenatum 0,93 0,92 GU479751 Floricola striata OTU_18 FJ439576 Fungus GMG_C2 OTU_3206 GU479755 Helicascus nypae 0,88 OTU_210 0,8 AY864822 Phoma herbarum 0,94 OTU_2661 0,92 0.03 AF164303 Chytriomyces annulatus AY635827 Rhizophidium sp. strain JEL354 isolate AFTOL-ID 41 0,98 OTU_2237 AY601711 Polychytrium aggregatum GQ995279 Uncultured Chytridiomycota clone T6P2AeF01 OTU_3335 0,89 HQ219392 Uncultured Chytridiomycota clone AY2009A5

0,99 JN090912 Uncultured eukaryote clone KRL02E73 HQ191293 Uncultured Chytridiomycota clone PA2009E9 OTU_55 HQ219354 Uncultured Chytridiomycota clone AY2009C5 AF164247 Gaertneriomyces semiglobifer Fungi JQ014011 Conidiobolus coronatus 0,89 1 0,95 AF368504 Basidiobolus haptosporus 0,86 OTU_1822 0,99 OTU_636 OTU_392 Entophlyctis 0,75 AY635824 sp. strain JEL174 isolate AFTOL-ID 38 0,8 OTU_2996

0,79 OTU_2718 HQ191313 Uncultured Chytridiomycota clone PA2009A3 OTU_814 OTU_740 0,52 0,96 0,99 JF730792 Uncultured eukaryote clone Ch8A2mG4 OTU_3241 AB695513 Uncultured eukaryote clone MPE2-18 OTU_1264 EF014367 Entophlyctis confervae-glomeratae AY635829 Rhizophlyctis rosea AY635830 Catenomyces sp. strain JEL342 isolate AFTOL-ID 47 GU568169 Uncultured soil fungus clone 8242 DQ536477 Spizellomycete sp. JEL355 isolate AFTOL-ID 637 DQ536480 Triparticalcar arcticum 0,86 X80341 Neocallimastix frontalis JN020240 Uncultured Chytridiomycota clone cher4_1E_68

0,8 HQ219449 Uncultured Chytridiomycota clone AY2009D3

0,96OTU_2781 EU162642 Uncultured Chytridiomycota clone PFH9SP2005 FJ354068 Uncultured eukaryote clone L73_ML_156 0,78 0,73 Y17504 Hyaloraphidium curvatum 0,98 OTU_3184 0,76 OTU_2787 OTU_817 0,8 0,87 OTU_2782 OTU_2168 0,82 0,94 GU067827 Uncultured fungus clone ESS220206.048 0,87 0,79 OTU_1773 0,78 OTU_813 0,77 OTU_1784 0,79 OTU_1803 GU568157 Uncultured soil fungus clone 8414 HQ901748 Powellomycetaceae sp. OTU_3343 0,81 HM486975 Uncultured fungus clone GA072 DQ322624 Olpidium brassicae 0,77 AY635826 Entophlyctis helioformis JF414197 Endogone sp. T13431 voucher OSC:T13431 clone 1 JF414222 Mucoromycotina sp. MIB 8352 vouvher M.I. Bidartondo:8352 0,84 AY635828 Mortierella sp. MS-6 isolate AFTOL-ID 42 0,87 EF025004 Uncultured eukaryote clone Elev_18S_4941 0,94 EU688965 Mortierella indohii

0,74 0,9OTU_12955 GX291382 Sequence 10 from patent US 7745198 0,79 EU736292 Mortierella minutissima 0,79 GU568171 Uncultured soil fungus clone 8469 0,91 AF157145 Mortierella verticillata JQ086387 Cantharellus sp. strain KX560JY AY180029 Uncultured fungus clone CCW64 0,73 291551841_Uncultured_fungus_partial_18S 1 OTU_1847 0,77 291551842_Uncultured_fungus_partial_18S 0,71 OTU_1854 0,99 EF100286 Uncultured eukaryote clone D3P05F02 0,88 OTU_3344 0,36 0,91 EF100411 Uncultured eukaryote clone D5P09A02 AY032608 Chytridium polysiphoniae OTU_459 Chytridiomycetes 0,73 0,86 EF638677 Uncultured Chytridiomycota clone MV5E2 JN986721 Uncultured Chytridiomycota clone MH2_NS7R_MV5E2_77 OTU_1824 0,8 0,97 AJ506000 Uncultured rhizosphere chytridiomycete clone RSC-CHU-18 0,91 OTU_1577 0,91 0,5 0,79 OTU_2930 HQ191401 Uncultured Chytridiomycota clone PA2009B5 JN705531 Uncultured microeukaryote clone 229Feb24_11F 0,87 0,93 OTU_471 0,7 0,85 OTU_1274 OTU_2662 0,76 EF443137 Chytridiales sp. isolate JEL369 0,82 EF443136 Chytridiales sp. isolate JEL178 OTU_3354 0,85 JF972676 Uncultured eukaryote clone ThJAR2B-48

0,74 OTU_3334 0,94 FJ804149 Mesochytrium penetrans 0,58 OTU_3230 0,77 FJ157331_1_Uncultured_eukaryote_clone_ko 0,94 0,85 GQ995414 Uncultured Chytridiomycota clone T5P2AeC07 OTU_1830 EU162643 Uncultured Chytridiomycota clone PFA12SP2005 0,82 OTU_374

0,86 OTU_806 OTU_901 0,98 EU162637 Uncultured Chytridiomycota clone PFD6SP2005 EF100328 Uncutured eukaryote clone D4P07E09 0,97 OTU_692 0,77 OTU_3242 0,91 OTU_1828 0,81 OTU_1882 0,76 OTU_3245 0,92 OTU_3329

1 OTU_3300 0,83 JN054675 Uncultured fungus clone C6 AY642725_1_Uncultured_eukaryotic_picopla

0,97 0,95 OTU_797 HQ191382 Uncultured Chytridiomycota clone PA2009B15 OTU_1302 0,92 OTU_3274 AY642695 Uncultured eukaryotic picoplankton clone P34.27 OTU_3273 AF372721 Uncultured chytrid clone BAQA128 OTU_3213 0,92 AY426915 Uncultured marine eukaryote clone BL010320 0,73 OTU_1859 EF023700 Uncultured Closteriaceae clone Amb_18S_970 AF164264 Rhizophidium sp. UGA-F16 OTU_62 0,79 OTU_2753 OTU_444

0,84 GQ995437 Uncultured Chytridiomycota clone T5P2AeG04

0,93 EU162636 Uncultured Chytridiomycota clone PFB4SP2005 0,67 DQ536478 Kappamyces leurelensis 0,87 JN207858 Uncultured eukaryote clone FP970 0,81 1 OTU_1870 0,91 HM135089 Uncultured fungus clone STFeb_269

0,91 HQ191301 Uncultured Chytrydiomycota clone PA2009E23 OTU_1578 0,76 OTU_1844 GQ995438 Uncultured Chytridiomycota clone T3P1AeE01 HM487003 Uncultured fungus clone GA113 0,83 AY635821 Rhizophydium sp. strain JEL317 isolate AFTOL-ID 35 AF372720 Uncultured chytrid clone LEMD195 OTU_1887 DQ536488 Rhizophydium sp. MP8 isolate AFTOL-ID 1538 0,73 OTU_801 0,97 DQ244013 Uncultured fungus clone PFD11AU2004 FN690503 Uncultured fungus clone 5-E11 AY601710 Rhizophydium sp. strain JEL136 0,76 0,92 OTU_2287 EF023374 Uncultured Boletaceae clone Amb_18S_707 DQ536492 Rhizophydium sp. strain JEL223 isolate AFTOL-ID 2007 AF164272 Rhizophlyctis harderi 0,84 OTU_2211 0,79 DQ536479 Rhizophlyctis elyensis DQ322622 Boothiomyces macrosporosum

Figure S. Approximate ML phylogenetic tree of 18S rDNA partial sequences of opisthokonts. A total of 342 unambiguously aligned positions were used to reconstruct the tree. Two amoebozoan sequences were used as outgroup to root the tree. Representative OTU sequences from our work are shown in bold green. Local support values greater than 0.5 are shown at nodes. Scale bar represents the number of estimated substitutions per position for a unit of branch length. 0,68 AB622285 Uncultured freshwater eukaryote clone K1JUL2009 OTU_10 JF730776 Uncultured eukaryote clone Ch8A2mE7 0,92 OTU_127 0,96 AB622282 Uncultured freshwater eukaryote clone K5JUN2009 FJ349632 Uncultured eukaryote clone 051011_T2S1_W_T_SDP_099 0,69 0,86 GU067921 Uncultured cercozoan clone ESS270706.028 0,68 OTU_352 AB721071 Uncultured freshwater eukaryote clone DW2_2011 0,94 OTU_2575 OTU_3336 AY642739 Uncultured eukaryotic picoplankton clone A50

0,96 OTU_930 Novel Clade 10 0,79 0,8 0,99 AB622289 Uncultured freshwater eukaryote clone K5JUL2009 OTU_3194 0,89 OTU_163 0,78 0,95 OTU_3198 AB622288 Uncultured freshwater eukaryote clone K4JUL2009 GU479952 Uncultured rhizarian clone PRS2_3E_116 0,63 OTU_1393 DQ243999 Uncultured freshwater cercozoan clone PCB7AU2004 0,92 OTU_2768 0,72 OTU_12980 1 0,8 HQ219347 Uncultured cercozoan clone AY2009C14 FJ153639 Uncultured marine eukaryote clone GoC1_F06 0,78 HQ121439 Cercozoa sp. strain ATCC 50530 0,93 EU567287 Uncultured cercozoan isolate Ib9 0,98 OTU_2162 Tremulida / Novel Clade 11 0,97 OTU_608 AJ418790 Tracheleuglypha dentata OTU_3118 0,88 OTU_1810 EU910610 Uncultured plasmodiophorid clone D20 OTU_3122 0,86 AF310898 Polymyxa graminis 1 AF310902 Polymyxa betae 0,77 OTU_1839 0,76 1 OTU_1863 OTU_2939 0,66 1 OTU_1837 0,9 OTU_3055 0,88 OTU_12985 Endomyxa 0,5 0,81 AF310901 Spongospora subterranea 0,76 OTU_816 0,89 OTU_717 0,98 OTU_660 EU701011 Plasmodiophora brassicae 0,84 OTU_1867 0,78 AY604173 Spongospora subterranea f. sp. subterranea AF3108990 Spongospora subterranea f. sp. subterranea EF024866 Thaumatomonadida environmental sample clone Elev_18S_1390 OTU_1825 EF032802 Uncultured eukaryote clone HAVOmat-euk24 EF024169 Athalamea environmental sample clone Elev_18S_508 0,6 EF023543 Athalamea environmental sample clone Amb_18S_1145 0,73 DQ243993 Uncultured freshwater cercozoan clone PCH11AU2004 1 EU5672550 Cercozoa sp. DDB-2008c 0,75 OTU_1024 0,68 0,8 0,84 AY082993 Uncultured eukaryote clone RT5iin4 0,87 AY620300 Uncultured cercozoan clone 13-2.7 DQ388459 Exuviaella pusilla 0,72 AY620265 Uncultured cercozoan clone 8-1.2 AY620268 Uncultured cercozoan clone 4-2.7 0,94 AF174374 Massisteria marina OTU_402 0,91 AY642693 Uncultured eukaryotic picoplankton clone P34.14 0,96 HQ121438 Cercozoa sp. ATCC 50407 0,58 0,96 AY748806 Cercomonas agilis 0,6 AY620286 Uncultured cercozoan clone 6-2.7 OTU_2954 0,89 GU479953 Uncultured Pseudodifflugiidae clone PR3_4E_92 OTU_3284 0,9 AY620274 Uncultured cercozoan clone 4-2.3 0,79 OTU_1230 0,85 AY620276 Uncultured cercozoan clone F7 0,58 AY642704 Uncultured eukaryotic picoplankton clone P34.13 0,55 AB275097 Uncultured eukaryote clone CYSGM-14 FJ824121 Protaspis obliqua 0,77 AY620281 Uncultured cercozoan clone F9 0,88 OTU_1628 0,93 AY620264 Uncultured cercozoan clone 8-1.1 0,88 0,84 EF024143 Uncultured eukaryote clone Elev_18S_460 EF023812 Uncultured eukaryote clone Amb_18S_1260 0,8 OTU_1650 0,85 HQ121430 Rhogostoma schuessleri 0,76 AY620295 Uncultured cercozoan clone 9-3.3 0,9 AB585966 Cercozoa sp. strain TGS3 JN090899 Uncultured eukaryote clone KRL01E39 0,52 OTU_1627 0,77 HQ121431 Rhogostoma sp. strain CCAP 1966/4 FJ824124 Protaspis sp. CC-2009a 0,75 OTU_637 0,86 JF730755 Uncultured eukaryote clone Ch8A2mA11 0,86 FN690380 Uncultured cercozoan clone 880 AF411273 Cercozoa sp. strain WHOI LI1-14 0,7 OTU_1924 0,88 HM135090 Uncultured cercozoan clone STFeb_183 0,93 GU822052 Uncultured rhizarian clone AI5F14RJ2E09 OTU_631 0,88 AY620278 Uncultured cercozoan clone PP2-2C 0,85 0,91 OTU_3037 0,99 AY821946 Uncultured cercozoan clone CV1_B1_11 0,76 GQ144681Thaumatomastix sp. isolate CC002-Boundary Bay OTU_3079 0,96 AY620310 Uncultured cercozoan clone 9-2.6 0,87 OTU_744 0,9 OTU_2178 0,88 OTU_12987 DQ211593 Gyromitus sp. strain HFCC94 0,8 EF024801 Thaumatomonadida environmental sample clone Elev_18S_1309 0,93 OTU_1298 0,88 AY496046 Thaumatomonas sp. strain SCCAP T1 0,99 EF455776Thaumatomonas seravini 0,88 OTU_2681 0,91 AF411262 Allas diplophysa 0,9 OTU_2359 OTU_3320 0,9 EF025018 Uncultured eukaryote clone Elev_18S_5891 EF023758 Thaumatomonadida environmental sample clone Amb_18S_1199 0,99 OTU_2067 0,91 HQ121434 Nudifila producta EF456749 Assulina seminulum AJ418785 Euglypha filifera 1 EF456752 Trinema lineare 0,55 OTU_1118 0,98 EF025028 Uncultured clone Elev_18S_6371 0,79 0,92 0,79 AJ418792 Trinema enchelys AJ418789 Trachelocorythion pulchellum AB505503 Uncultured eukaryote clone RM1-SGM46 AB252753 Uncultured eukaryote clone NAMAKO-13 0,98 OTU_349 0,72 AY620261 Uncultured cercozoan clone F2 1 HQ121435 Spongomonas sp. strain CCAP 1971/1 0,77 OTU_716 0,78 0,96 OTU_2502 0,75 AF372740 Uncultured cercozoan clone LEMD083 AB252754 Uncultured eukaryote clone NAMAKO-14 0,73 OTU_916 1 AY620331 Uncultured cercozoan clone C5 0,92 OTU_3327 0,89 FJ153695 Uncultured marine eukaryote clone GoC4_B04 0,86 AY620358 Uncultured cercozoan clone 12-3.3 OTU_1377 0,97 0,56 DQ199661Aurigomonas solis AB695519 Uncultured eukaryote gene clone MPE2-25 0,9 0,88 AY360723 Uncultured cercozoan clone 10-4Fu21 OTU_3260 EF024167 Cercomonadidae environmental sample clone Elev_18S_506 0,79 DQ243992 Uncultured cercozoan clone PCB12AU2004 0,99 Paracercomonas 0,87 HQ121440 sp. strain B148 0,97 AF411271 Cercomonas sp. (Tempisque) 0,93 AY965864 Soil flagellate isolate AND18 AM114800 Uncultured cercozoan clone WIM2 FJ790731 Paracercomonas saepenatans 0,92 0,99 OTU_278 0,92 FJ790730 Paracercomonas paralaciniaegerens 0,86 OTU_1624 FJ790725 Paracercomonas crassicauda 0,95 0,83 AY884341 Cercomonas sp. strain 19-5C 0,81 OTU_3267 0,74 FJ790724 Paracercomonas oxoniensis 0,78 AY620298 Uncultured cercozoan clone 9-6.4 OTU_1551 0,78 FJ790721 Paracercomonas producta Filosa 0,95 FJ790728 Paracercomonas ambulans FJ790727 Paracercomonas astra OTU_2949 0,77 AB695523 Uncultured eukaryote clone MPE2-29 OTU_2025 EF024284 Cercomonadidae environmental sample clone Elev_18S_681 FJ790723 Paracercomonas compacta OTU_3347 0,77 EF024516 Thaumatomonadida environmental sample clone Elev_18S_823 OTU_954 0,77 0,8 EF024999 Uncultured eukaryote clone Elev_18S_4451 EF023710 Thaumatomonadida environmental sample clone Amb_18S_989 0,74 OTU_3228 0,84 0,73 OTU_3359 1 EU709266 Uncultured cercozoan isolate HetAus17 0,85 AB534329 Uncultured eukaryote clone A_3_44 0,92 0,97 AB534349 Uncultured eukaryote clone A_3_96 OTU_3299 1 OTU_1657 OTU_686 0,78 0,92 0,97 OTU_1960 JF730833 Uncultured eukaryote clone LA8E2G6 0,75 0,92 OTU_198 HQ918170 Bodomorpha sp. strain B118 AF411276 Bodomorpha minima 0,93 OTU_3384 0,96 OTU_1361 0,76 OTU_228 0,78 AY620294 Uncultured cercozoan clone 9-4.6 0,91 EU709258 Uncultured cercozoan isolate HetPan3 0,56 OTU_672 EF023937 Uncultured eukaryote clone Amb_18S_1398 EF024871 Cercozoa environmental sample clone Elev_18S_1397 EF024853 Oxytrichidae environmental sample clone Elev_18S_1373 0,92 0,94 OTU_711 0,94 0,93 HQ918169 Allantion sp. strain CCAP 1906/1 AAYJ01000086Coxiella burnetii 0,84 0,74 OTU_1808 0,96 OTU_3049 0,81 OTU_12979 0,9 0,8 EF023859 Thaumatomonadida environmental sample clone Amb_18S_1311 OTU_3068 0,84 EU647174 Cercomonadida environmental sample clone D0570L03 AY962866 Soil flagellate isolate AND11 0,85 0,96 OTU_12961 1 EU709231 Cercozoa sp. B12ap57t2LS 0,96 OTU_2021 AB695472 Uncultured eukaryote clone MPE1-29 0,63 OTU_805 OTU_687 0,93 GU479955 Uncultured Heteromitidae clone PR2_3E_130 0,79 OTU_460 0,77 AB695526 Uncultured eukaryote clone MPE2-32 OTU_1283 0,76 OTU_1877 0,96 EF024589 Cercomonadida environmental sample clone Elev_18S_909 0,88 EF023523 Cercozoa environmental sample clone Amb_18S_1122 FJ592464 Uncultured eukaryote clone B07_SE1B 0,88 OTU_3108 OTU_1829 OTU_1988 0,8 OTU_1996 AY496048 Neocercomonas jutlandica 0,86 0,79 FJ790710 Cercomonas celer OTU_1591 DQ243998 Uncultured freshwater cercozoan clone PCF12AU2004 0,86 OTU_237 0,93 FJ790679 Cercomonas aff. longicauda FJ790702 Cercomonas braziliensis 0,91 OTU_1679 0,84 AF411268 Cercomonas plasmodialis 0.03 0,92 EF023152 Cercomonadidae environmental sample clone Amb_18S_440 EF024642 Cercomonadidae environmental sample clone Elev_18S_965 1 0,84 OTU_2065 0,86 FJ790700 Cercomonas fastiga FJ790701 Cercomonas rotunda HQ696567 Uncultured cercozoan clone 552 0,81 AY620263 Uncultured cercozoan clone 4-6.4 OTU_462 0,78 AF411267 Cercomonas alexieffi AY884316 Eocercomonas sp. strain 13-3.5 0,89 OTU_2123 0,87 AY884327 Eocercomonas sp. strain C-80 0,89 EF025030 Uncultured eukaryote clone Elev_18S_6781

0,77 OTU_1433 0,79 0,99 AF372741 Uncultured cercozoan clone LEMD079 FJ790715 Eocercomonas tribula FJ790716 Eocercomonas echina OTU_3331 EU709156 Uncultured cercozoan isolate HetPan22 EU709174 Cercozoa sp. WA49p88t2LS 0,94 OTU_1802 0,9 0,93 OTU_1270 0,91 OTU_3312 HQ918172 Cercozoa sp. ATCC PRA130 HQ918175 Cercozoa sp. B134 EU709182 Uncultured cercozoan isolate H25p79t5 0,84 OTU_286 EU709189 Cercozoa sp. WA28p82t6LS 0,83 EU709188 Uncultured cercozoan isolate H21p80t1 AY965866 Soil flagellate isolate AND21 EU709200 Uncultured cercozoan isolate H22p80t2 DQ211595 Bodomorpha sp. HFCC56 EU709160 Uncultured cercozoan isolate HetChi41 HQ918177 Cercozoa sp. strain B140 0,92 OTU_295 1 OTU_3305 EU709153 Cercozoa sp. W36p59t2LS

Figure S. Approximate ML phylogenetic tree of partial 18S rDNA sequences of cercozoans. A total of 353 unambiguously aligned positions were used to reconstruct the tree. Two ciliate sequences were used as an outgroup to root the tree. Representative sequences of OTUs from this work are shown in bold green. Local support values greater than 0.5 are shown at nodes. Scale bar represents the number of estimated substitutions per position for a unit branch length. DQ536492_1_Rhizophydium_sp_JEL223_isola

1 EF023700_1_Uncultured_Closteriaceae_clon AY635821_1_Rhizophydium_sp_JEL317_isola

0,7 AM920361 Staurodesmus brevispinus var. obversus AM920343 Euastrum trigibberum 0,91 AJ829653 Staurodesmus validus OTU_408 0,65 0,81 FR852619 Micrasterias tropica

0,99 OTU_239 1 AF352226 Closterium sp. strain NIES-52 isolate IB-4-9 OTU_3307 Streptophytes 1 AF352236 Closterium venus 0,75 0,93 OTU_2386 1 AF352222 Closterium moniliferum var. moniliferum 0,94 AF352235 Closterium pusillum var. maius 0,92 AF352241 Closterium spinosporum var. crassum 0,72 AJ553931 Spinoclosterium cuspidatum 1 X74754 Nephroselmis olivacea 1 OTU_1858 Nephroselmidophyceae 0,98 FN562436 Nephroselmis olivacea AJ629844 Crustomastix stigmatica

0,91 0,93 OTU_2462 OTU_3030 0,85 AB622308 Uncultured freshwater eukaryote clone K3OCT2009

0,78 OTU_3195 0,99 0,92 OTU_12925 0,92 AB622321 Uncultured freshwater eukaryote clone K4MAR2010 0,89 AB275081 Uncultured eukaryote clone DSGM-81 FN562449 Dolichomastix tenuilepis 0,9 OTU_142 Mamiellophyceae OTU_1227 0,86 0,88 OTU_2156 FN562445 Monomastix opisthostigma OTU_1790 0,75 0,68 OTU_29 0,88 HM135075 Uncultured Chlorophyta clone STFeb_126 0,96 GU067811 Uncultured Prasinophyceae clone ESS220206.032 0,93 OTU_2507 OTU_1869 JN592588 Pedinomonas minor 0,86 1 OTU_3086 Z28974 Microthamnion kuetzingianum HQ287929 Neocystis brevis

0,78 OTU_12978 0,58 Z68700 Trebouxia jamesii 1 0,97 EU091856 Uncultured Banisveld eukaryote clone P2-3m9 0,8 Z21553 Trebouxia asymmetrica 0,88 OTU_12964 0,81 0,8 1 OTU_1855 X68484 Scherffelia dubia M63002 Parietochloris pseudoalveolaris

0,9 OTU_3340 FR693368 Apatococcus lobatus 0,77 0,99 OTU_644 0,74 AF387148 Planctonema sp. strain M110-1 0,91 AY197626 Oocystaceae sp. isolate MDL6-7 1 GQ243428 Oocystaceae sp. isolate GSL021 0.7 OTU_1526 0.94 AF228689 Oocystis heteromucosa Trebouxiophyceae AJ306536 Koliella corcontica AB080306 Nannochloris sp. strain SAG 251-2

1 OTU_27 0,86 0,98 X89012 Choricystis minor HQ702326 Chlorella saccharophila 0,84 0,88 OTU_2177 0,94 FR865615 Chlorella reisiglii 0,86 EF595524 Heveochlorella hainangensis FM205847 Closteriopsis acicularis 0,87 0,91 OTU_426 HQ702312 Chlorella sp. strain KMMCC C-214 0,9 FM205842 Chlorellaceae sp. TP-2008 strain CCAP 283/1 X06425 Nanochlorum eucaryotum 0,89 OTU_6 0,89 FJ946890 Trebouxiophyceae sp. strain VPL1-3 HQ188978 Uncultured Viridiplantae clone E107_05C AM109906 Trichosarcina mucosa AF182816 Aphanochaete magna 0,98 OTU_1123 1 JF680955 Stigeoclonium helveticum JF930340 Mychonastes afer OTU_546 AF517098 Chlamydomonas bilatus 0,76 OTU_2293

0,91 0,94 U70787 Chlamydomonas noctigama 0,85 0,95 OTU_12927 OTU_3383 0,91 OTU_105 0,9 HQ219461 Uncultured Chlorophyta clone AY2009E16 0,77 Chlamydomonad 1 AY220571 sp. strain BogD6/3T-6w

0,84 OTU_12937 GU067821 Uncultured Volvocales clone ESS220206.042 0,86 0,77 HQ191399 Uncultured Chlorophyta clone PA2009B22 AY220576 Chlamydomonad sp. strain WTwin2/24P-1w OTU_738 0,98 HQ191352 Uncultured Chlorophyta clone PA2009C31 0,79 AB290341 Chlamydomonas sordida 0,99 GQ122368 Chlamydomonas hedleyi GQ122365 Chlorococcum minutum Chlorophytes 0,86 0,86 OTU_3249 0,77 AY180036 Uncultured chlorophyte clone CCW84 JN903992 Tetracystis aplanospora 0,88 0,95 OTU_3328 0,87 OTU_3057

0,77 OTU_3256 0,99 JF343798 Chlamydomonas raudensis 0,85 EF100372 Uncultured eukaryote clone D5P10D07 Atractomorpha echinata 0,93 U73470 OTU_1853 U63101 Bracteacoccus aerius 0,9 GQ375105 Radiococcus polycoccus 0,84 0,9 JQ411023 Bracteacoccus sp.strain GTD4a-1 Chlorotetraodon incus 0,96 AF288363 0.05 M63000 Characium hindakii OTU_3293 0,7 1 AF387160 Microspora sp. strain UTEX LB 472

0,83 X56100 Ankistrodesmus stipitatus Monoraphidium 0,88 DJ399992 Novel spc OTU_46 0,92 Y16939 Monoraphidium dybowskii 0,8 Y16937 Ankistrodesmus gracilis GQ985408 Dictyococcus varians OTU_386

0,76 0,56 AY780661 Pediastrum boryanum AY780669 Tetraedon caudatum 0,84 AB055800 Coelastrella saipanensis Scenedesmus parvus 0,95 FR865718

0,85 OTU_12940 1 Chlorophyceae Scenedesmus communis 0,99 0,7 X73994 EF024299 Uncultured Scenedesmaceae clone Elev_18S_700 0,88 AY195982 Coccoid green alga isolate JL 4-10 X65557 Spermatozopsis similis 0,84 AY195974 Pseudomuriella sp. isolate Itas 9/21 14-1d Carteria 0,88 GQ122380 sp. KMMCC FC-98 0,8 OTU_342 1 HQ219445 Uncultured Chlorophyta clone AY2009D25 OTU_726 AY220590 Chlamydomonad sp. strain Pic8/18P-1d 0,95 0,89 OTU_3313 FR865535 Chlamydomonas sp. strain CCAP 11/140 AJ410472 Oogamochlamys zimbabwiensis JX101906 Chlamydomonas noctigama JN982286 Lobochlamys segnis

0,74 0,8 OTU_3279 0,93 0,96 FR865555 Chlamydomonas sp. strain CCAP 11/161 DQ009753 Chlamydomonas sp. strain CCMP 233 0,82 OTU_1997 0,88 0,98 JN904004 Chloromonas subdivisa OTU_648

0,75 DQ009757 Chlamydomonas uva-maris JN090887 Uncultured eukaryote clone KRL01E27 0,88 0,78 AB058371 Chlamydomonas sp. strain MBIC10592 JN903987 Chlamydomonas sp. strain ISBAL 282

0,92 OTU_2756 0,86 AY220608 Chlamynomonadaceae sp. strain Mary9/21BT-1d

0,92 OTU_3185 AB548689 Chloromonas pseudoplatyrhyncha 0,78 U70791 Chloromonas clathrata 0,77 AF517091 Chloromonas palmelloides 0,86 OTU_2207 OTU_1092 0,86 U70596 Carteria olivieri 0,88 D86501 Carteria crucifera

0,97 OTU_3264

0,9 D86496 Hafniomonas reticulata

0,88 AB472272 Cephalomonas granulata AB218715 Neochlorosarcina negevensis 0,99 OTU_2442 0,88 0,77 AY220607 Chlamydomonadaceae sp. strain Tow8/18T-12w 0,81 OTU_800 FR865592 Chlamydomonas mexicana

0,98 0,93 AF408246 Paulschulzia pseudovolvox

0,93 AY220083 Chlamydomonadaceae sp. isolate Tow 2/24 P-8d OTU_1026 0,8 FR865524 Vitreochlamys incisa

0,78 OTU_12957 0,84 M32703 Chlamydomonas reinhardtii JN903975 Chlamydomonas debaryana 0,97 AY220086 Chlamydomonadaceae sp. isolate Pic 8/18 P-1w OTU_1836

Figure S. Approximate ML phylogenetic tree of partial 18S rDNA sequences of Archaeplastida. A total of 450 unambiguously aligned positions were used to reconstruct the tree. Two haptophyte sequences were used as an outgroup to root the tree. Representative sequences of OTUs from this work are shown in bold green. Local support values greater than 0.5 are shown at nodes. Scale bar represents the number of estimated substitutions per position for a unit branch length. AB092918 Pyrsonympha grandis AF244903 Trimastix pyriformis 1 OTU_1857 0,99 Excavates AF244905 Trimastix marina

1 DQ525696 Saccinobaculus ambloaxostylus 0,92 DQ525700 Saccinobaculus doroaxostylus OTU_2212 AY268038 Micronuclearia podoventralis

0,96 OTU_12959 0,99 Apusozoans 0,96 EU349233 Planomonas limna DQ207566 Ancyromonas sigmoides 0,9 EU349235 Planomonas howeae AF293900 Paraflabellula reniformis OTU_2417 EF455780 Planomonas micra 0,99 0,82 JQ340337 Ancyromonas indica

0,97 0,99 HM749955 Uncultured marine eukaryote clone BLACKSEA_cl_53 Apusozoans GU001166 Planomonas sp. 3b 0,76 EU349231 Planomonas mylnikovi JF791081 Ancyromonas micra AY635828 Mortierella sp. MS-6 isolate AFTOL-ID 42 0,93 1 FJ577822 Uncultured Amoebozoa clone Orly22 0,52 DQ229955 Platyamoeba oblongata 0,98 FN865764 Uncultured eukaryote clone O138006A05 0,78 OTU_3351 0,65 OTU_3341

0,98 EF024417 Uncultured eukaryote clone Elev_18S_1076 0.1 FJ544419 Ceratioxyxella tahitiensis

0,93 AY686576 Pseudoparamoeba pagei AF293895 Echinamoeba exundans Amoebozoans

0,96 OTU_3285 AY919786 Uncultured freshwater eukaryote clone LG30-03

0,85 AY960120 Protacanthamoeba bohemica HM159368 Acanthamoeba sp. strain WR 0,96 AB425954 Comandonia sp. strain E_5F 0,97 U07417 Acanthamoeba royreba U07413 Acanthamoeba castellanii AF251937 Acanthamoeba hatchetti

Figure S9. Approximate ML phylogenetic tree of partial 18S rDNA sequences of excavates, apusozoans and amoebozoans. A total of 339 unambiguously aligned positions were used to reconstruct the tree. Two bacterian sequences were used as an outgroup to root the tree. Representative sequences of OTUs from that work are shown in bold green. Local support values greater than 0.5 are shown at nodes. Scale bar represents the number of estimated substitutions per position for a unit branch length. Apicomplexa Cercozoa Ancyromonadida Haptophyta Ciliophora Streptophyta Apusomonadidae Katablepharida ABCDinophyta Chlorophyta Centroheliozoa Telonema Perkinsea Metamonada Cryptophyta Others Other Alveolata Others Others

MG MG MG

OrthoP OrthoP OrthoP Chlorophyll SR Chlorophyll SR Chlorophyll SR NH3 DOC NH DOC NH3 DOC LC 3 LC LC RSA RSA RSA TDS TDS TDS Temperature - Temperature - Temperature - NO2 NO2 NO2 O2 O2 O2 - - - NO3 pH NO3 pH NO3 pH

EV EV EV % 2 % 1 0 1 % 2 % 1 0 1 % 2 % 1 0 1

%2%1 0 1 2 %2%1 0 1 2 %2%1 0 1 2

Bacillariophyceae Oomyceta Discosea Bicosoecida Synurophyceae Other Amoebozoa D Chrysophyte Xanthophyceae E Fungi Labyrinthulida Other Stram Ichthyosporea MAST Others Choanoflagellida Others

MG MG

OrthoP OrthoP Chlorophyll Chlorophyll SR SRDOC NH DOC NH3 3 LC LC RSA RSA TDS TDS Temperature - Temperature - NO NO2 O2 2 O2 - - NO3 pH NO3 pH

EV EV % 2 % 1 0 1 % 2 % 1 0 1

%2%1 0 1 2 %2%1 0 1 2

Figure S10. Canonical Correspondance Analysis (CCA) plot. All plots are from the same analysis as in Fig. 6, with ellipses emphasizing the distribuQJ of JK1': C1: VR to alveolates (A), rhizarians, archaV]C:1R1 and excavates (B), cryptophytes, centroheliozoans, telonemids, haptophytes, katablepharids and apusozoans (C), stramenopiles (D) and unikonts (E). Projected 1JV`a on CCA1: 34.07% and on CA: 29.77%. Black dots correspond to OTUs. Grey triangles indicate the samples. SR, Saint Robert; EV, Etang des Vallées; MG, Mare Gabard; LC, La Claye; RSA, R u Sainte Anne. Replicate samples are superimposed. DOC, Dissolved Organic Carbon; OrthoP, Ortho Phosphate; TDS, Total Dissolved Solutes.