Proc. R. Soc. B (2005) 272, 2073–2081 doi:10.1098/rspb.2005.3195 Published online 17 August 2005 The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes Jan Sˇ lapeta, David Moreira and Purificacio´nLo´pez-Garcı´a* Unite´ d’Ecologie, Syste´matique et E´ volution, UMR CNRS 8079, Universite´ Paris-Sud, 91405 Orsay Cedex, France Classical studies on protist diversity of freshwater environments worldwide have led to the idea that most species of microbial eukaryotes are known. One exemplary case would be constituted by the ciliates, which have been claimed to encompass a few thousands of ubiquitous species, most of them already described. Recently, molecular methods have revealed an unsuspected protist diversity, especially in oceanic as well as some extreme environments, suggesting the occurrence of a hidden diversity of eukaryotic lineages. In order to test if this holds also for freshwater environments, we have carried out a molecular survey of small subunit ribosomal RNA genes in water and sediment samples of two ponds, one oxic and another suboxic, from the same geographic area. Our results show that protist diversity is very high. The majority of phylotypes affiliated within a few well established eukaryotic kingdoms or phyla, including alveolates, cryptophytes, heterokonts, Cercozoa, Centroheliozoa and haptophytes, although a few sequences did not display a clear taxonomic affiliation. The diversity of sequences within groups was very large, particularly that of ciliates, and a number of them were very divergent from known species, which could define new intra-phylum groups. This suggests that, contrary to current ideas, the diversity of freshwater protists is far from being completely described. Keywords: eukaryotes; environmental PCR; molecular ecology; protist diversity 1. INTRODUCTION applied to the study of protist diversity in a number of The accessibility of freshwater ecosystems has made of different ecosystems, including oceanic waters (Diez et al. these environments a privileged choice for protozoological 2001; Lo´pez-Garcı´a et al. 2001; Moon-van der Staay et al. studies for more than three centuries since Leeuwenhoek’s 2001; Romari & Vaulot 2004), deep-sea vents (Edgcomb times (Finlay & Esteban 2001). Protists are key com- et al. 2002; Lo´pez-Garcı´a et al. 2003), anoxic environ- ponents of aquatic food webs both as members of the ments (Dawson & Pace 2002; Stoeck et al. 2003), river phytoplankton (e.g. diatoms) and as major consumers of sediment (Berney et al. 2004) and one acidic river (Amaral bacterial biomass (e.g. heterotrophic flagellates and Zettler et al. 2002). In all these studies a large protist ciliates). In particular, nanoflagellates are abundant in diversity has been revealed and, although in general they planktonic communities, acting as the primary bacterial belong to well established eukaryotic kingdoms (Berney consumers and playing a cardinal role in nutrient cycling et al. 2004; Cavalier-Smith 2004), the lineages identified (Arndt et al. 2000; Weisse 2002). Aquatic ecologists have are very often highly divergent from known protist traditionally focused their attention on functional cat- sequences. This suggests that a large fraction of eukaryotic egories (trophic chain levels) rather than in species microbial communities remains to be discovered (Moreira composition, although there is a recognized increasing &Lo´pez-Garcı´a 2002). The existence of such a hidden interest in integrating the existing taxonomic data ( Weisse diversity would support detractors from the above- 2002; Finlay 2004). Indeed, many species have been mentioned idea that most protist species have already isolated and described, and taxonomic inventories of been described (Foissner 1999). freshwater protists have been constructed over many In order to see whether the results obtained by years. The recurrent observation of the same morphologi- molecular methods fit those accumulated by classical, cal types in freshwater systems from all over the world has morphological studies, we carried out a comprehensive led to the idea that the global protist species richness is survey of protist small subunit ribosomal DNA (SSU relatively low. Ciliates, easily distinguishable due to their rDNA) diversity from two different freshwater reservoirs. complex morphology, are used as a paradigmatic example. We analysed picoplanktonic and small nanoplanktonic With nearly 4000 species identified, it has been proposed (0.2–5 mm range), and larger planktonic (greater than that most ciliate species have already been discovered 5 mm) fractions as well as sediment samples from two (Finlay 1998). ponds, one oxic and one suboxic, located in the same Molecular methods based on the amplification and geographic region. Our results reveal a large diversity of sequencing of small subunit ribosomal RNA (SSU rRNA) lineages within known eukaryotic phyla, including some genes have opened the possibility of studying microbial phylotypes that are divergent from already known diversity independently of morphological identification sequences. This is particularly noticeable in the case of and cultivation. In recent years, these methods have been ciliates, which dominate the protist diversity in the suboxic system. These results support the idea that the inventory * Author for correspondence ([email protected]). of freshwater protists is far from complete. Received 19 January 2005 2073 q 2005 The Royal Society Accepted 27 April 2005 2074 J. Sˇlapeta and others The extent of protist diversity 2. MATERIAL AND METHODS were constructed in this way, including duplicate libraries for (a) Sampling both sediment and plankton samples. Clone inserts were Samples were collected in late autumn–early winter from a amplified with vector T7 and M13R primers, and inserts of suboxic pond (CV1) and from an oxic pool (CH1) located the expected size were sequenced directly using either specific a few kilometres south of Paris, France. The suboxic CV1 or vector primers by Genome-Express (Meylan, France). was an approximately 15 m diameter pond located at the campus of the Universite´ Paris-Sud (pH 6.5, water (c) Sequence and phylogenetic analyses temperature 11.5 8C). When stirred, its waters bubbled Initially, we obtained partial sequences from positive clones and smelled strongly of H2SandCH4, indicators of that were trimmed for ambiguities and used to construct a oxygen-depleted environments. ConOx probe ( WTW, local database, which was then analysed by internal BLAST to Weilheim) oxygen measurements yielded values ranging identify identical or highly similar sequences. Sequences K K from 0.38 mg l 1 (bottom) to 2.4 mg l 1 (surface). The exhibiting greater than 97% identity were considered to be 2 small (ca 30!50 m ), normally oxygenated (9.6 mg O2 per single phylotypes. In total, 377 eukaryotic sequences from 7 litre) shallow lake CH1 was located at the Chevreuse duplicate libraries (247 sequences corresponding to CV1 and locality in the Regional Natural Park of the Haute Valle´ede 130 to CH1) were included in our analysis (Electronic Chevreuse ( pH 7.6, water temperature 4 8C, a thin ice layer Appendix—table S1). Representative sequences of the covered the lake). In both cases, sediment and plankton difference (greater than 97%) identity groups were then were sampled. Dark brown/black sediments rich in organic selected and fully sequenced to obtain an almost complete detritus were collected from the bottom of the two ponds SSU rDNA. Full-length sequences were checked for the using sterile 50 ml Falcon tubes. A second, sandy, sediment presence of chimeras using CHIMERA_CHECK v. 2.7 at the sample was additionally collected from the edge of the Ribosomal Database Project II (Cole et al. 2003). For suboxic CV1 system. Water was collected into sterile 1 l suspicious sequences, we looked for incongruencies in glass bottles with a sterile gauze adapted to their openings. phylogenetic trees obtained from different parts of the Samples were processed (filtration and DNA extraction) sequence, and subsequently, multiple alignments were immediately after collection. Water samples were passed inspected visually to detect possible recombination sites through several layers of sterile gauze to filter out the between distant phylotypes. In addition, full sequence components larger than 50–100 mm in diameter. Approxi- datasets were analysed using BELLEROPHON (Huber et al. mately 0.5 l water was then filtered across a 5 mm-pore 2004), and putative chimeric clones thus identified were diameter filter (TMTP, Millipore), and the resulting filtrate manually checked. From the potential chimeras identified by was further passed across a 0.22 mm-pore diameter filter CHIMERA_CHECK and BELLEROPHON, only nine chimeras were (GTTP, Millipore). DNA was extracted from the biomass unambiguously verified (three in CV1 sample, six in CH1 retained on the 5 mm (CV1-5 and CH1-5) and 0.22 mm sample), which were excluded from our analyses. In total, we (CV1-2 and CH1-2) filters. obtained 83 almost complete eukaryotic SSU rDNA sequences, which were included in a local eukaryotic SSU (b) DNA extraction, PCR amplification, cloning rDNA database containing more than 5000 sequences. The and sequencing obtained sequences ranged from 1335 bp (CV1-B2-35) to The planktonic biomass retained on filters was suspended in 1956 bp (CH1-S2-16). The closest BLAST matches to our 500 ml PBS. Cells were then lysed in the presence of new sequences that were not included initially in this database 80 mgmlK1 proteinase K, 1% SDS, 1.4 M NaCl, 0.2% were retrieved from GenBank at NCBI, aligned using b-mercaptoethanol and 2% CTAB (final concentrations) at CLUSTALX v. 1.82 (Thompson et al. 1997), and the multiple 55 8C. DNA was then extracted once with phenol–chloro- alignment edited manually. Ambiguous positions in the form–isoamylalcohol, and once with chloroform–isoamylal- alignment were discarded from phylogenetic analyses. Trees cohol. Nucleic acids were concentrated by ethanol were reconstructed using both Bayesian and maximum precipitation. DNA from sediments was directly extracted likelihood methods. Bayesian phylogenetic analyses were by using the SoilMaster DNA Extraction Kit (Epicentre) done with MRBAYES v.