Diversity and distribution of planktonic protists in the ... - Oxford Journals

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Diversity and distribution of planktonic protists in the northern South China Sea LU-YAN LI 1, DUAN LIN 2, JIA-HUI CHEN 2, SHU-HUI WU 1, QIAO-JUAN HUANG 1, HUI ZHOU 1, LIANG-HU QU 1 AND YUE-QIN CHEN 1* 1

KEY LABORATORY OF GENE ENGINEERING OF THE MINISTRY OF EDUCATION, STATE KEY LABORATORY FOR BIOCONTROL, BIOTECHNOLOGY RESEARCH CENTER, 510275, PEOPLES REPUBLIC OF CHINA AND 2SOUTH CHINA SEA ENVIRONMENTAL MONITORING CENTER, STATE

SUN YAT-SEN UNIVERSITY, GUANGZHOU

OCEANIC ADMINISTRATION, GUANGZHOU

510300, CHINA

*CORRESPONDING AUTHOR: [email protected] Received December 19, 2009; accepted in principle July 23, 2010; accepted for publication September 2, 2010 Corresponding editor: John Dolan

To determine the molecular taxonomic affiliations of small planktonic protists in the northern South China Sea, we constructed phylogenetic trees by correlating the 18S rDNA sequences of known heterotrophic protists with those of unknown protists from four sites. There was a high diversity of these protists in the northern South China Sea that were not sampled by net collection. In addition, we discovered a surprisingly large number of novel radiolarian sequences that showed a unique biogeographic group for this location. Our new data on centrohelids confirmed recent studies showing that representatives of this group live in marine habitats and not only in fresh water. We also report two newly classified eukaryotic lineages (Telonemia and Katablepharidophyta) in the South China Sea for the first time. Furthermore, the phylogenetic relationships showed that the distribution of the novel protists in the northern South China Sea had distinct spatial variation. Our work extends knowledge about the diversity and habitats of planktonic protists in the South China Sea. KEYWORDS: protists; 18S rDNA; genetic diversity

I N T RO D U C T I O N Planktonic protists are abundant, ubiquitous members of the marine fauna. On cruises in the China Seas, planktonic protists are collected by vertical tows of 76-mm-mesh plankton nets, following standard methods (SAPRC, 2007). Regardless if this standard was followed (Zhang et al., 2008) or a smaller 2-um mesh was used (Huang et al., 2003), the focus of morphological taxonomists in the China Seas has been on ciliates (Zhang and Wang, 2001), tintinnids (Zhang et al., 2008) and marine flagellates (Huang et al., 2003). Before our work in the Nansha Sea area (Yuan et al., 2004), planktonic protists smaller than the net mesh size (76 mm) were poorly studied in the South China Sea. Their true diversity in the China Seas is unknown, as well as their spatial distribution patterns, spatial and temporal dynamics, and

ecological roles. Because of their colorless and fragile bodies, planktonic protists are much more difficult to identify than are microalgae. In addition, they are easily destroyed by fixatives, pressure of a cover slip or even by contact with the air– water interface. For these reasons, marine ecologists often ignore a large number of planktonic protists (Johannes, 1965). Molecular analyses based on the rRNA approach have provided an important method to study marine protists. By placing the sequences of this marker gene in a phylogenetic tree of eukaryotes, molecular taxonomy is a powerful tool for recovering evolutionary relationships among species (Massana et al., 2002). This has enabled definition of new plankton classes, including Pelagophyceae (Andersen et al., 1993) and Bolidophyceae (Guilou et al., 1999), as well as the identification of putatively novel

doi:10.1093/plankt/fbq125, available online at www.plankt.oxfordjournals.org. Advance Access publication October 5, 2010 # The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]


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planktonic organisms from major taxonomic groups, such as the picobiliphytes (Not et al., 2007b). Taxonomic affiliations of the phytoplankton sequences were easily determined by BLASTN from the large number of sequences of identified phytoplankton species in GenBank (Li et al., 2008); however, a substantial number of clones in our work were affiliated with marine eukaryotic groups uncharacterized by BLASTN. We determined the molecular taxonomic affiliation of planktonic protists by constructing phylogenetic trees and correlating the 18S rDNA sequences of the main eukaryotic protist groups with sequences of unknown protists collected from four sites in the northern South China Sea. The classification of eukaryotic diversity has changed rapidly in recent years. The four eukaryotic kingdoms, plants, animals, fungi and protists, have been transformed through numerous permutations into the current system of six “supergroups”: Opisthokonta, Amoebozoa, Excavata, Rhizaria, Archaeplastida and Chromalveolata (Cavalier-Smith, 1998, 2004). From the perspective of molecular taxonomy, the major revision is in the Kingdom Protista (Parfrey et al., 2006). The diverse singlecell eukaryotes generally had been placed in one group, the Protista; however, this historic distinction between macroscopic and microscopic eukaryotes does not adequately capture their complex evolutionary relationships or the vast diversity in the microbial world. The International Society of Protozoologists recently proposed a formal reclassification of eukaryotes into six supergroups, although acknowledging uncertainty in some groups (Parfrey et al., 2006). The most familiar phylogenetic groups of single-cell protists are as follows (Embley and Martin, 2006; Parfrey et al., 2006): alveolates and stramenopiles (both belonging to Chromalveolata); cercozoans and radiolarians (both belonging to Rhizaria); centroheliozoans (Cavalier-Smith and von der Heyden, 2007), katablepharidophytes and Telonemia (the basal groups just reported in the eukaryotic phylogenetic tree) (Reeb et al., 2009). The following sections discuss our results for these groups in the phylogenetic trees.

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Nucleic acid extraction The extraction of total community DNA involved several freeze-thaw cycles between liquid nitrogen and 658C after the filters had been soaked in algal DNA extraction buffer [4% CTAB, 1.4 mol L21 NaCl, 1% PVP, 100-mM Tris – HCl ( pH 8.0), 25-mM EDTA ( pH 8.0)]. Nucleic acids were extracted as previously described (Yuan et al., 2004; Li et al., 2008). Eukaryotic 18S rRNA genes were amplified by PCR with eukaryote-specific primers 18P1 (ACCTGGTTGATCC TGCCAGT) and 18P11R (TGATCCTTCYGCAG GTTCAC) designed (these degenerate primers were designed from representative eukaryotic sequences by Primer Premier 5.0) under the previously described condition (Yuan et al., 2004; Li et al., 2008). The PCR products, 18S rDNA genes were collected to construct libraries by use of the TA cloning kit (TaKaRa Biotechnology). Four libraries were constructed from coastal small plankton from the surface and 100-m depth and designated by the sampling sites and depths (“a” means 0.5 m, “b” means 100 m) (Table II).

Restriction fragment length polymorphism analysis and sequencing The positive transformants of rDNA libraries were screened by PCR amplification of inserts using the primers referred to above (18P1 and 18P11R). PCR amplification products containing the correct size of insert were digested with 1 U of restriction enzyme MspI mL21 for 6 – 12 h at 378C. The digested products were separated by electrophoresis at 120 V for 1.5–2 h in a 2.0% agarose gel. Representative clones of the library that showed unique restriction fragment length polymorphism patterns were selected and the plasmid DNA extracted and purified for sequencing. The sequences of our libraries were sequenced on 3730 DNA Sequencer by Invitrogen Biotech Co. Ltd. (Guangzhou, China). All of the sequences were sequenced from the direction of 18N1 through the region of the 18S rRNA gene by one sequencing reaction.

Phylogenetic analyses

Oceanographic sampling Samples were collected at four stations during the cruises of the South China Sea Environmental Monitoring Center (Fig. 1, Table I). Samples were collected on 0.45-mm Millipore filters from 2 L of seawater, which had passed through the 76-mm-mesh plankton net. The filters were frozen at 2208C before subsequent analysis.\

Preliminary taxonomic affiliation of the sequences was determined using BLASTN against the GenBank database (September 2009). Suspected chimeras were checked by use of the KeyDNAtools (http://KeyDNAtools.com). The sequences were aligned with the representative sequences of correlative groups by use of the Clustal X 1.8 program (Thompson et al., 1997). Alignments were manually checked by use of the

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PLANKTONIC PROTISTS IN THE SOUTH CHINA SEA

Fig. 1. Sites sampled for small planktonic protists in the South China Sea (created by ArcGis 8.3).

BioEdit 5.0.9 program. Difficult or poorly aligned positions for use in phylogenetic analyses were determined using the Gblocks method (Castresana, 2000) for selecting conserved blocks (minimum block length ¼ 5;

allowed gap positions ¼ with half) as well as manual elimination of non-homologous positions. Different nested models of DNA substitution and associated parameters were tested by use of the Modeltest2 (Posada,

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Table I: Sampling locations in the South China Sea, the environmental parameters and molecular libraries for small planktonic protists Site

Latitude (N)

Longitude (W)

Date

Bottom depths (m)

Depths (m)

Temperature (88 C)

Salinity (88 )

Clone

Sequence

Coverage (%)

N4 E1 B19 29

18 19.08 19.95 24.55

110 111.84 114.67 118.18

September 2006 June 2005 September 2005 May 2003

96 168 200 15

0.5 0.5 0.5 0.5

30.1 29.3 29.7 26.7

33.7 34.4 34.6 29.5

173 191

62 68

83.2 82.7

138

54

81.9

Table II: The percentages of small size protist clones affiliated with the main groups from four 18S rDNA libraries from the South China Sea Alveolata Apicomplexa Ciliates Group I Group II Stramenopiles Heterotrophic Novel MAST Radiolaria Acantharea Polycystinea Taxopodida Novel RAD Centroheliozoa Katablepharidophyta Telonemia

B19b (%)

N4a (%)

E1a (%)

29a (%)

3.6 7.3 18.1

1.7 4.6 2.3 21.4

0.5 11 6.3 14.7

27.8 3.8 0.9

5.8 8.1

1.6 0.5

0.9

1.6 1.1 8.5 3.2

1.9

11.6

7.3 6.5 1.5 35.5 1.2 0.7

into four major groups: Dino groups I and II (Lopez-Garcia et al., 2001; Groisillier et al., 2006; Guillou et al., 2008) , dinoflagellates, apicomplexans and ciliates (Fig. 2). No Dino group III, IV and V sequences (Guillou et al., 2008) were found in our work. Nearly all of our ciliate sequences were 97 – 100% similar to known ciliates species. The Dino group I sequences from the northern South China Sea fit within four clades of Dino group I (clades 1, 2, 4 and 5; Fig. 2) and were closely related to the sequences from the euphotic zone and deeper seawaters (2000 – 3000 m), but not from the sediments. Sixty-eight percent of the clones of Dino group II in our libraries belonged to the Amoebophrya clade. Except for the “Clade Amoebophrya”, other NAII-affiliated sequences in our work fit into Dino group II: clades 6, 7, 10/11, 14 and 20 (Fig. 2).

Molecular diversity of stramenopiles in the northern South China Sea

“a” means 0.5 m, “b” means 100 m.

2003). Settings given by Modeltest were used to perform the Bayesian analyses. Phylogenetic relationships were inferred by the neighbor-joining method with PAUP v.4.0b10 (Swofford, 2000) and Bayesian analysis with MrBayes ver. 3.1 (Huelsenbeck and Ronquist, 2001).

Nucleotide sequence accession numbers The sequences provided in this paper have been submitted to the NCBI Nucleotide Sequence Database under the Accession Numbers: EU333027 – EU333114 and HM769613 – HM769622.

R E S U LT S Molecular diversity of alveolates in the northern South China Sea Bayesian and NJ methods clearly separated all of our alveolate sequences in the northern South China Sea

Within the heterotrophic stramenopiles, sequences were gathered into four operational taxonomic units, which were closely similar to labyrinthulid and bicosoecida species (Fig. 3). Fifteen clones of the 16 heterotrophic stramenopile clones belonged to Labyrinthulida in the library of B19b. Most heterotrophic stramenopiles clones belonged to Bicosoecida in the library of N4a. There were no heterotrophic stramenopiles clones in the Xiamen harbor site (Table II). Our novel marine stramenopile (MAST) sequences were placed within the clades MAST-1, -4, -6, -7 and -9 (Fig. 3).

Radiolarian-affiliated sequences reported in the northern South China Sea The Radiolaria tree, composed of the Acantharea, Polycystinea and Taxopodida, achieved strong bootstrap support (Nikolaev et al., 2004). We obtained abundant radiolarian sequences in our 100-m depth libraries. Most of our radiolarian sequences were placed within RAD-III, as named in Not et al. (Not et al., 2007a). A number of others were classified under the Polycystinea

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Fig. 2. Phylogenetic tree of Alveolates, based upon the analysis of 177 partial 18S rDNA sequences, 656 bp in length. A GTRþIþG model was selected using the following parameters: Prset statefreqpr¼dirichlet(1,1,1,1); Lset nst¼6 rates¼invgamma. Bayesian posterior probabilities were computed by running Markov chain Monte Carlo search for 17 000 000 generations by using the program default priors on model parameters. Trees were sampled every 100 generations with the first 25% discarded as burn-in. Numbers at the nodes represent Bayesian and NJ support values, respectively. Sequences obtained from the northern South China Sea are printed in bold.

(Nassellaria and Spumellaria) and Acantharea, and some sequences were within or related to the Taxopodida, RAD-IV, as named in Not et al. (Not et al.,

2007a) (Fig. 4). In addition, we identified a particularly interesting group of South China Sea sequences (shaded in Fig. 4), composed of B19bC7, B19bD93 and

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Fig. 3. Phylogenetic tree of Stramenoplies, based upon the analysis of 71 partial 18S rDNA sequences, 555 bp in length. A GTRþIþG model was selected using the following parameters: Prset statefreqpr¼dirichlet(1,1,1,1); Lset nst¼6 rates¼invgamma. Bayesian posterior probabilities were computed by running Markov chain Monte Carlo search for 3 000 000 generations by using the program default priors on model parameters. Trees were sampled every 100 generations with the first 25% discarded as burn-in. Numbers at the nodes represent Bayesian and NJ support values, respectively. Sequences obtained from the northern South China Sea are printed in bold.

NS371B39. In the phylogenetic tree of Radiolaria constructed by Bayesian analysis (Fig. 4), this group of SCS sequences was basal to the Taxopodida and LC22_5EP_23 from venting fluids (Lopez-Garcia et al., 2007).

Novel marine centrohelid sequences reported from the northern South China Sea Our phylogenetic tree of Centroheliozoa (Fig. 5) was constructed based on the taxonomy of Pterocystina (Pterocystidae, Choanocystidae and Heterophryidae) and

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Fig. 4. Phylogenetic tree of Radiolaria, based upon the analysis of 44 partial 18S rDNA sequences, 456 bp in length. A GTRþG model was selected using the following parameters: Prset statefreqpr¼dirichlet(1,1,1,1); Lset nst¼6 rates¼invgamma. Bayesian posterior probabilities were computed by running Markov chain Monte Carlo search for 1 000 000 generations by using the program default priors on model parameters. Trees were sampled every 100 generations with the first 25% discarded as burn-in. Numbers at the nodes represent Bayesian and NJ support values, respectively. Sequences obtained from the northern South China Sea are printed in bold.

Acanthocystina (Acanthocystidae, Raphidiophryidae and Marophryidae) (Cavalier-Smith and Chao, 2003; Cavalier-Smith and von der Heyden, 2007b). The centrohelid sequences (E1a77, E1aE54 and N4aB2) we collected from the South China Sea clustered in the clade of Choanocystidae (CL3) (Fig. 5), which has a substantial mixture of marine and freshwater subclades as described by Cavalier-Smith and von der Heyden (Cavalier-Smith and von der Heyden, 2007).

First report of recently classified eukaryotic lineages (Telonemia and Katablepharidophyta) from the South China Sea In addition to the large variety of alveolate, stramenopile and radiolarian sequences, we identified two independent phylogenetic groups that could correspond to newly reported eukaryotic taxa, Telonemia and

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Fig. 5. Phylogenetic tree of Centroheliozoa, based upon the analysis of 34 partial 18S rDNA sequences, 448 bp in length. A SYMþIþG model was selected using the following parameters: Prset statefreqpr¼fixed (equal); Lset nst¼6 rates¼invgamma. Bayesian posterior probabilities were computed by running Markov chain Monte Carlo search for 1 000 000 generations by using the program default priors on model parameters. Trees were sampled every 100 generations with the first 25% discarded as burn-in. Numbers at the nodes represent Bayesian and NJ support values, respectively. Sequences obtained from the northern South China Sea are printed in bold. The marine sequences are marked with asterisks.

Katablepharidophyta (Fig. 6). Recent multigene analyses place Telonemia and Katablepharidophyta as chromalvoleate allies (Reeb et al., 2009). In our data, B19bB13, 29aB27, E1aB83, E1aC3 and E1aB50 fell into the lineage of Telonemia and 29aA03 belonged to katablepharids. Comparison of our sequences with a recent survey of Telonemia 18S rDNA by the Telonemia-specific PCR strategy (Bra˚tea et al., 2010) showed that E1aC3 was clustered within Tel 1a (Fig. 6) which included sequences all from the warm water areas: the Indian Ocean, the Mediterranean Sea near Spain and the Pacific Ocean near Hawaii (Bra˚tea et al., 2010). In our other three Tel 2 sequences, 29aB27 also clustered with an Indian Ocean

sequence, but did not have significant statistical support as a subgroup. B19aB13 belonged to Tel 2f and did not show this warm water character. The position of E1aB83 was difficult to distinguish. It belonged to the clade composed of subgroups 2b, 2c, 2d and 2e. This ambiguity may be because the genetic diversity of Telonemia in warm marine waters is still poorly known. We obtained katablepharid sequences from only one site (29) in the Xiamen harbor. The sequence 29aA03 did not cluster with other marine or freshwater environmental sequences. From the phylogenetic tree of Katablepharidophyta (Fig. 7), we confirmed it to be Leucocryptos marina sequence. The sequence 29aA03 had 18 clones in the library of site 29.

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Fig. 6. Phylogenetic tree of Telonemia, based upon the analysis of 80 partial 18S rDNA sequences, 564 bp in length. A SYMþG model was selected using the following parameters: Prset statefreqpr¼fixed (equal); Lset nst¼6 rates¼gamma. Bayesian posterior probabilities were computed by running Markov chain Monte Carlo search for 9 000 000 generations by using the program default priors on model parameters. Trees were sampled every 100 generations with the first 25% discarded as burn-in. Numbers at the nodes represent Bayesian and NJ support values, respectively. Sequences obtained from the northern South China Sea are printed in bold.

Genetic biodiversity of planktonic protists in the northern South China Sea The four 18S rDNA libraries were constructed from different geographic regions in the northern South China Sea, including continental slope areas (B19 and E1),

mainland continental edge (29) and coastal waters of Hainan Island (N4; Fig. 1). Sampling locations and their characteristics are given in Table I. Table II summarizes the abundance of key protist groups found in these clone libraries. The results show that the alveolates were the

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Fig. 7. Phylogenetic tree of Katablepharidophyta, based upon the analysis of 26 partial 18S rDNA sequences, 666 bp in length. A SYMþG model was selected using the following parameters: Prset statefreqpr¼fixed (equal); Lset nst¼6 rates¼gamma. Bayesian posterior probabilities were computed by running Markov chain Monte Carlo search for 1 000 000 generations by using the program default priors on model parameters. Trees were sampled every 100 generations with the first 25% discarded as burn-in. Numbers at the nodes represent Bayesian and NJ support values, respectively. Sequences obtained from the northern South China Sea are printed in bold. The marine sequences are marked with asterisks.

predominant groups in all libraries, except that radiolarians accounted for 50.7% of the sequences in B19b. In addition, the small protist communities in the 0.5-m samples were clearly different than in 100-m samples. The percentage of ciliates in samples from 0.5-m depth was higher than from 100-m depth. Also, 100-m depth libraries had significantly higher percentages of novel MAST cells and radiolarians. Alveolate group II showed no differences in site libraries except for the 29a library.

the Helgoland time series site in the German Bight (up to 45%) (Medlin et al., 2006). At site 29 with freshwater influence, the percentage of autotrophic groups was the highest, up to 64.62% (Li et al., 2008). The alveolates there were mainly diatoms (17.9%) and ciliates (27.8%). Although Amoebophrya sequences were reported in freshwater systems (Lefevre et al., 2008), novel alveolate groups usually were absent in freshwater or occurred in low percentages (Chen et al., 2008). These two groups may be restricted to marine environments (Guillou et al., 2008).

DISCUSSION High percentage of radiolarians Novel alveolate group may be marine protists Site 29 was located at the mainland edge where the Jiulong River delivers freshwater; thus, it was extremely different from the other coastal sites. Here, the presence of the alveolate group II was very low (only 0.9%) (Table II). In contrast, this group accounted for quite high percentages in other locations: other South China Sea libraries (15– 36%) and the summer library from

B19b had a high percentage of radiolarians (Table II). These uncultured radiolarian eukaryote sequences have been detected in the Nansha Sea area (Yuan et al., 2004), Mediterranean Sea (Marie et al., 2006), Sargasso Sea (Not et al., 2007a) and the Equatorial Pacific (Moon-van der Staay et al., 2001), but were not found in our near shore samples from the South China Sea (N4 and 29). A substantial fraction of clones from the open sea belong to the radiolarians (10% on average)

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(Massana and Pedros-Alio, 2008). The reason for high percentages of radiolarians at site B19 requires further study. The fact that many described radiolarian species have not been sequenced may be one reason why so many unknown radiolarian sequences occur in environmental surveys.

Phylogenetic relationships and distributions within the novel stramenopiles Massana et al. (Massana et al., 2004) described up to 12 clusters of probable heterotrophic stramenopiles. These MAST cells are widely distributed, mostly in the upper ( photic) ocean environment and account for a substantial fraction of heterotrophic flagellates globally. MAST-1, -4, and -7 were thought to be truly planktonic aerobic organisms distributed in almost all open sea and coastal waters (Massana et al., 2004). The majority of our MAST sequences in the northern South China Sea were also placed within MAST-1, -4 and -7, except for one clone in MAST-6 and two clones in MAST-9. Most sites previously surveyed yielded sequences from MAST-1, -2 and -4, suggesting a global distribution for these uncultured organisms (Massana and Pedros-Alio, 2008). Our results agreed with this conclusion. Both sequences in the northern South China Sea (E1ab60, N4aB91) and the Nansha Sea area (5bB266) were found in MAST-1 (Fig. 3). Previously, clone N4aB41 that clustered within MAST-9 sequences apparently occurred mostly in hydrothermal vents and was suggested to occur in anoxic or microoxic habitats (Massana et al., 2004; Not et al., 2007a; Lopez-Garcia et al., 2007). Our finding of N4aB41 in coastal waters of tropical islands (N4) suggested that MAST-9 also contained the sequences derived from oxic and mesophilic environment sequences, as for N4aB41 and BL010625.32 (Massana et al., 2004). The disagreement of phylogenetic distance and niches suggested that the different MAST clusters could represent different organisms with completely different physiological and ecological roles.

Possible presence of centrohelids Centrohelids are predominantly freshwater organisms, but some have been reported from marine or brackish waters (Cavalier-Smith and von der Heyden, 2007). Previous research reported exclusively marine, relatively ancient, multispecies clades and three other wellseparated marine lineages (CL3, CL4, and CL5) (Cavalier-Smith and von der Heyden, 2007). Our centrohelid sequences were from the continental slope areas (E1) and coastal waters of Hainan Island (N4), but

not from the low salinity site (29). 37bB28, which was sequenced in our previous work (Yuan et al., 2004), is also a centrohelid sequence. Site 37 was in the shallow equatorial shelf of the Nanshan Sea. The distribution of centrohelids also refuted prior assumptions that there are no truly marine centrohelids (Cavalier-Smith and von der Heyden, 2007).

Low-salinity attribute of two newly classified groups The discovery of two freshwater clades in Telonemia suggests that Telonemia have colonized freshwater habitats. We also found a freshwater clade in the tree of Katablepharidophyta (Fig. 7). But the Telonemia or katablepharid sequence from site 29 with freshwater influence did not show obvious relationship with these freshwater clades. This also agrees with the opinion that Telonemia and Katablepharidophyta adapted to the different environmental and ecological conditions independently (Bra˚tea et al., 2010). In conclusion, the environmental sequences in four 18S rDNA libraries suggest that some remarkable planktonic protists exist in the South China Sea that had not detected by net collection. The distribution of these protists showed a distinct spatial variation. This information suggests where they may be found in future research.

AC K N OW L E D G E M E N T S We thank all the participants of the South China Sea Environmental Monitoring Center for great assistance in sample collections during the summer cruises in 2005 and 2006. We especially thank Dr Medlin of the Alfred Wegener Institute for Polar and Marine Research in Germany for her comments on the manuscript.

FUNDING This study was supported by the National Natural Science Foundation of China (Grant No. U0631001), and funds from the Reserve Key Projects of Sun Yat-Sen University.

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