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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.
METHOD
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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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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 (Bråtea 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 (Bråtea 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 (Bråtea 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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