APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
July 2001, p. 2932–2941Vol. 67, No. 7
Study of Genetic Diversity of Eukaryotic Picoplankton in Different
Oceanic Regions by Small-Subunit rRNA Gene Cloning
BEATRIZ DI´EZ, CARLOS PEDRO ´S-ALIO ´,* AND RAMON MASSANA
Departament de Biologia Marina, Institut de Cie `ncies del Mar, CSIC,
E-08039 Barcelona, Catalunya, Spain
Received 10 January 2001/Accepted 11 April 2001
Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic
systems, often accounting for a significant fraction of the biomass and activity in a system. Their identity,
however, has remained elusive, since the small cells lack morphological features for identification. We deter-
mined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries
from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters. Picoplankton were obtained by
filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes. Genetic
libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis,
and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the
phylogenetic diversity in each library was rather great, and each library included many different OTUs and
members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal
classes, especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A
minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chry-
somonad Paraphysomonas, cercomonads, and fungi. There were two relatively abundant novel lineages, novel
stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism
that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel
stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the
importance of attempts to grow the small eukaryotic plankton in pure culture.
Marine picoeukaryotes (which are between 0.2 and 2 to 3
?m in diameter) (43) are probably the most abundant eu-
karyotes on Earth. They are found throughout the world’s
oceans at concentrations between 102and 104cells ml?1in the
photic zone, and they constitute an essential component of
microbial food webs, playing significant roles in global mineral
cycles (11, 21). Marine picoeukaryotes seem to belong to very
different phylogenetic groups. In fact, nearly every algal phy-
lum has picoplanktonic representatives (47), and in the last 10
years three novel algal classes have been described for picoeu-
karyotic isolates (3, 15, 27). However, the extent of the diver-
sity and the distribution and abundance of the different taxa in
situ remain unknown (33). In the open oceans most picoeu-
karyotes are coccoid or flagellated forms with chloroplasts
(phototrophic) or without chloroplasts (heterotrophic) and
with few morphologically distinct features (5, 44, 47). They can
hardly be discriminated, even at the class level, by conventional
optical microscopy (30). Electron microscopy generally allows
assignment to taxonomic classes (4), but most cells do not have
enough ultrastructural features for identification at lower tax-
onomic levels (38). Cultivation is the best possible way to
characterize a natural organism, and isolation of small picoeu-
karyotic strains is thus an important task. However, there is no
guarantee that organisms grown in culture are dominant or
important in the natural community (16, 23). Organisms be-
longing to different algal classes have different diagnostic
marker pigments that can be identified and quantified by high-
performance liquid chromatography (HPLC) (17). HPLC pig-
ment analysis is very useful for characterizing new isolates, but
it has some technical constraints when it is applied to natural
assemblages, since interpretation of the complex pigment pat-
terns of samples requires application of algorithms (20) which
generally involve untestable assumptions. At best, many of the
conventional characterization techniques have limited phylo-
genetic capacity and are cumbersome or time-consuming.
An alternative approach for characterizing the phylogenetic
diversity of marine picoeukaryotes is analysis of small-subunit
(SSU) rRNA genes (2, 33). During the last decade, cloning of
environmental rRNA genes has provided insight into the di-
versity of the marine prokaryotic picoplankton and has re-
vealed that this assemblage is dominated by novel lineages of
bacteria (13) and archaea (8, 12). Similar studies focusing on
marine picoeukaryotes are just beginning. Two very recent
papers described the diversity of picoeukaryotes as determined
by gene cloning and sequencing of ribosomal DNA (rDNA) in
one surface sample from the equatorial Pacific Ocean (29) and
several deep-sea samples from the Southern Ocean (24). Both
studies showed that the phylogenetic diversity of the assem-
blages was great and that novel lineages were present. Another
study analyzed the algal assemblages at two coastal sites by
using the plastidic genes found in bacterial SSU rRNA librar-
ies (39). Other molecular studies have focused on particular
taxonomic groups and have used a similar gene-cloning ap-
* Corresponding author. Mailing address: Departament de Biologia
Marina, Institut de Cie `ncies del Mar, CSIC, Passeig Joan de Borbo ´ s/n,
E-08039 Barcelona, Catalunya, Spain. Phone: 34-932216416. Fax: 34-
932217340. E-mail: firstname.lastname@example.org.
proach (16, 28) or taxon-specific rRNA probes in fluorescent in
situ hybridization experiments (22, 45). Finally, fingerprinting
techniques, such as denaturing gradient gel electrophoresis
(DGGE), have been used to quickly compare the compositions
of planktonic eukaryotic assemblages (9, 49).
The objective of this work was to study the diversity of
marine picoeukaryotes in different marine areas by gene clon-
ing and sequencing of eukaryotic rRNA genes. Clones derived
from five genetic libraries were analyzed by restriction frag-
ment length polymorphism (RFLP) analysis, and selected
clones were partially sequenced. With this approach we deter-
mined whether different picoeukaryotic groups were present in
different areas of oceans and estimated their relative abun-
MATERIALS AND METHODS
Sampling. Samples from different marine areas (Table 1) were collected with
Niskin bottles attached to a rosette and a conductivity, temperature, and depth
(CTD) probe. Seawater was transferred to 25-liter plastic containers that previ-
ously had been rinsed three times with the same water. Microbial biomass was
collected on 0.2-?m-pore-size Sterivex units (Durapore; Millipore) by filtering 10
to 20 liters of seawater through a prefilter and a Sterivex unit in succession with
a peristaltic pump at rates of 50 to 100 ml min?1. Different prefilters were used;
these prefilters included 5-?m-pore-size polycarbonate filters for the Mediter-
ranean sample, 2-?m-pore-size polycarbonate filters for the North Atlantic sam-
ples and 1.6-?m-pore-size GF/A glass fiber filters for the Antarctic samples. The
prefilters and the Sterivex units were covered with lysis buffer (40 mM EDTA, 50
mM Tris-HCl, 0.75 M sucrose) and frozen at ?70°C until nucleic acid was
extracted. An aliquot of seawater was fixed with glutaraldehyde to obtain epi-
fluorescence counts for heterotrophic flagellates (37). Subsamples of the whole
water and the filtrate after passage through the prefilter were used for chloro-
phyll (Chl a) and cytometry determinations. Approximately 100 ml of sample was
filtered through GF/F filters, and the Chl a concentration was determined by
measuring the fluorescence in acetone extracts with a Turner Designs fluorom-
eter (32). Subsamples used for flow cytometry counting were collected by fixing
1.2 ml of seawater with glutaraldehyde-paraformaldehyde (final concentration,
0.05 and 1%, respectively). Populations of Synechococcus, Prochlorococcus, and
photosynthetic picoeukaryotes were distinguished by their distinct size and pig-
ment properties by using a FACScalibur flow cytometer (Becton Dickinson) as
explained by Olson et al. (31). Strictly speaking, the picoeukaryotes comprise
organisms that are between 0.2 and 2 ?m in diameter, but here we use the term
loosely to include the larger organisms analyzed in the Mediterranean sample
(diameter, 0.2 to 5 ?m). Moreover, flow cytometry detects the most abundant
photosynthetic eukaryotes, often including organisms that are more than 2 ?m in
Nucleic acid extraction. Nucleic acid extraction started with addition of ly-
sozyme (final concentration, 1 mg ml?1) and incubation of the Sterivex units at
37°C for 45 min. Then, sodium dodecyl sulfate (final concentration, 1%) and
proteinase K (final concentration, 0.2 mg ml?1) were added, and the Sterivex
units were incubated at 55°C for 60 min. Lysates were recovered from the
Sterivex units with a syringe. Nucleic acids were extracted with phenol-chloro-
form-isoamyl alcohol (25:24:1), and the residual phenol was removed with chlo-
roform-isoamyl alcohol (24:1). Nucleic acid extracts were purified further, de-
salted, and concentrated with a Centricon-100 concentrator (Millipore). DNA
integrity was checked by agarose gel electrophoresis, and DNA yield was quan-
tified by a Hoechst dye fluorescence assay (35). Nucleic acid extracts were stored
at ?70°C until analysis.
Eukaryotic rDNA genetic libraries. Eukaryotic 18S rRNA genes were ampli-
fied by PCR with eukaryote-specific primers EukA, EukB (26), and 326f (22).
Most libraries were constructed with the 326f-EukB primer combination
(1,420-bp insert); the only exception was the ME1 library, which was constructed
with primers EukA and EukB (1,780-bp insert). The PCR mixtures (100 ?l) each
contained 10 to 100 ng of environmental DNA as the template, each de-
oxynucleoside triphosphate at a concentration of 200 ?M, 1.5 mM MgCl2, each
primer at a concentration of 0.3 ?M, 2.5 U of Taq DNA polymerase (Gibco
BRL), and the PCR buffer supplied with the enzyme. Reactions were carried out
in an automated thermocycler (Genius; Techne) with the following cycle: an
initial denaturation at 94°C for 3 min, 30 cycles of denaturation at 94°C for 45 s,
annealing at 55°C for 1 min, and extension at 72°C for 3 min, and a final
extension at 72°C for 5 min. Amplified rRNA gene products from several indi-
vidual PCRs were pooled (four 50-?l samples or two 100-?l samples), ethanol
precipitated, and resuspended in 20 ?l of sterile water. An aliquot of each
concentrated PCR product preparation was ligated into the prepared vector
(pCR 2.1) supplied with a TA cloning kit (Invitrogen) by following the manu-
facturer’s recommendations. Putative positive colonies were picked, transferred
to a multiwell plate containing Luria-Bertani medium and 7% glycerol, and
stored at ?70°C.
RFLP analysis. The presence of the 18S rDNA insert in colonies was checked
by PCR reamplification with primers 326f and EukB by using a small aliquot of
a culture as the template. PCR amplification products containing the right size of
insert were digested with 1 U of restriction enzyme HaeIII (Gibco BRL) ?l?1for
6 to 12 h at 37°C. The digested products were separated by electrophoresis at 80
V for 2 to 3 h in a 2.5% low-melting-point agarose gel. A 50-bp DNA ladder
(Gibco BRL) was included in each gel to aid in visual comparisons of the RFLP
patterns of clones appearing in different gels. When ambiguities appeared, clones
were electrophoresed simultaneously in the same agarose gel. Clones that pro-
duced the same RFLP pattern (DNA fragments of the same size) were grouped
together and were considered members of the same operational taxonomic unit
(OTU). Coverage values were calculated for each library by using the relative
distribution of OTUs and the equation described by Good (14).
rDNA sequencing. Double-stranded plasmid DNAs from selected clones were
extracted with a QIAprep miniprep kit (QIAGEN). Sequencing reactions were
performed with a Thermo SEQUENASE v.2 kit (Amersham, U.S. Biochemicals)
and an ABI PRISM model 377 (v. 3.3) automated sequencer. A single reaction
with primer 326f was performed for each clone, which resulted in a 550 to 750-bp
sequence. Sequences were subjected to a BLAST search (1) to determine the
first phylogenetic affiliation and to the CHECK-CHIMERA command (25) to
determine potential chimeric artifacts. Sequences were aligned with about 3,200
homologous eukaryotic 18S rRNA primary structures by using the automatic
alignment tool of the ARB program package (http://www.mikro.biologie.tu-
muenchen.de). Then partial sequences were inserted into the optimized tree
derived from complete sequence data by using the Quick add using parsimony
tool, which did not affect the initial tree topology. The resulting tree was pruned
to save space; only the closest relatives of our clones were retained. Since the
similarity value obtained in a BLAST analysis often is for only a fraction of the
sequence submitted, similarity values for new and database sequences were
calculated by using the aligned ARB file.
DGGE. Eukaryotic 18S rRNA genes were amplified with eukaryote-specific
primers Euk1A and Euk516r-GC, which amplify an approximately 560-bp frag-
ment (9). The PCR program included an initial denaturation at 94°C for 130 s
and 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 45 s, and
extension at 72°C for 130 s. The PCR products were quantified with a Low DNA
Mass Ladder (Gibco BRL) by performing agarose gel electrophoresis with a
DGGE-2000 system (CBS Scientific Company). A 0.75-mm-thick 6% polyacryl-
TABLE 1. Characteristics of the samples used to generate the five libraries of eukaryotic 18S rRNA genes
Library System CharacteristicsCoordinates
Chl a concn
Total In fraction
Mediterranean (Albora ´n Sea)
Antarctica (Weddell Sea)
Antarctica (Scotia Sea)
VOL. 67, 2001GENE CLONING ANALYSIS OF MARINE PICOEUKARYOTES 2933
amide gel was cast by mixing two stock solutions containing 45 and 65% DNA
denaturant agent (100% was defined as 7 M urea and 40% deionized form-
amide). Approximately 800 ng of PCR product was applied to each lane in the
gel. Electrophoresis was performed at 100 V and 60°C for 16 h in 1? TAE buffer
(40 mM Tris base, 20 mM sodium acetate, 1 mM EDTA; pH 7.4). The DGGE
gel was stained with GelStar (FMC BioProducts) for 30 min, rinsed with 1? TAE
buffer, and visualized with UV radiation by using a Fluor-S MultiImager and the
MultiAnalyst imaging software (Bio-Rad). The presence and intensity of DGGE
bands were estimated by image analysis as previously described (41).
Nucleotide sequence accession numbers. Nucleotide sequences determined in
this study have been deposited in the GenBank database under accession num-
bers AF363153 to AF363228.
RESULTS AND DISCUSSION
The aim of this study was to analyze the phylogenetic com-
position of the marine planktonic picoeukaryotes, a ubiqui-
tous, heterogeneous, poorly identified assemblage. We present
the results obtained with a molecular approach, gene cloning
and sequencing of SSU rRNA genes, that has been used very
successfully to identify marine bacteria and archaea (8, 13) and
has only recently been applied to marine eukaryotes (24, 29).
We analyzed clone libraries from five surface samples taken in
three distant marine regions, the Mediterranean Sea (library
ME1), the Southern Ocean (libraries ANT37 and ANT12),
and the North Atlantic Ocean (libraries NA11 and NA37).
These samples exhibited a wide range of in situ temperatures,
from 18°C in the Mediterranean Sea to ?1.8°C in the Weddell
Sea (Table 1). They also differed in terms of the composition
of the phototrophic picoplankton; picoeukaryotes were present
in all three systems, together with Synechococcus in the Med-
iterranean and Atlantic samples and Prochlorococcus in the
Mediterranean sample (Table 2). Therefore, the physical and
biological parameters of the five samples analyzed were very
In contrast to marine bacteria and archaea, the planktonic
eukaryotes cover a broad size spectrum; they vary from mi-
crons to millimeters in diameter. Therefore, the approach used
to collect picoeukaryotes is very important. Picoplanktonic bio-
mass was obtained by prefiltering a sample and collecting the
organisms that passed through the prefilter. The performance
of this size fractionation technique for the five samples used to
construct clone libraries was assessed by carrying out Chl a
(Table 1), flow cytometry (Table 2), and molecular fingerprint-
ing (Fig. 1) analyses. For the Mediterranean sample, filtration
through a 5-?m-pore-size filter resulted in a slight reduction in
the level of Chl a (Table 1) but no reduction in the level of
phototrophic picoeukaryotes (Table 2). For the North Atlantic
samples filtration through a 2-?m-pore-size filter resulted in a
significant reduction in the level of Chl a, but it had no effect
on phototrophic picoeukaryote abundance. For the Antarctic
samples, filtration through a 1.6-?m-pore-size filter resulted in
a dramatic decrease in the level of Chl a (only 3 to 7% passed
FIG. 1. DGGE gel separating eukaryotic 18S rDNA fragments
from the populations retained on prefilters and from the populations
appearing in filtrates from the five samples used to generate genetic
libraries. The filtrate samples analyzed by using genetic libraries are
TABLE 2. Concentrations of heterotrophic and phototrophic picoeukaryotes, Prochlorococcus and Synechococcus in whole water and in the
fractions passing through the prefilters for the samples used to generate eukaryotic genetic libraries
Concn (103cells ml?1) ofa:
aHeterotrophic picoeukaryotes were counted by the epifluorescence method, and phototrophic picoplankton (both eukaryotic and prokaryotic) were counted by the
flow cytometry method. P1, P2, and P3 are distinct phototrophic picoeukaryote populations (P1 cells are smaller than P3 cells).
bND, not determined.
2934 DI´EZ ET AL.APPL. ENVIRON. MICROBIOL.
through the filter) and in the level of phototrophic picoeu-
karyotes (5 to 31% passed through the filter). When possible,
distinct picoeukaryotic populations were distinguished on the
cytometry graph and analyzed separately (Table 2). The two
populations detected in the NA11 sample were not affected by
prefiltration, whereas the abundance of the three populations
detected in the Antarctic samples decreased after filtration and
there was a more pronounced effect on the largest of the three
populations (P3). Therefore, the fractions analyzed appeared
to contain all of the phototrophic picoeukaryotes for the Med-
iterranean and Atlantic samples and only a fraction of the
phototrophic picoeukaryotes for the Antarctic samples.
We then checked whether the eukaryotes that passed
through the prefilter (and thus were analyzed in the clone
library) were phylogenetically different from the eukaryotes
that were retained in the prefilter. It is well known that filters
allow passage of cells larger than their nominal pore sizes and
that filters can clog, which results in retention of smaller cells.
The DGGE fingerprints obtained with eukaryote-specific
primers for both size fractions were very different for the five
samples analyzed (Fig. 1). On average, 66% of the bands that
appeared in the larger-fraction fingerprint (accounting for
45% of the total band intensity) were not found in the smaller-
fraction fingerprint, indicating that many populations were to-
tally retained in the prefilter (Fig. 2). Conversely, on average,
one-half of the bands that appeared in the smaller-fraction
fingerprint (accounting for 32% of the band intensity) were
unique to this fraction, indicating that many populations com-
pletely passed through the prefilters used. Although the pre-
filtration method was not perfect and some other populations
appeared in both size fractions, this method appeared to enrich
the smallest cells and exclude most large eukaryotes. There-
fore, we were confident that we were analyzing mostly picoeu-
The five libraries of picoeukaryotic rRNA genes were first
screened by RFLP analysis, which grouped clones into discrete
OTUs (Table 3). An OTU comprising 33 clones in library ME1
was identical to the appendicularian Oikopleura, and an OTU
comprising 43 clones in library NA11 was similar to a copepod.
These metazoan OTUs were clearly artifacts of the prefiltra-
tion step and were thus excluded from further analyses. Apart
from these two OTUs, we analyzed a total of 225 clones, which
yielded 76 different OTUs. The coverage values, calculated
from the relative distribution of OTUs in each library, were
relatively high, ranging from 47% in library NA11, in which few
clones were obtained, to 82% in library ANT12 (Table 3).
These high values indicated that most of the diversity at the
level examined had been sampled; only a few more OTUs
would be recovered by analyzing more clones. About one-half
of the OTUs that appeared in each library were unique to that
library (Table 3), whereas the remaining OTUs appeared in
two or more libraries, indicating that there was potential over-
lap of picoeukaryotic phylotypes among samples.
In the ME1 and ANT37 libraries one clone of each OTU
was partially sequenced. When clones from different libraries
belonging to the same OTU were compared, they were found
to be very similar (average similarity for 10 cases examined,
98.6% [data not shown]). Thus, clones from the remaining
three libraries were affiliated with an OTU found in ME1 or
ANT37, and only clones representing new OTUs were se-
quenced. The affiliations of clones from each library with the
76 defined OTUs and the closest match in the database for the
clone representing each OTU are shown in Table 4. These
clones and database sequences are compared in a phylogenetic
tree in Fig. 3. All of the clones were affiliated with eukaryotes,
demonstrating the specificity of the eukaryotic primers used to
construct the clone libraries. The clones are also widely dis-
tributed on the eukaryotic tree, showing the ability of the
primers to recover distantly related phylogenetic groups. Some
clones were very similar to previously isolated organisms,
mainly organisms affiliated with groups having known picoeu-
karyotic representatives, such as prasinophytes, prymnesio-
phytes, and pelagophytes. Other clones seemed to represent
new phylotypes in well-defined phylogenetic groups or even
novel phylogenetic lineages.
The fact that clones belonging to the same OTU were sel-
dom identical indicated that we were underestimating the true
diversity by sequencing only one clone of each OTU. However,
this was the approach chosen since we were more interested in
FIG. 2. Numbers of bands that were unique to the filtrates, that
were shared, and that were unique to the prefilters for the five samples
used to generate genetic libraries after quantitative analysis of the
DGGE gel shown in Fig. 1. The values above the bars for unique bands
are the percentages of band intensity accounted for by these bands in
the DGGE profile.
TABLE 3. Results of RFLP analysis of the five genetic libraries
Library No. of clonesNo. of OTUs Coverage (%)
No. of OTUs
unique to library
No. of OTUs found
in libraries from the
No. of OTUs found in
libraries from different
aNA, not applicable.
VOL. 67, 2001GENE CLONING ANALYSIS OF MARINE PICOEUKARYOTES 2935
TABLE 4. Number of clones belonging to each OTU in genetic libraries and phylogenetic affiliations of the representative clones sequenced
No. of clones in libraries
Clone Closest relative (% similarity)
ME1 ANT37ANT12 NA11NA37
Ostreococcus tauri (98.1)
Mantoniella squamata (96.8)
Ostreococcus tauri (94.1)
Mantoniella squamata (99.9)
Mantoniella squamata (96.1)
Micromonas pusilla (99.0)
Micromonas pusilla (99.1)
Prymnesium patelliferum (94.3)
Phaeocystis antarctica (99.8)
Phaeocystis antarctica (97.6)
Emiliania huxleyi (96.0)
Emiliania huxleyi (95.6)
Papiliocellulus elegans (95.5)
Chaetoceros rostratus (96.7)
Skeletonema costatum (96.8)
Corethron criophilum (95.9)
Corethron criophilum (89.0)
Corethron criophilum (95.3)
Chaetoceros sp. (86.7)
Pseudonitzschia multiseries (98.6)
Lepidodinium viride (76.0)
Gymnodinium mikimotoi (96.7)
Lepidodinium viride (98.5)
Pelagophytes2416 ME1-27 Pelagomonas calceolata (100)
Cyanophora paradoxa (84.9)
Cyanophora paradoxa (80.0)
Cyanophora paradoxa (85.8)
Dictyocha speculum (90.3)
Dictyocha speculum (91.0)
Dictyocha speculum (91.5)
Cryptophytes 313 ME1-5Geminigera cryophila (98.5)
Eustigmatophytes 322 ME1-25 Nannochloropsis sp. (89.0)
Bolidophytes 331 ANT37-30 Bolidomonas pacifica (94.6)
Novel stramenopiles 34
Hyphochytrium catenoides (90.8)
Hyphochytrium catenoides (87.6)
Hyphochytrium catenoides (87.8)
Hyphochytrium catenoides (88.3)
Hyphochytrium catenoides (90.9)
Hyphochytrium catenoides (90.3)
Hyphochytrium catenoides (89.2)
Hyphochytrium catenoides (88.8)
Hyphochytrium catenoides (89.4)
Hyphochytrium catenoides (88.5)
Hyphochytrium catenoides (88.0)
Hyphochytrium catenoides (93.7)
Hyphochytrium catenoides (90.0)
Hyphochytrium catenoides (85.5)
Hyphochytrium catenoides (92.9)
Hyphochytrium catenoides (85.4)
Hyphochytrium catenoides (90.3)
Hyphochytrium catenoides (86.4)
Hyphochytrium catenoides (87.7)
Hyphochytrium catenoides (87.7)
Hyphochytrium catenoides (87.5)
Hyphochytrium catenoides (90.2)
Hyphochytrium catenoides (88.9)
Continued on following page
2936 DI´EZ ET AL.APPL. ENVIRON. MICROBIOL.
broad identification of the picoeukaryotic phylotypes present
in different marine environments than in a detailed list of
species. Moreover, only partial sequences were obtained (at
least one-third of the 18S rRNA gene), and the phylogenetic
affiliations of the clones and the percentages of similarity cal-
culated were not as precise as they would have been if we had
sequenced the whole gene. It is clear, however, that partial
sequences are sufficient to infer the positions of clones in a
given line of descent (46). Finally, the clonal representation of
a group does not necessarily reflect its precise abundance in
nature, given the potential biases inherent with PCR-based
methods (50). For this reason we refer here to the percentages
of clones in the libraries, which are useful values for comparing
the distributions of groups among libraries.
Phototrophic picoeukaryotic isolates generally belong to the
classes Prasinophyceae, Chlorophyceae, Prymnesiophyceae,
and Pelagophyceae (17, 38, 44, 47). The relevance of these
isolates in natural assemblages is uncertain, given the biases
that occur when microorganisms are cultured (2, 16, 23). A
significant number of clones in our libraries were affiliated with
these classes; however, there was a conspicuous absence of
chlorophytes. Our clones exhibited rather high similarities with
picoeukaryotic isolates (between 94.1 and 100%; average,
97.4%), suggesting that the cultures available are fair repre-
sentatives of natural phototrophic picoeukaryotes. The prasi-
nophyte group was the most abundant and widespread algal
group in our libraries and was represented by 46 clones and
seven OTUs. These clones were present in all libraries and
were dominant in ME1 and ANT37. The two Atlantic libraries
contained clones very similar to Micromonas pusilla. In the
other three libraries all clones were most similar to Mantoniella
squamata or Ostreococcus tauri. In particular, one OTU that
exhibited 96.8% similarity to M. squamata was represented 8
times in the Mediterranean library and 16 and 5 times in the
two Antarctic libraries. Clones belonging to this OTU obtained
from systems separated by thousands of kilometers were very
similar (98.8%), indicating that very similar phylotypes are
widely distributed. The second most abundant OTU had a
phylogenetic position between Ostreococcus and Mantoniella
and was also widely distributed. Finally, an OTU very similar to
O. tauri appeared six times, but only in the ME1 library. It is
perhaps not coincidental that O. tauri was described from a
Mediterranean lagoon (7). Our culture-independent data con-
firm the importance of prasinophytes in marine picoplankton,
in which their marker pigment prasinoxanthin is found widely
(18, 36), and indicate that their diversity is relatively high since
several phylotypes coexisted in the same sample.
The prymnesiophytes were represented by 28 clones belong-
ing to five OTUs. They were abundant in the two Antarctic
libraries but rare in the other three libraries. The most abun-
dant OTU, with 5 clones in ANT37 and 16 clones in ANT12,
was almost identical to Phaeocystis antarctica. The presence of
this organism is expected in Antarctic waters, where it fre-
quently forms large blooms, many times consisting of the co-
lonial form. Unicellular flagellated forms of P. antarctica were
probably responsible for the sequences detected. It must be
noted that prefiltration of the Antarctic samples removed
many phototrophic picoeukaryotes, and thus, a large fraction
of the natural assemblage remained undescribed. In the At-
lantic libraries two clones were moderately related to Emiliania
No. of clones in libraries
Clone Closest relative (% similarity)
ME1 ANT37ANT12NA11 NA37
571 ANT12-5Hyphochytrium catenoides (87.4)
Pentapharsodinium tyrrhenicum (87.2)
Heterocapsa triquetra (87.8)
Heterocapsa triquetra (89.3)
Heterocapsa triquetra (85.8)
Gymnodinium catenatum (89.2)
Lepidodinium viride (89.3)
Strombidium purpureum (93.3)
Oxytricha granulifera (92.2)
Oxytricha granulifera (87.5)
Oxytricha granulifera (91.3)
Oxytricha granulifera (88.1)
Oxytricha granulifera (93.5)
Paraphysomonas foraminifera (98.7)
Paraphysomonas foraminifera (95.3)1
Heteromita globosa (88.1)
Thaumatomonas sp. (82.9)
Thaumatomonas sp. (89.6)
Cercomonas strain ATCC 50318 (83.0)
Fungi 7623 ANT12-13 Stenocybe pullatula (80.0)
36 Oikopleura sp. (99.2)
Cancrincola plumipes (94.0)43
VOL. 67, 2001 GENE CLONING ANALYSIS OF MARINE PICOEUKARYOTES2937
FIG. 3. Phylogenetic tree for partial sequences of environmental clones and the most closely related cultured organisms. Environmental clones
are indicated by boldface type and each clone is designated by the library designation followed by a number. One clone representing each different
OTU detected by the RFLP analysis of the five genetic libraries is included. The bar indicates 10% estimated sequence divergence. The number
of clones in each genetic library belonging to each phylogenetic group is indicated on the right.
huxleyi. This prymnesiophyte accounted for up to 60% of the
phytoplankton biomass in the Atlantic samples (unpublished
results) but was obviously effectively removed by the prefiltra-
tion step. Finally, a few clones in the ME1 and ANT12 libraries
were moderately related to Prymnesium patelliferum.
Members of the Bacillariophyceae (diatoms) were repre-
sented by 18 clones belonging to eight OTUs. Most OTUs were
found only in one library, indicating that a different diatom
assemblage occurred in each marine region. In ANT37 most
diatom clones were affiliated with Corethron criophilum, and
one clone showed a very low level of similarity to Chaetoceros.
The three OTUs in ME1 exhibited relatively high levels of
similarity to Papiliocellulus elegans, Chaetoceros rostratus, and
Skeletonema costatum, and one clone in ANT12 was very sim-
ilar to Pseudonitzschia multiseries. Although most known dia-
toms tend to be larger than the size analyzed here, very small
diatoms have been described (47). In an HPLC study of the
distribution of size-fractionated pigments in the Arabian Sea,
Latasa and Bidigare (18) found that between 70 and 92% of
the marker pigment fucoxanthin occurred in the ?2-?m frac-
tion during the Spring Intermonsoon and between 26 and 85%
of this pigment occurred in this fraction during the Southwest
Monsoon in the two most open sea stations. The retrieval of
diatom genes in our study is consistent with the presence of the
marker pigment in the small-size fraction. Another possible
explanation is cell breakage during prefiltration or squeezing
of cells through the filter.
Dinoflagellates were represented by 14 clones in three dif-
ferent OTUs and accounted for a significant fraction of the
eukaryotic clones in the North Atlantic libraries. One OTU
that was most similar to Gymnodinium mikimotoi appeared in
all Antarctic and North Atlantic libraries. A single clone re-
covered from ANT37 was very similar to Lepidodinium viride,
whereas another clone in ME1 was very different from any
known dinoflagellate. Dinoflagellates tend to be large and con-
spicuous organisms, and there is not any known form of pico-
planktonic size. Dinoflagellates might be overrepresented in
genetic libraries because they have larger genomes than mem-
bers of other phytoplankton groups (40) and therefore poten-
tially higher rRNA gene copy numbers (6). Like diatoms, their
presence in genetic libraries might be due to inefficient prefil-
tration or the existence of unknown picodinoflagellates. In the
study mentioned above, Latasa and Bidigare (18) found that
often more than 50% (and up to 75%) of peridinin, the marker
pigment of dinoflagellates, appeared in the ?2-?m fraction.
The remaining phytoplankton groups were minor compo-
nents of our libraries (Table 4). One OTU represented by one
clone in ME1 and six clones in ANT12 was affiliated with the
Pelagophyceae, and the sequenced clone was 100% similar to
Pelagomonas calceolata. Three ME1 clones were very similar to
the cryptophyte Geminigera criophila, and a single clone in
ANT37 was moderately affiliated with the recently described
picoeukaryote Bolidomonas pacifica. The closest relative of
four clones in the Antarctic libraries was the glaucocystophyte
Cyanophora paradoxa, but the similarity was so low (80.0 to
85.8%) that even an affiliation with this algal class is uncertain.
The same is true for three clones in the ANT37 and Atlantic
libraries affiliated with Dictyocha speculum (similarities,
around 90%) and two ME1 clones distantly related to the
eustigmatophyte Nannochloropsis (89.0%).
Among the clearly heterotrophic groups we found clones
belonging to the Ciliophora, the cercomonads, and the fungi
(Fig. 3). Clones clustering with the ciliates were present in all
libraries (11 clones and six OTUs); one-half of them were in
the ME1 library, which was constructed by using the prefilter
with larger pores. These sequences were rather distantly re-
lated to database sequences (the levels of similarity were be-
tween 87.5 and 93.5%) and thus belong to new organisms. Five
clones representing four different OTUs were distantly related
to the cercomonads (the levels of similarity were between 83.0
and 89.6%), and low amounts of these clones were detected in
the three systems. Five Antarctic clones were affiliated with the
fungi and exhibited rather low levels of similarity to any known
organism (80.0%). Six clones found in the three systems were
affiliated with the class Chrysophyceae, which is known to
contain mostly phototrophic organisms but also some hetero-
trophs. These clones were closely related to Paraphysomonas
foraminifera and thus likely are heterotrophic flagellates and
not phototrophic organisms.
A significant number of clones in the libraries did not show
a close affiliation with any known class of organisms and
formed two novel phylogenetic lineages. Novel stramenopiles
were the more abundant lineages of the two (Fig. 3). These
sequences, representing 53 clones and 24 OTUs, accounted for
a significant fraction of the clones in each library: 19% in ME1,
19% in ANT37, 34% in ANT12, 36% in NA11, and 5% in
NA37. They clustered in the basal branches of the strameno-
pile line of descent; the sequence of the fungus-like organism
Hyphochytrium catenoides was the most similar sequence in the
database, but the levels of similarity were always very low. The
novel stramenopile clones were more similar to each other
than to any other sequence and showed a relatively high degree
of genetic diversity; separate clusters were apparent (Fig. 3).
These clusters did not necessarily correspond to different phy-
lotypes found in different samples. Instead, some phylotypes
had a wide geographic distribution; for instance, a clone from
the Mediterranean Sea (ME1-19) was almost identical (99.5%
similarity) to a clone from the North Atlantic (NA11-4).
The stramenopiles (34) form a monophyletic group that is
extremely diverse in terms of metabolism and cell type and
includes algal cells, fungus-like cells, and heterotrophic flagel-
lates. Phylogenetic relationships determined by using 18S
rDNA sequences suggest that stramenopiles were initially het-
erotrophic and acquired a chloroplast at a certain point in
evolution (19). Although the exact position of the novel stra-
branches could not be unambiguously resolved, our phyloge-
netic analyses indicated that these organisms appeared before
the chloroplast was acquired. Thus, the new sequences prob-
ably belong to heterotrophic organisms, and we hypothesize
that they account for the bulk of the heterotrophic flagellates
in the oceans (10). In open ocean waters, heterotrophic flagel-
lates are mainly cells less than 2 or 3 ?m in diameter (5, 42)
and might be as abundant as phototrophic picoeukaryotes.
Therefore, we expected these organisms to be included in our
clone libraries, but we detected very few clones related to
known heterotrophic flagellates (10, 48); only five clones were
distantly related to cercomonads and six clones were affiliated
with the chrysomonad Paraphysomonas. This is not surprising,
since in a previous study it was shown that Paraphysomonas
VOL. 67, 2001GENE CLONING ANALYSIS OF MARINE PICOEUKARYOTES 2939
imperforata systematically dominated enrichment cultures
from coastal samples but accounted for less than 1% of the
heterotrophic flagellates in the natural system (23). The novel
stramenopile sequences are also distantly related to known
heterotrophic flagellates, such as Developayella elegans or the
bicosoecids. In addition, there is reasonable agreement be-
tween the percentage of heterotrophic flagellate cells (based
on the total number of picoeukaryotic cells) and the percent-
age of novel stramenopile clones (based on the total number of
clones); these values are 10 and 19% in ME1, 11 and 34% in
ANT12, and 4 and 19% in ANT37, respectively.
Novel alveolates formed the second novel lineage that was
abundant in our libraries. They were represented by 19 clones
and six OTUs and were recovered only from the Mediterra-
nean and North Atlantic samples. Perhaps they were excluded
from the Antarctic samples by the more drastic prefiltration
technique used. The Mediterranean library contained the
greatest diversity of marine alveolates, with 12 clones and four
OTUs, whereas the Atlantic libraries (especially NA37) con-
tained large percentages of marine alveolates, given the low
number of clones analyzed. The novel alveolate sequences
clustered in the basal part of the dinoflagellate clade and
exhibited very low levels of similarity (76.0 to 89.3%) to
dinoflagellate sequences. Their intermediate position between
dinoflagellates and the newly established phylum Perkinsozoa,
which contains marine parasites, did not allow us to hypothe-
size about their role in planktonic systems. Similar sequences
have also been found in other picoeukaryotic genetic libraries
from a surface sample (29) and deep samples (24).
Our results uncovered several patterns related to the diver-
sity of the smallest eukaryotic plankton in the ocean. First, the
diversity of picoeukaryotes in a single sample was great, and
the organisms belonged to very different phylogenetic groups.
Second, prasinophytes were very important in all the libraries,
and this group may be the most widespread and abundant
group of small phytoplankton in the ocean. Although quanti-
tative data from PCR-based methods should be regarded with
caution (50), two additional PCR-based methods (involving
the use of different primers) applied to the Mediterranean
sample also showed the dominance of the prasinophytes (9).
Moreover, HPLC data also showed that there was a high pro-
portion of Chl b-containing algae (including prasinophytes) in
the same sample (9). Thus, the conclusion that prasinophytes
are abundant in the oceans seems to be robust. Third, a large
number of novel alveolate sequences (unrelated to known se-
quences) were relatively abundant in all libraries. And fourth,
clones belonging to novel lineages of stramenopiles were
present at very high frequencies in all libraries; these lineages
appeared to branch among the basal heterotrophic groups of
stramenopiles and may have important roles in the dynamics of
This work was funded by EU contracts MIDAS (MAS3-CT97-
00154) and PICODIV (EVK3-CT1999-00021). The North Atlantic
samples were collected during the ACSOE NAE cruise of the RRS
Discovery funded by the British NERC and Spanish CICYT through
grant MAR97-1885-E; the Mediterranean sample was collected during
the MATER cruise of the B/O Garcı ´a del Cid funded by EU grant
MATER (MAS3-CT96-0051); and the Antarctic samples were gath-
ered during the E-DOVETAIL cruise of the B.I.O. Hespe ´rides funded
by Spanish CICYT grant ANT96-0866.
We thank Marta Estrada and Mikel Latasa for helpful comments
and Josep M. Gasol for help with flow cytometry.
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