Environmental Microbiology (2009)

doi:10.1111/j.1462-2920.2009.02023.x

Eukaryotic diversity and phylogeny using smalland large-subunit ribosomal RNA genes from
environmental samples
emi_2023

1..10

William Marande, Purificacin Lpez-Garca and

David Moreira*
Unit dEcologie, Systmatique et Evolution, UMR
CNRS 8079, Univ. Paris-Sud 11, btiment 360, 91405
Orsay Cedex, France.
Summary
The recent introduction of molecular techniques in
eukaryotic microbial diversity studies, in particular
those based in the amplification and sequencing
of small-subunit ribosomal DNA (SSU rDNA), has
revealed the existence of an unexpected variety of
new phylotypes. The taxonomic ascription of the
organisms bearing those sequences is generally
deduced from phylogenetic analysis. Unfortunately,
the SSU rDNA sequence alone has often not enough
phylogenetic information to resolve the phylogeny of
fast-evolving or very divergent sequences, leading to
their misclassification. To address this problem, we
tried to increase the phylogenetic signal by amplifying the complete eukaryotic rDNA cluster [i.e. the SSU
rDNA, the internal transcribed spacers, the 5.8S rDNA
and the large-subunit (LSU) rDNA] from environmental samples, and sequencing the SSU and LSU rDNA
part of the clones. Using marine planktonic samples,
we showed that surveys based on either SSU or
SSU + LSU rDNA retrieved comparable diversity patterns. In addition, phylogenetic trees based on the
concatenated SSU + LSU rDNA sequences showed
better resolution, yielding good support for major
eukaryotic groups such as the Opisthokonta, Rhizaria
and Excavata. Finally, highly divergent SSU rDNA
sequences, whose phylogenetic position was impossible to determine with the SSU rDNA data alone,
could be placed correctly with the SSU + LSU rDNA
approach. These results suggest that this method can
be useful, in particular for the analysis of eukaryotic
microbial communities rich in phylotypes of difficult
phylogenetic ascription.
Received 22 August, 2009; accepted 26 June, 2009. *For correspondence. E-mail david.moreira@u-psud.fr; Tel. (+33) 169157608;
Fax (+33) 169154697.

2009 Society for Applied Microbiology and Blackwell Publishing Ltd

Introduction
The use of molecular methods has played a major role in
our understanding of microbial diversity. During the last
two decades, PCR amplification and sequencing of the
small-subunit ribosomal RNA gene (SSU rDNA) has been
extensively applied to survey the huge bacterial and
archaeal diversity found in many common and extreme
habitats (Barns et al., 1996; Hugenholtz et al., 1998).
Recently, this technique was also adopted for the analysis
of eukaryotic diversity and it revealed a variety of new
groups, such as the marine Group I alveolates (LpezGarca et al., 2001; Moon-van der Staay et al., 2001) or
the putative new algal clade picobyliphytes (Not et al.,
2007), as well as a large diversity within many well-known
groups such as the prasinophyte algae (Guillou et al.,
2004; Viprey et al., 2008). In addition, several SSU rDNA
sequences that branched deeply in the eukaryotic phylogeny were impossible to be specifically related to any
known group, even at the kingdom level, suggesting the
controversial existence of several novel eukaryotic
kingdom-level groups (Dawson and Pace, 2002;
Edgcomb et al., 2002).
A key component of all the SSU rDNA-based diversity
studies is the taxonomic classification of the organisms
whose sequences are retrieved, which is commonly
inferred by phylogenetic analysis of the novel environmental phylotypes. In addition to artefacts derived from
undetected chimeric sequences, single-marker phylogenies have a series of inherent methodological problems,
such as the lack of enough informative signal for the
resolution of deep nodes, which can engender artefactual misplacement in phylogenetic trees (Philippe and
Adoutte, 1998; Stiller and Hall, 1999; Philippe et al.,
2000). A typical phylogenetic reconstruction problem, the
long-branch attraction (LBA) artefact, often causes highly
divergent SSU rDNA sequences to be placed at the base
of phylogenetic trees, leading them to be considered as
potential novel eukaryotic lineages. Recently, it was demonstrated that from 28 published phylotypes representing
putative novel high-level eukaryotic taxa, only 11 were
actual potential new lineages (Berney et al., 2004;
Cavalier-Smith, 2004). For example, the phylotypes
DH145-EKD11 and CCW75, putative members of a new

W. Marande, P. Lpez-Garca and D. Moreira

eukaryotic clade (Lpez-Garca et al., 2001; Stoeck and

Epstein, 2003), were later recognized to be highly divergent phylotypes related to the very fast-evolving ciliates
Myrionecta rubra and Mesodinium pulex (Johnson et al.,
2004). This is probably the case for many other divergent
eukaryotic phylotypes (Berney et al., 2004; CavalierSmith, 2004).
A natural way to alleviate the misplacement of environmental sequences due to the intrinsic limitation of singlemarker data would be to increase the number of
phylogenetic informative sites in phylogenetic analyses by
concatenating large-subunit (LSU) and SSU rDNA
sequences. This strategy was applied recently, producing
eukaryotic phylogenetic trees with much better resolution
than the SSU rDNA alone and better statistical support for
the major eukaryotic groups (Moreira et al., 2007). The
LSU and SSU rRNA genes are generally adjacent in the
genome, so that both markers could be easily retrieved by
PCR amplification also from environmental DNA samples.
The feasibility of this approach has already been shown
for marine planktonic bacteria (Suzuki et al., 2001) but
never tried for eukaryotes. In this work, we analysed the
results of parallel SSU rDNA and SSU + LSU rDNA
surveys to check whether the use of this larger marker
retrieves comparable eukaryotic diversity profiles. In addition, we tested if the use of the SSU + LSU rDNA concatenations allows the correct placement of fast-evolving
phylotypes in eukaryotic phylogenetic trees.

Results and discussion

Diversity analysis and comparison between SSU rDNA
and complete rDNA cluster libraries
We successfully amplified the eukaryotic rDNA cluster
from DNA extracted from four different surface (25 m)
marine planktonic samples (Table 1), obtaining PCR
products of 56 kbp. To compare the genetic diversity
retrieved from the complete rDNA cluster amplification
with the classical SSU rDNA analysis, we generated in
parallel SSU rDNA libraries from the same four samples.
For the DH18, DH114 and Ma131 libraries, we selected
48 positive clones of each type (SSU and SSU + LSU),

whereas for the DH141 library, which appeared to show

the most even taxonomic diversity without any clearly
dominant group, we sequenced 96 additional clones (i.e.
144 clones in total) of each type in order to have a library
with a more exhaustive characterization for comparative
purposes. For each SSU or SSU + LSU rDNA clone, we
sequenced ~800 bp of the 5 region of the SSU rRNA
gene using the primer Euk-42F. A total of 508 partial
sequences were thus determined (see Table 1). The
overall taxonomic diversity retrieved was large, revealing
the presence of typical marine planktonic eukaryotic
groups in the samples, including alveolates, cryptophytes,
streptophytes, chlorophytes, haptophytes and heterokonts, with variable frequencies depending on the
sample analysed (Fig. 1). For each sample, we compared
the proportions of the different taxonomic groups for the
two types of libraries, SSU and SSU + LSU rDNA. We
detected the groups that normally dominate surface
marine environments (Dez et al., 2001) and observed
quite similar taxonomic compositions for the most abundant groups in both types of libraries (Fig. 1). Differences
between SSU and SSU + LSU rDNA libraries from a same
sample mostly concerned the less abundant groups
(Table 2). This likely reflected, at least in part, that the
library surveys were not exhaustive, namely that we had
not reached a plateau in the saturation curves for each
library (data not shown). However, this was not the only
reason. For example, we did not retrieve any haptophyte
sequence in the SSU + LSU rDNA libraries, despite the
fact that members of this widespread photosynthetic
group were identified, although in relatively small proportions, in all the corresponding SSU rDNA libraries.
An inspection of the available haptophyte LSU rDNA
sequences revealed that the eukaryotic universal primer
28S-4R, used for the construction of the SSU + LSU rDNA
libraries, showed two mismatches with those haptophyte
sequences, which probably explains the bias against the
haptophytes observed in the SSU + LSU rDNA libraries.
Nevertheless, this was the only clear bias that we could
identify in the taxonomic composition, considered at large
scale, in our libraries. Preferential PCR amplification and
cloning of rDNA clusters of small size is another possible
bias that may have escaped from our attention. It is known

Table 1. Sampling sites and number of clones sequenced.

Sample

Station

Coordinates

Depth (m)

Volume
filtered (l)

DH18

DHARMA 5
South Atlantic
DHARMA 32
South Atlantic
DHARMA 18b
South Atlantic
Marmara Sea

622211 533556

25

19

46/36

545944 582217

25

14

47/48

592245 554627

25

14

138/114

4050.295 N 281.397E

25

42/37

DH114
DH141
Ma131

Clones sequenced
(SSU/SSU + LSU)

2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

Eukaryotic diversity and phylogeny using SSU + LSU rDNA 3

90
80
70
60
50
40
30
20
10
0

i
ng
Fu

va
tes

ia

ca

lar

Ex

dio

Ra

im
a

ls

s
An

yte

ts

ph

on

pto

Ha

He

ter

ok

ola

tes

tes
ve

op

hy
tes
Ch

top
yp
Cr

SSU rDNA clones

lor

yte
ph

pto

Ma131

hy

Clo n e freq u en cy (% )

90
80
70
60
50
40
30
20
10
0

s
yte
ph
Ha

on
ts
re

pto

ok

St

He

ter

ola

tes

tes
ve

hy

Al

op
lor

Ch

yp

top

hy

tes

Clo n e freq u en cy (% )

DH18

DH114

Al

DH141

90
80
70
60
50
40
30
20
10
0

Cr

Cr
Clo n e freq u en cy (% )
yp
to
Ch phyt
lor es
op
hy
Al tes
ve
o
He lates
ter
ok
on
t
An s
im
Gl
a
au
Ch co ls
oa ph
no yte
s
fla
ge
Ha llate
pto s
ph
yte
s

Cr
Clo n e freq u en cy (% )
yp
top
Ch hy
lor tes
op
hy
Al tes
ve
o
He late
ter s
ok
Ha ont
pto s
ph
Te ytes
lon
em
i
Ce ds
rco
zo
a

90
80
70
60
50
40
30
20
10
0

SSU + LSU rDNA clones

Fig. 1. Eukaryotic diversity identified using SSU and SSU + LSU rDNA libraries. Histograms represent the percentage of clones belonging to
each eukaryotic group for a given environmental sample. For the number of clones analysed see Table 1.

that several eukaryotic groups, such as the foraminifers,

have very long rDNA clusters, but in most cases the
difficulties to amplify and clone their rDNAs are mostly
due to the fact that their sequences are not only long
but very divergent, decreasing the probability of primer
annealing unless taxon-specific primers are used (Habura
et al., 2004). Therefore, we think that primer bias is probably much more important than the amplicon-length bias,
as it is also the case for the classical SSU rDNA libraries.
To compare at a lower taxonomic level, we studied in
more detail the four studied samples. We calculated the
operational taxonomic units (OTU) composition of the corresponding SSU and SSU + LSU rDNA libraries for each
sample, by considering as members of a same OTU those
sequences with > 98% identity for the SSU rDNA region
(Table 2). We identified in that way a total of 41 OTUs
containing more than one sequence, 20 of which were
present in both the SSU and SSU + LSU rDNA libraries.
The 41 OTUs represented 85% of all the sequences; the
remaining 15% (76 sequences) were singletons (unique
sequences with less than 98% of identity with the rest of our
sequences). With the exception of the OTUs related to the
haptophytes (see above), all OTUs found in only one type

of library contained very few sequences (24), corresponding very likely to stochastic differences due to the incomplete exploration of the libraries. Our analysis showed that
for each of the most abundant OTUs, identical or very
closely related clones were retrieved with both markers
(Table 2). This was in agreement with the results already
reported for the SSU + LSU rDNA-based analysis of bacterial diversity in marine plankton (Suzuki et al., 2001). All
this suggested that the SSU + LSU rDNA approach is able
to retrieve a quite similar diversity than the SSU rDNA. This
opened the possibility to apply that approach to improve
the phylogenetic analysis of the environmental sequences
difficult to place on the basis of the SSU rDNA alone.
Phylogenetic reconstruction using SSU + LSU
rDNA concatenations
After confirming the feasibility of the construction of
SSU + LSU rDNA libraries for eukaryotic diversity description and their overall similarity when compared with the
classical SSU rDNA-based approach, we compared the
resolving power of these two data sets in phylogenetic
analysis. Given that the concatenation of SSU + LSU rDNA

2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

W. Marande, P. Lpez-Garca and D. Moreira

Table 2. OTUs retrieved in the eight marine surface SSU and SSU + LSU rDNA libraries.
Clones per library (SSU/SSU + LSU)
OTU
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Singletons

DH141

DH18

0/2
6/4
1/3
2/0
1/2
1/1
6/1
2/0
1/0
2/0
9/2

1/0
1/1
1/0
2/0
9/4
2/0

DH114

2/0
1/0
2/3
1/0
0/1
5/1

3/1

1/0
1/0
12/7
2/0
3/0
4/4

9/17
1/1

1/0

7/2

Ma131

Phylum

Taxonomy group, genusa

Alveolates

Dinoflagellates
Dinoflagellates, Karlodinium
Dinoflagellates
Dinoflagellates
Dinoflagellates, Lepidodinium
Dinoflagellates, Gyrodinium
Dinoflagellates, Pentapharsodinium
Dinoflagellates, Gymnodinium
Dinoflagellates, Karlodinium
Dinoflagellates, Karlodinium
Dinoflagellates
Dinoflagellates, Gyrodinium rubrum
Ciliates, Strombidium
Ciliates, Strombidium
Ciliates, Myrionecta
Ciliates, Strobilidium
Ciliates, Strombidium
Ciliates, Strombidium
Ciliates
Alveolates, Group I
Alveolates, Group I
Alveolates, Group I
Alveolates, Group I
Diatoms
Diatoms, Fragilariopsis
Dictyochophytes, Dictyocha
Mast1
Mast1
Mast1
Prasinophytes, Pyramimonas
Prasinophytes, Bathycoccus
Prasinophytes, Micromonas
Pyrenomonadales, Geminigera
Phaeocystales, Phaeocystis
Unclassified
Thaumatomonads, Protapsis
Picobiliphytes
Ascomycetes
Crustacea
Crustacea
Crustacea

1/0
1/1

1/0

3/7

0/3
3/9
2/3
3/0
0/2

1/0

2/5
1/2
0/2
0/1
14/11
3/0
67/63
8/0

0/2
0/2
0/2
0/2

Heterokonts

0/1
0/2
1/0
3/1
2/0
0/2

Chlorophytes
0/3
2/5
0/1
2/0

0/1

2/0
2/3
0/2
2/3
0/2
7/7

3/2

0/3
6/9

Cryptophytes
Haptophytes
Streptophytes
Cercozoa
Picobiliphytes
Fungi
Animals

28/14

a. OTUs are affiliated to a genus only if > 97% identical with a genus representative.

sequences yielded at least 2000 additional conserved

aligned sites for phylogenetic reconstruction, it was
expected that trees based on that combination of markers
should be significantly better resolved than those retrieved
from the SSU rDNA alone (Moreira et al., 2007). To test this
idea, we completely sequenced 22 new SSU + LSU rDNA
clones from our marine surface libraries and added their
sequences to an available 111-taxa SSU + LSU rDNA
concatenated alignment containing sequences representing the major eukaryotic groups (Moreira et al., 2007). With
those new sequences, we enriched the taxonomic sampling of that data set by incorporating sequences from
important missing groups, such as the polycystine and
acantharean radiolaria, prasinophyte green algae, nonphotosynthetic heterokonts and diverse alveolates. We

removed four groups composed exclusively of fastevolving species for which we did not retrieve representatives in our samples: Microsporidia, Diplomonadida,
Parabasalida and Amoebozoa. This allowed us including
~500 additional aligned sites, giving a total of 3247 conserved positions for phylogenetic reconstruction.
Maximum likelihood (ML) and Bayesian inference phylogenetic trees reconstructed from the SSU + LSU rDNA
alignment (Fig. 2) recovered a number of well-known
eukaryotic groups, in agreement with global eukaryotic
phylogenies published recently (Medina et al., 2003;
Rodrguez-Ezpeleta et al., 2005; 2007; Burki et al., 2007).
We found strong support for the monophyly of the
Opisthokonta, uniting Metazoa, Fungi and their unicellular
close relatives, the Choanozoa, Ichthyosporea and the

2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

Eukaryotic diversity and phylogeny using SSU + LSU rDNA 5

Spathidium composite
Paramecium tetraurelia
Alveolata
Tetrahymena thermophila Ciliophora
40/Euplotes aediculatus
DH114_3A07
96/1
DH18_2A73
100/1
69/1
DH18_2A74
Plasmodium falciparum
100/1
98/1
Theileria parva
Eimeria tenella
100/1
Sarcocystis neurona
99/1
Apicomplexa
Hammondia hammondi
100/1
100/1 Neospora caninum
100/1
84/1 Toxoplasma gondii
Chromera velia
Perkinsus andrewsi
Perkinsida
100/1
72/.91
Perkinsus sp.
Ma131_1A38
96/1
100/1
Ma131_1A45
Ma131_1A49
51/.71
Ma131_1A57
61/1
Gymnodinium aureolum
96/.56
Dinoflagellata
Prorocentrum micans
96/Pfiesteria piscicida
70/DH114_3A10
65/Alexandrium catenella
79/99/.91
Gonyaulax polyedra
polycystine-like clone HA2
Polycystinea
100/1
Ma_1A27
(1/4)
Rotaliid foram (composite)
100/1
Foraminifera
Sticholonche-like clone KW16
73/.92
Ma121_1A14
51/.91
Acantharia
100/1
100/1
Ma121_1A29
Gromia oviformis
Bigelowiella natans
92/1
Dimorpha-like
94/1
Cercozoa
Cercomonas longicauda
84/1
Thaumatomastix
sp.
99/1
RHIZARIA
88/1
Cercomonas sp.
96/1
100/1
C15_1A03
73/.93
Blastocystis hominis
Cafeteria roenbergensis
Heterokonta
DH18_4A30
100/1
DH22_2A48
85/1
Hyphochytrium catenoides
100/1 94/1
Hyaloperonospora parasitica Pseudofungi
100/1 Phytophthora megasperma
DH114_3A83
100/1
Rhizosolenia setigera
Cylindrotheca closterium
100/1
78/1
Skeletonema pseudocostatum
60/.97
100/1
DH22_2A36
Nannochloropsis salina
Glossomastix chrysoplasta
100/1
Pinguiococcus pyrenoidosus
99/1
53/1
Dictyocha speculum
100/1
Apedinella radians
100/1
Rhizochromulina marina
100/1
Aureococcus anophagefferens
Ochrophyta
100/1
Pelagomonas calceolata
/.59
DH114_3A43
97/1
81/1
Chrysolepidomonas dendrolepidota
Ochromonas danica
100/1
Mallomonas composite
97/1
Synura sphagnicola
Vacuolaria virescens
100/1
/.63
Chattonella subsalsa
64/.89
Heterosigma akashiwo
70/1
Fucus composite
100/1
Scytosiphon lomentaria
98/1
Tribonema aequale
100/1
Vaucheria bursata
Phaeocystis antarctica
Haptophyta
100/1
Prymnesium patelliferum
Pterocystis tropica
Heliozoa
100/1
Sphaerastrum fockii
67/.76
Jakobida
Reclinomonas americana
Ma131_1A46
90/1
(1/4)
(1/4)
Euglena gracilis
100/1
Crithidia fasciculata
Euglenozoa
54/.88
51/.7
(1/4)
Trypanosoma brucei
100/1
87/1
Trypanosoma cruzi
EXCAVATA
Ancyromonas sigmoides
Apusomonadida
79/1
Apusomonas sp.
Nucleariida
Nuclearia
simplex
87/99
92/1
Mucor racemosus
83/Pneumocystis carinii
100/1
Magnaporthe grisea Fungi
99/1
87/1
Saccharomyces cerevisiae
100/1
Ichthyophonus hoferi
80/1
Capsaspora owczarzaki
Choanozoa
79/1
Monosiga brevicollis
100/1
Salpingoeca infusionum
89/1
Atolla vanhoeffeni
100/1
Nectopyramis sp
70/.64
Leucosolenia sp.
100/1
Metazoa
Suberites ficus
61/.92
(1/4)
Drosophila melanogaster
OPISTHOKONTA
97/.92
100/1
Homo sapiens
Goniomonas sp.
Chilomonas paramecium
100/1
Cryptophyta
Guillardia theta
100/1
DH141_3A30N
Cyanophora paradoxa
Glaucophyta
75/.64
100/1
Glaucocystis nostochinearum
Cyanidioschyzon merolae
Corallina officinalis
67/1
100/1
Rhodogorgon carriebowensis
59/.64
100/1
Thorea violacea
Rhodophyta
Gelidium floridanum
100/1
Gracilaria verrucosa
54/.58
Marchantia polymorpha
Gnetum gnemon
100/1
Arabidopsis thaliana
100/1
100/1
Oryza sativa
100/1
Chlorella ellipsoidea
Viridiplantae
Chlamydomonas pulsatilla
100/1
100/1
Pediastrum duplex
81/.98
DH141_3A12
DH114_3A06
82/1
Ostreococcus lucimarinus
100/1
PLANTAE
70/.95
DH114_3A14
100/1

100/1

Fig. 2. ML phylogenetic tree constructed

with a SSU + LSU rDNA data set (129
taxa, 3247 positions). Several long
branches were shortened to one fourth
(labelled 1/4). Numbers at nodes are ML
bootstrap proportions and Bayesian
Inference posterior probabilities (only
those higher than 50% and 0.50 are
shown respectively).

0.06

2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

W. Marande, P. Lpez-Garca and D. Moreira

Nucleariida [100% ML bootstrap proportion (BP) and

Bayesian posterior probability (PP) of 1]. The Plantae,
which encompass the three primary photosynthetic
eukaryotic groups, Viridiplantae, Rhodophyta and Glaucophyta, branched together with moderate to strong
support (5992% ML BP and PP 0.640.92, depending on
the taxonomic sampling used). This result was very
encouraging as only phylogenomic analyses using large
multi-protein concatenations have been able to recover
their monophyly when haptophyte and cryptophyte
sequences were present in the analysis (Burki et al.,
2007), as in our data set.
The Excavata, represented in our tree by the Euglenozoa and the Jakobida, received high support (90% ML BP
and PP 1), in agreement with recent phylogenetic analyses
(Cavalier-Smith, 2002; Simpson, 2003; Hampl et al., 2005;
Moreira et al., 2007; Rodrguez-Ezpeleta et al., 2007).
Alveolata, Heterokonta and Rhizaria formed monophyletic
groups with 100% ML BP and PP 1. Within the Rhizaria, we
retrieved a strong relationship between Foraminifera and
Radiolaria, in agreement with their inclusion within the
Retaria (Moreira et al., 2007). Interestingly, the sisterhood
of Rhizaria and Heterokonta received strong support (88%
ML BP and PP 1), corroborating the recent multi-protein
phylogenomic analysis (~30 000 amino acid positions)
that proposed the new eukaryotic super-assemblage
S.A.R. (Stramenopile + Alveolates + Rhizaria) (Burki
et al., 2007). Those three groups, comprising the largest
diversity of known unicellular eukaryotes, were among
those for which we most significantly improved the taxonomic sampling, including new five basal heterokonts,
eight alveolates and four rhizarians, which most likely
helped to resolve the relationship between them in comparison with previous analyses (Moreira et al., 2007). In
contrast, we did not find support for the Chromalveolata,
originally defined as containing all the groups bearing
plastids derived from red algae, namely the haptophytes,
cryptophytes, heterokonts and alveolates (Cavalier-Smith,
1999). In our analysis, cryptophytes branched with Plantae
with moderate support (75% ML BP and PP 0.63), which
was most likely an artefactual result as recent phylogenomic analysis supported that cryptophytes are the sister
group of haptophytes (Burki et al., 2007). The position of
haptophytes and centrohelid heliozoa was unsupported in
all our analyses. Finally, we retrieved the monophyly of
Apusozoa (Ancyromonas sigmoides and Apusomonas
sp.) with relatively good support (75% ML and 1 PP).

topology of the trees based on our data set was in agreement with most of the accepted intra- and inter-group
relationships (see above). To test the hypothesis that the
SSU + LSU rDNA may be useful to infer the taxonomic
affiliation of fast-evolving, conflictive environmental
sequences, we analysed two phylotypes closely related to
the red-tide forming ciliate species M. rubra obtained
during our environmental diversity surveys. The very fastevolving SSU rDNA of this ciliate was sequenced only a
few years ago (Johnson et al., 2004), and it was shown
to affiliate with a number of divergent environmental
sequences that were originally thought to be members of
a potential novel eukaryotic phylum (Lpez-Garca et al.,
2001; Edgcomb et al., 2002; Savin et al., 2004). It was
shown that the extremely rapid evolutionary rate of those
SSU rDNA sequences made it impossible to retrieve their
correct ciliate affinity in SSU rDNA analyses because of a
LBA artefact.
Thanks to taxon-rich SSU rDNA phylogenetic analyses
including the SSU rDNA portion of our SSU + LSU phylotypes, we recognized that two of them (DH114-1A22
and DH18-4A88) were very closely related to M. rubra
(99% sequence identity). We incorporated the two
Myrionecta-like phylotypes to our data set and reconstructed phylogenetic trees with only the SSU rDNA
part (1221 positions) and the concatenated SSU + LSU
rDNA sequences (3247 positions). In the SSU rDNA
tree (Fig. 3), the Myrionecta-like phylotypes branched
together with the other long branches, represented by
the excavate species (Euglenozoa and Reclinomonas
americana), with moderate support (69% ML BP and PP
of 0.52), which testified for a LBA artefact. Interestingly,
in the tree reconstructed with the two concatenated
markers, the Myrionecta-like sequences correctly
branched within the Alveolata with maximal support
(100% ML BP and PP of 1), close to the ciliates (57%
ML BP, not retrieved with the Bayesian method). In addition, the overall topology of the SSU + LSU rDNA tree
was not affected by the addition of these two highly
divergent sequences (compare Figs 2 and 3) demonstrating the robustness of the SSU + LSU rDNA
approach. Therefore, even if the statistical support
remained moderate, the concatenation of the two rDNA
markers allowed identifying these highly divergent phylotypes as ciliates (and certainly as alveolates with
strong support), which was impossible when using the
SSU rDNA information alone.

Testing the SSU + LSU rDNA sequences to retrieve

correct taxonomic affiliations for fast-evolving
phylotypes: the Myrionecta case study

Conclusion

The improved SSU + LSU rDNA taxonomic sampling

covered all the major eukaryotic groups, and the general

For several years, the SSU rDNA has been used as the
preferred marker to explore the diversity of microbial
eukaryotic communities in a variety of environments,
leading to the discovery of a huge hidden diversity. Con-

2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

Eukaryotic diversity and phylogeny using SSU + LSU rDNA 7

SSU rDNA
69/.85
57/1

Dinoflagellata

100/1

Dinoflagellata

-/.76
97/1

Perkinsida

84/51/-

SSU + LSU rDNA

100/1
74/.94

Chromera velia

70/.84
98//.99

100/1

Apicomplexa

Perkinsida
Chromera velia

87/-

Ma131_1A49

100/1

Apicomplexa

100/1

Ma131_1A57
100/1

57/-

Ciliophora

99/1

Ciliophora
57*/-

100/.5

DH114_1A22

Heterokonta

(1/4)

89/1
97/1*
96/1
53/-

100/1
75/100/1

60/-

DH18_4A88

Rhizaria
Rhodophyta

100/1

50/1

100/1
77/-

Centrohelida

100/1
57/-

Cryptophyta

79/.92

Viridiplantae

100/1

100/1

93/.78

Opisthokonta

100/1

71/90/1

100/1

Opisthokonta

Ancyromonas sigmoides

80/1

Euglenozoa

Apusomonas sp.

Myrionecta rubra

100/1

62/.97
69/.52

Cryptophyta

72/.98

Ancyromonas sigmoides
(1/4)

Plantae

57/.92
72/.96

Apusomonas sp.

(1/4)

Haptophyta

Glaucophyta

67/.57

Rhizaria

100/1

(1/4)

Haptophyta

94/.98
100/1
81/-

Heterokonta

90/1

100/1

95/-

100/1

Centrohelida

DH114_1A22
100/1

(1/4)

DH18_4A88
Reclinomonas americana

Euglenozoa

100/1

89/1

Reclinomonas americana

0.06
0.06

Fig. 3. ML phylogenetic trees with Myrionecta-like phylotypes (grey boxes). The taxonomic sampling is the same as in Fig. 2, but large groups
have been collapsed. On the left, the tree based on the SSU rDNA alone (1200 positions); on the right, the tree based on the SSU + LSU
rDNA concatenation (3247 positions).

sequently, the number of environmental sequences has

increased exponentially and the necessity to place them
correctly in phylogenetic trees to make taxonomic inferences about the corresponding organisms has became
crucial (Berney et al., 2004; Cavalier-Smith, 2004). We
have shown here that amplifying the complete rDNA
cluster and using a combination of SSU + LSU rDNA
sequences allow the retrieval of quite similar diversity
profiles while significantly increasing the phylogenetic

signal, which is essential for correct taxonomic affiliation

of divergent phylotypes without the need of organism
isolation. The additional advantages of complete ribosomal DNA cluster analysis are that the existing large SSU
rDNA database remains exploitable (using the corresponding SSU rDNA portion of the cluster), but much
more additional information becomes available, not only
for phylogenetic or taxonomic purposes. For example, in a
comparable analysis where the complete rDNA cluster

2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

W. Marande, P. Lpez-Garca and D. Moreira

was amplified from marine bacterioplankton, the authors

used the ITS information to confirm the close relationship
among members of several clades and to identify recombination between different ITS regions (Suzuki et al.,
2001). Finally, as we have shown, with relatively modest
additional sequencing effort, the combined SSU + LSU
rDNA analyses provide more reliable eukaryotic phylogenetic trees and taxonomic affiliations of environmental
rDNA sequences.

Experimental procedures
DNA samples and library construction
We amplified rRNA genes from four DNA samples of smallsize plankton (0.25 mm size fraction) collected in various sea
surface (25 m) locations in the South Atlantic (DH18, DH114
and DH141) and in the Marmara Sea (Ma131) that were
available in the laboratory from previous studies (LpezGarca et al., 2001; Lara et al., 2009). For each sample, SSU
and SSU + LSU rDNA libraries were constructed by PCR
amplification and cloning. The SSU rDNA was amplified with
eukaryote specific primers, EK-42F (CTCAARGAYTAAGC
CATGCA) and EK-1498R (CACCTACGGAAACCTTGTTA),
and the PCR products were cloned with the Topo TA cloning
kit (Invitrogen) following the manufacturers instructions. The
protocol was slightly modified for the construction of the
SSU + LSU rDNA libraries because of the much larger size of
the fragments to be amplified and cloned. The size of a
complete eukaryotic ribosomal RNA gene cluster, i.e. the
SSU rDNA, the internal transcribed spacers (ITS1 and ITS2),
the 5.8S ribosomal RNA genes and the LSU rDNA, is of
around 5.5 kbp, so we used a long-run enzyme, the Ex
TaKaRa (TaKaRa) to avoid premature elongation end during
the PCR cycles. PCR reactions were carried out under the
following conditions: 35 cycles (denaturation at 94C for 15 s,
annealing at 55C for 30 s, extension at 72C for 6 min)
preceded by 2 min denaturation at 94C, and followed by
10 min extension at 72C using the SSU rDNA-specific
primer Euk-42F and the LSU rDNA primer 28S-4R (TTCTGACTTAGAGGCGTTCAG). The library construction was
made with the Topo TA XL kit (Invitrogen), designed for high
cloning efficiency of long PCR products, following the manufacturers instruction. Two additional libraries were constructed for the SSU + LSU rDNA marker only, from samples
DH22 (5 m depth South Atlantic) and Ma121 (500 m depth
Marmara Sea).

Diversity analysis
For each SSU and SSU + LSU rDNA library, we selected 48
positive clones and sequenced their 5 region of the SSU
rRNA gene using the eukaryotic primer Euk-42F. For preliminary phylogenetic affiliation, the sequences were blasted
against the nr GenBank database to identify their closest
relatives. Saturation analysis of our libraries was performed
with those partial sequences using the software DOTUR
(Schloss and Handelsman, 2005). The OTU identification
was carried out with Codon Code Aligner (Licor), by

clustering sequences with a minimum identity percentage of

98%.

SSU + LSU rDNA sequence data set construction

We sequenced the SSU and the LSU rRNA genes for 22
positive clones, selected to represent all the groups retrieved
in our libraries, using a set of internal primers, including
28S-568F (TTGAAACACGGACCAAGGAG), 28S-803R
(ACTTCGGAGGG AACCAGCTA), 28S-1909F (GGAGT
AACTATGACTCTCT),
28S-1965R
(TCTCGTTAATCCA
TTCATGC) and 28S-4R. In addition, we amplified and
sequenced the SSU and LSU rRNA genes from a culture of
Cafeteria roenbergensis isolated by Dr E. Lara from marine
sediment (1200 m depth) of the Marmara Sea (Turkey) from
the Cinarcik Basin (E. Lara, pers. comm.). We updated the
available data set of concatenated SSU + LSU rDNA
sequences (Moreira et al., 2007) with the new sequences
present in GenBank: Gymnodinium aureolum (DQ779991),
Chromera velia (EU106870), Hammondia hammondi
(AF101070) and two complete genome projects: Ostreococcus lucimarinus (Palenik et al., 2007) and Hyaloperonospora
parasitica (http://vmd.vbi.vt.edu/browse/cgi-bin/browserUI.
cgi). The LSU rDNA sequence of Blastocystis hominis was
generously provided by Drs F. Delbac and C. Vivares. This
sequence was obtained through the B. hominis genome
project conducted by the Genoscope (http://www.genoscope.
cns.fr/spip/Blastocystis-hominis-whole-genome.html).
We
checked for chimerical SSU rDNA and SSU + LSU rDNA
sequences manually by independent BLAST searches (Altschul et al., 1997) of the 5 and the 3 halves of the SSU rDNA
and by comparing the phylogenetic trees obtained with SSU
and LSU rDNA independently. We detected two potential
chimerical sequences in that way, which were removed from
our analyses. Sequences were deposited in GenBank under
Accession Numbers FJ032645FJ032660 and FJ032662
FJ032695.

Phylogenetic analysis
All new sequences were incorporated to the available data
set of concatenated SSU + LSU rDNA sequences and
aligned using the ED program of the package MUST (Philippe,
1993). Ambiguously aligned regions and gaps were excluded
in phylogenetic analyses. The ML phylogenetic analyses
were carried out with the program Treefinder (Jobb et al.,
2004) and bootstrap values were calculated from 1000 replicates. Bayesian trees were inferred using MrBAYES 3.1.
Markov chain Monte Carlo searches were run with four
chains for 1000 000 generations, with trees being sampled
every 100 generations. The first 5000 trees were discarded
as burnin, keeping only trees generated well after chain
parameters stabilized. For both the ML and the Bayesian
methods, we applied a GTR + G + I model of nucleotide
substitution, taking into account a proportion of invariable
sites, and a G-shaped distribution of the rates of substitution
among sites with four rate categories. Fast-evolving groups
(Microsporidia, Parabasalida, Diplomonadida and Amoebozoa), for which we did not retrieve closely related phylotypes
in our samples, were removed from our analyses. Sequence
alignments are available upon request.

2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

Eukaryotic diversity and phylogeny using SSU + LSU rDNA 9

Acknowledgements
This work was supported by a grant from the French Agence
Nationale de la Recherche (ANR JC05-44674) to D.M. We
thank E. Lara for providing the C. roenbergensis culture, and
C. Vivares, F. Delbac and the Genoscope for providing the
B. hominis sequences. We thank two anonymous reviewers
for constructive suggestions.

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