Widespread occurrence and genetic diversity of marineparasitoids belonging to Syndiniales (Alveolata)
L. Guillou,1* M. Viprey,1 A. Chambouvet,1
R. M. Welsh,2 A. R. Kirkham,3 R. Massana,4
D. J. Scanlan3 and A. Z. Worden21Station Biologique de Roscoff, UMR7144, CNRS etUniversité Pierre et Marie Curie, BP74, 29682 RoscoffCedex, France.2Monterey Bay Aquarium Research Institute, 7700Sandholdt Road, Moss Landing, CA 95039, USA.3Department of Biological Sciences, University ofWarwick, Coventry CV4 7AL, UK.4Institut de Ciències del Mar, CMIMA, Passeig Marítimde la Barceloneta 37-49, 08003 Barcelona, Catalonia,Spain.
Summary
Syndiniales are a parasitic order within the eukaryoticlineage Dinophyceae (Alveolata). Here, we analysedthe taxonomy of this group using 43655 18S rRNAgene sequences obtained either from environmentaldata sets or cultures, including 6874 environmentalsequences from this study derived from Atlantic andMediterranean waters. A total of 5571 out of the 43655sequences analysed fell within the Dinophyceae.Both bayesian and maximum likelihood phylogeniesplaced Syndiniales in five main groups (I–V), as amonophyletic lineage at the base of ‘core’ dinoflagel-lates (all Dinophyceae except Syndiniales), althoughthe latter placement was not bootstrap supported.Thus, the two uncultured novel marine alveolategroups I and II, which have been highlighted previ-ously, are confirmed to belong to the Syndiniales.These groups were the most diverse and highlyrepresented in environmental studies. Within each,8 and 44 clades were identified respectively.Co-evolutionary trends between parasitic Syndinialesand their putative hosts were not clear, suggestingthey may be relatively ‘general’ parasitoids. Based onthe overall distribution patterns of the Syndiniales-affiliated sequences, we propose that Syndiniales areexclusively marine. Interestingly, sequences belong-ing to groups II, III and V were largely retrieved from
the photic zone, while Group I dominated samplesfrom anoxic and suboxic ecosystems. Nevertheless,both groups I and II contained specific clades prefer-entially, or exclusively, retrieved from these latterecosystems. Given the broad distribution of Syndini-ales, our work indicates that parasitism may be amajor force in ocean food webs, a force that isneglected in current conceptualizations of the marinecarbon cycle.
Introduction
The Alveolata, one of the major eukaryotic lineages, iscomposed of four protist classes: the Ciliophora, theApicomplexa, the Perkinsea and the Dinoflagellata.Alveolata have adopted a large range of trophic modesand habitats. They can be important marine primaryproducers. For instance, about half of dinoflagellates arephotosynthetic (Lessard and Swift, 1986), with somebeing responsible for toxic algal blooms. Ciliates anddinoflagellates can also be active predators; others arecoral symbionts (e.g. the dinoflagellate Symbiodiniumspp.) and some ciliates even reside in mammalian guts(Williams and Coleman, 1992). Finally, a large number ofspecies are parasites, broadly distributed throughoutthese Alveolata classes. For instance, Apicomplexa iscomposed solely of obligate parasites. In addition to phy-logenetic relatedness based on gene sequence compari-sons, Alveolata are unified by specific morphologicalcharacters. These include the presence of membrane-bound flattened vesicles, termed alveoli (Cavalier-Smith,1993; Patterson, 1999), distinct pores called microporesthat pierce the outer membrane and the presence of amore or less developed apical complex apparatus usedby alveolate parasites to enter their host (reviewed byLeander and Keeling, 2003).
The discovery of novel marine alveolate (MALV) lin-eages in marine planktonic communities by culture-independent techniques, specifically MALV groups I and II(Díez et al., 2001a; López-García et al., 2001; Moon-vander Staay et al., 2001), has raised questions regardingfunctional roles of these diverse populations. Phyloge-netic analyses showed MALV Group II belongs to theSyndiniales, a dinoflagellate order exclusively composedof marine parasites. This assignment was based on theclose phylogenetic relatedness of the environmental 18S
Received 17 April, 2008; accepted 29 June, 2008. *Forcorrespondence. E-mail [email protected]; Tel. (+33) 2 98 29 2379; Fax (+33) 2 98 29 23 24.
Environmental Microbiology (2008) 10(12), 3349–3365 doi:10.1111/j.1462-2920.2008.01731.x
Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing LtdNo claim to original French government works
mailto:[email protected]
rRNA gene sequences (Moon-van der Staay et al., 2001;Skovgaard et al., 2005) with three previously describedgenera: Amoebophrya spp., Hematodinium spp. and Syn-dinium spp. More recently, 18S rRNA gene sequencesderived from Ichthyodinium chaberladi (Gestal et al.,2006) and Duboscquella sp. (Harada et al., 2007), twoparasitic Syndiniales genera, showed an affiliation withMALV Group I.
MALV groups I and II have been retrieved from variousmarine habitats, mainly from the picoplankton fraction (< 2or < 3 mm size fractionated samples). These groups fre-quently form the majority of sequences within marineenvironmental clone libraries (see López-García et al.,2001; Moon-van der Staay et al., 2001; Massana et al.,2004; Romari and Vaulot, 2004; Not et al., 2007). This hasled to a large increase in the number of MALV sequencesdeposited in GenBank over the last few years. In a previ-ous study, Groisillier and colleagues (2006) detected fivedistinct clades within MALV Group I, and 16 clades withinMALV Group II. However, in that study, many sequencescould not be clearly assigned to a specific clade, suggest-ing further discrete lineages might exist. Interestingly,some clades comprised sequences retrieved only fromvery specific habitats, such as anoxic environments ordeep sea hydrothermal vents, while other clades con-tained sequences obtained from widely varying habitats.
In the present work our aims were (i) to broaden repre-sentation of the different environments in which membersof the Alveolata are potentially encountered by construct-ing and sequencing 18S rRNA gene libraries; (ii) to clarifythe phylogeny of the Alveolata using recently publishedand the new 18S rRNA gene sequences for this group; (iii)to undertake a rigorous review of clade organizationwithin the above mentioned MALV groups I and II; (iv)to compare the genetic diversity of environmentalsequences derived from culture-independent PCRsurveys (targeting the 18S rRNA gene) to our presentknowledge of the taxonomy of Syndiniales; and (v) toextract general information on the preferred habitats anddistribution of specific members of the Syndiniales.
Results
Sample sites and environmental conditions
Most of the novel environmental sequences obtained inthis study were retrieved from coastal and oceanic watersin the Atlantic Ocean and Mediterranean Sea (Table 1).The collection sites varied in terms of season, depth andlevel of oligotrophy. For example, the Bermuda AtlanticTime-series Station (BATS) is relatively oligotrophic, andat the time of sampling was already strongly stratified. Incontrast, the northern Sargasso Sea Station, althoughrelatively close to BATS, was likely still influenced by deep
winter mixing that occurs in this area, bringing nutrients tosurface waters (see Cuvelier et al., 2008 for furtherdiscussion). The Florida Straits sites represent three dis-tinct water types. Station 1, on the western side of theFlorida Straits, was relatively coastal. Station 4 representsthe core of the Gulf Stream Current-forming waters, whichare highly oligotrophic, while Station 14 is also olig-otrophic but a shallow setting on the eastern side of theFlorida Straits (see Cuvelier et al., 2008). Other environ-mental sequences from the Atlantic Ocean were obtainedduring the Atlantic Meridional Transect (AMT 15, see alsoZwirglmaier et al., 2007), which extended from 48°N[south-west (SW) of the UK] to 40°S (SW of Cape Town,South Africa). Environmental sequences from the Medi-terranean Sea were collected in late summer along aMediterranean transect sampled during the PROSOPEcruise in 1999 (Garczarek et al., 2007), from high nutrientlevels in the Morocco upwelling to strong phosphoruslimitation in the eastern most basin. Although half of thegenetic libraries were built using a PCR approach biasedtowards green algae, dinoflagellate sequences wereretrieved in both data sets (the majority of them wereretrieved using general eukaryotic primers). We also useda PCR approach biased towards MALV Group II (usingthe specific primer ALV01) that we compared with the useof general eukaryotic primers from a coastal site (EnglishChannel, France). Most of genetic libraries were built onvery small size fractions (less than 2–3 mm), althoughsome were processed on larger size fractions and evenafter incubations (see genetic libraries from coastal sitesfrom the Mediterranean Sea). This heterogeneous dataset offered us a very large range of environmentalsequence origins. In total, 6874 new environmentalsequences were generated and screened for dinoflagel-late sequences.
Alveolate 18S rRNA gene sequence data set
The completed data set comprised 43655 eukaryotic 18SrRNA gene sequences obtained either from GenBank orfrom the environmental clone libraries described above.From marine environments, 351 environmental clonelibraries were analysed with major contribution from sites(Table 2) in the Atlantic (Díez et al., 2001a; Countwayet al., 2007; Not et al., 2007), Indian (Not et al., 2008),Arctic (Lovejoy et al., 2006; Stoeck et al., 2007) andPacific (Moon-van der Staay et al., 2001) Oceans, Medi-terranean Sea (Viprey et al., 2008), Antarctic (López-García et al., 2001), as well as several coastal sites(Massana et al., 2004; Romari and Vaulot, 2004; Medlinet al., 2006; Worden, 2006). These encompassed a rangeof marine habitats including the photic zone, sediments,hydrothermal vents and anoxic ecosystems (Table 2). Ter-restrial and continental ecosystems were also included
3350 L. Guillou et al.
Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 3349–3365No claim to original French government works
Tab
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tion
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and
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ple
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Tota
lnum
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Diversity of parasitic Syndiniales 3351
Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 3349–3365No claim to original French government works
Tab
le1.
cont
.
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6874
For
furt
her
info
rmat
ion
onth
epr
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sus
ed,
see
also
Tabl
eS
2.A
cces
sion
num
bers
are
prov
ided
for
dino
flage
llate
sequ
ence
son
ly.
3352 L. Guillou et al.
Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 3349–3365No claim to original French government works
(150 clone libraries, mostly from freshwater, sedimentsand soils). Taken together, this resulted in 4844 dinoflagel-late sequences from these environmental data sets, with1164 and 2435 belonging to MALV groups I and II respec-tively (Table 2). An additional 727 sequences werederived from dinoflagellate cultures including a few fromthe amplification of single cells (see Table 2), most ofthese belonging to marine photosynthetic dinoflagellates(a bias already pointed out by Murray et al., 2005). Themean length of the dinoflagellate sequences in the data-base was 950 nucleotides (range 108–1841 bp).
Phylogenetic analyses
Dinoflagellate sequences longer than 1600 bp (1018sequences in total, 291 retained for the final tree, see thecomplete list of sequences in the Table S1) were used toperform phylogenetic analyses. Sequences belonging tothe major Alveolata lineages were also included (Fig. 1).In order to avoid long-branch attraction artefacts, highlydivergent alveolate groups were removed after prelimi-nary phylogenetic analyses by neighbour joining (NJ).These divergent groups included Haemosporida (Apicom-plexa including the human parasite Plasmodium), twociliate groups, Mesodiniidae (Myrionecta and Mesod-inium) and Ellobiopsidae (Silberman et al., 2004). Highlydivergent dinoflagellates, such as members of Noctilu-cales and Oxyrrhis marina, were also excluded. Bayesianphylogeny, using 1137 positions in the 18S rRNA genesequence alignments, delineated four primary lineageswithin the Alveolata (Fig. 1), the ciliates, the Apicomplexa,the Perkinsea and the Dinophyceae. As previouslyobserved (Leander and Keeling, 2003; Groisillier et al.,2006), ciliates fell in the basal region of the tree, followedby Apicomplexa (Fig. 1). Perkinsea are the closest rela-tives of dinoflagellates. As frequently found in 18S rRNAgene phylogenies, many of these backbone nodes did notretain bootstrap support. At the basal part of Dino-phyceae, several distinct taxa were placed in the baye-sian analysis, here termed Syndiniales groups I–V, as amonophyletic lineage (Fig. 1). The general tree topologyobtained with maximum likelihood (ML) was similar (datanot shown). The existence of environmental MALV GroupI and II was previously described (López-García et al.,2001; Moon-van der Staay et al., 2001), but hererenamed (Syndiniales groups I and II) given more defini-tive placement within the Syndiniales. The genetic diver-sity and clade nomenclature of Syndiniales groups I and IIare described in detail in separate analyses using partialsequences (Figs 2 and 3). For the definition of clades, wemodified the general criteria chosen by Groisillier andcolleagues (2006) so that a clade must (i) contain envi-ronmental sequences from at least 2 different clone librar-ies, and (ii) be bootstrap supported, at the defining nodes,Ta
ble
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Syn
dini
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.
Diversity of parasitic Syndiniales 3353
Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 3349–3365No claim to original French government works
by values greater than 60% in NJ and maximum parsi-mony (MP) analysis. Some exceptions to these rules weremade as detailed below. The mean sequence identitywithin a clade was 87% for Syndiniales Group I (rangingfrom 76.6% for clade 5 to 91.9% for clade 7) and 93.5%for Syndiniales Group II (ranging from 80.8% for clade 8 to99.4% for clades 35 and 43, see also Fig. S1). Syndini-ales Group I contained eight different clades (Fig. 2).Clades 1–5 were described previously by Groisillier andcolleagues (2006), while three new clades emerged fromthis study. All of these eight clades are supported bybootstrap values > 60%, except for Clade 3, which is onlysupported by MP, bootstrap analyses (74%). Neverthe-less, the tree topology is identical with the two differentmethods (NJ and MP) and minimal sequence identitieswithin this clade are inside the range of other clades
belonging to this group (see Fig. S1). Ichthyodinium chab-erladi, a parasitoid of fish eggs, belongs to Group I Clade3, while Duboscquella spp., a parasitoid of tintinnides,belongs to Group I Clade 4 (Harada et al., 2007). Syndini-ales sequences retrieved by single-cell PCR on radiolar-ian isolates (Dolven et al., 2007) belong to Clade 1(DQ916408, though not in the tree because this sequencecontained several nucleotide ambiguities) and Clade 2(DQ916404–DQ916407 and DQ916410). Clades 5–8 areonly composed of environmental sequences. Somesequences within clades 1–4 (highlighted in grey in Fig. 2)were retrieved exclusively from suboxic and anoxicecosystems. Together, clades 1 and 5 were the mostcommonly retrieved from environmental clone libraries,representing � 75% of sequences belonging to Syndini-ales Group I.
DQ504327 LC22-5EP-44
DQ925237 CsH1AF421184 Hematodinium sp.EF065718 Hematodinium pereziEF065717 Hematodinium pereziEF172791 SSRPB76
DQ504356 LC22-5EP-37EU793192 E1-80m.27DQ146406 Syndinium sp.DQ146404 Syndinium turboDQ146403 Syndinium turboDQ146405 Syndinium turbo
AF286023 Hematodinium sp.
IV
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EU818259 FS04F028_85mEU817911 OC413NSS_Q031_15m
EU793593 ED-15m.135EU793324 E5-25m.204DQ918792 ENVP36162.00253
AY664993 SCM15 C23EU818113 FS04G196_5mEU818541 FS14N090_58m
EU818542 FS14N072_58mEU561731 IND31.60EU818431 FS14K050_5mEU818080 FS04G102_5m
EU780604 AMT15_1B_36EF539059 MB01.33
EU818673 FS01D005_65mAY295467 RA000609.43EU818141 FS04GA80_5mAY426937 BL010625.44EU818430 FS14K031_5m
EU818672 FS01D055_65mEU793987 EU-30m.99
EU818014 OC413BATS_O095_75mEU793515 E9-65m.113
EU818655 FS01D056_65mDQ918274 ENVP21819.00021
EU818152 FS04H034_89mAJ402349 OLI11005
DQ918653 ENVP223.00266EF526769 NIF 4C5
DQ310226 FV23 1E1DQ918940 ENVP366.00147
DQ918434 ENVP21819.00364EU818349 FS14I039_70mEU793615 ED-15m.57
EU793164 E1-30m.67EU793978 EU-30m.74EU793475 E9-5m.53EU793818 EM-110m.215
EF539065 TH07.15DQ647514 CD8.10EU562048 IND70.56EU780607 AMT15_1B_40
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2%
III
Otherdinoflagellates
II
Syndinium turboHematodinium spp. III
IV
PerkinseaColpodellidae
CoccidiaPiroplasmida
Cryptosporidium spp.
Ciliates
Apicomplexa
IV
10%
AY665063 SCM38 C14 AF133909 Parvilucifera
Und. Apicomplexa 1
GregariniaColpodellidae Und. Apicomplexa 2
Outgroups
100/100
100/100
80/-
98/95
100/99
64/-
100/100
100/100
100/100100/100
99/82
100/99
Syndiniales
Dinophyceae
Fig. 1. Phylogeny of dinoflagellates using near-complete 18S rRNA gene sequences. Left: Bayesian phylogeny of alveolates based onanalysis of 291 near full-length 18S rRNA gene sequences. Five sequences of Bolidophyceae (stramenopiles) were used as an outgroup.Gblock retained 1137 positions for the phylogenetic analyses. Bootstrap values, given at the principal nodes of the tree, correspond toneighbour-joining and maximum parsimony analyses respectively (1000 replicates, values > 60% shown). For neighbour-joining bootstrapping,a GTR + G + I model was selected with the following parameters: Lset Base = (0.2757 0. 1787 0.2494), Nst = 6, Rmat = (1.0746 2.96001.2514 1.0820 4.7079), Rates = gamma, Shape = 0.7091, Pinvar = 0.2330. The scale bar corresponds to 10% sequence divergence. Inset (atthe right): Details of groups III to VI, based upon analysis of partial (500 bp) sequences (59 sequences including outgroups). The scale barcorresponds to 2% sequence divergence. The two sequences in grey belonging to Group IV are from hydrothermal vents.
3354 L. Guillou et al.
Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 3349–3365No claim to original French government works
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Phy
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Syn
dini
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Gro
upI.
Nei
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inin
gph
ylog
eny
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cted
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edi
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ighb
our-
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ing
and
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imum
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imon
yan
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show
n).
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ence
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rgen
ce.
The
tree
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der
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tter
sepa
rate
the
maj
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inth
efig
ure.
Seq
uenc
esin
grey
are
from
hydr
othe
rmal
and
subo
xic
ecos
yste
ms.
Diversity of parasitic Syndiniales 3355
Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 3349–3365No claim to original French government works
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Fig
.3.
Phy
loge
nyof
Syn
dini
ales
Gro
upII.
Nei
ghbo
ur-jo
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ylog
eny
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nces
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red
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udin
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odel
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cted
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gth
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llow
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ers:
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oots
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cipa
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rent
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es,
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ndto
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ony
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yses
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ectiv
ely
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teris
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icat
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es).
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verg
ence
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ascu
tin
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rse
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teth
em
ajor
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esin
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figur
e.In
set:
asm
allp
ortio
nof
the
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hbou
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tain
edus
ing
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Age
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sed
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ysis
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th(in
clud
ing
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roup
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ence
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rgen
ce.
Boo
tstr
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Seq
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grey
are
from
hydr
othe
rmal
and
subo
xic
ecos
yste
ms.
3356 L. Guillou et al.
Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 3349–3365No claim to original French government works
By comparison, Syndiniales Group II is genetically morediverse than Group I. In addition to clades 1–16 (seeGroisillier et al., 2006), 28 new clades emerged from thepresent study (clades 17–44, Fig. 3). Some of theseclades are not supported by bootstrap analyses obtainedwith one of the two phylogenetic methods tested (clades2, 6, 10+11, 14, 16, 23, 25 and 26), but in such cases thetree topologies are identical between the two methods.Clade 10 and Clade 11 described by Groisillier and col-leagues (2006) are now merged into a single clade(herein named Clade 10+11), which includes cloneBL10320.32 (a singleton in the work of Groisillier et al.,2006). However, Clade 10+11 is not supported by boot-strap analyses. Numerous environmental sequences dis-playing long branches (labelled ‘close to 10+11?’ in Fig. 3)also appear to be closely related to Clade 10+11.
Only a single genus within Syndiniales Group II hasbeen formally described, the Amoebophrya. AllAmoebophrya sequences obtained from cultures andsingle-cell analyses formed a monophyletic group as rec-ognized by Groisillier and colleagues (2006). This mono-phyletic group now also includes clades 1–5 as well asclades 25, 33, 39, 41 and 42 (Fig. 3). Recently, Kim andcolleagues (2008) recognized nine different subgroupswithin Amoebophrya. Subgroup 1 (from the analysis ofKim and colleagues) corresponds primarily with our Clade1 (except sequence AF290077 which belongs to the novelClade 25) while subgroups 3, 4, 5 and 6 correspond toclades 4, 3, 5 and 2 respectively. The recognition of sub-group 2 (identified in Kim et al., 2008) is probably skewedby inclusion of a sequence we identified to be a chimera(DQ186527). Thus, sequence AY260468, obtained fromthe direct amplification of Ceratium tripos infected withAmoebophrya, likely remains a singleton. SequenceAY295690 (subgroup 8) is also a probable chimera,whereas sequences included within subgroups 7 and 9belong to Clade 33 in the present study. SequenceDQ916402, obtained from the direct amplification of a cellof spumellarida Hexacontium gigantheum (Radiolaria), isclosely related to Clade 6. This sequence was removedfrom our global analyses due to the high number of nucle-otide ambiguities it contained. Clades 15 and 9 are pri-marily composed of environmental sequences fromanoxic or suboxic ecosystems (highlighted in grey inFig. 3). Both clades also include sequences retrievedfrom deep-sea methane cold seeps (DSGM-16 and 17,Takishita et al., 2007) and deep oceanic waters (Count-way et al., 2007), but these sequences were not includedin this analysis due to their short length. Clades originallycontaining only sequences from coastal systems (i.e.clades 2, 4, 5, 8, 12 and 13; see Groisillier et al., 2006) areshown here to also include clones from other oceanicregions, including oligotrophic waters. This highlights theimportance of gaining sequence representation from a
broader array of geographical locations. Finally, in termsof the number of sequences, the most commonly foundclades in rank order were clades 10+11, 7, 6, 1, 19, 3, 22and 16.
Other Syndiniales groups (III–V) also emerged from thepresent study (detailed in Fig. 1). Syndiniales Group IIIcontains 71 environmental sequences, including cloneOLI11005 (AJ402349) that was previously placed outsideGroup II (see Groisillier et al. 2006), as well as numerousenvironmental sequences retrieved from various oceanicsurface waters (Mediterranean Sea, Indian Ocean, Sar-gasso Sea), coastal waters [Southern Taiwan Strait,Blanes Bay (Spain) and the English Channel] and from asupersulfidic anoxic fjord (DQ310226). Syndiniales GroupIV contains the genera Syndinium and Hematodinium,five closely related environmental sequences, a sequenceretrieved from the Mediterranean Sea, E1–80m.27,closely related to Syndinium turbo and a clade allied withthe genus Hematodinium and composed of sequencesfrom the Sargasso Sea (EF172791 and DQ918583)and from deep sea hydrothermal vents (DQ504327 andDQ504356). Syndiniales Group V contains 67 environ-mental sequences, all collected within the euphotic zone,but includes clones retrieved from very different oceanicecosystems (Indian Ocean, Atlantic Ocean, Mediterra-nean Sea and Sargasso Sea). Sequence BL000921.23from Blanes also belongs to Syndiniales Group V (notincluded here due to sequence length dissimilarities).Finally, two partial environmental sequences could not beassigned to any specific group: EU793524 (E9–65m.123)and EU818559 (F14DEC+), but phylogenetic analysisplaced them closely related, although separated fromthese Syndiniales groups.
Ecological distributions
Dinoflagellate sequences represent less than 1% ofenvironmental sequences from continental ecosystems(aquatic and terrestrial), whereas they represent close to30% of the sequences obtained from marine ecosystems.With respect to aquatic environments (both marine andfreshwater), dinoflagellates sequences as a whole wereobtained primarily from the plankton, as opposed to otherenvironments such as sediments (Table 2). Sequencesbelonging to Syndiniales groups I and II were absent insamples collected to date from continental ecosystems,however, they represent the largest portion of dinoflagel-late sequences from marine systems. Syndiniales GroupII alone represented about half of all environmentaldinoflagellate sequences (Table 2).
We divided marine ecosystems included in oursequence database into various categories: (i) anoxic andsuboxic ecosystems, (ii) ecosystems with hydrothermalactivities, (iii) sediments, or (iv) the water column
Diversity of parasitic Syndiniales 3357
Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 3349–3365No claim to original French government works
(wherever possible, we also specified whether sequenceswere derived from the aphotic or euphotic zone; Fig. 4). Incases where these categories overlapped (e.g. anoxicsediments collected from deep hydrothermal vents, oranoxic water columns), environmental sequences wereincluded in both categories. Considering all dinoflagellatesequences, Syndiniales Group II sequences were moreabundant in clone libraries derived from the water column(planktonic ecosystems) than in other habitats, whilethe relative importance of the remaining dinoflagellatesequences (including Syndiniales Group I) was greater inanoxic environments, hydrothermal vents and sediments(Fig. 4). Within planktonic ecosystems, the relative contri-bution to clone libraries of Syndiniales Group I and IIcompared with other dinoflagellate sequences was similarbetween euphotic and aphotic waters (Fig. 4).
Environmental sequences derived from the euphoticzone, both coastal and oceanic waters, have generallybeen recovered using three different primer setsEuk328/Euk329, EukA/EukB and EukA/EukB′ (for primerreferences see Table S2). Although the contribution ofdinoflagellate sequences to the total number of clones is
very similar using either the EukA/EukB or EukA/EukB′primer sets, the relative contribution of Group II is higherusing the Euk328f/Euk329r primer set (Fig. 5). However,the distribution of Syndiniales Group I and II cladeswithin the euphotic zone, as evidenced from these clonelibraries, is quite similar with the different primer sets(Fig. 6). According to clone library composition, sunlitsurface marine waters are dominated by SyndinialesGroup I clades 1, 4 and 5, and Group II clades 1, 6, 7and 10+11 (Fig. 6). Comparing sequences obtainedusing the same primer sets, Syndiniales clade distribu-tions within the aphotic zone are quite different fromsurface waters, mainly dominated by Syndiniales GroupI clades 1, 2 and 3 and by Syndiniales Group II clades6 and 7 (Fig. 6).
Discussion
Our phylogenetic analysis of available 18S rRNA genesequences from described par