Widespread occurrence and genetic diversity of marine ... · photosynthetic (Lessard and Swift, 1986), ... quently form the majority of sequences within marine ... 3349–3365 No

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. Worden2

1Station 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

le1.

Des

crip

tion

ofth

esa

mpl

ing

site

san

dch

arac

teris

tics

ofcl

one

libra

ries

cons

truc

ted

for

this

stud

y.

Env

ironm

ent

and

crui

seS

tatio

nan

dS

iteC

oord

inat

esD

ate

Cod

elib

rary

Dep

th(m

)P

rimer

Sam

ple

deta

ils(m

m)

Tota

lnum

ber

ofcl

ones

with

inse

rtsc

reen

ed

Med

iterr

anea

nS

eaP

RO

SO

PE

Oce

anic

euph

otic

zone

Acc

essi

onnu

mbe

rsfr

omE

U79

2921

toE

U79

3941

+E

U84

8494

toE

U84

8495

See

also

Vip

rey

etal

.(2

008)

St

1S

trai

tof

Gib

ralta

r38

°08′

N05

°18′

W14

-Sep

-99

C1-

3030

Euk

328f

-CH

LO02

<3

246

E1-

3030

Euk

328f

-Euk

329r

<3

135

C1-

8080

Euk

328f

-CH

LO02

<3

195

E1-

8080

Euk

328f

-Euk

329r

<3

69S

t3

Alg

eria

nB

asin

37°9

8′N

03°8

3′E

16-S

ep-9

9C

3-5

5E

uk32

8f-C

HLO

02<

316

5E

3-5

5E

uk32

8f-E

uk32

9r<

346

C3-

2525

Euk

328f

-CH

LO02

<3

23E

3-25

25E

uk32

8f-E

uk32

9r<

356

C3-

9595

Euk

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LO02

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173

E3-

9595

Euk

328f

-Euk

329r

<3

27S

t5

Str

ait

ofS

icily

36°4

7′N

13°3

2′E

18-S

ep-9

9C

5-25

25E

uk32

8f-C

HLO

02<

315

1E

5-25

25E

uk32

8f-E

uk32

9<

316

8C

5-55

55E

uk32

8f-C

HLO

02<

318

5E

5-55

55E

uk32

8f-E

uk32

9r<

313

1S

tM

IOIo

nian

Bas

in33

°98′

N22

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E20

-Sep

-99

CM

-55

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328f

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LO02

<3

173

EM

-55

Euk

328f

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329r

<3

62C

M-5

050

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328f

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171

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uk32

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uk32

9r<

310

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1011

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uk32

8f-E

uk32

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311

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41°8

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10°4

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310

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322

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9S

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Bas

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N07

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CD

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uk32

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02<

317

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515

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118

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320

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196

Eng

lish

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num

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from

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7850

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7852

55

Ast

an48

°45′

N04

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W21

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urfa

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uk52

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uk32

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5301

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28-F

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U78

5302

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U78

5340

See

also

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etal

.(2

006)

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nes

Bay

41°4

0′N

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trat

ion

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4S

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cuba

tion

afte

rfil

trat

ion

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21

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ntic

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RO

SO

PE

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stal

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zone

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essi

onnu

mbe

rsfr

omE

U79

3942

toE

U79

3987

See

also

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rey

etal

.(2

008)

St

UP

WM

oroc

coup

wel

ling

31°0

2 ′N

10°0

3′W

09-S

ep-9

9C

U-3

030

Euk

328f

-CH

LO02

<3

53

EU

-30

30E

uk32

8f-E

uk32

9r<

312

6

Atla

ntic

Mer

idio

nalT

rans

ect

(AM

T15

)O

cean

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phot

iczo

neA

cces

sion

num

bers

from

EU

7805

89to

EU

7806

36S

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soZ

wirg

lmai

eret

al.

(200

7)

Nor

ther

nco

asta

lS

tatio

n1

48°7

4′N

7°08

′W19

-Sep

t-04

AM

T15

-110

Euk

328f

-Euk

329r

<3

217

Upw

ellin

gS

tatio

n15

17°8

3′N

20°8

9′W

04-O

ct-0

4A

MT

15-1

510

Euk

328f

-Euk

329r

<3

207

Sou

ther

ngy

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tatio

n27

23°5

6′S

17°4

9′W

18-O

ct-0

4A

MT

15-2

730

Euk

328f

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329r

<3

119

Sou

ther

nte

mpe

rate

Sta

tion

3337

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S1°

23′E

24-O

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4A

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15-3

35

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328f

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329r

<3

234

Diversity of parasitic Syndiniales 3351


Tab

le1.

cont

.

Env

ironm

ent

and

crui

seS

tatio

nan

dS

iteC

oord

inat

esD

ate

Cod

elib

rary

Dep

th(m

)P

rimer

Sam

ple

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ils(m

m)

Tota

lnum

ber

ofcl

ones

with

inse

rtsc

reen

ed

Flo

rida

Str

aits

,Atla

ntic

Oce

anC

oast

aleu

phot

iczo

neA

cces

sion

num

bers

from

EU

8185

67to

EU

8186

91an

dE

U83

6584

toE

U83

6597

Sta

tion

125

°30′

N80

°04′

W31

-Mar

-05

FS

01B

5E

uk32

8f-E

uk32

9r<

290

25°3

0′N

80°0

4′W

31-M

ar-0

5F

S01

C70

Euk

328f

-Euk

329r

<2

85

25°3

0′N

80°0

3′W

01-A

ug-0

5F

S01

A5

Euk

328f

-Euk

329r

<2

85

25°3

0′N

80°0

3′W

01-A

ug-0

5F

S01

D65

Euk

328f

-Euk

329r

<2

85

Flo

rida

Str

aits

,Atla

ntic

Oce

anO

cean

iceu

phot

iczo

neA

cces

sion

num

bers

from

EU

8180

78to

EU

8185

66an

dE

U83

6598

toE

U83

6612

Sta

tion

425

°30′

N79

°57′

W31

-Mar

-05

FS

04E

5E

uk32

8f-E

uk32

9r<

281

25°3

0′N

79°5

7′W

31-M

ar-0

5F

S04

F85

Euk

328f

-Euk

329r

<2

88

25°3

0′N

79°5

7′W

01-A

ug-0

5F

S04

G5

Euk

328f

-Euk

329r

<2

90

25°3

0′N

79°5

7′W

01-A

ug-0

5F

S04

H89

Euk

328f

-Euk

329r

<2

169

Sta

tion

1425

°29′

N79

°20′

W30

-Mar

-05

FS

14I

70E

uk32

8f-E

uk32

9r<

274

25°2

9′N

79°2

0′W

30-M

ar-0

5F

S14

J5

Euk

328f

-Euk

329r

<2

90

25°2

9′N

79°2

0′W

31-J

ul-0

5F

S14

K5

Euk

328f

-Euk

329r

<2

69

25°2

9′N

79°2

0′W

31-J

ul-0

5F

S14

L80

Euk

328f

-Euk

329r

<2

83

25°2

9′N

79°2

0′W

Dec

-08

FS

14M

58E

uk32

8f-E

uk32

9r<

283

25°2

9′N

79°2

0′W

Dec

-08

FS

14N

58E

uk32

8f-E

uk32

9r�

281

Sar

gass

oS

eaA

tlant

icO

cean

Oce

anic

euph

otic

zone

Acc

essi

onnu

mbe

rsfr

omE

U81

7966

toE

U81

8077

and

EU

8366

13to

EU

8366

15

Ber

mud

aA

tlant

icTi

me-

Ser

ies

Sta

tion

31°3

9′N

64°3

7′W

29-M

ay-0

5O

C41

3BA

TS

_O75

Euk

328f

-Euk

329r

<2

90

01-J

un-0

5O

C41

3BA

TS

_P15

Euk

328f

-Euk

329r

<2

87

Sar

gass

oS

ea,A

tlant

icO

cean

Oce

anic

euph

otic

zone

Acc

essi

onnu

mbe

rsfr

omE

U81

7882

toE

U81

7965

+E

U83

6616

toE

U83

6617

35°0

9′N

66°3

3′W

05-J

un-0

5O

C41

3NS

S_Q

15E

uk32

8f-E

uk32

9r<

284

08-J

un-0

5O

C41

3NS

S_R

70E

uk32

8f-E

uk32

9r<

283

Tota

lnum

ber

ofcl

ones

6874

For

furt

her

info

rmat

ion

onth

epr

imer

sus

ed,

see

also

Tabl

eS

2.A

cces

sion

num

bers

are

prov

ided

for

dino

flage

llate

sequ

ence

son

ly.



(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

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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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EU793593 ED-15m.135EU793324 E5-25m.204DQ918792 ENVP36162.00253

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2%

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CoccidiaPiroplasmida

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Ciliates

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AY665063 SCM38 C14AF133909 Parvilucifera

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Outgroups

100/100

100/100

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98/95

100/99

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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.



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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



(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 parasitic genera (Amoe-bophrya, Ichthyodinium, Duboscquella, Syndinium, Hema-todinium) and many environmental sequences, resolvedfor the first time the Syndiniales as a monophyletic lineageat the base of the more classical dinoflagellates. The basalposition is not supported by bootstrap analyses, but thesame topology was generated by Bayesian and ML phy-logenies. This topology agrees with historical descriptionsof the Duboscquodinida as a tribe (a taxonomic categorybetween a genus and a subfamily) by Cachon (1964), andSyndiniales as an Order by Loeblich (1976). However, athigher taxonomic levels, Syndiniales are still difficult toplace. Historically, Syndiniales have been retained withindinoflagellates because their short-lived dispersal stages,called dinospores, have a classic naked gymnoid morphol-ogy while also their nucleus has the chromatin condensedduring part of the life cycle, like the dinokaryon ofdinoflagellates. Syndiniales have trichocysts, a typicalfeature for Dinophyceae. However, Loeblich (1976) sepa-rated them from the rest of Dinophyceae (named also‘core’ dinoflagellate) and placed Syndiniales inside theclass Syndiniophyceae due to peculiarities of nuclear divi-sion in Syndiniales (Ris and Kubai, 1974; Soyer, 1974).Nevertheless, for all Dinophyceae and Syndiniales, chro-

60%

80%

100%

N = 113 N = 102 N = 3589

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40%

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Fig. 4. The relative contribution of dinoflagellate sequences withinenvironmental clone libraries. Left: relative contribution (inpercentage) from (i) low oxygenated (L.O.) ecosystems (sedimentsor deep waters), (ii) Hydrothermal (H) vent ecosystems (collecteddirectly in the hydrothermal chimney, on collectors or on sedimentsclose to the chimney), (iii) sediments (S) that can be oxic or anoxic,deep or more coastal, and (iv) plankton from the water column (W).For this last category, two additional conditions were compared(right side of Fig. 4): whether the water sample was collected fromthe euphotic or the aphotic layer. N = Total number ofenvironmental sequences considered.

Fig. 5. Proportion of dinoflagellate sequencesretrieved using different primer sets. Relativeproportion of environmental sequencesbelonging to Syndiniales (I, II and others =III–VI) and the rest of dinoflagellates (= CoreDinoflagellates) compared with othereukaryotes retrieved using different primersets during PCR amplification. For descriptionof primers, see Table S2. Only clone librariesconstructed from the euphotic zone of marinewaters have been considered. N = the totalnumber of sequences.

EukA/EukB'Euk328f/Euk329r EukA/EukB

Core Di fl ll

OtherSyndiniales

III

Dinoflagellates

N = 3478 N = 1133N = 1568

OtherEukaryotes



mosomes stay anchored to the persistant nuclear enve-lope during mitotic division, but their segregation is drivenby microtubules through the nuclear envelope in Dino-phyceae while centrioles participate in the formation of themitotic apparatus in Syndiniales (Hollande, 1974; Ris andKubai, 1974). Another Syndiniales peculiarity is their lowchromosome number, 4–10 for Syndiniales compared with>100 for some Dinophyceae members such as Gonyaulaxand Ceratium (see Loeblich, 1976). Taking all this together,it seems reasonable that these last two features (peculiari-ties in nuclear division and low chromosome number) arederived from an initial dinoflagellate model (Xiao-Ping andJing-Yan, 1986), being forced by the obligately parasiticlifestyle of Syndiniales. Controversially, some recent phy-logenies using 28S rRNA gene sequences, have placedthe Syndiniales Ichthyodinium chaberladi (Group I Clade3) within dinoflagellates (Gestal et al., 2006) and an envi-ronmental sequence belonging to Group I clade 1 close toPerkinsus (Massana et al., 2008). More sequences areneeded to obtain a LSU rRNA phylogeny robust enough toprovide accurate affiliations for the Syndiniales. Proteincoding genes, such as actin and b- and g-tubulins may alsobe a better alternative to resolve basal branches of thedinoflagellate lineage (Saldarriaga et al., 2003), includingseveral deep genera such as Perkinsus, Oxyrrhis andNoctiluca, which are almost impossible to place usingphylogenies based upon ribosomal genes.

Within Syndiniales, five main families have beendescribed: the Sphaeriparidae and the Coccidinidae (nosequences available to date for both), the Amoebophry-idae, the Duboscquellidae and the Syndinidae (Table S3).As Ichthyodinium (Syndinidae) and Duboscquella(Duboscquellidae) are clearly allied within Group I by 18S

rRNA gene analyses, while Hematodinium and Syndinium(Syndinidae) are members of Group IV and quite distinctfrom Group I, it is likely that the taxonomy of Syndinidaewill soon be modified. Nevertheless, it is possible thatAmoebophryidae will remain synonymous with Syndini-ales Group II. To date, seven different species of Amoe-bophrya have been described (see Table S3). However,this clearly does not reflect the genetic diversity observedin Group II; herein we find at least 44 distinct clades.Unfortunately, only one of the described species,A. ceratii, has been genetically characterized to date.Amoebophrya ceratii is a parasitoid of many (if not all)dinoflagellates, including heterotrophic species (for areview see Park et al., 2004) and was most recentlyreported as being a ‘complex species’ rather than a realspecies (see Coats et al., 1996). Currently, available SSUrRNA gene sequences from this complex species arefrom strains able to infect photosynthetic dinoflagellates,in accordance with the fact that this group is largelyretrieved from the euphotic zone in environmental studies.Nevertheless, A. ceratii was also described as being ableto parasitize other Amoebophrya species (e.g. it is fre-quently observed in A. leptodisci) or other Syndiniales(such as the genus Keppenodinium; see Cachon 1964).Consequently, despite recent additions of a substantialnumber of new sequences, all Amoebophrya SSU rRNAgene sequences fall within a monophyletic lineage, whichcontains at least 10 different clades. However, the taxo-nomic correspondence of each clade is still not clear.Within Hematodinium and Syndinium, SSU rRNA genesequences from different species are almost identical,and species can only be distinguished using more vari-able genetic regions (ITS, see Hudson and Adlard, 1996;

Fig. 6. Syndiniales clades retrieved usingdifferent primer sets, in the euphotic andaphotic layers of marine waters. The relativeproportion of clades belonging to Syndiniales(groups I and II) using different sets ofprimers is indicated. Only clone librariesconstructed from the euphotic zone of marinewaters have been included (as in Fig. 5).N = the total number of sequences.

88

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EukA/EukB +EukA/EukB’



Skovgaard et al., 2005). In the case of Amoebophrya,genetic variability is much more evident, even using con-served markers such as the SSU rRNA gene.

All described Syndiniales are parasitoids, and obli-gately kill their host. A potential exception is Sphaeriparacatenata, which in some cases only causes castration ofits host, but not death (Cachon and Cachon-Enjumet,1964). Described Syndiniales generally complete their lifecycle in less than 3 days, leading to the release of hun-dreds of free-living dispersive dinospores. Environmentalsequences obtained from genetic libraries likely resultfrom such dinospores. In general, Syndiniales producetwo different types of dinospores, the micro and mac-rospores that can both be very small (from 1 to 12 mm indiameter depending on the species). There is a generalagreement that dinospores can only survive a few daysafter their release (e.g. Coats and Park, 2002). Hence, thefact that they systematically dominate clone libraries fromcoastal and oceanic waters, whatever the season consid-ered, suggests that their production is fairly constant.Alternatively, it could mean that other more resistantplanktonic stages are produced. To our knowledge, cystproduction has never been reported within Syndiniales,with the notable exception of cyst-like cells described innatural populations of Duboscquella cachoni infecting thetintinnid Eutintinnus pectinis (Coats, 1988). In this particu-lar case, cyst-like formation was completely independentof dinospore production, and the cysts formed were non-motile. Within dinoflagellates, permanent cysts are pro-duced by the fusion of two gametes. Sexuality, issuedfrom different individuals (anisogamous and heterothal-lic), has only been observed once in Coccidinidae, withthe production of a planozygote-like body (Chatton andBiecheler, 1936). Nevertheless, cyst production was notobserved.

The recent reports of Amoebophrya spp. in freshwatersystems (Lefèvre et al., 2008) are based on placement oftwo environmental sequences incorrectly labelled uncul-tured Amoebophrya Clone E and F (Di Giuseppe and Dini,2004). These sequences are in fact closely related to thegenus Cryptocaryon (Ciliates, Protosmatea). Thus, todate, environmental sequences of Syndiniales have beenretrieved solely from marine ecosystems. This supportsthe ‘marine-ness’ of this group, together with the fact thatall described Syndiniales species are marine. In contrast,other unicellular alveolate parasites, such as Perkinsea,Colpodellids or even other parasitic dinoflagellates, haveboth marine and continental species. This is interestinggiven that almost all known Syndiniales hosts havespecies well adapted to continental ecosystems (withinciliates, dinoflagellates, cercozoa and crabs).

Although the distribution of Syndiniales is relativelyhomogeneous at the group level (note their similar con-tribution to both euphotic and aphotic zones), communi-

ties are quite different at the clade level. Some cladesare uniformly distributed vertically through the watercolumn (e.g. Group I clade 1, Group II clade 6), whereasothers (e.g. Group I clades 2 and 3, Group II clade 7)are more prevalent in aphotic layers. We speculate thatthese ‘deeper’ clades are able to parasitize well-knowndeep planktonic organisms such as Phaeodarea (Cerco-zoa), Acantharea (Radiolaria) and Polycystinea (Radi-olaria). Recent analyses of environmental sequencesbelonging to these groups highlight the increasing con-tribution of radiolarian environmental sequences withdepth (Not et al., 2007), in particular sequences belong-ing to Spumellarida (Polycystinea). Interestingly, radi-olarians are also exclusively marine, and well known tobe oceanic, or blue-water, organisms. Hence, both Syn-diniales and radiolarians may have shared a commonevolutionary history inhabiting similar ecosystems asdiscussed by Not and colleagues (2007). Sequencesretrieved from single radiolarian cells (Dolven et al.,2007), likely infected by Syndiniales, belong to bothGroup I and Group II (Fig. 3). Nevertheless, it is difficultto assign an identity to these sequences as Syndinialesable to infect radiolarians are described within three ofthe five described families (the Amoebophryidae, theDuboscquellidae and the Syndinidae). Much more infor-mation on host specificity is provided by data from thegenus Amoebophrya. Phylogenies of Amoebophryaclades (Fig. 3) indicate that host–parasite co-evolution isnot a central feature driving the observed diversity. Para-sites of the same host genus, but of different species,belong to completely separate clades. In fact, within the‘A. ceratii complex’, host specificity as assessed in cul-tures, is highly strain dependent, with some beingextremely specific (Coats and Park, 2002), while othershave broader host ranges (Kim, 2006). This fact couldmask clear co-evolutionary patterns. The same conclu-sion can be drawn when the entire Syndiniales order isconsidered. For example, all described Syndiniales thatoperate as parasitoids of metazoans are not closelyrelated. The genus Ichthyodinium, a parasite of fisheggs, is more closely related to parasites that infect cili-ates (i.e. Duboscquella) belonging to Group I than toGroup IV, which contains Syndinium and Hematodinium(two genera which infect metazoans). Thus, evenbetween groups, Syndiniales and their hosts do notseem to co-evolve. Consequently, we expect that para-sites within Syndiniales are generalists or opportunistswith respect to host range. This is also congruent withthe detection of clades widely distributed in all exploredmarine ecosystems. Nevertheless, the presence of morespecialized clades or subclades, related with depth orspecifically retrieved from extreme environments, suchas Group I clades 3 and 4, and Group II clades 9 and15, confirms that specific interactions may exist.



Conclusions

Based on the integration of a large number of environ-mental sequences, we propose a new phylogeny forSyndiniales. This order should be considered a mono-phyletic sister group of the rest of Dinophyceae basedupon 18S rRNA gene phylogenies. The Syndiniales arecomposed of five main groups, which include environ-mental sequences formerly referred to as novel marinealveolate groups I and II. All Syndiniales speciesdescribed to date are parasitic marine organisms. Theexclusively marine lifestyle of this order as a whole,including sequences from uncultured organisms, is con-firmed by the fact that their 18S rRNA gene sequenceshave not been retrieved from terrestrial or freshwaterPCR surveys, but rather come solely from marine eco-systems. Syndiniales appear to colonize all the marinehabitats investigated thus far, from oceanic surfacewaters to sediments. Some clades are likely adapted tofairly specific habitats (and/or hosts that reside only inspecific habitats). However, these parasites and theirhosts do not appear to share co-evolutionary trends.This suggests that at least some Syndiniales are highlyopportunistic, with a capacity to infect very differentmarine hosts, from many trophic levels within marinefood webs. Even so, we cannot completely exclude thatsome members of the Syndiniales may have retainedspecific ancestral traits still found among the differentalveolate lineages, e.g. phagotrophy and/or the inherit-ance of photosynthetic genes. In fact, proteins charac-teristic of secondary endosymbiont plastids haverecently been detected from both the non-photosyntheticgenera Oxyrrhis and Perkinsus (Matsuzaki et al., 2008;Slamovits and Keeling, 2008). Hence, the ecologicalsuccess of the Syndiniales may yet have a more sur-prising and complex explanation. That said, it is certainlystill the case that the overall role of parasitism in marinesystems requires further investigation and incorporationinto food web and carbon flow models.

Experimental procedures

Environmental sampling and library processing

Materials and methods for each of the libraries publishedherein varied by site. Thus, details are provided separately foreach of the regions sampled. See also Table 1 for additionaldetails on the clone libraries constructed.

English Channel, France (RA). Ten litres of surface seawater was sampled weekly at the ASTAN site (SOMLITstation, English Channel, France, see http://www.domino.u-bordeaux.fr/somlit_national/) from the end of June throughto the middle of September 2004. Water samples were size-fractionated using a peristaltic pump (with the flow rate fixed

at 100 ml min-1) through sequential 47-mm-diameter poly-carbonate filters (12 and 3 mm, Osmonic Poretics Products)in filter housings connected in series, terminated by a 0.2-mm-pore-size Sterivex unit (Millipore). Following sample col-lection, filters were then submerged in DNA lysis buffer(0.75 M sucrose, 40 mM EDTA, 50 mM Tris-HCl, pH 8),immediately frozen in liquid nitrogen and stored at -80°C.DNA was extracted using a 3% (w/v) CTAB (Cetyltrimethy-lammonium bromide) extraction procedure (Doyle andDoyle, 1987). PCR conditions for amplifying the 18S rRNAgene were as described in Moon-van der Staay and col-leagues (2001). PCR products were cloned using theTOPO-TA cloning kit (Invitrogen). Clones (96 randomlyselected) were partially sequenced using the primerEuk528f for RA1 and the primer ALV01 (for primer refer-ences see Table S2) for both RA2 and RA3 on an ABIPrism 3100 (Applied Biosystems).

Barcelona Harbour, Mediterranean Sea, Spain (BAFRACT).Fifty litres of sea water was collected on 28 February 2001from Barcelona harbour (Spain) and pre-filtered through a50 mm mesh. On return to the laboratory (less than one hour),the sample was then fractionated through seven differentpore-sizes (20, 14, 10, 5, 3, 1 and 0.6 mm), using similar filterhousings connected in series as described above. Samplestorage and DNA extraction were as described above.Genetic diversity in each size fraction was determined byDGGE (not shown), as described by Díez and colleagues(2001b). Clone libraries were performed for size fractionsdisplaying the most ‘contrasting’ DGGE band patterns. PCRconditions were as in Díez and colleagues (2001a). Cloningwas as described above. Restriction fragment length poly-morphism (RFLP) analysis was used to detect polymor-phisms among the retrieved clones. Briefly, PCR productswere digested with 1 U ml-1 of restriction enzyme HaeIII(Gibco BRL) for 6–12 h at 37°C. The digested products wereseparated by electrophoresis at 80 V for 2–3 h in a 2.5%low-melting-point agarose gel. One clone per unique RFLPpattern was partially sequenced using the internal primerEuk528f. Sequencing was performed by Qiagen GenomicsSequencing Services (Germany).

Blanes Bay. Surface water was collected in Blanes Bay(Catalan coast, north-western Mediterranean Sea). Seawater was rapidly transported to the laboratory (less than2 h), and gently filtered by gravity through 2- and 5-mm-pore-size polycarbonate filters. One litre of each size fraction wasincubated both in the light [around 100 (mmol quanta) m-2 s-1]and in the dark (four bottles in total). All the incubations werecarried out at 20°C, close to in situ temperature (18°C).Samples (100 ml) were filtered through 0.2-mm-pore-sizeDurapore filters for DNA analysis over a course of 5 days.DNA was then extracted following the protocol described inDíez and colleagues (2001b). Abundance and genetic diver-sity of eukaryotes were monitored by epifluorescence micros-copy and DGGE (using the same protocol as describedabove, data not show). 18S rRNA genes were amplified fromthe initial day and after 4 days of incubation both in the lightand dark. PCR products were cloned using the TOPO-TAcloning kit. Clones were sequenced by the Qiagen GenomicsSequencing Services (Germany).



http://www.domino.u-bordeaux.fr/somlit_national/

http://www.domino.u-bordeaux.fr/somlit_national/

Atlantic Meridional Transect (AMT15). Both the samplingstrategy, DNA extraction procedure and the PCR amplifica-tion protocol used have been previously published (Zwirgl-maier et al., 2007). Cloning was as described above.Restriction fragment length polymorphism analysis was usedto screen for distinct inserts by digesting with HaeIII in a totalvolume of 20 ml using 5 U of HaeIII at 37°C for 2 h. Fragmentswere resolved by electrophoresis in a 2.5% agarose gel at60 V for 3 h. At least two representatives of each RFLPpattern were sequenced in entirety following purification ofthe PCR product using the QIAquick PCR Purification Kit(Qiagen), on an ABI 3130xl Genetic Analyser (AppliedBiosystems).

Florida Straits and Sargasso Sea. Sea water was collectedfrom both the surface and deep chlorophyll maximum onthree transect cruises across the Straits of Florida in 2005 asdescribed in Cuvelier and colleagues (2008). Briefly, 1 lsamples were first gravity filtered through 47 mm diameter,2-mm-pore-size filters (GE Osmonics, Minnetonka, Minne-sota, USA) and then onto a 0.2-mm-pore-size Supor filter (PallGelman, Ann Arbor, Michigan, USA) using approximately 5psi vacuum. Filters were placed in cryovials and frozen inliquid nitrogen. DNA was extracted using the DNeasy kitaccording to the manufacturer’s recommendations (Qiagen,Germantown, MD, USA). PCR conditions have been pub-lished elsewhere (Cuvelier et al., 2008). The PCR productwas ligated into vector pCR2.1 (Invitrogen, Carlsbad, CA,USA) and transformed. Clones were then sequenced on anABI 3730xl sequencer (Applied Biosystems).

Samples were also collected on a cruise from Woods Hole,MA USA to the BATS. Two regions were sampled, BATS anda site north-west of BATS. Samples were collected and pro-cessed as for the Florida Straits clone libraries (as reported inCuvelier et al., 2008).

Development of sequence database

The 18S rRNA gene database was built using all publishedenvironmental sequences deposited in GenBank up toDecember 2007. We also integrated the data sets generatedherein and a number of other libraries which are beingpublished independently, but without specific attention tothe analysis of Alveolata sequences. The complete list ofsequences used in this study and their annotation is availableupon request (Excel file).

Sequence group assignments

The software package, KeyDNAtools, was used to (i) detectsequences belonging to dinoflagellates within a large data-base including more than 42000 eukaryotic SSU rRNA genesequences; (ii) remove potential chimeras; and (iii) automati-cally assign sequences to a specific clade. Details of thesoftware development will be described elsewhere. Briefly,environmental sequences were annotated using small oligo-nucleotides ‘probes’ (15 bp length), named ‘keys’, which weregenerated in silico. The specificity of each key, from the Orderto the clade level (where possible) was automaticallydeduced from a reference database containing as many

eukaryotic lineages as possible as outgroup and at leasttwo sequences from each taxa created within marinedinoflagellates. In this study, more than 110000 keys weregenerated, each being specific at least to the level of Order.Annotations of 18S rRNA genes were deduced from thespecificity of each key when they matched a sequence. As anexample, sequence EF172938 was annotated by 175 differ-ent keys, providing converging annotation at the order leveland covering positions 1–1289 along the sequences(Table 3). Thus, this sequence was considered as a Syndini-ales, Dino-Group II, and belonging to Dino-Group II-Clade 7(this last annotation is provided by 47 keys). The 18S rRNAgene sequences belonging to the majority of Dinophyceaewere not variable enough to allow a clear separation ofsequences even at the Order level. Thus, keys specific tomost of the Dinophyceae are labelled as Dinophyceae at theClass and the Order levels (see annotation of sequenceEU087283). Wherever possible, more precise information isgiven (e.g. at the Family, Genus, Species and Clade levels;see annotation of sequence EU087283). New groups/cladeswere often detected by conflicting annotation (caused byincorrect deduction of specificity by the keys) in a particulartaxonomic field. Chimeras were detected when annotationsconflicted for separate parts of an individual sequence. As anexample (Table 3), sequence AJ965100 was detected as achimera at the class level, a false gene sequence combiningpart of a Ciliophora (annotation provided by 25 keys coveringpositions 44–155) gene sequence with part of a Dinophyceae(31 keys covering positions 213–474) gene sequence. Auto-matic annotation of eukaryotic 18S rRNA environmental genesequences using the final generated keys can be freelytested on the following web site: http://KeyDNAtools.com.

Phylogenetic analyses

Sequences were aligned using the slow and iterative refine-ment method FFT-NS-i with Mafft 5.8 software (Katoh et al.,2007). Using the BioEdit sequence editor (Hall, 1999) forvisualization of the alignment, we deduced the secondarystructures by hand using previously published studies (seefor example Lange et al., 1996). For complete sequencealignments only, poorly aligned and highly variable regions ofthe alignments were automatically removed using Gblocks(Castresana, 2000) with the following parameters: allowinggaps in half positions and the minimum length of a block = 5.Different nested models of DNA substitution and associatedparameters were tested using Modeltest v.3.06 (Posada andCrandall, 1998). Settings given by Modeltest were used toperform the NJ and the ML analyses. The NJ, ML and MPanalyses were performed using PAUP 4.0b10 (Swofford,2002). A heuristic search procedure using the tree bisection/reconnection branch swapping algorithm (setting as in MP)was performed to find the optimal ML tree topology (with70 000 re-arrangements). Bootstrap values for NJ and MPwere estimated from 1000 replicates. For MP, the number ofre-arrangements was limited to 5000 for each bootstrapreplicate. Starting trees were obtained by randomized step-wise addition (number of replicates = 20). Additionally, weused Bayesian reconstruction for the analysis of completesequences with MrBayes, v.3.0b4 (Huelsenbeck and Ron-quist, 2001). The GTR model of substitution was used, taking



http://KeyDNAtools.com

into account a gamma-shaped distribution of the rates ofsubstitution among sites. The chains were run for 1000000generations. Trees were sampled every 100 generations. Thefirst 5000 sampled trees, corresponding to the initial phasebefore the chains became stationary, were discarded(burn in).

Acknowledgements

We thank R. Cowen, B.J. Binder and E. Goetze for cruisespace as well as the captain and crews of the R/V Oceanus,R/V Walton Smith; R/V Atalante, R/V Melville, RRS Discov-ery; M.L. Cuvelier, A. Engman, C. Guigand, J.A. Hilton and F.Not for cruise assistance; J. Heidelberg, A. Ortiz, M. Peren-nou, C. Cormier-Caillault, J. Winkler and A. Gobet for helpwith clone libraries and sequencing; and P. Zimmermann forhelp with sequence annotation. This work was supported bythe GIS génomique marine and the ANR AQUAPARADOXprojects (to L.G.), the Gordon and Betty Moore Foundationand National Science Foundation grant OCE-0836721 (toA.Z.W.) and by the NERC (to A.R.K., D.J.S.).

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Supporting information

Additional Supporting Information may be found in the onlineversion of this article:Fig. S1. Mean sequence identities within the different cladesbelonging to Syndiniales groups I and II.Table S1. Details of the sequences used for the phyloge-netic analyses described in Fig. 1.Table S2. Primer sequences used or cited in this study.Table S3. Taxonomy of Syndiniales (= Syndinida) and theirhosts modified from Loeblich (1976).

Please note: Blackwell Publishing are not responsible for thecontent or functionality of any supporting materials suppliedby the authors. Any queries (other than missing material)should be directed to the corresponding author for the article.

