ARTICLE IN PRESS

http://www.elsevier.de/protisPublished online date 20 January 2006

1

Correspondinfax +1 617 373e-mail s.epste

& 2005 Elsevdoi:10.1016/j

157, 31—43, January 2006

Protist, Vol.

ORIGINAL PAPER

A Multiple PCR-primer Approach to Access theMicroeukaryotic Diversity in EnvironmentalSamples

Thorsten Stoecka, Brett Haywardb, Gordon T. Taylorc, Ramon Varelad, andSlava S. Epsteinb,e,1

aDepartment of Ecology, Technical University Kaiserslautern, Kaiserslautern, GermanybMarine Science Center, Northeastern University, Nahant, MA 01908, USAcMarine Sciences Research Center, Stony Brook University, Stony Brook, NY 11794, USAdEstacion de Investigaciones Marinas de Margarita, Fundacion la Salle de Ciencias Naturales, NuevaEsparta, Venezuela

eBiology Department, Northeastern University, Boston, MA 02115, USA

Submitted June 28, 2005; Accepted October 30, 2005Monitoring Editor: Michael Melkonian

The Cariaco Basin off the Venezuelan coast in the Caribbean Sea is the world’s largest truly marinebody of anoxic water. The first rRNA survey of microbial eukaryotes in this environment revealed anumber of novel lineages, but sampled only a fraction of the entire diversity. The goal of this study wasto significantly improve recovery of protistan rRNA from the Basin. This was achieved by a systematicapplication of multiple PCR primer sets and substantially larger sequencing efforts. We focused on themost diverse habitat in the basin, anoxic waters E100 m below the oxic—anoxic interface, anddetected novel lineages that escaped the single PCR primer approach. All clones obtained provedunique. A 99% sequence similarity cut-off value combined these clones into operational taxonomicunits (OTUs), over 75% of which proved novel. Some of these OTUs form deep branches withinestablished protistan groups. Others signify discovery of novel protistan lineages that appearunrelated to any known microeukaryote. Surprisingly, even this large-scale multi-primer rRNAapproach still missed a substantial part of the samples’ rRNA diversity. The overlap between thespecies lists obtained with different primers is low, with only 4% of OTUs shared by all three libraries,and the number of species detected only once is large (55%). This strongly indicates that, at least inanoxic environments, protistan diversity may be much larger than is commonly thought. A singlesample appears to contain thousands of largely novel protistan species. Multiple PCR primercombinations may be needed to capture these species.& 2005 Elsevier GmbH. All rights reserved.

Introduction

The extent of microeukaryotic diversity on ourplanet is unknown (Patterson 1999), and so is the

g author;3724in@neu.edu (S.S. Epstein)

ier GmbH. All rights reserved..protis.2005.10.004

true extent of its ecological and evolutionaryimportance (Countway et al. 2005; Dawson andPace 2002). The prevailing view is that it farexceeds the diversity known from classical works,and that molecular techniques represent ourbest tool for detecting the ‘‘missing’’ part of

ARTICLE IN PRESS

32 T. Stoeck et al.

microeukaryotic diversity (Baldauf 2003). Eventhough light microscopy approaches for docu-menting the diversity and distribution of protistshave contributed significantly to our currentknowledge, these techniques are mainly restrictedto fairly large (generally 420 mm) organisms with adistinctive morphology. Additionally, the traditionalapproaches are hampered by the fact that mostprotists cannot be presently cultivated. Thisuncultivated majority may have escaped micro-scopic analyses (Dawson and Pace 2002; Moreiraand Lopez-Garcıa 2002). It was not until recentlythat first applications of an rRNA approach tomicroeukaryotic discovery provided a strongindication that an enormous reservoir of protistsmay indeed remain to be discovered (Amaral-Zettler et al. 2002; Countway et al. 2005; Dawsonand Pace 2002; Diez et al. 2001; Edgcomb et al.2002; Lopez-Garcıa et al. 2001, 2003; Massanaet al. 2004a,b; Moon-van der Staay et al. 2001;Romari and Vaulot 2004; Stoeck and Epstein2003; Stoeck et al. 2003a). All these studiesreported a high number of novel phylotypes thatcould not be linked to any morphologicallydescribed taxa. However, the extent of molecularstudies of microeukaryotic diversity is still dwarfedby that in prokaryotic microbiology, and the 18SrRNA of microbial eukaryotes has been sampledonly in a handful of natural environments. Inparallel, the studies of 16S rRNA gene diversityabundantly showed the shortcomings of thisapproach, including PCR biases (Acinas et al.2004; Caron et al. 2004). It is very reasonable toassume that a single PCR primer set does notamplify equally well the 18S rRNA gene from allprotistan taxa (Caron et al. 2004), but so far onlyone study has considered the ramifications ofsuch a bias (Dawson and Pace 2002), and nonesystematically applied multiple primer sets. There-fore, even in those few environments that havebeen investigated with respect to their 18S rRNAgene diversity, the sequence recovery was likely tobe biased, and the degree of undersampling maybe even higher than suggested by the rarefactioncurves (Chao et al. 2005; Colwell et al. 2004; Hecket al. 1975). In fact, it may be so high that theresults of the earlier studies, including ours, mayor may not be viewed as informative inventories,and may or may not be meaningfully compared.

Recently, we applied the rRNA approach tostudy the diversity of microbial eukaryotes in theanoxic waters of the Cariaco Basin off the coast ofVenezuela in the Caribbean Sea with a singleprimer set (Stoeck et al. 2003a). The findingsincluded new clades unrelated to any known

eukaryotes, as well as deep branches withinestablished protistan groups, but this study didnot escape the limitations identified above. Thedominance of lineages that occurred only once inour 18S rRNA environmental clone libraries, andthe biases associated with the single primer setused, made it extremely likely that we capturedonly an insignificant portion of the entire protistandiversity in the Cariaco Basin (Stoeck et al.2003a). To address this shortcoming, we returnedto the Cariaco Basin and re-sampled its richesthabitat (anoxic waters below the oxic/anoxicinterface). We doubled the amount of 18S rRNAsequence information by employing a multi-primerPCR approach together with an amplificationartifact-reducing protocol to minimize the PCRbiases (Acinas et al. 2004; Caron et al. 2004). Asexpected, we discovered a number of novelprotistan lineages. However, unexpectedly, wedid not come any closer to a more completeinventory. The multi-primer approach tripled thenumber of species detected in the environment,but it also showed that the target community wassignificantly more diverse than that whichemerged from the traditional single primer pair-based data sets. It seems that as the detectionapproaches improve, the estimates of the overalldiversity grow, with the diversity of protists in atleast anoxic environments appearing to be trulyspectacular.

Results and Discussion

The three clone libraries, constructed using theprimer sets E528F/Univ1391, E528F/Univ1492,and E528F/Univ1517, contained 241, 119, and137 clones respectively. The clustering, based ona 99% sequence identity criterion, produced 24,31, and 36 OTUs unique to the respective library,with most of them (35—65%) detected only once.Some of these OTUs were shared between theindividual libraries, decreasing the study-widenumber of unique OTUs to 72. Collectively, thisalmost triples the number of protistan OTUsreported previously from the same depth in theCariaco Basin (Stoeck et al. 2003a). Takentogether, with over 500 clones obtained in theabove study, the total of more than 1000 18SrRNA gene clones represents one of the largestprotistan clone libraries obtained to date from asingle environment.

Do the multiple-primer PCR and more traditionalsingle primer pair-based PCR approaches qualita-tively recover different uncultured protists or does

ARTICLE IN PRESS

33Multiple PCR-primer Approach

the use of different PCR primers simply reveal‘‘more of the same’’? Direct comparison is onlypossible when the respective clone librariescontain all recoverable rRNA species, but thiscondition is not met in this or any other studies todate. However, the two rates of recovery can becompared indirectly, e.g., by considering theproportion of species detected only once by eachprimer set (‘‘singletons’’). The logic is that as thesize of the clone library grows, so does thenumber of species captured and rare speciesstart getting recaptured, consequently decreasingthe proportion of singletons. It follows that if PCRprimers exhibited little selection, then the threelibraries constructed here would represent differ-ent subsamples from essentially a single pool ofPCR products. If so, the combined data set shouldcontain fewer singletons than the individual(smaller) libraries. However, this is not what weobserved. Figure 1 illustrates the OTU frequenciesin three individual clone libraries and in thecombined data set. The proportion of singletonsin the former vs. the latter is essentially the same(0.5370.16 and 0.55 respectively). This meansthat the three libraries are likely to be subsamplesfrom somewhat different pools of PCR products,and the likeliest explanation for this is a PCR bias.This in turn means that the clone libraries obtainedhere, and the fourth obtained earlier (Stoeck et al.2003a), recovered different parts of the targetcommunity. It follows that each primer set, andthus each library, bring 18S rRNA gene sequencesnot recoverable with other primer pairs. Inciden-tally, this tentative conclusion makes one specificprediction: the lists of species recovered by the

Figure 1. Operational taxonomic units (OTUs)shared among the three 18S rRNA gene librariesconstructed with different primer sets (see Methods).The area of the ovals area is proportional to the sizeof the respective clone library and the overlap area isproportional to the number of OTUs shared by therelevant libraries.

individual libraries should overlap little, if at all.Indeed, only three OTUs were shared between allthree clone libraries (Fig. 1).

Remarkably, even the single primer set-basedapproach revealed so much of uncultured proti-stan diversity that the existing inventories werecalled ‘‘the tip of the iceberg’’ (Moreira and Lopez-Garcıa 2002). The level of diversity emerging fromthe multiple-primer approach is even larger, andpoints to a truly spectacular richness of protists. Itnow seems very likely that all 18S rRNA surveysconducted to date, most of them based on asingle primer set amplification, have missed evenmore substantial portions of protistan biodiversityof the respective environments than are deducedfrom rarefaction curves.

Not surprisingly, all rRNA gene sequencesobtained here are unique and do not match anysequences in the public databases, includingthose detected previously in the same environ-ment (Stoeck et al. 2003a). Grouped on the basisof the 99% cut-off value, these sequences form 72OTUs, of which 61 proved novel, i.e. they do notinclude any previously known rRNA sequence.Below, we describe the OTUs detected anddiscuss their significance.

Generally, the Cariaco OTUs can be divided intotwo subgroups. The first is represented by OTUswhose closest relatives are named organismsfrom established taxa. The most divergent OTUin this group is formed by the clone T37E6 (96.0%similarity to Strombidium sp. SNB99-2 [Strueder-Kypke and Lynn 2003]) (Fig. 2). The least divergentOTUs in this group are over 99% similar to oneor another named organism, and thus are merevariants within previously described OTUs(or species). An example of such a sequence isour clone P13F2 that is 99.35% similar toPentapharsodinium tyrrhenicum (Saunders et al.1997) (Fig. 2).

Overall, the OTUs in this subset are likely to benew strains within established species, or newspecies within the described genera and families.Their characteristics are likely to be predictable byextension of what is known about their describedclose relatives. The number of such OTUs is ratherlow (o25%). Examples include five diatom OTUsand one unclassified flagellate (Fig. 3), five fungalOTUs (Fig. 4), three ciliate OTUs, one dinoflagel-late (Fig. 2), one bicosoecid, and one novel OTUwithin the Cercozoa (Fig. 5).

The second subgroup of OTUs, while falling intoestablished clades, has its closest relatives fromamong environmental sequences rather thannamed organisms. This group encompasses the

ARTICLE IN PRESS

0.005 substitutions/site

Engelmanniella mobilisp13F1

E28 (Cariaco)T37E6

Strombidium sp. SNB99T37F5 (p15D10, T41A8)

CCW111 (Sippewisset)Eutintinnus pectinis

T41D1CCI60 (Sippewisset)

Aspidisca steiniT41H4T37D5

p15G08p15E10

T41D10 (p15B06)H1b (Cariaco)

CCA31 (Sippewisset)Nyctotherus ovalis

Metopus palaeformisStentor coeruleus

Blepharisma americanumGeleia simplex 2000 Nahant

Parduczia orbisToxoplasma gondii

Sarcocystis neuronaPentapharsodinium tyrrhenicumAmphidinium longum

p15C03p13F2

T41F8T37F4 (p14B3)C1_E005 (Guaymas)

E131 (Cariaco)OLI11011 (equatorial Pacific)

C2_E007 (Guaymas)UEPACDp5 (coastal Pacific)

T60A6UEPACQp5 (coastal Pacific)

D75 (Cariaco)p15F05

T45F12DH147-EKD18 (Antarctic Deep-Sea)

AT4-47 (Mid-Atlantic Ridge)D188 (Cariaco)

p15H09C1_E020 (Guaymas)

AT4-42 (Mid-Atlantic Ridge)T53D7T53H5 (T45A2, T60A4)C2_E017 (Guaymas)

DH144-EKD3 (Antarctic Deep-Sea)OLI11511

UEPAC36p4 (coastal Pacific)CS_E023 (Guaymas)

T41A6H67 (Cariaco)T53B4

T41G1UEPACLp4 (coastal Pacific)

p15D01OLI11023 (equatorial Pacific)

Amoebophrya sp.OLI11012 (equatorial Pacific)

97

9956

70

82

10098

66 96100

100

100

10088

100

8755100

67

64

87

55

56

51

6160

96

85

94

75

83

69

6198

62

86

65

100

85

9295

67

100

34 T. Stoeck et al.

ARTICLE IN PRESS

35Multiple PCR-primer Approach

majority (75%) of the newly detected OTUs. Mostof them belong to the clades known exclusively by18S environmental signatures, with some repre-senting deep branches within, or sister groups of,the established clades. Selected examples in-clude the following:

AlveolatesA group of eight OTUs falls within the ubiquitous

uncultured marine alveolates Group I (Fig. 2). Thisgroup was first described by Lopez-Garcıa et al.(2001) and has since been detected in, forexample, the Guaymas Basin (Edgcomb et al.2002), the Mid-Atlantic Ridge (Lopez-Garcıa et al.2003), the coastal and equatorial Pacific (Moon-van der Staay et al. 2001; Worden, unpubl.sequence data from GenBank), as well as in ourearlier study of the Cariaco Basin (Stoeck et al.2003a). This phylogenetically diverse and cosmo-politan group of organisms contains no namedspecies and represents an important target forcultivation efforts.

We detected a group of five OTUs that fall intothe candidate ciliate class CAR-H, a cladeobserved first in our previous rRNA survey of theCariaco area (Stoeck et al. 2003a) (Fig. 2). Thenovelty of the CAR-H clade and degree ofseparation from known ciliate classes suggest adivergence at the class level. This is remarkablebecause the Phylum Ciliophora is among the beststudied Protozoa, and claims have been madethat most species of ciliates have been largelydiscovered by now (Finlay and Fenchel 1999; forcriticism, see Foissner 1999; Richards and Bass2005). The proposed clade, if confirmed bycultivation studies and p-taxonomy, would re-present the first new ciliate class-level groupdiscovered in decades. An additional interestingfeature of this clade is that its occurrence seemsto be limited to anoxic environments.

We also detected a pair of OTUs clusteringwithin the marine alveolate Group II (Lopez-Garcıaet al. 2001). This group is well supported by 100%

Figure 2. Minimum evolution phylogenetic tree of eukalveolate clones detected in the Cariaco basin. The treeby using a TIM+I+G DNA substitution model with the va0.7613, the proportion of invariable sites at 0.2213 and bmodel as suggested by Modeltest (see Methods), basebootstrap values over 50% from an analysis of 1000 boThree acantharean sequences were chosen as outgroupSequences marked in red refer to library 1 (primer se(primer set Euk528F/U1492R), and sequences in bluesequences in brackets indicate clones from different li99% to the sequence included in the phylogeny (repre

bootstrap (Fig. 2), but its relationship to otheralveolates, including dinoflagellates, is unclear.The clade is ubiquitously distributed and may bequite important ecologically, but little can be saidabout the biology of this group as it is composedalmost exclusively of environmental sequences.The only cultured representative is Amoebophryasp. Phylogenetic analyses placed this organism atthe base of the dinoflagellates (Gunderson et al.1999). However, based on morphological, struc-tural, and life cycle characteristics, Grasse (1952)classifies Amoebophrya as a flagellate incertaesedis. The author does not assign Amoebophrya aprecise systematic position and cannot evenaffirm that Amoebophrya is a flagellate at all. Thisis supported by the phylogenetic studies ofMoreira and Lopez-Garcıa (2002) showing thatAmoebophrya might be the first described mem-ber of a new group of alveolates. However, moresequence data of the order Syndiniales andcultured representatives of the uncultured marinealveolate Group II will be required to clarify thisissue. The phylogenetic position of clone T53B4,closely related to another clone (H67) previouslyfound in Cariaco, is uncertain (bootstrap 51%).Using a slightly modified alignment and different aand I, these sequences appeared branchingclearly within the uncultured marine alveolateGroup II.

StramenopilesWe identified 14 diatom OTUs, out of which

eight contain environmental sequences detectedpreviously (Stoeck and Epstein 2003; Stoeck et al.2003a) and six are novel. Such abundance ofdiatom 18S rRNA sequences in an aphotic andanoxic environment is remarkable. While it ispossible that all these sequences belong tosinking and dying (or dead) cells, we cannotexclude the possibility that some of them in factrepresent genuinely anaerobic heterotrophic dia-toms. This interpretation is consistent with a highabundance of live diatoms in marine anoxic

aryotic small-subunit rRNA showing the position ofwas constructed under maximum-likelihood criteria

riable-site gamma distribution shape parameter (a) atase frequencies and a rate matrix for the substitutiond on 727 unambiguously aligned positions. Distanceotstrap replicates are given at the respective nodes.. Sequences retrieved in this study are color-coded.

t Euk 528F/U1517), sequences in green to library 2to library 3 (primer set Euk528F/U1391R). Colored

braries that shared a sequence similarity of at leastsenting an operational taxonomic unit, OTU).

ARTICLE IN PRESS

E233 (Cariaco)T60H4 (T45D10)T60A7 (p15D9)

CCA67 (Sippewisset)BOLA250 (Bolinas tidal flat)

Nanofrustulum shiloi

Thalassionema nitzschioidesp14G3

E171 (Cariaco)T37A12 (p14C2, p15B03)

H65 (Cariaco)

0.01 substitutions/site

p15E5T53H1

E108 (Cariaco)Thalassiosira rotulaE29 (Cariaco)

T49E9C2 E024 (Guaymas)p15F11

B04D2 (Cariaco)T60D2 (p13E1)

p15C05p15E06

Skeletonema pseudocostatumD88 (Cariaco)

T53B7

Amphora montanaCCW50 (Sippewisset)

T45C5 (T60C6)

Phaeodactylum tricornutum

T45A8 (p14D5)

Pseudonitzschia multiseriesE77 (Cariaco)

T60G2p13B5

C3_E024 (Guaymas)D67 (Cariaco)

p15B01Chaetoceros gracilis

Nannochloropsis limneticaEustigmatos magna

Ochromonas danicaParaphysomonas foraminifera

Costaria costataZonaria diesingiana

Hyphochytrium catenoidesRhizidiomyces apophysatus

T41F1 (p15F01)D179 (Cariaco)C1_E009 (Guaymas)

BL010625 31 (Mediterranean)Aplanochytrium stocchinoi

Thraustochytrium multirudimentaleANT12-11 (Antarctica)

BAQA232 (coastal Pacific)T53A1

D85 (Cariaco)UEPACRp5 (coastal Pacific)HE000427.21 (North Sea)NA11-5 (North Atlantic)

p14A1T41B10

T45C1Lagenidium giganteum

Apodachlya brachynemap13B12

p14D7D184 (Cariaco)

Cafeteria roenbergensisSiluania monomastiga

T41H2D101 (Cariaco)

Blastocystis hominisp15SBG2

Ancyromonas sigmoides

36 T. Stoeck et al.

ARTICLE IN PRESS

37Multiple PCR-primer Approach

sediments, and the knowledge of obligate andfacultative heterotrophy for at least some diatoms(Admiraal and Peletier 1979; Hellebust and Lewin1977; Li and Volcani 1987).

Four novel Cariaco clones (T53A1, p14A1,T41B10, T45C1), which branch among the hetero-trophic stramenopiles, are not related to namedspecies (Fig. 3). In our analysis, these sequencesare affiliated with other environmental sequencesfrom different oceanic regions, such as theuncultured marine stramenopile cluster MAST-3(Massana et al. 2004b). This group consistentlyappears as a separate clade in several publishedstudies, though sufficient statistical support forthis clade is lacking (Fig. 3; Dawson and Pace2002; Diez et al. 2001; Massana et al. 2004b;Stoeck et al. 2003a).

Furthermore, we discovered two labyrinthulidclones closely related to other environmentalsequences from the Cariaco Basin and elsewhere(Edgcomb et al. 2002; Massana et al. 2004a;Stoeck et al. 2003a) (Fig. 3). Labyrinthulids aremarine osmoheterotrophs that occur globallytypically prospering in oxygen-depleted deep-sea environments (Raghukumar 2002), and someare known to be anaerobes (Naqvi 1994).

RhizariaWithin the Rhizaria (Radiolaria, Foraminifera,

Cercozoa), we detected OTUs belonging to theAcantharea, Polycystinea and Cercozoa (Fig. 5).Nine acantharean OTUs obtained include two thatbranch within the Acantharea, and seven formingits sister clade. This sister clade is without acultured representative, which indicates that theAcantharea as a group are likely to be under-sampled and more diverse than currently thought.These organisms are free-floating planktonicheterotrophs with cosmopolitan distribution. Theyare rarely found in coastal waters and appear tooccupy species-specific positions in the watercolumn, often at considerable depth (Edgcomb etal. 2002; Lopez-Garcıa et al. 2001; Stoeck et al.2003a). The phylogenetic position of the acanthar-eans was considered rather enigmatic for a longtime (Amaral-Zettler et al. 1997). Our analysessupport the more recent findings that both

Figure 3. Minimum evolution phylogenetic tree of eukstramenopile clones detected in the Cariaco basin. Themodel based on 985 unambiguously aligned positions. Dof 1000 bootstrap replicates are given at the respectiveoutgroup. The final positions of the major heterotrophalternative models suggested by Modeltest (see Methobootstrap support. The legend is the same as for Figur

Acantharea and Polycystinea (belonging to theRadiolaria) form monophyletic sister groups tothe Cercozoa and branch within the radiation ofthe crown groups (Bass et al. 2005; Lopez-Garcıaet al. 2002; Nikolaev et al. 2004; Polet et al. 2004).

EuglenozoaThe phylum Euglenozoa consists of three dis-

tinct protozoan groups: the euglenoids, diplone-mids and kinetoplastids (Cavalier-Smith 1993;Simpson 1997). Two of the new Cariaco clones(T53C3, T53F7), together with two previouslydetected Cariaco clones (Stoeck et al. 2003a),show a close and well-supported (98% bootstrap)affiliation to Rhynchopus sp., a diplonemid flagel-late (Fig. 6). This sequence clade is of specialinterest, as the phylogenetic position of diplone-mids within the euglenozoa remains unsettled(Busse and Preisfeld 2002; Cavalier-Smith 2003;Maslov et al. 1999; Moreira et al. 2001; Simpsonet al. 2002; Von der Heyden et al. 2004). Also inour analysis, the phylogenetic position of diplone-mids is statistically not supported. Taxonomicsampling was shown to be a major factor affectingthe topology of the SSU rRNA euglenozoan tree(Moreira et al. 2001). Since only few diplonemidsequences are available, concerted efforts are inorder to enlarge the diplonemid sampling andretrieve novel diplonemid sequences is a highpriority in euglenozoan phylogeny.

Several conclusions follow from the aboveobservations.

First, the true extent of protistan diversity in ourstudy site is unknown, and it is difficult to estimatewith any certainty what portion of this diversity hasbeen captured by our clone libraries. It appearshowever that this portion may be insignificant, andeven with 41000 clones from four clone librariesconstructed with four different primer sets, themajority of protists inhabiting this anoxic environ-ment might still have escaped detection. This maybe typical of most, if not all, 18S rRNA surveysconducted to date.

Second, the protistan diversity in anoxic watersof the Cariaco Basin includes lineages of sub-stantial phylogenetic novelty. The 18S rRNA genesequence information obtained strongly support

aryotic small-subunit rRNA showing the position oftree topology was obtained under a TrN substitutionistance bootstrap values over 50% from an analysisnodes. Three alveolate sequences were chosen asstramenopile clades are varying depending on theds); the degree of instability is indicated by a lowere 2.

ARTICLE IN PRESS

T41

A11

Mic

rodo

chiu

m n

ival

e

p15C

07

T45

B5

(p14

A7)

0.01

sub

stitu

tions

/site

Pen

icil

lium

nam

yslo

wsk

ii

p15D

12

Par

apha

eosp

haer

ia s

p.

T37

G8

A1_

E03

1(G

uaym

as)

Cla

dosp

oriu

m c

lado

spor

ioid

es

T37

A10

p15H

03

p15A

06

Chy

trid

ium

pol

ysip

honi

ae

Phy

soph

ora

hydr

osta

tica

Phy

sali

a ph

ysal

is

Zea

may

s

Ory

za s

ativ

a

Chl

orel

la m

inut

issi

ma

Nan

ochl

orum

euk

aryo

tum

Cho

anoe

ca p

erpl

exa

Salp

ingo

eca

infu

sion

um

Ani

mal

s

Lan

d Pl

ants

Gre

en A

lgae

Cho

anof

lage

llate

s

Fung

i

73

100

53

100

90

60

100

90

56

62 82

100

100

100

50

99

100

100

Fig

ure

4.

Min

imum

evo

lutio

np

hylo

genetic

tree

of

eukary

otic

sm

all-

sub

unit

rRN

Asho

win

gth

ep

ositio

no

ffu

ng

alclo

nes

dete

cte

din

the

Cariaco

basin

.T

he

tree

was

co

nstr

ucte

dund

er

aG

TR

+I+

GD

NA

sub

stitu

tio

nm

od

el

with

the

variab

le-s

ite

gam

ma

dis

trib

utio

nshap

ep

ara

mete

r(a

)at

0.5

874,

the

pro

po

rtio

no

fin

variab

lesites

at

0.2

632

and

base

freq

uencie

sand

ara

tem

atr

ixfo

rth

esub

stitu

tio

nm

od

ela

ssug

geste

db

yM

od

eltest

(see

Meth

od

s),

based

on

936

unam

big

uo

usly

alig

ned

po

sitio

ns.

Dis

tance

bo

ots

trap

valu

es

over

50%

fro

man

analy

sis

of

1000

bo

ots

trap

rep

licate

sare

giv

en

at

the

resp

ective

no

des.

Thre

ealv

eo

late

seq

uences

were

cho

sen

as

outg

roup

.O

nly

seq

uences

of

the

clo

sest

rela

tives

of

the

Cariaco

clo

nes,

and

sele

cte

dre

pre

senta

tives

of

oth

er

taxo

no

mic

gro

up

sare

sho

wn.

The

leg

end

isth

esam

eas

for

Fig

ure

2.

0.01

sub

stitu

tions

/site

T41

D11

C3_

E01

6 (G

uyam

as)

p15C

2

Cha

unac

anth

id s

p. 2

17b

Sym

phya

cant

hid

sp. 2

11

Aca

ntho

met

ra s

p.

Art

hrac

anth

id s

p. 2

06

E16

(C

aria

co)

T45

B2

C1_

E04

5 (G

uaym

as)

T37

C4

p14C

4

E19

1 (C

aria

co)

p15D

07

T41

E10

T41

A2

Rha

phid

ozou

mac

ufer

um

Col

lozo

um p

elag

icum

T41

F3 AT8

-54

(Mid

-Atla

ntic

Rid

ge)

E10

7 (C

aria

co)

Siph

onos

phae

racy

athi

na

Col

losp

haer

agl

obul

aris

huxl

eyi

Pha

gom

yxa

odon

tell

ae

Pol

ymyx

a b

etae

Hap

losp

orid

ium

nel

soni

Uro

spor

idiu

m c

resc

ens

Cry

othe

com

onas

aest

ival

is

T41

D6 BO

LA

383

(Bol

inas

tida

l fla

t)

p15D

09

Cer

com

onas

long

icau

da

CC

W29

(Si

ppew

isse

t)

T37

A2

Eug

lyph

a ro

tund

a

Tra

chel

ocor

ythi

on

pulc

hell

um

Coe

lode

ndru

mra

mos

issi

mum

Aul

osph

aera

trig

onop

aPh

aeod

area

Cer

cozo

aHap

losp

orid

ia

Phyt

omyx

ea

Poly

cyst

inea

Aca

ntha

rea

uncu

lture

dR

hiza

ria

56 68 98

100

100

65

100

82

96

59

5110

0

100

100

96

92

99

84

7853

62

99

9850

74

58

Fig

ure

5.

Min

imum

evo

lutio

np

hylo

genetic

tree

ofeukary

otic

sm

all-

sub

unit

rRN

Asho

win

gth

ep

ositio

no

frh

izarian

clo

nes

reco

vere

dfr

om

the

Cariaco

Basin

.T

he

tree

was

co

nstr

ucte

dusin

ga

TrN

+G

DN

Asub

stitu

tio

nm

od

el

with

the

variab

le-

site

gam

ma

dis

trib

utio

nshap

ep

ara

mete

r(a

)at

0.5

721

as

sug

geste

db

yM

od

eltest

(see

Meth

od

s),

based

on

713

unam

big

uo

usly

alig

ned

po

sitio

ns.

Dis

tance

bo

ots

trap

valu

es

over

50%

fro

man

analy

sis

of

1000

bo

ots

trap

rep

licate

sare

giv

en

at

the

resp

ective

no

des.

Thre

ecili

ate

seq

uences

were

cho

sen

as

outg

roup

.T

he

leg

end

isth

esam

eas

for

Fig

ure

2.

ARTICLE IN PRESS

Naegleria minor

Vahlkampfia lobospinosaHeterolobosea

Trypanosoma cruzi

Trypanosoma dionisii

Bodo caudatus

Trypanoplasma borreli

Rhynchomonas nasuta

Bodo saliens

Actuariola framvarensis FV18-8TS

Kinetoplastida

AT4-56

Ichtyobodo necator

Rhynchopus sp.

E220 (Cariaco)

H25 (Cariaco)

T53F7

T53C3

Distigma proteus

Gyropaigne lefevrei

Lepocindis ovum

Euglena gracilis

Petalomonas cantuscygni

Giardia inestinalis

Hexamita inflata

Encephalitozoon cuniculi

Nosema necatrix

Pentatrichomonides scroa

Trichomonas tenax

0.05 substitutions/site

Diplonemida

Euglenida

Euglenozoa

Trichomonads

Microsporidia

Diplomonads

98

98

100

100

9855

10062

100

60

52

53

100

100

93

99

100

54

76

62

82

62

Figure 6. Minimum evolution phylogenetic tree of eukaryotic small-subunit rRNA showing the position ofeuglenozoan clones recovered from the Cariaco Basin. The tree was constructed under maximum-likelihoodcriteria by using a GTR+I+G DNA substitution model with the variable-site gamma distribution shapeparameter (a) at 0.8883, the proportion of invariable sites at 0.1861 and base frequencies and a rate matrix forthe substitution model as suggested by Modeltest (see Methods), based on 670 unambiguously alignedpositions. Distance bootstrap values over 50% from an analysis of 1000 bootstrap replicates are given at therespective nodes. Three archaean sequences were chosen as outgroup. The legend is the same as forFigure 2.

39Multiple PCR-primer Approach

ARTICLE IN PRESS

40 T. Stoeck et al.

the reality of previously detected, deeply rootedprotistan clades within the stramenopiles andalveolates, their sister, or ancestral groups. Itwould be particularly interesting to gain accessto representatives of these clades to assess theirmorphological and physiological uniqueness aswell as ecological and evolutionary importance.We are in the process of recovering suchrepresentatives using a novel methodology thatcombines fluorescence in situ hybridization andlight and electron microscopy (Stoeck et al.2003b).

Methods

Study site and sampling: The Cariaco Basin isthe world’s largest truly marine body of anoxicwater and represents a large natural sedimenttrap, collecting sinking debris and biota fromsurface waters. Detailed descriptions of thisenvironment can be found elsewhere (Muller-Karger et al. 2001; Richards and Vaccaro 1956;Taylor et al. 2001). The sampling procedure isdescribed in Stoeck et al. (2003a). In short, a 2.3 lseawater sample was collected just below theoxic/anoxic interface at a water depth of 340 m,and cells were collected on 47 mm Duraporemembranes (0.65mm pore-size) (Millipore, Bill-erica, MA) under gentle vacuum (o40 ml min�1).Sampling and sample processing protocols weredesigned to prevent exposure to the atmosphere(Stoeck et al. 2003a). Membranes were placed incryovials containing 1 ml of DNA extraction buffer(Stoeck et al. 2003a) to which 5ml of proteinase Kwas added and then returned to the United Statesfrozen in a block of ice.

DNA isolation, PCR amplification, cloning,and sequencing: The nucleic acids were ob-tained as described previously (Stoeck et al.2003a). We amplified E1000—1300 bp fragmentsof the 18S rRNA gene using three primer sets,consisting of a single forward primer E528F(50-CGGTAATTCCAGCTCC-30) (Edgcomb et al.2002) and three different universal reverse primersUniv1391RE (50-GGGCGGTGTGTACAARGRG-30)(Dawson and Pace 2002), Univ1492RE (50-AC-CTTGTTACGRCTT-30) (Edgcomb et al. 2002), andUniv1517 (50-ACGGCTACCTTGTTACGACTT-30,modified from Univ1492RE). The first two primersets successfully amplified the rRNA gene usingthe sample’s DNA extract as a template. The latterprimer set was successful only with a nested PCR,with the template for the second PCR providedby the first reaction with primers Euk A

(50-AACCTGGTTGATCCTGCCAGT-30) and Euk B(50-TGATCCTTCTGCAGGTTCACCTAC-30) (Medlinet al. 1988). The PCR protocol employed HotStartTaq DNA polymerase (Qiagen, Valencia, CA) in allcases; this protocol consisted of an initial hot-startincubation (15 min at 95 1C) followed by 30identical amplification cycles (denaturing at 95 1Cfor 45 s, annealing at 55 1C for 1 min, and exten-sion at 72 1C for 2.5 min), and final extension at72 1C for 7 min. The PCR products were cloned,separately for each primer set, using the TAcloning kit (Invitrogen, Carlsbad, CA) accordingto the manufacturer’s instructions. Plasmids wereisolated from overnight cultures by using theMachery—Nagel NucleoSpin Robot-96 PlasmidKit (Easton, PA) or amplified from plate coloniesvia the Templiphi 100 Amplification Kit (AmershamBiosciences, Piscataway, NJ) according to themanufacturers’ instructions. The presence of thetarget insert was confirmed by PCR reamplifica-tion as described above. Clones were initiallygrouped into operational taxonomic units (OTUs)based on partial sequences using M13F sequen-cing primer considering only sequences withca. 600—800 Phred20 quality checked bp (se-quence similarity cutoff for OUT definition ¼ 99%).All in all, we partially sequenced nearly 600 clones,of which 497 proved to be protistan targets, whichwere considered for further analyses. The OTUgrouping was then checked using HaeIII (NewEngland Biolabs, Beverly, MA) digestion patternsof all clones (ARDRA — amplified ribosomal DNArestriction analyses). At least one clone of eachOTU ðn ¼ 91Þ was sequenced bidirectionally at theUniversity of Maine DNA Sequencing Facility.Sequence variability within ARDRA patterns(OTUs) was also assessed by random completebidirectional sequencing of multiple clones withinARDRA patterns (OTUs) ðn ¼ 7Þ. Sequence com-parison revealed that sequences within a singledigestion pattern do not diverge by more than 1%.

Phylogenetic analyses: We followed the pro-cedure described earlier (Stoeck et al. 2003a). Inshort, the 18S rRNA gene sequences werecompared to those in GenBank and to 45000prealigned eukaryotic small-subunit rRNA in theRibosomal Database Project (Maidak et al. 2001).Multiple alignments were obtained and manuallyrefined according to conserved secondary struc-tures using ClustalX (Thompson et al. 1994),MacClade v. 4.06 (Maddison and Maddison2000), and the secondary-structure predictiontool of the Vienna RNA Package (Cannone et al.2002; Hofacker et al. 1994). Further phylogeneticanalyses included only conserved regions.

ARTICLE IN PRESS

41Multiple PCR-primer Approach

Minimum-evolutionary-distance trees and max-imum-likelihood trees (where applicable) wereconstructed by using the PAUP* software package4.0b10 (Swofford 2001). We used the programModeltest (Posada and Crandall 1998) to choosethe models of DNA substitution that best fit ourdata sets from among 56 possible models andassessed the relative stability of tree topologies byusing 1000 bootstrap replicates.

The gene sequences from this study have beendeposited in the GenBank database (accessionnumbers AY882442—AY882540).

Acknowledgements

We thank the captain and crew of the B/OHermano Gines and the staff of the Estacion deInvestigaciones Marinas (EDIMAR) de Margarita(Fundacion la Salle de Ciencias Naturales, Puntade Piedras, Isla de Margarita, Venezuela) for theirhelp in the field. The authors are grateful to thescientists of the CARIACO time series program,particularly F. Muller-Karger and Y. Astor forlogistical field support and ancillary data. We thankDr. E. Jarroll (Northeastern University, Boston, MA,USA) for his untiring support of this project. Manythanks to Ted Maney (Marine Science Center,Northeastern University, Nahant, MA, USA) foreffective management of logistical matters. Themanuscript was substantially improved by twoanonymous reviewers and by additional sugges-tions by the monitoring editor. This work wasfunded by the Deutsche ForschungsgemeinschaftGrants STO414/2-1 and STO414/2-2 to TS and USNational Science Foundation Grants to GT(OCE9730278) and SE (MCB-0348341).

This is contribution # 255 of the Marine ScienceCenter of Northeastern University, Nahant, MA, USA

References

Acinas SG, Klepac-Ceraj V, Hunt DE, Pharino C,Ceraj I, Distel DL, Polz MF (2004) Fine-scalephylogenetic architecture of a complex bacterialcommunity. Nature 430: 551—554

Admiraal W, Peletier H (1979) Influence of organiccompounds and light limitations on the growth rateof estuarine benthic diatoms. Br Phycol J 14:197—206

Amaral-Zettler LA, Sogin ML, Caron DA (1997)Phylogenetic relationships between the Acanthareaand the Polycystinea: a molecular perspective on

Haeckel’s Radiolaria. Proc Natl Acad Sci USA 94:11411—11416

Amaral-Zettler LA, Gomez F, Zettler E, KeenanBG, Amils R, Sogin ML (2002) Eukaryotic diversityin Spain’s River of Fire. Nature 417: 137

Baldauf SL (2003) The deep roots of eukaryotes.Science 300: 1703—1706

Bass D, Moreira D, Lopez-Garcıa P, Polet S, ChaoEE, von der Heyden S, Pawlowski J, Cavalier-Smith T (2005) Polyubiquitin insertions and thephylogeny of Cercozoa and Rhizaria. Protist 156:149—161

Busse I, Preisfeld A (2002) Phylogenetic position ofRhynchopus sp. and Diplonema ambulator asindicated by analyses of euglenozoan small subunitribosomal DNA. Gene 284: 83—91

Cannone JJ, Subramanian S, Schnare MN, CollettJR, D’Souza LM, Du Y, Feng B, Lin N, MadabusiLV, Muller KM, Pande N, Shang Z, Yu N, Gutell RR(2002) The Comparative RNA Web (CRW) Site: anonline database of comparative sequence andstructure information for ribosomal, intron, and otherRNAs. BMC Bioinformatics 3: 2

Caron DA, Countway P, Brown MV (2004) Thegrowing contributions of molecular biology andimmunology to protistan ecology: molecular signa-tures as ecological tools. J Eukaryot Microbiol 51:38—48

Cavalier-Smith T (1993) Kingdom protozoa and its18 phyla. Microbiol Rev 57: 953—994

Cavalier-Smith T (2003) The excavate protozoanphyla: Grasse emend (Anaeromonadea, Parabasalia,Carpediemonas, Eopharyngia) and Loukozoaemend. (Jakobea, Malawimonas): their evolutionaryaffinities and new higher taxa. Int J Syst EvolMicrobiol 53: 1741—1758

Chao A, Chazdon RL, Colwell RK, Shen T-J (2005)A new statistical approach for assessing composi-tional similarity based on incidence and abundancedata. Ecol Lett 8: 148—159

Colwell RK, Mao CX, Chang J (2004) Interpolating,extrapolating, and comparing incidence-basedspecies accumulation curves. Ecology 85:2717—2727

Countway PD, Gast RJ, Savai P, Caron DA (2005)Protistan diversity estimates based on 18S rDNAfrom seawater incubations in the western NorthAtlantic. J Eukaryot Microbiol 52: 1—12

Dawson SC, Pace NR (2002) Novel kingdom-leveleukaryotic diversity in anoxic environments ProcNatl Acad Sci USA 99: 8324—8329

ARTICLE IN PRESS

42 T. Stoeck et al.

Diez B, Pedros-Alio C, Massana R (2001) Study ofgenetic diversity of eukaryotic picoplankton indifferent oceanic regions by small-subunit rRNAgene cloning and sequencing. Appl Environ Micro-biol 67: 2932—2941

Edgcomb VP, Kysela DT, Teske A, de Vera GomezA, Sogin ML (2002) Benthic eukaryotic diversity inthe Guaymas Basin hydrothermal vent environment.Proc Natl Acad Sci USA 99: 7658—7662

Finlay BJ, Fenchel T (1999) Divergent perspectiveson protist species richness. Protist 150: 229—233

Foissner W (1999) Protist diversity: estimates of thenear-imponderable. Protist 150: 363—368

Grasse PP (1952) Zooflagelles de Position Systema-tique Incertaine (Flagellate Incertae Sedis). In:Grasse PP (ed) Traite de Zoologie, Vol. 1. Masson& Cia, Paris, pp 1005—1022

Gunderson J, Goss S, Coats DW (1999) Phyloge-netic position of Amoebophrya sp. from Gymnodi-nium sanguineum. J Eukaryot Microbiol 46: 194—197

Heck Jr KL, van Belle G, Simberloff D (1975)Explicit calculation of the rarefaction diversity mea-surement and the determination of sufficient samplesize. Ecology 56: 1459—1461

Hellebust JA, Lewin J (1977) Heterotrophic Nutri-tion. In: Werner D (ed) The Biology of Diatoms.Blackwell, Oxford, pp 169—197

Hofacker IL, Fontana W, Stadler PF, BonhoefferLS, Tacker M, Schuster M (1994) Fast folding andcomparison of RNA secondary structures. MonatshChem 125: 167—188

Li C-W, Volcani BE (1987) Four new apochloroticdiatoms. Br Phycol J 22: 375—382

Lopez-Garcıa P, Rodriguez-Valera F, Moreira D(2002) Towards the monophyly of Haeckel’s Radi-oloaria: 18S rRNA environmental data support thesisterhood of Polycystinea and Acantharea. Mol BiolEvol 19: 118—121

Lopez-Garcıa P, Philippe H, Gail F, Moreira D(2003) Autochthonous eukaryotic diversity in hydro-thermal sediment and experimental microcolonizersat the Mid-Atlantic Ridge. Proc Natl Acad Sci USA100: 697—702

Lopez-Garcıa P, Rodriguez-Valera F, Pedros-AlioC, Moreira D (2001) Unexpected diversity of smalleukaryotes in deep-sea Antarctic plankton. Nature409: 603—607

Maddison D, Maddison W (2000) MacClade 4:Analysis of Phylogeny and Character Evolution, CD-ROM. Sinauer Associates, Sunderland, MA

Maidak BL, Cole JR, Lilburn TG, Parker Jr CT,Saxman PR, Farris RJ, Garrity GM, Olsen GJ,Schmidt TM, Tiedje JM (2001) The RDP-II (Ribo-somal Database Project). Nucleic Acids Res 29:173—174

Maslov DA, Yasuhira S, Simpson L (1999) Phylo-genetic affinities of Diplonema within the Euglenozoaas inferred from the SSU rRNA gene and partial COIprotein sequences. Protist 150: 33—42

Massana R, Balague V, Guillou L, Pedros-Alio C(2004a) Picoeukaryotic diversity in an oligotrophiccoastal site studied by molecular and culturingapproaches. FEMS Microbiol Ecol 50: 231—243

Massana R, Castresana J, Balague V, Guillou L,Romari K, Groisillier A, Valentin K, Pedros-Alio C(2004b) Phylogenetic and ecological analysis ofnovel marine stramenopiles. Appl Environ Microbiol70: 3528—3534

Medlin L, Elwood HJ, Stickel S, Sogin ML (1988)The characterization of enzymatically amplified eu-karyotic 16S-like rRNA-coding regions. Gene 71:491—499

Moon-van der Staay SY, De Wachter R, Vaulot D(2001) Oceanic 18S rDNA sequences from pico-plankton reveal unsuspected eukaryotic diversity.Nature 409: 607—610

Moreira D, Lopez-Garcıa P (2002) The molecularecology of microbial eukaryotes unveils a hiddenworld. Trends Microbiol 10: 31—38

Moreira D, Lopez-Garcıa P, Rodriguez-Valera F(2001) New insights into the phylogenetic positionof diplonemids: G+C content bias, differencesof evolutionary rate and a new environmentalsequence. Int J Syst Evol Microbiol 51:2211—2219

Muller-Karger F, Varel R, Thunell R, Scranton M,Bohrer R, Taylor GT, Capelo J, Asto Y, Tappa E,Ho T-Y, Walsh JJ (2001) Annual cycle of primaryproduction in the Cariaco Basin: Response toupwelling and implications for vertical export.J Geophys Res 106: 4527—4542

Naqvi SWA (1994) Denitrification processes in theArabian Sea. Proc Ind Acad Sci (Earth Planet Sci)103: 279—300

Nikolaev SI, Berney C, Fahrni JF, Bolivar I, PoletS, Mylnikov AP, Aleshin VV, Petrov NB, PawlowskiJ (2004) The twilight of Heliozoa and rise of Rhizaria,an emerging subgroup of amoeboid eukaryotes.Proc Natl Acad Sci USA 101: 8066—8071

Patterson DJ (1999) The diversity of eukaryotes. AmNat 154: 96—124

ARTICLE IN PRESS

43Multiple PCR-primer Approach

Polet S, Berney C, Fahrni J, Pawlowski J (2004)Small-subunit ribosomal RNA gene sequences ofPhaeodarea challenge the monophyly of Haeckel’sRadiolaria. Protist 155: 53—63

Posada D, Crandall KA (1998) MODELTEST: testingthe model of DNA substitution. Bioinformatics 14:817—818

Raghukumar S (2002) Ecology of the marineprotists, the Labyrinthulomycetes (Thraustochytridsand Labyrinthuloids). Europ J Protistol 38: 127—145

Richards FA, Vaccaro RF (1956) The CariacoTrench, an anaerobic basin in the Caribbean.Deep-Sea Res 3: 214—228

Richards TA, Bass D (2005) Molecular screening offree-living microbial eukaryotes: diversity and dis-tribution using a meta-analysis. Curr Opin Microbiol8: 240—252

Romari K, Vaulot (2004) Composition and temporalvariability of picoeukaryote communities at a coastalsite of the English Channel from 18S rDNA se-quences. Limnol Oceanogr 49: 784—798

Saunders GW, Hill DR, Sexton JP, Andersen RA(1997) Small-Subunit Ribosomal RNA Sequencesfrom Selected Dinoflagellates: Testing ClassicalEvolutionary Hypotheses with Molecular SystematicMethods. In: Bhattacharya D (ed) Origins of Algaeand their Plastids. Springer-Verlag Wien, New York,pp 237—259

Simpson AGB (1997) The identity and compositionof the Euglenozoa. Arch Protistenkd 148: 318—328

Simpson AGB, Lukes J, Roger AJ (2002) Theevolutionary history of kinetoplastids and theirkinetoplasts. Mol Biol Evol 19: 2071—2083

Stoeck T, Epstein SS (2003) Novel eukaryoticlineages inferred from SSU rRNA analyses in oxy-

gen-depleted marine environments. Appl EnvironMicrobiol 69: 2657—2663

Stoeck T, Taylor G, Epstein SS (2003a) Noveleukaryotes from a permanently anoxic CariacoBasin (Caribbean Sea). Appl Environ Microbiol 69:5656—5663

Stoeck T, Fowle W, Epstein SS (2003b) Methodol-ogy of protistan discovery: from rRNA detection toquality SEM images. Appl Environ Microbiol 69:6856—6863

Strueder-Kypke MC, Lynn DH (2003) Sequenceanalyses of the small subunit rRNA gene confirm theparaphyly of oligotrich ciliates sensu lato andsupport the monophyly of the subclasses Oligotri-chia and Choreotrichia (Ciliophora, Spirotrichea).J Zool (London) 260: 87—97

Swofford DL (2001) PAUP*: Phylogenetic analysisusing parsimony (*and other methods) v4.0b6.Sinauer Associates, Sunderland, MA

Taylor GT, Scranton MI, Iabichella M, Ho T-Y,Thunell RC, Muller-Karger F, Varela R (2001)Chemoautotrophy in the redox transition zone ofthe Cariaco Basin: a significant midwater source oforganic carbon production. Limnol Oceanogr 46:148—163

Thompson JD, Higgins DG, Gibson TJ (1994)CLUSTAL W: improving the sensitivity of progressivemultiple sequence alignment through sequenceweighting, position-specific gap penalties andweight matrix choice. Nucleic Acids Res 22:4673—4680

Von der Heyden S, Chao EE, Vickerman K,Cavalier-Smith T (2004) Robosomal RNA phylogenyof bodonid and diplonemid flagellates and theevolution of euglenozoa. J Eukaryot Microbiol 51:402—416