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Protist, Vol. 158, 173—180, April 2007 http://www.elsevier.de/protis Available online 3 January 2007 ORIGINAL PAPER Analysis of Environmental 18S Ribosomal RNA Sequences reveals Unknown Diversity of the Cosmopolitan Phylum Telonemia Kamran Shalchian-Tabrizi a,1 , Ha ˚ vard Kauserud b , Ramon Massana c , Dag Klaveness d , and Kjetill S. Jakobsen a a Department of Biology, Centre for Ecological and Evolutionary Synthesis, University of Oslo, N-0316 Oslo, Norway b Department of Biology, Program for Molecular Ecology and Biosystematics, University of Oslo, N-0316 Oslo, Norway c Institut de Cie `ncies del Mar, CMIMA, Passeig Marı ´tim de la Barceloneta 37-49, 08003 Barcelona, Catalonia, Spain d Department of Biology, Program for Plankton Biology, University of Oslo, N-0316 Oslo, Norway Submitted July 9, 2006; Accepted October 21, 2006 Monitoring Editor: Robert A. Andersen Telonemia has recently been described as a new eukaryotic phylum with uncertain evolutionary origin. So far, only two Telonemia species, Telonema subtilis and Telonema antarcticum, have been described, but there are substantial variations in size and morphology among Telonema isolates and field observations, indicating a hidden diversity of Telonemia-like species and populations. In this study, we investigated the diversity and the global distribution of this group by analyzing 18S rDNA sequences from marine environmental clone libraries published in GenBank as well as several unpublished sequences from the Indian Ocean. Phylogenetic analyses of the identified sequences suggest that the Telonemia phylum includes several undescribed 18S rDNA phylotypes, probably corresponding to a number of different species and/or populations. The Telonemia phylotypes form two main groups, here referred to as Telonemia Groups 1 and 2. Some of the closely related sequences originate from separate oceans, indicating worldwide distributions of various Telonemia phylotypes, while other phylotypes seem to have limited geographical distribution. Further investigations of the evolutionary relationships within Telonemia should be conducted on isolated cultures of Telonema-like strains using multi-locus sequencing and morphological data. & 2006 Elsevier GmbH. All rights reserved. Key words: 18S rDNA diversity; environmental sequences; Telonema; Telonemia; phylogeny. Introduction Phylogenetic studies of environmental sampled DNA have uncovered a large hidden diversity of eukaryotes from marine and freshwater environ- ments (´ez et al. 2001; Lopez-Garcia et al. 2001; Moon-van der Staay et al. 2001; Richards et al. 2005). Few sequences, however, seem to con- stitute entirely new phyla or kingdoms, indicating that the vast majority of the extant eukaryotic ARTICLE IN PRESS 1 Corresponding author; fax +47 22854001 e-mail [email protected] (K. Shalchian-Tabrizi). & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2006.10.003

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Page 1: Analysis of Environmental 18S Ribosomal RNA Sequences reveals Unknown Diversity of the Cosmopolitan Phylum Telonemia

ARTICLE IN PRESS

http://www.elsevier.de/protisAvailable online 3 January 2007

1

Correspondinfax +47 22854e-mail kamran

& 2006 Elsevdoi:10.1016/j

158, 173—180, April 2007

Protist, Vol.

ORIGINAL PAPER

Analysis of Environmental 18S Ribosomal RNASequences reveals Unknown Diversity of theCosmopolitan Phylum Telonemia

Kamran Shalchian-Tabrizia,1, Havard Kauserudb, Ramon Massanac, Dag Klavenessd, andKjetill S. Jakobsena

aDepartment of Biology, Centre for Ecological and Evolutionary Synthesis, University of Oslo,N-0316 Oslo, Norway

bDepartment of Biology, Program for Molecular Ecology and Biosystematics, University of Oslo,N-0316 Oslo, Norway

cInstitut de Ciencies del Mar, CMIMA, Passeig Marıtim de la Barceloneta 37-49, 08003 Barcelona,Catalonia, Spain

dDepartment of Biology, Program for Plankton Biology, University of Oslo, N-0316 Oslo, Norway

Submitted July 9, 2006; Accepted October 21, 2006Monitoring Editor: Robert A. Andersen

Telonemia has recently been described as a new eukaryotic phylum with uncertain evolutionary origin.So far, only two Telonemia species, Telonema subtilis and Telonema antarcticum, have beendescribed, but there are substantial variations in size and morphology among Telonema isolates andfield observations, indicating a hidden diversity of Telonemia-like species and populations. In thisstudy, we investigated the diversity and the global distribution of this group by analyzing 18S rDNAsequences from marine environmental clone libraries published in GenBank as well as severalunpublished sequences from the Indian Ocean. Phylogenetic analyses of the identified sequencessuggest that the Telonemia phylum includes several undescribed 18S rDNA phylotypes, probablycorresponding to a number of different species and/or populations. The Telonemia phylotypes formtwo main groups, here referred to as Telonemia Groups 1 and 2. Some of the closely relatedsequences originate from separate oceans, indicating worldwide distributions of various Telonemiaphylotypes, while other phylotypes seem to have limited geographical distribution. Furtherinvestigations of the evolutionary relationships within Telonemia should be conducted on isolatedcultures of Telonema-like strains using multi-locus sequencing and morphological data.& 2006 Elsevier GmbH. All rights reserved.

Key words: 18S rDNA diversity; environmental sequences; Telonema; Telonemia; phylogeny.

Introduction

Phylogenetic studies of environmental sampledDNA have uncovered a large hidden diversity of

g author;[email protected] (K. Shalchian-Tabrizi).

ier GmbH. All rights reserved..protis.2006.10.003

eukaryotes from marine and freshwater environ-ments (Dıez et al. 2001; Lopez-Garcia et al. 2001;Moon-van der Staay et al. 2001; Richards et al.2005). Few sequences, however, seem to con-stitute entirely new phyla or kingdoms, indicatingthat the vast majority of the extant eukaryotic

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174 K. Shalchian-Tabrizi et al.

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175Telonemia Diversity

groups are already described (Berney et al. 2004;Cavalier-Smith 2004; Lopez-Garcia et al. 2001;Moon-van der Staay et al. 2001; Richards et al.2005). An exception to this pattern is the identi-fication of a new lineage composed of a fewphylotypes and two described species, Telonemasubtilis Griessmann, 1913 and Telonema antarc-ticum Thomsen (Klaveness et al. 2005; Massanaet al. 2004; Romari and Vaulot 2004). Recently,this lineage was identified as a new phylumTelonemia (Shalchian-Tabrizi et al. 2006).

The origin of Telonemia is uncertain, but ultra-structure, morphology, and multigene phylogeniesmay indicate evolutionary affinity to cryptophytesand haptophytes (Shalchian-Tabrizi et al. 2006).Electron and light microscope observations ofTelonema-like organisms from distant oceansclearly suggest a worldwide distribution of thephylum (Klaveness et al. 2005; Lee and Patterson1998). However, since the size and the shape ofthe directly identified Telonema cells vary quiteextensively depending upon author and localities(T. subtilis between 2—6� 4—14mm and T.antarcticum 8—16� 6—12 mm; Klaveness et al.2005), it is unclear whether they belong to eitherT. subtilis or T. antarcticum, or constitute assem-blages of cryptic species. Besides being widelydistributed, Telonemia species are likely to play asignificant ecological role, as they are regularlyobserved in marine and brackish waters (Lee andPatterson 1998; Vørs 1992a, b) and occasionallyas a dominant heterotrophic species in surveys ofprotist plankton (Klaveness et al. 2005).

Despite the evolutionary and ecological impor-tance of Telonemia, little is known about thespecies diversity, intraspecific divergence, andpopulation structure within the group, although afew sequences in independent environmentalmolecular surveys have been suggested asTelonema or Telonema-like sequences (Klavenesset al. 2005; Lovejoy et al. 2006; Romari and Vaulot2004; Shalchian-Tabrizi et al. 2006). In this study,we investigated the phylogenetic diversity and theglobal distribution of Telonemia by assemblingTelonemia-related 18S rDNA sequences from thepublic GenBank sequence database, picoeukar-yote clone libraries from the Indian Ocean, andunamended incubation of 3mm-filtered seawaternear Svalbard. Altogether, 36 sequences werefound to be related to Telonemia, and two main

Figure 1. 18S rDNA phylogeny of Telonemia-like senucleotide database. The tree is inferred with the maxim470% are indicated with thick lines.

branches in Telonemia were identified and referredto here as Telonemia Groups 1 and 2.

Results and Discussion

Identification of Telonemia-relatedSequences

Using Telonema antarcticum 18S rDNA as querysequence in BLAST searches against the NCBInrnucleotide database, we identified 33 differentpartial and full-length 18S rDNA sequences withhigh similarity to Telonemia from various environ-mental surveys of eukaryotic diversity. Some ofthese have previously been presented as Telonema-like sequences in separate papers (Klavenesset al. 2005; Lovejoy et al. 2006; Romari and Vaulot2004; Shalchian-Tabrizi et al. 2006). In addition,we found two sequences from several 18S rDNAclone libraries from the Indian Ocean (IND31.100and IND33.54) and one sequence from anunamended incubation in the Barents Sea atSvalbard (PD6.20) with high similarity to Telonemasequences; altogether 36 sequences. The newlyidentified sequences were imported to a 18SrDNA alignment, including most of the eukaryoticmain lineages and three previously characterizedsequences from T. antarcticum and T. subtilis. Theassembled sequence data set (altogether 175taxa and 1159 characters) was analyzed using themaximum-likelihood method. The overall treetopology and most of the basal branches dividingthe main eukaryotic groups were weakly sup-ported, but largely in accordance with otherrecently published eukaryote phylogenies (Fig. 1;Berney et al. 2004; Cavalier-Smith 2004; Richardset al. 2005; Shalchian-Tabrizi et al. 2006). Theinternal Telonemia topology was in general weaklysupported, except for a clade of eight sequencesincluding the T. subtilis sequences (91% bootstrapsupport). Nevertheless, the phylogenetic analysisconfirmed that BLAST searches against GenBankand sequencing of 18S rDNA from the IndianOcean and arctic Svalbard (Spitsbergen) identifiedsequences with an evolutionary relationship toTelonemia. When removing characters withmissing data from the partial Telonemia-likesequences (i.e. analyzing 323 characters), the 36Telonema sequences were still clustered together,

quences identified with BLASTn against NCBI nrum-likelihood method using GARLI. Bootstrap values

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176 K. Shalchian-Tabrizi et al.

suggesting that the mixture of partial and full-length sequences in the data is not causingsubstantial artifacts in the phylogenetic recon-structions.

Telonemia constitutes Two New Groups andSeveral Undetermined 18S Phylotypes

In a further phylogenetic analysis, we excludedsequences that showed no clear relationship tothe Telonemia lineage, allowing inclusion of more

Figure 2. Phylogeny of Telonemia-like sequences inferfor Figure 1. Bootstrap values 450% from two maximPAUP* are indicated above and under the internal bransequences are identified (Groups 1 and 2) which includespecies or populations.

unambiguously aligned sites. The alignment con-tained 36 sequences and 1805 characters in total;missing characters in the partial sequences werefilled with question marks. Phylogenetic analysisusing maximum-likelihood methods, divided theTelonemia-like sequences in two main clades,hereafter referred to as Groups 1 and 2, with100% bootstrap support (Fig. 2). Two sequencesfrom T. subtilis and six other closely relatedphylotypes were placed together in Group 1, whileT. antarcticum clustered together with 28 relatedsequences in Group 2. The former group was also

red with fewer sequences and more characters thanum-likelihood analyses implemented in GARLI andches respectively. Two main branches of Telonemiasmaller subgroups that may correspond to separate

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supported (91%) in analysis of all eukaryotes(Fig. 1). The root was inferred to be at the longestinternal branch separating Groups 1 and 2 whenusing haptophytes as outgroup (results notshown). Groups 1 and 2 had no polymorphic sitesin common (i.e. all were fixed in one group orboth). Thus, a complete lineage sorting hashappened among Groups 1 and 2, indicating along lasting separation.

Group 1 was the smallest group and includedsequences from the English Channel (both cul-tures and environmental DNA) and the MariagerFjord, Denmark; all from coastal regions (Table 1).Based on the internal branching pattern and

Table 1. Description of Telonemia-like sequences used

Sample localization Accession numbe

Canada Basin, Arctic Ocean DQ119996Canada Basin, Arctic Ocean DQ119997Canada Basin, Arctic Ocean DQ119998Canada Basin, Arctic Ocean DQ119999Canada Basin, Arctic Ocean DQ120000Canada Basin, Arctic Ocean DQ120001English Channel, Roscoff, France AY295644English Channel, Roscoff, France AY295513English Channel, Roscoff, France AY295498English Channel, Roscoff, France AY295476English Channel, Roscoff, France AY295470English Channel, Roscoff, France AY295583English Channel, Roscoff, France AY295352English Channel, Roscoff, France AY295531English Channel, Roscoff, France AY295501English Channel, Roscoff, France AY295375English Channel, Roscoff, France AJ564771Helgoland, Germany AJ965240Orkney Islands; United Kingdom AJ965244Indian Ocean AM418563Indian Ocean AM418562Mariager Fjord, Denmark DQ103882Mariager Fjord, Denmark DQ103827Mariager Fjord, Denmark DQ103867Mediterranean Sea, Spain AY426930Oslo fjord, Norway AJ564773Sargasso Sea AY665027Sargasso Sea AY665042Sargasso Sea AY665041Sargasso Sea AY665040Sargasso Sea AY665043Sargasso Sea AY665037Svalbard DQ119994Svalbard DQ119995Svalbard DQ119993Svalbard DQ647533

bootstrap support values, it appears that Group1 comprises two subgroups plus two otherseparate independent sequences (hereafter calledSubgroups 1a—1d for simplicity; Fig. 2).

Group 2 sequences were obtained from world-wide locations including Canada Basin ArcticOcean stations, English Channel (off Roscoff,France), Helgoland (Germany), Indian Ocean,Mediterranean Sea (off Blanes, Spain), Oslo Fjord(Oslo, Norway), Sargasso Sea, and Svalbard(arctic region, north of Norway). Eight sub-groups were separated from the other phylo-types with high bootstrap support (Subgroups2a—2h).

in the phylogenetic analysis.

rs Sequencelength

Clone numbers/speciesnames

809 NW614.31843 NW614.39808 NW414.01843 NW414.42841 NW414.45813 NW617.20550 RA010516.38545 RA000907.47544 RA000907.26547 RA000609.57550 RA000609.49550 RA010412.17550 Telonema subtilis (RCC358)750 RA001219.10546 RA000907.3546 RA000412.136

1831 Telonema subtilis (RCC404.5)551 He000427_29608 OR000415_188

1795 IND31.1001798 IND33.54974 M2_18A10

1658 M2_18G121801 M3_18B12725 BL010625.25

1787 Telonema antarcticum1748 SCM16C391751 SCM27C31751 SCM27C121753 SCM27C231751 SCM27C461751 SCM38C20785 NOR26.35778 NOR46.11780 NOR26.38

1568 PD6.20

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It is unclear which taxonomic rank the 12recognized subgroups should have, but wehypothesize that they represent different speciesor populations. For example, Subgroups 2d and 2fshared non-polymorphic sites, indicating a longlasting separation. Recent works on other protistmorphospecies have uncovered cryptic species(Slapeta et al. 2006). To further analyze speciesdelimitations, gene genealogies from independentDNA markers should be compared to revealwhether recombination occurs between the var-ious lineages.

Although Group 2 seems to have a wider globaldistribution than Group 1, the majority of all theidentified species/populations show geographicstructuring; for instance, Subgroups 1a—1d arerestricted to Denmark and the English Channel,while Subgroups 2b, c, and d+e are restricted tothe Canadian Arctic Ocean and the Sargasso Seaonly. In contrast, Subgroups 2a and 2f arecomposed of sequences from more distantregions (i.e. Canadian Arctic Ocean, Svalbard,English Channel, and Indian Ocean). The widegeographic dispersal of Telonemia-like sequencesshows that the phylum as a whole is cosmopoli-tan, confirming previous field observations ofT. antarcticum and T. subtilis (Klaveness et al.2005; Lee and Patterson 1998). Nonetheless, thephylogenetic inferences also suggest that Telone-mia species/populations may be more geographi-cally restricted than hitherto known. The spatialdistribution of Telonemia sequences should befurther investigated applying other DNA samplingstrategies as a substantial number of environ-mental surveys have so far focused on thediversity of pico-sized eukaryotes (Lopez-Garciaet al. 2001; Moon-van der Staay et al. 2001, 2000;Richards et al. 2005; Romari and Vaulot 2004). Webelieve several other Telonemia sequences arelikely to be found if the 18S rDNA libraries wereprepared from larger cell-size fractions and differentlocalities. A key issue would be to rigorouslyexamine whether Telonema species/populationsfollow the classical morphology-based doctrinethat microeukaryotes comprise relatively few cos-mopolitan species (‘‘everything is everywhere’’; seefor instance Finlay 1998; Lee and Patterson 1998;Pedros-Alio 2006) or if the molecular data canuncover new species within this phylum.

Methods

Indian Ocean environmental libraries were fromsamples collected at station INO2 at surface

(IND31) and 74 m (IND33) as explained in Massanaet al. (2006, 2006a). Picoplanktonic biomass(between 0.2 and 3 mm) was collected on filtersand community DNA was extracted for thesesamples. Almost complete 18S rDNA genes werePCR-amplified with the eukaryotic-specific pri-mers EukA and EukB, and the PCR products werecloned. Between 50 and 100 clones were partiallysequenced with primer 528f in each library. Detailsof filtering setup, DNA extraction protocol, primersused, and PCR and cloning conditions aredescribed elsewhere (Dıez et al. 2001; Massanaet al. 2004). One additional sequence derives froman unamended incubation of 3 mm-filtered sea-water near Svalbard after 6 d in the dark thatpromoted the growth of heterotrophic flagellates(Massana et al. 2006b).

Identification of Telonemia-related sequencesfrom public databases and environmentalclone libraries: 18S rDNA from previously pub-lished T. antarcticum Thomsen in Klaveness et al.2005 was used as query in BLASTn search againstNCBInr nucleotide database on July 5, 2006. Allbest hits with undetermined eukaryotic sequenceswere downloaded and added manually to a 18Salignment composed of a large diversity ofeukaryotic groups and environmental 18S rDNAsequences with no clear affinity (Berney et al.2004; Cavalier-Smith 2004). In addition, 18S rDNAsequences with high similarity to Telonema se-quences were identified with BLAST from clonelibraries from the Indian Ocean and incubatedseawater from Svalbard. Ambiguously alignedsites were subsequently removed and missingsites were filled with question marks. Sequenceaccession and clone numbers are given in Table 1.

Phylogenetic analysis: Two different 18S se-quence alignments were made to infer the evolu-tionary relationship of the identified Telonema-likesequences from clone libraries and GenBank(sequence alignments are available upon requestfrom KST). The largest data set contained 175taxa and 1159 characters. Based on phylogeneticinferences of the alignment (Fig. 1), a subset ofsequences with phylogenetic affinity to T. antarc-ticum Thomsen in Klaveness et al. 2005 andT. subtilis Griessmann, 1913 were further analyzedby including additional unambiguously alignedsites (in total, 36 taxa and 1805 characters).Question marks were used as missing charactersin both ends of partial Telonemia-like sequences.Both data sets were analyzed using the max-imum-likelihood method implemented in GARLIversion 0.942 (www.zo.utexas.edu/faculty/anti-sense/garli/Garli.html). The most likely GARLI tree

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topology was inferred from 10 independent runsfrom random starting trees. The stoptime andstopgen parameters were set to 5—15,000,000and other parameters were set to default values.Bootstrap analyses were done with 300 replicateswith the same parameters as the initial treesearch. The phylogeny of the smallest data setwas also reconstructed with the maximum-like-lihood method using PAUP* (Swofford 1998).PAUP* trees were inferred using 10 heuristicsearches, NNI branch swapping, and the followingparameters (estimated from a Kimura-2-para-meter tree): GTR substitution matrix, base fre-quencies, gamma distribution of site rates (G), andproportion of invariable sites (I). One hundredbootstrap analyses were carried out with the sameparameters and one heuristic search per pseudor-eplicate. All inferences were done on the freelyavailable University of Oslo Bioportal (www.bio-portal.uio.no). Shared, fixed, and unique numberof mutations between Groups 1 and 2 andsubgroups (with more than three sequences) werecalculated in DnaSP 4.0 (Rozas et al. 2003).

Acknowledgements

We thank two anonymous reviewers for helpfulcomments, Tellef Rygh for initial BLAST searches,and the Bioinformatics team at the University ofOslo Bioportal for assistance with installationand testing of applications. We thank CedricBerney for 18S alignment of eukaryote super-groups. This work was supported by the Norwe-gian Research Council to KSJ (No. 166555 and159822) and a grant from the French GIS(PICOCEAN).

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