PHYLOGENETIC ANALYSIS AND GENOME SIZE OF OSTREOCOCCUS TAURI (CHLOROPHYTA, PRASINOPHYCEAE)

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J. Phycol. 34, 844–849 (1998)

PHYLOGENETIC ANALYSIS AND GENOME SIZE OF OSTREOCOCCUS TAURI(CHLOROPHYTA, PRASINOPHYCEAE)1

Claude Courties2

O. O. B., Laboratoire Modeles en Biologie Cellulaire et Evolution, CNRS/INSU/UPMC, B.P. 44, 66651 Banyuls-sur-Mer, France

Roland PerassoLaboratoire de Biologie Cellulaire 4, Universite Paris Sud, Batiment 444, Faculte des Sciences, 91405 Orsay, France

Marie-Josephe Chretiennot-DinetO. O. B., Laboratoire d’Oceanographie Biologique, CNRS/INSU/UPMC, B.P. 44, 66651 Banyuls-sur-Mer, France

Manolo GouyLaboratoire de Biometrie Genetique et Biologie des populations, UMR CNRS 5558, Universite Claude Bernard - Lyon I, 43 Bd du 11

Novembre 1918, 69622 Villeurbanne Cedex, France

Laure GuillouStation Biologique, CNRS/INSU/UPMC, BP 74, place Georges Tessier, 29682, Roscoff, France

and

Marc TroussellierLaboratoire d’Hydrobiologie Marine, UMR CNRS 5556, Universite de Montpellier II, place Eugene Bataillon, 34095 Montpellier

Cedex 5, France

ABSTRACT

Ostreococcus tauri Courties et Chretiennot-Dinet is thesmallest described autotrophic eukaryote dominating thephytoplanktonic assemblage of the marine MediterraneanThau lagoon (France). Its taxonomic position was partlyelucidated from ultrastructure and high-pressure liquidchromatography (HLPC) pigment analysis. The sequenceanalysis of the 18S rDNA gene of O. tauri measured hereis available in EMBL Nucleotide Sequence Database (ac-cession number: Y15814) and allowed to clarify its phy-logenetic position. O. tauri belongs to the Prasinophyceaeand appears very close to Mantoniella, a typical scalyPrasinophyceae, morphologically very different from the na-ked and coccoid Ostreococcus. An electrophoretic analy-sis of O. tauri shows that the nucleus contains 10.20 mbp.This small genome, fragmented into 14 chromosomes rang-ing in size from 300 to 1500 kbp, confirms the minimalistcharacteristics of Ostreococcus tauri.

Key index words: chromosome; eukaryote; genome size;molecular phylogeny; Ostreococcus; Prasinophyceae; 18SrDNA gene sequence

Ostreococcus tauri Courties et Chretiennot-Dinet(Chretiennot-Dinet et al. 1995), a new abundantautotrophic eukaryote discovered in the Mediterra-nean Thau lagoon (France), was described as the

1 Received 16 January 1998. Accepted 19 June 1998.2 Author for reprint requests; e-mail courties@arago.obs-ban-

yuls.fr.

smallest eukaryotic organism (Courties et al. 1994).Barely visible by light microscopy, this species mea-suring less than 1 mm was detected by flow cyto-metry, a technique that is now widely used for thestudy of aquatic microorganisms (Shapiro 1995).Several techniques, i.e. electron microscopy, pig-ment analysis, and gene sequence analyses, have tobe used to classify such small organisms into classes,orders, or families (Chretiennot-Dinet and Courties1997, Potter et al. 1997).

Transmission electron microscope examinationshows a very simple cellular organization of O. tauri:a relatively large nucleus, a single chloroplast with astarch granule, a mitochondrion, a Golgi body, anda very reduced cytoplasm. The presence of a starchgranule clearly indicates the belonging of O. taurito the Chlorophyta. The absence of flagella and/orscales, a distinctive character between Prasinophy-ceae and Chlorophyceae, was in this case misleadingin favor of Chlorophyceae.

Pigment signatures of green coccoid cells havebeen studied in detail and appear to be discriminant(Hooks et al. 1988). In O. tauri, HPLC measure-ments revealed the presence of a chl c-like pigmentindicative of affinities with Prasinophyceae, identi-fied as Mg 3,8 DVPa5 (Magnesium 3,8 divinyl-phaeo-porphyrin a5, carbon atoms numbered according toIUPAC convention; see Goericke and Repeta 1992).

Ribosomal DNA sequencing of marine picopro-karyotes from the Sargasso Sea proved that analysisof rDNA genes is a useful tool to determine taxo-nomic position (Giovannoni et al. 1990, Britschgi

845PHYLOGENY OF OSTREOCOCCUS TAURI

and Giovannoni 1991), and molecular techniquesare now used to address taxonomic affinities anddefine species limits (Medlin et al. 1995). However,most picoplanktonic eukaryotic cells known todaywere discovered during the last decade (Chretien-not-Dinet and Courties 1997), and only a few ofthem have been gene sequenced and appear in re-cent phylogenetic trees. They generally belong togreen algae (Kantz et al. 1990, Steinkotter et al.1994, Friedl 1995, 1996, Krienitz et al. 1996, Potteret al. 1997), although some are placed in a recentlyintroduced new class, the Pelagophyceae (Andersenet al. 1993, DeYoe et al. 1995). Because of the lackof morphological characters that could help in plac-ing O. tauri within Prasinophyceae, we present herean analysis of its 18S rDNA gene sequence. It is com-pared to gene sequences of other marine Prasino-phyceae to which we have added gene sequences oftwo undescribed strains, CCMP 1407 and CCMP1220, accessible in EMBL/GenBank.

In addition, O. tauri can be considered a goodcandidate for biological models such as cell divisionand/or genome sequencing studies. Thus, genomesize and chromosome number are also described inrelation to its status as smallest eukaryotic cell.

MATERIALS AND METHODS

Cell isolation and cultures. The Thau lagoon (38369 E, 438249 N),spreading over 75 km2, is connected to the Mediterranean Sea bythree channels crossing the off-shore bar. The mean depth is 5m. Temperatures and salinities vary from 48 to 278 C and from24‰ to 38‰, respectively. Ostreococcus tauri was isolated from thephytoplanktonic assemblage of the Thau lagoon by two successivefiltrations by gravity through 0.8-mm nuclepore filters. This pro-cedure allowed the isolation of two strains, OTTH 0394 (now lost)and OTTH 0595 in culture at AQUAMER (Meze, France). Cellspassing through these membranes were first cultured in a modi-fied F/2 medium (Guillard 1975) close to the nutrient composi-tion of the lagoon (Chretiennot-Dinet et al. 1995). In a secondstep, a Conway medium (Walne 1966) was used to increase thefinal cell density. Cultures were grown in test tubes and sterilebottles (10 mL, 200 mL, and 1 L) at 60 mmol quanta·m22·s21

irradiance on a 16:8 L:D photocycle at 188 C. Cell cultures wereused for transmission electron microscopy and for gene sequenceanalysis. Isolates of O. tauri and cultures were checked using aBruker-Spectrospin 1400 SP (Wissembourg, France) according toTroussellier et al. (1993) or a FacsCalibur (Becton-Dickinson, SanJose, California) flow cytometer. The blue light excitation fromthe mercury lamp (470–490 nm) or from the argon laser (488nm) allowed cell type identification according to cell light scatter(mainly related to size and refractive index) and red fluorescencedepending on chlorophyll content (Olson et al. 1989, Li 1997).Fluorescent 0.96-mm YG beads (Polysciences, Warrington, Penn-sylvania) were used as internal standard and simultaneously ana-lyzed with cultured cells to calibrate the instrument and to stan-dardize flow cytometric data. Accurate cell counts were measuredby flow cytometric analysis of known sample volumes (from 100to 500 mL).

Gene sequencing. Cells from 100 mL of a dense culture (1.5 108

cells mL21) were harvested by centrifugation at 2500 3 g for 20min. The pellet was resuspended and lysed in Tris-HCl 10 mM(pH 8), EDTA 5 mM, SDS 1%, NaCl 100 mM, and incubated at508 C for 4 h with proteinase K (1 mg·mL21). DNA extractionand purification were performed using standard phenol-chloro-form protocol and ethanol precipitation (Sambrook et al. 1989).The sense and antisense primers commercially synthesized (Bio-probe, France) are based on the conserved domains located at

the 59 and 39 ends of the 18S rDNA gene. These phosphorylatedprimers are, respectively: 59ACCTGGTTGATCCTGCC39 and59GCTACCTTGTTACGACTT39. Amplification was performed us-ing 50 ng DNA in 50 mL of 10 mM Tris-HC1 at pH 8.3, 1.5 mMMgCl2, 200 mM deoxyribonucleotide triphosphates, and 1 mM ofeach primer and was topped with mineral oil. After a 5-min de-naturation step at 948 C in a programmable thermalcycler (TRIO-Thermoblock Biometra, Germany), two units of Taq polymerase(Bioprobe) were added, and 35 cycles were carried out as follows:1 min at 948 C, 1 min at 528 C, and 3 min at 728 C. After a finalelongation step at 728 C, the PCR product was checked by agarosegel electrophoresis. The 1.7-kb fragment obtained was then pu-rified by electroelution and cloned into the Sma I site of the plas-mid vector pUC 18 (Pharmacia, France). Dideoxy double-strandsequencing was performed using T7 DNA polymerase (Amer-sham, France) and standard procedures (Sambrook et al. 1989).Two clones of O. tauri were sequenced: OTTH 0394 and OTTH0595. The sequence is now available under the accession numberY15814 in the EMBL Nucleotide Sequence Database.

Genome size. Cells were collected by centrifugation and resus-pended in 1 mL of a solution containing 0.5 M EDTA and 10mM Tris-HC1 pH 8. After quickly mixing with 1 mL of 1% low-melting agarose in 0.125 M EDTA and 10 mM Tris-HC1 (pH 8)solution at 508 C, the cell suspension was poured into plug molds.The plugs were incubated at 378 C for 24 h in 10 mL of 10 mMTris-HCl (pH 8) 0.5 M EDTA, 1% lauroyl-sarcosinate, and pro-teinase K (1 mg mL21). After washing three times in 0.5 M EDTA(pH 8) at 378 C for 2 h, the plugs were stored at 48 C in the samesolution. Pulsed field electrophoresis was performed using theCHEF Mapper XA System (Bio-Rad S. A. France). The gels were1% agarose in 0.5 3 TBE (45 mM Tris, 45 mM boric acid, 1 mMEDTA, pH 8.3). The electrophoresis conditions were: 24 h runtime, 40 to 120 s switch time ramp, and 6 V cm21 (148 C). Chro-mosomes of Saccharomyces cerevisiae (MegaBase I from GibcoBRL,France) were used as size standards. After electrophoresis, thegels were stained with ethidium bromide (0.51 mg·mL21) for 20min and washed in distilled water for 30 min.

Phylogenetic analysis. Ostreococcus tauri gene sequence (EMBL ac-cession number: Y15814) was aligned using DCSE software (deRijk and de Wachter 1993) with the following organisms withEMBL/GenBank accession numbers: Scherffelia dubia: X68484, Te-traselmis convolutae: U05039, Tetraselmis striata: X70802, Mantoniellasquamata: X73999, Pseudoscourfieldia marina: X75565, Nephroselmisolivacea: X74754, CCMP 1220 (unidentified coccoid prasinophy-te): U40920, CCMP 1407 (Prasinococcus cf. capsulatus): U40919,Chlamydornonas reinhardtii: M32703, Volvox carteri: X53904, Neo-chloris aquatica: M62861, Scenedesmus communis: X73994, Nanochlo-rum eucaryotum: X06425, Chlorella minutissima: X56102, Trebouxiaimpressa: Z21551, Prototheca wickerharmii: X56099. Cyanophora par-adoxa: X68483 was used as an outgroup, taking into considerationthat Glaucocystophyta are in relation with the theory of the evo-lution of eukaryotic cells (Kies and Kremer 1989). Phylogeneticalanalyses were made on 1606 sites.

Neighbor-Joining (NJ) distance analysis (Saitou and Nei 1987)was employed with the Kimura (1980) correction. The NJ, themaximum likelihood (ML) (Felsenstein 1981), and the parsimo-ny (Felsenstein 1993) analyses were conducted with the PHYLIPprogram package (Felsenstein 1993) included in the PHY-LOpWIN software (Galtier et al. 1996). Phylogenetic bootstrap-ping with 100 replicates (50 for the ML method) were executedto measure the confidence of the nodes (Felsenstein 1985).

RESULTS AND DISCUSSION

Growth in culture. With the modified Guillard me-dium, cell concentrations of the OTTH 0394 strainvaried between 106 and 107 cells mL21. Use of theConway medium for the OTTH 0595 strain in-creased cell concentrations up to 4·108 cells mL21.In this case, growth rates, expressed in division perday, were high (4.23 div. d21, SD 5 1.44 div. d21, n5 10) compared to growth rates already measured

846 CLAUDE COURTIES ET AL.

FIG. 1. Evolutionary relation-ships between Ostreococcus tauriand other chlorophytes deducedfrom Neighbor-Joining analysisof complete 18S rDNA sequenc-es. The tree was obtained withthe maximum likelihood analysis.Bootstrapping analyses have beentested on the Neighbor-Joining,parsimony, and maximum likeli-hood methods to examine theconfidence of nodes. They wereplaced at the internal branchesin this order (values . 70% dis-played). Ostreococcus tauri belongsto the Prasinophyceae and isclosely related to Mamiellaleswith 100% bootstrap values withthe three methods used. Scale 50.1 divergence.

for other marine picoeukaryote species (from 1.2 to2.1 div. d21; Simon 1995).

Gene sequence analysis. The total 18S rDNA of Os-treococcus tauri was sequenced, except for the twoshort parts external to the region delimited by theprimer sites used for amplification. The sequenceobtained is 1741 nucleotides long including primerswith 46.5% GC content. Similar percentages werefound in other coccoid Prasinophyceae, although bya different method (Simon et al. 1994).

Phylogenetic analysis. The phylogenetic analysis ofthe O. tauri 18S rDNA sequence (Fig. 1), comparedto homologous sequences of Chlorophytes, unam-biguously shows that O. tauri belongs to the Prasi-nophyceae and is most closely related to Mantoniellasquamata, with 100% bootstrap values using thethree methods: NJ, Parsimony, and ML. The treealso shows that Prasinophyceae are represented byfour clades, with at least two different species thatbelong, for the first three clades, to orders suggestedby Melkonian (1989), i.e. the Chlorodendrales, thePseudoscourfieldiales, and the Mamiellales, includ-ing now O. tauri. The fourth uncharacterized cladeis represented by two oceanic coccoid strains: CCPM

1407 and CCMP 1220. Strain CCPM 1407 (accessionnumber: U40919) has been identified as Prasinococ-cus cf. capsulatus from thin sections by M-J. C-D andL.G. (unpubl.). Strain CCPM 1220 (accession num-ber: U40920) is still morphologically unidentified.The prasinophycean coccoid species described to-day are phylogenetically characterized from rbcL or18S rDNA data. Pycnococcus provasolii Guillard et al.,first classified among the Mamiellales according toultrastructural features, is most closely affiliated toPseudoscourfieldiales behind rbcl (Daugbjerg et al.1995) and partial 18S rDNA (Knauber et al. 1996)data. The Prasinococcus genus Miyashita et Chihara(Miyashita et al. 1993) and Prasinoderma colonialeHasegawa et Chihara (Hasegawa et al. 1996) wereclassified in the Pycnococcaceae Guillard emendedMiyashita et Chihara among the Mamiellales. Our18S rDNA gene sequence of the CCMP 1407 strainshows that Prasinococcus capsulatus is clearly separat-ed from Mamiellales and should be included in anew order. Our tree (Fig. 1) complements the pra-sinophycean lineages from literature and also con-firms the usefulness of molecular data to clarify thetaxonomy of close coccoid species. This phyloge-

847PHYLOGENY OF OSTREOCOCCUS TAURI

FIG. 2. Pulsed field electrophoresis of Ostreococcus tauri DNA.Agarose gel has been stained with ethidium bromide. Lane ‘‘O.t.’’: Ostreococcus tauri chromosomes; Lane ‘‘Y’’: Saccharomyces cer-evisiae chromosomes. Band size markers in mb.

netic analysis is still indicative of the paraphyleticorigin of the Prasinophyceae as previously reportedby Steinkotter et al. (1994) and Daugbjerg et al.(1995).

Morphological data. Within Prasinophyceae, as cir-cumscribed by Melkonian (1989), different morpho-logical types are present. The most typical represen-tatives are found in Pseudoscourfieldiales with thepresence of two layers of organic scales on the fla-gella as well as body scales. Chlorodendrales have amodified cell covering, appearing as a theca, but stillhave two types of scales on their flagella. Mamiella-les, sensu Moestrup (Moestrup 1984), are structur-ally the most simple prasinophytes. A reduction offlagella appears in this order, with Mantoniella squa-mata, which has a little developed second flagellum,and Bathycoccus prasinos, which has lost its flagellabut is still covered by scales. In O. tauri, the lack ofscales and flagella is interpreted as two losses ofmorphological structures. A similar situation isfound in Chlorophyceae (i.e. reduction of flagella)with Nanochlorum eucaryotum (Wilhelm et al. 1982),a coccoid form that, although surrounded by asmooth and thin cell wall, is placed in the orderChlorococcales from molecular data (Schreiner etal. 1995). The phylogenetical analysis proposed here(Fig. 1) also shows that the coccoid morphologywithin Prasinophyta presents a paraphyletic originand is not restricted to Mamiellales.

Genome size. Pulsed field gel electrophoresis al-lowed the separation of 14 chromosomal bands inO. tauri’s genome (Fig. 2). The size of the bandsranged from 0.3 to 1.5 MegaBase (mb). They aredistributed among three size classes. The first classpresents three very small chromosomes rangingfrom 0.3 to 0.42 mb. The second class contains 10medium-sized bands ranging from 0.5 to 1.0 mb.The remaining band is related to one larger chro-mosome measuring 1.5 mb. The total nuclear ge-nome size for O. tauri is 10.2 mb, which is a quarterof that of Chlorella (Higashiyama and Yamada 1991).Several unicellular eukaryotes, such as Cyanidioschy-zon merolae, a red alga living in an acidic thermalenvironment (Maleska 1993), or the diplomonadGiardia lamblia (Fan et al. 1991), show a similar re-duced genome, with 11.7 mb and values from 10.6to 11.9 mb, respectively. Genomes of microsporidi-an species like Spraguea lophii and Encephalitozooncuniculi are smaller, with 6.2 and 2.9 mb, respectively(Biderre et al. 1994, 1995), but they are parasiticand, according to this particular physiological andecological status, some parts of their genetic con-tents may have been secondarily lost. When com-pared with prokaryotes, the genome size of O. tauriis only two or three times higher than that of Esch-erichia coli or the autotrophic Synechocystis strainPCC6803, which have only one chromosome mea-suring 4.7 and 3.51 mb, respectively (Kaneko et al.1996). The genome configuration is typically eu-karyotic in O. tauri with a fragmentation in several

chromosomes, some being very short. Two of them,measuring 0.30 and 0.35 mb, respectively, are small-er than the smallest chromosome described in C.merolae (0.41 mb, Maleska 1993).

CONCLUSION

Ostreococcus tauri is a prasinophycean coccoid, anew member of the Mamiellaceae without flagellaand scales, with an elementary ultrastructure and agenome appearing as one of the smallest amongfree-living organisms. Even very small, this genomeis fragmented in 14 short pieces, and such a frag-mentation could represent a functional nuclear con-figuration for a free-living autotrophic eukaryote. Inthe same way, Maleska (1993) suggested for C. mer-olae that genome fragmentation could be an adap-tative mechanism to its thermal-acidic environment.Potter et al. (1997) argue that ‘‘a tiny coccoid mor-phology confers some adaptative advantage in theopen ocean.’’ A similar hypothesis might also be val-id for O. tauri, which has a high growth rate and isnumerically dominant in the Thau lagoon through-out the year (Courties et al. 1994; Vaquer et al.1996). Finally, because it is now well established thatalgae emerged late in the eukaryotic tree (Perassoet al. 1989) and that members of the family Ma-miellaceae are secondary reduced forms (Daugbjerget al. 1995), these unique characters of O. tauri (i.e.small genome and cell size, elementary cell organi-zation) must be interpreted as evolutionarily derivedrather than as ancestral.

848 CLAUDE COURTIES ET AL.

We are grateful to Eric Causse and Bruno Baroux from AQUA-MER Company (Meze, France) as well as Jean Claude Baccou(University of Montpellier II, France) for culture monitoring.Many thanks to Nathalie Simon (Station Biologique of Roscoff,France) and to two anonymous referees for their fruitful com-ments. This work was supported by the Reseau Biodiversite(CNRS, ACC-SV 7) and the IFREMER URM n8 5 French pro-grams.

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