Vestigial Chloroplasts in Heterotrophic Stramenopiles Pteridomonas danica and Ciliophrys infusionum (Dictyochophyceae)

Protist, Vol. 153, 157–167, June 2002 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/protistPublished online 29 May 2002

Introduction

Organisms that secondarily lost photosynthetic abil-ity are known in various algal groups, including theChlorophyceae (e.g. Polytoma), Cryptophyceae (e.g.Chilomonas), Euglenophyceae (e.g. Astasia) andChrysophyceae (e.g. Spumella). Interestingly, mostof these non-photosynthetic algae retain the chloro-plast as a vestigial form known as the leucoplast.

It seems to be a common phenomenon in mostlineages of photosynthetic eukaryotes that organ-isms retain the chloroplast in a vestigial form, eventhough they lost photosynthetic ability. This is true

even in the Apicomplexa which lost the leucoplastand became obligate parasites as a whole taxon.Apicomplexans such as Plasmodium and Toxo-plasma retain reduced chloroplasts referred to asapicoplasts (Hopkins et al. 1999; McFadden et al.1996). When the chloroplast is retained as a leu-coplast, the chloroplast genome is also retained in areduced form. Astasia longa, a non-photosyntheticeuglenoid, retains a 73 kb leucoplast genome(Siemeister and Hachtel 1990) and apicomplexanshave been demonstrated to have a 35 kb genome inthe apicoplasts (Köhler et al. 1997; McFadden et al.1996). The complete sequences of the vestigialchloroplast genome have been determined forEpifagus virginiana (Orobanchaceae, land plant;

Vestigial Chloroplasts in Heterotrophic Stramenopiles Pteridomonas danicaand Ciliophrys infusionum (Dictyochophyceae)

Hiroshi Sekiguchi, Mayumi Moriya, Takeshi Nakayama, and Isao Inouye1

Institute of Biological Sciences, University of Tsukuba, 1-1-1, Tennoudai, Tsukuba, Ibaraki 305-8572, Japan

Submitted January 30, 2002; Accepted March 22, 2002Monitoring Editor: Robert A. Andersen

Two heterotrophic members of the Dictyochophyceae (stramenopiles), Pteridomonas danica and Cil-iophrys infusionum, were investigated. An undescribed organelle bounded by four membranes andclosely associated with the nucleus was detected in P. danica. The outermost membrane was contin-uous with the outer nuclear membrane. These features strongly suggested that this organelle was avestigial chloroplast. A photosynthetic gene, rbcL, was successfully amplified by polymerase chainreaction (PCR) from P. danica and C. infusionum. These sequences were readily and well aligned withthose of photosynthetic stramenopiles. Phylogenetic trees of 18S rDNA and rbcL were constructed.In all the trees obtained, P. danica and C. infusionum appeared in two different clades, the Pedinel-lales clade and the Ciliophryales/Rhizochromulinales clade, each of which contained photosyntheticmembers as well as heterotrophic members. The results indicated that the loss of photosyntheticability occurred independently in P. danica and C. infusionum. This is the first report of the presenceof a vestigial chloroplast (leucoplast) in colorless dictyochophytes.

ORIGINAL PAPER

1 Corresponding author;fax 81-298-53-6614e-mail [email protected]

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Wolfe et al. 1992), A. longa (Gockel and Hachtel2000) and Plasmodium falciparum (Wilson et al.1996). In contrast, the leucoplast genome is not wellinvestigated in other algal groups and nothing hasbeen reported for the stramenopiles.

In the stramenopiles, the leucoplast is known inthe Chrysophyceae, Spumella spp., Paraphyso-monas spp. and Anthophysa vegetans (Belcher andSwale 1972, 1976; Mignot 1977; Preisig and Hib-berd 1982b). In each case, the leucoplast isbounded by four membranes and the outermostmembrane is continuous with the nuclear mem-brane as seen in the typical chloroplast of the stra-menopile algae (= heterokont algae). In contrast,several members of the stramenopiles such as thebicosoecids, labyrinthulids, oomycetes, protero-monads and Developayella have neither the chloro-plast nor the leucoplast. These organisms are be-lieved to be ancestral lineages of the stramenopilesthat diverged before the ancestor of the heterokontalgae acquired the chloroplast via secondary en-dosymbiosis. Their ancestral phylogenetic positionhas been suggested by molecular phylogeneticstudies (e.g., Guillou et al. 1999; Moriya et al. 2000).Therefore, heterotrophic stramenopiles should fallinto two different categories, those that never ac-quired the chloroplast and those that did but thensecondarily lost their photosynthetic ability.

The Dictyochophyceae is a distinct class of thestramenopiles and contains both photosynthetic andheterotrophic members. The heterotrophic membersinclude four genera, Actinomonas, Pteridomonas,Parapedinella (Pedinellales) and Ciliophrys (Cilio-phryales). It has been thought that these organismscompletely lack the chloroplast and that they haveno trace of a leucoplast (Cavalier-Smith 1992; Cava-lier-Smith et al. 1995/96; Patterson 1989) so thatthey had once been considered as a candidate of theancestor of the heterokont algae (Patterson 1986).However, recent phylogenetic analyses of nuclear-encoded 18S rDNA suggested that the heterotrophicmembers of the Dictyochophyceae are not an earlydivergence of the stramenopiles, but they have beenderived from photosynthetic members by the loss ofphotosynthetic ability (Cavalier-Smith 1995; Cava-lier-Smith and Chao 1996). If this is true, the het-erotrophic dictyochophytes are exceptions of thecommon phenomenon that the chloroplast remainsas a vestigial form after the loss of photosyntheticability. Have these dictyochophytes truly lost thechloroplast completely? Has the chloroplast genomealso been lost entirely? There has not been any evi-dence to answer these questions and the “com-plete” loss of the chloroplasts in the heterotrophicdictyochophytes is still questionable.

In this paper, ultrastructural observations andmolecular analyses were conducted on two het-erotrophic dictyochophytes, Pteridomonas danicaPatterson & Fenchel 1985 (Pedinellales) and Cilio-phrys infusionum Cienkowki 1875 (Ciliophryales) inorder to answer the above questions.

158 H. Sekiguchi et al.

Figures 1–4. Pteridomonas danica. 1. An attachedcell with posterior stalk, showing anterior tentacles(Tc) and flagellum (F)(DIC). scale bar = 3 µm. 2. Flagel-lum (F) bearing tripartite flagellar hairs and flagellarscales (arrow)(uranyl acetate stained material). Scalebar = 200 nm. 3. Longitudinal section of the cell show-ing proximal helix (arrows) situated below transitionalplate. Scale bar = 200 nm. 4. Transverse section ofanterior region of the cell, showing proximal helix(arrow) and microtubular triads (arrowheads). Scalebar = 500 nm.

ResultsGeneral Characteristics and IdentificationPteridomonas danicaThe organism is spherical to oval, 4–6 µm in lengthand 3–5 µm in width. In the anterior view, the cellshows a radially symmetrical architecture. A singleflagellum arises from the anterior end of the cell, andabout 8 to 10 tentacles are situated around the flag-ellum (Fig. 1). A fine stalk of variable length arisesfrom the posterior end of the cell (Fig. 1). At the lightmicroscopical level, there is no indication of chloro-plasts, and no autofluorescence of chlorophylls isdetected under the fluorescence microscope. Thecell is usually free-swimming but sometimes sessile,attaching to the substratum with the posterior stalk.Small annular flagellar scales (about 80 nm in diame-ter) are present (Fig. 2). These scales are indistin-guishable from those of Pteridomonas, Actinomonasand Parapedinella reported previously (Larsen 1985;Moestrup 1995; Pedersen et al. 1986). In the flagellar

transition region, there is a helical structure proximalto the basal plate (proximal helix in Honda et al.1995), a diagnostic feature to distinguish the genusPteridomonas from Actinomonas (Larsen 1985; Pat-terson and Fenchel 1985; Figs 3, 4). These featuresare congruent with those of Pteridomonas danica(Patterson and Fenchel 1985). In addition, sequencecomparison of 18S rDNA for the aligned regionshowed 98% similarity between P. danica (L37204;Cavalier-Smith et al. 1995) and the organism exam-ined here. Furthermore, the 18S rDNA phylogenetictree showed that these two organisms formed aclade with 100% bootstrap support (Fig. 5). The or-ganism was therefore assigned to P. danica.

Ciliophrys infusionumThe organism is amoeboid, about 6–12 µm long,and fine pseudopodia radiate from the cell surface(Fig. 6). There is no sign of chloroplasts under thelight microscope. Pseudopodia are unbranched,and there is no intercellular association of pseu-

Dictyochophycean Leucoplast 159

Figure 5. NJ tree based on 18S rRNA sequences (HKY85 model) of dictyochophycean algae. Bootstrap valuesare given in order (MP/NJ/ML). The bootstrap values are represented by “–” for less than 50% clades. Asterisksrepresent the organisms sequenced in this study. The substitution model for ML analysis was TrN+I+G model,determined by MODELTEST.

dopodia (Fig. 6), that is, cells are always solitary.Amoeboid movement does not occur. Bead-likestructures are present on the pseudopodia (Fig. 6).They do not move along the pseudopodia. Theamoeboid cell possesses one flagellum that eitherdoes not move or undulates very slowly (Fig. 6). Theflagellum often shows the shape of “8“.

The amoeboid cell transforms into the motile cellfollowing mechanical stimuli such as tapping on aglass slide. The motile cell is elongated, teardropshaped and about 7–18 µm in length. It has a singleflagellum arising from the acute anterior tip of thecell (Fig. 7). Flagellar scales are absent (Figs 8, 9).These features are congruent with general featuresof Ciliophrys infusionum (Cienkowski 1875; David-son 1982). The 18S rDNA sequences (1814 bases)of the organism showed 95% similarity for thealigned regions with those of the organism identifiedas C. infusionum (L37205) (Cavalier-Smith et al.1995). The phylogenetic tree showed that these twoorganisms formed a clade with 100% bootstrapsupport (Fig. 5). Therefore, the organism was as-signed to Ciliophrys infusionum.

Undescribed Organelle in Pteridomonas danica

In the cell of Pteridomonas danica, an undescribedstructure bounded by four membranes is presentalong the nucleus (Fig. 10). It is oval, about 400 nmin length and 200 nm in width (Figs. 10, 11). Theinner two membranes are closely appressed to eachother and arranged in parallel, and form an oval pro-file of the structure (Fig. 11). The space enclosed bythese two membranes contains small granules ofabout 8 nm (Fig. 12). These are slightly smaller thannuclear and cytoplasmic ribosomes and about thesame size as mitochondrial ribosomes so that theseare identified as ribosomes. No other distinct struc-ture has been detected in this organelle (Fig. 12).The third membrane, lying outside these two mem-branes, is often protruding to the nucleus (Fig. 11).Vesicular structures are present between the sec-ond and the third membranes (Fig. 13). The fourth(outermost) membrane is rough and continuous withthe outer nuclear envelope (Figs 10, 11).

We tried to detect a similar organelle in Ciliophrysinfusionum using electron microscopy but withoutsuccess.

rbcL in Pteridomonas and Ciliophrys

Using the primers for rbcL, DNA was amplified fromtwo heterotrophic dictyochophytes, Pteridomonasdanica and Ciliophrys infusionum. We determined1154 bases of the amplified DNA of P. danica and1421 bases of C. infusionum. These sequences werealigned with rbcL sequences of phototrophic dicty-ochophytes, Apedinella radians, Pseudopedinellaelastica, Rhizochromulina CCMP 237 and Pedinellasp. The rbcL sequences of both P. danica and C. infu-


Figures 6–9. Ciliophrys infusionum. 6. Amoeboid cellshowing radiating pseudopodia (Ps) and emergentflagellum (F). Arrow shows bead-like structures on theflagellum (Video image) scale bar = 5 µm. 7. Swimmingcell showing flagellum (F) (Video image). Scale bar = 3µm. 8. Whole-mount preparation of swimming cell,showing tripartite flagellar hairs (uranyl acetate stainedmaterial). Scale bar = 2 µm. 9. High magnification offlagellum and tripartite flagellar hairs. Note that flagellarscale are absent (uranyl acetate stained material).Scale bar = 200 nm.


Figures 10–13. Pteridomonas danica. 10. Transverse section of the cell showing leucoplast (asterisk) and majororganelles; nucleus (N), mitochondrion (M) and Golgi body (G). The nuclear envelope and the outermost leu-coplast membrane are continuous (arrowheads). Microtubular triads extend from the nuclear envelope (arrow).Scale bar = 1 µm. 11. Leucoplast appressed to inner two membranes. (Large arrows: innermost membrane, largearrowheads: second membrane.) Amorphous mass between the second and third membrane (small arrows) isvisible (asterisk). The outermost membrane (small arrowheads) is continuous with nuclear envelope. Scale bar =500 nm. 12. Leucoplast, containing the ribosomes (small arrows). Scale bar = 200 nm. 13. Vesicular structure sit-uated between the second and third membrane (arrow). Scale bar = 500 nm.

sionum aligned very well with those of other dicty-ochophytes, suggesting that they are not pseudo-genes and that they do not contain introns or a stopcodon. Amino acid sequences were inferred fromthese DNA sequences. With respect to the amino acidsequences, P. danica was most similar to Pedinellasp. (97% similarity, for the aligned region), and C. infu-sionum was most similar to Rhizochromulina CCMP237 (96% similarity, for the aligned region).

Phylogenetic Analysis

18S rDNA sequences were determined forPteridomonas danica, Ciliophrys infusionum andPedinella sp. and aligned with available sequencesof six dictyochophytes. MP, NJ and ML trees of 18SrDNA and rbcL were constructed (Figs 5, 14).Topologies were different between 18S rDNA andrbcL trees, though these were the same betweenthe trees of each gene constructed by differentmethods. NJ trees of 18S rDNA and rbcL are givenin Figures 5 and 14.

In the 18S rDNA tree, Dictyocha speculum formsan independent clade from all other dicty-ochophytes. Except for D. speculum, both 18SrDNA and rbcL trees generated two clades in thedictyochophytes. One corresponded to the orderPedinellales and the other to the Ciliophryales andRhizochromulinales. Reliability of these two cladeswas 100% in 18S rDNA trees but not high in rbcLtrees. Pteridomonas danica and Ciliophrys infu-sionum appeared separately in these two clades.

Topology within the Pedinellales clade was differ-ent between 18S rDNA and rbcL trees. In the 18SrDNA tree, Pteridomonas danica was the sister toApedinella radians (100% bootstrap support). Pseu-dopedinella elastica and Pedinella sp. became anoutgroup of the clade of P. danica and A. radians(Fig. 5). In rbcL trees, P. danica was most closely re-lated to Pedinella sp. (MP: 99%, NJ: 92%, ML: –),and A. radians and Pseudopedinella elasticashowed close relationship supported by high bootstrap values (NJ: 96%, MP: 100%, ML: 96%; Fig. 14).


Figure 14. NJ tree based on rbcL sequences (HKY85 model) of dictyochophycean algae. Bootstrap values aregiven in order (MP/NJ/ML). The bootstrap values are represented by “–” for less than 50% clades. Asterisks rep-resent the organisms sequenced in this study. The substitution model for ML analysis was GTR+I+G model, de-termined by MODELTEST.

Discussion

The organelle lying along the nucleus and boundedby four membranes in Pteridomonas danica is un-doubtedly a vestigial chloroplast (leucoplast), be-cause it possesses various features common withthe leucoplast of heterotrophic chrysophytes(Belcher and Swale 1972, 1976; Mignot 1977;Preisig and Hibberd 1982b) and the chloroplasts ofheterokont algae in general (Hibberd 1976; Preisigand Hibberd 1982b). The chloroplast of heterokontalgae is typically encircled by four membranes. Theinner two are chloroplast membranes (inner chloro-plast membrane (ICM) and outer chloroplast mem-brane (OCM) and the outer two are simply calledchloroplast ER or in different terms, periplastidmembrane (PPM) or chloroplast endoplasmic reticu-lum (CER). The CER is continuous with the nuclearenvelope and between the OCM and PPM are tubu-lar structures called periplastidal reticula (Cavalier-Smith 1989; Gibbs 1981). The leucoplasts of het-erotrophic chrysophytes, including Paraphyso-monas spp. (Preisig and Hibberd 1982b), Spumellaspp. (Belcher and Swale 1976; Mignot 1977) andAnthophysa vegetans (Müller) Stein (1878)(Belcherand Swale 1972) are also encircled by four mem-branes and the outermost one is continuous withthe nuclear envelope. In the leucoplast of these het-erotrophic chrysophytes, there is no trace of thy-lakoids, but small granules assignable to ribosomesare present (see Fig. 14 in Belcher and Swale 1972;Fig. 11 in Belcher and Swale 1976; Figs 8F, 9D, 10Band 16A in Preisig and Hibberd 1982b). In the leu-coplast of A. vegetans and Spumella elongata(Stokes) Belcher & Swale, periplastidal reticula(PPR) are present in the periplastidal compartment(Belcher and Swale 1972, 1976). Similar structuresare also present in the organelle of P. danica be-tween the second and the third membrane (Fig. 13).In all the aspects mentioned above, the undescribedorganelle detected in Pteridomonas danica resem-bles the leucoplast of Paraphysomonas spp., A. vegetans and Spumella spp. Therefore, this struc-ture is concluded to be a vestigial chloroplast (leu-coplast) that has been overlooked in previous stud-ies probably because of its small size (ca. 400 nmlong).

Because rbcL is located in the chloroplastgenome in all the photosynthetic eukaryotes so farinvestigated, it is likely that rbcL is also localized inthe leucoplast genome in Pteridomonas danica andCiliophrys infusionum. Moreover, we used theprimers, NDrbcL2 and DPrbcL7, designed by Daug-bjerg and Andersen (1997) for the first PCR. Theprimer DPrbcL7 was designed to anneal at the end

of rbcS (Daugbjerg and Andersen 1997). The factthat these primers amplified rbcL in P. danica and C.infusionum indicates that rbcS is also present nextto rbcL in the same genome. In all the heterokontalgae so far investigated, rbcL and rbcS are en-coded in the chloroplast genome (Delaney et al.1995). It would therefore be reasonable to concludethat, in P. danica and C. infusionum, rbcL is encodedin the chloroplast genome, and the genome shouldbe located in the leucoplast.

The morphological and molecular evidence pre-sented here indicate that both Pteridomonas danicaand Ciliophrys infusionum lost photosynthetic abilitysecondarily as suggested from nuclear-encodedgene analyses (18S rDNA) (Cavalier-Smith and Chao1996; Cavalier-Smith et al. 1995), and that, at leastPteridomonas danica still retains the chloroplast in avestigial form. A previous statement about “com-plete” loss of the chloroplast in dictyochophytesshould therefore be corrected. We have not yet suc-ceeded to detect a vestigial chloroplast in Ciliophrysinfusionum. However, more careful observationsshould prove that the leucoplast is also present inthis organism.

The 18S rDNA trees clearly indicated that the dic-tyochophytes examined in this study were membersof two distinct clades, the Pedinellales clade and theCiliophryales/Rhizochromulinales clade, andPteridomonas danica was included in the Pedinel-lales clade and Ciliophrys infusionum was in the Cil-iophryales/Rhizochromulinales clade. These twoclades are clearly distinct and both contain photo-synthetic taxa. This implies that these two het-erotrophic dictyochophytes lost their photosyntheticability independently.

Actinomonas spp. and Parapedinella reticulataPedersen et al. (1986) are heterotrophic taxa placedin the Pedinellales. Because molecular data are notavailable for these dictyochophytes, it is not easy todiscuss whether or not they also lost photosyntheticability independently. However, our cladistic analy-sis based on light microscopical and ultrastructuraldata as well as behavioral properties (e.g. method ofprey capture) suggested they are closely related toPteridomonas danica (Sekiguchi et al. in prep.). Itconsequently suggests monophyly of these het-erotrophic taxa and a single loss of photosyntheticability in the Pedinellales clade. The flagellar scalesof indistinguishable morphology may be a synapo-morphy of the clade. Therefore, it is likely that pho-tosynthetic ability was lost twice in the Dicty-ochophyceae, once in the Pedinellales clade andonce in the Ciliophryales/Rhizochromulinales clade.

Recently, complete genomes of vestigial chloro-plast (leucoplast genomes) have been determined


for several taxa (Gockel and Hachtel 2000; McFad-den et al. 1997; Wolfe et al. 1992). From these stud-ies, it is clear that the coded genes in the leucoplastgenome are largely different depending on the taxa.In Epifagus virginiana (L.) Barton, an epiphytic landplant, the 70 kbp leucoplast genome containshouse-keeping genes, and the genes related to pho-tosynthesis are retained as pseudogenes or werelost (DePamphilis and Palmer 1989; Wolfe et al.1992). The apicomplexan Toxoplasma gondii has a35 kb leucoplast (apicoplast) genome. 68 genes areencoded in the apicoplast, and most of them (about60) are house-keeping genes. Genes related to pho-tosynthesis are completely lost in T. gondii (Wilsonet al. 1996). It has recently been demonstrated inthese organisms that the apicoplast is responsiblefor fatty acid synthesis and the shikimate pathway(Roberts et al. 1998; Waller et al. 1998). In Astasialonga, the leucoplast genome is 73 kbp. The 16SrRNA gene in one of three copies of the tandem re-peat (rrnC) is the only pseudogene in the genome(Gockel and Hachtel 2000), and transcripts of vari-ous genes such as rbcL and tufA have been de-tected (Siemeister et al. 1990). Such considerabledifferences of encoded genes among the leucoplastgenomes suggest that the leucoplasts have differentroles in these organisms.

Genes related to photosynthesis are usually lostor become nonfunctional pseudogenes even if theleucoplast is retained. The rbcL gene is an excep-tion. It is retained intact in various organisms thatlost their photosynthetic ability. In a holoparasiticland plant, Lathraea clandestina, rbcL is expressedand functional RuBisCO is formed (Lusson et al.1998). Wolfe and DePamphilis (1997) addressedseveral possibilities about the presence of Ru-BisCO: carbon fixation may proceed to a minimumextent; RuBisCO may act as an oxydase, withglycine and serine forming via the glycolate path-way; RuBisCO genes may be on the way to beinglost or to becoming pseudogenes. Astasia longa isthe sole example among algae that has an intactrbcL and forms functional RuBisCO. Although itsactual role is uncertain, it is likely that, in L. clandes-tina and A. longa, the RuBisCO in the leucoplast isinvolved in some cellular activity other than photo-synthesis. Pteridomonas danica and Ciliophrys infu-sionum also retain intact rbcL. Their amino acid se-quences are highly conserved: similarity is 97% be-tween P. danica and Pedinella sp. and 96% betweenC. infusionum and Rhizochromulina CCMP237. Whythese heterotrophic organisms still retain rbcL is asenigmatic as in L. clandestina and A. longa. How-ever, the presence of highly conserved intact rbcL inPteridomonas danica and Ciliophrys infusionum

suggests that RuBisCO also exists and has a certainrole in the leucoplast. This should be studied inten-sively in the future.

Methods

Materials: Pteridomonas danica was collected fromYokohama Bay, Kanagawa Prefecture, Japan inMay, 1995. Ciliophrys infusionum was collectedfrom Otaru Port Hokkaido, Japan in August, 1996.Pedinella sp. (a detailed description of this organismwill be published elsewhere) was collected from off-shore of Kinkazan (Pacific Ocean), Miyagi Prefec-ture, Japan in September 1993.

The organisms isolated with micropipettes wereincubated into 20 ml test tubes or 100 ml Erlen-meyer flasks using URO-YT medium (Moriya et al.2000) for Pteridomonas danica and Ciliophrys infu-sionum and ESM medium (Watanabe et al. 2000) forPedinella sp. Cultures were maintained at 20 °Cunder light of 15 µEm–2s–1 and in a 14:10 hr light:dark regime. The culture of Ciliophrys infusionumwas recently lost. Pteridomonas danica is availablefrom the authors upon request.

Light microscopy: For light microscopy of flagel-late cells, samples were mixed with an equal volumeof 5% glutaraldehyde (GA) prepared in 0.2 Msodium cacodylate buffer (SCB) (pH 7.2) immedi-ately before observations were made. Cells wereobserved with an OPTIPHOT XF-NT with Nomarskidifferential interference contrast optics (DIC) (Nikon,Tokyo, Japan).

Electron microscopy: Samples fixed in 5% GAin seawater were mounted on mesh-grids and leftfor 5 min to allow cells settle on the formvar film. Theexcess medium was removed with filter paper, and asmall amount of 2% uranyl acetate was added. 30sec later, the uranyl acetate was removed with filterpaper. Samples were then dried and used for obser-vation.

For thin sections, a fixative containing 5% GA, 0.8M sucrose and 10 mM EGTA prepared in 0.2 M SCBwas mixed with a sample of equal volume and themixture was kept at 4 °C for 6 hrs. Cells were har-vested by centrifugation at 3000 rpm for 10 min andrinsed three times in SCB (5 min each). After the re-moval of SCB, 1% OsO4 in 0.2 M SCB was added tothe sample, and cells were post-fixed for 2 hrs at 4 °C. Samples were rinsed, dehydrated using agraded ethanol series (1hr each for 30 and 50%ethanol; 30 min each for 75, 90 and 95% ethanol; 15 min for absolute ethanol, repeated four times).The sample was then transferred to Spurr’s resin


(Spurr 1969) through immersion in propylene oxideand polymerized overnight at 70 °C. Sections weremade with an EM-ULTRACUT-S ultramicrotome(Reichert, Germany), double-stained with 2% uranylacetate and Reynolds’ lead. Observations weremade with a JEM-1010 transmission electron micro-scope (JEOL, Tokyo, Japan).

DNA extraction and sequencing: Cells ofPteridomonas danica, Ciliophrys infusionum, andPedinella sp. were harvested by centrifugation andlysed by UNSET buffer (Garriga et al. 1984). TotalDNA was extracted by a standard phenol/chloro-form method (Sambrook et al. 1989). Then the DNAwas amplified by polymerase chain reaction (PCR)(Saiki et al. 1988). The primers for 18S rDNA werethe same as in Nakayama et al. (1998). We adoptedsome primers for rbcL analysis reported by Daugb-jerg and Andersen (1997); however, two primerswere designed to replace NDrbcL4 and NDrbcL5(Daugbjerg and Andersen 1997). These were rbcLF(5′-CWGCWTCWATYAWYGGWAACG-3′) and rbcL5′(5′-CWCAASCWTTYATGCG-3′). DNA amplificationwas done with a thermal cycler QTP-1 (Nippon Ge-netica Corp., Tokyo, Japan) for 28 repetitions at 93°C for 1 min, 50 °C for 2 min and 72 °C for 3 min. The second PCR was done under the sameconditions, and the primer sets were the same asNakayama et al. (1998) for 18S rDNA and Daugbjergand Andersen (1997) for rbcL. After removing theprimer from the PCR products using polyetheleneglycol (PEG), the double-stranded PCR productswere sequenced directly by ABI Prism 377 DNA Se-quencer (Perkin-Elmer Corp., United Kingdom)using Dye-Terminator Cycle Sequencing Core Kit(Perkin-Elmer Corp., United Kingdom) according tothe manufacturer’s instructions. Accession numbersdeposited in the DDBJ/EMBL/GenBank nucleotidesequence database were as follows (given in order18S rDNA and rbcL): Pteridomonas danica(AB081640/AB081642), Ciliophrys infusionum(AB081641/AB081643) and Pedinella sp.(AB081517/ AB081639).

Sequence analyses: The sequences obtainedwere aligned with available sequence data usingCLUSTAL X computer program (Thompson et al.1997). The sequences used are as follows (numbersin parentheses are accession numbers of 18S rDNAand rbcL): Pseudopedinella elastica (U14387/U89899), Apedinella radians (U14384/AF015573),Pteridomonas danica (L37204/this study), Ciliophrysinfusionum (L37205/this study), Rhizochromulina cf.marina (U14388/-), Rhizochromulina sp. CCMP237(-/AF015574), Dictyocha speculum (U14385/-).

Pelagomonas calceolata (U14389/U89898), Chro-mulina nebulosa (AF123285/AF155876) and Rhi-zosolenia setigera (M87329/ AF015568) were cho-sen as an outgroup. In 18S rDNA analyses, ambigu-ous sites were excluded. The alignment is availablefrom the corresponding author upon request.

Three kinds of algorithms were employed to con-struct phylogenetic trees, neighbor-joining (NJ)(Saitou and Nei 1987), maximum parsimony (MP)and maximum likelihood (ML) methods. For MLanalyses, the substitution model was determinedusing MODELTEST (version 3.06; Posada and Cran-dall 1998). Each tree was constructed with PAUP*(version 4.0.8) computer program (Swofford 2001).Bootstrap analyses (Felsenstein 1985) were carriedout for each analysis with 100 replications.

Acknowledgements

We especially thank Dr. Masanobu Kawachi, Na-tional Institute for Environmental Studies, Tsukubafor providing Pedinella sp., and Dr. Daiske Honda,Konan University, Kobe for helpful suggestions. Thisstudy was supported by JSPS grants JSPS-RFTF00L0162 and 12440239.

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