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Vestigial Chloroplasts in Heterotrophic Stramenopiles Pteridomonas danica and Ciliophrys infusionum (Dictyochophyceae)
Protist
Protist, Vol. 153, 157–167, June 2002 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/protist Published online 29 May 2002
ORIGINAL PAPER
Vestigial Chloroplasts in Heterotrophic Stramenopiles Pteridomonas danica and 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, 2002 Monitoring Editor: Robert A. Andersen
Two heterotrophic members of the Dictyochophyceae (stramenopiles), Pteridomonas danica and Ciliophrys infusionum, were investigated. An undescribed organelle bounded by four membranes and closely associated with the nucleus was detected in P. danica. The outermost membrane was continuous with the outer nuclear membrane. These features strongly suggested that this organelle was a vestigial chloroplast. A photosynthetic gene, rbcL, was successfully amplified by polymerase chain reaction (PCR) from P. danica and C. infusionum. These sequences were readily and well aligned with those 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 Pedinellales clade and the Ciliophryales/Rhizochromulinales clade, each of which contained photosynthetic members as well as heterotrophic members. The results indicated that the loss of photosynthetic ability occurred independently in P. danica and C. infusionum. This is the first report of the presence of a vestigial chloroplast (leucoplast) in colorless dictyochophytes.
Introduction Organisms that secondarily lost photosynthetic ability are known in various algal groups, including the Chlorophyceae (e.g. Polytoma), Cryptophyceae (e.g. Chilomonas), Euglenophyceae (e.g. Astasia) and Chrysophyceae (e.g. Spumella). Interestingly, most of these non-photosynthetic algae retain the chloroplast as a vestigial form known as the leucoplast. It seems to be a common phenomenon in most lineages of photosynthetic eukaryotes that organisms retain the chloroplast in a vestigial form, even though they lost photosynthetic ability. This is true 1 Corresponding author; fax 81-298-53-6614 e-mail [email protected]
even in the Apicomplexa which lost the leucoplast and became obligate parasites as a whole taxon. Apicomplexans such as Plasmodium and Toxoplasma retain reduced chloroplasts referred to as apicoplasts (Hopkins et al. 1999; McFadden et al. 1996). When the chloroplast is retained as a leucoplast, the chloroplast genome is also retained in a reduced form. Astasia longa, a non-photosynthetic euglenoid, retains a 73 kb leucoplast genome (Siemeister and Hachtel 1990) and apicomplexans have been demonstrated to have a 35 kb genome in the apicoplasts (Köhler et al. 1997; McFadden et al. 1996). The complete sequences of the vestigial chloroplast genome have been determined for Epifagus virginiana (Orobanchaceae, land plant; 1434-4610/02/153/02-157 $ 15.00/0
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Wolfe et al. 1992), A. longa (Gockel and Hachtel 2000) and Plasmodium falciparum (Wilson et al. 1996). In contrast, the leucoplast genome is not well investigated in other algal groups and nothing has been reported for the stramenopiles. In the stramenopiles, the leucoplast is known in the Chrysophyceae, Spumella spp., Paraphysomonas spp. and Anthophysa vegetans (Belcher and Swale 1972, 1976; Mignot 1977; Preisig and Hibberd 1982b). In each case, the leucoplast is bounded by four membranes and the outermost membrane is continuous with the nuclear membrane as seen in the typical chloroplast of the stramenopile algae (= heterokont algae). In contrast, several members of the stramenopiles such as the bicosoecids, labyrinthulids, oomycetes, proteromonads and Developayella have neither the chloroplast nor the leucoplast. These organisms are believed to be ancestral lineages of the stramenopiles that diverged before the ancestor of the heterokont algae acquired the chloroplast via secondary endosymbiosis. Their ancestral phylogenetic position has been suggested by molecular phylogenetic studies (e.g., Guillou et al. 1999; Moriya et al. 2000). Therefore, heterotrophic stramenopiles should fall into two different categories, those that never acquired the chloroplast and those that did but then secondarily lost their photosynthetic ability. The Dictyochophyceae is a distinct class of the stramenopiles and contains both photosynthetic and heterotrophic members. The heterotrophic members include four genera, Actinomonas, Pteridomonas, Parapedinella (Pedinellales) and Ciliophrys (Ciliophryales). It has been thought that these organisms completely lack the chloroplast and that they have no trace of a leucoplast (Cavalier-Smith 1992; Cavalier-Smith et al. 1995/96; Patterson 1989) so that they had once been considered as a candidate of the ancestor of the heterokont algae (Patterson 1986). However, recent phylogenetic analyses of nuclearencoded 18S rDNA suggested that the heterotrophic members of the Dictyochophyceae are not an early divergence of the stramenopiles, but they have been derived from photosynthetic members by the loss of photosynthetic ability (Cavalier-Smith 1995; Cavalier-Smith and Chao 1996). If this is true, the heterotrophic dictyochophytes are exceptions of the common phenomenon that the chloroplast remains as a vestigial form after the loss of photosynthetic ability. Have these dictyochophytes truly lost the chloroplast completely? Has the chloroplast genome also been lost entirely? There has not been any evidence to answer these questions and the “complete” loss of the chloroplasts in the heterotrophic dictyochophytes is still questionable.
In this paper, ultrastructural observations and molecular analyses were conducted on two heterotrophic dictyochophytes, Pteridomonas danica Patterson & Fenchel 1985 (Pedinellales) and Ciliophrys infusionum Cienkowki 1875 (Ciliophryales) in order to answer the above questions.
Figures 1–4. Pteridomonas danica. 1. An attached cell with posterior stalk, showing anterior tentacles (Tc) and flagellum (F)(DIC). scale bar = 3 µm. 2. Flagellum (F) bearing tripartite flagellar hairs and flagellar scales (arrow)(uranyl acetate stained material). Scale bar = 200 nm. 3. Longitudinal section of the cell showing proximal helix (arrows) situated below transitional plate. Scale bar = 200 nm. 4. Transverse section of anterior region of the cell, showing proximal helix (arrow) and microtubular triads (arrowheads). Scale bar = 500 nm.
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Results General Characteristics and Identification Pteridomonas danica The organism is spherical to oval, 4–6 µm in length and 3–5 µm in width. In the anterior view, the cell shows a radially symmetrical architecture. A single flagellum arises from the anterior end of the cell, and about 8 to 10 tentacles are situated around the flagellum (Fig. 1). A fine stalk of variable length arises from the posterior end of the cell (Fig. 1). At the light microscopical level, there is no indication of chloroplasts, and no autofluorescence of chlorophylls is detected under the fluorescence microscope. The cell is usually free-swimming but sometimes sessile, attaching to the substratum with the posterior stalk. Small annular flagellar scales (about 80 nm in diameter) are present (Fig. 2). These scales are indistinguishable from those of Pteridomonas, Actinomonas and Parapedinella reported previously (Larsen 1985; Moestrup 1995; Pedersen et al. 1986). In the flagellar
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transition region, there is a helical structure proximal to the basal plate (proximal helix in Honda et al. 1995), a diagnostic feature to distinguish the genus Pteridomonas from Actinomonas (Larsen 1985; Patterson and Fenchel 1985; Figs 3, 4). These features are congruent with those of Pteridomonas danica (Patterson and Fenchel 1985). In addition, sequence comparison of 18S rDNA for the aligned region showed 98% similarity between P. danica (L37204; Cavalier-Smith et al. 1995) and the organism examined here. Furthermore, the 18S rDNA phylogenetic tree showed that these two organisms formed a clade with 100% bootstrap support (Fig. 5). The organism was therefore assigned to P. danica. Ciliophrys infusionum The 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 the light microscope. Pseudopodia are unbranched, and there is no intercellular association of pseu-
Figure 5. NJ tree based on 18S rRNA sequences (HKY85 model) of dictyochophycean algae. Bootstrap values are given in order (MP/NJ/ML). The bootstrap values are represented by “–” for less than 50% clades. Asterisks represent the organisms sequenced in this study. The substitution model for ML analysis was TrN+I+G model, determined by MODELTEST.
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dopodia (Fig. 6), that is, cells are always solitary. Amoeboid movement does not occur. Bead-like structures are present on the pseudopodia (Fig. 6). They do not move along the pseudopodia. The amoeboid cell possesses one flagellum that either does not move or undulates very slowly (Fig. 6). The flagellum often shows the shape of “8“.
The amoeboid cell transforms into the motile cell following mechanical stimuli such as tapping on a glass slide. The motile cell is elongated, teardrop shaped and about 7–18 µm in length. It has a single flagellum arising from the acute anterior tip of the cell (Fig. 7). Flagellar scales are absent (Figs 8, 9). These features are congruent with general features of Ciliophrys infusionum (Cienkowski 1875; Davidson 1982). The 18S rDNA sequences (1814 bases) of the organism showed 95% similarity for the aligned regions with those of the organism identified as C. infusionum (L37205) (Cavalier-Smith et al. 1995). The phylogenetic tree showed that these two organisms formed a clade with 100% bootstrap support (Fig. 5). Therefore, the organism was assigned to Ciliophrys infusionum.
Undescribed Organelle in Pteridomonas danica In the cell of Pteridomonas danica, an undescribed structure bounded by four membranes is present along the nucleus (Fig. 10). It is oval, about 400 nm in length and 200 nm in width (Figs. 10, 11). The inner two membranes are closely appressed to each other and arranged in parallel, and form an oval profile of the structure (Fig. 11). The space enclosed by these two membranes contains small granules of about 8 nm (Fig. 12). These are slightly smaller than nuclear and cytoplasmic ribosomes and about the same size as mitochondrial ribosomes so that these are identified as ribosomes. No other distinct structure has been detected in this organelle (Fig. 12). The third membrane, lying outside these two membranes, is often protruding to the nucleus (Fig. 11). Vesicular structures are present between the second and the third membranes (Fig. 13). The fourth (outermost) membrane is rough and continuous with the outer nuclear envelope (Figs 10, 11). We tried to detect a similar organelle in Ciliophrys infusionum using electron microscopy but without success.
rbcL in Pteridomonas and Ciliophrys Figures 6–9. Ciliophrys infusionum. 6. Amoeboid cell showing radiating pseudopodia (Ps) and emergent flagellum (F). Arrow shows bead-like structures on the flagellum (Video image) scale bar = 5 µm. 7. Swimming cell showing flagellum (F) (Video image). Scale bar = 3 µm. 8. Whole-mount preparation of swimming cell, showing tripartite flagellar hairs (uranyl acetate stained material). Scale bar = 2 µm. 9. High magnification of flagellum and tripartite flagellar hairs. Note that flagellar scale are absent (uranyl acetate stained material). Scale bar = 200 nm.
Using the primers for rbcL, DNA was amplified from two heterotrophic dictyochophytes, Pteridomonas danica and Ciliophrys infusionum. We determined 1154 bases of the amplified DNA of P. danica and 1421 bases of C. infusionum. These sequences were aligned with rbcL sequences of phototrophic dictyochophytes, Apedinella radians, Pseudopedinella elastica, Rhizochromulina CCMP 237 and Pedinella sp. The rbcL sequences of both P. danica and C. infu-
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Figures 10–13. Pteridomonas danica. 10. Transverse section of the cell showing leucoplast (asterisk) and major organelles; nucleus (N), mitochondrion (M) and Golgi body (G). The nuclear envelope and the outermost leucoplast 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, large arrowheads: second membrane.) Amorphous mass between the second and third membrane (small arrows) is visible (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 situated between the second and third membrane (arrow). Scale bar = 500 nm.
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sionum aligned very well with those of other dictyochophytes, suggesting that they are not pseudogenes and that they do not contain introns or a stop codon. Amino acid sequences were inferred from these DNA sequences. With respect to the amino acid sequences, P. danica was most similar to Pedinella sp. (97% similarity, for the aligned region), and C. infusionum was most similar to Rhizochromulina CCMP 237 (96% similarity, for the aligned region).
Phylogenetic Analysis 18S rDNA sequences were determined for Pteridomonas danica, Ciliophrys infusionum and Pedinella sp. and aligned with available sequences of six dictyochophytes. MP, NJ and ML trees of 18S rDNA and rbcL were constructed (Figs 5, 14). Topologies were different between 18S rDNA and rbcL trees, though these were the same between the trees of each gene constructed by different methods. NJ trees of 18S rDNA and rbcL are given in Figures 5 and 14.
In the 18S rDNA tree, Dictyocha speculum forms an independent clade from all other dictyochophytes. Except for D. speculum, both 18S rDNA and rbcL trees generated two clades in the dictyochophytes. One corresponded to the order Pedinellales and the other to the Ciliophryales and Rhizochromulinales. Reliability of these two clades was 100% in 18S rDNA trees but not high in rbcL trees. Pteridomonas danica and Ciliophrys infusionum appeared separately in these two clades. Topology within the Pedinellales clade was different between 18S rDNA and rbcL trees. In the 18S rDNA tree, Pteridomonas danica was the sister to Apedinella radians (100% bootstrap support). Pseudopedinella elastica and Pedinella sp. became an outgroup of the clade of P. danica and A. radians (Fig. 5). In rbcL trees, P. danica was most closely related to Pedinella sp. (MP: 99%, NJ: 92%, ML: –), and A. radians and Pseudopedinella elastica showed 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 are given in order (MP/NJ/ML). The bootstrap values are represented by “–” for less than 50% clades. Asterisks represent the organisms sequenced in this study. The substitution model for ML analysis was GTR+I+G model, determined by MODELTEST.
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Discussion The organelle lying along the nucleus and bounded by four membranes in Pteridomonas danica is undoubtedly a vestigial chloroplast (leucoplast), because it possesses various features common with the leucoplast of heterotrophic chrysophytes (Belcher and Swale 1972, 1976; Mignot 1977; Preisig and Hibberd 1982b) and the chloroplasts of heterokont algae in general (Hibberd 1976; Preisig and Hibberd 1982b). The chloroplast of heterokont algae is typically encircled by four membranes. The inner two are chloroplast membranes (inner chloroplast membrane (ICM) and outer chloroplast membrane (OCM) and the outer two are simply called chloroplast ER or in different terms, periplastid membrane (PPM) or chloroplast endoplasmic reticulum (CER). The CER is continuous with the nuclear envelope and between the OCM and PPM are tubular structures called periplastidal reticula (CavalierSmith 1989; Gibbs 1981). The leucoplasts of heterotrophic chrysophytes, including Paraphysomonas spp. (Preisig and Hibberd 1982b), Spumella spp. (Belcher and Swale 1976; Mignot 1977) and Anthophysa vegetans (Müller) Stein (1878)(Belcher and Swale 1972) are also encircled by four membranes and the outermost one is continuous with the nuclear envelope. In the leucoplast of these heterotrophic chrysophytes, there is no trace of thylakoids, but small granules assignable to ribosomes are present (see Fig. 14 in Belcher and Swale 1972; Fig. 11 in Belcher and Swale 1976; Figs 8F, 9D, 10B and 16A in Preisig and Hibberd 1982b). In the leucoplast of A. vegetans and Spumella elongata (Stokes) Belcher & Swale, periplastidal reticula (PPR) are present in the periplastidal compartment (Belcher and Swale 1972, 1976). Similar structures are also present in the organelle of P. danica between the second and the third membrane (Fig. 13). In all the aspects mentioned above, the undescribed organelle detected in Pteridomonas danica resembles the leucoplast of Paraphysomonas spp., A. vegetans and Spumella spp. Therefore, this structure is concluded to be a vestigial chloroplast (leucoplast) that has been overlooked in previous studies probably because of its small size (ca. 400 nm long). Because rbcL is located in the chloroplast genome in all the photosynthetic eukaryotes so far investigated, it is likely that rbcL is also localized in the leucoplast genome in Pteridomonas danica and Ciliophrys infusionum. Moreover, we used the primers, NDrbcL2 and DPrbcL7, designed by Daugbjerg and Andersen (1997) for the first PCR. The primer DPrbcL7 was designed to anneal at the end
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of rbcS (Daugbjerg and Andersen 1997). The fact that these primers amplified rbcL in P. danica and C. infusionum indicates that rbcS is also present next to rbcL in the same genome. In all the heterokont algae so far investigated, rbcL and rbcS are encoded in the chloroplast genome (Delaney et al. 1995). It would therefore be reasonable to conclude that, in P. danica and C. infusionum, rbcL is encoded in the chloroplast genome, and the genome should be located in the leucoplast. The morphological and molecular evidence presented here indicate that both Pteridomonas danica and Ciliophrys infusionum lost photosynthetic ability secondarily as suggested from nuclear-encoded gene analyses (18S rDNA) (Cavalier-Smith and Chao 1996; Cavalier-Smith et al. 1995), and that, at least Pteridomonas danica still retains the chloroplast in a vestigial form. A previous statement about “complete” loss of the chloroplast in dictyochophytes should therefore be corrected. We have not yet succeeded to detect a vestigial chloroplast in Ciliophrys infusionum. However, more careful observations should prove that the leucoplast is also present in this organism. The 18S rDNA trees clearly indicated that the dictyochophytes examined in this study were members of two distinct clades, the Pedinellales clade and the Ciliophryales/Rhizochromulinales clade, and Pteridomonas danica was included in the Pedinellales clade and Ciliophrys infusionum was in the Ciliophryales/Rhizochromulinales clade. These two clades are clearly distinct and both contain photosynthetic taxa. This implies that these two heterotrophic dictyochophytes lost their photosynthetic ability independently. Actinomonas spp. and Parapedinella reticulata Pedersen et al. (1986) are heterotrophic taxa placed in the Pedinellales. Because molecular data are not available for these dictyochophytes, it is not easy to discuss whether or not they also lost photosynthetic ability independently. However, our cladistic analysis based on light microscopical and ultrastructural data as well as behavioral properties (e.g. method of prey capture) suggested they are closely related to Pteridomonas danica (Sekiguchi et al. in prep.). It consequently suggests monophyly of these heterotrophic taxa and a single loss of photosynthetic ability in the Pedinellales clade. The flagellar scales of indistinguishable morphology may be a synapomorphy of the clade. Therefore, it is likely that photosynthetic ability was lost twice in the Dictyochophyceae, once in the Pedinellales clade and once in the Ciliophryales/Rhizochromulinales clade. Recently, complete genomes of vestigial chloroplast (leucoplast genomes) have been determined
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for several taxa (Gockel and Hachtel 2000; McFadden et al. 1997; Wolfe et al. 1992). From these studies, it is clear that the coded genes in the leucoplast genome are largely different depending on the taxa. In Epifagus virginiana (L.) Barton, an epiphytic land plant, the 70 kbp leucoplast genome contains house-keeping genes, and the genes related to photosynthesis are retained as pseudogenes or were lost (DePamphilis and Palmer 1989; Wolfe et al. 1992). The apicomplexan Toxoplasma gondii has a 35 kb leucoplast (apicoplast) genome. 68 genes are encoded in the apicoplast, and most of them (about 60) are house-keeping genes. Genes related to photosynthesis are completely lost in T. gondii (Wilson et al. 1996). It has recently been demonstrated in these organisms that the apicoplast is responsible for fatty acid synthesis and the shikimate pathway (Roberts et al. 1998; Waller et al. 1998). In Astasia longa, the leucoplast genome is 73 kbp. The 16S rRNA gene in one of three copies of the tandem repeat (rrnC) is the only pseudogene in the genome (Gockel and Hachtel 2000), and transcripts of various genes such as rbcL and tufA have been detected (Siemeister et al. 1990). Such considerable differences of encoded genes among the leucoplast genomes suggest that the leucoplasts have different roles in these organisms. Genes related to photosynthesis are usually lost or become nonfunctional pseudogenes even if the leucoplast is retained. The rbcL gene is an exception. It is retained intact in various organisms that lost their photosynthetic ability. In a holoparasitic land plant, Lathraea clandestina, rbcL is expressed and functional RuBisCO is formed (Lusson et al. 1998). Wolfe and DePamphilis (1997) addressed several possibilities about the presence of RuBisCO: carbon fixation may proceed to a minimum extent; RuBisCO may act as an oxydase, with glycine and serine forming via the glycolate pathway; RuBisCO genes may be on the way to being lost or to becoming pseudogenes. Astasia longa is the sole example among algae that has an intact rbcL and forms functional RuBisCO. Although its actual role is uncertain, it is likely that, in L. clandestina and A. longa, the RuBisCO in the leucoplast is involved in some cellular activity other than photosynthesis. Pteridomonas danica and Ciliophrys infusionum also retain intact rbcL. Their amino acid sequences are highly conserved: similarity is 97% between P. danica and Pedinella sp. and 96% between C. infusionum and Rhizochromulina CCMP237. Why these heterotrophic organisms still retain rbcL is as enigmatic as in L. clandestina and A. longa. However, the presence of highly conserved intact rbcL in Pteridomonas danica and Ciliophrys infusionum
suggests that RuBisCO also exists and has a certain role in the leucoplast. This should be studied intensively in the future.
Methods Materials: Pteridomonas danica was collected from Yokohama Bay, Kanagawa Prefecture, Japan in May, 1995. Ciliophrys infusionum was collected from Otaru Port Hokkaido, Japan in August, 1996. Pedinella sp. (a detailed description of this organism will be published elsewhere) was collected from offshore of Kinkazan (Pacific Ocean), Miyagi Prefecture, Japan in September 1993. The organisms isolated with micropipettes were incubated into 20 ml test tubes or 100 ml Erlenmeyer flasks using URO-YT medium (Moriya et al. 2000) for Pteridomonas danica and Ciliophrys infusionum and ESM medium (Watanabe et al. 2000) for Pedinella sp. Cultures were maintained at 20 °C under light of 15 µEm–2s–1 and in a 14:10 hr light: dark regime. The culture of Ciliophrys infusionum was recently lost. Pteridomonas danica is available from the authors upon request. Light microscopy: For light microscopy of flagellate cells, samples were mixed with an equal volume of 5% glutaraldehyde (GA) prepared in 0.2 M sodium cacodylate buffer (SCB) (pH 7.2) immediately before observations were made. Cells were observed with an OPTIPHOT XF-NT with Nomarski differential interference contrast optics (DIC) (Nikon, Tokyo, Japan). Electron microscopy: Samples fixed in 5% GA in seawater were mounted on mesh-grids and left for 5 min to allow cells settle on the formvar film. The excess medium was removed with filter paper, and a small amount of 2% uranyl acetate was added. 30 sec later, the uranyl acetate was removed with filter paper. Samples were then dried and used for observation. For thin sections, a fixative containing 5% GA, 0.8 M sucrose and 10 mM EGTA prepared in 0.2 M SCB was mixed with a sample of equal volume and the mixture was kept at 4 °C for 6 hrs. Cells were harvested by centrifugation at 3000 rpm for 10 min and rinsed three times in SCB (5 min each). After the removal of SCB, 1% OsO4 in 0.2 M SCB was added to the sample, and cells were post-fixed for 2 hrs at 4 °C. Samples were rinsed, dehydrated using a graded 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
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(Spurr 1969) through immersion in propylene oxide and polymerized overnight at 70 °C. Sections were made with an EM-ULTRACUT-S ultramicrotome (Reichert, Germany), double-stained with 2% uranyl acetate and Reynolds’ lead. Observations were made with a JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan). DNA extraction and sequencing: Cells of Pteridomonas danica, Ciliophrys infusionum, and Pedinella sp. were harvested by centrifugation and lysed by UNSET buffer (Garriga et al. 1984). Total DNA was extracted by a standard phenol/chloroform method (Sambrook et al. 1989). Then the DNA was amplified by polymerase chain reaction (PCR) (Saiki et al. 1988). The primers for 18S rDNA were the same as in Nakayama et al. (1998). We adopted some primers for rbcL analysis reported by Daugbjerg and Andersen (1997); however, two primers were designed to replace NDrbcL4 and NDrbcL5 (Daugbjerg and Andersen 1997). These were rbcLF (5′-CWGCWTCWATYAWYGGWAACG-3′) and rbcL5′ (5′-CWCAASCWTTYATGCG-3′). DNA amplification was done with a thermal cycler QTP-1 (Nippon Genetica 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 same conditions, and the primer sets were the same as Nakayama et al. (1998) for 18S rDNA and Daugbjerg and Andersen (1997) for rbcL. After removing the primer from the PCR products using polyethelene glycol (PEG), the double-stranded PCR products were sequenced directly by ABI Prism 377 DNA Sequencer (Perkin-Elmer Corp., United Kingdom) using Dye-Terminator Cycle Sequencing Core Kit (Perkin-Elmer Corp., United Kingdom) according to the manufacturer’s instructions. Accession numbers deposited in the DDBJ/EMBL/GenBank nucleotide sequence database were as follows (given in order 18S rDNA and rbcL): Pteridomonas danica (AB081640/AB081642), Ciliophrys infusionum (AB081641/AB081643) and Pedinella sp. (AB081517/ AB081639). Sequence analyses: The sequences obtained were aligned with available sequence data using CLUSTAL X computer program (Thompson et al. 1997). The sequences used are as follows (numbers in parentheses are accession numbers of 18S rDNA and rbcL): Pseudopedinella elastica (U14387/ U89899), Apedinella radians (U14384/AF015573), Pteridomonas danica (L37204/this study), Ciliophrys infusionum (L37205/this study), Rhizochromulina cf. marina (U14388/-), Rhizochromulina sp. CCMP237 (-/AF015574), Dictyocha speculum (U14385/-).
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Pelagomonas calceolata (U14389/U89898), Chromulina nebulosa (AF123285/AF155876) and Rhizosolenia setigera (M87329/ AF015568) were chosen as an outgroup. In 18S rDNA analyses, ambiguous sites were excluded. The alignment is available from the corresponding author upon request. Three kinds of algorithms were employed to construct phylogenetic trees, neighbor-joining (NJ) (Saitou and Nei 1987), maximum parsimony (MP) and maximum likelihood (ML) methods. For ML analyses, the substitution model was determined using MODELTEST (version 3.06; Posada and Crandall 1998). Each tree was constructed with PAUP* (version 4.0.8) computer program (Swofford 2001). Bootstrap analyses (Felsenstein 1985) were carried out for each analysis with 100 replications.
Acknowledgements We especially thank Dr. Masanobu Kawachi, National Institute for Environmental Studies, Tsukuba for providing Pedinella sp., and Dr. Daiske Honda, Konan University, Kobe for helpful suggestions. This study was supported by JSPS grants JSPSRFTF00L0162 and 12440239.
References Belcher JH, Swale EMF (1972) The morphology and fine structure of the colourless colonial flagellate Anthophysa vegetans (O. F. Müller) Stein. Br Phycol J 7: 335–346 Belcher JH, Swale EMF (1976) Spumella elongata (Stokes) nov. comb., a colourless flagellate from soil. Arch Protistenkd 118: 215–220 Cavalier-Smith T (1989) The Kingdom Chromista. In Green JC, Leadbeater BSC, Diver WL (eds) The Chromophyte Algae. Clarendon Press, Oxford, pp 381–407 Cavalier-Smith T (1992) The origin, losses and gains of chloroplasts. In Lewin RA (eds) Origins of Plastids. Chapman & Hall, New York, London, pp 291–348 Cavalier-Smith T (1995) Evolutionary protistology comes of age: biodiversity and molecular cell biology. Arch Protistenkd 145: 145–154 Cavalier-Smith T, Chao EE (1996) 18S rRNA sequence of Heterosigma carterae (Raphidophyceae), and the phylogeny of heterokont algae (Ochrophyta). Phycologia 35: 500–510 Cavalier-Smith T, Chao EE, Allsopp MTEP (1995) Ribosomal RNA evidence for chloroplast loss within Heterokonta: Pedinellid relationships and revised classification of ochristan algae. Arch Protistenkd 145: 209–220
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Cavalier-Smith T, Chao EE, Thompson CE, Hourihane SL (1995/96) Oikomonas, a distinctive zooflagellate related to chrysomonads. Arch Protistenkd 146: 273–279
Hopkins J, Fowler R, Krishna S, Wilson I, Mitchell G, Bannister L (1999) The plastid in Plasmodium falciparum asexual blood stages: a three-dimensional ultrastructural analysis. Protist 150: 283–295
Cienkowski L (1875) Über einge Rhizopoden und verwandte Organismen. Arch Mikroskop Anat 12: 15–50
Köhler S, Delwiche CF, Denny PW, Tilney LG, Webster P, Wilson RJM, Palmer JD, Roos DS (1997) A plastid of probable green algal origin in apicomplexan parasites. Science 275: 1485–1489
Daugbjerg N, Andersen RA (1997) A molecular phylogeny of the heterokont algae based on analyses of chloroplast-encoded rbcL sequence data. J Phycol 33: 1031–1041 Davidson LA (1982) Ultrastructure, behavior, and algal flagellate affinities of the helioflagellate Ciliophrys marina, and the classification of the helioflagellates (Protista, Actinopoda, Heliozoa). J Protozool 29: 19–29 Delaney TP, Hardison LK, Cattolico RA (1995) Evolution of Plastid Genomes: Inferences from Discordant Molecular Phylogenies. In Sandgren CD, Smol JP, Kristiansen J (eds) Chrysophyte Algae: Ecology, Phylogeny and Development. Cambridge University Press, Cambridge, pp 25–45 DePamphilis CW, Palmer JD (1989) Evolution and Function of Plastid DNA: a Review with Special Reference to Nonphotosynthetic Plants. In Boyer CD, Shannon JC, Hardison RC (eds) Physiology, Biochemistry, and Genetics of Nongreen Plastids. The American Society of Plant Physiologists, Rockville, pp 182–202 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791 Garriga GH, Bertrandt H, Lambowitz A (1984) RNA splicing in Neurospora mitochondria: nuclear mutants defective in both splicing and 3’ end synthesis of large rRNA. Cell 36: 623–634 Gibbs SP (1981) The chloroplast endoplasmic reticulum: structure, function, and evolutionary significance. Int Rev Cytol 72: 49–99 Gockel G, Hachtel W (2000) Complete gene map of the plastid genome of the nonphotosynthetic euglenoid flagellate Astasia longa. Protist 151: 347–351 Guillou L, Chrétiennot-Dinet MJ, Boulben S, Moonvan der Staay SY, Vaulot D (1999) Symbiomonas scintillans gen. et sp. nov. and Picophagus flagellatus gen. et sp. nov. (Heterokonta): two new heterotrophic flagellates of picoplanktonic size. Protist 150: 383–398 Hibberd DJ (1976) The ultrastructure and taxonomy of the Chrysophyceae and Prymnesiophyceae (Haptophyceae): a survey with some new observations on the ultrastructure of the Chrysophyceae. Bot J Linn Soc 72: 55–80 Honda D, Kawachi M, Inouye I (1995) Sulcochrysis biplastida gen. et sp. nov.: cell structure and absolute configuration of the flagellar apparatus of an enigmatic chromophyte alga. Phycol Res 43: 1–16
Larsen J (1985) Ultrastructure and taxonomy of Actinomonas pusilla, a heterotrophic member of the Pedinellales (Chrysophyceae). Br Phycol J 20: 341–355 Lusson NA, Delavault PM, Thalouarn PA (1998) The rbcL gene from the non-photosynthetic parasite Lathraea clandestima is not transcribed by a plastid-encoded RNA polymerase. Curr Genet 34: 212–215 McFadden GI, Reith ME, Munholland J, Lang-Unnasch N (1996) Plastid in human parasites. Nature 381: 482 McFadden GI, Waller RE, Reith ME, Lang-Unnasch N (1997) Plastids in apicomplexan parasites. Plant Syst Evol 11: 261–287 Mignot JP (1977) Etude ultrastructurale d’un flagellé du genre Spumella Cienk. (= Heterochromonas Pascher = Monas O. F. Müller), Chrysononadine leucoplastidiée. Protistologica 13: 219–231 Moestrup Ø (1995) Current Status of Chrysophyte ‘Splinter Groups’: Synurophytes, Pedinellids, Silicoflagellates. In Sandgren CD, Smol JP, Kristiansen J (eds) Chrysophyte Algae: Ecology, Phylogeny and Development. Cambridge University Press, Cambridge, pp 75–91 Moriya M, Nakayama T, Inouye I (2000) Ultrastructure and 18S rDNA sequence analysis of Wobblia lunata gen. et sp. nov., a new heterotrophic flagellate (Stramenopiles, Incertae Sedis). Protist 151: 41–55 Nakayama T, Marin B, Kranz HD, Surek B, Huss VAR, Inouye I, Melkonian M (1998) The basal position of scaly green flagellates among the green algae (Chlorophyta) is revealed by analyses of nuclear-encoded SSU rRNA sequences. Protist 149: 367–380 Patterson DJ (1986) The Actinophryid Heliozoa (Sarcodina, Actinopoda) as Chromophytes. In Kristiansen J, Andersen RA (eds) Chrysophytes: Aspects and Problems. Cambridge University Press, Cambridge, pp 49–67 Patterson DJ (1989) Stramenopiles: Chromophytes from a Protistan Perspective. In Green JC, Leadbeater BSC, Diver WL (eds) The Chromophyte Algae, Problems and Perspectives. Clarendon press, Oxford, pp 357–379 Patterson DJ, Fenchel T (1985) Insights into the evolution of heliozoa (Protozoa, Sarcodina) as provided by ultrastructural studies on a new species of flagellate from the genus Pteridomonas. Biol J Linn Soc 34: 381–403
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Pedersen SM, Beech PL, Thomsen HA (1986) Parapedinella reticulata gen. et sp. nov. (Chrysophyceae) from Danish waters. Nord J Bot 6: 507–513 Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817–818 Preisig HR, Hibberd DJ (1982a) Ultrastructure and taxonomy of Paraphysomonas (Chrysophyceae) and related genera 1. Nord J Bot 2: 397–420 Preisig HR, Hibberd DJ (1982b) Ultrastructure and taxonomy of Paraphysomonas (Chrysophyceae) and related genera 3. Nord J Bot 3: 695–723 Roberts F, Roberts CW, Johnson JJ, Kyle DE, Krell T, Coggins JR, Coombs GH, Milhous WK, Tzipori S, Ferguson DJP, Chakrabarti D, McLeod R (1998) Evidence for the shikimate pathway in apicomplexan parasites. Nature 393: 801–805 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Ehrlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425 Sambrook J, Fritch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York Siemeister G, Buchholz C, Hachtel W (1990) Genes for the plastid elongation factor Tu and ribosomal protein S7 and six tRNA genes on the 73 kb DNA from Astasia longa that resembles the chloroplast DNA of Euglena. Mol Gen Genet 220: 425–432 Siemeister G, Hachtel W (1990) Structure and expression of a gene encoding the large subunit of ribulose1,5–bisphosphate carboxylase (rbcL) in the colourless
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euglenoid flagellate Astasia longa. Plant Mol Biol 14: 825–833 Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastr Res 26: 31–43 Swofford DL (2001) PAUP* 4.0b8. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts Thompson JD, Gibson TJ, Plewniak K, Jeanmougin F, Higgins DG (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24: 4876–4882 Waller RF, Keeling PJ, Donald RGK, Striepen B, Handman E, Lang-Unnasch N, Cowman AF, Besra GS, Roos DS, McFadden GI (1998) Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. Proc Natl Acad Sci USA 95: 12352–12357 Watanabe MM, Kawachi M, Hiroki M, Kasai F (2000) NIES-Collection List of Strains. Microalgae and Protozoa. NIES, Japan Wilson RJM, Denny PW, Preiser PR, Rangachari K, Roberts K, Roy A, Whyte A, Strath M, Moore DJ, Moore PW, Williamson DH (1996) Complete gene map of the plastid-like DNA of the malaria parasite Plasmodium falciparum. J Mol Biol 261: 155–172 Wolfe AD, DePamphilis W (1997) Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche. Plant Mol Biol 33: 965–977 Wolfe KH, Morden CW, Palmer JD (1992) Function and evolution of a minimal plastid genome from a nonphotosynthetic parasitic plant. Proc Natl Acad Sci USA 89: 10648–10652
Protist, Vol. 153, 157–167, June 2002 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/protist Published online 29 May 2002
ORIGINAL PAPER
Vestigial Chloroplasts in Heterotrophic Stramenopiles Pteridomonas danica and 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, 2002 Monitoring Editor: Robert A. Andersen
Two heterotrophic members of the Dictyochophyceae (stramenopiles), Pteridomonas danica and Ciliophrys infusionum, were investigated. An undescribed organelle bounded by four membranes and closely associated with the nucleus was detected in P. danica. The outermost membrane was continuous with the outer nuclear membrane. These features strongly suggested that this organelle was a vestigial chloroplast. A photosynthetic gene, rbcL, was successfully amplified by polymerase chain reaction (PCR) from P. danica and C. infusionum. These sequences were readily and well aligned with those 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 Pedinellales clade and the Ciliophryales/Rhizochromulinales clade, each of which contained photosynthetic members as well as heterotrophic members. The results indicated that the loss of photosynthetic ability occurred independently in P. danica and C. infusionum. This is the first report of the presence of a vestigial chloroplast (leucoplast) in colorless dictyochophytes.
Introduction Organisms that secondarily lost photosynthetic ability are known in various algal groups, including the Chlorophyceae (e.g. Polytoma), Cryptophyceae (e.g. Chilomonas), Euglenophyceae (e.g. Astasia) and Chrysophyceae (e.g. Spumella). Interestingly, most of these non-photosynthetic algae retain the chloroplast as a vestigial form known as the leucoplast. It seems to be a common phenomenon in most lineages of photosynthetic eukaryotes that organisms retain the chloroplast in a vestigial form, even though they lost photosynthetic ability. This is true 1 Corresponding author; fax 81-298-53-6614 e-mail [email protected]
even in the Apicomplexa which lost the leucoplast and became obligate parasites as a whole taxon. Apicomplexans such as Plasmodium and Toxoplasma retain reduced chloroplasts referred to as apicoplasts (Hopkins et al. 1999; McFadden et al. 1996). When the chloroplast is retained as a leucoplast, the chloroplast genome is also retained in a reduced form. Astasia longa, a non-photosynthetic euglenoid, retains a 73 kb leucoplast genome (Siemeister and Hachtel 1990) and apicomplexans have been demonstrated to have a 35 kb genome in the apicoplasts (Köhler et al. 1997; McFadden et al. 1996). The complete sequences of the vestigial chloroplast genome have been determined for Epifagus virginiana (Orobanchaceae, land plant; 1434-4610/02/153/02-157 $ 15.00/0
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Wolfe et al. 1992), A. longa (Gockel and Hachtel 2000) and Plasmodium falciparum (Wilson et al. 1996). In contrast, the leucoplast genome is not well investigated in other algal groups and nothing has been reported for the stramenopiles. In the stramenopiles, the leucoplast is known in the Chrysophyceae, Spumella spp., Paraphysomonas spp. and Anthophysa vegetans (Belcher and Swale 1972, 1976; Mignot 1977; Preisig and Hibberd 1982b). In each case, the leucoplast is bounded by four membranes and the outermost membrane is continuous with the nuclear membrane as seen in the typical chloroplast of the stramenopile algae (= heterokont algae). In contrast, several members of the stramenopiles such as the bicosoecids, labyrinthulids, oomycetes, proteromonads and Developayella have neither the chloroplast nor the leucoplast. These organisms are believed to be ancestral lineages of the stramenopiles that diverged before the ancestor of the heterokont algae acquired the chloroplast via secondary endosymbiosis. Their ancestral phylogenetic position has been suggested by molecular phylogenetic studies (e.g., Guillou et al. 1999; Moriya et al. 2000). Therefore, heterotrophic stramenopiles should fall into two different categories, those that never acquired the chloroplast and those that did but then secondarily lost their photosynthetic ability. The Dictyochophyceae is a distinct class of the stramenopiles and contains both photosynthetic and heterotrophic members. The heterotrophic members include four genera, Actinomonas, Pteridomonas, Parapedinella (Pedinellales) and Ciliophrys (Ciliophryales). It has been thought that these organisms completely lack the chloroplast and that they have no trace of a leucoplast (Cavalier-Smith 1992; Cavalier-Smith et al. 1995/96; Patterson 1989) so that they had once been considered as a candidate of the ancestor of the heterokont algae (Patterson 1986). However, recent phylogenetic analyses of nuclearencoded 18S rDNA suggested that the heterotrophic members of the Dictyochophyceae are not an early divergence of the stramenopiles, but they have been derived from photosynthetic members by the loss of photosynthetic ability (Cavalier-Smith 1995; Cavalier-Smith and Chao 1996). If this is true, the heterotrophic dictyochophytes are exceptions of the common phenomenon that the chloroplast remains as a vestigial form after the loss of photosynthetic ability. Have these dictyochophytes truly lost the chloroplast completely? Has the chloroplast genome also been lost entirely? There has not been any evidence to answer these questions and the “complete” loss of the chloroplasts in the heterotrophic dictyochophytes is still questionable.
In this paper, ultrastructural observations and molecular analyses were conducted on two heterotrophic dictyochophytes, Pteridomonas danica Patterson & Fenchel 1985 (Pedinellales) and Ciliophrys infusionum Cienkowki 1875 (Ciliophryales) in order to answer the above questions.
Figures 1–4. Pteridomonas danica. 1. An attached cell with posterior stalk, showing anterior tentacles (Tc) and flagellum (F)(DIC). scale bar = 3 µm. 2. Flagellum (F) bearing tripartite flagellar hairs and flagellar scales (arrow)(uranyl acetate stained material). Scale bar = 200 nm. 3. Longitudinal section of the cell showing proximal helix (arrows) situated below transitional plate. Scale bar = 200 nm. 4. Transverse section of anterior region of the cell, showing proximal helix (arrow) and microtubular triads (arrowheads). Scale bar = 500 nm.
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Results General Characteristics and Identification Pteridomonas danica The organism is spherical to oval, 4–6 µm in length and 3–5 µm in width. In the anterior view, the cell shows a radially symmetrical architecture. A single flagellum arises from the anterior end of the cell, and about 8 to 10 tentacles are situated around the flagellum (Fig. 1). A fine stalk of variable length arises from the posterior end of the cell (Fig. 1). At the light microscopical level, there is no indication of chloroplasts, and no autofluorescence of chlorophylls is detected under the fluorescence microscope. The cell is usually free-swimming but sometimes sessile, attaching to the substratum with the posterior stalk. Small annular flagellar scales (about 80 nm in diameter) are present (Fig. 2). These scales are indistinguishable from those of Pteridomonas, Actinomonas and Parapedinella reported previously (Larsen 1985; Moestrup 1995; Pedersen et al. 1986). In the flagellar
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transition region, there is a helical structure proximal to the basal plate (proximal helix in Honda et al. 1995), a diagnostic feature to distinguish the genus Pteridomonas from Actinomonas (Larsen 1985; Patterson and Fenchel 1985; Figs 3, 4). These features are congruent with those of Pteridomonas danica (Patterson and Fenchel 1985). In addition, sequence comparison of 18S rDNA for the aligned region showed 98% similarity between P. danica (L37204; Cavalier-Smith et al. 1995) and the organism examined here. Furthermore, the 18S rDNA phylogenetic tree showed that these two organisms formed a clade with 100% bootstrap support (Fig. 5). The organism was therefore assigned to P. danica. Ciliophrys infusionum The 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 the light microscope. Pseudopodia are unbranched, and there is no intercellular association of pseu-
Figure 5. NJ tree based on 18S rRNA sequences (HKY85 model) of dictyochophycean algae. Bootstrap values are given in order (MP/NJ/ML). The bootstrap values are represented by “–” for less than 50% clades. Asterisks represent the organisms sequenced in this study. The substitution model for ML analysis was TrN+I+G model, determined by MODELTEST.
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dopodia (Fig. 6), that is, cells are always solitary. Amoeboid movement does not occur. Bead-like structures are present on the pseudopodia (Fig. 6). They do not move along the pseudopodia. The amoeboid cell possesses one flagellum that either does not move or undulates very slowly (Fig. 6). The flagellum often shows the shape of “8“.
The amoeboid cell transforms into the motile cell following mechanical stimuli such as tapping on a glass slide. The motile cell is elongated, teardrop shaped and about 7–18 µm in length. It has a single flagellum arising from the acute anterior tip of the cell (Fig. 7). Flagellar scales are absent (Figs 8, 9). These features are congruent with general features of Ciliophrys infusionum (Cienkowski 1875; Davidson 1982). The 18S rDNA sequences (1814 bases) of the organism showed 95% similarity for the aligned regions with those of the organism identified as C. infusionum (L37205) (Cavalier-Smith et al. 1995). The phylogenetic tree showed that these two organisms formed a clade with 100% bootstrap support (Fig. 5). Therefore, the organism was assigned to Ciliophrys infusionum.
Undescribed Organelle in Pteridomonas danica In the cell of Pteridomonas danica, an undescribed structure bounded by four membranes is present along the nucleus (Fig. 10). It is oval, about 400 nm in length and 200 nm in width (Figs. 10, 11). The inner two membranes are closely appressed to each other and arranged in parallel, and form an oval profile of the structure (Fig. 11). The space enclosed by these two membranes contains small granules of about 8 nm (Fig. 12). These are slightly smaller than nuclear and cytoplasmic ribosomes and about the same size as mitochondrial ribosomes so that these are identified as ribosomes. No other distinct structure has been detected in this organelle (Fig. 12). The third membrane, lying outside these two membranes, is often protruding to the nucleus (Fig. 11). Vesicular structures are present between the second and the third membranes (Fig. 13). The fourth (outermost) membrane is rough and continuous with the outer nuclear envelope (Figs 10, 11). We tried to detect a similar organelle in Ciliophrys infusionum using electron microscopy but without success.
rbcL in Pteridomonas and Ciliophrys Figures 6–9. Ciliophrys infusionum. 6. Amoeboid cell showing radiating pseudopodia (Ps) and emergent flagellum (F). Arrow shows bead-like structures on the flagellum (Video image) scale bar = 5 µm. 7. Swimming cell showing flagellum (F) (Video image). Scale bar = 3 µm. 8. Whole-mount preparation of swimming cell, showing tripartite flagellar hairs (uranyl acetate stained material). Scale bar = 2 µm. 9. High magnification of flagellum and tripartite flagellar hairs. Note that flagellar scale are absent (uranyl acetate stained material). Scale bar = 200 nm.
Using the primers for rbcL, DNA was amplified from two heterotrophic dictyochophytes, Pteridomonas danica and Ciliophrys infusionum. We determined 1154 bases of the amplified DNA of P. danica and 1421 bases of C. infusionum. These sequences were aligned with rbcL sequences of phototrophic dictyochophytes, Apedinella radians, Pseudopedinella elastica, Rhizochromulina CCMP 237 and Pedinella sp. The rbcL sequences of both P. danica and C. infu-
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Figures 10–13. Pteridomonas danica. 10. Transverse section of the cell showing leucoplast (asterisk) and major organelles; nucleus (N), mitochondrion (M) and Golgi body (G). The nuclear envelope and the outermost leucoplast 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, large arrowheads: second membrane.) Amorphous mass between the second and third membrane (small arrows) is visible (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 situated between the second and third membrane (arrow). Scale bar = 500 nm.
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sionum aligned very well with those of other dictyochophytes, suggesting that they are not pseudogenes and that they do not contain introns or a stop codon. Amino acid sequences were inferred from these DNA sequences. With respect to the amino acid sequences, P. danica was most similar to Pedinella sp. (97% similarity, for the aligned region), and C. infusionum was most similar to Rhizochromulina CCMP 237 (96% similarity, for the aligned region).
Phylogenetic Analysis 18S rDNA sequences were determined for Pteridomonas danica, Ciliophrys infusionum and Pedinella sp. and aligned with available sequences of six dictyochophytes. MP, NJ and ML trees of 18S rDNA and rbcL were constructed (Figs 5, 14). Topologies were different between 18S rDNA and rbcL trees, though these were the same between the trees of each gene constructed by different methods. NJ trees of 18S rDNA and rbcL are given in Figures 5 and 14.
In the 18S rDNA tree, Dictyocha speculum forms an independent clade from all other dictyochophytes. Except for D. speculum, both 18S rDNA and rbcL trees generated two clades in the dictyochophytes. One corresponded to the order Pedinellales and the other to the Ciliophryales and Rhizochromulinales. Reliability of these two clades was 100% in 18S rDNA trees but not high in rbcL trees. Pteridomonas danica and Ciliophrys infusionum appeared separately in these two clades. Topology within the Pedinellales clade was different between 18S rDNA and rbcL trees. In the 18S rDNA tree, Pteridomonas danica was the sister to Apedinella radians (100% bootstrap support). Pseudopedinella elastica and Pedinella sp. became an outgroup of the clade of P. danica and A. radians (Fig. 5). In rbcL trees, P. danica was most closely related to Pedinella sp. (MP: 99%, NJ: 92%, ML: –), and A. radians and Pseudopedinella elastica showed 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 are given in order (MP/NJ/ML). The bootstrap values are represented by “–” for less than 50% clades. Asterisks represent the organisms sequenced in this study. The substitution model for ML analysis was GTR+I+G model, determined by MODELTEST.
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Discussion The organelle lying along the nucleus and bounded by four membranes in Pteridomonas danica is undoubtedly a vestigial chloroplast (leucoplast), because it possesses various features common with the leucoplast of heterotrophic chrysophytes (Belcher and Swale 1972, 1976; Mignot 1977; Preisig and Hibberd 1982b) and the chloroplasts of heterokont algae in general (Hibberd 1976; Preisig and Hibberd 1982b). The chloroplast of heterokont algae is typically encircled by four membranes. The inner two are chloroplast membranes (inner chloroplast membrane (ICM) and outer chloroplast membrane (OCM) and the outer two are simply called chloroplast ER or in different terms, periplastid membrane (PPM) or chloroplast endoplasmic reticulum (CER). The CER is continuous with the nuclear envelope and between the OCM and PPM are tubular structures called periplastidal reticula (CavalierSmith 1989; Gibbs 1981). The leucoplasts of heterotrophic chrysophytes, including Paraphysomonas spp. (Preisig and Hibberd 1982b), Spumella spp. (Belcher and Swale 1976; Mignot 1977) and Anthophysa vegetans (Müller) Stein (1878)(Belcher and Swale 1972) are also encircled by four membranes and the outermost one is continuous with the nuclear envelope. In the leucoplast of these heterotrophic chrysophytes, there is no trace of thylakoids, but small granules assignable to ribosomes are present (see Fig. 14 in Belcher and Swale 1972; Fig. 11 in Belcher and Swale 1976; Figs 8F, 9D, 10B and 16A in Preisig and Hibberd 1982b). In the leucoplast of A. vegetans and Spumella elongata (Stokes) Belcher & Swale, periplastidal reticula (PPR) are present in the periplastidal compartment (Belcher and Swale 1972, 1976). Similar structures are also present in the organelle of P. danica between the second and the third membrane (Fig. 13). In all the aspects mentioned above, the undescribed organelle detected in Pteridomonas danica resembles the leucoplast of Paraphysomonas spp., A. vegetans and Spumella spp. Therefore, this structure is concluded to be a vestigial chloroplast (leucoplast) that has been overlooked in previous studies probably because of its small size (ca. 400 nm long). Because rbcL is located in the chloroplast genome in all the photosynthetic eukaryotes so far investigated, it is likely that rbcL is also localized in the leucoplast genome in Pteridomonas danica and Ciliophrys infusionum. Moreover, we used the primers, NDrbcL2 and DPrbcL7, designed by Daugbjerg and Andersen (1997) for the first PCR. The primer DPrbcL7 was designed to anneal at the end
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of rbcS (Daugbjerg and Andersen 1997). The fact that these primers amplified rbcL in P. danica and C. infusionum indicates that rbcS is also present next to rbcL in the same genome. In all the heterokont algae so far investigated, rbcL and rbcS are encoded in the chloroplast genome (Delaney et al. 1995). It would therefore be reasonable to conclude that, in P. danica and C. infusionum, rbcL is encoded in the chloroplast genome, and the genome should be located in the leucoplast. The morphological and molecular evidence presented here indicate that both Pteridomonas danica and Ciliophrys infusionum lost photosynthetic ability secondarily as suggested from nuclear-encoded gene analyses (18S rDNA) (Cavalier-Smith and Chao 1996; Cavalier-Smith et al. 1995), and that, at least Pteridomonas danica still retains the chloroplast in a vestigial form. A previous statement about “complete” loss of the chloroplast in dictyochophytes should therefore be corrected. We have not yet succeeded to detect a vestigial chloroplast in Ciliophrys infusionum. However, more careful observations should prove that the leucoplast is also present in this organism. The 18S rDNA trees clearly indicated that the dictyochophytes examined in this study were members of two distinct clades, the Pedinellales clade and the Ciliophryales/Rhizochromulinales clade, and Pteridomonas danica was included in the Pedinellales clade and Ciliophrys infusionum was in the Ciliophryales/Rhizochromulinales clade. These two clades are clearly distinct and both contain photosynthetic taxa. This implies that these two heterotrophic dictyochophytes lost their photosynthetic ability independently. Actinomonas spp. and Parapedinella reticulata Pedersen et al. (1986) are heterotrophic taxa placed in the Pedinellales. Because molecular data are not available for these dictyochophytes, it is not easy to discuss whether or not they also lost photosynthetic ability independently. However, our cladistic analysis based on light microscopical and ultrastructural data as well as behavioral properties (e.g. method of prey capture) suggested they are closely related to Pteridomonas danica (Sekiguchi et al. in prep.). It consequently suggests monophyly of these heterotrophic taxa and a single loss of photosynthetic ability in the Pedinellales clade. The flagellar scales of indistinguishable morphology may be a synapomorphy of the clade. Therefore, it is likely that photosynthetic ability was lost twice in the Dictyochophyceae, once in the Pedinellales clade and once in the Ciliophryales/Rhizochromulinales clade. Recently, complete genomes of vestigial chloroplast (leucoplast genomes) have been determined
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for several taxa (Gockel and Hachtel 2000; McFadden et al. 1997; Wolfe et al. 1992). From these studies, it is clear that the coded genes in the leucoplast genome are largely different depending on the taxa. In Epifagus virginiana (L.) Barton, an epiphytic land plant, the 70 kbp leucoplast genome contains house-keeping genes, and the genes related to photosynthesis are retained as pseudogenes or were lost (DePamphilis and Palmer 1989; Wolfe et al. 1992). The apicomplexan Toxoplasma gondii has a 35 kb leucoplast (apicoplast) genome. 68 genes are encoded in the apicoplast, and most of them (about 60) are house-keeping genes. Genes related to photosynthesis are completely lost in T. gondii (Wilson et al. 1996). It has recently been demonstrated in these organisms that the apicoplast is responsible for fatty acid synthesis and the shikimate pathway (Roberts et al. 1998; Waller et al. 1998). In Astasia longa, the leucoplast genome is 73 kbp. The 16S rRNA gene in one of three copies of the tandem repeat (rrnC) is the only pseudogene in the genome (Gockel and Hachtel 2000), and transcripts of various genes such as rbcL and tufA have been detected (Siemeister et al. 1990). Such considerable differences of encoded genes among the leucoplast genomes suggest that the leucoplasts have different roles in these organisms. Genes related to photosynthesis are usually lost or become nonfunctional pseudogenes even if the leucoplast is retained. The rbcL gene is an exception. It is retained intact in various organisms that lost their photosynthetic ability. In a holoparasitic land plant, Lathraea clandestina, rbcL is expressed and functional RuBisCO is formed (Lusson et al. 1998). Wolfe and DePamphilis (1997) addressed several possibilities about the presence of RuBisCO: carbon fixation may proceed to a minimum extent; RuBisCO may act as an oxydase, with glycine and serine forming via the glycolate pathway; RuBisCO genes may be on the way to being lost or to becoming pseudogenes. Astasia longa is the sole example among algae that has an intact rbcL and forms functional RuBisCO. Although its actual role is uncertain, it is likely that, in L. clandestina and A. longa, the RuBisCO in the leucoplast is involved in some cellular activity other than photosynthesis. Pteridomonas danica and Ciliophrys infusionum also retain intact rbcL. Their amino acid sequences are highly conserved: similarity is 97% between P. danica and Pedinella sp. and 96% between C. infusionum and Rhizochromulina CCMP237. Why these heterotrophic organisms still retain rbcL is as enigmatic as in L. clandestina and A. longa. However, the presence of highly conserved intact rbcL in Pteridomonas danica and Ciliophrys infusionum
suggests that RuBisCO also exists and has a certain role in the leucoplast. This should be studied intensively in the future.
Methods Materials: Pteridomonas danica was collected from Yokohama Bay, Kanagawa Prefecture, Japan in May, 1995. Ciliophrys infusionum was collected from Otaru Port Hokkaido, Japan in August, 1996. Pedinella sp. (a detailed description of this organism will be published elsewhere) was collected from offshore of Kinkazan (Pacific Ocean), Miyagi Prefecture, Japan in September 1993. The organisms isolated with micropipettes were incubated into 20 ml test tubes or 100 ml Erlenmeyer flasks using URO-YT medium (Moriya et al. 2000) for Pteridomonas danica and Ciliophrys infusionum and ESM medium (Watanabe et al. 2000) for Pedinella sp. Cultures were maintained at 20 °C under light of 15 µEm–2s–1 and in a 14:10 hr light: dark regime. The culture of Ciliophrys infusionum was recently lost. Pteridomonas danica is available from the authors upon request. Light microscopy: For light microscopy of flagellate cells, samples were mixed with an equal volume of 5% glutaraldehyde (GA) prepared in 0.2 M sodium cacodylate buffer (SCB) (pH 7.2) immediately before observations were made. Cells were observed with an OPTIPHOT XF-NT with Nomarski differential interference contrast optics (DIC) (Nikon, Tokyo, Japan). Electron microscopy: Samples fixed in 5% GA in seawater were mounted on mesh-grids and left for 5 min to allow cells settle on the formvar film. The excess medium was removed with filter paper, and a small amount of 2% uranyl acetate was added. 30 sec later, the uranyl acetate was removed with filter paper. Samples were then dried and used for observation. For thin sections, a fixative containing 5% GA, 0.8 M sucrose and 10 mM EGTA prepared in 0.2 M SCB was mixed with a sample of equal volume and the mixture was kept at 4 °C for 6 hrs. Cells were harvested by centrifugation at 3000 rpm for 10 min and rinsed three times in SCB (5 min each). After the removal of SCB, 1% OsO4 in 0.2 M SCB was added to the sample, and cells were post-fixed for 2 hrs at 4 °C. Samples were rinsed, dehydrated using a graded 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
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(Spurr 1969) through immersion in propylene oxide and polymerized overnight at 70 °C. Sections were made with an EM-ULTRACUT-S ultramicrotome (Reichert, Germany), double-stained with 2% uranyl acetate and Reynolds’ lead. Observations were made with a JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan). DNA extraction and sequencing: Cells of Pteridomonas danica, Ciliophrys infusionum, and Pedinella sp. were harvested by centrifugation and lysed by UNSET buffer (Garriga et al. 1984). Total DNA was extracted by a standard phenol/chloroform method (Sambrook et al. 1989). Then the DNA was amplified by polymerase chain reaction (PCR) (Saiki et al. 1988). The primers for 18S rDNA were the same as in Nakayama et al. (1998). We adopted some primers for rbcL analysis reported by Daugbjerg and Andersen (1997); however, two primers were designed to replace NDrbcL4 and NDrbcL5 (Daugbjerg and Andersen 1997). These were rbcLF (5′-CWGCWTCWATYAWYGGWAACG-3′) and rbcL5′ (5′-CWCAASCWTTYATGCG-3′). DNA amplification was done with a thermal cycler QTP-1 (Nippon Genetica 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 same conditions, and the primer sets were the same as Nakayama et al. (1998) for 18S rDNA and Daugbjerg and Andersen (1997) for rbcL. After removing the primer from the PCR products using polyethelene glycol (PEG), the double-stranded PCR products were sequenced directly by ABI Prism 377 DNA Sequencer (Perkin-Elmer Corp., United Kingdom) using Dye-Terminator Cycle Sequencing Core Kit (Perkin-Elmer Corp., United Kingdom) according to the manufacturer’s instructions. Accession numbers deposited in the DDBJ/EMBL/GenBank nucleotide sequence database were as follows (given in order 18S rDNA and rbcL): Pteridomonas danica (AB081640/AB081642), Ciliophrys infusionum (AB081641/AB081643) and Pedinella sp. (AB081517/ AB081639). Sequence analyses: The sequences obtained were aligned with available sequence data using CLUSTAL X computer program (Thompson et al. 1997). The sequences used are as follows (numbers in parentheses are accession numbers of 18S rDNA and rbcL): Pseudopedinella elastica (U14387/ U89899), Apedinella radians (U14384/AF015573), Pteridomonas danica (L37204/this study), Ciliophrys infusionum (L37205/this study), Rhizochromulina cf. marina (U14388/-), Rhizochromulina sp. CCMP237 (-/AF015574), Dictyocha speculum (U14385/-).
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Pelagomonas calceolata (U14389/U89898), Chromulina nebulosa (AF123285/AF155876) and Rhizosolenia setigera (M87329/ AF015568) were chosen as an outgroup. In 18S rDNA analyses, ambiguous sites were excluded. The alignment is available from the corresponding author upon request. Three kinds of algorithms were employed to construct phylogenetic trees, neighbor-joining (NJ) (Saitou and Nei 1987), maximum parsimony (MP) and maximum likelihood (ML) methods. For ML analyses, the substitution model was determined using MODELTEST (version 3.06; Posada and Crandall 1998). Each tree was constructed with PAUP* (version 4.0.8) computer program (Swofford 2001). Bootstrap analyses (Felsenstein 1985) were carried out for each analysis with 100 replications.
Acknowledgements We especially thank Dr. Masanobu Kawachi, National Institute for Environmental Studies, Tsukuba for providing Pedinella sp., and Dr. Daiske Honda, Konan University, Kobe for helpful suggestions. This study was supported by JSPS grants JSPSRFTF00L0162 and 12440239.
References Belcher JH, Swale EMF (1972) The morphology and fine structure of the colourless colonial flagellate Anthophysa vegetans (O. F. Müller) Stein. Br Phycol J 7: 335–346 Belcher JH, Swale EMF (1976) Spumella elongata (Stokes) nov. comb., a colourless flagellate from soil. Arch Protistenkd 118: 215–220 Cavalier-Smith T (1989) The Kingdom Chromista. In Green JC, Leadbeater BSC, Diver WL (eds) The Chromophyte Algae. Clarendon Press, Oxford, pp 381–407 Cavalier-Smith T (1992) The origin, losses and gains of chloroplasts. In Lewin RA (eds) Origins of Plastids. Chapman & Hall, New York, London, pp 291–348 Cavalier-Smith T (1995) Evolutionary protistology comes of age: biodiversity and molecular cell biology. Arch Protistenkd 145: 145–154 Cavalier-Smith T, Chao EE (1996) 18S rRNA sequence of Heterosigma carterae (Raphidophyceae), and the phylogeny of heterokont algae (Ochrophyta). Phycologia 35: 500–510 Cavalier-Smith T, Chao EE, Allsopp MTEP (1995) Ribosomal RNA evidence for chloroplast loss within Heterokonta: Pedinellid relationships and revised classification of ochristan algae. Arch Protistenkd 145: 209–220
166
H. Sekiguchi et al.
Cavalier-Smith T, Chao EE, Thompson CE, Hourihane SL (1995/96) Oikomonas, a distinctive zooflagellate related to chrysomonads. Arch Protistenkd 146: 273–279
Hopkins J, Fowler R, Krishna S, Wilson I, Mitchell G, Bannister L (1999) The plastid in Plasmodium falciparum asexual blood stages: a three-dimensional ultrastructural analysis. Protist 150: 283–295
Cienkowski L (1875) Über einge Rhizopoden und verwandte Organismen. Arch Mikroskop Anat 12: 15–50
Köhler S, Delwiche CF, Denny PW, Tilney LG, Webster P, Wilson RJM, Palmer JD, Roos DS (1997) A plastid of probable green algal origin in apicomplexan parasites. Science 275: 1485–1489
Daugbjerg N, Andersen RA (1997) A molecular phylogeny of the heterokont algae based on analyses of chloroplast-encoded rbcL sequence data. J Phycol 33: 1031–1041 Davidson LA (1982) Ultrastructure, behavior, and algal flagellate affinities of the helioflagellate Ciliophrys marina, and the classification of the helioflagellates (Protista, Actinopoda, Heliozoa). J Protozool 29: 19–29 Delaney TP, Hardison LK, Cattolico RA (1995) Evolution of Plastid Genomes: Inferences from Discordant Molecular Phylogenies. In Sandgren CD, Smol JP, Kristiansen J (eds) Chrysophyte Algae: Ecology, Phylogeny and Development. Cambridge University Press, Cambridge, pp 25–45 DePamphilis CW, Palmer JD (1989) Evolution and Function of Plastid DNA: a Review with Special Reference to Nonphotosynthetic Plants. In Boyer CD, Shannon JC, Hardison RC (eds) Physiology, Biochemistry, and Genetics of Nongreen Plastids. The American Society of Plant Physiologists, Rockville, pp 182–202 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791 Garriga GH, Bertrandt H, Lambowitz A (1984) RNA splicing in Neurospora mitochondria: nuclear mutants defective in both splicing and 3’ end synthesis of large rRNA. Cell 36: 623–634 Gibbs SP (1981) The chloroplast endoplasmic reticulum: structure, function, and evolutionary significance. Int Rev Cytol 72: 49–99 Gockel G, Hachtel W (2000) Complete gene map of the plastid genome of the nonphotosynthetic euglenoid flagellate Astasia longa. Protist 151: 347–351 Guillou L, Chrétiennot-Dinet MJ, Boulben S, Moonvan der Staay SY, Vaulot D (1999) Symbiomonas scintillans gen. et sp. nov. and Picophagus flagellatus gen. et sp. nov. (Heterokonta): two new heterotrophic flagellates of picoplanktonic size. Protist 150: 383–398 Hibberd DJ (1976) The ultrastructure and taxonomy of the Chrysophyceae and Prymnesiophyceae (Haptophyceae): a survey with some new observations on the ultrastructure of the Chrysophyceae. Bot J Linn Soc 72: 55–80 Honda D, Kawachi M, Inouye I (1995) Sulcochrysis biplastida gen. et sp. nov.: cell structure and absolute configuration of the flagellar apparatus of an enigmatic chromophyte alga. Phycol Res 43: 1–16
Larsen J (1985) Ultrastructure and taxonomy of Actinomonas pusilla, a heterotrophic member of the Pedinellales (Chrysophyceae). Br Phycol J 20: 341–355 Lusson NA, Delavault PM, Thalouarn PA (1998) The rbcL gene from the non-photosynthetic parasite Lathraea clandestima is not transcribed by a plastid-encoded RNA polymerase. Curr Genet 34: 212–215 McFadden GI, Reith ME, Munholland J, Lang-Unnasch N (1996) Plastid in human parasites. Nature 381: 482 McFadden GI, Waller RE, Reith ME, Lang-Unnasch N (1997) Plastids in apicomplexan parasites. Plant Syst Evol 11: 261–287 Mignot JP (1977) Etude ultrastructurale d’un flagellé du genre Spumella Cienk. (= Heterochromonas Pascher = Monas O. F. Müller), Chrysononadine leucoplastidiée. Protistologica 13: 219–231 Moestrup Ø (1995) Current Status of Chrysophyte ‘Splinter Groups’: Synurophytes, Pedinellids, Silicoflagellates. In Sandgren CD, Smol JP, Kristiansen J (eds) Chrysophyte Algae: Ecology, Phylogeny and Development. Cambridge University Press, Cambridge, pp 75–91 Moriya M, Nakayama T, Inouye I (2000) Ultrastructure and 18S rDNA sequence analysis of Wobblia lunata gen. et sp. nov., a new heterotrophic flagellate (Stramenopiles, Incertae Sedis). Protist 151: 41–55 Nakayama T, Marin B, Kranz HD, Surek B, Huss VAR, Inouye I, Melkonian M (1998) The basal position of scaly green flagellates among the green algae (Chlorophyta) is revealed by analyses of nuclear-encoded SSU rRNA sequences. Protist 149: 367–380 Patterson DJ (1986) The Actinophryid Heliozoa (Sarcodina, Actinopoda) as Chromophytes. In Kristiansen J, Andersen RA (eds) Chrysophytes: Aspects and Problems. Cambridge University Press, Cambridge, pp 49–67 Patterson DJ (1989) Stramenopiles: Chromophytes from a Protistan Perspective. In Green JC, Leadbeater BSC, Diver WL (eds) The Chromophyte Algae, Problems and Perspectives. Clarendon press, Oxford, pp 357–379 Patterson DJ, Fenchel T (1985) Insights into the evolution of heliozoa (Protozoa, Sarcodina) as provided by ultrastructural studies on a new species of flagellate from the genus Pteridomonas. Biol J Linn Soc 34: 381–403
Dictyochophycean Leucoplast
Pedersen SM, Beech PL, Thomsen HA (1986) Parapedinella reticulata gen. et sp. nov. (Chrysophyceae) from Danish waters. Nord J Bot 6: 507–513 Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817–818 Preisig HR, Hibberd DJ (1982a) Ultrastructure and taxonomy of Paraphysomonas (Chrysophyceae) and related genera 1. Nord J Bot 2: 397–420 Preisig HR, Hibberd DJ (1982b) Ultrastructure and taxonomy of Paraphysomonas (Chrysophyceae) and related genera 3. Nord J Bot 3: 695–723 Roberts F, Roberts CW, Johnson JJ, Kyle DE, Krell T, Coggins JR, Coombs GH, Milhous WK, Tzipori S, Ferguson DJP, Chakrabarti D, McLeod R (1998) Evidence for the shikimate pathway in apicomplexan parasites. Nature 393: 801–805 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Ehrlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425 Sambrook J, Fritch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York Siemeister G, Buchholz C, Hachtel W (1990) Genes for the plastid elongation factor Tu and ribosomal protein S7 and six tRNA genes on the 73 kb DNA from Astasia longa that resembles the chloroplast DNA of Euglena. Mol Gen Genet 220: 425–432 Siemeister G, Hachtel W (1990) Structure and expression of a gene encoding the large subunit of ribulose1,5–bisphosphate carboxylase (rbcL) in the colourless
167
euglenoid flagellate Astasia longa. Plant Mol Biol 14: 825–833 Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastr Res 26: 31–43 Swofford DL (2001) PAUP* 4.0b8. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts Thompson JD, Gibson TJ, Plewniak K, Jeanmougin F, Higgins DG (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24: 4876–4882 Waller RF, Keeling PJ, Donald RGK, Striepen B, Handman E, Lang-Unnasch N, Cowman AF, Besra GS, Roos DS, McFadden GI (1998) Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. Proc Natl Acad Sci USA 95: 12352–12357 Watanabe MM, Kawachi M, Hiroki M, Kasai F (2000) NIES-Collection List of Strains. Microalgae and Protozoa. NIES, Japan Wilson RJM, Denny PW, Preiser PR, Rangachari K, Roberts K, Roy A, Whyte A, Strath M, Moore DJ, Moore PW, Williamson DH (1996) Complete gene map of the plastid-like DNA of the malaria parasite Plasmodium falciparum. J Mol Biol 261: 155–172 Wolfe AD, DePamphilis W (1997) Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche. Plant Mol Biol 33: 965–977 Wolfe KH, Morden CW, Palmer JD (1992) Function and evolution of a minimal plastid genome from a nonphotosynthetic parasitic plant. Proc Natl Acad Sci USA 89: 10648–10652