EVOLUTIONARY ORIGIN OF GLOEOMONAS (VOLVOCALES,CHLOROPHYCEAE), BASED ON ULTRASTRUCTURE OF CHLOROPLASTS AND MOLECULAR PHYLOGENY1

Gloeomonas is a peculiar unicellular volvocalean genus because it lacks pyrenoids in the chloroplasts under the light microscope and has two flagellar bases that are remote from each other. However, ultrastructural features of chloroplasts are very limited, and no molecular phylogenetic analyses have been carried out in Gloeomonas. In this study, we observed ultrastructural features of chloroplasts of three species of Gloeomonas and Chloromonas rubrifilum (Korshikov ex Pascher) Pröschold, B. Marin, U. Schlösser et Melkonian SAG 3.85, and phylogenetic analyses were carried out based on the combined data set from 18S rRNA, ATP synthase beta‐subunit, and P700 chl a–apoprotein A2 gene sequences to deduce the natural phylogenetic positions of the genus Gloeomonas. The present EM demonstrated that the chloroplasts of the three Gloeomonas species and C. rubrifilum SAG 3.85 did not have typical pyrenoids with associated starch grains, but they possessed pyrenoid matrices that protruded interiorly within the stroma regions of the chloroplast. The pyrenoid matrices were large and broad in C. rubrifilum, whereas those of the three Gloeomonas species were recognized in only the small protruded regions of the chloroplast lobes. The present multigene phylogenetic analyses resolved that the three species of Gloeomonas belong to the Chloromonas lineage or Chloromonadinia of the Volvocales, and Chloromonas insignis (Anakhin) Gerloff et H. Ettl NIES‐447 and C. rubrifilum SAG 3.85, both of which have pyrenoids without associated starch grains, were positioned basally to the clade composed of the three species of Gloeomonas. Therefore, Gloeomonas might have evolved from such a Chloromonas species through reduction in pyrenoid matrix size within the chloroplast and by separating their two flagellar bases.     

PHYLOGENYOF CHLOROMONAS (CHLOROPHYCEAE): A STUDY OF 185 RIBOSOMAL RNA GENE SEQUENCES1

The unicellular, biflagellate genus Chloromonas differs from its ally, Chlamydomonas, primarily by the absence of pyrenoids in the vegetative stage of the former. As with most green flagellate genera, little is known about phylogenetic affinities within and among Chloromonas species. Phylogenetic analyses of nuclear-encoded small-subunit ribosomal RNA gene sequences demonstrate that a sampling of five Chloromonas taxa, obtained from major culture collections, do not form a monophyletic group. However, only three of these isolates, Chloromonas clathrata, Chloromonas serbinowi, and Chloromonas rosae, are diagnosable morphologically as Chloromonas species by the absence of a pyrenoid in the vegetative stage. The three diagnosable Chloromonas taxa form an alliance with two pyrenoid-bearing chlamydomonads, Chlamydomonas augustae and Chlamydomonas macrostellata. With the exception of Chloromonas serbinowi, which represents the basal lineage within the clade, each of the diagnosable Chloromonas taxa and their pyrenoidbearing Chlamydomonas allies were isolated originally from mountain soils, snow, or cold peat. These observations suggest that habitat, independent of pyrenoid status, may be most closely linked to the natural history of this clade of chlamydomonad flagellates.     

A COMBINED 18S rDNA AND rbcL PHYLOGENETIC ANALYSIS OF CHLOROMONAS AND CHLAMYDOMONAS (CHLOROPHYCEAE,VOLVOCALES) EMPHASIZING SNOW AND OTHER COLD‐TEMPERATURE HABITATS1

An extensive phylogenetic analysis of the biflagellate genera, Chlamydomonas Ehrenberg and Chloromonas Gobi emend. Wille, was undertaken using 18S rDNA and rbcL gene sequence analysis. Emphasis was placed on 21 cold‐tolerant taxa of which 10 are from snow. These taxa occurred in four distinct clades each in the 18S rDNA and rbcL phylogenies, and when taken together suggest at least five distinct origins in cold habitats. Most of these taxa occur in a single clade (A), and all snow species occurred in this clade. In the rbcL and combined rbcL–18S rDNA analyses, the snow taxa fell into three groups. Two groups occurred in subclade 1: Chlamydomonas augustae Skuja CU, Chlamydomonas augustae UTEX, and Chlamydomonas sp.‐A and Chloromonas clathrata Korshikov, Chloromonas rosae Ettl CU, and Chloromonas rosae v. psychrophila var. nov. The third snow group, subclade 2, included three species with unique cell divisions, Chloromonas brevispina (Fritsch) Hoham, Roemer et Mullet, Chloromonas pichinchae (Lagerheim) Wille, and Chloromonas sp.‐D, and the basal Chloromonas nivalis (Chodat) Hoham et Mullet with normal cell divisions. This suggests that the snow habitat has been colonized at least twice and possibly three times in the history of these biflagellates. In the 18S rDNA tree, one cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa (Pascher et Jahoda) Gerloff et Ettl. In the rbcL tree, three cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa, Chloromonas sp.‐ANT1, and Chloromonas sp.‐ANT3. These results support previous findings that pyrenoids have been gained and lost several times within this complex.     

Presence of the CO2-concentrating mechanism in some species of the pyrenoid-less free-living algal genus Chloromonas (Volvocales, Chlorophyta)

Physiological and morphological characteristics related to the CO₂-concentrating mechanism (CCM) were examined in several species of the free-living, unicellular volvocalean genus Chloromonas (Chlorophyta), which differs morphologically from the genus Chlamydomonas only by lacking pyrenoids. The absence of pyrenoids in the chloroplasts of Chloromonas (Cr.) rosae UTEX 1337, Cr. serbinowii UTEX 492, Cr.␣clatharata UTEX 1970, Cr. rosae SAG 26.90, and Cr. palmelloides SAG 32.86 was confirmed by light and electron microscopy. In addition, immunogold electron microscopy demonstrated that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were distributed almost evenly throughout the chloroplasts in all five Chloromonas strains. However, Chloromonas exhibited two types of physiological characteristics related to the CCM depending on the species or strains examined. Chloromonas rosae UTEX 1337 and Cr. serbinowii had high photosynthetic affinities for CO₂ in cells grown in culture medium bubbled with air (low-CO₂ cells), compared with those grown in medium bubbled with 5% CO₂ (high-CO₂ cells), indicating the presence of the low-CO₂-inducible CCM. In addition, these two Chloromonas strains exhibited low-CO₂-inducible carbonic anhydrase (CA; EC 4.2.1.1) activity and seemed to have small intracellular inorganic carbon pools. Therefore, it appears that Cr. rosae UTEX 1337 and Cr. serbinowii possess the CCM as in pyrenoid-containing microalgae such as Chlamydomonas reinhardtii. By contrast, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides showed low photosynthetic affinities for CO₂ when grown under both CO₂ conditions. Moreover, these three strains exhibited an apparent absence of intracellular inorganic carbon pools and lacked low-CO₂-inducible CA activity. Thus, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides, like other pyrenoid-less algae (lichen photobionts) reported previously, seem to lack the CCM. The present study is the first demonstration of the CCM in pyrenoid-less algae, indicating that pyrenoids or accumulation of Rubisco in the chloroplasts are not always essential for the CCM in algae. Focusing on this type of CCM in pyrenoid-less algae, the physiological and evolutionary significance of pyrenoid absence is discussed. Received: 1 May 1997 / Accepted: 11 September 1997     

Role of pyrenoids in the CO2-concentrating mechanism: comparative morphology, physiology and molecular phylogenetic analysis of closely related strains of Chlamydomonas and Chloromonas (Volvocales)

The morphology of the pyrenoid and the physiology of the CO₂-concentrating mechanism (CCM) were investigated in Chlamydomonas (Cd.) mutabilis Gerloff UTEX 578, Cd. radiata Deason et Bold UTEX 966, Cd. augustae Skuja UTEX 1969, Cd. macrostellata Lund SAG 72.81, Cd. bipapillata Bourrelly SAG 11-47, and Chloromonas (Cr.) insignis Gerloff et Ettl NIES-447, all of which are closely related phylogenetically to the pyrenoid-less strains of Chloromonas. In the chloroplasts of Cd. mutabilis UTEX 578, Cd. radiata UTEX 966, Cd. augustae UTEX 1969, and Cd. macrostellata SAG 72.81, a typical, spheroidal, electron-dense pyrenoid matrix surrounded by starch granules was present, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were highly concentrated in the pyrenoid matrix. On the other hand, while the pyrenoid matrix of Cr. insignis NIES-447 was electron-dense that of Cd. bipapillata SAG 11-47 was not, and neither was surrounded by starch granules. The pyrenoid matrices of these two species exhibited a higher concentration of Rubisco molecules than the thylakoid region (thylakoid and stroma) of the chloroplasts; however, the densities of Rubisco molecules in these pyrenoid matrices were low compared with those of the other four Chlamydomonas strains examined in this study and that of Cd. reinhardtii Dangeard. In all six strains examined, the presence of the CCM was indicated by relatively high photosynthetic affinities for CO₂ (low values of K_0.5(CO₂)). However, differences in the inorganic carbon (Ci) pools were recognized in relation to the differences in pyrenoid morphology among the strains. In the typical pyrenoid-containing strains. Cd. mutabilis UTEX 578 and Cd. radiata UTEX 966, the ratio of internal to external inorganic carbon was about 20, while in Cr. insignis NIES-447 and Cd. bipapillata SAG 11-47 the ratio was only 2–3 similar to the two pyrenoid-less, CCM-containing strains of Chloromonas previously examined (E. Morita et al., 1998, Planta 204: 269–276). It is thus speculated that the presence of typical pyrenoids with a high concentration of Rubisco molecules is related to the formation of large Ci pools in the CCM. Detailed phylogenetic relationships among these Chlamydomonas/Chloromonas strains and the pyrenoid-less Chloromonas strains previously investigated were inferred based on the sequence of rbcL, the gene for the large subunit of Rubisco. Two monophyletic groups were resolved with high bootstrap values. Based on the tree topology resolved, it was inferred that loss of the typical pyrenoids accompanied by a decrease in intracellular Ci pools might have taken place independently in the two groups. Received: 21 August 1998 / Accepted: 30 November 1998     

The fine structure of Synura sphagnicola (Korsh.) Korsh. (Chrysophyceae)

S. sphagnicola resembles other species of Synura previously described by electron microscopy in most features of structure but differs in possessing pyrenoids and up to five cylindrical stacks of smooth cisternae which occur between the pyrenoids and leucosin vesicles. Each stack is surrounded by a tubular cisterna which bears ribosomes on its distal face but there are no clear permanent connections between this and the chloroplast ER. Other features apparently unique to this species previously known from light microscopy are described. These include the axial position of the chloroplasts; the peripheral position of the leucosin vesicles; and the loose attachment of the scales. The structure of the body scales is described for the first time from sections. The flagellar scales are formed in the swollen edges of the Golgi cisternae and appear to pass to the cell surface in large vesicles.     

Characterization of linear-oblong pyrenoids with cp-DNA along their sides in Nitzschia sigmoidea (Bacillariophyceae)

In the past 10 years Nitzschia sigmoidea (Nitzsch) W. Sm. has begun to occur in Japanese rivers in various areas. It is a common diatom in Europe but was previously absent in Japan. Each chloroplast of N. sigmoidea contains many unusual linear‐oblong structures. The internal structure of the chloroplast in this species was observed using epifluorescence and electron microscopy with immunolocalization techniques. The linear‐oblong structures in the chloroplasts could hardly be observed by conventional light microscopy of living cells, but were obvious in cells stained with propionocarmine. Transmission electron microscopy showed that the cross sections of this structure were lanceolate to fusiform with penetration by a single thylakoid. In cells stained with DAPI, chloroplast DNA was detected along both sides of the linear‐oblong structures, and DNA fibrils were detected by electron microscopy. Immunofluorescence microscopy of sectioned cells and also immunoelectron microscopy revealed specific localization of Rubisco between these DNA‐containing areas, which divided at the same time as the chloroplast. Our observations confirmed that the linear‐oblong structures are pyrenoids. The diversity of localization patterns of chloroplast DNA in diatoms is discussed.     

THE OPTIMUM PH OF FOUR STRAINS OF ACIDOPHILIC SNOW ALGAE IN THE GENUS CHLOROMONAS (CHLOROPHYTA) AND POSSIBLE EFFECTS OF ACID PRECIPITATION1

Four axenic strains of snow algae were examined for optimum pH under laboratory conditions using M-1 growth medium. Growth was measured using cell counts, cell measurements and absorbance readings at 440 nm. Strains C204 and C479A of Chloromonas sp. from the Adirondack Mountains, New York, grew optimally at pH 4.0 to 5.0. Strains C381F and C381G, Chloromonas polyptera (Fritsch) Hoh., Mull. & Roem. from the White Mountains, Arizona, grew optimally at pH 4.5 to 5.0. Growth was significantly higher at pH 4.0 in the northeastern species (Chloromonas sp.), but no significant difference was observed in final growth at pH 4.5, 5.0 and 5.5 between species. It is postulated that the more acidic precipitation in the northeastern United States may be selecting for strains of snow algae with greater tolerance to acidity than in strains from the southwestern United States or that the different pH optima reported are simply species differences. New York strain C204 was also grown in heavily buffered AM medium where it had an optimum pH of 5.0, but cells became irregularly shaped and tended to clump at pH 6.0 to 7.0. Growth of C204 in AM medium was significantly lower than in M-1 medium for snow algae. These findings justify the use of M-1 medium for this type of experimentation.     

Die Feinstruktur vonChloromonas rosae Ettl

Zusammenfassung Die Feinstruktur vonChloromonas rosae wird sowohl mit dem Lichtals auch mit dem Elektronenmikroskop untersucht und verglichen. Diese Art ist lichtoptisch schon lange gut bekannt, so daß ein Vergleich der taxonomischen Merkmale mit den elektronenmikroskopischen Aufnahmen ohne Schwierigkeiten möglich ist. Im Elektronenmikroskop kommen viele Details zum Vorschein, vor allem die Struktur der Membran und der Papille, die Geißelinsertion und die Anordnung der übrigen Organellen. Diese Details können aber die Taxonomie der GattungChloromonas vorläufig nicht weitgehend beeinflussen. Für die Taxonomie bleibt auch weiterhin die mit dem Lichtmikroskop feststellbare Zellmorphologie maßgebend. Untersuchungen mit dem Elektronenmikroskop können jedoch bei äußerst kleinen Arten von besonderem Wert sein, wo es gilt, Organellen deutlicher zu zeigen.

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Summary The fine structure ofChloromonas rosae as shown by light optical as well as electron optical investigations is compared. Light microscopically, this species has been known well for a long time, so that taxonomic features may be compared to electron microscopic photographs without difficulty. In the electron microscope many details show up, especially the structures of membrane and papilla, the insertion of the flagella and the arrangement of the other organelles. For the present, however, these details cannot have much influence on the taxonomy of the genusChloromonas. Cell morphology as established by light microscopy will be decisive for taxonomy until further notice. It is with extremely small species that electron microscopic investigations can be of special value, where the main point is to show organelles more clearly.

     

Characterization of the unarmored dinoflagellate Pseliodinium pirum (Ceratoperidiniaceae) from Jiaozhou Bay,China

A strain of Pseliodinium pirum was isolated from Jiaozhou Bay, China, identified based on a recently emended classification, and further characterized for its morphology using light, scanning electron, and transmission electron microscopy, and phylogeny based on SSU and partial LSU rDNA sequences. The pigment composition, life history, and potential effects on aquatic animals were also examined. We observed the typical characteristics of Ceratoperidiniaceae, in which its apical structure complex (ASC) formed a circular loop, and the ASC of our isolate comprised four to five rows of vesicles, and connected with sulcal intrusion. Epifluorescence and transmission electron microscopy revealed a bean‐shaped, centrally located nucleus, with at least 76 chromosomes. Numerous rod‐shaped chloroplasts were in connection to the irregularly shaped pyrenoids. Pigment analysis showed that peridinin was the most abundant among all carotenoids and other pigments. Phylogenetic analyses using maximum likelihood and Bayesian inference indicated that our isolate is conspecific with the entity Cochlodinium cf. helix (accession No. KF245459), but different from Ceratoperidinium, Kirithra, and other unidentified species of the family Ceratoperidiniaceae. Pseliodinium pirum could produce sexual, thin‐walled cyst, with subspherical and spherical shape and smooth surface (without spines or rough projections). The cyst could germinate within 3 days. Bioassays did not show adverse effects of P. pirum on the finfish Oryzias melastigma and the rotifer Brachionus plicatilis, indicating it may not be a harmful species.     

LIGHT AND ELECTRON MICROSCOPIC CHARACTERIZATION OF TWO TYPES OF PYRENOIDS IN GONIUM (GONIACEAE,CHLOROPHYTA)1

The single, basal pyrenoids of Gonium quadratum Pringsheim ex Nozaki and G. pectorale Müller (Goniaceae, Chlorophyta) differed in appearance when vegetative colonies were cultured photoheterotrophically in medium containing sodium acetate. Chloroplasts of G. quadratum had distinct pyrenoids when grown in medium without major carbon compounds. However, the pyrenoids degenerated and were markedly reduced in size when such cells were inoculated into a medium containing 400 mg·L^?1 of sodium acetate. No pyrenoids were visible under the light microscope; however, with electron microscopy small pyrenoids and electron-dense bodies were visible within the degenerating chloroplasts, which had only single layers of thylakoid lamellae at the periphery. The chloroplasts subsequently developed distinct pyrenoids and several layers of thylakoid lamellae as the culture aged. In contrast, vegetative cells of G. pectorale always showed distinct pyrenoids when cells were inoculated into medium containing sodium acetate, sodium pyruvic acid, sodium lactate, and/or yeast extract. Therefore, we propose two terms, “unstable pyrenoids” and “stable pyrenoids,” for pyrenoids of G. quadratum and G. pectorale, respectively. Chloroplasts of the colonial green flagellates should thus be examined under various culture conditions in order to determine whether their pyrenoids are unstable or stable when pyrenoids are used as taxonomic indicators. Immunogold electron microscopy showed that the ratios of gold particle density of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) between pyrenoid matrix and chloroplast stroma in G. quadratum grown in medium with or without sodium acetate were lower than those of G. pectorale. Heavy labeling by anti-RuBisCO was observed in both the electron-dense bodies and pyrenoid matrix of G. quadratum. This is the first electron microscopic demonstration of degeneration and development of both pyrenoids and thylakoid lamellae in the chloroplast as a function of culture condition in green algae.     

Differences in pyrenoid morphology are correlated with differences in the rbcL genes of members of the Chloromonas lineage (volvocales,chlorophyceae)

Chloromonas is distinguished from Chlamydomonas primarily by the absence of pyrenoids, which are structures that are present in the chloroplasts of most algae and are composed primarily of the CO2-fixing enzyme Rubisco. In this study we compared sequences of the rbcL (Rubisco large subunit-encoding) genes of pyrenoid-less Chloromonas species with those of closely related pyrenoid-containing Chlamydomonas species in the "Chloromonas lineage" and with those of 45 other green algae. We found that the proteins encoded by the rbcL genes had a much higher level of amino acid substitution in members of the Chloromonas lineage than they did in other algae. This kind of elevated substitution rate was not observed, however, in the deduced proteins encoded by two other chloroplast genes that we analyzed: atpB and psaB. The rates of synonymous and nonsynonymous nucleotide substitutions in the rbcL genes indicate that the rapid evolution of these genes in members of the Chloromonas lineage is not due to relaxed selection (as it preasumably is in parasitic land plants). A phylogenetic tree based on rbcL nucleotide sequences nested two Chlamydomonas species as a "pyrenoid-regained" clade within a monophyletic Chloromonas "pyrenoid-lost" clade. Character-state optimization with this tree suggested that the loss and the regain of pyrenoids were accompanied by eight synapomorphic amino acid replacements in the Rubisco large subunit, four of which are positioned in the region involved in its dimerization. However, both the atpB and the psaB sequence data gave robust support for a rather different set of phylogenetic relationships in which neither the "pyrenoid-lost" nor the "pyrenoid-regained" clade was resolved. The appearance of such clades in the rbcL-based tree may be an artifact of convergent evolutionary changes that have occurred in a region of the large subunit that determines whether Rubisco molecules will aggregate to form a visible pyrenoid.     

Observations on the morphology and vegetative cell division of the diatomDonkinia recta

Wild and cultured material ofDonkinia recta (Donk.) Grun. has been examined using light microscopy, and both the structure of the living cell and its vegetative cell division are described. Unlike most naviculoid diatoms,D. recta has four chloroplasts per cell, each with four oblong pyrenoids, and division of the chloroplasts follows rather than precedes mitosis and cell division. These two unusual features are probably linked. The species is briefly discussed in relation to other naviculoid taxa in the light of these findings.     

The first report of the potentially harmful diatom Pseudo‐nitzschia caciantha from Australian coastal waters

The diatom Pseudo‐nitzschia is a significant component of coastal waters worldwide and a producer of the potent neurotoxin, domoic acid. Sixteen species belonging to this genus have been reported from Australian waters, but the potentially toxic species P. caciantha has not been previously known from this region. Two clonal strains of P. caciantha were isolated from Coogee Beach, south‐east Australia, and the morphological, molecular and toxicological evidence for this species delineation were examined using light and transmission electron microscopy, phylogenetic analysis based on sequences of the second internal transcribed spacer and domoic acid production as measured by liquid chromatography–mass spectrometry. The results unambiguously confirmed that these isolates are the potentially toxic species P. caciantha , being only the second report of this species in the Southern Hemisphere. The potential for further hidden Pseudo‐nitzschia diversity in these waters is considerable.     

CACHONINA ILLDEFINA SP. NOV. (DINOPHYCEAE): CHLOROPLAST TUBULES AND DEGENERATION OF THE PYRENOID1

A new species of the dinoflagellate genus Cachonina, C. illdefina sp. nov., was isolated from a red tide off El Capitan State Park, Santa Barbara County, California, in October 1973. The organism is light yellowgreen in color with deeply incised girdle and sulcal grooves. Electron microscopy of the organism, revealed a typical dinokaryotic nucleus. The chloroplasts of the organism are connected, and often contain microtubule-like elements, 25 nm diam. The pyrenoids are characterized as excluding chloroplast thylakoids and ribosomes, although containing an amorphous matrix and numerous tubular invaginations from the cytoplasm. The pyrenoids become detached from the chloroplasts and degenerate into small vesicles. C. illdefina is not bioluminescent.     

Description of a New Planktonic Mixotrophic Dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. from the Coastal Waters off Western Korea: Morphology,Pigments, and Ribosomal DNA Gene Sequence

ABSTRACT. The mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU) rDNA and photosynthetic pigments are reported. The episome is conical, while the hyposome is hemispherical. Cells are covered with polygonal amphiesmal vesicles arranged in 16 rows and containing a very thin plate‐like component. There is neither an apical groove nor apical line of narrow plates. Instead, there is a sulcal extension‐like furrow. The cingulum is as wide as 0.2–0.3 × cell length and displaced by 0.2–0.3 × cell length. Cell length and width of live cells fed Amphidinium carterae were 8.4–19.3 and 6.1–16.0 μm, respectively. Paragymnodinium shiwhaense does not have a nuclear envelope chamber nor a nuclear fibrous connective (NFC). Cells contain chloroplasts, nematocysts, trichocysts, and peduncle, though eyespots, pyrenoids, and pusules are absent. The main accessory pigment is peridinin. The sequence of the SSU rDNA of this dinoflagellate (GenBank AM408889) is 4% different from that of Gymnodinium aureolum, Lepidodinium viride, and Gymnodinium catenatum, the three closest species, while the LSU rDNA was 17–18% different from that of G. catenatum, Lepidodinium chlorophorum, and Gymnodinium nolleri. The phylogenetic trees show that this dinoflagellate belongs within the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers, NFC, and an apical groove. Unlike Polykrikos spp., which have a taeniocyst–nematocyst complex, P. shiwhaense has nematocysts without taeniocysts. In addition, P. shiwhaense does not have ocelloids in contrast to Warnowia spp. and Nematodinium spp. Therefore, based on morphological and molecular analyses, we suggest that this taxon is a new species, also within a new genus.     

Occurrence of the gametophyte of Agarum clathratum (Laminariales, Phaeophyceae) as an endophyte in Orculifilum denticulatum (Gigartinales, Rhodophyceae)

An endophytic filamentous brown alga, growing in the red alga Orculifilum denticulatum Lindstrom, was collected from the north‐east Pacific, near Juneau, Alaska. Within the host tissue, its branched filaments formed a network in the space between the filaments of the host tissues embedded in the host intercellular substances. Cells of the filaments contained many discoid chloroplasts without pyrenoids. Neither microscopic morphological observation nor culturing was sufficient to reveal the specific identity or even the familial affinity of the alga; in contrast, molecular phylogenetic analysis of its rbcL gene and rDNA ITS sequences showed that it was the gametophyte of Agarum clathratum Dumortier (Laminariales). There are few reports of laminarialean gametophytes in nature; this is the first report actually identifying the species of laminarialean gametophyte in a red alga.     

STRUCTURE AND ASEXUAL REPRODUCTION OF THE ENIGMATIC CHAROPHYCEAN GREEN ALGA ENTRANSIA FIMBRIATA (KLEBSORMIDIALES,CHAROPHYCEAE)1

As the closest relatives of embryophytes, the charophycean green algae (sensu Mattox and Stewart) may reveal the evolutionary history of characters in this lineage. Recent molecular phylogenetic analysis indicates that the little‐known species Entransia fimbriata Hughes is a member of the charophycean order Klebsormidiales. In this study LM and EM were used to identify and describe additional structural characters of Entransia so that comparisons could be made with Klebsormidium and with other charophycean algae outside the order Klebsormidiales. Features that Entransia shares with various members of the genus Klebsormidium include cylindrical cells in unbranched filaments that may spiral, parietal chloroplasts that cover only part of the circumference of the cell, H‐shaped cross walls, and vegetative reproduction by both fragmentation and formation of zoospores or aplanospores. Among the characteristics that distinguish Entransia from Klebsormidium are a highly lobed chloroplast with multiple pyrenoids; a single large vacuole; short cells that die and collapse, apparently facilitating filament fragmentation; and germinating filaments with condensed adhesive at the base and a tapering spine at the tip. Although Entransia has sometimes been tentatively considered to be a member of the Zygnemataceae, the presence of a flagellate life history stage distinguishes Entransia from this group. The pyrenoids of Entransia are typical of those of charophycean algae in having traversing membranes and surrounding starch. Presence of multiple such pyrenoids in each chloroplast of Entransia supports the hypothesis that the common ancestor of charophycean algae and embryophytes had a single chloroplast with multiple pyrenoids.     

Phylogeny and taxonomy of the Raphidophyceae (Heterokontophyta) and Chlorinimonas sublosa gen. et sp. nov., a new marine sand-dwelling raphidophyte

A new flagellate of the Raphidophyceae, Chlorinimonas sublosa gen. et sp. nov., collected from Wakayama Prefecture, Japan is described based on morphological observations, microspectrophotometry of chloroplasts, and phylogenetic analysis of SSU rDNA sequences. The cell was usually elliptical, sometimes spherical, oval or slender, and possessed two subequal heterodynamic flagella emerging from a subapical pit. Greenish yellow discoidal chloroplasts, 15–25 per cell, were situated at the periphery of the cell. The alga is very similar to the genus Heterosigma, but distinct in that there is no invagination of thylakoids into the pyrenoids and no typical girdle lamella in the chloroplast, and the chloroplasts are greenish yellow. Phylogenetic analysis of SSU rDNA revealed that this alga forms a sister clade with the clade of Chattonella and Heterosigma. Based on these results, we propose a new genus Chlorinimonas with Chlorinimonas sublosa as the type species. In addition, this paper is the first report of molecular data covering all genera of the Raphidophyceae. The phylogenetic analysis suggests that the intrusion to freshwater habitat has occurred only once in the Raphidophyceae.