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Marine phytoplankton are major primary producers, and their growth is primarily limited by nitrogen in the oligotrophic ocean environment. The haptophyte Braarudosphaera bigelowii possesses a cyanobacterial endosymbiont (UCYN-A), which plays a major role in nitrogen fixation in the ocean. However, host-symbiont interactions are poorly understood because B. bigelowii was unculturable. In this study, we sequenced the complete genome of the B. bigelowii endosymbiont and showed that it was highly reductive and closely related to UCYN-A2 (an ecotype of UCYN-A). We succeeded in establishing B. bigelowii strains and performed microscopic observations. The detailed observations showed that the cyanobacterial endosymbiont was surrounded by a single host derived membrane and divided synchronously with the host cell division. The transcriptome of B. bigelowii revealed that B. bigelowii lacked the expression of many essential genes associated with the uptake of most nitrogen compounds, except ammonia. During cultivation, some of the strains completely lost the endosymbiont. Moreover, we did not find any evidence of endosymbiotic gene transfer from the endosymbiont to the host. These findings illustrate an unstable morphological, metabolic, and genetic relationship between B. bigelowii and its endosymbiont.

Rapidly accumulating genetic data from environmental sequencing approaches have revealed an extraordinary level of unsuspected diversity within marine phytoplankton, which is responsible for around 50% of global net primary production.However, the phenotypic identity of many of the organisms distinguished by environmental DNA sequences remains unclear. The rappemonads represent a plastid-bearing protistan lineage that to date has only been identified by environmental plastid 16S rRNA sequences.The phenotypic identity of this group, which does not confidently cluster in any known algal clades in 16S rRNA phylogenetic reconstructions, has remained unknown since the first report of environmental sequences over two decades ago. We show that rappemonads are closely related to a haptophyte microalga, Pavlomulina ranunculiformis gen. nov. et sp. nov., and belong to a new haptophyte class, the Rappephyceae. Organellar phylogenomic analyses provide strong evidence for the inclusion of this lineage within the Haptophyta as a sister group to the Prymnesiophyceae. Members of this new class have a cosmopolitan distribution in coastal and oceanic regions. The relative read abundance of Rappephyceae in a large environmental barcoding dataset was comparable to, or greater than, those of major haptophyte species, such as the bloom-forming Gephyrocapsa huxleyi and Prymnesium parvum, and this result indicates that they likely have a significant impact as primary producers. Detailed characterization of Pavlomulina allowed for reconstruction of the ancient evolutionary history of the Haptophyta, a group that is one of the most important components of extant marine phytoplankton communities., 20年以上謎だった生物の正体が判明 --光合成生物進化解明のカギに--. 京都大学プレスリリース. 2021-03-29.

Coastal areas in the Tohoku region were severely damaged by the tsunami that followed the March 2011 earthquake. Consequently, many lands have become non-arable. To assess the use of these non-arable lands for the production of algal biomass, we carried out experimental cultivations of native algal communities in Minamisoma city, Fukushima Prefecture, located in the cool temperate zone of Japan. Productivity of the native algal community was measured using 1-m2 raceway ponds (120 L) and open vessel-type bioreactors (50 L–500 L), with different hydraulic retention times (HRTs) and in the presence or absence of sodium acetate. Maximum productivity in 1-m2 raceway ponds (10-cm water depth) was observed with an HRT of 4 days and sodium acetate supplementation in June 2015 (13.2 g/m2/day); average productivity throughout the year was 10.6 g/m2/day. In the case of deep-water cultivation (80-cm water depth) in vessel-type cultures, the highest productivity (> 29 g/m2/day) was obtained between January and September 2015. The dominant native microalgal species in the cultivations were Desmodesmus sp. and Scenedesmus acuminatus. Heterotrophic organisms, native bacteria, and protista, such as ciliates and amoeba, were also observed. Species composition and abundance varied depending on the medium content and seasonal conditions.

A novel Diplophrys-like organism, Fibrophrys columna, was isolated from Hiuchigaike Pond in Japan. F. columna showed a nearly orbicular or broadly elliptical cell shape and has fine filamentous, branching ectoplasmic elements emanating from both polar ends of the cell. Cells also contain orange, amber, or colorless lipid bodies. Although its whole cell morphology resembles that of the genus Diplophrys, Fibrophrys is clearly distinct from Diplophrys on the basis of 18S rDNA sequences. Molecular phylogenetic analysis showed a close relationship of F. columna with Amphifila marina, and its sequence is similar to many environmental stramenopile sequences. The cells of F. columna measured 5.0–8.3 × 5.6–10.3 μm and sometimes possessed hernia-like prongs instead of filamentous ectoplasmic elements. An axis-like electron-dense body was observed in the mitochondria. We also studied the ultrastructure of another Fibrophrys strain, Fibrophrys sp. E-1, which is different from the type strain of F. columna. A ladder-like pattern was recognized in the outer part of unidentified cytoplasmic membranes connected with the mitochondria. The unidentified cytoplasmic membranes were connected to the nuclear, lipid body, and mitochondrial outer membranes. We propose a new genus, Fibrophrys, and a new species, F. columna, based on these ultrastructural and molecular features.

The Fukushima 1 Nuclear Power Plant accident in March 2011 released an enormously high level of radionuclides into the environment, a total estimation of 6.3 × 1017 Bq represented by mainly radioactive Cs, Sr, and I. Because these radionuclides are biophilic, an urgent risk has arisen due to biological intake and subsequent food web contamination in the ecosystem. Thus, urgent elimination of radionuclides from the environment is necessary to prevent substantial radiopollution of organisms. In this study, we selected microalgae and aquatic plants that can efficiently eliminate these radionuclides from the environment. The ability of aquatic plants and algae was assessed by determining the elimination rate of radioactive Cs, Sr and I from culture medium and the accumulation capacity of radionuclides into single cells or whole bodies. Among 188 strains examined from microalgae, aquatic plants and unidentified algal species, we identified six, three and eight strains that can accumulate high levels of radioactive Cs, Sr and I from the medium, respectively. Notably, a novel eustigmatophycean unicellular algal strain, nak 9, showed the highest ability to eliminate radioactive Cs from the medium by cellular accumulation. Our results provide an important strategy for decreasing radiopollution in Fukushima area. Electronic supplementary material The online version of this article (doi:10.1007/s10265-013-0596-9) contains supplementary material, which is available to authorized users.

Both 132,173-cyclopheophorbide a and b enols were produced along with ingestion of green microalgae containing chlorophylls a and b by a centrohelid protist (phycophagy). The results suggest that chlorophyll b as well as chlorophyll a were directly degraded to colored yet non-phototoxic catabolites in the protistan phycophagic process. Such a simple process by the predators makes a contrast to the much sophisticated chlorophyll degradation process of land plants and some algae, where phototoxicity of chlorophylls was cancelled through the multiple enzymatic steps resulting in colorless and non-phototoxic catabolites.

Life cycle and perennation of a colorless chrysophyte, Spumella sp., isolated from an ephemeral ditch were investigated. From a single resting cyst (statospore), only one nonmotile cell germinated. Shortly after germination, the cell generated flagella, started to swim, and formed a gelatinous sphere. The cell itself retained the ability to swim within the sphere. Cells fed on bacteria inhabiting the sphere and grew by longitudinal binary cell division very rapidly. The gelatinous sphere gradually enlarged as the number of cells increased. When it reached maximum size (∼500 μm in diameter), the gelatinous substance of the sphere weakened, and the sphere gradually broke into several pieces, forming cleavages between them. Cells swam away through the cleavages. Five to ∼40 swimming cells soon gathered and formed a swarm. In the swarm, some cells cannibalized other sibling cells and enlarged, resulting in giant cells that were two to three times larger in diameter than ordinary cells. The giant cells soon started statospore formation. Statospore formation was independent of any changes of environmental factors, such as increase or decrease in temperature or changes in nutrient or light levels, which are known to induce resting-cyst formation in other groups of algae and protists. Statospore formation started when cells divided 15 to 16 times after germination. This is congruent with the idea that statospore formation in planktonic chrysophytes directly depends on cell density. An extraordinarily high growth rate and cannibalism involved in the initiation of statospore formation are interpreted as adaptations to achieve the perennation in ephemeral aquatic environments.

Chlorophylls are essential components of the photosynthetic apparati that sustain all of the life forms that ultimately depend on solar energy. However, a drawback of the extraordinary photosensitizing efficiency of certain chlorophyll species is their ability to generate harmful singlet oxygen. Recent studies have clarified the catabolic processes involved in the detoxification of chlorophylls in land plants, but little is understood about these strategies in aquatic ecosystem. Here, we report that a variety of heterotrophic protists accumulate the chlorophyll a catabolite 13 2 ,17 3 -cyclopheophorbide a enol (cPPB- a E) after their ingestion of algae. This chlorophyll derivative is nonfluorescent in solution, and its inability to generate singlet oxygen in vitro qualifies it as a detoxified catabolite of chlorophyll a . Using a modified analytical method, we show that cPPB- a E is ubiquitous in aquatic environments, and it is often the major chlorophyll a derivative. Our findings suggest that cPPB- a E metabolism is one of the most important, widely distributed processes in aquatic ecosystems. Therefore, the herbivorous protists that convert chlorophyll a to cPPB- a E are suggested to play more significant roles in the modern oceanic carbon flux than was previously recognized, critically linking microscopic primary producers to the macroscopic food web and carbon sequestration in the ocean.

The biomass and lipid productions by a newly isolated Aurantiochytrium strain, 4W-1b, were investigated at different temperatures and glucose concentrations. The maximum biomass was produced at 15–25°C. The biomass, lipid, and fatty acid productions were the maximum in a 6% glucose medium. The lipid and fatty acid productions were estimated to be approximately 11g/L and 9g/L, respectively, with the highest yields of docosahexaenoic acid (22:6; 1.5g/L) and palmitic acid (16:0; 4.8g/L). The 4W-1b strain is considered to have a high potential for uses in various industrial sectors, including fuel, health supplements, soap, and food oil companies.

The effect of ocean acidification, caused by the increase in pCO2 in seawater, on phytoplankton population and on related organic nitrogen production was experimentally examined by use of a natural coastal microbial population. pCO2 and pH were controlled by aeration with air in which pCO2 was at the current level (control), for which ambient air was used, and with air in which pCO2 was “800” and “1200” ppm, in 500-L culture vessels. The experiment was continued for 15 days after addition of the inorganic nutrients such as nitrate, phosphate, and silicate. During most of the experimental period, a minor increase in phytoplankton biomass was noted, probably because of low irradiance, an increase in phytoplankton biomass was observed at the end of the experiment. Flow cytometric and microscopic observations revealed that this increase was because of Chrysochromulina sp. (Haptophyceae). The growth of Chrysochromulina sp. was most obvious in the control vessel, and tended to be obscured by increasing pCO2 (decrease in pH), indicating the possibility that ocean acidification inhibits the growth of specific phytoplankton groups, for example Chrysochromulina sp. Production of particulate organic nitrogen (PON), determined by the 15N tracer method, also diminished under acidified conditions compared with that at the current level.

Iida H., Nishii I. and Inouye I. 2011. Embryogenesis and cell positioning in Platydorina caudata (Volvocaceae, Chlorophyta). Phycologia 50: 530–540. DOI: 10.2216/10-80.1 The colony formation of Platydorina caudata (Volvocaceae, Chlorophyta) was studied in detail using time-lapse video and scanning and transmission electron microscopy. A unique feature of P. caudata, a horseshoe-shaped flattened colony comprising 16 cells arranged in a single twisted layer, is formed by ‘intercalation’, the rearrangement of embryo cells within the colony, which proceeds subsequent to the embryo inversion that brings flagellar ends of cells from the concave to the convex surface. Cell fate is precisely decided in colony formation, suggesting that intercalation is a precisely regulated process in which given cells are intercalated into particular parts of the colony. The presence of flask-shaped cells with an elongated stalk and cytoplasmic bridges during the inversion process strongly suggests that a similar mechanism known...

Most photosynthetic dinoflagellates possess plastids containing chlorophylls a+c, but species belonging to the genus Lepidodinium are unique in bearing non-canonical plastids containing chlorophylls a+b. According to the pioneering works on pigment composition data, it has been proposed that Lepidodinium plastids were derived from a prasinophyte species, though this hypothesis was not supported by a recent phylogenetic analysis based on an alignment comprised of eight plastid proteins (Takishita et al. 2008, Gene 410: 26-26). This “8-protein” analysis however was insufficient to clarify the origin of Lepidodinium plastids for two major reasons: First, the alignment lacked sufficient evolutionary information to resolve the precise origin of Lepidodinium plastids. Second, the taxa considered did not well represent the diversity of Chlorophyta. Particularly, prasinophytes were poorly sampled in the alignment. In this study, we sequenced plastid-encoded genes from L. chlorophorum, one pedinophyte species, one ulvophyte species, and six prasinophyte species. The 85 sequences newly determined in this study and recent progress in plastid genome sequencing enabled us to prepare an alignment comprised of 11 plastid proteins from green algal taxa that appropriately cover the diversity of Chlorophyta. All the analyses of the 11-protein data set robustly grouped L. chlorophorum with members of the “core chlorophytes.” Thus, we here propose that Lepidodinium plastids are of core chlorophyte origin.

Treatment of symbiotic alga-bearing Paramecium bursaria cells with a protein synthesis inhibitor, cycloheximide, induces synchronous swelling of all perialgal vacuoles at about 24 h after treatment under a constant light condition. Subsequently, the vacuoles detach from the host cell cortex. The algae in the vacuoles are digested by the host's lysosomal fusion to the vacuoles. To elucidate the timing of algal degeneration, P. bursaria cells were treated with cycloheximide under a constant light condition. Then the cells were observed using transmission electron microscopy. Results show that algal chloroplasts and nuclei degenerated within 9 h after treatment, but before the synchronous swelling of the perialgal vacuole and appearance of acid phosphatase activity in the perialgal vacuole by lysosomal fusion. Treatment with cycloheximide under a constant dark condition and treatment with chloramphenicol under a constant light condition induced neither synchronous swelling of the vacuoles nor digestion of the algae inside the vacuoles. These results demonstrate that algal proteins synthesized during photosynthesis are necessary to maintain chloroplastic and nuclear structures, and that inhibition of protein synthesis induces rapid lysis of these organelles, after which synchronous swelling of the perialgal vacuole and fusion occur with the host lysosomes.

The genus Nephroselmis (Nephroselmidophyceae), which had been placed in the Prasinophyceae, is one of the primitive green flagellates that are important to our understanding of the early evolution of green plants. We studied a new species of Nephroselmis isolated from Japan, Fiji and South Africa. This species has been known for a long time as undescribed species ‘N. viridis.’ N. viridis possesses some ultrastructural characters shared with only the freshwater type species N. olivacea, including a disc-like structure beneath the pyrenoid and bipolar spiny body scales with 1-5-8-5-1 spines. Molecular phylogenetic analysis based on 18S rDNA also supports a sister relationship between N. viridis and N. olivacea. However, N. viridis is distinguishable from N. olivacea by the shape of its starch sheath, its scales, its pigment composition and its habitat. In this paper, we designate the formal description of N. viridis sp. nov. We also describe variability in the 18S rDNA introns of various N. viridis strains. This detailed study of N. viridis provides some insights into the evolution of Nephroselmis.

Ultrastructural and molecular phylogenetic evidence indicate that the Parabasalia consists of seven main subgroups: the Trichomonadida, Honigbergiellida, Hypotrichomonadida, Tritrichomonadida, Cristamonadida, Spirotrichonymphida, and Trichonymphida. Only five species of free-living parabasalids are known: Monotrichomonas carabina, Ditrichomonas honigbergii, Honigbergiella sp., Tetratrichomonas undula, and Pseudotrichomonas keilini. Phylogenetic analyses show that free-living species do not form a clade and instead branch in several different positions within the context of their parasitic relatives. Because the diversity of free-living parabasalids is poorly understood, the systematics of these lineages is in a significant state of disarray. In order to better understand the phylogenetic distribution of free-living parabasalids, we sequenced the small subunit rDNA from three different strains reminiscent of P. keilini; the strains were isolated from different geographical locations: (1) mangrove sediments in Japan and (2) sediments in Cyprus. These data demonstrated that the free-living parabasalids P. keilini and Lacusteria cypriaca n. g., n. sp., form a paraphyletic assemblage near the origin of a clade consisting mostly of parasitic trichomonadids (e.g. Trichomonas vaginalis). This paraphyletic distribution of similar morphotypes indicates that free-living trichomonadids represent a compelling example of morphostasis that provides insight into the suite of features present in the most recent free-living ancestor of their parasitic relatives.

A new species of unicellular opisthokont protist, Nuclearia thermophila sp. nov., was isolated from the warm spring water of Yunoko Lake, Japan, and has been described using light and electron microscopy. It exists as a spherical floating form and a flattened amoeboid form showing various shapes. The cells occasionally extended as branches or knobbed filopodia. The spherical form when suspended in medium measured 20–40 μm in diameter (excluding filopodia). The amoeboid form may exceed 65 μm along the longest axis. A nucleus with an obvious spherical nucleolus, dictyosomes, mitochondria with flat cristae, food vacuoles, and lipid droplet-like vacuoles with homogeneous contents were observed; no extracellular matrix or bacterial endosymbionts were present. The cells ingested flour particles. No cysts were seen. The molecular phylogenetic tree constructed on the basis of the small subunit ribosomal DNA revealed the novelty of N. thermophila and its relationships with previously known nucleariids.

A new heterokontophyte alga, Aurearena cruciata gen. et sp. nov., was isolated from sandy beaches in Japan. Isolates were characterized by light and electron microscopy, spectroscopy of pigment composition, and molecular phylogenetic analyses using 18S rDNA and rbcL. The alga usually possessed a cell wall but also retained two heterokont flagella beneath the cell wall. Each walled cell first produced only a single flagellate cell that subsequently divided into two flagellate cells. Electron-opaque vesicles, possibly associated with cell wall formation, were observed beneath the cell membrane. The chloroplast consisted of two compartments, each enclosed by a chloroplast envelope and the inner membrane of the chloroplast endoplasmic reticulum; these two compartments were surrounded by a common outer membrane of chloroplast endoplasmic reticulum. Molecular phylogenetic trees suggested that this alga was a new and independent member of the clade that included the Phaeophyceae and Xanthophyceae (PX clade). A new class, Aurearenophyceae classis nova was proposed for A. cruciata.

Nephroselmis astigmatica sp. nov. is described based on light and electron microscope observations of cultured material, originally collected and isolated from the Natal South Coast, Republic of South Africa. It is characterized by (1) large cell size, (2) absence of a stigma, (3) markedly differentiated anterior part of the cell, (4) possessing two types of flagellar scales in addition to hair scales, (5) possessing four types of body scales and (6) the presence of characteristic pit scales in the flagellar pit. Scale morphology was compared with previously described species, and the morphology of spiny (or stellate) body scales thought to be one of the most useful diagnostic characters in delineating species within the genus. The origin of pit scales is discussed and a similar origin for the third layer of flagellar scales of the type species, N. olivacea Stein is suggested. N. astigmatica shares many ultrastructural features with the type species, including the microtubular flagellar root system consisting of three different roots, one of which is multilayered. The validity of this root system as a generic character is suggested.

Life cycle and perennation of a colorless chrysophyte, Spumella sp., isolated from an ephemeral ditch were investigated. From a single resting cyst (statospore), only one nonmotile cell germinated. Shortly after germination, the cell generated flagella, started to swim, and formed a gelatinous sphere. The cell itself retained the ability to swim within the sphere. Cells fed on bacteria inhabiting the sphere and grew by longitudinal binary cell division very rapidly. The gelatinous sphere gradually enlarged as the number of cells increased. When it reached maximum size (∼500 μm in diameter), the gelatinous substance of the sphere weakened, and the sphere gradually broke into several pieces, forming cleavages between them. Cells swam away through the cleavages. Five to ∼40 swimming cells soon gathered and formed a swarm. In the swarm, some cells cannibalized other sibling cells and enlarged, resulting in giant cells that were two to three times larger in diameter than ordinary cells. The giant cells soon started statospore formation. Statospore formation was independent of any changes of environmental factors, such as increase or decrease in temperature or changes in nutrient or light levels, which are known to induce resting-cyst formation in other groups of algae and protists. Statospore formation started when cells divided 15 to 16 times after germination. This is congruent with the idea that statospore formation in planktonic chrysophytes directly depends on cell density. An extraordinarily high growth rate and cannibalism involved in the initiation of statospore formation are interpreted as adaptations to achieve the perennation in ephemeral aquatic environments.

A new species of Nephroselmis, Nephroselmis anterostigmatica, is described from coastal waters of the Palau Islands and the southern part of Japan. The long flagellum was stretched out at angle to the cell and showed a characteristic sigmoid line. A cup-shaped yellowish green chloroplast with two narrow sinuses included a pyrenoid with tubular invaginations of thylakoids. In contrast to other species of Nephroselmis, the eyespot was located at the anterior side of the cell. The transitional region of the flagellum possessed a single transitional plate and a stellate structure. The cell body was covered by two types of square scales (windmill/Maltese cross scales absent), small stellate scales and two types of large spiny scales. The flagellar surface was covered by inner square and outer rod-shaped scales, and ornamented with T- and Pl-hairs. Tip hairs are absent. Nephroselmis anterostigmatica is superficially similar to Nephroselmis pyriformis (N. Carter) Ettl, but a detailed comparison of morph...

Hatena arenicola gen. et sp. nov., an enigmatic flagellate of the katablepharids, is described. It shows ultrastructural affinities to the katablepharids, including large and small ejectisomes, cell covering, and a feeding apparatus. Although molecular phylogenies of the 18S ribosomal DNA support its classification into the katablepharids, the cell is characterized by a dorsiventrally compressed cell shape and a crawling motion, both of which are unusual within this group. The most distinctive feature of Hatena arenicola is that it harbors a Nephroselmis symbiont. This symbiosis is distinct from previously reported cases of ongoing symbiosis in that the symbiont plastid is selectively enlarged, while other structures such as the mitochondria, Golgi body, cytoskeleton, and endomembrane system are degraded; the host and symbiont have developed a morphological association, i.e., the eyespot of the symbiont is always at the cell apex of Hatena arenicola; and only one daughter cell inherits the symbiont during cell division, resulting in a symbiont-bearing green cell and a symbiontlacking colorless cell. Interestingly, the colorless cells have a feeding apparatus that corresponds to the location of the eyespot in symbiont-bearing cells, and they are able to feed on prey cells. This indicates that the morphology of the host depends on the presence or absence of the symbiont. These observations suggest that Hatena arenicola has a unique ‘‘half-plant, half-predator’’ life cycle; one cell divides into an autotrophic cell possessing a symbiotic Nephroselmis species, and a symbiont-lacking colorless cell, which later develops a feeding apparatus de novo. The evolutionary implications of Hatena arenicola as an intermediate step in plastid acquisition are discussed in the context of other examples of ongoing endosymbioses in dinoflagellates. & 2006 Elsevier GmbH. All rights reserved.

A haptophyte alga bearing hyaline but conspicuous scales was discovered in surface water samples of Shiribeshi Seamount, Japan. X-ray elemental analysis confirmed that silica was the major element in these scales. These scales were hat-shaped, ellipsoidal in top view, 4-6mum wide and 5-7mum long, perforated by several small pores, and were deposited on cells in several layers. Beneath the siliceous scale layers, organic scales were present, which are typical of haptophytes. The cells were non-motile despite having two short flagella hidden in the scale case. The haptonema, extended over the scale case, and one and a half times the cell length. The intracellular features were typical of haptophytes, including the peripheral endoplasmic reticulum (PER), Golgi cisternae with peculiar dilations, and the flagellar apparatus. The siliceous scales were produced in vesicles in the posterior region of the cell. Motile cells lacking silica scales were observed occasionally when cultures were maintained at lower temperatures and under oligotrophic conditions. This alga was described as Hyalolithus neolepis gen. et sp. nov. Phylogenetic analysis using the SSU rDNA and rbcL gene sequences indicated that Hyalolithus is a member of the Prymnesiales and falls in a clade including Prymnesium, Platychrysis, and Chrysochromulina polylepis. Based on these results, the evolutionary implications of the presence of silicified scales in haptophytes is discussed.

We investigated the phylogenetic relationships and the taxonomy of 20 sarcinoid strains by their morphological features and using 18S rDNA sequence data. Nineteen strains of Chlorosarcinopsis (Gerneck) Herndon and allied genera were divided into four groups (A-D). Group A, comprising eight species, was concordant with the description of Chlorosarcinopsis and divided into four polyphyletic subgroups. Group B included two species of Chlorosarcinopsis and two strains of Desmotetra stigmatica (Deason) Deason et Floyd, all of which had parallel thylakoid membranes in pyrenoid matrices, and zoospores bearing apical stigma and subapical flagellar apparatus. The definition of Desmotetra Deason et Floyd was emended to include these features, and Chlorosarcinopsis delicata S. Watanabe was transferred to this genus. Group C comprised Chlorosarcina stigmatica Deason (ASIB.T105), for which we proposed Sarcinochlamys gen. nov., a genus distinct from Chlorosarcina Gerneck in having walled zoospores. Group D, comprising six species, corresponded to Neochlorosarcina and was divided into two subgroups. The presence of thin cell walls in Neochlorosarcina S. Watanabe was ascertained as a valid feature for circumscribing the sarcinoid genera. The physiological experiments on the species of Chlorosarcinopsis and Chlorosarcina by Groover and Bold (1968) were assessed based on the phylogenetic results. Groups (A, B, D) were roughly characterized by these features: algal mass color, utilization of selected nitrogen compounds and carbon sources under light/dark and aerobic/anaerobic conditions, and requirement for vitamin B 12 . Molecular analysis revealed that Chlorosphaeropsis alveolata Herndon had closer affinity with solitary Protosiphon Klebs and Spongiochloris Starr than with sarcinoid members.

A cell-fusing hyperthermophilic archaeon was isolated from hydrothermal fluid obtained from Suiyo Seamount of the Izu-Bonin Arc. The isolate, TS1T, is an irregular coccus, usually 0·5–2 μm in diameter and motile with a polar tuft of flagella. Cells in the exponential phase of growth fused at room temperature in the presence of DNA-intercalating dye to become as large as 5 μm in diameter. Fused cells showed dark spots that moved along in the cytoplasm. Large cells with a similar appearance were also observed upon culture at 87 °C, suggesting the occurrence of similar cell fusions during growth. Transmission electron microscopy revealed that cells in the exponential phase possessed a thin and electron-lucent cell envelope that could be lost subsequently during culture. The fragile cell envelope must be related to cell fusion. The cells grew at 57–90 °C, pH 5·2–8·7 and at NaCl concentrations of 1·5–4·5 %, with the optima being 87 °C, pH 6·5 and 2·5 % NaCl. The isolate was an anaerobic chemo-organotroph that grew on either yeast extract or tryptone as the sole growth substrate. The genomic DNA G+C content was 53·9 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the isolate was closely related to Thermococcus species. However, no significant DNA–DNA hybridization was observed between genomic DNA of strain TS1T and phylogenetically related Thermococcus species. We propose that isolate TS1T represents a novel species, Thermococcus coalescens sp. nov., with the name reflecting the cell fusion activity observed in the strain. The type strain is TS1T (=JCM 12540T=DSM 16538T).

Amoeboid protists are major targets of recent molecular phylogeny in connection with reconstruction of global phylogeny of eukaryotes as well as the search for the root of eukaryotes. The Centrohelida are one of the major groups of Heliozoa, classified in the Actinopodida, whose evolutionary position is not well understood. To clarify the relationships between the Centrohelida and other eukaryotes, we sequenced SSU rRNA, alpha-tubulin, and beta-tubulin genes from a centroheliozoan protist, Raphidiophrys contractilis. The SSU rRNA phylogeny showed that the Centrohelida are not closely related to other heliozoan groups, Actinophryida, Desmothoracida, or Taxopodida. Maximum likelihood analyses of the combined phylogeny using a concatenate model for an alpha- + beta-tubulin + actin data set, and a separate model for SSU rRNA, alpha- and beta-tubulin, and actin gene data sets revealed the best tree, in which the Centrohelida have a closer relationship to Rhodophyta than to other major eukaryotic groups. However, both weighted Shimodaira-Hasegawa and approximately unbiased tests for the concatenate protein phylogeny did not reject alternative trees in which Centrohelida were constrained to be sisters to the Amoebozoa. Moreover, alternative trees in which Centrohelida were placed at the node branching before and after Amoebozoa or Viridiplantae were not rejected by the WSH tests. These results narrowed the possibilities for the position of Centrohelida to a sister to the Rhodophyta, to the Amoebozoa, or to an independent branch between the branchings of Amoebozoa and Rhodophyta (or possibly Plantae) at the basal position within the bikonts clade in the eukaryotic tree.

The katablepharids are a morphologically well-defined group of heterotrophic flagellates. Since their original description in 1939, they have been classified in the Cryptophyceae (Cryptophyta) based on their similar cell shape, flagellar orientation, and the presence of ejectisomes visible by light microscopy. However, electron microscopy suggests that the katablepharids are distinct from cryptomonads. A possible affinity with the Alveolata has been proposed which is mainly based on the resemblance of their feeding apparatus to the apical complex of the Apicomplexa or to the tentacles of the Ciliophora. In this study, we provide the first SSU rDNA and beta-tubulin molecular sequence data for two katablepharids: Katablepharis japonica sp. nov. and Leucocryptos marina. We reveal that the katablepharids are not closely related to the Alveolata; rather, phylogenetic reconstruction analyses of SSU rDNA and beta-tubulin suggest that the katablepharids are a distant sister group of the Cryptophyta. We therefore conclude that the katablepharids should be a group equivalent to the Cryptophyta and propose Katablepharidophyta divisio nova (ICBN)/Kathablepharida phylum novum (ICZN).

We isolated a novel haptophyte alga from the coastal waters of the northern part of Japan. The cell is asymmetrical spindle-to knife-shaped and possesses two nearly equal flagella and a haptonema from the anterior tip. Two yellowish chloroplasts including immersed pyrenoids are situated asymmetrically. Two types of organic scales, small elliptical and large rhomboidal scales, cover the cell. The rhomboidal scale has two tubular projections at the longitudinal poles and closely touched to each other. The transition region of the flagellum includes only one transitional plate, which is probably homologous with the distal plate in other prymnesiophyceans. The flagellar apparatus of this alga has basic components found in other prymnesiophyceans. Developed root 1 (R1) is divided into two components that join again. The R1 extends toward the posterior tip of the cell with associated endoplasmic reticulum (ER) and forms a cytoplasmic tongue. A fibrous root, which has only been reported in some coccolit...

Carotenoid compositions were analyzed for ten strains of Nephroselmis (Prasinophyceae) containing four described and three undescribed species. Based on the distribution pattern of the siphonaxanthin series, five carotenoid types were recognized in the examined strains/species: type I (N. astigmatica Inouye et Pienaar, N. pyriformis (N. Carter) Ettl, and Nephroselmis sp1. MBIC 11158) had siphonaxanthin C12:1 and C14:1 esters as well as 6′-OH siphonaxanthin C12:1 and C14:1 esters, type II (Nephroselmis sp2. MBIC 11149) had siphonaxanthin C8:1 ester, type III (Nephroselmis sp3. NIES 486, NIES-PS 535, and MBIC 10871) had 19-methoxy siphonaxanthin and siphonaxanthin C12:1 and C14:1 esters, type IV (N. spinosa Suda) had only a small amount of siphonaxanthin C12:1 ester, and type V (N. olivacea Stein) had lutein as a major carotenoid but completely lacked the siphonaxanthin series. 19-Methoxy siphonaxanthin was a novel and very unique carotenoid, that is, it contains a methoxy group and was found for the first time in photosynthetic eukaryotes. Additionally, carotenoids containing a primary methoxy group had previously never been found in any group of organisms. Siphonaxanthin C8:1 ester, which was only known as a trace carotenoid in Chlamydomonas parkeae Ettl, was first discovered as a major carotenoid in Nephroselmis sp2. (MBIC 11149). Based on these results and comparison of the phylogenetic relationships of the Nephroselmis species used, we discuss the taxonomic significance of the carotenoid types and evolutionary process of the photosynthetic antenna systems in green plants.

From a phylogenetic perspective, most plant biodiversity lie in the algae, which comprise nine divisions distinct in cell architecture. In the past decade or so, molecular phylogenies have revealed that many algal divisions are only distantly related, and belong to five different supergroups of eukaryotes. The scattered and distant distributions of algae are interpreted as the result of separate endosymbioses that occurred in various lineages of eukaryotes. Endosymbiosis is a major driving force of algal evolution and diversification, and therefore, is a key process in understanding plant evolution. In this paper, the process of plastid acquisition via endosymbiosis is considered, focusing on the evolution of protein transport machinery, which is indispensable for establishing new lineages of algae, and simultaneous lateral gene transfer from the symbiont to the host nucleus. The current understanding of protein transport diversity is reviewed in relation to the membrane topologies of plastids. The endosymbioses found in various algae and protists are natural occurrences in plant evolution and diversification, and exhibit intermediate stages of plastid acquisition. The conditions necessary for plastid establishment are also considered, using several examples of ongoing endosymbioses.

Eukaryotic autotrophs, generally assembled as plants, are polyphyletic and they appear across at least five super groups of eukaryotes, which indicate that photosynthesis has been horizontally transferred between different lineages of eukaryotes. This situation is explained by secondary endosymbioses that should have occurred between heterotrophic eukaryotes and primary plants (red and green algae). Evolutionary process generating new algal lineage can be considered by comparison of dinoflagellates and Hatena, a flagellate we recently found, all of which are likely under intermediate stages of secondary endosymbioses. Evolution of protein transport machinery to send nuclear encoded proteins back into the plastids was also considered referring recent reports of molecular biology.

Photosynthetic pigments were analyzed by HPLC for 27 samples of the Cladophorales (Ulvophyceae, Chlorophyta). The carotenoid compositions of the examined algae were classified into three types based on the final compound of biosynthesis of the α-carotene branch: lutein type, characterized by containing lutein as a major carotenoid and lacking loroxanthin and siphonaxanthin; loroxanthin type, characterized by containing loroxanthin and lacking siphonaxanthin; and siphonaxanthin type, characterized by containing siphonaxanthin. We constructed molecular phylogenetic tree of the species examined in the present study using 18S rRNA gene sequences and mapped the carotenoid types of the species onto the tree. The molecular phylogenetic analysis divided the Cladophorales into two major clades, clade 1 and Aegagropila-clade (clade 2), and divided clade 1 into subclade 1-1 and subclade 1-2. All the examined species positioned in the Aegagropila-clade and those of the subclade 1-1 belonged to the loroxanthin type, whereas both lutein type and siphonaxanthin type appeared only in the subclade 1-2. The clades delineated by molecular phylogenetic analysis were congruent with distribution of the carotenoid types, indicating that the carotenoid types are of taxonomic significance in the Cladophorales. Considering the distribution pattern of these carotenoid types and minimum state changes in the Cladophorales, we concluded that the loroxanthin type was the primitive (plesiomorphic) state and the siphonaxanthin type and lutein type appeared in the subclade 1-2 as advanced (apomorphic) state within this order and suggested that the cladophoralean siphonaxanthin type would have been secondarily acquired.

SUMMARY The electron microscopy of zygote formation and the early stages of zygote germination in Nephroselmis olivacea Stein are presented. Although the gametes differ behaviorally during the early stages of gamete fusion, the alga is isogamous. The minus gamete settled on the substrate, and attached with its left side. The plus gamete swam to the minus gamete, attached ventral to the right side of the minus gamete, while slightly on its left side, and plasmogamy started. No specialized organelle for gamete fusion was seen using either scanning or transmission electron microscopy. Gametic fusion was uniform; the right side of the minus gamete always fused with the ventral, slightly left side of the plus gamete, which suggests the participation of the d-rootlets of the flagellar apparatus of the two gametes. Body scales were retained throughout the entire sexual process. Before karyogamy, a network of endoplasmic reticulum developed between the nuclei. This position corresponded to the contractile vacuole of the plus gamete. Fusion proceeded as the minus gamete was drawn to the plus gamete and resulted in a hemispherical zygote. Fibrous material appeared on the cell surface, embedding the body scales to form a layer that thickened and contributed to the strong adhesion of the zygote to the substrate. During this stage, karyogamy was completed. A thick zygotic wall composed of two layers, an electron-dense outer layer and a straticulate electron-lucent inner layer developed beneath the layer of fibrous material and scales. Zygote germination was induced. After the first meiotic division, the layer of fibrous material and scales ruptured and the inner layer of the zygotic wall thinned, allowing the emergence of two germ cells. They had newly formed scales and two starch grains, but no typical pyrenoid.

The holococcolith Calyptrosphaera sphaeroidea Schiller was collected at Miyake-jima Island, Japan and unialgal cultures established. Alternation of the holococcolith and heterococcolith phases was induced using new culture media (MNK, TR, and LO). Cells synchronized in the holococcolith phase were transferred into TR medium to induce a life cycle change. The heterococcolith phase, which has never been reported before, appeared after more than 40 days. The heterococcoliths were very small elliptical discs, about 0.5 μm wide and 1 μm long. Typical diploid-type organic scales on the cell surface were observed. This phase was very stable in culture and was tolerant of unfavorable conditions. To reverse the life phase, cells in the heterococcolith phase were transferred into cold LO medium and exposed to low temperature (4° C) and low light (2 μmol photons.m - 2 .s - 1 ) for 30min before culturing at normal conditions (22.5° C and 20 μmol photons. m - 2 .s - 1 ). The swimming behavior of the holococcolith cells seemed to be an indicator of the life cycle phase transition. This article reports for the first time a set of conditions that could control the transition of a coccolithophorid from one life phase to the other. Selected vitamins and trace metals induced the heterococcolith phase, whereas a slightly higher concentration of components in the basic medium along with concomitant stresses of light and temperature induced the holococcolith phase. Based on the results, we propose a hypothesis that the alternation of coccolithophorid life phases is regulated by changes between pelagic and coastal environments coupled with changes in seasonal conditions.

This study reports a hitherto undescribed endophytic diatom that forms dense unialgal colonies in the mucilage that fills the internodes of a red alga, Coelarthrum opuntia. The diatom is very similar to Gyrosigma tenuissimum, in that the valve outline is delicate and linear, although it has a central raphe with externally unusual, overlapping central fissures. Based on morphological features and its unusual habitat, this endophyte is described as a new species: G. coelophilum Okamoto & Nagumo sp. nov. Gyrosigma coelophilum was found only in the internodes of C. opuntia, not on the thallus surface, indicating that G. coelophilum is an endophyte and not an epiphyte; this situation was true of the thalli collected from several different localities. Furthermore, we measured the light transmittance of the cortex of C. opuntia to estimate the light condition inside the internodes where G. coelophilum lives as an endophyte. It was shown that more than half the light that G. coelophilum can potentially e...

The marine prasinophycean flagellate Cymbomonas tetramitiformis has been established in culture from Japan and Australia and is examined in detail by electron microscopy. The results are described as a series of reports, the first of which deals with the general cell structure and the structure and morphogenesis of the organic scales that cover the surface of the cell and the flagella. The cell produces seven types of organic scales, all of which are manufactured in the cisternae of the Golgi apparatus. Two scale types are deposited on the cell body and cover the cell in three close-fitting layers, two types are confined to the flagellar pit region, and three types cover the flagellar surfaces. Cymbomonas is quadriflagellate and closely related to another quadriflagellate, Pyramimonas, in scale structure and scale ontogeny. It differs markedly in cell symmetry, swimming behavior, and in details of the flagellar apparatus. The flagellar end of the cell with the flagellar pit is surrounded by a horseshoe-shaped rim, which is very different from the usually four-lobed anterior end of Pyramimonas. Cymbomonas also shows resemblance in scale structure to members of the order Mamiellales, notably Mamiella, and to the phycoma-producing genera Pterosperma and Halosphaera. The Japanese isolate of Cymbomonas formed cysts for a short period, and the cyst appears to be homologous with the so-called phycoma stage of Pterosperma and Halosphaera. Since two chloroplasts were seen in the cysts, the cysts probably arise as a result of sexual reproduction. Concomitant with the presence of cysts in the culture, tiny uniflagellate cells were also present, and they probably represent gametes. Cymbomonas was described from the Adriatic in 1913 and was not reported again anywhere until 1986 and 1987 when Throndsen refound it in the Gulf of Naples in southern Italy. It has subsequently been found in large parts of the world and is now readily recognized by its characteristic scales and mode of swimming. It is often common but does not appear to form blooms.Key words: Prasinophyceae, marine nanoplankton, ultrastructure, Cymbomonas.

The photosynthetic pigment composition of Mesostigma viride Lauterborn, a primitive green alga, was determined. This alga contained chl a and b, lycopene, lutein, siphonaxanthin, γ-carotene, (3-carotene, antheraxanthin, violaxanthin, neoxanthin, and two novel carotenoid fatty acid esters, siphonaxanthin C12:0 ester and siphonaxanthin C14:0 ester. The esters were saturated, whereas all previously identified siphonaxanthin and loroxanthin esters have been mono-unsaturated (trans-Δ2). Neoxanthin was the all-trans form. This is the first such case detected in the chloroplasts of green plants. The 9'-cis form of neoxanthin is believed to be universally present in the chloroplasts of green plants (Streptophyta and Chlorophyta) and is a precursor of abscisic acid. However, the 9'-cis form was not found in M. viride. Based on these results, we discuss the phylogenetic implications and early evolution of the antenna pigment system in green plants.

Pedinella squamata Sekiguchi, Kawachi, Nakayama & Inouye sp. nov. (Pedinellales, Dictyochophyceae) was obtained from an offshore water sample collected in Miyagi Prefecture, Japan. The organism had a ring of tentacles around the flagellum and a stalk that extended posteriorly and was capable of attaching to the substratum. In anterior view, the cell was radially symmetrical, with six chloroplasts that possessed bulging pyrenoids. Plate scales covered the cell surface. Cells were mixotrophic, capturing food particles with the anterior tentacles and ingesting them from the anterior end of the cell. A second alga with three chloroplasts, anterior tentacles and a coiled posterior stalk was also investigated. A proximal helix occurred in the basal bodies of this alga. It was assigned to Helicopedinella Sekiguchi, Kawachi, Nakayama & Inouye gen. nov. as H. tricostata (Roukhiyajnen) Sekiguchi, Kawachi, Nakayama & Inouye comb. nov. A cladistic analysis was performed for 14 dictyochophycean species (11 ge...

Summary 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, rbc L, 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 rbc L 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.

Summary A marine flagellate resembling Cafeteria roenbergensis (bicosoecids, stramenopiles) in cell shape and behavior of the cell while attached to substratum was collected from the coast of Japan. The flagellate was examined by light and electron microscopy, and the 18S rDNA was sequenced to elucidate its taxonomic and phylogenetic position. Ultrastructural features suggested that the flagellate is not a bicosoecid, but a relative of the recently described stramenopile, Wobblia lunata. 18S rDNA phylogenetic trees also revealed that the flagellate forms a monophyletic clade with W. lunata and that it is distantly related to Cafeteria and other bicosoecids. The flagellate differs from W. lunata due to its lack of wobbling motion as well as intracellular features such as the number of mitochondria, flagellar apparatus architecture, the presence of a paranuclear body and cytoplasmic microtubules. The similarity of 18S rDNA sequences was 81% between the flagellate and W. lunata. This new flagellate was described as Placidia cafeteriopsis gen. et sp. nov. Because the phylogenetic lineage comprised of W. lunata and P. cafeteriopsis was one of the major, deep-branching clades of the stramenopiles, the class Placididea (= Placidiophyceae) classis nova was proposed.

Carotenoid composition and spectroscopic characteristics were analyzed for Pterosperma cristatum Schiller, one of the most primitive known members of green algae. This alga contained a substantial amount of carotenoid esters, siphonaxanthin C14:1 trans-Δ2 ester and 6′-OH siphonaxanthin C14:1 trans-Δ2 ester, but lacked lutein. This is the first report of carotenoid C14:1 trans-Δ2 esters from phototrophic organisms. In vivo absorption spectra and excitation spectra of the cells revealed that these carotenoids absorbed blue-green light and could transfer energy to chl a. These carotenoids were concluded to function as antenna pigments in P. cristatum.

This study investigated the taxonomic affiliation of the algal strain nak-9, which has been reported to absorb radioactive cesium with high efficiency, using light and electron microscopy, and molecular phylogenetic analysis based on 18S ribosomal RNA gene (rDNA) sequences. This alga is spherical and coccoid, with a smooth cell wall, large vacuole, crystalline structure, reddish globule, and refractile granules (lamellate vesicles). The cells possess one to several greenish parietal chloroplasts with a bulging pyrenoid surrounded by lamellate vesicles. The chloroplasts include orderly thylakoid lamellae but no girdle lamella. Molecular phylogenetic analysis suggests that strain nak-9 is a member of the eustigmatophycean clade, which includes Goniochloris, Pseudostaurastrum, and Trachydiscus. On the basis of these results, we propose that strain nak-9 (NIES-2860) comprises a new species and new genus of the Eustigmatophyceae, Vacuoliviride crystalliferum gen. et sp. nov.

Cells of five unarmoured kleptoplastidic dinoflagellates, Amphidinium latum, Amphidinium poecilochroum, Gymnodinium amphidinioides, Gymnodinium acidotum and Gymnodinium aeruginosum were observed under light and/or scanning electron microscopy and subjected to single-cell PCR. The SSU rDNA and the partial LSU rDNA of all the examined species were sequenced, and the SSU rDNA of G. myriopyrenoides was sequenced. Phylogenetic analyses revealed that the unarmoured kleptoplastidic species formed a monophyletic clade within the Gymnodinium-clade sensu Daugbjerg et al. (2000). The sister taxa for this clade were Gymnodinium palustre and Spiniferodinium galeiforme, both of which possess brown-coloured chloroplasts. The results indicated that acquisition of kleptoplastidy in these unarmoured dinoflagellates was a single event and that these unarmoured kleptoplastidic dinoflagellates may have evolved from a form with permanent chloroplasts. Molecular trees suggested that the acquisition of kleptoplastidy took place in a marine habitat and later some species colonized the freshwater habitat. Because these unarmoured kleptoplastidic dinoflagellates are monophyletic and characterized by distinct morphological and cytological features (including the presence of the same type of apical groove, absence of nuclear chambers in the nuclear envelope, absence of genuine chloroplasts, and the possession of kleptochloroplasts), we propose the establishment of a new genus, Nusuttodinium, to accommodate all these dinoflagellates.

A phylogeny of 21 haptophyte algae was inferred by maximum parsimony, neighbor-joining, and maximum likelihood analyses of sequences of the plastid-encoded gene, rbcL. Sequence variation in the spacer region of the RUBISCO operon was also investigated. In all the rbcL trees constructed, the haptophytes form two distinct clades: one includes the Pavlovales and the other includes the Prymnesiales, Coccosphaerales, and Isochrysidales (all sensuParke and Green 1976. This relationship coincides with the recent taxonomic treatment splitting the division into two subclasses, the Prymnesidae and Pavlovidae (Cavalier-Smith 1989) or the Prymnesiophycidae and the Pavlovophycidae using botanical suffixes ( Jordan and Green 1994), or into two classes, the Patelliferea and the Pavlovea (Cavalier-Smith 1993). In the Prymnesiophycidae, all the coccolithophorids examined are placed in a single clade, which suggests a single origin of the coccolithophorids and the ability of coccolith formation in the haptophytes. The genus Chrysochromulina is polyphyletic. Species of Chrysochromulina with a very long haptonema and a compressed cell body (typical of species including the type C. parva Lackey) form a clade, including Imantonia, that is often classified in the Isochrysidales in the neighbor-joining tree, whereas some species possessing a nontypical cell body and cell covering form a clade with Prymnesium and Platychrysis in all trees. It is suggested that loss of the haptonema in Imantonia and the reduction in Prymnesium and Platychrysis occurred secondarily and independently in two different lineages. Within the coccolithophorids, four clades are recognized: Pleurochrysis, Calyptrosphaera-Cruciplacolithus-Calcidiscus-Umbilicosphaera, Helicosphaera, and Emiliania-Gephyrocapsa. A non-coccolith-bearing haptophyte, Isochrysis, is an ingroup of the Emiliania-Gephyrocapsa clade, suggesting its secondary loss of the ability to form a coccolith. Sequence comparison of the spacer region of RUBISCO operon supports most results obtained in the analysis of rbcL sequences. Monophyly of the Prymnesiales sensu Parke and Green is still unclear because of low (

The physiological responses of alkenone unsaturation indices to changes in growth status of E. huxleyi and G. oceanica strains isolated from a water sample of the NW Pacific were examined using an isothermal batch culture system. In both E. huxleyi and G. oceanica the unsaturation index U37K′ changed during the growth period, but the effects of this change were different. This suggests that genotypic variation rather than the adaptation of the strains to the geographical environment of the sampling location is a major factor in determining the physiological responses to U37K′. Changes of U37K′ were associated with those of the unsaturation indices of C38 and C39 alkenones, the abundance ratios of lower to higher homologues of alkenones, the abundance ratios of saturated to polyunsaturated n-fatty acids, the abundance ratio of ethyl alkenoate to alkenones, and sterol contents. These associations might be attributable to the physiological response of lipids for maintaining their fluidity. The degree of unsaturation both in alkenones and n-fatty acids increased at day 8, possibly due to nutrient depletion. The ethyl alkenoate/total alkenone and ethyl alkenoate/C37 alkenone ratios increased abruptly at day 8 in both strains. These ratios should be useful in clarifying the relationship between the marine environment and its corresponding growth phase of batch culture. E. huxleyi and G. oceanica can be effectively distinguished using the U37K′-U38EtK diagram.

A new prasinophycean flagellate, Crustomastix didyma Nakayama, Kawachi & Inouye gen. & sp. nov., is described from the northwestern Pacific. This alga is elongate and bean-shaped and has two ventrally inserted, subequal flagella, with three types of hair scales. The entire cell and flagella are covered by a thin coat, which is probably derived from the Golgi apparatus and released through the duct system. The yellowish chloroplast is divided into two lobes connected by a narrow bridge. There is no pyrenoid or eyespot. The flagellar apparatus comprises the distal and proximal fibres, the Id and Is microtubular roots, and a duct fibre. A multilayered structure (MLS) is associated with the Id root. Some features, such as the swimming behaviour, the transitional region, and the absence of microtubular roots associated with the number 2 basal body, suggest a close relationship to the Mamiellales, but this alga differs from other members of the Mamiellales in having a MLS and a duct system, which are f...