Isao Inouye

The phylogenetic relationship between the Chlamydomonadales and Chlorococcales inferred from 18SrDNA sequence data

Nuclear‐encoded small subunit ribosomal RNA gene (185rDNA) sequences were determined for Chlamydomonas moewusii Gerloff and five chlorococcalean algae (Chlorococcum hypnosporum Starr; Chlorococcum oleofaciens Trainor et Bold; Chlorococcum sp.; Tetracystis aeria Brown et Bold; Protosiphon botryoides (Kützingl Klebs). All these algae are characterized by a clockwise CCW) flagellar apparatus. Phylogenetic trees were constructed from sequences from these algae together with 20 green algae. All algae with a CW flagellar apparatus form a monophyletic clade (CW group). Three principal clades can be recognized in the CW group, although no morphological character supports monophyly of any of these three clades. The 185rDNA trees clearly demonstrate the non‐monophyly of the Chlamydomonadales and Chlorococcales, suggesting that vegetative morphology does not reflect phylogenetic relationships in the CW group. The paraphyly or polyphyly of the genus Chlamydomonas and Chlorococcum are also revealed. Present analysis suggests that the presence or absence of a zoospores cell wall and the multinucleate condition have limited taxonomic values at higher taxonomic ranks.

The Basal Position of Scaly Green Flagellates among the Green Algae (Chlorophyta) is Revealed by Analyses of Nuclear-Encoded SSU rRNA Sequences

The prasinophytes comprise a morphologically heterogeneous assembly of mostly marine flagellates and coccoid taxa, which represent an important component of the nano- and picoplankton, and have previously figured prominently in discussions about the origin and phylogeny of the green plants. To evaluate their putative basal position in the Viridiplantae and to resolve the phylogenetic relationships among the prasinophyte taxa, we determined complete nuclear-encoded SSU rRNA sequences from 13 prasinophyte taxa representing the genera Cymbomonas, Halosphaera, Mamiella, Mantoniella, Micromonas, Pterosperma, Pycnococcus, and Pyramimonas. Phylogenetic analyses of SSU rRNA sequences using distance, parsimony and likelihood methods revealed four independent prasi.nophyte lineages (clades) which constitute the earliest divergences among the Chlorophyta. In order of their divergence these clades are represented by the genera Cymbomonas, Halosphaera, Pterosperma, Pyramimonas (clade I), Mamiella, Mantoniella, Micromonas (clade II), Pseudoscourfieldia (strain CCMP 717), Nephroselmis (clade III), and Tetraselmis, Scherffelia (clade IV). The coccoid Pycnococcus provasolii diverged after clade II, but before clade III. Since no other coccoid prasinophyte taxa were analyzed in this study, the phylogenetic status of this taxon is presently unresolved. Our analyses provide further evidence for the basal phylogenetic position of the scaly green flagellates among the Chlorophyta and raise important questions concerning the class-level classification of the Chlorophyta.

A green dinoflagellate with chlorophylls A and B: morphology, fine structure of the chloroplast and chlorophyll composition

A green‐colored marine unicell has been grown in unialgal culture and its morphology, chloroplast fine structure, and chlorophyll composition investigated. The organism is typical of dinoflagellates in its shape, flagellation, nucleus, mitochondria, and trichocysts. It is similar to Gymnodinium but possesses fine body scales. Chloroplasts and two kinds of vesicles bounded by double membranes, but no organelles obviously identifiable as nuclei or mitochondria, are associated in ribosome‐dense cytoplasm separated by a double membrane from the dinophycean cytoplasm. The chloroplasts are unlike any previously reported for dinoflagellates. Each is enclosed by an envelope consisting of a double membrane. Chloroplast lamellae consist of three appressed thylakoids. Interlamellar pyrenoids are present. Pigment analysis reveals chlorophylls a and b but not chlorophyll c. It seems likely that the organism is an undescribed dinoflagellate containing an endosymbiont with chlorophylls a and b and that the reduction of the endosymbiont nucleus and mitochondria has permitted a more initmate symbiosis.

INDUCED DIMORPHIC LIFE CYCLE OF A COCCOLITHOPHORID, CALYPTROSPHAERA SPHAEROIDEA (PRYMNESIOPHYCEAE, HAPTOPHYTA)1

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 30 min 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.

MOLECULAR PHYLOGENY OF THE HAPTOPHYTA BASED ON THE rbcL GENE AND SEQUENCE VARIATION IN THE SPACER REGION OF THE RUBISCO OPERON

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 sensu Parke 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 (<50%) bootstrap support for this group.

Physiological responses of lipids in Emiliania huxleyi and Gephyrocapsa oceanica (Haptophyceae) to growth status and their implications for alkenone paleothermometry

Abstract 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.

Thraustochytrid Aurantiochytrium sp. 18W-13a Accummulates High Amounts of Squalene

Here we report on the 18W-13a strain of Aurantiochytrium sp., which accumulates very high amounts of squalene. The squalene contents and production at 4 d of culture were 198 mg/g and 1.29±0.13 g/L, respectively, exceptionally high values compared to previous reports.

High-speed video analysis of the flagellar beat and swimming patterns of algae: possible evolutionary trends in green algae

SummaryThe flagellar beat and swimming patterns of flagellated cells of 22 green plants, including 17 green flagellates (volvocalean and prasinophyte algae), motile cells of three seaweeds,Bryopsis, Caulerpa, andUlva, sperms of a liverwort,Marchantia, and a fern,Athyrium, were examined using a high-speed video system. So-called breast-stroke is widely distributed in green plants, and occurs rarely in prasinophyte flagellates and ulvophycean algae; in these algal groups flagellar beat similar to that found in animal sperm is common, both during forward and backward swimming. Different types of swimming patterns were observed in prasinophytes. The results indicate evolutionary trends of flagellar beat and swimming patterns in green plants such as change from backward to forward swimming, from flagellar to ciliary beating and from uni-directional (parallel) to radial-directional (cruciate) beating. Such trends are shown in two prasinophyte groups, thePyramimonas-lineage andTetraselmis-lineage.

Optimization of culture conditions of the thraustochytrid Aurantiochytrium sp. strain 18W-13a for squalene production

Optimum conditions of temperature, salinity and glucose concentration were investigated for squalene production of the strain of Aurantiochytrium sp. 18 W-13a, with a high content of squalene. Squalene production by this strain was optimum at 25 °C, 25-50% seawater concentration and 2-6% glucose concentration. When this strain was grown in the optimum condition, the squalene content and production of approximately 171 mg/g dry weight and 0.9 g/L were much higher than that previously reported in thraustochytrids, plants and yeasts, respectively. Therefore, 18 W-13a could be used as an alternative source of commercial squalene.

What is new in coccolithophore biology

Knowledge of the biology of coccolithophores has progressed considerably in recent years thanks to culture studies and meticulous observations of coccospheres in wild samples. It has been confirmed that holococcolithophores and other “anomalous” coccolithophores are not autonomous but stages in the life cycle of oceanic heterococcolithophores. The existence of such heteromorphic life cycles linking former “species” has far reaching consequences on the taxonomy and nomenclature of coccolithophores and should foster research on the environmental factors triggering phase changes. The cytological characteristics of coccolithophores are reviewed in detail with special attention to the cell covering, coccolitho-genesis and the specificity of appendages in this group. There have been comparatively few recent studies concerning the cytology of oceanic representatives. Important issues such as status of aplastidic groups, mode of synthesis of holococcoliths/nannoliths and details of the flagellar apparatus need to be addressed. Such morphological data will enable a more natural classification of modern coccolithophores in a phylogenetic perspective.