Yoshihito Takano

ACQUIRING SCANNING ELECTRON MICROSCOPICAL, LIGHT MICROSCOPICAL AND MULTIPLE GENE SEQUENCE DATA FROM A SINGLE DINOFLAGELLATE CELL1

We have developed a useful method to obtain light and scanning electron micrographs of a single dinoflagellate cell, prior to applying the cell to the single cell PCR technique. This method allows us to record detailed morphological information on any cell used for sequencing, which can be extremely important for the future identification of the organism, because cells used for single cell PCR usually cannot be retained. Furthermore, by applying multiple sets of PCR primers at the same time, we have successfully amplified and sequenced multiple genes (and DNA regions) simultaneously, even from a single cell. In this note, we demonstrate the methods of this technique by using two different types of dinoflagellates, i.e. an armored freshwater species, Peridinium willei Huitfeld‐Kaas, and an unarmored marine species, Akashiwo sanguinea (Hirasaka) Hansen and Moestrup. By rotating the cell, photographs of all aspects of a single cell can be taken even using the SEM. The genes and DNA regions sequenced in these examples include a region of the ribosomal DNA (SSU, ITS1, 5.8S, ITS2, and part of the LSU) as well as part of the mitochondrial DNA‐encoded gene, cox1. This technique can be applied to both photosynthetic and heterotrophic dinoflagellates and will accelerate biodiversity studies.

Surface ultrastructure and molecular phylogenetics of four unarmored heterotrophic dinoflagellates, including the type species of the genus Gyrodinium (Dinophyceae)

Small subunit rRNA gene sequences were determined for four unarmored heterotrophic dinoflagellates (Gyrodinium spirals, the type species of the genus Gyrodinium, as well as G. fusiforme, Gymnodinium rubrum and the freshwater species G. helveticum) using a single‐cell polymerase chain reaction (PCR) technique. For identification and record keeping, each cell was carefully observed and photographed using a light microscope under high magnification, prior to single‐cell PCR. G. rubrum and G. helveticum possess an elliptical apical groove and longitudinal striations similar to those of G. spirale and G. fusiforme, and molecular phylogenetic studies reveal that the four species form a single clade. We therefore propose the following new combinations: Gyrodinium rubrum (Kofoid et Swezy) Takano et Horiguchi comb. nov. and Gyrodinium helveticum (Penard) Takano et Horiguchi comb. nov.

NEW EVIDENCE FOR MORPHOLOGICAL AND GENETIC VARIATION IN THE COSMOPOLITAN COCCOLITHOPHORE EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) FROM THE COX1b-ATP4 GENES

Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler is a cosmopolitan coccolithophore occurring from tropical to subpolar waters and exhibiting variations in morphology of coccoliths possibly related to environmental conditions. We examined morphological characters of coccoliths and partial mitochondrial sequences of the cytochrome oxidase 1b (cox1b) through adenosine triphosphate synthase 4 (atp4) genes of 39 clonal E. huxleyi strains from the Atlantic and Pacific Oceans, Mediterranean Sea, and their adjacent seas. Based on the morphological study of culture strains by SEM, Type O, a new morphotype characterized by coccoliths with an open central area, was separated from existing morphotypes A, B, B/C, C, R, and var. corona, characterized by coccoliths with central area elements. Molecular phylogenetic studies revealed that E. huxleyi consists of at least two mitochondrial sequence groups with different temperature preferences/tolerances: a cool‐water group occurring in subarctic North Atlantic and Pacific and a warm‐water group occurring in the subtropical Atlantic and Pacific and in the Mediterranean Sea.

Serial replacement of a diatom endosymbiont in the marine dinoflagellate Peridinium quinquecorne (Peridiniales, Dinophyceae)

To infer the phylogeny of both the host and the endosymbiont of Peridinium quinquecorne Abé, the small subunit (SSU) ribosomal DNA (rDNA) from the host and two genes of endosymbiont origin (plastid‐encoded rbcL and nuclear‐encoded SSU rDNA) were determined. The phylogenetic analysis of the host revealed that the marine dinoflagellate P. quinquecorne formed a clade with other diatom‐harbouring dinoflagellates, including Kryptoperidinium foliaceum (Stein) Lindeman, Durinskia baltica (Levander) Carty et Cox and Galeidinium rugatum Tamura et Horiguchi, indicating a single endosymbiotic event for this lineage. Phylogenetic analyses of the endosymbiont in these organisms revealed that the endosymbiont of P. quinquecorne formed a clade with a centric diatom (SSU data indicated it to be closely related to Chaetoceros), whereas the endosymbionts of other three dinoflagellates formed a clade with a pennate diatom. The discrepancy between the host and the endosymbiont phylogenies suggests a secondary replacement of the endosymbiont from a pennate to a centric diatom in P. quinquecorne.

Serial Replacement of Diatom Endosymbionts in Two Freshwater Dinoflagellates, Peridiniopsis spp. (Peridiniales, Dinophyceae)

Y. Takano, G. Hansen, D. Fujita and T. Horiguchi. 2007. Serial replacement of diatom endosymbionts in two freshwater dinoflagellates, Peridiniopsis spp. (Peridiniales, Dinophyceae). Phycologia 47: 41–53. DOI: 10.2216/07-36.1 Two freshwater armoured dinoflagellates, Peridiniopsis cf. kevei from Japan and Peridiniopsis penardii from Japan and Italy, were examined by means of light, scanning and transmission electron microscopy. Morphological studies indicated that the two dinoflagellates had similar type of cellular structure and possessed an endosymbiotic diatom. The diatom endosymbiont, which contained a eukaryotic nucleus, chloroplasts and mitochondria, was separated from the dinoflagellate cytoplasm by a single unit membrane. The dinoflagellate cytoplasm contained a triple-membrane-bound eyespot, in addition to typical dinoflagellate organelles. Molecular phylogenetic analyses based on small subunit ribosomal RNA gene (SSU rDNA) revealed a close relationship between these two dinoflagellates. They formed a clade with other dinoflagellates possessing a diatom endosymbiont, suggesting a single origin of diatom-harbouring dinoflagellates. On the contrary, the phylogenetic analyses based on plastid-encoded rbcL and nuclear-encoded SSU rDNA of the endosymbionts included the endosymbiont of these two freshwater dinoflagellates in the Thalassiosira/Skeletonema-clade (Centrales), whereas the endosymbionts of other diatom-containing dinoflagellates, except for Peridinium quinquecorne, were closely related to members of the Bacillariaceae (Pennales), most likely a Nitzschia-like diatom. The discrepancy between the host phylogeny and the endosymbiont phylogeny suggested, as in the case of Peridinium quinquecorne, that there was a serial replacement of endosymbionts from original a pennate Nitzschia-like diatom to a centric diatom, such as Thalassiosira (or possibly Skeletonema).

A new heterotrophic dinoflagellate from the North-eastern Pacific, Protoperidinium fukuyoi: cyst–theca relationship, phylogeny, distribution and ecology

The cyst–theca relationship of Protoperidinium fukuyoi n. sp. (Dinoflagellata, Protoperidiniaceae) is established by incubating resting cysts from estuarine sediments off southern Vancouver Island, British Columbia, Canada, and San Pedro Harbor, California, USA. The cysts have a brown‐coloured wall, and are characterized by a saphopylic archeopyle comprising three apical plates, the apical pore plate and canal plate; and acuminate processes typically arranged in linear clusters. We elucidate the phylogenetic relationship of P. fukuyoi through large and small subunit (LSU and SSU) rDNA sequences, and also report the SSU of the cyst‐defined species Islandinium minutum (Harland & Reid) Head et al. 2001. Molecular phylogenetic analysis by SSU rDNA shows that both species are closely related to Protoperidinium americanum (Gran & Braarud 1935) Balech 1974. Large subunit rDNA phylogeny also supports a close relationship between P. fukuyoi and P. americanum. Three subgroups in total are further characterized within the Monovela group. The cyst of P. fukuyoi shows a wide geographical range along the coastal tropical to temperate areas of the North‐east Pacific, its distribution reflecting optimal summer sea‐surface temperatures of ~14–18 °C and salinities of 22–34 psu.

Phylogeny of five species of Nusuttodinium gen. nov. (Dinophyceae), a genus of unarmoured kleptoplastidic dinoflagellates.

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.

Encystment and Excystment under Laboratory Conditions of the Nontoxic Dinoflagellate Alexandrium fraterculus (Dinophyceae) Isolated from the Seto Inland Sea, Japan

S. Nagai, G. Nishitani, Y. Takano, M. Yoshida and H. Takayama. 2009. Encystment and excystment under laboratory conditions of the nontoxic dinoflagellate Alexandrium fraterculus (Dinophyceae) isolated from the Seto Inland Sea, Japan. Phycologia 48: 177–185. DOI: 10.2216/08-43.1. The sexuality (i.e. encystment and excystment) of the nontoxic dinoflagellate Alexandrium fraterculus, isolated from the Seto Inland Sea, Japan, was clarified for the first time under laboratory conditions. Sexual reproduction was by conjugation of isogametes, and plasmogamy was completed in 25–60 min after the conjugation started and produced a planozygote with one transverse and two longitudinal flagella, then a cyst was formed. Cysts were round or elliptical. The cyst size was 37.5–50.0 µm in diameter. The surface of cysts was smooth, and there was no paratabulation. Encystment through sexual reproduction was observed in two of 28 pairs, which included seven self-crossings, by use of seven nonaxenic clonal strains. No planozygote formation or encystment was found in any of the self-crossings, indicating that this species is heterothallic. Successful excystment was observed when the cysts, which had been preserved at 10°C in the dark for 3 mo, were incubated under light conditions at 25°C, thus showing that this species has a similar life cycle as reported in A. tamarense/catenella/tamiyavanichii.

Comparative study of the validity of three regions of the 18S‐rRNA gene for massively parallel sequencing‐based monitoring of the planktonic eukaryote community

The nuclear 18S‐rRNA gene has been used as a metabarcoding marker in massively parallel sequencing (MPS)‐based environmental surveys for plankton biodiversity research. However, different hypervariable regions have been used in different studies, and their utility has been debated among researchers. In this study, detailed investigations into 18S‐rRNA were carried out; we investigated the effective number of sequences deposited in international nucleotide sequence databases (INSDs), the amplification bias, and the amplicon sequence variability among the three variable regions, V1–3, V4–5 and V7–9, using in silico polymerase chain reaction (PCR) amplification based on INSDs. We also examined the primer universality and the taxonomic identification power, using MPS‐based environmental surveys in the Sea of Okhotsk, to determine which region is more useful for MPS‐based monitoring. The primer universality was not significantly different among the three regions, but the number of sequences deposited in INSDs was markedly larger for the V4–5 region than for the other two regions. The sequence variability was significantly different, with the highest variability in the V1–3 region, followed by the V7–9 region, and the lowest variability in the V4–5 region. The results of the MPS‐based environmental surveys showed significantly higher identification power in the V1–3 and V7–9 regions than in the V4–5 region, but no significant difference was detected between the V1–3 and V7–9 regions. We therefore conclude that the V1–3 region will be the most suitable for future MPS‐based monitoring of natural eukaryote communities, as the number of sequences deposited in INSDs increases.

Living fossils in the Indo-Pacific warm pool: A refuge for thermophilic dinoflagellates during glaciations

How important are refugia for plankton biogeography? Here for the first time we report living cysts of the fossil dinoflagellate Dapsilidinium pastielsii from Southeast Asia: Shioya Bay (Okinawa, Japan), Koror (Palau), Ambon (Indonesia), East Vietnam Sea (Vietnam), and Masinloc (the Philippines). This species, thought to have become extinct in the early Pleistocene, is the last survivor of a major early Cenozoic lineage. Its disappearance from the Atlantic following the early Pleistocene implies cooling, and the discovery of living D. pastielsii in the Indo-Pacific warm pool suggests that this unique environment with stable temperatures served as an important refuge for thermophilic dinoflagellates with a >50 m.y. lineage. This is the first record of a refugium plankton species within the Indo-Pacific warm pool.