Mitsunori Iwataki

Chlorella variabilis and Micractinium reisseri sp. nov. (Chlorellaceae, Trebouxiophyceae): Redescription of the endosymbiotic green algae of Paramecium bursaria (Peniculia, Oligohymenophorea) in the 120th year

Symbiotic algae of the ciliate Paramecium bursaria (Ehrenberg) Focker are key species in the fields of virology and molecular evolutionary biology as well as in the biology of symbiotic relationships. These symbiotic algae were once identified as Zoochlorella conductrix Brandt by the Dutch microbiologist, Beijerinck 120 years ago. However, after many twists and turns, the algae are today treated as nameless organisms. Recent molecular analyses have revealed several different algal partners depending on P. bursaria strains, but nearly all P. bursaria contains a symbiont belonging to either the so‐called ‘American’ or ‘European’ group. The absence of proper names for these algae is beginning to provoke ill effects in the above‐mentioned study areas. In the present study, we confirmed the genetic autonomy of the ‘American’ and ‘European’ groups and described the symbionts as Chlorella variabilis Shihira et Krauss and Micractinium reisseri Hoshina, Iwataki et Imamura sp. nov., respectively (Chlorellaceae, Trebouxiophyceae).

Cochlodinium fulvescens sp. nov. (Gymnodiniales, Dinophyceae), a new chain-forming unarmored dinoflagellate from Asian coasts

Cellular morphology and the phylogenetic position of a new unarmored photosynthetic dinoflagellate Cochlodinium fulvescens Iwataki, Kawami et Matsuoka sp. nov. were examined by light microscopy and molecular phylogenetic analyses based on partial large subunit ribosomal DNA (LSU rDNA) and small subunit ribosomal DNA (SSU rDNA) sequences. The cells of C. fulvescens closely resemble C. polykrikoides, one of the most harmful red tide forming dinoflagellates, due to it possessing a cingulum encircling the cell approximately twice, a spherical nucleus positioned in the anterior part of the cell and an eyespot‐like orange pigmented body located in the dorsal side of the epicone, as well as formation of cell‐chains. However, this species is clearly distinguished from C. polykrikoides based on several morphological characteristics, namely, cell size, shape of chloroplasts and the position of narrow sulcus situated in the cell surface. The sulcus of C. fulvescens is located at the intermediate position of the cingulum in the dorsal side, whereas that of C. polykrikoides is situated immediately beneath the cingulum. LSU rDNA phylogenies indicated that C. fulvescens is clearly distinct from, but closely related to C. polykrikoides among dinoflagellates.

Reconstruction of historical nutrient levels in Korean and Japanese coastal areas based on dinoflagellate cyst assemblages.

Dinoflagellate cysts acquired from sediment cores were analyzed in order to reconstruct historical nutrient levels in Gamak Bay, Korea and Ariake Bay, Japan. Dinoflagellate cyst assemblages in Gamak Bay were characterized by high proportions of heterotrophic cysts such as Brigantedinium spp., Protoperidinium americanum and Polykrikos cysts, which suggested that nutrients levels may have already been high before 1970s, and then increased further to the hypertrophic conditions of the 1990s. In contrast, dinoflagellate cyst assemblages in Ariake Bay were characterized by high relative abundances of Lingulodinium machaerophorum and Spiniferites spp., which suggested that nutrient levels in Ariake Bay had increased gradually since the mid 1960s, and may have been significantly enhanced by the mid 1980s. Dinoflagellate cyst assemblages reflecting environmental changes in the two bays are contrasting, perhaps due to different nutrient enrichment mechanisms. This suggests that the indicators of nutrient levels encoded in dinoflagellate cyst assemblages may exhibit site-specific information.

Proposal of identification criteria for resting spores of Chaetoceros species (Bacillariophyceae) from a temperate coastal sea

Ishii K.-I., Iwataki M., Matsuoka K. and Imai I. 2011. Proposal of identification criteria for resting spores of Chaetoceros species (Bacillariophyceae) from a temperate coastal sea. Phycologia 50: 351–362. DOI: 10.2216/10-36.1 Chaetoceros is a large, ecologically important genus of marine planktonic diatoms. Many species of Chaetoceros form resting spores to survive environmental stress. Species identification based on morphological characteristics of resting spores is difficult. In the present study, we proposed a flow diagram for the identification of Chaetoceros species based on morphology. Resting spores of 18 Chaetoceros species were identified in water and sediment samples from southern Japan and the East China Sea based on morphological characteristics of resting spores and/or the vegetative cells containing them. A single ring of puncta was found to be the most important morphological feature of resting spores for species identification, by which the positions of primary and secondary valves were correctly determined. Other morphological characteristics such as features of the primary valve face, margins of the primary and secondary valve mantles, and secondary valve face are also useful for species identification. A combination of these morphological characteristics was used to construct a flow diagram for identification of the 18 Chaetoceros species found in our samples with light microscopy. This flow diagram is most applicable to Japanese waters, but the approach should be refined and improved to understand the biology and ecology of Chaetoceros species in any marine ecosystem.

Further examination of the cyst-theca relationship of Protoperidinium thulesense (Peridiniales, Dinophyceae) and the phylogenetic significance of round brown cysts

K. Matsuoka, H. Kawami, R. Fujii and M. Iwataki. 2006. Further examination of the cyst-theca relationship of Protoperidinium thulesense (Peridiniales, Dinophyceae) and the phylogenetic significance of round brown cysts. Phycologia 45: 632–641. DOI: 10.2216/05-42.1 The heterotrophic armored dinoflagellate Protoperidinium thulesense has an unusual combination of morphological characters, i.e. the thecal plate arrangement of the motile cell resembles a typical Protoperidinium, whereas the shape and archeopyle of the cyst are like the diplopsalids. We have re-examined the cyst-motile relationship of P. thulesense by cyst incubation and thecal plate analysis together with a molecular phylogenetic study based on small subunit (SSU) rDNA sequences. Five isolates of P. thulesense, including motile cells and cysts, and three Protoperidinium and three diplopsalid species were examined by using the single cell PCR method. The thecal plate arrangement of the motile cells isolated from field samples and those germinated from cysts were identical. The plate formula was: Po, X, 3′, 3a, 7″, 3c+t, 4s, 5′″, 2″″. The cysts of P. thulesense were round and brown with a theropylic archeopyle, and rather similar to the diplopsalid species, Diplopsalis lenticula, D. lebourae, Gotoius abei and others. SSU rDNA sequence analysis reveals P. thulesense is closely related to the subgenus Protoperidinium, especially species of the section Conica, and distant from the diplopsalid species. The phylogenetic and taxonomic significances of Protoperidinium and diplopsalid round brown cysts with a theropylic archeopyle are discussed.

Ultrastructure of the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) with reference to the apical groove and Flagellar apparatus.

ABSTRACT. The external and internal ultrastructure of the harmful unarmored dinoflagellate Cochlodinium polykrikoides Margalef has been examined with special reference to the apical groove and three‐dimensional structure of the flagellar apparatus. The apical groove is U‐shaped and connected to the anterior sulcal extension on the dorsal side of the epicone. The eyespot is located dorsally and composed of two layers of globules situated within the chloroplast. A narrow invagination of the plasma membrane is associated with the eyespot. The nuclear envelope has normal nuclear pores similar to other eukaryotes but different from the Gymnodinium group with diagnostic nuclear chambers. The longitudinal and transverse basal bodies are separated by approximately 0.5–1.0 μm and interconnected directly by a striated basal body connective and indirectly by microtubular and fibrous structures. Characteristic features of the flagellar apparatus are as follows: (1) a nuclear extension projects to the R1 (longitudinal microtubular root) and is connected to the root by thin fibrous material; (2) fibrillar structures are associated with the longitudinal and transverse flagellar canal; and (3) a striated ventral connective extends toward the posterior end of the cell along the longitudinal flagellar canal. We conclude, based on both morphological and molecular evidence, that Cochlodinium is only distantly related to Gymnodinium.

Investigations of body scales in twelve Heterocapsa species (Peridiniales, Dinophyceae), including a new species H. pseudotriquetra sp. nov.

Abstract The body scales of 12 Heterocapsa species including a new species, H. pseudotriquetra Iwataki, Gert Hansen & Fukuyo, sp. nov. were investigated by transmission electron microscopy to clarify ultrastructural differences. All species had scales with a body-scale structure consisting of a triradiate basal plate and a three-dimensional construction composed of vertically standing uprights or spines and horizontal bars. This basic scale structure is considered to be a generic characteristic. Finer scale details such as the shape and presence of a central hole in the basal plate, the numbers of uprights, bars and spines are considered to be species-specific. Two types of scales were found within clonal cultures of H. arctica and H. circularisquama and it is suggested that these represent ‘mature’ and ‘immature’ body scales. Intraspecific variation of the basal plate ‘reticulation’ was also observed. This variation was particularly evident in prolonged cultures of H. horiguchii and H. rotundata. The body-scale structure of 11 described species can be distinguished. However, two species, namely H. triquetra and H. pseudotriquetra, have the same scale morphology and are distinguished by differences in cell shape and LSU rDNA (25S rDNA) sequences.

Heterocapsa psammophila sp. nov. (Peridiniales, Dinophyceae), a new sand‐dwelling marine dinoflagellate

A new armored dinoflagellate species, Heterocapsa psammophila Tamura, Iwataki et Horiguchi sp. nov. is described from Kenmin‐no‐hama beach, Hiroshima, Japan using light and electron microscopy. This dinoflagellate possesses the typical thecal plate arrangement of the genus Heterocapsa, Po, cp, 5′, 3a, 7′′, 6c, 5s, 5′′′, 2′′′′; and the 3‐D body scales of Heterocapsa on the plasma membrane. The cell shape is ovoidal. The spherical nucleus and the pyrenoid are situated in the hypotheca and the epitheca, respectively. The ultrastructure of H. psammophila is typical of dinoflagellates and the pyrenoid is invaginated by cytoplasmic tubules. H. psammophila is distinguished from all other hitherto‐described Heterocapsa species by the cell shape, the relative position of the nucleus and pyrenoid and the structure of the body scale. The habitat and behavior of this new species in culture suggest that the organism is truly a sand‐dwelling species.

Research Note: Molecular phylogenetic affiliations of Dissodinium pseudolunula, Pheopolykrikos hartmannii, Polykrikos cf. schwartzii and Polykrikos kofoidii to Gymnodinium sensu stricto species (Dinophyceae)

Partial large subunit ribosomal DNA (LSU rDNA) sequences of Dissodinium pseudolunula Swift ex Elbrächter et Drebes, Pheopolykrikos hartmannii (Zimmermann) Matsuoka et Fukuyo, Polykrikos cf. schwartzii Bütschli and Polykrikos kofoidii Chatton were analyzed to reveal their phylogenetic status. The four athecate dinoflagellate species fell within the clade of Gymnodinium sensu G. Hansen et Moestrup supported by high likelihood values without apparent phylogenetic relationships to neither Gymnodinium species nor each other. Their genetic affiliations to typical Gymnodinium species were morphologically supported by the loop‐shaped apical grooves in Pheopolykrikos and Polykrikos species and the Gymnodinium‐like motile stage at least once during pleomorphic life cycles in D. pseudolunula and Ph. hartmannii.

Morphology of two marine woloszynskioid dinoflagellates, Biecheleria brevisulcata sp. nov. and Biecheleriopsis adriatica (Suessiaceae, Dinophyceae), from Japanese coasts

Abstract: The morphology of two marine woloszynskioid dinoflagellates from the Japanese coast was examined by light, scanning and transmission electron microscopy, and phylogenetic positions were inferred on the basis of partial nuclear-encoded large-subunit ribosomal (r)DNA (D1–D3) sequences. Both species had a single elongate apical vesicle (EAV) on the anterior end, a diagnostic feature of the Suessiaceae, and were identified as Biecheleriopsis adriatica and an undescribed species of Biecheleria. Cells of the undescribed Biecheleria were spherical to ellipsoidal with parietal chloroplasts, conspicuous pyrenoids, an eyespot, and a nucleus located anteriorly. Transmission electron microscope observations revealed penetrations of the thylakoid into the pyrenoid matrix and an eyespot composed of a stack of cisternae containing several brick-like materials (type E). This species had a total of nine to 10 latitudinal series of amphiesmal vesicles (AVs), including three to fo ur epiconal, three cingular, and three hypoconal series. Nineteen small globular knobs were present on the EAV. This species was distinguished from other marine woloszynskioids by cell size, position of nucleus, and the number of AV series and EAV knobs, i.e. previously reported Biecheleria have more than 25 knobs. We describe a new marine woloszynskioid dinoflagellate Biecheleria brevisulcata sp. nov. The Japanese strains of Bps. adriatica had 11–12 series of AVs in total, including four to five epiconal, three cingular, and four hypoconal series, and there were 32 knobs on the EAV. The features of these strains basically coincided with those of the original description of Bps. adriatica, but differed in the larger number of EAV knobs. Molecular phylogeny also indicated their affinity to the Suessiaceae. Strains of B. brevisulcata formed a clade related to B. baltica and B. cincta. The two genera, Biecheleria and Biecheleriopsis, have a strong morphological resemblance to one another, and they were distinguished only by rDNA sequences and the presence of a fibrous flagellar root (nuclear connective). This study also showed a morphological difference between the two genera; vesicles of the third row of cingular vesicles were larger and the lower cingular margin had a zigzag line in Biecheleria, whereas they were smaller and formed a straight line in Biecheleriopsis, especially on the dorsal side.