ULTRASTRUCTURE OF VEGETATIVE AND DIVIDING CELLS OF THE UNICELLULAR RED ALGAE RHODELLA VIOLACEA AND RHODELLA MACULATA1

Rhodella violacea (Kornmann) Wehrmeyer and Rhodella maculata Evans were investigated for ultrastructural details of vegetative and dividing cells. Rhodella violacea has a nuclear projection into the pyrenoid similar to that found in R. maculata, although the nuclear projection in R. maculata traverses a starch-lined area before contacting the pyrenoid. Unlike most, red algae, the two Rhodella species lack a peripheral encircling thylakoid in the chloroplast and have dictyosomes associated solely with endoplasmic reticulum (ER) instead of with both mitochondria and ER. Both species also have a well-developed peripheral system of ER connected to the plasmalemma by tubules, a situation found only in red algal unicells, Cell division was studied primarily in R. violacea; a less thorough examination of R. maculata showed no essential differences. Both have small, double-ringed, nucleus-associated organ files (NAOs) surrounded by moderately electron-dense material, metaphase–anaphase polar gaps in the nuclear envelope, absence of perinuclear ER. and short interzonal spindles. This pattern of mitosis is similar in most respects to that reported in the unicell Flintiella. Following mitosis, microtubules extend from the region of each NAO to its associated nucleus and to the undivided pyrenoid. The NAOs appear to apply tension to the nuclei and the pyrenoid and may be the mechanism for ensuring equal partitioning of both organdies. Two different forms of pyrenoid-nucleus association occur during mitosis. Nuclear projections into the pyrenoid, prevalent during interphase and early stages of mitosis, recede at metaphase. Then, the pyrenoid extends protrusions into the nuclear polar areas, forming a cup that partially surrounds the nucleus. Cell division and vegetative characters confirm the close taxonomic affinity of these two species of Rhodella and support their separation from the genus Porphyridium.     

STRUCTURE AND FUNCTION OF THE ALGAL PYRENOID. I. ULTRASTRUCTURE AND CYTOCHEMISTRY DURING ZOOSPOROGENESIS OF TETRACYSTIS EXCENTRICA1

The fine structure of the pyrenoid in the mature vegetative cell of Tetracystis excentrica Brown and Bold is described. During zoosporogenesis, the pyrenoid undergoes regression, and the ultrastructure of this process is described in detail. The ground substance undergoes dissolution, and reticulate fibrillar structures appear as well as intruding chloroplast thylakoids. Pyrenoid-associated starch plates diminish, and quantities of starch not associated with the pyrenoid are produced. New pyrenoids appear late in the division cycle after all other major organelles associated with the motile cell have been formed. Zoospore pyrenoids develop in thylakoid-free spaces of the chloroplast which are similar to the DNA-containing regions. The new pyrenoid ground substance, which is loosely fibrillar, arises in close proximity to starch grains which may be formed in the stroma. Then the zoospore pyrenoid produces 2 hemispherical starch plates identical to those in the mature vegetative cell. Zoospore pyrenoids lack the 2 convoluted thylakoids between the starch plates and the ground substance characteristic of those in the mature vegetative cell. Instead, the thylakoids are identical to those of the chloroplast at first, and then develop into a convoluted state in the vegetative cell. Cytochemical tests for DNA, RNA, and protein were made for the cytoplasm, nucleus, nucleolus, and pyrenoid. Conclusive evidence is presented for the presence of RNA in the cytoplasm and nucleolus, DNA in the nucleus, and protein in the pyrenoid. The tests did not conclusively demonstrate the presence or absence of DNA and RNA in the pyrenoid; however, they suggested that small amounts of both DNA and RNA may be present.     

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.     

SPECIES-SPECIFIC DIFFERENCES OF PYRENOIDS IN CHLORELLA (CHLOROPHYTA)1

The structure of the pyrenoid supports the separation of Chlorella species into two groups based on cell wall chemistry and suggests evolutionary relationships. Chlorella species with a glucan-type wall exhibit quite diverse pyrenoid structures, which may indicate that these species are not closely related. Those species with glucosamine cell walls (C. kessleri, C. sorokiniana, C. vulgaris) are virtually identical in pyrenoid morphology, indicating a closer evolutionary relationship. In the species with glucosamine walls, the thylakoid that penetrates into the pyrenoid matrix, is unijormly double-layered. Pyrenoids in the species with glucan walls show various features: 1) a pyrenoid matrix only, 2) a pyrenoid traversed by a few discs of double thylakoids with many adhering pyrenoglobuli, 3) a pyrenoid penetrated with tubelike structures or 4) a pyrenoid penetrated with many single undulating thylakoids. The pyrenoid structure of the symbiotic Chlorella in Paramecium bursaria resembles those of free-living Chlorella with glucosamine walls.     

COMPARATIVE STUDIES OF PYRENOID ULTRASTRUCTURE IN ALGAE OF THE MONOSTROMA COMPLEX123

The pyrenoid structure in 15 species of the Monostroma complex is very diverse us revealed by a study of the morphology of the pyrenoid matrix, associated starch shell, and pattern of intrapyrenoidalthylakoid bands. From these characteristics 8 types of pyrenoid structure were classified. The variation of pyrenoid structure was shown not only among the species studied, but also between the alternation of generations (M. angicava and M. nitidum). In M. fuscum var. splendens, M. groenlandicum, M. undulatum, and M. zostericola pyrenoid structure is the same throughout the life cycle. The pyrenoid matrix of M. zostericola is surrounded by a double membrane that prevents the direct connection of the pyrenoid matrix with chloroplast thylakoids. The pyrenoid also lacks a starch shell. These findings support the establishment of a new genus Kornmannia by Bliding to include M. zostericola. In addition, similarities in pyrenoid ultrastructure suggest an affinity of Capsosiphon fulvescens with M. groenlandicum.     

Corynoplastis japonica gen. et sp. nov. and Dixoniellales ord. nov. (Rhodellophyceae,Rhodophyta) based on morphological and molecular evidence

A new unicellular red alga, Corynoplastis japonica gen. et sp. nov., is described from Tobishima, Japan. Cells are spherical, 18–33 µm in diameter, pale purple to brownish red and surrounded by a mucilaginous sheath. A single chloroplast with many lobes extends from the cell periphery to the cell center. A peripheral thylakoid is present. A pyrenoid occurs at each innermost chloroplast lobe end and one or two thylakoids are present in the pyrenoid matrix. The nucleus is eccentric to peripheral and Golgi bodies are scattered throughout the cell and associated with endoplasmic reticulum. Cells have a slow random gliding motility. The low molecular weight carbohydrate mannitol is present in the cells. Molecular phylogenetic analysis indicates that this alga is closely related to members of the genus Rhodella. A new order, Dixoniellales, is established for Dixoniella, Neorhodella and Glaucosphaera based on molecular and ultrastructural evidence (Golgi bodies associated only with the nucleus). The redefined order Rhodellales in which Rhodella and Corynoplastis are placed is characterized ultrastructurally by Golgi bodies scattered throughout the cytoplasm and associated with endoplasmic reticulum.     

PYRENOID FORMATION ASSOCIATED WITH THE CELL CYCLE IN THE BROWN ALGA,SCYTOSIPHON LOMENTARIA (SCYTOSIPHONALES,PHAEOPHYCEAE)1

Vegetative cells of the brown alga Scytosiphon lomentaria (Lyngbye) Link characteristically have only one chloroplast with a prominent protruding pyrenoid, whereas zygotes have both paternal and maternal chloroplasts. In zygotes, before cell and chloroplast division, each chloroplast has an old and a new pyrenoid. In this study, we raised a polyclonal antibody to RUBISCO and examined the distribution of RUBISCO by immunofluorescence microscopy, focusing on new pyrenoid formation in vegetative cells of gametophytes and zygotes in Scytosiphon. In interphase, only one old pyrenoid was positively indicated by anti‐RUBISCO antibody in vegetative cells of gametophytes. From mid‐S phase, small fluorescence aggregates reflecting RUBISCO localization started to appear at stroma positions other than adjacent to the old protruding pyrenoid. The fluorescent spots eventually coalesced into a protrusion into the adjacent cytoplasm. We also used inhibitors to clarify the relationship between the cell cycle and new pyrenoid formation, using zygotes after fertilization. When DNA replication was blocked by aphidicolin, new pyrenoid formation was also inhibited. Washing out aphidicolin permitted new pyrenoid formation with the progression of the cell cycle. When mitosis was prolonged by nocodazole, which disrupted the spindle microtubules, the fluorescent masses indicating RUBISCO localization continued to increase when compared with pyrenoid formation in untreated zygotes. During treatment with chloramphenicol, mitosis and cytokinesis were completed. However, there was no occurrence of new RUBISCO localization within the chloroplast stroma beyond the old pyrenoid. From these observations, it seems clear that new pyrenoid formation in the brown alga Scytosiphon depends on the cell cycle.     

NEW PYRENOID FORMATION IN THE BROWN ALGA,SCYTOSIPHON LOMENTARIA (SCYTOSIPHONALES,PHAEOPHYCEAE)1

The Scytosiphon lomentaria (Lyngbye) Link cell characteristically has only one chloroplast with a prominent protruding pyrenoid. We observed the appearance of a new pyrenoid in each chloroplast during first mitosis in zygotes of S. lomentaria, using the freeze substitution technique. At first, a pyrenoid matrix appeared within the outermost stroma, in which thylakoid triplets and ribosomes were absent. At this time, the surface of this part remained smooth. The old pyrenoid was covered with a pyrenoid cap on the cytoplasmic side, whereas there was no pyrenoid cap on the new pyrenoid before protrusion. Irregularly shaped membranous sacs containing fine granular materials associated with the cytoplasmic side of the new pyrenoid. The sacs fused with each other and changed conformation and finally transformed into the pyrenoid cap. The new pyrenoid gradually protruded toward the cytoplasm, and the new pyrenoid cap became curved along the surface of pyrenoid. Cytokinesis occurred, and each chloroplast had two prominent protruding pyrenoids in two‐celled zygotes. We examined immunolocalization of β‐1,3‐glucans within the pyrenoid cap with a monoclonal antibody, using EM. Gold particles indicating localization of β‐1,3‐glucans were detected in vacuoles but never in the pyrenoid cap. This observation suggests that the pyrenoid cap in brown algae contains no photosynthetic products such as polysaccharide.     

The compound internal pyrenoid in cultured cells of the green algaMonoraphidium griffithii (Berkel.) Komar.-Legner.

Summary The chloroplast ultrastructure ofMonoraphidium griffithii (Berkel.) Komar.-Legner. has been studied in axenic cultures of various ages. The algae have grown in a complete nutrient solution (illumination about 3,000 lx) and on its agar medium (illumination about 600 lx).The large parietal cup-shaped chloroplast of the cells includes a multiformed compound internal pyrenoid that is situated, especially in older cells, in the central part of the chloroplast opposite to the dictyosome and the nucleus. The chloroplast thylakoids either reach the edge of the pyrenoid or penetrate its matrix and run there parallel in more or less long bits. Starch grains were not found to form any sheath around the pyrenoid regions. The number of starch grains increased with the age of the cell.     

Diatom motility: An explanation and a problem

Except for the lack of a centriole, interphase cell morphology and cell division in Stichococcus is similar to that in Klebsormidium. The cell in Stichococcus is largely filled by a chloroplast and pyrenoid, at the side of which are two mitochondria and one small peroxisome. The chloroplast/pyrenoid cleaves early in prophase, probably completely, and the nucleus is inserted between the two halves. A band of 3–5 microtubules always encircles the prophase nucleus; these disappear by metaphase. The spindle is open, the daughter nuclei remain far apart at telophase and during cytokinesis, and vacuoles collect between them; no phycoplast is associated with the cleavage furrow.

These results indicate a close phyletic relationship between Stichococcus and Klebsormidium, two organisms which are now considered to be more closely related to the progenitors of the higher land plants than most of the other members of the Ulotrichales.     

ALGAL SYMBIONT OF A SOFT CORAL1

The algal symbiont of the soft coral Sarcothelia edmondsoni (Verrill), is identified as the dinoflagellate Gymnodinium microadriaticum (Freudenthal) Taylor on the basis of its cytology apparent in electron micrographs. The character of the nucleus, peripheral lobed chloroplast with a vase-shaped pyrenoid body and other inclusions form the basis for identification.     

A light and electron microscopical study of the green flagellateSpermatozopsis similis spec. nova

The green flagellateSpermatozopsis similis spec. nova has been studied in culture by light and electron microscopy. The flagellate bears two flagella, is naked and has a characteristic crescent and spirally twisted cell shape. The two flagella are of subequal length, each with a prominent hair-point. Each cell contains two contractile vacuoles, a single chloroplast with an anterior eyespot but lacking a pyrenoid, an anteriorly located nucleus, a single dictyosome associated with the posterior end of the nucleus, a single mitochondrion posterior to the nucleus and associated with a small microbody, some conspicuous vacuoles, and a greater number of secondary cytoskeletal microtubules which probably are responsible for maintaining the peculiar shape of this species. SinceS. similis in culture is only biflagellate, it cannot be accommodated within the quadriflagellate, but otherwise very similar speciesS. exsultans. Spermatozopsis similis is compared with other green flagellates and is shown to share common ultrastructural characters withChlamydomonas-type green algae.     

Observations on the ultrastructure of representatives of the genera Hemiselmis and Chroomonas (Cryptophyceae)

Hemiselmis rufescens Parke and three species of the genus Chroomonas have been examined by electron microscopy. They demonstrate certain characteristic features of the Cryptophyceae, such as the presence of trichocysts and the arrangement of thylakoids in pairs in the plastid. The prominent pyrenoid of the genus Chroomonas is penetrated longitudinally by a tongue of cytoplasmic matrix which originates from between the two pairs of plastid membranes. In the genus Hemiselmis, however, the pyrenoid is traversed by a pair of thylakoids. The nucleus, Golgi apparatus and Corps de Maupas also occupy characteristic positions. These features indicate a close relationship between these two genera and the other major genus Cryptomonas, and support the suggestion that the Cryptophyceae is a discrete taxonomic group.     

Heterocapsa circularisquama sp. nov. (Peridiniales, Dinophyceae): A new marine dinoflagellate causing mass mortality of bivalves in Japan

Heterocapsa circularisquama Horiguchi sp. nov. is described from Ago Bay, central Japan. The dinoflagellate produced large-scale red tides in the bays of central and western Japan and caused mass mortality of bivalves, notably the pearl oysters. The cell is small and is composed of a conical epitheca and a hemi-spheroidal hypothecs. The chloroplast is single and is connected to the single pyrenoid. The nucleus is elongated and is located in the left side of the cell. Thecal plate arrangement has been determined as: Po, cp, 5′, 3a, 7″, 6c, 5s, 5″′, 2″″. Heterocapsa circularisquama is morphologically very similar to Heterocapsa illdefina and it is almost impossible to distinguish these two species at light microscopical level. The characteristics which can be used to distinguish these two species are the morphology of body scales and the ultrastructure of the pyrenoid matrix. The body scales of H. circularisquama possess six radiating ridges on the circular basal plate; no such ridges can be observed on the roughly triangular basal plate of the scales of H. illdefina. Furthermore, the scales of the latter species possess substantially shorter spines compared to those of H. circularisquama. The pyrenoid matrix of H. circularisquama is hardly perforated by cytoplasmic tubules, while in H. tlldefina the pyrenoid matrix is always penetrated by many cytoplasmic tubules. Based on the arrangement of thecal plates, morphology of body scales, and ultra-structure of the pyrenoid, I am placing H. circularisquama sp nov. into the genus Heterocapsa.     

Abnormal Ultrastructural Features of a Marine Dinoflagellate Adapted to Grow Successfully in the Presence of Inhibitory Fluoride Concentration1

ABSTRACT. Amphidinium carteri was unable to grow on nutrient-enriched seawater in the presence of 200 μg/ml fluoride (F) but could be adapted to grow successfully on this F concentration when repeatedly cultured with stepwise increases in sub-inhibitory F concentration. Electron microscopic investigation of the F-adapted dinoflagellate cells showed abnormal ultrastructural features in the chloroplast (especially the pyrenoid), mitochondria, and nucleus. Simultaneous comparison with the F-inhibited dinoflagellate cells showed that thylakoid formation was extremely disorganized by fluoride and that F-adaptation conferred a prolamellar-like configuration on the thylakoids in the center of the pyrenoid. This unexpected appearance of lamellae formation in the F-adapted cells suggested that the pyrenoid may be a center for thylakoid assembly. Such cells also showed large, intensely osmiophilic inclusions in the mitochondria. Microbodies are found in close juxtaposition to the mitochondria and chloroplast, suggesting an increased metabolic dependence on photorespiration. The F-adapted nucleus showed dark and light concentric rings in the nucleolus region, accompanied by other signs of mitotic activity, which were not observed in the F-inhibited cells. It was inferred that the F-adaptation may have required some form of genetic change resulting presumably in the development of a phenotype mutant.     

ULTRASTRUCTURE OF THE PYRENOID OF TREBOUXIA IN RAMALINA MENZIESII TUCK.1,2

Trebouxia sp., the phycobiont of the lichen Ramalina menziesii Tuck., was examined with the electron microscope. Its pyrenoid is characterized structurally as being permeated by interconnected vesiculate membrane systems associated with osmiophilic globules termed pyrenoglobuli. The variety of membrane structure associated with the pyrenoid has been documented with electron micrographs. A model based on serial sections was constructed to show the extent of vesiculation, to demonstrate that the pyrenoglobuli within the pyrenoid matrix are invariably adjacent to the thylakoid membranes within that, matrix, and to emphasize the recurrent thylakoid membrane to pyrenoglobuli relationship. A comparison of the glutaraldehyde-osmium fixation image of the pyrenoglobuli and pyrenoid matrix with the permanganate fixation image has been included. The structure of the pyrenoid matrix in thin section was examined and appears essentially granular.     

FINE STRUCTURE OF CALONEIS AMPHISBAENA (BACILLARIOPHYCEAE)1

The structure of the motile pennate diatom Caloneis amphisbaena Cleve is described, with emphasis on the lateral, lobed pyrenoid with neither a limiting membrane nor penetration by thylakoids, an interphase nucleus with centers of condensed chromatin, paired dictyosomes, and mitochondria cradled within the chambers of the valve. Microfilaments forming two bundles which lie beneath each raphe slit are of the same size and appearance as actin microfilaments associated with other motile systems.     

ULTRASTRUCTURE OF THE CONCHOCELIS STAGE OF THE MARINE RED ALGA PORPHYRA LEUCOSTICTA1

The ultrastructure of the Conchocelis or filamentous stage of Porphyra leucosticta was investigated. Each cell contains 1 or 2 parietal, stellate chloroplasts with a single pyrenoid in each chloroplast. The centrally located nucleus is irregularly shaped and contains 1–2 nucleoli. The cytoplasm has typical floridean starch grains and nonmernbrane-bound lipid bodies. The cell wall is divided into an outer and an inner wall. Many lomasomes are associated with the cell membrane. Pit connections are found between cells, and their taxonomic significance is discussed.     

Comparative ultrastructure of pyrenoids inTrebouxia (Microthamniales,Chlorophyta)

Pyrenoid ultrastructure has been investigated from cultures of all 26 species ofTrebouxia with the aim of establishing pyrenoids as a taxonomic character. Different arrangements and forms of thylakoid lamellae within the pyrenoid matrix allow eight pyrenoid types to be distinguished. Each type is characteristic of a group of species. Thegigantea- andimpressa-type are similar, differing only in the form of the tubules: short, branched tubules mark thegigantea-type; ± long and straight invaginations theimpressa-type. Thearboricola-type is characterized by meandering pyrenoid membranes developing from lamellae parallel with each other in young autospores. Pyrenoids of thegelatinosa-type are traversed by thin parallel-arranged tubules. Few thylakoids with a curved profile are typical of theirregularis-type. Thecorticola-type is different from all others in having a distinct starch sheath closely connected with the pyrenoid matrix and no pyrenoglobuli being associated with the pyrenoid membranes. No true pyrenoids have been found inT. magna andT. erici. Within the chloroplast, they have indistinct areas with pyrenoglobuli, but without differentiated thylakoids. Pyrenoid morphology is stable in culture on different media as well as in phycobionts within lichen thalli. Comparing the pyrenoid of a lichenizedTrebouxia with that from cultured species, the identification of the phycobiont within the lichen thallus is possible, without the need of culturing the algae. This has been shown in species ofParmelia andHypogymnia. New aspects for the taxonomy and systematics ofTrebouxia are discussed.Dedicated to Prof. DrLothar Geitler on the occasion of the 90th anniversary of his birthday.     

Pinguiococcus pyrenoidosus gen. et sp. nov. (Pinguiophyceae), a new marine coccoid alga

A coccoid marine alga, collected from an aquaculture tank and maintained in culture as CCMP1144, was examined using light and electron microscopy. Young, rapidly growing cells were mostly spherical in shape, approximately 4–6 μm in diameter. Older cells often produced protrusions and pseudopodia‐like extensions, giving cells an amoeboid‐like appearance, but no amoeboid movement was observed and the pseudopodia‐like extensions exhibited no active movement. The single chloroplast had a typical photosynthetic stramenopile ultrastructure. A large stalked pyrenoid was easily observed by light microscopy. Ultrastructurally, the granular portion of the pyrenoid was divided into sections by a penetrating chloroplast envelope. A mitochondrion was often, but not always, adjacent to the pyrenoid, and in some cases the mitochondrion formed a ‘cap’ over the protruding pyrenoid. The Golgi cisternae were (when viewed in cross‐section) curved toward the nucleus. A peripheral network of anastomosing tube‐like membranes was located immediately beneath the plasmalemma. Two centrioles were located adjacent to the nuclear envelope. Lipid‐like and electron transparent vacuoles were present. Based on this investigation and data published elsewhere (large percentage of eicosapentaenoic acid, 18S rRNA and rbcL genes), this alga was described as Pinguiococcus pyrenoidosus gen. et sp. nov.