Light and electron microscopy and molecular phylogenetic analyses of Chloromonas pseudoplatyrhyncha (Volvocales,Chlorophyceae)

A strain of Chloromonas pseudoplatyrhyncha (Pascher) P. C. Silva, which has not been studied previously using cultured material, was established from a soil sample collected in Japan and examined by light microscopy, transmission electron microscopy, and molecular phylogenetic analyses. The chloroplasts of this species showed no pyrenoids under light microscopy. However, transmission electron microscopy and the staining methods with carmine after fixation in an acidified hypochlorite solution revealed that Chloromonas pseudoplatyrhyncha actually had multiple, atypical pyrenoids (pyrenoid matrices without associated starch grains) that were angular in shape and distributed in the interior regions of the lobes of the chloroplasts. Although some other species of Chloromonas have atypical pyrenoids in the chloroplast, such angular pyrenoids have not previously been reported within the Volvocales. The present molecular phylogenetic analysis, based on 18S ribosomal RNA, adenosine triphosphate synthase β‐subunit, and P700 chlorophyll a‐apoprotein A2 gene sequences, demonstrated that Chloromonas pseudoplatyrhyncha belonged to the Chloromonas lineage or Chloromonadinia, in which it occupied a basal position outside a robust, large monophyletic group consisting of 13 species of Chloromonas and Gloeomonas.     

Observations on the micro-anatomy of crystallolithus hyalinus gaarder and markali

Summary The fine structure of Crystallolithus hyalinus (Gaarder and Markali), known to be the motile stage of Coccolithus pelagicus (Wallich) Schiller (Parke and Adams 1960), has been investigated for purposes of comparison with Chrysochromulina and Prymnesium, recently recognised as probably related to it. Anoptral contrast light microscopy of living cells and electron microscopy of thin sections and shadow-cast whole mounts show that there is a close resemblance to species of Chrysochromulina not only in cell structure but also in the details of haptonema structure and scale structure. The origin of the scales from internal vesicles has been traced as in other genera but the holococcoliths (crystalloliths) do not appear to arise in this way. The mode of origin of the crystalloliths is thus unknown, but it is suggested that they may be developed outside the cell from calcite secreted in solution into the appropriate cavity. Their constant position between a layer of scales and an outer enveloping skin is consistent with this and one unfamiliar cytoplasmic organelle, composed of peculiar thick-walled tubes within some large superficial vesicles, is described as perhaps involved in this process. Apart from these special features the resemblance to both Chrysochromulina and Prymnesium is sufficient to add considerable weight to Christensen's suggestion (Christensen 1962) that a new group should be defined to accommodate these genera.     

New observations on the fine structure of Chrysochromulina strobilus Parke and Manton with special reference to some unusual features of the haptonema and scales

Summary A new isolate of C. strobilus Parke and Manton has been investigated with light and electron microscopy to add significant details not previously studied. With the aid of a ciné record, to be described more fully elsewhere, some of the more important haptonema movements have been accurately timed. With the aid of improved technical methods of electron microscopy, new observations have been made on the haptonema base, and on the structure and mode of origin of the scales. The presence of 6 microtubules (instead of the more usual 7) in the very long haptonema of this species has been confirmed but the elaborate changes in tubule number and arrangement which take place in the haptonema base are shown to be essentially like those of other species. The morphology and arrangement of the two types of scale present on the cell are as previously described though information on several new and taxonomically valuable surface features have been added. The cup-shaped scales in particular are shown to be more different from those of other species now known in the genus than might previously have been recognised. This difference extends to the position of nascent scales within the Golgi system which is described in a preliminary way. Further discussion is deferred until some other new isolates representing related taxa have been described and named.     

Observations on the morphology and vegetative cell division of the diatomDonkinia recta

Wild and cultured material ofDonkinia recta (Donk.) Grun. has been examined using light microscopy, and both the structure of the living cell and its vegetative cell division are described. Unlike most naviculoid diatoms,D. recta has four chloroplasts per cell, each with four oblong pyrenoids, and division of the chloroplasts follows rather than precedes mitosis and cell division. These two unusual features are probably linked. The species is briefly discussed in relation to other naviculoid taxa in the light of these findings.     

CACHONINA ILLDEFINA SP. NOV. (DINOPHYCEAE): CHLOROPLAST TUBULES AND DEGENERATION OF THE PYRENOID1

A new species of the dinoflagellate genus Cachonina, C. illdefina sp. nov., was isolated from a red tide off El Capitan State Park, Santa Barbara County, California, in October 1973. The organism is light yellowgreen in color with deeply incised girdle and sulcal grooves. Electron microscopy of the organism, revealed a typical dinokaryotic nucleus. The chloroplasts of the organism are connected, and often contain microtubule-like elements, 25 nm diam. The pyrenoids are characterized as excluding chloroplast thylakoids and ribosomes, although containing an amorphous matrix and numerous tubular invaginations from the cytoplasm. The pyrenoids become detached from the chloroplasts and degenerate into small vesicles. C. illdefina is not bioluminescent.     

Characterization of linear-oblong pyrenoids with cp-DNA along their sides in Nitzschia sigmoidea (Bacillariophyceae)

In the past 10 years Nitzschia sigmoidea (Nitzsch) W. Sm. has begun to occur in Japanese rivers in various areas. It is a common diatom in Europe but was previously absent in Japan. Each chloroplast of N. sigmoidea contains many unusual linear‐oblong structures. The internal structure of the chloroplast in this species was observed using epifluorescence and electron microscopy with immunolocalization techniques. The linear‐oblong structures in the chloroplasts could hardly be observed by conventional light microscopy of living cells, but were obvious in cells stained with propionocarmine. Transmission electron microscopy showed that the cross sections of this structure were lanceolate to fusiform with penetration by a single thylakoid. In cells stained with DAPI, chloroplast DNA was detected along both sides of the linear‐oblong structures, and DNA fibrils were detected by electron microscopy. Immunofluorescence microscopy of sectioned cells and also immunoelectron microscopy revealed specific localization of Rubisco between these DNA‐containing areas, which divided at the same time as the chloroplast. Our observations confirmed that the linear‐oblong structures are pyrenoids. The diversity of localization patterns of chloroplast DNA in diatoms is discussed.     

Cochliopodium arabianum n. sp. (Amorphea,Amoebozoa)

A new species of Cochliopodium isolated from freshwater at Arabia Lake in Lithonia, GA, USA is described based on light microscopic morphology, fine structure, and molecular genetic evidence. Cochliopodium arabianum n. sp., previously labeled as “isolate Con1” in prior publications, has been shown to group within the genus Cochliopodium in our molecular phylogenetic analysis. Light microscopy and fine structure evidence indicates the new isolate not only shares characters of the genus but also unique distinctive features. Cochliopodium arabianum n. sp. is typically round when stationary; or oval to sometimes broadly flabellate or triangular in shape during locomotion, with average length of 35 μm and breadth of 51 μm. Fine structure evidence indicates C. arabianum n. sp. has tower‐like scales, lacking a terminal spine, sharing high similarity with its closest relative C. actinophorum. However, the scales of C. arabianum n. sp. are unique in height and the breadth of the base plate. Both morphological and molecular data, including SSU‐rDNA and COI, indicate that this new species falls in a clade sufficiently different from other species to suggest that it is a valid new species.     

Golgi apparatus activity and membrane flow during scale biogenesis in the green flagellateScherffelia dubia (Prasinophyceae). I: Flagellar regeneration

Summary Cells ofScherffelia dubia regenerate flagella with a complete scale covering after experimental flagellar amputation. Flagellar regeneration was used to study Golgi apparatus (GA) activity during flagellar scale production. By comparing the number of scales present on mature flagella with the flagellar regeneration kinetics, it is calculated that each cell produces ca. 260 scales per minute during flagellar regeneration. Flagellar scales are assembled exclusively in the GA and abstricted from the rims of thetrans-most GA cisternae into vesicles. Exocytosis of scales occurs at the base of the anterior flagellar groove. The central portion of thetrans-most cisterna, containing no scales, detaches from the stack of cisternae and develops a coat to become a coated polygonal vesicle. Scale biogenesis involves continuous turnover of GA cisternae, and scale production rates indicate maturation of four cisternae per minute from each of the cells two dictyosomes. A possible model of membrane flow routes during flagellar regeneration, which involves a membrane recycling loop via the coated polygonal vesicles, is presented.     

The pyrenoid is the site of ribulose 1,5-bisphosphate carboxylase/oxygenase accumulation in the hornwort (Bryophyta: Anthocerotae) chloroplast

Summary Chloroplasts of many species of hornworts (Anthocerotae) have a structure that resembles the pyrenoid of green algae but whether these two structures are homologous has not been determined. We utilized immunogold labelling on thin sections to determine the distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the major protein of algal pyrenoids, in sixteen hornwort species with and without pyrenoids. Several species (Phaeoceros laevis, Anthoceros punctatus, A. formosae, A. laminiferus, Folioceros fuciformis, Folioceros sp.,Dendroceros tubercularis, D. japonicus, D. validus, Notothylas orbicularis, N. temperata, andSpaerosporoceros adscendens) have uniplastidic (or primarily uniplastidic) cells with large prominent multiple pyrenoids. In all of these species, the labelling is found exclusively in the pyrenoid and, with the exception of theFolioceros, Dendroceros, andNotothylas species, the labelling is randomly distributed throughout the pyrenoid. In the exceptional species, the pyrenoids have prominent pyrenoglobuli or other inclusions that are unlabelled. InMegaceros flagellaris andM. longispirus, the cells are multiplastidic (with the exception of the apical cell and some epidermal cells) and the chloroplasts lack pyrenoids.Anthoceros fusiformis andPhaeoceros coriaceus have primarily uniplastidic cells but the chloroplasts lack pyrenoids; only an area of stroma in the center of the plastid devoid of starch, reminiscent of a pyrenoid, is found. In all of the species lacking pyrenoids, RuBisCo is found throughout the stroma, including the stromal spaces made by the so-called channel thylakoids. No preferential accumulation of RuBisCo is found in the pyrenoid-like region inA. fusiformis andP. coriaceus. These data indicate that 1) the hornwort pyrenoid is homologous to algal pyrenoids in the presence of RuBisCo; 2) that at least some of the RuBisCo in the pyrenoid must represent an active form of the enzyme; and 3) that, in the absence of pyrenoids, the RuBisCo is distributed throughout the stroma, as in higher plants.Abbreviations RuBisCo ribulose 1,5-bisphosphate carboxylase/oxygenase     

Semicystic spermatogenesis and biflagellate spermatozoon ultrastructure in the Nile electric catfish Malapterurus electricus (Teleostei: Siluriformes: Malapteruridae)

Spermatogenesis and spermatozoon ultrastructure in the Nile electric catfish Malapterurus electricus are described using scanning and transmission electron microscopy. Although the testis organization conforms to the ‘unrestricted’ spermatogonial type, the species has a rare type of spermatogenesis not previously described among catfishes, ‘semicystic’, in which the cyst ruptures before the spermatozoon stage. Spermiogenesis also involves some peculiar features such as condensation of the chromatin in the posterior part of the nucleus to form a compact electron‐dense mass with some irregular electron‐lucent lacunae, while the uppermost part of the nucleus is a loose electron‐lucent area, absence of the nuclear rotation and, as a consequence, the centriolar complex and the initial segment of each flagellum arise directly in a position perpendicular to the basal pole of the nucleus, and occurrence of numerous vesicles in the midpiece. In addition, spermiogenesis includes migration of the diplosome and mitochondria to the basal pole of the nucleus, formation of two moderate nuclear fossae, each of which contains the centriolar complex, development of two independent flagella and elimination of the excess cytoplasm. The mature spermatozoon has a more or less round head with no acrosome or acrosomal vesicle, a long midpiece with numerous mitochondria and vesicles and two long tails or flagella having the classical axoneme structure of 9 + 2 microtubular doublet pattern and with no lateral fins and membranous compartment. These findings suggest that the ultrastructural features of spermiogenesis and spermatozoa of M. electricus are synapomorphies of types I and II spermiogenesis and spermiogenesis is closely similar to the type described in the Nile catfish Chrysichthys auratus.     

The golgi-mediated scale production in the marine haptophycean alga Ochrosphaera neapolitana Schussnig

Abstract

Some ultrastructural features of cells of the marine haptophycean alga, Ochrosphaera neapolitana Schussnig in the palmelloid stage were examined. Chloroplasts which are contained in a compartment isolated from the cytoplasm by ER profiles and nuclear envelope, display trilamellated thylakoids running along the major axis. The stalked pyrenoid with inner bilamellated thylakoids, protrudes in a large membrane-bounded vacuole. Other structures, as the haptonematic and flagellar bases, autophagic vacuoles and mitochondria, are typical of the chrysophycean and haptophycean genera so far investigated.

The Golgi apparatus is represented by a single dictyosome composed of stacked cisternae fonctioning in a way that they form organic scales which constitute the main part of the cell covering. The scales, build up of microfibrils disposed parallel each to other, lie in cisternal lumina of the dictyosomal maturing face; scaly cisternae are numerous in the peripheral cytoplasm and are observed merging in the plasma membrane and discharging the content outside the protoplast.

Dictyosomal activity is evidenced morphologically by massive vesicle production. Three kinds of membrane-bounded vesicles were identified in the present material: i) inner-granulated vesicles, arising from the maturation face; ii) coated vesicles, scattered in the cytoplasm or at the periphery of the golgi body, and iii) dense-cored vesicles, present in the proximity of the maturation face. The possible functional relationships related to scale production and assembly outside the protoplast, and between the nucleus and dictyosome are discussed.     

LIGHT AND ELECTRON MICROSCOPIC CHARACTERIZATION OF TWO TYPES OF PYRENOIDS IN GONIUM (GONIACEAE,CHLOROPHYTA)1

The single, basal pyrenoids of Gonium quadratum Pringsheim ex Nozaki and G. pectorale Müller (Goniaceae, Chlorophyta) differed in appearance when vegetative colonies were cultured photoheterotrophically in medium containing sodium acetate. Chloroplasts of G. quadratum had distinct pyrenoids when grown in medium without major carbon compounds. However, the pyrenoids degenerated and were markedly reduced in size when such cells were inoculated into a medium containing 400 mg·L^?1 of sodium acetate. No pyrenoids were visible under the light microscope; however, with electron microscopy small pyrenoids and electron-dense bodies were visible within the degenerating chloroplasts, which had only single layers of thylakoid lamellae at the periphery. The chloroplasts subsequently developed distinct pyrenoids and several layers of thylakoid lamellae as the culture aged. In contrast, vegetative cells of G. pectorale always showed distinct pyrenoids when cells were inoculated into medium containing sodium acetate, sodium pyruvic acid, sodium lactate, and/or yeast extract. Therefore, we propose two terms, “unstable pyrenoids” and “stable pyrenoids,” for pyrenoids of G. quadratum and G. pectorale, respectively. Chloroplasts of the colonial green flagellates should thus be examined under various culture conditions in order to determine whether their pyrenoids are unstable or stable when pyrenoids are used as taxonomic indicators. Immunogold electron microscopy showed that the ratios of gold particle density of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) between pyrenoid matrix and chloroplast stroma in G. quadratum grown in medium with or without sodium acetate were lower than those of G. pectorale. Heavy labeling by anti-RuBisCO was observed in both the electron-dense bodies and pyrenoid matrix of G. quadratum. This is the first electron microscopic demonstration of degeneration and development of both pyrenoids and thylakoid lamellae in the chloroplast as a function of culture condition in green algae.     

Gymnodinium smaydae n. sp., a New Planktonic Phototrophic Dinoflagellate from the Coastal Waters of Western Korea: Morphology and Molecular Characterization

The marine phototrophic dinoflagellate Gymnodinium smaydae n. sp. is described from cells prepared for light, scanning, and transmission electron microscopy. Also, sequences of the small (SSU) and large subunits (LSU) and the internal transcribed spacer region (ITS1–5.8S–ITS2) of ribosomal DNA were analyzed. This newly isolated dinoflagellate possessed nuclear chambers, nuclear fibrous connective, an apical groove running in a counterclockwise direction around the apex, and a major accessory pigment peridinin, which are four key features for the genus Gymnodinium. The epicone was conical with a round apex, while the hypocone was ellipsoid. Cells growing photosynthetically were 6.3–10.9 μm long and 5.1–10.0 μm wide, and therefore smaller than any other Gymnodinium species so far reported except Gymnodinium nanum. Cells were covered with polygonal amphiesmal vesicles arranged in 11 horizontal rows, and the vesicles were smaller than those of the other Gymnodinium species. This dinoflagellate had a sharp and elongated ventral ridge reaching half way down the hypocone, unlike other Gymnodinium species. Moreover, displacement of the cingulum was 0.4–0.6 × cell length while in other known Gymnodinium species it is less than 0.3 × cell length. In addition, the new species possessed a peduncle, permanent chloroplasts, pyrenoids, trichocysts, pusule systems, and small knobs along the apical furrow, but it lacked an eyespot, nematocysts, and body scales. The sequence of the SSU, ITS1–5.8S–ITS2, and LSU rDNA region differed by 1.5–3.8%, 6.0–17.4%, and 9.1–17.5%, respectively, from those of the most closely related species. The phylogenetic trees demonstrated that the new species belonged to the Gymnodinium clade at the base of a clade consisting of Gymnodinium acidotum, Gymnodinium dorsalisulcum, Gymnodinium eucyaneum, etc. Based on morphological and molecular data, we suggest that the taxon represents a new species, Gymnodinium smaydae n. sp.     

Fine Structure of Yellow-Brown Symbionts (Prymnesiida) in Solitary Radiolaria and Their Comparison with Similar Acantharian Symbionts1

Yellow-brown, algal symbionts varying in diameter from approximately 5 μ m to 20 μ m, associated with solitary Radiolaria with spongiose skeletons (i.e. Spongodrymus sp.), exhibit fine structural features resembling the Prymnesiida (botanical class, Prymnesiophyceae). A large central vacuole is surrounded by a thin layer of cytoplasm containing plastids with lamellae composed of three thylakoids and granular pyrenoids with internal tubules immersed between the thylakoids. The pyrenoids lack internal thylakoid membranes. The nucleus is surrounded by a dilated cisterna of the nuclear envelope that also encloses the plastids and gives rise to saccules of the endoplasmic reticulum. The algal symbionts appear coccoid; hence no flagella nor surface scales were observed. The symbiont fine structure is compared to similar yellow-brown symbionts associated with Acantharia. Thus far, three kinds of algal symbionts have been observed to be associated with solitary Radiolaria: dinoflagellate, prasinomonad, and this apparent prymnesiomonad.     

ULTRASTRUCTURE OF PRYMNESIUM NEMAMETHECUM SP. NOV. (PRYMNESIOPHYCEAE)1

Based on material collected from Cape Town, a new sand-dwelling, marine species of Prymnesium is described. Using light and electron microscopy, Prymnesium nemamethecum sp. nov. has been found to resemble other species of the genus in size, organelle arrangement, and swimming behavior. It differs from other described species in that it has three types of scales, one of which is confined to the region of appendage insertion and forms a sheath of simple plate scales over the haptonema. In addition, the scales constituting the proximal body scale layer(s) are unusual because they are not simple plate scales but are specifically ornamented.     

Pinguiochrysis pyriformis gen. et sp. nov. (Pinguiophyceae), a new picoplanktonic alga isolated from the Pacific Ocean

Pinguiochrysis pyriformis gen. et sp. nov. is a brown, naked, non‐motile, marine picoplankton. A culture was established from a surface sample collected in 1991 from the tropical Western Pacific Ocean. Typical cells of P. pyriformis are distinctively pear‐shaped and have one ovoid chloroplast; these two features distinguish this species from the other picophytoplankton species. However, the pyriform morphology is not consistent and cells frequently change to a subspherical shape. The chloroplast and mitochondrion ultrastructure confirm that this species belongs to the photosynthetic stramenopiles (chromophytes). Additional distinctive ultrastructural characteristics of P. pyriformis include (i) a chloroplast envelope forming a tubular invagination that penetrates into the pyrenoid; (ii) thylakoid lamellae consisting of more than three layers in some cells; (iii) the lack of basal bodies and centrioles; and (iv) the lack of scales or other extracellular structures. Based on the morphological features, this picoplanktonic species was described as a new species and placed in the Pinguiophyceae on the basis of the molecular phylogenetic analysis and biochemical data published elsewhere.     

Simultaneous visualization of the actin plate and new cell partition membrane during cytokinesis in the brown alga Sphacelaria rigidula (Sphacelariales,Phaeophyceae)

In many brown algae, cytokinesis is accomplished through the centrifugal expansion of the membrane structure formed by the fusion of Golgi vesicles and flat cisternae. In contrast, it has been reported that cytokinesis in Sphacelaria rigidula progresses centripetally by adding Golgi vesicles and flat cisternae to cleaving furrows of the plasma membrane. The reason why this cytokinetic pattern was observed only in Sphacelaria species is unknown. In either cytokinesis pattern, a plate-like actin structure (the actin plate) coincides with the cytokinetic plane between the daughter nuclei. However, it is unclear how the actin plate is related to cytokinesis progression. In this study, we re-examined cytokinesis in the apical cells of S. rigidula using transmission electron microscopy. Double staining of the actin plate and the developing membrane was followed by fluorescence microscopy analysis to determine the relationship between these two formations. The results showed that cytokinesis in S. rigidula, as in many brown algae, was completed by centrifugal growth of the new cell partition membrane. A furrow of the plasma membrane was observed at the beginning of cytokinesis; however, further invagination did not occur. The actin plate arose at the center of the cytokinetic plane before membrane fusion and extended parallel to the expansion of the new cell partition membrane. When cytokinesis was slow due to insufficient Golgi vesicle supply to the cytokinetic plane in the cells under brefeldin A treatment, the extension of the actin plate was also suspended. In this study, the spatiotemporal relationship between the occurrence and expansion of the actin plate and the new cell partition membrane was revealed. These observations indicate that the actin plate might promote membrane fusion or lead to the growth of a new cell partition membrane.     

The origin of vacuoles in young embryos of Pelargonium x Hortorum Bailey

Summary The different mechanisms of vacuole formation in embryonic tissues of Pelargonium are described. Some vacuoles are formed by mechanisms widely reported in a variety of plant species and plant tissues, but other vacuoles are initiated as differentiated zones of the cytoplasm around which the tonoplast is gradually built up form vesicles and small cisternae.     

Laboratory culture and taxonomy ofHymenomonas coronata andOchrosphaera verrucosa (Class Prymnesiophyceae) from the Northwest Pacific

Hymenomonas coronata andOchrosphaera verrucosa, both members of the coccolithophorids, Class Prymnesiophyceae, have been studied by means of electron microscopy and with the aid of laboratory culture. Living specimens of these two species were collected in temperate and subtropical regions of Japan, including the Kii Peninsula and the Ryukyu Islands, and unialgal cultures were established in the laboratory. Their life histories are fundamentally identical, and consist of a non-motile vegetative stage that produces motile cells. The vegetative stage is either unicellular, or a packet consisting of a few cells. Both the non-motile cells and the motile cells are covered with two kinds of scales: these are thin scales of unmineralized nature and coccoliths. These two species differ from each other in the shape of the coccoliths and in the presence or absence of visible rudimentary haptonema, and they have been in separate families. The present study reveals that both species are fundamentally identical in the structure and the distribution of major organelles, especially with respect to two opposed pyrenoids which bulge from chloroplasts, each being traversed by two thylakoid bands, and a group of microtubules forming a flagellar root. On the basis of these characteristics, it would appear more logical to place these two species in the same family, namely the Hymenomonadaceae.