Lotharella reticulosa sp. nov.: a highly reticulated network forming chlorarachniophyte from the Mediterranean Sea

A new chlorarachniophyte Lotharella reticulosa sp. nov. is described from a culture isolated from the Mediterranean Sea. This strain is maintained as strain RCC375 at the Roscoff Culture Collection, France. This species presents a multiphasic life cycle: vegetative cells of this species were observed to be coccoid, but amoeboid cells with filopodia and globular suspended cells were also present in the life cycle, both of which were not dominant phases. Flagellate cells were also observed but remained very rare in culture. The vegetative cells were 9-16 μm in diameter and highly vacuolated, containing several green chloroplasts with a projecting pyrenoid, mitochondria, and a nucleus. The chloroplast was surrounded by four membranes possessing a nucleomorph in the periplastidial compartment near the pyrenoid base. According to ultrastructural observations of the pyrenoid and nucleomorph, the present species belongs to the genus Lotharella in the phylum Chlorarachniophyta. This taxonomic placement is consistent with the molecular phylogenetic trees of the 18S rRNA gene and ITS sequences. This species showed a unique colonization pattern. Clusters of cells extended cytoplasmic strands radially. Then, amoeboid cells being born proximately moved distally along the cytoplasmic strand like on a "railway track". Subsequently the amoeboid cell became coccoid near the strand. In this way, daughter cells were dispersed evenly on the substratum. We also observed that the present species regularly formed a structure of filopodial nodes in mid-stage and later-stage cultures, which is a novel phenotype in chlorarachniophytes. The unique colonization pattern and other unique features demonstrate that RCC375 is a new chlorarachniophyte belonging to genus Lotharella, which we describe as Lotharella reticulosa sp. nov.     

Taxonomic studies on the Chlorarachniophyta. II. Generic delimitation of the chlorarachniophytes and description of Gymnochlora stellata gen. et sp. nov. and Lotharella gen. nov.

A new chlorarachniophytan alga, Gymnochlora stellata Ishida et Y. Hara gen. et sp. nov., has been isolated from Anae Island in Guam. It is a green, star-shaped, unicellular, amoeboid organism with several filopodia that do not form a reticulopodial network. Neither zoospores nor walled coccoid cells have been observed throughout the life cycle. The chloroplast ultrastructure is similar to those of described species; however, the pyrenoid matrix, which is invaded by many tubular structures originating from the inner membrane of the chloroplast envelope, is unique. A classification system is proposed for the Chlorarachniophyta. In this system, the ultrastructural features of the pyrenoid and the location of the nucleomorph in the periplastidial compartment are used as generic criteria, while the morphological features of the vegetative cells and life cycle patterns are used for species criteria. The described species, except for Cryptochlora perforans Calderon-Saenz et Schnetter, are also reassessed under the new system, and consequent nomenclatural requirements for the genus Chlorarachnion are dealt with in this paper. The taxonomic rank of a previously described species, Chlorarachnion globosum Ishida et Y. Hara, is elevated and Lotharella globosa (Ishida et Y. Hara) Ishida et Y. Hara gen. nov. et comb. nov. is proposed.     

Lotharella amoeboformis sp. nov.: A new species of chlorarachniophytes from Japan

The ultrastructure, morphology and life cycle of a new chlorarachniophyte alga collected from Okinawa in Japan have been studied. The life cycle of this alga consists of amoeboid, wall‐less round, coccoid and flagellated cells in culture condition; however, the coccoid and flagellate cells are very rare. The pyrenoid ultra‐structure of this alga is the same as that of a previously described species, Lotharella globosa. Since pyrenoid ultrastructure has been adopted as the main criterion for the generic classification of the chlorarachniophytes, the present alga is placed in Lotharella. However, the present alga has a dominant amoeboid cell stage and a reduced walled‐cell stage in the life cycle, while in L. globosa, the walled‐cell stage is dominant and there is no amoeboid cell stage. Therefore the present alga is described as a new species of Lotharella: Lotharella amoeboformis Ishida et Y. Hara sp. nov.     

Kalinella bambusicola gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a novel coccoid Chlorella-like subaerial alga from Southeast Asia

The traditional green algal genus Chlorella , which comprised coccoid algae surrounded by a smooth cell wall and reproducing solely by autosporulation, has proved to be polyphyletic and extremely diverse in phylogenetic terms. We studied a new subaerial Chlorella -like strain CAUP H7901 and morphological, ultrastructural, and molecular phylogenetic investigations indicated that it represents a new lineage of the trebouxiophycean Watanabea clade, dissimilar from other members of this group. The alga has globular coccoid cells with a single parietal pyrenoid-bearing chloroplast. The pyrenoid is transected by multiple radial thylakoid bands. The alga reproduces exclusively by means of asexual autospores of unequal size. In 18S rDNA sequence phylogenies, it was nested within the Watanabea clade close to lineages containing Chlorella saccharophila , Chlorella luteoviridis , Heveochlorella hainangensis , and two uncharacterized strains, but alternative positions within the Watanabea clade could not be rejected by an approximately unbiased (AU) test. Here we describe this organism as a new genus and species Kalinella bambusicola gen. et sp. nov. Furthermore, we describe Heterochlorella gen. nov. to accommodate a species previously referred to as Chlorella luteoviridis .     

Reconsideration of the taxonomy of ellipsoidal species of Chlorella (Trebouxiophyceae, Chlorophyta), with establishment of Watanabea sen. nov.

Subcultures of SAG 211–9b and 1AM C-211, ultimately derived from CCAP 211/9b, a strain isolated by Pringsheim in 1939 and identified as Chlorella sac-charophila (Kruger) Migula were observed using light and electron microscopy. Their morphology proved to be basically identical. Both have two forms of cells, one (E-form) narrowly to broadly ellipsoidal, the other (S-form) ovoid to spheroidal. The cell wall of both forms is composed of a single smooth layer. The chloroplast of young cells is trough-like or saucer-shaped with a smooth margin, while that of mature cells is band- or cup-shaped with deep incisions. The thylakoid lamellae are loosely stacked and neither form has a pyrenoid. Both types of cells are capable of producing autospores: eight to 16 in E-form cells, two to four in S-form cells. These morphological features are different from those of C. saccharophila, which has a pyrenoid and produces only one form of autospores. In the absence of any existing genus that includes Chlorella-like algae with a simple cell wall, no pyrenoid, and two forms of mature cells and autospores, a new genus, Watanabea, is proposed with the type species W. reniformis.     

Lotharella vacuolata sp. nov., a new species of chlorarachniophyte algae, and time-lapse video observations on its unique post-cell division behavior

A new species of chlorarachniophyte alga, Lotharella vacuolata Ota et Ishida sp. nov., is described. This alga has been maintained as strain CCMP240 at the Provasoli‐Guillard National Center for Culture of Marine Phytoplankton at Bigelow Laboratory for Ocean Sciences. We examined in detail its morphology, ultrastructure and life cycle, using light microscopy, transmission electron microscopy and time‐lapse videomicroscopy. The dominant stage in the life cycle was represented by coccoid cells; however, amoeboid and flagellated stages were also observed. This alga showed unique post‐cell division behavior: one of the two daughter cells became amoeboid and escaped through a pore on the parental cell wall; the other daughter cell remained within the parental cell wall. Pyrenoid ultrastructure and nucleomorph location, which are used as the main generic criteria of chlorarachniophytes, confirmed that the strain CCMP240 is a member of Lotharella. This alga, however, was clearly distinguished from other known Lotharella species by the presence of large vacuoles, unusual post‐cell division behavior and some unique ultrastructural characters.     

TRADITIONAL GENERIC CONCEPTS VERSUS 18S rRNA GENE PHYLOGENY IN THE GREEN ALGAL FAMILY SELENASTRACEAE (CHLOROPHYCEAE, CHLOROPHYTA)

Coccoid green algae of the Selenastraceae were investigated by means of light microscopy, TEM, and 18S rRNA analyses to evaluate the generic concept in this family. Phylogenetic trees inferred from the 18S rRNA gene sequences showed that the studied species of autosporic Selenastraceae formed a well-resolved monophyletic clade within the DO group of Chlorophyceae. Several morphological characteristics that are traditionally used as generic features were investigated, especially the arrangement of autospores in the mother cells, colony formation, and pyrenoid structure. The parallel arrangement of autospores was confirmed for the genera Ankistrodesmus , Podohedriella , and Quadrigula. In mother cells of Monoraphidium and Kirchneriella , the autospores were arranged serially. Colony formation was either stable ( Quadrigula ) or variable ( Ankistrodesmus , Podohedriella ) within genera. All strains studied possessed naked or starch-covered pyrenoids within the chloroplast. The pyrenoid matrix was homogenous or penetrated by thylakoids. In contrast to considerations of traditional systematics, the present study showed that the presence and structure of pyrenoids are unsuitable for differentiation of genera in Selenastraceae. Furthermore, the molecular analyses showed that any morphological criterion considered so far is not significant for the systematics of the Selenastraceae on the generic level. Species assigned to different genera such as Ankistrodesmus and Monoraphidium were not monophyletic and therefore not distinguishable as separate genera. Species of Monoraphidium appeared in four different lineages of the Selenastraceae. Our phylogenetic analyses support earlier discussions to abandon the common practice of conceiving "small" genera (i.e. genera that are differentiated from other genera by only a few diacritic characteristics and that contain only a small number of species) and to reestablish "large" genera of Selenastraceae such as Ankistrodesmus.     

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.     

OBSERVATIONS ON THE STRUCTURE OF SOME FORMS OF GOMPHONEMA PARVULUM KÜTZ. II. THE INTERNAL ORGANIZATION1

The diatom species Gomphonema parvulum Kütz. was isolated, grown in culture, and the details of the internal organization studied by light and electron microscopy. The organism is usually identified from its frustular morphology, but members of the genus Gomphonema can be separated from other naviculoid forms by the H-shaped chromatophore, whose lobes are connected on 3 sides across the center of the cell, and also from the position of the pyrenoid. This is situated, on the inner side of the chromatophore and is not embedded in the center of the chromatophore. New observations of this pennate diatom include details of the periplastidial network, of the Golgi vesicular activity, and of the storage products.     

MOLECULAR,ENZYMATIC AND ULRASTRUCURE CHARACTERIZATION OF THE PYRENOID OF THE SCALY GREEN MONAD MICROMONAS SQUAMATA1,2

Pyrenoid material of micromonas squamata Manton & Parke was obtained free of cell and subcellular particle conamination by differential centrifugation of brei from osmoically lysed cells. The isolated pyrenoid particles were characterized by transmission and scanning electron microscopy. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of pyrenoid extracts revealed a compled polypeptide composition with major components of 12, 54 and 66 kilodalton mol wt. Whole pyrenoids possessed the enzymatic properties of ribulose diphosphate carboxylase and fixed carbon dioxide with specific activity 10 times greaer than that of a pyrenoid-free high speed supenaant fracion of cell brei. Energy dispersive X-ray microanalysis revealed he presence of copper in masses of cryo-impacted pyrenoid material. Ultrastrucural cytochemistry was employed o determine he chemical nature of the reserve carbohydrate shell. Also, the pyrenoid of the intact cell was characterized by transmission electron microscopy.     

The ultrastructure of Mychonastes ruminatus gen. et sp. nov., a new member of the Chlorophyceae isolated from brackish water

A new monotypic genus of the Chlorophyceae isolated from brackish water of the Chesapeake Bay, Maryland, U.S.A. is described as Mychonastes ruminatus. The alga is compared with similar members of the Oocystaceae. Based on the presence of a thick ruminate cell wall, the lack of discernible pyrenoids, and other vegetative characters, Mychonastes can be delineated from other green coccoid unicells.

The cellular life cycle of Mychonastes was studied by electron microscopy and compared to published studies of Chlorella. The genera are differentiated by wall and chloroplast structure. Mychonastes cell wall is sculptured and changes during the life cycle from a highly irregular appearance when young, to a less irregular state when mature. Mychonastes chloroplast lacks pyrenoids at all times. Chlorella has a smooth wall and pyrenoids which are apparent just after release from the mother cell until division. Organelles of both genera migrate in a similar pattern throughout the life cycle, with Mychonastes producing two or four autospores, and Chlorella producing four or more (rarely two).     

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.     

HALOSTYLODINIUM ARENARIUM, GEN ET SP. NOV. (DINOPHYCEAE), A COCCOID SAND-DWELLING DINOFLAGELLATE FROM SUBTROPICAL JAPAN

A new genus and species of marine coccoid dinoflagellate from subtropical Japan, Halostylodinium arenarium Horiguchi et Yoshizawa-Ebata, gen. et sp. nov., is described. The dominant stage of the dinoflagellate is a nonmotile ovoidal to spheroidal cell with a distinct stalk. The stalk consists of an upper thick tubule, a lower thin tubule, and a discoidal holdfast. The dinoflagellate possesses a yellowish-brown chloroplast with multiple lobes radiating from a central pyrenoid. It reproduces by the formation of two motile cells, which swim for a short period and then transform directly into the stalked nonmotile cell. The stalk is produced during transformation from the apical stalk complex present in the apex of the motile cell. The apical stalk complex consists of a double-folded apical pore plate and doughnut-shaped holdfast-building material. The ultrastructure of the apical stalk complex is compared with those of Bysmatrum arenicola and Stylodinium littorale. Halostylodinium arenarium possesses delicate thecal plates, and the thecal plate formula is Po, 5', 2a, 7", 7c, 6s, 5"', 1p, 2"". A phylogenetic study based on the 18S ribosomal RNA gene did not show any clear affinities between this organism and any species included in the analysis.     

The effect of light color on the nucleocytoplasmic and chloroplast cycle of the green chlorococcal algaScenedesmus obliquus

The color of light (white, red, blue, and green) had a significant effect on the growth and reproductive processes (both in the nucleocytoplasmic and chloroplast compartment of the cells) in synchronous cultures of Scenedesmus obliquus. This effect decreased in the order red > white > blue > green. In the same order, the light phase of the cell cycle (time when first autospores started to be released) was prolonged. The length of dark phase (time when 100 % of daughters were allowed to release from mothers) was not influenced and was the same for all colors. Critical cell size for cell division in green light was shifted to a smaller size (compared with cells grown in other lights) and so was the size of released daughters. The nuclear cycle was slowed in blue and even in green light, contrary to cells grown in red and white light. At the beginning of the cell cycle, one-nucleus daughters possess approximately 10 nucleoids; during the cell cycle their number doubled in all variants before the division of nuclei. Both events were delayed in cultures grown more slowly most markedly in green light. Smaller daughters in the green variant possessed a lower number of nucleoids. Motile cells released in continuous green or blue lights but not in red one were rarely observed.     

TAXONOMIC STUDY OF BIGELOWIELLA LONGIFILA SP. NOV. (CHLORARACHNIOPHYTA) AND A TIME‐LAPSE VIDEO OBSERVATION OF THE UNIQUE MIGRATION OF AMOEBOID CELLS1

A new species of a chlorarachniophyte alga, Bigelowiella longifila sp. nov., is described. It is classified as a member of Bigelowiella as flagellate cells constitute the main stage of the life cycle. However, this alga is different from the only described species of the genus, B. natans Moestrup, in having a unique amoeboid stage in the life cycle. We observed an interesting behavior of amoeboid daughter cells after cell division: One of the two daughter cells inherits the long filopodium of the parental cell, and it subsequently transports its cell contents through the filopodium to develop at its opposite end. The other daughter cell forms a new filopodium. This unequal behavior of daughter cells may have evolved before the chlorarachniophytes and some colorless cercozoans diverged.     

Ultraviolet Inactivation and Photoreactivation in Synchronized Chlorella

Synchronous cultures of Chlorella pyrenoidosa have been used in studies of the action of UV-irradiation during different stages in the life cycle on cell division capacity. These experiments show that there is a considerably higher sensitivity to UV-irradiation during the first hours of the life cycle. The variations in photoreactivating capacity of UV-damaged cells during a life cycle have been investigated. The results from these investigations show that the photoreactivating capacity varies and that it is higher in young daughter cells (autospores) and 12–15 hour cells. These variations can be due to variations in the activity of the photoreactivating enzyme during the life cycle.     

Endomyces tetrasperma, sp. n

A new fungal species has been described and placed in the genus Endomyces. Endomyces tetrasperma forms a true septate, multinucleate mycelium which breaks up into arthrospores. Ascus formation occurs after isogamous copulation between sexual protuberances which develop at the ends of arthrospores or between two cells, adjacent mycelial cells, or arthrospores. The asci which dehisce at maturity release two to four smooth, ovoid, thick-walled spores, each containing two oil droplets. The proposed life cycle is based on morphological and cytological observations.     

Pyramidodinium atrofuscum gen. et sp. nov. (Dinophyceae), a new marine sand-dwelling coccoid dinoflagellate from tropical waters

A new marine sand‐dwelling coccoid dinoflagellate Pyramidodinium atrofuscum Horiguchi et Sukigara gen. et sp. nov. is described from Jellyfish Lake, Republic of Palau. The dinoflagellate alternates a non‐motile vegetative stage with a motile gymnodinioid stage within its life cycle. The non‐motile stage is dominant in the life cycle and the dinoflagellate reproduces itself by means of the production of two motile cells. The released motile cell swims only for a short period and is directly transformed into the non‐motile cell. The non‐motile cell is sessile, pyramidal in shape, with a single longitudinal ridge and a double transverse ridge. The surface of the cell wall is covered with many processes. The motile cell has a Gymnodinium‐like morphology, but no apical groove is present. An ultrastructural study revealed that the dinoflagellate possesses typical dinoflagellate organelles. Based on the unique morphology of the vegetative non‐motile stage, we propose a new genus Pyramidodinium for this dinoflagellate, with the type species Pyramidodinium atrofuscum Horiguchi et Sukigara, gen. et sp. nov.     

A New Species of Dysmorphococcus (Phacotaceae,Volvocales) and Its Life Cycle

This paper deals with the morphology and life cycle of a new species of Dysmorphococcus oblatus Yu et Wei (Phacotaceae, Volvocales) from Africa. The alga seems to be morphologically rather distinct from the other members (lorica, protoplast and number of pyrenoid). Moreover, a comparison is made of overall morphological and reproductive characteristics of the present taxon with the other 8 known species of this genus. The cultural experiment of the life cycle of this alga reveals certain attributes unrecorded for early described species of this genus. The alga is homothallic, and heterogametic and sexual reproduc-tion produces 32-64 male gametes and 2 female gametes.