Physiological and biochemical contributions to the taxonomy of the genus Chlorella

Starch hydrolysis, i.e., the production of extracellular amylolytic enzymes, was found to be a specific character for most species of the genus Chlorella. C. fusca var. vacuolata, C. spec. 211-30, and C. spec. 211-11r hydrolyse starch, whereas C. vulgaris, C. fusca var. rubescens, C. zofingiensis, C. fusca var. fusca, C. minutissima, C. homosphaera, C. kessleri, C. luteoviridis, and C. protothecoides are unable to hydrolyse starch. Only C. sorokiniana and C. saccharophila appear heterogenous; within C. sorokiniana, 7 strains hydrolyse starch and 9 do not; and within C. saccharophila, 6 strains exhibit amylolytic activity and 2 do not. — A key for the identification, according to 9 easily determined physiological and biochemical characters, of the Chlorella species is presented.     

Species-specific ability ofChlorella strains (Chlorophyceae) to form stable symbioses withHydra viridis

46 strains ofChlorella, identified by physiological and biochemical characters, were examined for their ability to form stable symbioses with aposymbioticHydra viridis. It was found to be a species-specific characteristic. Among the 15 taxa studied, onlyC. saccharophila var.ellipsoidea, C. saccharophila var.saccharophila, C. fusca var.vacuolata, C. kessleri, C. luteoviridis, andC. protothecoides formed stable symbioses withHydra viridis. Among the 11 known physiological and biochemical characters of theseChlorella species, only acid tolerance seems to be correlated with symbiosis: All symbiotic species are capable of growing at or below pH 4.0.     

Physiological and biochemical contributions to the taxonomy of the genus Chlorella

The products of glucose fermentation were studied in 87 strains of the genus Chlorella. Lactic acid, acetic acid, formic acid, glycerol, ethanol, H2 and CO2 were identified. The lactic acid was shown to be D(minus)lactic acid. The pattern of fermentation produces is species-specific and can therefore be used as a taxonomic character. Lactic acid was found in C. fusca (varieties vacuolata, fusca, and rubescens), C. zofingiensis, C. vulgaris (var. vulgaris and f.tertia), and C. protothecoides. Formic acid and H2 appeared in those species which contain hydrogenase. Rather large amounts of glycerol were produced only by the most salt-tolerant species C. luteoviridis, C. saccharophila, and C. protothecoides.     

Physiological and biochemical contributions to the taxonomy of the genus Chlorella

1. Rabbit antisera to soluble cytochrome c-553 and to ferredoxin were characterized by double immunodiffusion, the antiserum to cytochrome c-553 also by immuno-electrophoresis and by the quantitative precipitation reaction.
2. Soluble cytochromes c-553 were isolated from 11 strains of Chlorella belonging to 8 species and from one strain of Scenedesmus. Their reactivity with antiserum to soluble cytochrome c-553 from Chlorella fusca var. vacuolata 211-8b was studied.
3. With the double immunodiffusion method, the soluble cytochromes c-553 from C. fusca var. vacuolata 211-8b, C. fusca var. rubescens 232/1, C. fusca var. fusca 343, and Scenedesmus obliquus 276-3b appear immunologically identical.
4. The soluble cytochromes c-553 from C. zofingiensis 211-14a, 211-14b, and 211-14c show in the cross reaction a partial immunological relationship with that from C. fusca var. vacuolata 211-8b.
5. The soluble cytochromes c-553 from C. sorokiniana 211-8k, C. vulgaris 211-11c and 211-19, C. saccharophila 211-1 a, and C. homosphaera 211-8e (Ca.) do not show a cross reaction with that from C. fusca var. vacuolata 211-8b.
6. These results indicate that not only C. fusca var. fusca, but also C. fusca var. vacuolata, C. fusca var. rubescens, and possibly C. zofingiensis might be more closely related to the genus Scenedesmus than to the Chlorella species of the “C. vulgaris group” (i.e., C. sorokiniana, C. vulgaris, and C. saccharophila).
7. An application of the double immunodiffusion method with an antiserum to ferredoxin from C. vulgaris 211-8m revealed that the ferredoxins from 20 strains of 9 Chlorella species and from one strain of Scenedesmus do not exhibit any immunological differences as compared to the homologous antigen.

     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

DNA hybridization techniques showed Chlorella fusca var. vacuolata and C. kessleri to be homogeneous species with DNA homologies of 90–100% C. fusca var. fusca and var. rubescens, however, have only about 15% DNA homology with C. fusca var. vacuolata and should no longer be regarded as varieties. A good correlation was found so far between biochemical and physiological characters used in the taxonomy of Chlorella and DNA relatedness. Mutant strains of Chlorella were tested for DNA homologies to prove the reliability of the taxonomical interpretation.     

Physiologische und biochemische Beiträge zur Taxonomie der Gattung Chlorella

Zusammenfassung Die 15 verfügbaren Stämme von Chlorella vulgaris f. tertia Fott et Nováková (früher als Chlorella III, zwei auxotrophe Stämme als Chlorella VI, bezeichnet), die im Gegensatz zu Chlorella vulgaris Beijerinck Hydrogenase enthalten, wurden auf Thermophilie geprüft. Sie erwiesen sich sämtlich als thermophil und erreichen die obere Grenze des Wachstums bei 38–42°C. Damit erscheinen die beiden klassischen Hochtemperatur-Chlorellen (die Stämme 7-11-05 und 1-9-30 von Sorokin) als Angehörige eines ziemlich weit verbreiteten thermophilen Taxons. Unter den 19 verfügbaren Stämmen von Chlorella vulgaris wurden demgegenüber keine Hochtemperatur-Stämme gefunden. Auch bei C. homosphaera, C. fusca var. rubescens, C. luteoviridis, C. zofingiensis, C. sacharophila, C. minutissima und C. protothecoides konnten wir keine thermophilen Stämme nachweisen, während sich bei C. fusca und C. kessleri eine leichte Tendenz zur Thermophilie zeigte. Chlorella vulgaris f. tertia ist somit anscheinend ein Taxon ökologischer Spezialisten, die zu einer Existenz bei erhöhten Temperaturen, unter anaeroben Bedingungen oder auch als Symbionten in Tieren (zwei Stämme sind Zoochlorellen aus dem Süßwasser-Schwamm Spongilla) befähight sind. Wir nehmen an, daß es sich dabei um eine primitive, phylogenetisch alte Gruppe von Organismen handelt, aus der sich die morphologisch gleiche, typische Chlorella vulgaris durch den Verlust von Hydrogenase und Thermophilie entwickelt haben könnte.

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Physiological and biochemical contributions to the taxonomy of the genus Chlorella VII. The thermophily of Chlorella vulgaris f. tertia Fott et Nováková

Summary The 15 available strains of Chlorella vulgaris f. tertia Fott et Nováková (previously designated as Chlorella III, two auxotrophic strains as Chlorella VI) contain hydrogenase, in contrast to Chlorella vulgaris Beijerinck. In addition, all strains of C. vulgaris f. tertia were now found to be thermophilic, i.e., able to grow at up to 38–42°C. Thus the two classical high-temperature Chlorellae (strains 7-11-05 and 1-9-30 of Sorokin) appear as members of a rather common thermophilic taxon. Among the 19 available strains of Chlorella vulgaris, by contrast, no hightemperature strains were observed. Likewise, we did not find any thermophilic strains of C. homosphaera, C. fusca var. rubescens, C. luteoviridis, C. zofingiensis, C. saccharophila, C. minutissima, and C. protothecoides. C. fusca and C. kessleri, on the other hand, show a slight tendency towards thermophily. Chlorella vulgaris f. tertia therefore seems to be a taxon of ecological specialists, equipped for the existence at elevated temperatures, in anaerobic environments, or as symbionts in animals (two strains are Zoochlorellae isolated from the fresh-water sponge, Spongilla). We assume that it represents a primitive, phylogenetically old group of organisms. The morphologically identical, typical Chlorella vulgaris might have evolved from it through loss of hydrogenase and thermophily.

     

BIOCHEMICAL TAXONOMY AND MOLECULAR PHYLOGENY OF THE GENUS CHLORELLA SENSU LATO (CHLOROPHYTA)

A multimethod approach was used to characterize unicellular green algae that were traditionally assigned to the genus Chlorella Beijerinck and to resolve their phylogenetic relationships within the Chlorophyta. Biochemical, physiological, and ultrastructural characters, together with molecular data such as DNA base composition and DNA hybridization values, were compared with a molecular phylogeny based on complete 18S rRNA sequences. Our results show that Chlorella taxa are dispersed over two classes of chlorophytes, the Trebouxiophyceae and the Chlorophyceae. We propose that only four species should be kept in the genus Chlorella (Chlorophyta, Trebouxiophyceae): C. vulgaris Beijerinck, C. lobophora Andreyeva, C. sorokiniana Shih. et Krauss, and C. kessleri Fott et Nováková. Common characteristics of these taxa are glucosamine as a dominant cell wall component and the presence of a double thylakoid bisecting the pyrenoid matrix. Norspermine, norspermidine, and secondary carotenoids are never produced. Other "Chlorella" species belong to different taxa within the Trebouxiophyceae ( "C." protothecoides = Auxenochlorella protothecoides [Krüger] Kalina et Punčochářová, "C." ellipsoidea, "C." mirabilis, "C." saccharophila, and "C." luteoviridis ) and Chlorophyceae ( "C." zofingiensis and "C." homosphaera = Mychonastes homosphaera Kalina et Punčochářová). The latter taxa can easily be recognized by the production of secondary carotenoids under nitrogen-deficient conditions.     

Deoxyribonucleic acid reassociation and interspecies relationships of the genusChlorella (Chlorophyceae)

Phylogenetic relationships within the genusChlorella were studied by means of DNA/DNA hybridization under both optimal and relaxed reassociation conditions as well as by determination of the thermal stability of hybrid DNA duplexes. The results indicate a relationship betweenC. fusca var.fusca, C. fusca var.rubescens, C. fusca var.vacuolata, and the genusScenedesmus. In addition, the strains endosymbiotic withParamecium bursaria seem to be related with theC. vulgaris/sorokiniana group. The relations between most other species, however, could not be sufficiently resolved by the above methods. This implies considerable phylogenetic divergency within the genusChlorella.     

Excretion of Sugars by Chlorella Species Capable and Incapable of Symbiosis with Hydra viridis

The excretion of some sugars (maltose, glucose, and glucose-6-phosphate) was studied at pH 2.5–6.0 in 38 strains of Chlorella belonging to 15 species of which 7 are capable and 8 incapable of symbiosis with Hydra viridis. A high rate of maltose excretion below pH 4.0 (Cernichiari et al., 1969) was found only in C. vulgaris (non-symbiotic) and C. mirabilis (non-symbiotic). The other Chlorella species are characterized by quite different patterns of sugar excretion. C. spec. (= “C. paramecii”; symbiotic) excretes very high amounts of maltose in the whole range from pH 2.5–6.0. C. kessleri (symbiotic), C. luteoviridis (symbiotic), and C. fusca var. fusca (non-symbiotic) show a predominant excretion of glucose-6-phosphate from pH 2.5–6.0. Some strains also exhibit a high excretion of glucose above pH 4.0 (C. spec. = “C. paramecii”) or below pH 3.0 (C. fusca var. vacuolata). Several species, e.g. C. saccharophila var. saccharophila (symbiotic), C. sorokiniana (non-symbiotic), and C. protothecoides (symbiotic), excrete only very small amounts of sugars. There is no obvious correlation between sugar excretion and the ability or inability of the Chlorella species to form stable symbioses with Hydra viridis.     

Infectivity of Chlorella species for the ciliate Paramecium bursaria is not based on sugar residues of their cell wall components, but on their ability to localize beneath the host cell membrane after escaping from the host digestive vacuole in the early infection process

Summary. Paramecium bursaria cells harbor several hundred symbiotic algae in their cytoplasm. Algae-free cells can be reinfected with algae isolated from algae-bearing cells or cultivated Chlorella species through the digestive vacuoles. To determine the relationship between the infectivity of various Chlorella species and the nature of their cell wall components, algae-free P. bursaria cells were mixed with 15 strains of cultivated Chlorella species and observed for the establishment of endosymbiosis at 1 h and 3 weeks after mixing. Only 2 free-living algal strains, C. sorokiniana C-212 and C. kessleri C-531, were maintained in the host cells, whereas free-living C. sorokiniana C-43, C. kessleri C-208, C. vulgaris C-27, C. ellipsoidea C-87 and C-542, C. saccharophila C-183 and C-169, C. fusca var. vacuolata C-104 and C-28, C. zofingiensis C-111, and C. protothecoides C-150 and C-206 and the cultivated symbiotic Chlorella sp. strain C-201 derived from Spongilla fluviatilis could not be maintained. These infection-incapable strains could escape from the host digestive vacuole but failed to localize beneath the host cell membrane and were eventually digested. Labeling of their cell walls with Alexa Fluor 488-conjugated wheat germ agglutinin, GS-II, or concanavalin A, with or without pretreatment with 0.4 N NaOH, showed no relationship between their infectivity and the stainability with these lectins. Our results indicate that the infectivity of Chlorella species for P. bursaria is not based on the sugar residues on their cell wall and on the alkali-insoluble part of the cell wall components, but on their ability to localize just beneath the host cell membrane after escaping from the host digestive vacuole. Correspondence and reprints: Environmental Science and Engineering, Graduate School of Science and Engineering, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Comparative studies onChlorella cell walls: Induction of protoplast formation

Among 12 strains ofChlorella ellipsoidea, C. vulgaris, andC. saccharophila tested, 4 strains (1,C. ellpsoidea; 2,C. vulgaris; 1,C. saccharophila) formed osmotically labile protoplasts after treatment with mixtures of polysaccharide degrading enzymes. The relationship between enzymatical digestibility and structure or composition ofChlorella cell walls were studied by electron microscopy and staining techniques with some specific dyes. The cell wall structures of the 12Chlorella strains were grouped into three types: (1) with a trilaminar outer layer, (2) with a thin outer monolayer, and (3) without an outer layer. Protoplasts were formed only from the strains with a cell wall of Type 2. In the strains with a cell wall of Type 1, the outer layer protected the inner major microfibrillar layer against enzymatic digestion. The cell wall of Type 3 was totally resistant to the enzymes; the chemical composition of the cell wall would be somewhat different from that of other types.     

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.     

Physiologische und biochemische Beiträge zur Taxonomie der Gattung Chlorella

Zusammenfassung Es wurde die Verwendbarkeit von Acetat, Glucose, Fructose, Galactose, Saccharose und Lactose als Kohlenstoffquelle für das Wachstum von 72 Chlorella-Stämmen, die 10 autotrophen Taxa angehören, untersucht. Im Dunkeln zeigen mit Acetat 34 Stämme und mit Glucose 37 Stämme gutes Wachstum (Chlorella kessleri, die meisten Stämme von C. vulgaris und C. vulgaris f. tertia, sowie einige wenige Stämme von C. fusca), während Fructose von 21 Stämmen verwertet wird (C. kessleri, die meisten Stämme von C. luteoviridis und C. saccharophila, sowie einige Stämme von C. fusca und C. zofingiensis). Gutes Wachstum mit Galactose wurde bei 11 Stämmen gefunden (C. kessleri und einige Stämme von C. vulgaris). Saccharose und Lactose ermöglichen dagegen kein intensives Wachstum. Die Verwendbarkeit der 6 geprüften organischen Verbindungen für heterotrophes Wachstum ist als taxonomisches Merkmal zur Charakterisierung von Chlorella-Arten nicht geeignet. Lediglich Chlorella kessleri Fott et Nováková, die allgemein die ausgeprägteste Fähigkeit zu heterotrophem Wachstum besitzt, unterscheidet sich durch gute Verwertung von Galactose von den übrigen Arten.

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Physiological and biochemical contributions to the taxonomy of the genus Chlorella VI. Utilization of organic carbon compounds

Summary The utilization of acetate, glucose, fructose, glactose, saccharose, and lactose as sources of carbon for growth in the dark of 72 Chlorella strains belonging to 10 autotrophic taxa was studied. 34 strains grow well with acetate and 37 strains with glucose (Chlorella kessleri, most strains of C. vulgaris and C. vulgaris f. tertia, and a few strains of C. fusca), and fructose is utilized by 21 strains (C. kessleri, most strains of C. luteoviridis and C. saccharophila, and some strains of C. fusca and C. zofingiensis). 11 strains show good growth with galactose (C. kessleri and some strains of C. vulgaris). Saccharose and lactose, on the other hand, do not support vigorous growth. Utilization of the 6 organic compounds cannot serve as a taxonomic character in the genus Chlorella. However, Chlorella kessleri Fott et Nováková shows the most pronounced ability for heterotrophic growth and differs in its good growth with galactose from the other species.

     

Physiological,Biochemical, and Molecular Characters for the Taxonomy of the Subgenera of Scenedesmus (Chlorococcales,Chlorophyta)

Biochemical and physiological properties of 16 Scenedesmus species representing the three subgenera Scenedesmus, Acutodesmus, and Desmodesmus are not suitable for species differentiation. All Scenedesmus species studied thus far produce secondary carotenoids, e.g. astaxanthin and canthaxanthin, under nitrogen-deficient conditions. In addition, with the exception of one strain, hydrogenase activity under anaerobic conditions is generally present. Sequence analyses of ribosomal 18S RNAs indicate that the subgenus Desmodesmus is phylogenetically well separated from the other subgenera, whereas the separation of Scenedesmus and Acutodesmus appears less convincing and is dismissed in favour of a single subgenus, Scenedesmus. Three taxa formerly assigned to the genus Chlorella are shown to be unicellular species of the genus Scenedesmus. “Chlorella” fusca var. vacuolata and “C.” fusca var. rubescens, which is closely related to S. obliquus, belong to the Scenedesmus/‘Acutodesmus group. “C.” fusca var. fusca is closely related to S. communis and thus belongs to the subgenus Desmodesmus. Inclusion of Kermatia pupukensis into the genus Scenedesmus is also strongly supported by the RNA data which furthermore indicate a relationship with the subgenus Desmodesmus.     

Upper limits of temperature for growth inChlorella (Chlorophyceae)

The upper limit of temperature for growth is a species-specific character in the genusChlorella. The limits of 14Chlorella species range from 26–30°C (C. saccharophila) to 38–42°C (C. sorokiniana), withC. fusca var.vacuolata (34°C) andC. kessleri (34–36°C) assuming an intermediate position. Thus, there is no wide gap in the temperature limits between the normal (“low-temperature”) species ofChlorella and the “high-temperature” species,C. sorokiniana.     

Ribulose Diphosphate Carboxylase Synthesis in Euglena: III. Serological Relationships of the Intact Enzyme and its Subunits

Ribulose 1,5-diphosphate carboxylase was isolated from Euglena gracilis Klebs strain Z Pringsheim, Chlorella fusca var. vacuolata, and Chlamydobotrys stellata, and the subunits from each enzyme were separated and purified by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulfate. Rabbit antibody was elicited against purified Euglena ribulose 1,5-diphosphate carboxylase whole enzyme and the isolated large and small subunits. Euglena ribulose 1,5-diphosphate carboxylase showed partial immunological identity on Ouchterlony gels with the Chlorella and Chlamydobotrys carboxylases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates between antibody to the Euglena large subunit and the isolated large subunits of the Chlorella and Chlamydobotrys enzymes showed this was due to determinants on the large subunit. There was no serological affinity between the small subunits of the Euglena, Chlorella, and Chlamydobotrys carboxylases, and NH₂-terminal amino acid analyses provided further evidence of variability in the structure of the small subunits.     

The impact of cell wall carbohydrate composition on the chitosan flocculation of Chlorella

The carbohydrate composition of the algal cell wall was investigated for its role in cell flocculation. Cultures of Chlorella variabilis NC64A, which were found to have different levels of neutral sugar, uronic acid and amino sugar in the cell wall when cultured in different nitrogen sources and concentrations, were subjected to flocculation with chitosan at dosages of 0–69.6 mg/l and pH values of 5.5, 7 and 8.5. In addition, flocculations of another three strains of Chlorella, which have different levels of cell wall components, were tested. Flocculation improved for all strains at pH 8.5 suggesting that inter molecular forces such as hydrogen bonding might be more important than charge neutralization in the flocculation of Chlorella. Total carbohydrate content in the cell wall was the most significant factor positively affecting the flocculation efficiency of C. variabilis NC64A cells with different cell wall compositions and the other Chlorella strains. The results presented in this study suggest that chitosan flocculation can be improved by optimizing the cell culture conditions to achieve higher cell wall polysaccharide content or selecting an algal strain with higher cell wall polysaccharide content.     

RELATIONSHIP BETWEEN PRESENCE OF A MOTHER CELL WALL AND SPECIATION IN THE UNICELLULAR MICROALGA NANNOCHLORIS (CHLOROPHYTA)1

The cell division mechanisms of seven strains from six species of Nannochloris Naumann were analyzed and compared with those of three species of Chlorella Beijerinck and Trebouxia erici Ahmadjian using differential interference microscopy and fluorescence microscopy. Nannochloris bacillaris Naumann divides by binary fission and N. coccoides Naumann divides by budding. Distinct triangular spaces or mother cell walls were found in the dividing autosporangia of the other five strains from four species of Nannochloris, three species of Chlorella, and T. erici. In an attempt to infer an evolutionary relationship between nonautosporic and autosporic species of Nannochloris, we constructed a phylogenetic tree of the actin genes using seven strains from six species of Nannochloris, three species of Chlorella, and T. erici. Nannochloris species were polyphyletic in the Trebouxiophyceae group. Two nonautosporic species of N. bacillaris and N. coccoides were monophyletic and positioned distally. Moreover, to determine their phylogenetic position within the Trebouxiophyceae, we constructed phylogenetic tree of 18S rRNA genes adding other species of Trebouxiophyceae. Nannochloris species were polyphyletic in the Trebouxiophyceae and appeared in two different lineages, a Chlorella–Nannochloris group and a Trebouxia–Choricystis group. The nonautosporic species, N. bacillaris and N. coccoides, and three autosporic species of Nannochloris belonged to the Chlorella–Nannochloris group. Nannochloris bacillaris and N. coccoides were also monophyletic and positioned distally in the phylogenetic tree of 18S rRNA genes. These results suggest that autosporulation is the ancestral mode of cell division in Nannochloris and that nonautosporulative mechanisms, such as binary fission and budding, evolved secondarily.     

TAXONOMIC REVISION OF THE CONJUGATOPHYCEAN FAMILY PENIACEAE ON THE BASIS OF CELL WALL ULTRASTRUCTURE1

An ultrastructural investigation of the cell wall of Penium silvae-nigrae Raban. and P. spinospermum Josh. showed that these species possess true pores with a pore apparatus and overlapping semi-cell walls. It follows that these two taxa belong not to the Peniaceae, but to the Desmidiaceae sensu stricto; they are referred to the genus Actinotaenium Teil. on account of the shape of their cells and chloroplasts. Two other species previously included in Penium Bréb. are referred to Actinotaenium. Although their cell wall structure could not be studied, they are distinguished from “typical” representatives of Penium by the following photomicroscopically observable complex of features: (pseudo-) girdle bands none, cell wall pores in longitudinal rows, zygospores not globose but of irregular shape. The following new combinations ensued: Actinotaenium borgeanum (Skuja), A. phymatosporum (Nordst.), A. silvae-nigrae (Raban.), A. silvaenigrae var. parallelum (Krieger) and A. spinospermum (Josh.). In addition the diagnosis of the genus Penium was emended and P. margaritaceum (Ehr.) ex Bréb. was selected as the lectotype species. The family Gonatozy-gaceae is merged into the Peniaceae on the basis of cell wall structure.     

ASSESSING THE RELATIONSHIPS OF AUTOSPORIC AND ZOOSPORIC CHLOROCOCCALEAN GREEN ALGAE WITH 18S rDNA SEQUENCE DATA1

We examined the relationships of autosporic and zoosporic taxa in the Chlorococcales by analyzing available complete nuclear-encoded small-subunit (18S) rRNA gene sequences along with two new sequences from Hydrodictyon reticulatum (L.) Lagerh. and Neochloris vigenis Archibald. Some autosporic taxa grouped with the coenobial hydrodictyacean algae and related unicellular organisms having directly opposed basal bodies. These include Scenedesmus obliquus (Turp.) Kütz. and Chlorella fusca var. vacuolata Shihira et Krauss. Other autosporic organisms, including Chlorella kessleri Fott et Novakova, C. minutissima Fott et Novakova, C. pro-tothecoides Krug., C. vulgaris Beij., Nanochlorum eucaryotum Wilhelm, Eisenbeis, Wild, et Zahn, and Prototheca wickerhamii Soneda et Tubaki, form a separate group. Of the zoosporic taxa examined, this group would appear to have the greatest affinity to organisms having a counterclockwise displacement of basal bodies, the Pleurastrophyceae. Beyond the fact that Ankistrodesmus stipitatus (=A. falcatus var. stipitatus (Chodat) Lemm.) does not group with the latter organisms, its position remains in doubt. None of the autosporic taxa appear to be closely related to chlorophycean organisms possessing a clockwise basal body displacement.