SUGAR COMPOSITION OF THE CELL WALL AND THE TAXONOMY OF CHLORELLA (CHLOROPHYCEAE)1

Cell walls of forty Chlorella strains covering all species of the Algal Collection of Göttingen (C. fusca var. vacuolata, C. kessleri, C. luteoviridis, C. minutissima, C. protothecoides, C. saccharophila, C. sorokiniana, C. vulgaris, and C. zofingiensis) were compared. The nine species were divided into two groups according to the major sugar in the rigid wall. The first group had a glucose-mannose-rigid wall and included C. fusca var. vacuolata, C. luteoviridis, C. minutissima, C. protothecoides, C. saccharophila, and C. zofingiensis. The second group, with a glucosamine-rigid wall, included C. kessleri, C. sorokiniana, and C. vulgaris. Chlorella strains of the nine species were further classified by constituent sugars, ruthenium red stainability, and anisotropy of the cell walls.     

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.     

CILIATE‐SYMBIONT SPECIFICITY OF FRESHWATER ENDOSYMBIOTIC CHLORELLA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

The nature of Chlorella symbioses in invertebrates and protists has attracted much interest, but the uncertain taxonomy of the algal partner has constrained a deeper ecological understanding of this symbiosis. We sequenced parts of the nuclear 18S rDNA, the internal transcribed spacer (ITS)‐1 region, and the chloroplast 16S rDNA of several Chlorella isolated from pelagic ciliate species of different lakes, Paramecium bursaria symbionts, and free‐living Chlorella to elucidate phylogenetic relationships of Chlorella‐like algae and to assess their host specificity. Sequence analyses resulted in well‐resolved phylogenetic trees providing strong statistical support for a homogenous ‘zoochlorellae’ group of different ciliate species from one lake, but clearly different Chlorella in one of those ciliate species occurring in another lake. The two Chlorella strains isolated from the same ciliate species, but from lakes having a 10‐fold difference in underwater UV transparency, also presented a distinct physiological trait, such as the ability to synthesize UV‐absorbing substances known as mycosporine‐like amino acids (MAAs). Algal symbionts of all P. bursaria strains of different origin resolved in one clade apart from the other ciliate symbionts but split into two distinct lineages, suggesting the existence of a biogeographic pattern. Overall, our results suggest a high degree of species specificity but also hint at the importance of physiological adaptation in symbiotic Chlorella.     

The Occurrence of the Glucose-inducible Transport Systems for Glucose,Proline, and Arginine in Different Species of Chlorella

Incubation of the green alga Chlorella vulgaris (strain K, Tanner and Kandler, 1967) with glucose leads to the induction of a glucose transport system and of two amino acid transport systems. Because it was not clear whether the regulation of 3 different transport systems by glucose is specific to our strain of Chlorella or whether it is a general property of the genus Chlorella, 11 other free living and symbiotic Chlorella species and strains were tested for glucose-inducible glucose, arginine and proline transport. It was found that nearly all Chlorella species possess glucose and amino acid uptake systems. Often they were constitutive, although in some species they were induced or stimulated by glucose. According to the transport activities of the different Chlorella species and strains, a physiological classification of Chlorella was constructed, resulting in 3 groups: the C. fusca vacuolata, the C. vulgaris and the symbiotic Chlorella group. Our Chlorella (strain K) obviously belongs to the C. vulgaris group and forms a link to symbiotic Chlorella strains. This suggests that the possession of the glucose-regulated transport systems is of advantage for Chlorella in symbiotic situations, whereas the constitutive systems are useful for free living Chlorella.     

THE FEEDING OF AMEBAE ON ALGAE IN CULTURE1

Amebae differed greatly in their ability to prey on algae. Species of Chlamydomonas, Pandorina, Anabaena, Ourococcus, Ankistrodesmus, and Gloeocystis were utilized by 3 or all 4 of the test protozoa, while strains of Staurastrum and Chlorella would not support growth of the predators. Heating the cells of Chlorella sp. rendered them available to the 4 amebae and made Pandorina morum a better nutrient for some of the protozoa. Heating Chlamydomonas oblonga had no effect on its availability to Tetramituis rostratus and reduced its suitability as a food source for Amoeba discoides. The rate of protozoan feeding was also influenced by temperature, pH, and age of the algal prey.     

Chlorella species as hosts for genetic engineering and expression of heterologous proteins: Progress,challenge and perspective

The species of Chlorella represent a highly specialized group of green microalgae that can produce high levels of protein. Many Chlorella strains can grow rapidly and achieve high cell density under controlled conditions and are thus considered to be promising protein sources. Many advances in the genetic engineering of Chlorella have occurred in recent years, with significant developments in successful expression of heterologous proteins for various applications. Nevertheless, a lot of obstacles remain to be addressed, and a sophisticated and stable Chlorella expression system has yet to emerge. This review provides a brief summary of current knowledge on Chlorella and an overview of recent progress in the genetic engineering of Chlorella, and highlights the advances in the development of a genetic toolbox of Chlorella for heterologous protein expression. Research directions to further exploit the Chlorella expression system with respect to both challenges and perspectives are also discussed. This paper serves as a comprehensive literature review for the Chlorella community and will provide valuable insights into future exploration of Chlorella as a promising host for heterologous protein expression.     

Molecular characterization of Chlorella cultures of the National Institute for Environmental Studies culture collection with description of Micractinium inermum sp. nov., Didymogenes sphaerica sp. nov., and Didymogenes soliella sp. nov. (Chlorellaceae,Trebouxiophyceae)

Chlorella Beijerinck (Chlorellaceae, Trebouxiophyceae) strains from the collection of the National Institute for Environmental Studies (NIES) were characterized using gene sequence data. The misidentification of a number of strains was rectified. Chlorella vulgaris Beijerinck NIES‐2173 was reclassified as C. sorokiniana Shihira et Krauss. Chlorella sp. NIES‐2171 was described as a new species in the genus Micractinium Fresinius, M. inermum Hoshina et Fujiwara. Chlorella sorokiniana NIES‐2167 and Chlorella sp. NIES‐2330 were found to be phylogenetically related to Didymogenes Schmidle. We propose these two strains be transferred to the genus Didymogenes and given new names: D. sphaerica Hoshina et Fujiwara and D. soliella Hoshina et Fujiwara. Taxonomic decisions were primarily based on small subunit‐internal transcribed spacer ribosomal DNA phylogeny for genus assignment and ITS2 sequence‐structure to determine species autonomy. Our findings suggest that this strategy is the most effective way to use the species concept among autosporic coccoids.     

Unterschiedliche Wirkung von Captan auf das Wachstum einiger Stämme von Chlorella und Scenedesmus

Zusammenfassung Das Fungicid Captan erwies sich bei 37 Stämmen von Chlorella als ein wirksamer Wachstumshemmstoff. Nur 3 Chlorella-Stämme besitzen eine gewisse Captanresistenz. Das Merkmal Captanresistenz scheint in der Gattung Chlorella nicht artspezifisch verteilt zu sein.Bei Scenedesmus acutus f. alternans Hortob. und Scenedesmus armatus (Chod.) Smith wird Captan noch in einer Dosis von 50 mg/l vertragen, ohne daß Anzeichen einer Hemmwirkung zu erkennen sind.

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Differential action of captan on the growth of some strains of Chlorella and Scenedesmus

Summary The fungicide captan was found to inhibit strongly the photoautotrophic growth of 37 Chlorella strains. Only 3 strains are fairly resistant to captan. In the genus Chlorella resistance to captan does not seem to be species specific.Two strains of Scenedesmus (Scenedesmus acutus f. alternans Hortob. and Scenedesmus armatus (Chod.) Smith) tolerate captan up to 50 mg/l without being inhibited at all.

     

CYTOGEOGRAPHY AND PHYLOGENY OF THE NORTH AMERICAN SPECIES OF VERBENA

Lewis , Walter H., and Royce L. Oliver . (Stephen F. Austin State Coll., Nacogdoches, Texas.) Cytogeography and phylogeny of the North American species of Verbena. Amer. Jour. Bot. 48(7): 638–643. Illus. 1961.—Chromosome numbers of 26 Verbena species in North America, separable into 2 series, are reported. The x = 5 series has no diploid representative, few tetraploids, and numerous widespread hexaploid species, while the x = 7 series has many pandemic diploid species and few tetraploid and hexaploid taxa. When compared with the data available for the South American verbenas, these frequencies and distributions were found to be almost opposite. The origin of the 2 groups from an x = 6 prototype followed by ascending and descending aneuploidy is suggested. Migration via tropical land bridges is proposed to explain the occurrence of both groups in the 2 continents. The following taxa are proposed: subgenus Verbenaca (Schauer) Lewis, and subgenus Glandularia (Gmelin) Lewis.     

Physiologische und biochemische Beiträge zur Taxonomie der Gattung Chlorella

Zusammenfassung Es wurde die Verwendbarkeit von Glutaminsäure, Glutamin, Nicotinsäure, Nicotinsäureamid und Purin als Stickstoffquelle für die Kultur von 71 autotrophen Chlorella-Stämmen, die 8 Arten angehören, untersucht. Im Licht zeigen mit Glutaminsäure 63 und mit Glutamin 67 Stämme gutes Wachstum. Nicotinsäure wird dagegen nur von 1 Stamm, Nicotinsäureamid von 9 und Purin von 16 Stämmen verwertet. Die Verwendbarkeit der 5 geprüften organischen Stickstoffverbindungen als N-Quelle ist innerhalb der Gattung Chlorella als taxonomisches Merkmal zur Charakterisierung von Arten nicht geeignet. Lediglich die 7 Stämme von Chlorella kessleri unterscheiden sich durch gutes Wachstum mit Nicotinsäureamid von der sonst recht ähnlichen Chlorella luteoviridis sowie von den übrigen Arten.

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Physiological and biochemical contributions to the taxonomy of the genus Chlorella IV. Utilization of organic nitrogen compounds

Summary The utilization of glutamic acid, glutamine, nicotinic acid, nicotinamide, and purine as sources of nitrogen for growth in the light of 71 autotrophic Chlorella strains belonging to 8 species was studied. Whereas 63 strains grow well with glutamic acid and 67 with glutamine, nicotinic acid is used by 1, nicotinamide by 9, and purine by 16 strains only. Utilization of the 5 organic nitrogen compounds tested cannot serve as a taxonomic character in the genus Chlorella. However, the 7 strains of Chlorella kessleri differ in their good growth with nicotinamide from the otherwise rather similar Chlorella luteoviridis and from the other species.

     

THE OCCURRENCE OF CHLOROPHYLLS C1 AND C2 IN THE CHRYSOPHYCEAE1

We analyzed 34 strains representing 25 species of Chrysophyceae for chlorophylls c₁ and c₂ using thin-layer chromatography. Most organisms had both chlorophylls c₁ and c₂ in addition to chlorophyll a but 17 strains of 9 species of Synura and Mallomonas possessed only chlorophylls a and c₁. These are the first chlorophyll c-bearing algae which lack chlorophyll c₂. We postulate that at least some of the silica-scaled algae including Mallomonas and Synura may be distinct from other Chrysophyceae based upon pigmentation and other characters described in the literature.     

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.     

POLYPHYLETIC ORIGIN OF THE DICTYOSPHAERIUM MORPHOTYPE WITHIN CHLORELLACEAE (TREBOUXIOPHYCEAE)1

The green algal Dictyosphaerium morphotype is characterized by spherical or oval cells connected by gelatinized strands to microscopic colonies, which are covered by prominent mucilaginous envelopes. Combined SSU and ITS rRNA gene sequence analyses revealed that this morphotype evolved independently both in the Chlorella and Parachlorella clades of the Chlorellaceae. It was shown that strains exhibiting the morphology of the type species Dictyosphaerium ehrenbergianum Nägeli established a sister lineage to Parachlorella. The strain D. ehrenbergianum CCAP 222/1A was designated as an authentic strain for establishing the epitype of the genus Dictyosphaerium. The comparison of this strain with the authentic strain of Parachlorella beijerinckii Krienitz, E. Hegewald, Hepperle, V. Huss, T. Rohr et M. Wolf (SAG 2046) showed considerable differences in the secondary structure of the ITS region. Within the whole ITS‐1 and ITS‐2 region, 27 compensatory base changes (CBCs) were recognized. In the conserved Helix III of the ITS‐2, five CBCs/HemiCBCs were detected. This is a conclusive argument for separation of these two species. The clear definition of Dictyosphaerium is intended to be the necessary starting point of taxonomic reevaluation of Dictyosphaerium‐like algae within different evolutionary lineages of the Chlorellaceae.     

CHROMOSOME NUMBERS IN THE LEGUMINOSAE. III. SPECIES OF THE SOUTHWESTERN UNITED STATES AND MEXICO

Turner, B. L., and Olin S. Fearing. (U. Texas, Austin.) Chromosome numbers in the Leguminosae. III. Species of the Southwestern United States and Mexico. Amer. Jour. Bot. 47(7) : 603–608. Illus. 1960.—Chromosome counts for 43 species of the Leguminosae from the southwestern United States and Mexico have been reported. These include first reports for 42 taxa of which 16 are for the subfamily Mimosoideae. Olneya tesota (2n = 18) is the only new generic count listed. Chromosome reports of particular significance include a single polyploid count for a North American species of Acacia, as well as diploid and tetraploid counts for closely related taxa in this genus. Four species of the genus Schrankia were found to be diploid with In = 26, indicating a base of x = 13 instead of the x = 8 reported by some previous workers. Leucaena pulverulenta was found to have a diploid count of 2n = 56 indicating a base of x = 14.     

ANALYSIS OF A PLASTID MULTIGENE DATA SET AND THE PHYLOGENETIC POSITION OF THE MARINE MACROALGA CAULERPA FILIFORMIS (CHLOROPHYTA)1

Molecular phylogenetic relationships within the Chlorophyta have relied heavily on rRNA data. These data have revolutionized our insight in green algal evolution, yet some class relationships have never been well resolved. A commonly used class within the Chlorophyta is the Ulvophyceae, although there is not much support for its monophyly. The relationships among the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae are also contentious. In recent years, chloroplast genome data have shown their utility in resolving relationships between the main green algal clades, but such studies have never included marine macroalgae. We provide partial chloroplast genome data (～30,000 bp, 23 genes) of the ulvophycean macroalga Caulerpa filiformis (Suhr) K. Herig. We show gene order conservation for some gene combinations and rearrangements in other regions compared to closely related taxa. Our data also revealed a pseudogene (ycf62) in Caulerpa species. Our phylogenetic results, based on analyses of a 23‐gene alignment, suggest that neither Ulvophyceae nor Trebouxiophyceae are monophyletic, with Caulerpa being more closely related to the trebouxiophyte Chlorella than to Oltmannsiellopsis and Pseudendoclonium.     

THE ROLE OF POPULATIONAL DIFFERENTIATION IN EXPERIMENTAL INFECTION OF PROSOPIS BY PHORADENDRON

Experimental infection of Prosopis species with the mistletoe Phoradendron tomeniosum subsp. tomentosum in a uniform garden demonstrated that differences in infection success between species, populations, and individuals can be caused by genetically-based differences in both hosts and parasites. Prosopis glandulosa var. glandulosa was least resistant to infection; P. laevigata was more resistant; and P. torreyana was most resistant. A population of P. glandulosa var. glandulosa grown from seeds collected in the lower Rio Grande valley was significantly more resistant to infection than three populations of the same variety from central and northern Texas. The higher resistance of the Rio Grande valley population appears to be due to introgression with P. laevigata. The experimental population of Phoradendron tomentosum subsp. tomentosum from northeastern Mexico and the population of the same subspecies from southern Texas had greater infectivity than did those from central and northern Texas, corresponding to the greater resistance of the southern Prosopis populations.     

INTERPOPULATION DIFFERENCES IN HOST PREFERENCE AND THE EVOLUTION OF LEARNING IN THE BUTTERFLY,BATTUS PHILENOR

The host-discrimination behavior of the adult female pipevine swallowtail butterfly (Battus philenor) was investigated for two populations, an east Texas population that uses two host species with different leaf shapes and a Virginia montane population that uses one host species with a single leaf shape. While Texas and Virginia females exhibited similar chemotactile responses after landing on various host species, butterflies from each population landed more frequently on certain host species used by that particular population. Despite this difference in searching behavior, Texas and Virginia populations were equally capable of learning to search for the leaf shape of a particular host species in artificial enclosure arrays. In addition, learning of leaf-shape preference was confounded equally when each population was introduced into arrays containing all the host species found in both populations. The lack of differentiation in learning of leaf-shape preference between populations that differ in host breadth and host preference argues against the specific hypothesis that learning of leaf-shape preference represents a local adaptation that permits foraging butterflies in the east Texas population to respond to seasonal changes in the relative quality or abundance of host species that differ in leaf shape. Three hypotheses are proposed to explain the apparent absence of interpopulation genetic variation in learning traits. One hypothesis supposes that learning of leaf-shape discrimination is mediated by the same physiological mechanisms that permit females to learn to discriminate among suitable and unsuitable conspecific plants. Selection for restriction of learning of leaf-shape preference in the Virginia montane population may therefore be constrained by selection for learning of other types of discrimination behavior.     

Deep genomic analysis of the Chlorella sorokiniana SAG 211-8k chloroplast

The chloroplast genome contains information that is applicable in many scientific fields, such as plant systematics, phylogenetic reconstruction and biotechnology, because its features are highly conserved among species. To date, several complete green algal chloroplast genomes have been sequenced and assembled. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Chlorella sorokiniana SAG 211-8k is reported and compared for the first time to the chloroplast genomes of 10 Chlorellaceae. The recently updated Chlorella sorokiniana cpDNA sequence, assembled as a circular map of 109?811 bp, encodes 113 genes. Similar to other Chlorella strains, this chloroplast genome does not show a quadripartite structure and lacks the large rRNA operon-encoding Inverted Repeat (IR). The Chlorella sorokiniana plastid encodes the tRNA(Ile)-lysidine synthetase (tilS), which is responsible for modifying the CAU anticodon of a unique tRNA. Gene ordering and clustering highlight the close relationships among Chlorella clade members and the preservation of crucial gene clusters in photosynthetic strains. The features of Chlorella sorokiniana presented here reinforce the monophyletic character of Chlorellaceae and provide important information that sheds light on chloroplast genome evolution among species of Chlorella.