CELL DIVISION AND COLONY FORMATION IN THE GREEN ALGA COELASTRUM (CHLOROCOCCALES)1

Multinucleate cells of Coelastrum undergo precisely directed cytokinesis, guided by phycoplast microtubules, to form a number of uninucleate daughter cells which subsequently adhere to form characteristically patterned aggregates. As there is no movement of the daughter cells relative to one another before their adhesion, the disposition of cells in daughter colonies reflects the pattern of cytokinesis of parent cells. Centrioles lie at the poles of the mitotic nuclei which are partially enclosed by a perinuclear envelope of endoplasmic reticulum. The centrioles disappear at the time of cytokinesis of the parental cell and apparently reform de novo once the daughter cells have acquired a cell wall following their adhesion. The trilaminar layer of cell wall, often termed the pectic layer, does not stain with ruthenium red and resists acetolysis suggesting that it contains sporopollenin rather than pectin.     

GRAZER-INDUCED COLONY FORMATION IN SCENEDESMUS ACUTUS (CHLOROPHYCEAE): ECOMORPH EXPRESSION AT DIFFERENT TEMPERATURES

Scenedesmus acutus Meyen was cultured at four temperatures (9.5°, 16.5°, 24°, and 29° C) in standard medium or in medium with filtered water from a Daphnia culture. Growth was significantly reduced at low temperatures. At 9.5° C it took more than a week before formation of eight-celled coenobia occurred in both the absence and presence of water from a Daphnia culture. At higher temperatures, formation of four- and eight-celled coenobia occurred more rapidly and was already observed in the presence of Daphnia water within 2 days. As cultures aged, also in the absence of Daphnia water, four-celled coenobia became dominant. At cold temperature, cell volume initially was significantly larger but declined after 3–4 weeks. Grazer-induced colony formation had occurred independent of the incubation temperature, but the number of cells per colony was increased with declining temperature. The morphological expression may be interpreted as a cyclomorphosis driven by nutrients, temperature, and chemical cues from grazers.     

ULTRASTRUCTURE OF CELL DIVISION AND REPRODUCTIVE DIFFERENTIATION OF MALE PLANTS IN THE FLORIDEOPHYCEAE (RHODOPHYTA): CELL DIVISION IN POLYSIPHONIA1

Cell division in the marine red algae Polysiphonia harveyi Bailey and P. denudata (Dillwyn) Kutzing was studied with the electron microscope. Cells comprising the compact spermatangial branches of male plants were used exclusively because of their small size, large numbers and the ease with which the division planes can be predetermined. Some features characterizing mitosis in Polysiphonia confirm earlier electron microscope observations in Membranoptera, the only other florideophycean algae in which mitosis has been studied in detail. Common to both genera are a closed, fenestrated spindle, perinuclear endoplasmic reticulum, a typical metaphase plate arrangement of chromosomes, conspicuous, layered kinetochores, chromosomal and non-chromosomal microtubules, and nucleus associated organelles (NAOs) known as polar rings (PRs) located singly in large ribosome-free zones of exclusion at division poles in late prophase. However, other features, unreported in Membranoptera, were observed consistently in Polysiphonia. These include the presence of PR pairs in interphase-early prophase cells, the attachment of PRs to the nuclear envelope during all mitotic stages, the migration of a single PR to establish the division axis, a prominent, nuclear envelope protrusion (NEP) at both division poles at late prophase, the prometaphase splitting of PRs into proximal and distal portions, and the reformation of post-mitotic nuclei by the separation of an elongated interzonal nuclear midpiece at telophase. During cytokinesis, cleavage furrows impinge upon a central vacuolar region located between the two nuclei and eventually pit connections are formed in a manner basically similar to that reported for other red algae. Diagrammatic sequences of proposed PR behavior during mitosis are presented which can account for events known to occur during cell division in Polysiphonia. Mitosis is compared with that reported in several other lower plants and it is suggested that features of cell division are useful criteria to aid in the assessment of phylogenetic relationships of red algae.     

CELL DIVISION IN THE FILAMENTOUS PLEURASTRUM AND ITS COMPARISON WITH THE UNICELLULAR PLATYMONAS (CHLOROPHYCEAE)1

At prophase in Pleurastrum, extranuclear spindle microtubules develop from the region of centrioles, which lie lateral to the nucleus midway between the future sites of the metaphase spindle poles. The microtubules then move laterally to overarch the nucleus and finally become incorporated into the spindle. The centrioles do not migrate and therefore lie in the same plane as the chromosomes at metaphase. At telophase, 2, more different systems of microtubules develop from the vicinity of the centrioles—a phycoplast and extensive arrays of microtubules that ensheath the daughter nuclei. Cell division in the filamentous Pleurastrum is compared to that in the green flagellate, Platymonas. The similarities between cell division in the 2 algae are interpreted as evidence: (i) that rhizoplasts (which in Platymonas resemble myofibrils) are somehow homologous to microtubules; and, (ii) that cell division in Pleurastrum differs from cell division in other examined filamentous chlorophycean genera because Pleurastrum has an independent evolutionary origin from a monad with Platymonas-like characteristics.     

THE ROLE OF UNICELLS IN THE POLYMORPHIC SCENEDESMUS ARMATUS (CHLOROPHYCEAE)1

The UTEX 2193 strain of Scenedesmus armatus (Chod.) Chod, when cultured in any of several media (whether natural or artificial, concentrated or dilute) produced a variety of colonial morphologies as well as a unicell population. Morphological expression was related to culture ape. When the initial cell density was just a feu1 hundred cells per mL. the culture first produced a unicell population, then spiny colonies, and as stationary phase was approached, spine-less colonies. Two classes of spiny colonies were detected. Type I colonies had elongate cells with the terminal cells shorter than median cells. Spines were longer than cell length. The wider, oval, grainy cells of Type II colonies were uniform m length. Spines were shorter and thicker than those on Type I colonies. Only Type I colonies produced unicells: the latter appeared as two morphs. The smaller unicell was obovoid with four delicate spines: the larger had ovate cells bearing four thicker spines. Control of unicell development in all media was achieved by carefully monitoring colony type and cell number used for the inoculum. A unicellular population developed in batch culture in defined media, both concentrated and dilute, when the initial cell density (either Type I or Type II colonies) was low (below 1000 cells-mL^?1), as well as in synchronous cultures. With higher initial cell densities, e.g. 2 × 10⁴ cells·mL^?1, the inoculum had to contain Type I colonies to produce unicells. Unicells were also produced in water from Agronomy Pond, where the strain originated. We discuss the role of unicell populations in the distribution of Scenedesmus.     

MITOSIS AND CELL DIVISION IN CYLINDROCAPSA GEMINELLA (CHLOROPHYCEAE)1

Mitosis and cell division were studied in the green alga Cylindrocapsa geminella Wolle with transmission electron microscopy. Vegetative cells possess a parietal, lobed chloroplast, and a central pyrenoid. Prophase and metaphase nuclei are surrounded by 1–3 layers of perinuclear endoplasmic reticulum. At early prophase a small number of perinuclear microtubules (MTs) are present while at late prophase MTs are concentrated at the presumptive spindle poles. At the same time, MTs begin to appear in the nucleoplasm. Metaphase spindles are diamond-shaped and centric. During telophase, centrioles migrate towards the center of the equatorial zone, presumably guided by a small group of perinuclear MTs. A second system of MTs develops in the equatorial plane, initially consisting of randomly orientated microtubular elements. Later they tend to run in a predominantly radial direction although a common MT focal point or organizing center is not apparent. The two centriole complexes remain at the center of the equatorial plane until well into interphase, facing each other across the newly formed transverse septum. Centrioles are associated with root templates and connecting fibers. The present observations corroborate the view that C. geminella does not form a true filament in the ulotrichalean or chaetophoralean sense, but rather consists of a row of autospores. Its affinity with other “pseudo-filamentous” green algae and the Chlorococcales is discussed. The interpretation of the cytokinetic MTs in C. geminella as a phycoplast appears to be problematic.     

EFFECT OF GRAZING-ASSOCIATED INFOCHEMICALS ON GROWTH AND MORPHOLOGICAL DEVELOPMENT IN SCENEDESMUS ACUTUS (CHLOROPHYCEAE)

Populations of the phenotypically plastic alga Scenedesmus acutus Meyen were cultured in standard medium or in medium with filtered water from a Daphnia culture to examine the expression of ecomorphs in both water types. A rapid formation of four- and eight-celled coenobia was observed in the presence of Daphnia water, but not in standard medium. Moreover, cell dimensions were increased in the Daphnia water containing medium. Population growth rates were comparable in both water types; however, the carrying capacity appeared significantly lower in Daphnia water. Daphnia needed to feed on digestible food to produce the colony-inducing chemicals; medium from starved animals and Daphnia fed polystyrene beads appeared inactive. Neither suspensions of homogenate of Scenedesmus and Cryptomonas nor auxins affected colony formation or growth rate in S. acutus. The colony-inducing infochemicals are probably not constituents of the algae themselves; however, modification of algal constituents in the digestive system of daphniids may be involved.     

CULTURING,ULTRASTRUCTURE AND COLONY FORMATION IN PECTODICTYON CUBICUM (CHLOROPHYCEAE,CHLOROCOCCALES) 12

Pectodictyon cubicum Taft collected in California develops typical eight-celled, cuboidal colonies only in alkaline media. Vegetative cell ultrastructure is similar to that in other genera of the Chlorococcales, except for an extensive endoplasmic reticulum system paralleling the plasmalemma (termed a paramural ER). Autosporogenesis proceeds inside the parent cell wall by three mitotic divisions producing eight nuclei in two tiers of four. Cleavage furrows following paths delineated by long ER segments and indistinct microtubules bisect and then radically separate the protoplast into eight pyramidal units. Walls comprised of a thick, presumably cellulose layer and a thinner trilaminar layer (not acetolysis resistant) form as cells become rounded, except where touching. A gelatinous matrix is produced around and between cells with concentration at the three sites of prior cell contact 90 degrees apart. Pulsed production of mucilage in three solid strands forces cells to separate, and the expanding colony becomes a hollow cube with a cell at each corner.     

LAGERHEIMIA HINDAKII IS THE UNICELLULAR STAGE OF A SCENEDESMUS1

Extensive variability in spine number and position was emphasized in the protologue for the spiny unicell Lagerheimia hindakii Hegew. et A. Schmidt. Using an axenic clone established from culture Hindk 1975/95, which provided the type material of that organism, we initiated a morphogenetic study. While in the unicellular stage, three short spines were usually formed at each cell pole. A colonial morph was first observed during the initial isolation procedure after unicells were dispersed on firm agar and allowed to grow. We propose that L. hindakii is a Scenedesmus, close to S. subspicatus R. Chod. This organism is unlike most representatives of the genus Scenedesmus because colonies do not readily appear in dilute media; they also fail to form in Bristol's liquid medium unless there is an organic supplement, e.g. glucose. Hindk 1975/95 may be more closely related to truly unicellular taxa than to other Scenedesmus.     

COENOBIAL CELL NUMBER IN SCENEDESMUS QUADRICAUDA (CHLOROPHYCEAE) AS A FUNCTION OF GROWTH RATE IN NITRATE-LIMITED CHEMOSTATS1

The relative numbers of cells growing as coenobia of different cell number are functions of growth rate when Scenedesmus quadricauda (Turp.) Bréb. is grown axenically in nitrate-limited, steady-state chemostats in continuous light. Unicells decreased from a maximum fraction of 0.04–0.05 of the total number of cells at 0.1 day^?1 to 0.01 or less as growth rate increased. The fraction of cells that grew as two-celled coenobia decreased from about 0.2 to 0.01–0.02. The fraction that grew as 4-celled coenobia increased from about 0.7–0.8 at 0.1 day^?1 to near unity at 0.5–0.6 day^?1, and then decreased sharply. The fraction of 8-celled coenobial cells increased from very small values below 0.6 day^?1, to ca. 0.9 at 1.0 day^?1.     

COLONY FORMATION AND SEXUAL MORPHOGENESIS IN THE COCCOLITHOPHORE PLEUROCHRYSIS SP. (HAPTOPHYTA)1

Pleurochrysis sp. formed two types of symmetrical, diploid colonies on solid media: (i) single‐cell lineage (SCL) colonies and (ii) aggregation (AG) colonies. The first division of a single mother cell was asymmetric in ～54% of SCL colonies. These colonies developed at a slower rate than AG colonies. Diffusible molecules released from the cells acted like morphogens enhancing formation of AG colonies; their influence on chemotaxis of aggregating cells was dependent on concentration of the inoculum. Nitrogen depletion of diploid colonies induced sexual morphogenesis and colony patterning into inner and outer regions. The smaller innermost cells were surrounded by outer larger cells. Developmental mechanisms of colony formation were examined in relation to the heteromorphic, haplo‐diploid life cycle.     

REGULATION OF GAMETOGENESIS IN SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE)1

The effects of nutrients, temperature and light on gametogenesis in Scenedesmus obliquus (Turp.) Kütz, were studied in culture. Concentrations of nitrogen in the medium employed showed a marked influence on gamete production. Gametogenesis is inhibited by N excess but is not a response to N starvation or depletion. A drop in N level from that of the growth medium is not required, nor does it per se trigger gametogenesis. The N concentration satisfying growth requirements insufficiently low to permit sexual differentiation. Nitrogen level in the growth medium has no effect on subsequent N to maintain a typical culture. Number of gametes present at maximum production time is inversely related to N concentration, but neither time of onset of gametogenesis, nor time of maximum gamete production is affected by N concentration. Cultures incubated at 15 C in medium lacking N take a minimum of 20—24 h to develop cells irreversibly committed to gamete formation. At the concentrations tested, no medium component other than the N-containing salt affected gametogenesis. Temperature influences both time of maximum production and numbers present at maximum production time. Time of maximum production is inversely related to incubation temperature; a 15 C incubation temperature yielded highest gamete production. Light enhances gametogenesis but gamete formation can occur in absence of light. Achievement of a light-saturated response is dependent upon illumination given at two critical periods: one occurs shortly after N withdrawal; the other occurs later, when cells are becoming irreversibly committed to gamete formation. Ability to produce gametes diminished with prolonged laboratory culture.     

TWO TYPES OF CYTOPLASMIC CLEAVAGE IN THE COENOCYTIC SOIL ALGA PROTOSIPHON BOTRYOIDES (CHLOROPHYCEAE)1

Cytokinesis in the coenocytic green alga Protosiphon botryoides (Kütz.) Klebs was studied with transmission electron microscopy. In vegetative cells, nuclei with associated basal bodies and dictyosomes are scattered throughout the cytoplasm. Mature cells may develop either multinucleate resting spores (coenocysts) or uninucleate zoospores. Cytokinesis may be preceded by contraction of the protoplast due to the disintegration of vacuoles that are present in larger, siphonous cells. The formation of coenocysts in ageing, siphonous cells, is signalled by cleavage of the chloroplast and the development of arrays of phycoplast microtubules in one or more transversely oriented planes through the cell. Nuclei with associated basal apparatuses stay dispersed throughout the cytoplasm; the basal bodies apparently are not involved in organization of the phycoplast. The plasma membrane invaginates, resulting in a centripetal cleavage of the protoplast into two or more multinucleate daughter protoplasts. Simultaneously, wall material is deposited along the outside of the daughter protoplasts by dictyosome-derived vesicles, and finally two or more thick-walled coenocysts are formed. The formation of zoospores, on the other hand, is signalled by clustering of the nuclei in one or more groups depending on the shape of the mother cell. The nuclei become arranged with the associated basal apparatuses facing toward the center of the cluster. Bundles of phycoplast microtubules develop between the nuclei, radiating from the center of a cluster toward the plasma membrane; basal apparatuses or associated structures apparently are involved in organization of the phycoplast. Cleavage furrows grow out centrifugally along these bundles of micro-tubules, fed by dictyosome-derived vesicles. No wall material is deposited. An additional mitotic division occurs during cleavage, and finally numerous uninucleate, wall-less, biflagellate zoospores are formed. The ultrastructural features of the two different types of cytoplasmic cleavage associated with two different types of daughter cells have not previously been reported for chlorophycean algae.     

PHENOTYPIC PLASTICITY IN SCENEDESMUS COMMUNIS (CHLOROPHYCEAE). I. RELATIONSHIP OF S. COMMUNIS TO S. KOMAREKII1

When scenedesmus communis Hegew. (UTEX 76) was transferred daily in dilute media and a low cell density was maintained (ca. 1000 cells · mL^?1), up to 30% unicells were produced in that population. Unlike previously described uncell-coenobium-unicell transformation with other species, these unicells never produced S. communis coenobia (large coenobium type, LCT) but rather small coenobium type (SCT) resembling S. komarekii Hegew. Growth and morphological development of the paratype strain of S. komarekii (UTEX 1236) was compared with an isolated SCT strain (SCT 76–8). SCT 76–8 never produced LCTs and grew significantly faster than UTEX 1236. Both SCT 76–8 and UTEX 1236 produced uncells at low cell densities. Coenobia formed when cell densities increased over time in batch cultures. SCT 76–8 and UTEX 1236 did not differ morphologically when viewed with the light microscope. Under scanning electron microscopy, an outer opaque layer covered an inner warty layer on unicells. The outer layer was reduced or absent in coenobia from batch cultures in stationary growth. In addition, long spikelets, not present on the walls of unicells, were prominent on coenobial walls. The spikelets of UTEX 1236 appeared smaller and more uniformly distributed than in strain 76–8. In contrast, the surface wall morphology of LCT S. communis was composed of an outer reticulate layer supported by spikelets and appeared as a pentagonal meshwork covering the cell walls. This phenotypic plasticity, as demonstrated by SEM and light microscopy, provides further evidence needed for an understanding of Scenedesmus evolution.     

SEQUENCE ANALYSIS OF THE ITS-2 REGION: A TOOL TO IDENTIFY STRAINS OF SCENEDESMUS (CHLOROPHYCEAE)

The genetic distances between several strains of Scenedesmus obliquus (Turp.) Kütz., S. acutus Hortobagyi, and S. naegelii Chod. calculated from ITS-2 sequences were found to be smaller than the genetic distances within other strains of Scenedesmus —that is, in S. acuminatus (Lagerh.) Chod. and S. pectinatus Meyen. These results confirm that the studied strains were not properly identified and should be renamed S. obliquus , as already suggested in other studies.     

THE ULTRASTRUCTURE OF SCENEDESMUS (CHLOROPHYCEAE). I. SPECIES WITH THE “RETICULATE” OR “WARTY” TYPE OF ORNAMENTAL LAYER1

The ultrastructure of the wall layers and ornamentative features of Scenedesmus pannonicus and S. longus are described using carefully correlated freeze-etched replicas, thin sections and scanning electron micrographs. The two species arc enclosed by different types of ornamented layers, S. pannonicus by the tightly filling, “warty” layer and S. longus by the loosely fitting, “reticulate” layer, held off the coenobium by 2 types of tubular propping spikelets and rosettes. The reticulate layer has an intricate substructure, especially when studied with freeze-etching. Its inner and outer surfaces appear different, as is its attachment to the 2 types of spikelets. Whole cells of S. longus subjected to acetolysis lack the cellulose wall and cytoplasm, but all other surface features survive, including the Trilaminar Sheath (TLS); this ornamentation cannot be “pectic.” The cellulose wall and ornamentation is unaffected by boiling water alone. Boiling in 6n NaOH removes the surface ornamentation, but the TLS and wall remain; the possibility that these features contain silica is discussed. The terminal spines of both species consist of closely packed spikelets enclosed within a skin of hexagonally-packed subunits. Similar subunits are seen in the propping spikelets of S. longus, and in the rows or “combs” of laterally fused spikelets of S. pannonicus. The warty layer of S. pannonicus is tightly appressed to the TLS except close to where the cells are joined, where it is suspended free. It is composed of a layer of globular subunits, and small indentations form the warts. Single, evenly distributed warts characterize the freely suspended sections of the warty layer, and the layer that encloses young coenobia soon after they have been formed: in contrast, the warts are clumped over the surface of older and larger colonies. Some of the single warts form characteristic double rows, but these latter remain single even on older cells. The surface structure of the warty layer, TLS, and plasmalemma are revealed by the freeze-etching process.     

GRAZER-INDUCED COENOBIAL FORMATION IN CLONAL CULTURES OF SCENENDESMUS OBLIQUUS (CHLOROCOCCALES, CHLOROPHYCEAE)

Five strains of the phenotypically plastic alga Scenedesmus obliquus (Turpin) Kützing were cultured in standard medium or in medium containing filtered water from a Daphnia culture to examine grazer-induced coenobia formation. Strains varied considerably in their response to Daphnia chemicals. A rapid formation of four- and eight-celled coenobia was observed in the presence of Daphnia water in three strains. One strain showed numerous irregular aggregates with more than eight cells per colony, and no colony formation occurred in a fifth strain. In standard medium, all cultures remained unicellular. Growth rates were affected by Daphnia water in four strains and were different among strains. The chl a concentration was significantly different among strains but was not influenced by Daphnia water. The specific absorption coefficient was reduced at larger colony sizes, suggesting a possible cost associated with colony formation. The phenomenon of grazer-induced coenobia formation seems widespread but not universal within the species S. obliquus.     

ULTRASTRUCTURE OF MITOSIS AND CYTOKINESIS IN KLEBSORMIDIUM MUCOSUM NOV. COMB., FORMERLY ULOTHRIX VERRUCOSA (CHLOROPHYTA)1

A transmission electron microscopy study of dividing cells of Ulothrix verrucosa Lokhorst has provided clear evidence that this species differs in many respects from other Ulothrix Kützing species. These differences include the presence of a microtubular sheath around the prophase nucleus, the complete disintegration of the nuclear envelope coinciding with the proliferation of extranuclear microtubules into the prometaphase nucleus and the intrusion of vacuoles into the interzonal spindle region in between the widely separated telophase nuclei. This necessitates the transfer of Ulothrix verrucosa to the charophycean genus Klebsormidium Silva, Mattox and Blackwell. The new combination Klebsormidium mucosum is proposed. On account of its mitotic pattern, this species can be placed in the (charophycean) evolutionary line towards the higher plants. However, because of its cytokinesis (annular centripetal ingrowth of the plasmalemma) this species probably should be considered as a blind offshoot of this line. It is emphasized that furrowing green algae with a persistent interzonal spindle at telophase (including the presently studied alga) often show an ill-defined cytokinetic microtubular system.     

SURVIVAL OF SCENEDESMUS ACUMINATUS (CHLOROPHYCEAE) IN DARKNESS1

Survival of the green alga Scenedesmus acuminatus Lagerh. in complete darkness was studied in axenic batch cultures at 7°C and 22°C for three months. The decrease in cell numbers was insensitive to temperature and slower than the loss of dry weight. However, the lag phase before cells began to lyse was more than twice as long at 7° C than at 22°C. The decline in cellular carbohydrates and proteins occurred in two phases. During the first 3-4 days, the decrease in cellular carbohydrate levels was significantly accelerated and temperature-sensitive. Pyrenoids disappeared within 5 days of darkness. Proteins showed 20-fold higher degradation rates at 22°C than at 7°C during the first 4 days. Thereafter, the rates of carbohydrate and protein decomposition were slow and temperature-independent. By contrast, lipids degraded only little at virtually constant and temperature-insensitive rates over the entire experimental period. After three months of dark incubation, about 40% of the remaining cells had retained their growth potential. However, the lag phase, after which cell division was resumed when exposed to light, increased with the duration of the previous dark period. The decrease in photo synthetic potential, which was more pronounced at 22° C than at 7° C, was apparent both in declining maximum assimilation numbers and maximum quantum yields. Cellular chlorophyll a concentrations in surviving cells decreased only slightly. We conclude that the primary means by which S. acuminatus survives extended dark periods is by reduction of catabolic reactions. This was suggested by the slow loss of cell weight. No evidence of significant heterotrophic acetate uptake was found. The initial temperature-dependence of most observed processes indicates that in natural environments chances for survival of algae are augmented by the prevailing low water temperatures.     

FINE STRUCTURE OF MITOSIS AND CLEAVAGE IN FRIEDMANNIA ISRAELENSIS (CHLOROPHYCEAE: CHLOROSARCINACEAE)1

Observations on the ultrastructure of Friedmannia israelensis Chantanachat & Bold revealed the presence of a phycoplast and zoospores with cruciate rootlets. During mitosis, the nuclear envelope partially disintegrates and the basal bodies remain at the cell surface on either side of the developing cleavage furrow. The events during mitosis and cleavage in Friedmannia resemble those reported in the other green algae, Platymonas and Pleurastrum.