THE OPTIMUM PH OF FOUR STRAINS OF ACIDOPHILIC SNOW ALGAE IN THE GENUS CHLOROMONAS (CHLOROPHYTA) AND POSSIBLE EFFECTS OF ACID PRECIPITATION1

Four axenic strains of snow algae were examined for optimum pH under laboratory conditions using M-1 growth medium. Growth was measured using cell counts, cell measurements and absorbance readings at 440 nm. Strains C204 and C479A of Chloromonas sp. from the Adirondack Mountains, New York, grew optimally at pH 4.0 to 5.0. Strains C381F and C381G, Chloromonas polyptera (Fritsch) Hoh., Mull. & Roem. from the White Mountains, Arizona, grew optimally at pH 4.5 to 5.0. Growth was significantly higher at pH 4.0 in the northeastern species (Chloromonas sp.), but no significant difference was observed in final growth at pH 4.5, 5.0 and 5.5 between species. It is postulated that the more acidic precipitation in the northeastern United States may be selecting for strains of snow algae with greater tolerance to acidity than in strains from the southwestern United States or that the different pH optima reported are simply species differences. New York strain C204 was also grown in heavily buffered AM medium where it had an optimum pH of 5.0, but cells became irregularly shaped and tended to clump at pH 6.0 to 7.0. Growth of C204 in AM medium was significantly lower than in M-1 medium for snow algae. These findings justify the use of M-1 medium for this type of experimentation.     

SECONDARY CAROTENOID ACCUMULATION IN SCENEDESMUS KOMAREKII (CHLOROPHYCEAE, CHLOROPHYTA)

As Scenedesmus komarekii Hegewald was cultured under high light intensity and nitrogen limitation, the color of cells progressed from green to brown, and finally through orange to brick red. The secondary carotenoids astaxanthin and canthaxanthin, as well as apolar carotenoids, were detected in the brown and orange cells. These carotenoids were contained in lipoidal globules that were first formed at the periphery of the cell and progressively propagated toward its inside, eventually filling most of it. The chloroplast was single and parietal in the green cells. As the cells turned brown, the chloroplast divided into several small lobes and was pushed toward the interior by the accumulating lipoidal globules. Sometimes the outer layer of the wall of the brown cell developed one or two diametrically opposed swellings. Once the cells became orange or red, neither lipoidal globules nor any major organelles were distinguishable. The cell wall in the orange cells became thick because of the formation of electron-dense granules between its outer and inner layers. The mode of the secondary carotenoid accumulation in S. komarekii differs from that of Haematococcus, an alga well known for its ability to accumulate secondary carotenoids, but resembles that of " Chlorella " zofingiensis (= Mychonastes zofingiensis (Dönz) Kalina et Punčochářová).     

FUNCTIONAL ASPECTS OF SECONDARY CAROTENOIDS IN HAEMATOCOCCUS LACUSTRIS (VOLVOCALES). III. ACTION AS A “SUNSHADE”1

We investigated the protection from photoinhibition by different developmental stages of Haematococcus lacustris [Girod] Rostafinski using chlorophyll fluorescence measurements of single cells and suspensions. An overall correlation between higher cellular content of secondary carotenoids and the capacity to withstand excessive irradiation was observed in flagellated cells and aplanospores of H. lacustris. Low-light-reversible spreading of extra-chloroplastic secondary carotenoids occurred in the periphery of the cell during strong irradiation. This process resulted in increased shading of the cup-shaped chloroplast as demonstrated by a decrease in chlorophyll fluorescence. Extrachloroplastic accumulation of secondary carotenoids in H. lacustris can be interpreted as a specific adaptation to habitats that exhibit strong insolation.     

TAXONOMIC SIGNIFICANCE OF SECONDARY CAROTENOID FORMATION IN NEOSPONGIOCOCCUM (CHLOROCOCCALES,CHLOROPHYTA)1

The pigmentation of 23 species of Neospongiococcum Deason was studied in the stationary phase of nitrogen-limited cultures on agar with or without glucose or in liquid medium. Fourteen species gradually developed an orange or brown color, and in all, secondary carotenoids were detected by thin-layer chromatography. Among these, the keto-carotenoids echinenone, canthaxanthin and esterified astaxanthin were identified, although the last named was not always present. When aging on glucose agar, the remaining nine species developed only a yellow-green or yellow color and a pronounced tendency to bleach. In these, the aforementioned keto-carotenoids were lacking, and β-carotene and lutein lucre dominant. From this study it is not clear whether secondary carotenoid formation is species-specific in Neospongiococcum.     

THE LIPID COMPOSITION OF THORACOSPHAERA HEIMII: EVIDENCE FOR INCLUSION IN THE DINOPHYCEAE1

The fatty acid, sterol and chlorophyll composition of the calcified, unicellular alga Thoracosphaera heimii (Lohmann) Kamptner are reported. The presence of 4,23,24-termethyl-5α-cholest-22E-en-3β-ol (dinosterol), 4,23,24-trimethyl-5α-cholest-22E-en-3-one (dinosterone) and the predominance of C₁₈, C₂₀ and C₂₂ unsaturated fatty acids, including the acid 18:5ω3, indicates that T. heimii is a dinoflagellate. The fatty acid: sterol ratio (1.3), is typical of dinoflagellates. The geochemical significance of dinosterone, the high relative concentration of 4-desmethyl-5α-stanols and the role of 23-methyl-5α-cholest-22E-en-3β-ol in the biosynthesis of dinosterol in T. heimii are also discussed.     

CAROTENOID COMPOSITION OF MARINE RED ALGAE1

Photosynthetic organisms possess carotenoids that function either as accessory, photoprotective, or structural pigments. Therefore, the carotenoid profile provides information about certain photoacclimation and photoprotection responses. Carotenoids are also important chemosystematic markers because specific enzymes mediate each step of carotenoid biosynthesis. For red algae, diverse and often contradictory carotenoid compositions have been reported. As a consequence, it is difficult to infer the physiological importance of carotenoids in Rhodophyta. To characterize the relationship between carotenoid composition, rhodophycean phylogeny, and the presence of potentially photoprotective pigments, we analyzed the carotenoid composition of 65 subtropical species from 12 orders and 18 rhodophyte families. Our results showed that red algae do not present a unique carotenoid profile. However, a common profile was observed up to the level of order, with exception of the Ceramiales and the Corallinales. The main difference between profiles is related to the xanthophyll that represents the major carotenoid. In some species lutein is the major carotenoid while in others it is substituted by zeaxanthin or antheraxanthin. The presence of this epoxy carotenoid together with the presence of violaxanthin that are xanthophyll cycle (XC)‐related pigments was found in four of the 12 analyzed orders. The carotenoid pigment profiles are discussed in relation to Rhodophyta phylogeny, and it is suggested that the xanthophyll cycle‐related pigments appeared early in the evolution of eukaryotic phototrophs.     

PHOTOSYNTHESIS IN THE SNOW: THE ALGA CHLAMYDOMONAS NIVALIS (CHLOROPHYCEAE)1

Red blooms of snow algae consisting almost exclusively of large spherical red cells of Chlamydomonas nivalis (Bauer) Wille are widespread during the summer in the Beartooth Mountains in Montana and Wyoming. Field studies designed to examine the effects of temperature, light, and water potential on algal activity were performed with natural populations using photosynthetic ¹⁴C-HCO₃- or ¹⁴CO₂ incorporation as a measure of activity. The algae photo-synthesized optimally at 5.4 × 10⁴ lx, but were not inhibited by increased light intensity up to 8.6 × 10⁴ lx, the maximum observed in the field. Photosynthesis was sensitive to a reduction in water potential, and since low water potentials develop in snow at temperatures below 0 C, it is unlikely that significant algal activity occurs at the sub-0 temperatures which occur throughout winter. Photosynthesis was much lower following melting of the snow, but this was probably due to decreased diffusion of CO₂. The optimal temperatures varied considerably among the different algal populations. Most samples photo-synthesized optimally at 10 or 20 C but retained substantial activity at temperatures as low as 0 or -3 C. Exceptional samples photosynthesized optimally at 0 or -3 C. It is proposed that the varied temperature responses reflect the presence of different temperature strains. Taken together, the data suggest that development of the snow algae can occur only during the summer months.     

A MOLECULAR PHYLOGENY OF THE HETEROKONT ALGAE BASED ON ANALYSES OF CHLOROPLAST-ENCODED rbcL SEQUENCE DATA1

Nearly complete ribulose-1,5-bisphosphate carboxylase/ oxygenase (rbcL)sequences from 27 taxa of heterokont algae were determined and combined with rbcL sequences obtained from GenBank for four other heterokont algae and three red algae. The phylogeny of the morphologically diverse haterokont algae was inferred from an unambiguously aligned data matrix using the red algae as the root, Significantly higher levels of mutational saturation in third codon positions were found when plotting the pair-wise substitutions with and without corrections for multiple substitutions at the same site for first and second codon positions only and for third positions only. In light of this observation, third codon positions were excluded from phylogenetic analyses. Both weighted-parsimony and maximum-likelihood analyses supported with high bootstrap values the monophyly of the nine currently recognized classes of heterokont algae. The Eustigmatophyceae were the most basal group, and the Dictyochophyceae branched off as the second most basal group. The branching pattern for the other classes was well supported in terms of bootstrap values in the weightedparsimony analysis but was weakly supported in the maximum-likelihood analysis (<50%). In the parsimony analysis, the diatoms formed a sister group to the branch containing the Chrysophyceae and Synurophyceae. This clade, charactetized by siliceous structures (frustules, cysts, scales), was the sister group to the Pelagophyceae/Sarcinochrysidales and Phaeo-/Xantho-/ Raphidophyceae clades. In the latter clade, the raphido-phytes were sister to the Phaeophyceae and Xanthophyceae. A relative rate test revealed that the rbcL gene in the Chrysophyceae and Synurophyceae has experienced a significantly different rate of substitutions compared to other classes of heterokont algae. The branch lengths in the maximum-likelihood reconstruction suggest that these two classes have evolved at an accelerated rate. Six major carotenoids were analyzed cladistically to study the usefulness of carotenoid pigmentation as a class-level character in the heterokont algae. In addition, each carotenoid was mapped onto both the rbcL tree and a consensus tree derived from nuclear-encoded small-subunit ribosomal DNA (SSU rDNA) sequences. Carotenoid pigmentation does not provide unambiguous phylogenetic information, whether analyzed cladistically by itself or when mapped onto phylogenetic trees based upon molecular sequence data.     

SNOW ALGAE OF THE WINDMILL ISLANDS, CONTINENTAL ANTARCTICA: DESMOTETRA AUREOSPORA, SP. NOV. AND D. ANTARCTICA, COMB. NOV. (CHLOROPHYTA)

Two cryophilic Desmotetra species, D. aureospora , sp. nov., and D. antarctica (Fritsch) Ling appear to be unique to the southern hemisphere snow ecosystem, or at least to the Windmill Island region, Antarctica. They have not been encountered in previous extensive studies of the Arctic and northern alpine regions. Also unusual are the higher pH (6.8 and 7.8) and conductivities of 279 μS·cm⁻¹ and 426 μS·cm⁻¹ for habitat conditions of D. antarctica that can be attributed to the influence of penguin guano. Both species are characterized by cells enveloped in individual mucilage layers, 1–3 contractile vacuoles, and a cup-shaped chloroplast containing a diffuse pyrenoid. The cells divided in three planes to form cubical loosely aggregated green cell packages embedded in mucilage. Vegetative cells of the two species cannot be distinguished with certainty; however, their zygospores are very different. Desmotetra aureospora has spherical, smooth-walled, golden zygospores, whereas D. antarctica has pale, yellow green, aereolate zygospores. Mucilage stalk morphology of cells in stationary-phase cultures can also be used to separate the two species. Zygospores of D. antarctica have previously been identified as the snow alga Trochiscia antarctica Fritsch. Both species are currently maintained in culture at the Australian Antarctic Division. The cultures did not grow at temperatures above 15° C. The two species are compared with the soil alga D. stigmatica (Deason) Deason et Floyd, the only other species in the genus, and also with Chlorosarcina stigmatica Deason strain T105. Results show that the three Desmotetra species form a natural group and that the absence or presence of a wall on the zoospore is of dubious value in classifications of green algal taxa above the species level.     

AN UNUSUAL METHOD FOR CULTURE OF UNICELLULAR MARINE ALGAE1

A method for the axenic culture of marine microscopic algae that utilizes various types of paper as solid substrates for growth is described. Investigations into the growth of 39 algal, strains on various paper substrates were conducted. Applications of the method to bioassay are discussed.     

N-METHYL-N'-NITRO-N-NITROSOGUANIDINE AS A MUTAGEN FOR BLUE-GREEN ALGAE: EVIDENCE FOR REPAIR1

Survival and mutagenesis have been examined in the marine coccoid blue-green alga, Agmenellum quadruplicatum, after treatment with the chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (NTG). When cells were immediately returned to growth conditions following NTG treatment, the survival level was consistently low and essentially independent of the treatment conditions. The types of mutants found paralleled those previously described in the freshwater coccoid, Anacystis nidulans. If, however, cells were kept under very low light intensity, a nongrowth condition, following NTG treatment, viable cell recovery was dramatically increased. This “dim light” repair in A. quadruplicatum has characteristics similar to those reported for the dark repair systems of bacteria and yeast.     

ASTAXANTHIN ACCUMULATION IN HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) AS AN ACTIVE PHOTOPROTECTIVE PROCESS UNDER HIGH IRRADIANCE1

Green cells of Haematococcus pluvialis Flotow accumulate the ketocarotenoid astaxanthin under stress conditions, such as high irradiance, nutrient deficiency, high salinity, and high temperature. Though some photoprotective mechanisms have been suggested, the function of astaxanthin in red cysts is still questioned. We studied the role of astaxanthin in photoprotection by inducing its formation in logarithmically growing cultures by high irradiance, thus avoiding unrelated processes that can occur in H. pluvialis when carotenogenesis is induced by other stresses. On exposure to high irradiance, the green Haematococcus culture turned red as lipid globules loaded with astaxanthin esters were formed and concentrated at the periphery of the cell. During this phase of induction, the photosynthesis rates remained high, but the amount of the D1 protein of PSII was significantly reduced. The decline in D1 protein content stopped after 1 day; the level then increased, returning to normal after 5 days. The response of the D1 protein was indicative of a transitional phase in the acclimation of Haematococcus to high light. The formation and deposition of astaxanthin seemed to prevent further reduction in D1 protein level, thus enabling the cell to maintain PSII function and structural integrity. This result seems to be a clear indication of the light screening by astaxanthin, which absorbs light in the blue region, thus protecting the photosynthetic apparatus. When the cells recovered from the high light stress, the astaxanthin globules concentrated around the nucleus, indicating that the pigment also serves as a physicochemical barrier, protecting the replicating DNA from oxidation as the cells divide.     

Desmids of the Staurastrum tetracerum-group from a eutrophic lake in mid-wales

Seven desmids have been found in the phytoplankton of a markedly eutrophic lake in mid Wales. All have grown well in laboratory cultures and so an opportunity has been provided to explore the taxonomy and morphological variability of four species, Staurastrum tetracerum, S. irregulare, S. bibrachiatum and S. pseudotetracerum, previously described inadequately. Suggestions for nomenclatural changes are made for S. irregulare and the very variable S. bibrachiatum, which is recorded from the British Isles for the first time. Reasons for the occurrence of the seven desmid species in the eutrophic lake plankton are discussed.     

PHOTOPHYSIOLOGICAL EVIDENCE OF NUTRIENT LIMITATION OF PLATELET ICE ALGAE IN MCMURDO SOUND, ANTARCTICA

Seasonally changing photophysiological and biochemical characteristics of sea ice microalgae are interpreted with respect to light availability and measurements of nutrient concentration made at high vertical resolution (12.5 cm) during a dense bloom in the platelet ice layer of McMurdo Sound during a 6-week study in austral spring of 1989. Platelet ice algae remained highly shade adapted throughout the spring as shown by their low photoadaptive index (E_k, 3.7–8.4 μmol photons·m⁻²·s⁻¹), low mean specific absorption coefficient (<0.009 m² mg⁻¹ Chl a), high optical cross-sectional area of photosystem II (σ_PSII, 3.0–8.2), and high molar ratio of fucoxanthin:chlorophyll a (mean = 1.62 ± 0.15 SD). Between 24 October and 8 November, the algae exhibited a photoacclimative response that was marked by a 30% decrease in photosynthetic efficiency (α^B), a 75% decrease in maximum photosynthetic rate (P^B/_m), and a 60% increase in σ_PSII. The photochemical conversion efficiency at photosystem II (F_v/F_m= ca. 0.5) and the quantum yield of photosynthesis (Ø_C= 0.062– 0.078 mol C mol⁻¹ photons) were ca. 80% of their maximal values. After 8 November, changes in algal photophysiology and biochemistry, which were inconsistent with a photoacclimation response, suggest that the platelet ice algae near the platelet/congelation ice interface became increasingly nutrient limited. The number of pennate diatoms increased threefold to 150 × 10⁹ cells m⁻³ between 8 and 14 November, then remained unchanged throughout the remainder of the field season. Following the increase in cell number, F_v/F_m, Ø_C, and C:Chla decreased by >40%, σ_PSII increased by 70%; and the biochemical ratios C:N and C:Si increased 25%–30%. Nutrient depletion was apparent from the high-resolution vertical profiles, but nutrient concentrations limiting algal growth were not observed. However, nutrient concentrations at the likely site of nutrient limitation near the platelet/congelation ice interface were not measured, indicating that higher resolution sampling is necessary to fully characterize this highly variable habitat.     

EFFECTS OF HYDROXAMATE SIDEROPHORES (STRONG Fe (III) CHELATORS) ON THE GROWTH OF ALGAE1

Varying concentrations of Fe were tested with three hydroxamate siderophores to demonstrate the interactions affecting growth of Chlamydomonas reinhardtii and Chlorella vulgaris. Schizokinen was purified from the excretions of the blue-green alga Anabaena sp. grown in low-Fe medium. Chlamydomonas reinhardtii was inhibited by schizokinen when in molar excess of the Fe concentration; the inhibition was overcome with excess Fe. The growth of C. vulgaris was not affected by this chelator. Results with desferrioxamine were similar. A weaker chelator, rhodorulic acid, did not inhibit the growth of either alga. Low concentrations of the chelators may stimulate algal growth when Fe precipitates are hydrolyzed. Since different algae respond differently to the presence of the chelators, the observed interactions could be important in determining competitive relationships when Fe is limiting. If an alga can excrete a strong chelating agent, as does Anabaena, algae lacking the ability to compete with the chelator may not grow.     

ORGANIC COMPOUNDS RELEASED FROM A NATURAL POPULATION OF EPIBENTHIC BLUEGREEN ALGAE1

Extracellular release of dissolved organic compounds by the bluegreen algal community of a brackish marsh was studied using ¹⁴C techniques. Mannitol and trehalose were identified as the most commonly released compounds. The proportions of these two extracellular compounds varied in response to light intensity and the water potential of the environment. The presence of mannitol, in particular, suggests that excretion of organic compounds in natural situations is a function of osmotic adjustment.     

ALGAE AS COMPETITORS FOR GLUCOSE WITH HETEROTROPHIC BACTERIA1

Dissolved organic carbon (DOC) constitutes the bulk of organic carbon in aquatic environments. The importance of DOC utilization by mixotrophic algae is unclear since heterotrophic bacteria are regarded as more efficient users. We tested the hypothesis that algae decrease the DOC concentration in the light to lower levels than in darkness resulting in competitive exclusion of heterotrophic bacteria according to the mechanistic competition theory. We investigated (a) the uptake kinetics of glucose as a model substrate by two cultured algae and mixed bacteria populations, (b) the competition for glucose between algae and bacteria in chemostats, (c) the effect of discontinuous glucose supply in chemostats, and (d) the minimum glucose concentrations achieved in cultures of algae and bacteria. Bacteria showed higher specific‐glucose‐uptake rates than algae. In chemostats, algae became extinct in the dark and coexisted in the light where they decreased bacteria to lower densities. Discontinuous glucose supply promoted the algae compared to continuous substrate addition. Several algae consumed glucose to lower concentrations in the dark than in the light and showed lower or equal residual glucose concentrations than bacteria. Residual concentrations were not related to allometric traits (cell volume) and photosynthetic potential (chl content). Overall, the hypothesis was not supported, and mechanisms of competition for DOC obviously differed from those for particulate prey. However, since some algae showed lower or equal residual glucose concentrations than bacteria, algal dark uptake of DOC may be important in deep layers of many waters.     

SEASONAL DIFFERENCES IN THE TEMPERATURE RANGES OF GROWTH OF VIRGINIA ALGAE1

One hundred and fifteen clonal, unialgal strains were isolated and tested for their ability to grow over a range of temperatures from 2 to 40° C. Responses of 63 strains isolated from habitats that were 6° C when sampled and 52 strains isolated from habitats that were 20° C when sampled showed trends toward increasing adaptation to cold or warm temperatures commensurate with their seasonal in situ temperatures. Based on temperature-growth responses alone, 24% of the plankton isolates and 17% of the periphyton isolates could be perennial within the natural habitats. At 5° C, 56% of the warm water plankton isolates and 48% of the warm water periphyton isolates were incapable of growth and, therefore, probably could not be important components of the winter algal community. Likewise at 25° C, 25% of the cold water plankton isolates and 13% of the cold water periphyton isolates were incapable of growth. Thus, temperature alone probably is an important variable regulating seasonal changes in algal community structure. Pollution of these habitats by a thermal enrichment averaging + 5° C year-round could effect a pronounced change in algal species composition because many more taxa could be perennial and more taxa would be incapable of growth during naturally warm periods.     

FINE STRUCTURE OF THE SNOW ALGA (CHLAMYDOMONAS NIVALIS) AND ASSOCIATED BACTERIA1

Chlamydomonas nivalis (Bau.) Wille is present in red snow as large spherical resting cells. Fine structural studies reveal an abundance of clear granules in the cytoplasm and occasional starch grains in the chloroplast. Individual cells display a thick cell wall with a smooth outer surface. Cells may be surrounded by a loose fibrous network in which encapsulated bacteria are seen. The bacteria have a characteristic Gram-negative cell wall and constrictive mode of division. The algal-bacterial association appears to be characteristic of red snow populations.     

TWO SNOW SPECIES OF THE QUADRIFLAGELLATE GREEN ALGA CHLAINOMONAS (CHLOROPHYTA,VOLVOCALES): ULTRASTRUCTURE AND PHYLOGENETIC POSITION WITHIN THE CHLOROMONAS CLADE1

The quadriflagellate snow alga Chlainomonas Christen, distributed in New Zealand and North America, has several unusual structural attributes. A process assumed to be cytokinesis involves extrusion of protoplasm from the parent through a narrow canal, C. kolii (J. T. Hardy et Curl) Hoham produces a net‐like outer envelope rather than a cell wall, and the flagellar basal apparatus of Chlainomonas consists of two semi‐independent pairs of basal bodies. Structural connections between basal body pairs appear minimal, but a connecting system different from that observed in other genera exists within each pair. Phylogenetic analysis using rbcL sequences places Chlainomonas in the Chloromonas clade, other known members of which are all biflagellate. Chlainomonas is split into two robust lineages, with New Zealand collections sharing an origin with northern North American collections. Although the quadriflagellate condition is regarded as ancestral in the Chlorophyceae, we speculate—based on ultrastructural and molecular data presented here—that Chlainomonas represents a derived form that has arisen from fusion of two ancestral biflagellate cells. Other explanations (for example, that Chlainomonas represents a diploid form of a biflagellate species) are remotely possible but are presently at odds with extensive observations of field material. Improvements in techniques for experimental manipulation of these sensitive cryophiles will be required to fully characterize their structure and progress our understanding of their biology.