COMPARATIVE ULTRASTRUCTURE OF THE VALVES OF SOME CYCLOTELLA SPECIES (BACILLARIOPHYCEAE)1

Cyclotella atomus Hust., C. meneghiniana Kütz., C. comta (Ehr.) Kütz., C. antiqua W. Sm., C. michiganiana Skv., C. ocellata Pant., C. glomerata Bachmann, C. pseudostelligera Hust., and C. stelligera (Cleve et Grun.) V. H., were selected for ultrastructure investigations on the basis of their ready availability. Specimens were examined employing both transmission and scanning electron microscopy. Strutted processes, labiate processes, spines and other special features were surveyed in all species. On the basis of these observations the species of Cyclotella observed seem to be in one of 3 morphological groups, the meneghiniana group, the comta group, and the stelligera group.     

GROWTH RESPONSES OF THE DIATOM,CYCLOTELLA CRYPTICA (BACILLARIOPHYCEAE), TO GIBBERELLIC ACID1

The plant hormone, gibberellic acid (GA), stimulated growth of a marine diatom, Cyclotella cryptica Reimann, Lewin and Guillard. Four concentrations of GA (5 × 20 × 25 × and 35 × 10^?6 g/mL) were added to axenic cultures of C. cryptica. Changes in cell densities, measured by cell counts and turbidimetric readings, confirmed that GA at 20 × 10^?6 g/mL produced maximum stimulation. There was an increase in the total number of cells produced and a shorter lag phase of growth at this concentration. Coulter counter measurements of cell size, as well as ocular micrometer measurements, indicated there was no significant variation in cell volumes of GA grown cells over that of the controls.     

COPPER SORPTION AND RELEASE BY CYCLOTELLA MENEGHINIANA (BACILLARIOPHYCEAE) AND CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Initial Cu⁺⁺ sorption by Cyclotella meneghiniana Kütz. (Cu⁺⁺-sensitive) and Chlamydomonas reinhardtii Dangeard (Cu⁺⁺-resistant) was rapid in the first 5 min of Cu⁺⁺ incubation with little sorption after 2 h. On a cell to cell basis, Cyclotella sorbed ca. five times more Cu⁺⁺ from the medium than Chlamydomonas. In MBL medium with EDTA Cyclotella and Chlamydomonas cells sorbed 21.0 and 4.41 nM Cu⁺⁺/10⁶ cells respectively in 6 h with 0.3 mg Cu⁺⁺/l in the medium. Proportionally similar quantities of Cu⁺⁺ were sorbed when the cells were Cu⁺⁺ incubated in MBL + citrate or filtered lake water. Cleaned cell walls of Cyclotella sorbed little Cu⁺⁺ (1.7 nM/10⁶ cells) as compared to living cells (17.5 nM Cu⁺⁺/10⁶ cells) in 3 h. Therefore, in living Cyclotella most of the Cu⁺⁺ taken up must be absorbed by the protoplasm or perhaps by the organic layer surrounding the silica wall. Cleaned cell walls of Chlamydomonas sorbed 3.5 nM Cu⁺⁺/10⁶ cells and living Chlamydomonas cells sorbed 2.6 nM Cu⁺⁺/10⁶ cells. This indicates that most of the Cu⁺⁺ sorbed by Chlamydomonas cells remained bound to the cell wall and probably did not readily enter into the protoplasm: When placed in Cu⁺⁺ free medium after Cu⁺⁺ incubation, Cyclotella and Chlamydomonas cells released 46 and 59% respectively of the Cu⁺⁺ sorbed.     

EFFECTS OF SILICON DEFICIENCY ON LIPID COMPOSITION AND METABOLISM IN THE DIATOM CYCLOTELLA CRYPTICA1

The effects of silicon deficiency on the metabolism and composition of lipids in Cyclotella cryptica T13L Reimann, Lewin, and Guillard were examined. Silicon-deficient cells had higher levels of neutral lipids (primarily triacylglycerols) and higher proportions of saturated and monounsaturated fatty acids than silicon-replete cells. After 4 h of silicon deficiency, the percentage of newly assimilated NaH¹⁴CO₃ partitioned into lipids increased from 27.6% to 54.1%, whereas the percentage partitioned into chrysolaminarin decreased from 21.6% to 10.6%. In addition, pulse-chase experiments with NaH¹⁴CO₃ indicated that the amount of ¹⁴C in the total cellular lipid fraction increased by 32% after 12 h of silicon deficiency despite the absence of additional photoassimilable ¹⁴C. Therefore, the accumulation of lipids in response to silicon deficiency appears to be due to two distinct processes: (a) an increase in the proportion of newly assimilated carbon partioned into lipids, and (2) a slow conversion of previously assimilated carbon from non-lipid compounds into lipids     

AUTOGAMOUS AUXOSPORULATION IN PINNULARIA NODOSA (BACILLARIOPHYCEAE)1

Auxosporulation of the freshwater epipelic diatom Pinnularia nodosa (Ehrenb.) W. Sm. was studied in a clonal culture. Interphase cells possessed two chloroplasts with invaginated pyrenoids. The nucleus contained a single small body of heterochromatin at one end, also visible during most of meiotic prophase. During auxosporulation, induced by transfer of stationary‐phase cells to fresh medium and suppressed by high nitrogen (N), an unpaired mother cell produced a single auxospore. Although meiosis II and nuclear fusion were not observed, indirect evidence indicated that auxosporulation was autogamous (rarely reported in pennate diatoms), rather than apomictic; paedogamy was excluded. The protoplast produced after meiosis either (1) matured into a “pseudozygote,” via an asymmetrical contraction after meiosis I to form a single spherical cell at one end of the mother cell (pathway 1); or (2) constricted into two spherical cells (pathway 2). In pathway 2, the “pseudogametes” never fused and only one or none developed into a pseudozygote and then into an auxospore. Pathway 2 could be suppressed by continuous light. During metamorphosis of the spherical pseudozygote into an elongate young auxospore, a complete covering of thin siliceous incunabular strips was formed, separate from the organic wall formed around the pseudozygote when first formed and from the perizonium. Mature auxospores produced via pathway 2 had 60% of the volume as those produced via pathway 1 and had smaller chloroplasts (through loss of fragments during protoplast cleavage), but they achieved exactly the same lengths, suggesting that absolute length is monitored during expansion.     

CORRELATED EVOLUTION OF GENOME SIZE AND CELL VOLUME IN DIATOMS (BACILLARIOPHYCEAE)1

A correlation between genome size and cell volume has been observed across diverse assemblages of eukaryotes. We examined this relationship in diatoms (Bacillariophyceae), a phylum in which cell volume is of critical ecological and biogeochemical importance. In addition to testing whether there is a predictive relationship across extant species, we tested whether evolutionary divergences in genome size were correlated with evolutionary divergences in cell size (using independent contrasts). We estimated total DNA content for 16 diatom species using a flow cytometer and estimated cell volumes using critical dimensions with scaling equations. Our independent contrast analyses indicated a significant correlated evolution between genome size and cell volume. We then explored the evolutionary and ecological implications of this evolutionary relationship. Diatom cell volume is an important component of the global carbon cycle; therefore, understanding the mechanisms that drive diatom genome evolution has both evolutionary and ecological importance.     

THE NATURAL LIFE CYCLE IN WILD POPULATIONS OF DIATOMA MONILIFORMIS (BACILLARIOPHYCEAE) AND ITS DISRUPTION IN AN ABERRANT ENVIRONMENT1

We studied how size variation in populations of Diatoma moniliformis Kütz. was influenced by environmental effects on the diatom life cycle. One of the two populations sampled monthly in the northern Baltic Sea grew under natural conditions; the other population was in a cooling water discharge channel of a nuclear power plant, where the temperature and flow rate of the water were artificially higher. The life cycle was synchronous at the natural site, with sexual reproduction occurring in the winter; most of the initial cells were found in March-April. After this, a reduction in cell size occurred, and the vegetative life cycle consisted of two parts. During the first part, cell volume decreased, whereas the surface area to volume ratio increased, and during the second part of the cycle, both of these parameters decreased. No direct evidence was found for the existence of a supra-annual life cycle in D. moniliformis, as convincing modes fm large cells were lacking in the size-frequency distributions. It was concluded from extrapolations of the data that the natural life cycle of D. moniliformis probably lasts 2 or 3 years. The changes in cell proportions during the life cycle fit well with annual growth cycles of D. moniliformis at the natural site (i.e. the cells had high surface area to volume ratios during the period of optimal growth in late spring [May-June]). At the site affected by cooling water discharge, the synchronization of the natural life cycle was disrupted, but some seasonal size variation did occur. Under natural conditions, auxosporulation is probably triggered by a combination of small cell size, low water temperature (0–3° C), and rapidly increasing light intensity or daylength in late winter to early spring. When these conditions were not met (e.g. at the heated site, the required low temperature was absent), auxosporulation did not occur simultaneously. This paper also presents scanning electron photomicrographs showing the typical shape and fine structure of the initial cell of D. moniliformis. These cells are semispherical in cross section, possess a pronounced curvature along the longitudinal axis, and are bent in the perualvar plane.     

UPTAKE OF ORGANIC SUBSTRATES BY CYCLOTELLA CRYPTICA (BACILLARIOPHYCEAE): EFFECTS OF IONS,IONOPHORES AND METABOLIC AND TRANSPORT INHIBITORS1,2

The uptake of glucose and amino acids by the euryhaline diatom Cyclotella cryptica Reimann, Lewin & Guillard does not appear to be related to proton gradients. Instead, the transport systems for these organic solutes show a strong requirement for the presence of NaCl. The relationship between uptake and NaCl concentration is hyperbolic, with optimal uptake rates being approached at 100 mM NaCl. High concentrations of KCl cause strong reductions in uptake rates. The (Na⁺, K⁺)-stimulated ATPase inhibitor ouabain has no effect on glucose uptake, whereas the diphenolic glucoside phlorizin and its aglucone phloretin are strongly inhibitory. The proton translocating uncoupler CCCP (carbonylcyanide m-chlorophenyl hydrazone) and the ATPase inhibitor DCCD (dicyclohexylcarbodiimide) both almost completely abolish glucose transport, and low concentrations of the ionophares monensin and valenomycin strongly inhibit glucose uptake by the diatom. The requirement of high external NaCl concentrations for glucose transport, and the inhibitory effect an transport of the Na⁺-specific ionophore monensin are consistent with a coupling of Na⁺ and organic substrate transport, but could also be explained by a Na⁺ requirement for glucose binding to a transport carrier, and/or a possible interference with energy producing reactions associated with a monensin-induced collapse of the normal Na⁺ gradient.     

EFFECT OF SILICATE LIMITATION ON VALVE MORPHOLOGY IN THALASSIOSIRA AND COSCINODISCUS (BACILLARIOPHYCEAE)1

Coscinodiscus radiatus Ehrenb. and Thalassiosira eccentrica (Ehrenb.) Cleve were grown in a silicate-limited chemostat at silicate concentrations below 1 μg-atoms · l^?1. The resulting abnormal valves of C. radiatus lacked a thickened ring around the foramina; their pore membranes were thinner and their loculi shallower than those in normal cells. Abnormal valves of T. eccentrica had a fasciculate areolae pattern; they lacked a silica covering over the foramina and some tangential areolae walls. Neither abnormal valve could be termed a new species.     

SEXUALITY, INCOMPATIBILITY, SIZE VARIATION, AND PREFERENTIAL POLYANDRY IN NATURAL POPULATIONS AND CLONES OF SELLAPHORA PUPULA (BACILLARIOPHYCEAE)

The capitate and rectangular demes of the freshwater epipelic diatom Sellaphora pupula (Kütz.) Mereschk. are dioecious, the first such report for any freshwater diatom. Sexual differentiation, which is probably determined genetically, involves recognition at the cell surface as well as differences in gamete behavior (one gametangium produces an active "male" gamete, the other a passive "female" gamete). In culture, successful sexual reproduction occurs only when compatible clones are mixed. All cells of a clone behave identically in interclonal crosses, being either male or female, regardless of the stage of the life cycle, in contrast to the sequential hermaphroditism of centric diatoms. Males and females have identical frustule morphology. As in other diatoms, there is an upper size threshold for sexual reproduction, below which cells become progressively easier to sexualize. In culture, sexual interactions occur in cells much smaller than those ever seen in natural populations, so that in nature the sexual size range is effectively open. Natural populations almost always contain sexualizable cells; often, most of the cells are below the upper sexual size threshold. Male gametangia are, on average, slightly larger than females in the capitate deme, which may be produced by preferential polyandry, depleting the population of males and making them younger at mating. Rarely, selfing occurs producing zygotes, but these abort before producing initial cells. The sizes of the gametangia and initial cells are correlated but this does not invalidate the use of "cardinal points" of the life cycle in taxonomy. No interbreeding occurs between the rectangular and capitate demes. However, when males of one deme are mixed with females of the other, there is a stimulation of activity, as during the early stages of pairing in compatible intrademic crosses.     

PHYSIOLOGICAL AND ULTRASTRUCTURAL RESPONSES OF CYCLOTELLA MENEGHINIANA (BACILLARIOPHYTA) TO LIGHT INTENSITY AND NUTRIENT LIMITATION1

Photosynthetic characteristics and chloroplast ultrastructure of Cyclotella meneghiniana Kütz. were quantified while the organism was simultaneously adjusting to light and nutrient stress. Cells were grown in batch culture at either low or high light intensity on medium with a nitrogen/phosphorus molar ratio of 2:1 as a control, or with nitrogen or phosphorus deleted from the medium to create nutrient deficiencies. Analysis of variance indicated that light intensity, nutrient deficiency and duration of nutrient deficiency all had significant effects on cell growth, chlorophyll (Chl) concentration/cell, cellular fluorescence capacity (CFC), chloroplast volume and thylakoid surface density. Because interactions existed among nutrient deficiency, extent of nutrient deficiency, and light intensity, all three must be considered together in order to describe accurately the physiology and chloroplast ultrastructure of the diatom. Significant correlations were found between the Chl/cell or CFC/cell and chloroplast volume and thylakoid surface density. Through an increase in Chi concentration, chloroplast volume and thylakoid surface density, the cells successfully adapted to the conditions of low light intensity even while under nutrient stress. In contrast, less Chl/cell, smaller chloroplast volume and less thylakoid surface density were found at high light intensity.     

FIVE NEW GOMPHONEMA SPECIES (BACILLARIOPHYCEAE) FROM RIVERS IN SOUTH AFRICA AND SWAZILAND1

Diatom material from South Africa and Swaziland was examined with light and scanning electron microscopy. Five new taxa are proposed: Gomphonema crocodilei, G. quasicrocodilei, G. venusta, G. latistigmata, and G. cholnokyi. Gomphonema venusta has been previously misidentified as Gomphonema clevei Fricke and G. cholnokyi as G. subclavatum (Grunow) Grunow. All of the species possess biseriate striae, areolae with external vela, a true stigma, and marginal laminae underlying the valve margin and mantle. Compared to the other members of gomphonemoid lineage, the five new species show greatest similarity to Gomphoneis mesta Passy-Tolar & Lowe, G. magna Kociolek & Stoermer, and G. rhombica (Fricke) Merino, García, Hernández-Mariné, & Fernández and to the Herculeana lineage of Gomphoneis sensu Kociolek and Stoermer. The most recent phylogenetic analysis of the genus Gomphoneis showed that the two lineages of the genus. Herculeana and Elegans, are not monophyletic. The Herculeana lineage is more closely allied to Gomphonema than to the Elegans lineage. Since the Elegans lineage contains the generitype of Gomphoneis (G. elegans (Grunow) Cheve), and the species described here are more closely related to the Herculeana lineage, we have assigned them to Gomphonema.     

TEMPERATURE-DEPENDENT CHANGES IN COLONY SIZE OF THE FRESHWATER PENNATE DIATOM ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE) AND THEIR POSSIBLE ECOLOGICAL IMPLICATIONS1

Changes in colony size (cell number per colony) of Asterionella Formosa Hass. were experimentally evaluated in relation to water temperature using two types of clones having colony sizes of four or eight cells. The clones were isolated from two different temperate freshwater lakes. Both clones showed the same general trend with changing temperature. Most of the colonies were normal in size at low temperatures, but colony size was twice as large at high temperatures. Variable colony sizes were present at low percentages. Colony separation occurred at the oldest connection within the colony after cell division. Culture experiments showed that the rates of specific growth and colony separation were balanced except for a rather short period of time when the temperature was changed. Optical and scanning electron micrography did not show any distinctive morphological structure at the point of connection except for porelli and mucilage pads. Seasonal changes in colony size of A. formosa observed in a freshwater lake are discussed based on these temperature results.     

CARBONIC ANHYDRASE IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

The zinc metalloenzyme carbonic anhydrase plays a critical role in inorganic carbon acquisition in marine diatoms, thus conferring on zinc a key role in oceanic carbon cycling. As a first step in determining the location and function of carbonic anhydrase (CA) in Bacillariophyceae, we purified and partially sequenced CA from T. weissflogii (Gru) Fryxell et Hasle (TWCA1) and cloned the corresponding cDNA (twca1). The twca1 sequence is different from other known algal carbonic anhydrase genes, and encodes a protein of roughly 34 kDa. The amino terminal amino acids sequenced from purified TWCA1 are 72 residues downstream of the putative starting methionine predicted by twca1. This difference may be due to the presence of a short-lived signal sequence designed to guide the enzyme to the correct cellular location. The absence of any homology between TWCA1 and previously sequenced CAs from Chlorophyceae may indicate either convergent evolution or that carbon acquisition represents a fundamental physiological difference among algal phyla.     

MODULATION OF THE HEAT SHOCK UBIQUITIN POOL IN SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

Immunoblotting experiments performed with an anti-ubiquitin antibody revealed that Skeletonema costatum (Grev.) Cleve cells contained free ubiquitin as well as ubiquitin conjugated to various endogenous proteins. A temperature shift from 18° to 30°C greatly increased the total amount of ubiquitin and particularly the ubiquitin fraction in high molecular mass conjugates. A solid-phase immunoassay indicated values of 0.031 ± 0.004 pmol·10^?6 cells for free ubiquitin and 0.046 ± 0.004 pmol·10^?6 cells for conjugated ubiquitin for cells grown at 18°C, and 0.056 ± 0.008pmol·10^?6cells and 0.21 ± 0.03 pmol·10^?6cells, respectively, after a temperature increase from 18° to 30°C. Cell-free extracts of S. costatum were equally able to form thiol ester linkages with ¹²⁵I-ubiquitin in an adenosine triphosphate–dependent manner at 18° C and at 30°C. Cell-free extracts were also able to conjugate ¹²⁵I-ubiquitin to endogenous proteins, but the ubiquitin conjugation rate at 30°C was lower than at 18°C. Incubation of S. costatum for 3 h at 30°C and then for 3 h at 18°C resulted in the formation of high amounts of ubiquitin conjugates, suggesting that partially inactive or denaturated proteins accumulate during heat stress. These denaturated proteins are then conjugated to ubiquitin very efficiently when the physiological temperature is restored. Thus, S. costatum cells contain ubiquitin and an active ubiquitin conjugation system responding to stress conditions (temperature stress). The intracellular concentration of ubiquitin conjugates is most likely limited by the availability of protein substrates to be conjugated rather than by ubiquitin-conjugating activity.     

QUANTITATIVE GENETICS OF FATTY ACID VARIATION IN ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Fatty acid variation among culture collection strains and 40 new isolates of Isochrysis galbana Parke was analyzed by quantitative genetic methods. Fatty acid variation among strains and among isolates was highly significant for major fatty acids showing the existence of a genetic component in the determination of differences in fatty acid content. The heritabilities for the major fatty acids ranged between 0.68 and 0.99 among collection strains and between 0.31 and 0.43 among isolates. Eicosapentaenoic acid (EPA) had the highest heritability in I. galbana, but the majority of remaining fatty acids also showed high heritability values. A similar experiment with five UTEX strains of Phaeodactylum tricornutum also showed the presence of a genetic component in four out of seven major fatty acids. Nevertheless, the UTEX strains did not differ significantly in EPA content, although they showed a heritability of 0.40 for this fatty acid. An additional experiment culturing the same isolates of I. galbana in larger volumes of media showed that there was a high significant positive linear relation between EPA content in different volumes. Therefore, EPA content in small volume cultures was an unbiased indicator of EPA content in larger volume cultures. Our results provide support for the genetic determination of fatty acid content in microalgae and suggest that selection, and mutation and selection, are likely to improve EPA content in I. galbana and probably in many other microalgae. Such a selection program can be carried out in small-volume cultures with high confidence.     

TAXONOMY,LIFE CYCLE,AND AUXOSPORULATION OF NITZSCHIA FONTICOLA (BACILLARIOPHYTA)1

Nitzschia fonticola (Grunow) Grunow is a member of Nitzschia sect. Lanceolatae, a group of taxonomically intractable but ecologically important and widespread diatoms. We investigated the morphology and life cycle in three clones of N. fonticola and all exhibited reduced sexuality, with pedogamous production of auxospores in unpaired gametangia. The auxospores of all clones contained tangles of striplike elements that lay outside the perizonium and were distinct from it in structure and ontogeny. We introduce a new term, incunabula, to refer to such components of the auxospore wall. Semicryptic variation was detected: one clone differed from the other two in valve size and shape, stria density, and fibula density, as well as its nuclear large subunit ribosomal DNA (LSU rDNA) sequence. The implications of reduced sexuality for the taxonomy of sect. Lanceolatae are discussed. A lectotype is designated for N. fonticola from among original material of Grunow, and the application of the name is clarified further by designating illustrations and the LSU sequence AM182191 from one of our clones as epitypes.     

PHOSPHATE AND SILICATE GROWTH AND UPTAKE KINETICS OF THE DIATOMS ASTERIONELLA FORMOSA AND CYCLOTELLA MENEGHINIANA IN BATCH AND SEMICONTINUOUS CULTURE1

Information on the nutrient kinetics of Asterionella formosa Hass. and Cyclotella meneghiniana Kutz. under either phosphate or silicate limitation was obtained for use in a Monod model and in a variable internal stores model of growth. Short-term batch culture growth experiments were fit to the Monod model and long-term semicontinuous culture experiments and short-term uptake experiments were fit to the variable internal stores model. Mathematical analysis indicates that the parameters of the 2 models may be expressed in terms of each other at steady state. The qualitative results of both batch and steady state culture methods agree. For limiting phosphate experiments. A. formosa is better able to grow at low PO₄-P concentrations than C. meneghiniana, as shown by its lower K for PO₄-P limited growth. The k_Q of A. formosa compared to C. meneghiniana found in long-term semicontinuous culture indicates that A. formosa is almost an order of magnitude more efficient at using internal phosphate for growth. The qualitative results under silicate-limited growth of C. meneghiniana is less than that of A. formosa. The k_Q from semicontinuous culture experiments indicates that C. meneghiniana is the more efficient at using internal silicate for growth. Nutrient uptake experiments showed more variability from a Michaelis-Menten relationship than short-term growth experiments. There were no significant differences between the 2 species in half saturation constants for either phosphate or silicate uptake. We observed a marked dependence of the coefficient of luxury consumption (R) of phosphate on the steady state growth rate. A. formosa has a higher R than C. meneghiniana.     

PSEUDO-NITZSCHIA PUNGENS AND P. MULTISERIES (BACILLARIOPHYCEAE): NOMENCLATURAL HISTORY,MORPHOLOGY, AND DISTRIBUTION1

Pseudo-nitzschia pungens f: multiseries is raised in rank from form to species based on morphological, physiological, and genetic features. Distinctive details of the valve face striae, the valve mantle, and the girdle of P. pungens and P. multiseries are outlined. The nomenclatural history and the distribution of the two species and their relationship to other species of the genus are discussed.