SOME ASPECTS OF MORPHOLOGICAL VARIATION IN STEPHANODISCUS NIAGARAE (BACILLARIOPHYCEAE)1

Valves of S. niagarae var. niagarae Ehr. and S. niagarae var. magnifica Fricke from geographically dispersed sediment and plankton collections were observed by light microscopy and scanning electron microscopy (LM and SEM). Measurements made by LM can be arranged so that means and ranges of diameter, areolar density, or strial density intergrade from one population into the next. Mean diameter is negatively related to increasing areolar and strial densities. No unique features observable by SEM distinguish the two described varieties. Thus, S. niagarae var. magnifica, having large diameter valves with low areolar and strial densities, may represent one end of a trend in overall variation in S. niagarae. At the opposite extreme are the populations with small valves and high areolar densities which are often erroneously referred to as S. astraea. Type material of S. niagarae lies intermediate to these forms. Three populations considered in this study have distinct morphological characteristics. Valves from Yellowstone Lake sediments have spine placements distinctly different from valves of all other populations. Specimens from Lake Superior have nearly flat central areas. Ribs of valves from Grand Traverse Bay (Lake Michigan) are covered with granules.     

MORPHOLOGICAL VARIATION OF THE ATTACHED DIATOM: COCCONEIS SCUTELLUM VAR. SCUTELLUM: (BACILLARIOPHYCEAE)1

The morphology and sexual reproduction of the attached diatom Cocconeis scutellum Ehr. var. scutellum have been investigated using field and culture materials. The diatom grew well at 14–22° C and in a medium using enriched full strength seawater or 50% diluted seawater. One gamete was formed without cytokinesis in a gametangium and one auxospore arose from paired gametangia. The size of gametangia and initial cells were 17.8–29.9 μm and 43.3–59.5 μm, respectively. Valve width decreased linearly with decreasing valve length. The regression coefficient between them was invariable with the change of the environment. The transapical striation densities were 7–12 and 7.5–13.5 in 10 μm in araphid and raphid valves, respectively. The density showed a tendency to increase with decreasing valve length in both valves. The densities in both valves, except the small araphid valve, are invariable with the change of the environment. The relationships between valve length and width, and between valve length and striation density have been compared with those of taxa allied to C. scutellum var. scutellum. It is concluded that C. scutellum var. scutellum, as usually delimited, is a heterogenous taxon.     

NITZSCHIA OVALIS (BACILLARIOPHYCEAE) MONO LAKE STRAIN ACCUMULATES 1,4/2,5 CYCLOHEXANETETROL IN RESPONSE TO INCREASED SALINITY1

The growth of microalgae in hypersaline conditions requires that cells accumulate osmoprotectants. In many instances, these are polyols. We isolated the diatom Nitzschia ovalis H. J. Arn. from the saline and alkaline water body Mono Lake (CA, USA). This isolate can grow in salinities ranging from 5 to 120 parts per thousand (ppt) of salt but normally at 90 ppt salinity. In this report, we identified the major polyol osmoprotectant as 1,4/2,5 cyclohexanetetrol by electron ionization‐mass spectrometry (EI–MS), ¹H, ¹³C nuclear magnetic resonance spectroscopy (NMR), and infrared (IR) and showed an increase in cellular concentration in response to rising salinity. This increase in the cyclitol concentration was evaluated by gas chromatography of the derived tetraacetylated cyclohexanetetrol obtaining an average of 0.7 fmol · cell^?1 at 5 ppt and rising to 22.5 fmol · cell^?1 at 120 ppt. The 1,4/2,5 cyclohexanetetrol was also detected in the red alga Porphyridium purpureum. Analysis of the free amino acid content in N. ovalis cultures exposed to changes in salinity showed that proline and lysine also accumulate with increased salinity, but the cellular concentration of these amino acids is about 10‐fold lower than the concentration of 1,4/2,5 cyclohexanetetrol. The comparison of amino acid concentration per cell with cyclitol suggests that this polyol is important in compensating the cellular osmotic pressure due to increased salinity, but other physiological functions could also be considered.     

EFFECT OF SALINITY ON PSEUDO‐NITZSCHIA SPECIES (BACILLARIOPHYCEAE) GROWTH AND DISTRIBUTION1

Salinity varies widely in coastal areas that often have a high abundance of Pseudo‐nitzschia H. Peragallo. Pseudo‐nitzschia is abundant in Louisiana waters, and high cellular domoic acid has been observed in natural samples but no human illness has been reported. To assess the threat of amnesic shellfish poisoning (ASP), we examined the effect of salinity on Pseudo‐nitzschia occurrence in the field and growth in the laboratory with special emphasis on the salinity range where oysters are harvested (10–20 psu). In Louisiana coastal waters, Pseudo‐nitzschia spp. occurred over a salinity range of 1 to >35 psu, but they occurred more frequently at higher rather than lower salinities. Seven species were identified, including toxigenic species occurring at low salinities. In culture studies, seven clones of three species grew over a salinity range of 15 to 40 psu, some grew at salinities down to 6.25 psu, and most grew at salinities up to 45 psu. Tolerance of low salinities decreased from Pseudo‐nitzschia delicatissima (Cleve) Heiden to P. multiseries (Hasle) Hasle to P. pseudodelicatissima (Hasle) Hasle emend. Lundholm, Hasle et Moestrup. In conclusion, although Pseudo‐nitzschia was more prevalent in the field and grew better in the laboratory at higher salinities, it grew and has been observed at low salinities. Therefore, the probability of ASP from consumption of oysters harvested from the low salinity estuaries of the northern Gulf of Mexico is low but not zero; animal mortality events from toxin vectors other than oysters at higher salinity on the shelf are more likely.     

A MORPHOLOGICAL AND TAXONOMIC CONSIDERATION OF TABELLARIA (BACILLARIOPHYCEAE) FROM THE NORTHCENTRAL UNITED STATES1,2

The taxonomy, distribution, and ecology of the diatom genus Tabellaria Ehr. from a portion of the northcentral United States were studied from collections made from August 1968 to October 1971. Using the transmission electron microscope with carbon replicas of the frustule surfaces, taxonomically significant characters were identified and related to those visible with the light microscope, four morphologically distinguishable taxa were recognized. The frustular morphology of T. fenestrata (Lyngbye) kütz. em. Knudson var. fenestrata was confirmed by both light and electron microscopy. T. flocculosa (Roth) Kütz. var. flocculosa is composed of 3 morphologically distinguishable strains. One of which is sufficiently different from the nominate variety to be described as T. flocculosa var. linearis var. nov. Also, a planktonic ecotype of one of the strains was identified.     

INFLUENCE OF SALINITY AND SULFATE ON THE TOXICITY OF CHROMIUM(VI) TO THE ESTUARINE DIATOM THALASSIOSIRA PSEUDONANA1

The acute toxicity of Cr(VI) to the diatom Thalassiosira pseudonana (Hasle and Heimdal) clone 3H was determined in artificial media of 3.2 and 0.32 ppt salinity and with variations of sulfate concentration in the media independent of salinity. Inhibitory concentrations of Cr(VI) ranged from 6.6 μM for growth rate and 4.9 μM for cell yield at 3.2 ppt salinity and 2.8 μM sulfate to 0.04 μM for growth rate and 0.02 μM for cell yield at 0.32 ppt salinity and 0.019 mM sulfate. The inhibition by Cr(VI) was a function of the ratio of Cr(VI) to sulfate. Inhibition occurred when-this ratio exceeded about 500:1. It is suggested that the mechanism for the toxicity of Cr(VI) to diatoms and perhaps other aquatic organisms involves a site at which sulfate and chromate compete.     

PHOTOSYNTHETIC PIGMENT ORGANIZATION IN DIATOMS (BACILLARIOPHYCEAE)1

The isolation and characterization of six pigment-protein complexes from five diatom species (Phaeodactylum tricornutum Bohlin, Chaetoceros gracilis Schutt, Nitzschia sp. Mono Lake, Nitzschia laevis Hust. and Thalassiosira pseudonana (Hust.) Hasle and Heimdal) was accomplished by membrane dissociation with digitonin followed by gel electrophoresis. Six analogous complexes obtained from all species were correlated in spectral characteristics and relative mass with complexes from higher plants obtained by the same procedure. The largest of these complexes, comprising about 15% of the total Chl a, contained reaction centers of Photosystem I (PSI) and antenna pigments (LHC₁).Some PSI complexes also separated from LHC₁ in the gel. For the first time in diatoms, a Photosystem II complex was isolated and identified from its position in the gels, absorption and fluorescence spectra, lack of P700, and enrichment carotene. Three antenna pigment-protein complexes in addition to LHC₁ occurred in varying proportion under different experimental conditions but in sum, they accounted for 70% of the total Chl a. All three bands were highly enriched in Chl c and fucoxanthin, although the ratio of Chl c/ xanthophyll decreased between the slowest migrating LHC₂ and fastest moving LHC₄ LHC₃ contained the highest proportio of pigment-protein and was composed primarily of polypeptides of about 18,000 D. Essentially all α- and β-carotene was bound to the reaction center complexes. The Nitzschia from Mono Lake differed from the other species in that PSI complexes could not be readily dissociated from its membrane by digitonin treatment, a characteristic which may reflect a different chloroplast membrane structure in this alga.     

DIVERSITY IN THE GENUS SKELETONEMA (BACILLARIOPHYCEAE): III. PHYLOGENETIC POSITION AND MORPHOLOGICAL VARIABILITY OF SKELETONEMA COSTATUM AND SKELETONEMA GREVILLEI,WITH THE DESCRIPTION OF SKELETONEMA ARDENS SP. NOV.1

Skeletonema costatum (Grev.) Cleve emend. Zingone et Sarno and S. grevillei Sarno et Zingone were known only from the type material collected from Hong Kong waters more than a century ago. Both species have now been collected as live material, and their morphology and phylogenetic position are investigated in this study. Eight Skeletonema strains isolated from Florida, USA; Uruguay; and Brazil are attributed to S. costatum, while one strain from Oman is ascribed to S. grevillei based on morphological similarity to the type material of these species. In addition, a new Skeletonema species, S. ardens Sarno et Zingone, is described for a strain from Singapore and two from northern Australian waters. Skeletonema ardens has terminal fultoportula processes ending in a tapered, undulate protrusion and long intercalary fultoportulae with 1:1 junctions. The rimoportula of terminal valves is located at the margin of the valve face. No major morphological variations were observed within S. grevillei and S. ardens along a salinity gradient, whereas in S. costatum, the processes shortened and the valves came into close contact at low salinities, as already described for S. subsalsum (Cleve) Bethge. Consistent with their morphology, Skeletonema costatum and Skeletonema subsalsum also had similar rDNA sequences. Skeletonema grevillei and S. ardens were distinct in the large subunit (LSU) phylogeny. Skeletonema ardens exhibited consistent intraspecific genetic differences in both the LSU and small subunit (SSU) rDNA.     

CELL ENLARGEMENT IN SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

Skeletonema costatum (Grev.) Cl. exhibits an asexual means, of increasing cell size that is more common than auxosporulation. This phenomenon accounts for the genetic stability of S. costatum in culture and for the deficiency of heterozygotes in natural populations, and has important implications far the life history of this species.     

OCCURRENCE OF THREE NITZSCHIA (BACILLARIOPHYCEAE) TAXA WITHIN COLONIES OF TUBE-FORMING DIATOMS1

In benthic samples from the unchannelized Missouri River, the diatoms Nitzschia dissipata (Kütz.) Grun., N. filiformis (W. Sm.) Schütt and N. pseudofonticola Hust. were observed within the mucilage tubes of four tube-forming diatoms: Cymbella prostrata (Berk.) Cl., C. prostrata var. auerswaldii (Rabh.) Reim., Navicula tripunctata var. schizonemoides (V.H.) Patr., and Nitzschia filiformis. Microscopical observations of live and preserved specimens indicated that “invasion” by Nitzschia occurred primarily in older tubes. Data are presented on the environmental conditions in which the tube-formers and their cohabitants have been found.     

OCCURRENCE OF THREE NITZSCHIA (BACILLARIOPHYCEAE) TAXA WITHIN COLONIES OF TUBE-FORMING DIATOMS1

In benthic samples from the unchannelized Missouri River, the diatoms Nitzschia dissipata (Kütz.) Grun., N. filiformis (W.Sm.) Schütt and N. pseudofonticola Hust. were observed within the mucilage tubes of four tube-forming diatoms: Cymbella prostrata (Berk.) Cl., C. prostrata var. auerswaldii (Rabh.) Reim., Navicula tripunctata var. schizonemoides (V.H.) Patr., and Nitzschia filiformis. Microscopical observations of live and preserved specimens indicated that “invasion” by Nitzschia occurred primarily in older tubes. Data are presented on the environmental conditions in which the tube-formers and their cohabitants have been found.     

THE INLAND CHAETOCEROS (BACILLARIOPHYCEAE) SPECIES OF NORTH AMERICA1

Five taxa of Chaetoceros occur in inland waters of North America. These most commonly occur in waters with elevated total dissolved solids in arid regions of the western United States and Canada. Chaetoceros amanita Cleve-Euler is characterized by consistently forming relatively long chains of cells and having very spinose primary resting spore valves. Chaetoceros elmorei Boyer also forms long chains of cells which are connected by evident valvar processes; spores are nearly always smooth. Chaetoceros muelleri Lemm. may form short chains with processes between sibling valves, but also produces solitary cells lacking processes. Chaetoceros muelleri var. subsalsum (Lemm.)Johansen et Rushforth is similar to the nominate but never produces cells with Processes. Both of the C. muelleri varieties produce spores with smooth primary valves. Chaetoceros simplex Ostenfeld is characterized by a noncolonial habit, cells lacking processes and the production of resting spores with warty to some what spinose primary valves.     

USING QUANTITATIVE PCR TO DETERMINE THE DISTRIBUTION OF A SEMICRYPTIC BENTHIC DIATOM,NAVICULA PHYLLEPTA (BACILLARIOPHYCEAE)1

Evidence has accumulated during the last decade showing that many established diatom morpho‐species actually consist of several semicryptic or truly cryptic species. As these species are difficult or even impossible to differentiate by microscopic analysis, there is virtually no information on how they behave in natural environments. In this study, we developed a quantitative real‐time PCR (qPCR) assay using TaqMan probes^® targeted to the internal transcribed spacer 1 (ITS1) to assess the spatial distribution and seasonal dynamics of an important component of the microphytobenthos of intertidal sediments. Navicula phyllepta Kützing is a brackish‐marine morpho‐species with a cosmopolitan distribution. Axenic clones of this species were isolated from natural assemblages of benthic diatoms at different intertidal stations in the Westerschelde estuary (The Netherlands). At least two distinct semicryptic species of N. phyllepta were present, as shown by differences in the quantity of DNA per cell, the ITS1 sequences and the copy number of ITS per cell. DNA and chl a concentrations extracted from sediment surface samples were closely correlated, showing that the DNA used for subsequent analysis mostly belonged to the microalgal community. The results of real‐time qPCR from sites throughout the estuary and over several seasons agreed well with microscopic counts. Additionally, the seasonal pattern of the two forms of N. phyllepta showed an overlapping, but unique distribution along the estuary.     

OPTIMAL GROWTH CONDITIONS AND ANTIOXIDATIVE ACTIVITIES OF CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE)1

We isolated the unialgal strain of Cylindotheca closterium (Ehrenb.) Reimann et J. C. Lewin and produced an axenic strain using an antibiotic cocktail of enriched f/2 artificial seawater medium. The optimal growth conditions were estimated under 27 different combinations of temperature, salinity, and nutrients, and mass culture was performed based on the best specific growth conditions. Its antioxidant activities were determined from the extracts of methanol, water, and enzymes (proteases and carbohydrases). The maximum specific growth rate (μ_max) varied from 0.63 to 0.97 · d^?1. The maximum cell density was 7.20 × 10⁴ cells · mL^?1, while the μ_max was 0.82 · d^?1 in culture conditions of 20°C, 30 psu (practical salinity unit), and “F” nutrient concentrations on day 10 of the culture period. The scavenging rates for 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free radical were 72.5% and 69.4% from Viscozyme and methanol extracts, respectively. The enzymatic extracts of C. closterium prepared by the hydrolyses of Amyloglucosidase (AMG) and Viscozyme showed 45.8% and 45.5% nitric‐oxide‐scavenging rates, slightly lower than the activity of alpha‐tocopherol (α‐tocopherol) but similar to butylated hydroxytoluene (BHT). The extract from methanol and water showed 44.8% and 44.4% scavenging rates, statistically similar with BHT. The metal‐chelating activities of the Kojizyme, Alcalase, methanol, Viscozyme, and Neutrase extracts were 67.1, 53.9, 53.2, 52.1, and 50.2 %, respectively, five to six times higher than the commercial antioxidants. The AMG, Viscozyme, and Neutrase extracts showed a remarkable linoleic acid peroxidation inhibition, which was higher than BHT and statistically similar with α‐tocopherol.     

FRUSTULAR MORPHOLOGY AND TAXONOMIC AFFINITIES OF NAVICULA COMPLANATOIDES (BACILLARIOPHYCEAE)1

Live and prepared cells of the marine pennate diatom Navicula complanatoides Hust. were examined with light and electron microscopy. It has narrowly lanceolate valves (26–55 μm long, 4–5 μm wide) and girdles 10–24 μm in depth. Striae are parallel at the center of the valve (24–28 in 10 μm), becoming slightly convergent toward the apices. Electron microscopy revealed that the external valve surface presents a longitudinally ribbed appearance (20–28 parallel ribs at its maximum width), whereas internally, rectangular areolae are occluded by ricae. The raphe slit lies in a narrow axial area, and one side of the raphe sternum is deeper and folds over the other, obscuring the internal opening. Internally, the central virga on one side of the raphe and two virgae on the other are somewhat broader. A conspicuous pore (stigma) is present between the two broadened virgae. The girdle consists of valvocopulae, copulae, and pleurae. There are 16–20 bands per cingulum. The valvocopulae and copulae are hollow tube-like structures, with inner and outer portions contrsting in morphology. They decrease in diameter in an abvalvar direction. There are four pleurae. These are flat bands which facilitate overlap of the epicingulum and hypocingulum. Fundamental features of the valve and girdle reveal the distinctness of this species within Navicula. The areolae, external longitudinal ribs, and raphe structure suggest affinities with Pleurosigma, Gyrosigma, and Haslea. It is hypothesized that they share a derived state which indicates a recent common ancestor for these taxa. N. complanatoides and related species of the Naviculae microstigmatacae are distinctive enough to merit their own genus within the Naviculaceae.     

DIEL PERIODICITY OF PHOTOSYNTHESIS AND CELL DIVISION COMPARED IN THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

Diel patterns of photosynthesis and cell division were examined in Thalassiosira weissflogii Grun. (clone Actin) grown in nitrogen-limited cyclostat culture. Ammonia (NH₄+) was either supplied continuously or as a daily pulse to cultures grown in constant light or in a light: dark cycle. When either nitrogen or light was supplied periodically, both cell division and photosynthetic capacity were periodic. When both nitrogen and light were supplied periodically, cell division was coupled to the N-pulse whereas periodicities of photosynthetic capacity were modified but remained coupled to the light-dark cycle. Diel oscillations in photosynthesis were i) largely independent of cellular pigmentation and ii) similar for light-limiting and saturating irradiances. Periodicity in photosynthetic capacity also persisted following transfer of non-dividing batch cultures to constant light. Results suggest that photosynthesis but not cell division was coupled to a circadian clock in T. weissflogii. A circadian rhythm of photosynthesis may optimize carbon assimilation in phytoplankton exposed to intermittent nutrient supply by ensuring that maximum photosynthetic capacity occurs during the day.     

ISOLATION AND PHYSIOLOGICAL STUDIES ON THREE ISOLATES OF AMPHORA (BACILLARIOPHYCEAE)1

Three clones of the diatom Amphora were euryhaline, able to grow autotrophically at 160 lx (0.001 ly/min) and heterotrophically on glucose and fructose. Furthermore 2 clones grew on glutamate and feast extract. Light-limited growth of individual clones was stimulated by glycerol, galactose, lactate, acetate, aspartate and asparagine, although mannose torn inhibitory at low and high light levels. The half-saturation constant for growth of A. coffeaefomis var. perpusilla Grunow (Cleve) with glucose was 25 μM. Heterotrophic growth rate of this organism became saturated with respect to glucose at 0.5 mM.