Purification of α-1,4-glucan lyase from red algae and fungi has previously been reported. The α-glucan lyase converts α-glucans to 1,5-anhydro-D-fructose. In this study, an endophyte was detected between the algal cells of Gracilariopsis sp., but not penetrating the cell walls. Histological staining was consistent with the endophyte being fungal and immunohistochemistry revealed that it possessed an α-1,4-glucan lyase differing from that of the host. The endophyte α-glucan lyase was recognized by antisera against both fungal and red algal α-glucan lyases, whereas the α-glucan lyase found in the algal tissue was recognized only by the antisera against algal α-glucan lyase. The lyases occur independently in the host and in the endophyte, and were sometimes detected simultaneously. The endophyte was only observed in Gracilariopsis sp. collected in China; no endophyte could be detected in Gracilariopsis sp. from California or in Gracilaria chilensis.

The development of weighted averaging models for diatoms and lake-water temperature is reviewed in relation to other factors controlling diatom abundance in lakes. It is argued that diatom–temperature models are weaker than those developed for salinity, pH and phosphorus. Moreover, even where a statistically significant model is developed for water temperature, its application over long time periods requires caution. For example, over long time periods (such as the Holocene), soil and vegetation will have changed considerably, altering lake-catchment hydrochemistry and hence the lake biota. Applications of diatom-temperature models in these instances may result in inferred temperatures that are merely artefacts of changing lake-water chemistry. However, palaeolimnological studies using variance partitioning analysis of diatom profiles in sediment cores and independent climate data show that climate signals are recorded in lake sediments. It is suggested, therefore, that the link between changing climate and diatoms be addressed via an alternative method than that of temperature-inference models. The situation demands a more complete understanding of the interactions between lake hydrodynamics and nutrient inputs from catchments over a variety of timescales.

Growth parameters of Mesophyllum alternans and Lithophyllum frondosum were studied in the northwestern Mediterranean. Growth and shrinkage rates, in terms of area gained or lost per month, were measured for both crustose corallines over a 2 year period by photographic and computer-assisted image analysis. No seasonal pattern in growth or shrinkage was found for either species. M. alternans grew and shrank at significantly higher rates than L. frondosum. Growth rates for both species were over an order of magnitude lower than those previously reported for other Mediterranean and tropical species, and were similar to reports for crustose corallines in Arctic and temperate waters. Almost 50% of monitored M. alternans thalli underwent at least one fission or fusion event over the 2 year monitoring period, while in L. frondosum these events were rarely observed. Interspecific differences in rates of growth, shrinkage, fission and fusion appear to depend on differences in thallus thickness (M. alternans is thin and L. frondosum is thick). These differences were interpreted as distinct biological strategies of persistence and the occupation of primary substratum.

Daily integrals of photosynthesis by a cyanobacterial bloom in the Baltic Sea, during the summer of 1993, were calculated from the vertical distributions of light, temperature and the organisms in the water column and from photosynthesis/irradiance curves of picoplanktonic and diazotrophic cyanobacteria isolated from the community. The distribution of chlorophyll a in size-classes < 20 μm and > 20 μm was monitored over 9 days that included a deep mixing event followed by calm. Picocyanobacteria formed 70% of the cyanobacterial biomass and contributed 56% of the total primary production. Of the filamentous diazotrophs that formed the other 30%, Aphanizomenon contributed 28% and a Nodularia-containing fraction 16% of the primary production. For the whole population there was little change in standardized photosynthetic O2 production, which remained at about 31 mmol m−2 before and after the mixing event. There were differences, however, between the classes of cyanobacteria: in picocyanobacteria primary production hardly changed, while in Aphanizomenon it increased by 2·6 and in Nodularia it fell below zero. Total phytoplankton photosynthesis was strongly dependent on total daily insolation with the compensation point at a photon insolation of 22·7 mol m−2 d−1. Similar analyses of N2 fixation showed much less dependence on depth distribution of light and biomass: Aphanizomenon fixed about twice as much N2 as Nodularia their; their fixation exceeded their own N demand by about 12%. Together, these species contributed 49% of the total N demand of the phytoplankton population. Computer models based on the measured light attenuation and photosynthetic coefficients indicate that growth of the cyanobacterial population could occur only in the summer months when the critical depth of the cyanobacteria exceeds the depth of mixing.

Repair of DNA damage induced by ultraviolet-B radiation (UVBR) was investigated in the sublittoral red alga Rhodymenia pseudopalmata at different temperatures, using immunofluorescent detection of thymine dimers. Photoreactivation of thymine dimers was completed within about 3 h at 6, 12 and 18 °C in the presence of ultraviolet-A radiation (UVAR) and photosynthetically active radiation (PAR) but no repair was found in dark-incubated fragments after 16–17 h. In plants previously exposed to a low UVBR dose (biologically effective dose: BEDDNA300 = 1·6 kJ m−2; unweighted irradiance = 0·72 W m−2 for 4 h), photoreactivation started within the first hour under UVAR+PAR at 18 °C but repair only started after 1–2 h at 12 and 6 °C. At 6 °C, repair was more efficient after exposure to a high UVBR dose (BEDDNA300 = 3·9 kJ m−2; unweighted irradiance = 0·78 W m−2 for 5 h) than to a lower dose (BEDDNA300 = 1·6 kJ m−2); no such difference was found at 18 °C. It is concluded that R. pseudopalmata is able to repair DNA damage induced by high UVBR doses efficiently.

The effect of N- and P-limitation on the growth and paralytic shellfish toxin content of Alexandrium fundyense was studied in a series of batch cultures in which the N[ratio ]P supply mass ratio was varied from less than 1 up to 160 (cf. Redfield ≡ 7·2). N- and P-limitation of growth was observed at the lower and higher ratios respectively, with dual limitation between. Cellular parameters were similar during the exponential phase of growth, regardless of external N[ratio ]P ratio. Upon nutrient exhaustion cell yields and internal pools were affected, although these also varied according to the N-source (nitrate vs. ammonium). Generally N-limitation alone led to a higher C[ratio ]N ratio, lower C[ratio ]P and N[ratio ]P ratios and a decreased toxin content in stationary-phase cells. P-limitation led to increased cell size and cell quotas, lower C[ratio ]N ratios, and higher C[ratio ]P and N[ratio ]P ratios (to maxima of 350 and 35 respectively). Complex internal P-fractionation schemes were found unnecessary to determine the P-status of cells and extraction using potassium persulphate, cold water and cold HCl alone were adequate. Severe P-limitation led to increased toxin content but only when N was also limiting, suggesting a synergistic effect of N and P availability on toxin synthesis and turnover. A positive relationship was found between toxin content and the intracellular concentration of arginine, but this varied with the nutrient status (in particular P-status) of the cells.

Bacteria associated with toxic algae in culture have been implicated in the enhancement of algal toxin production and auto-toxigenesis. Toxigenic and non-toxigenic bacterial isolates from Alexandrium spp. have been determined to belong predominantly to the α- and γ- subclasses of the class Proteobacteria. Within these subclasses, the isolates were further classified into two clades that were phylogenetically affiliated with (1) the genus Roseobacter, a newly identified group that is of major significance within the α-Proteobacteria and (2) the genus Alteromonas. Specific 16S rRNA sequence signatures were identified for these clades as well as for three pairs of isolates and for one subcluster within the Roseobacter clade and for three clusters representing eight isolates in the Alteromonas clade. Oligonucleotide probes complementary to these regions were designed and their specificities were tested using dot-blot and whole-cell in situ hybridizations of target and non-target strains. The empirically determined dissociation temperatures of the probes ranged between 55 and 65 making them applicable as a set for screening environmental samples. In situ hybridization of bacteria in cultures of Alexandrium spp. with the clade-specific probes showed a loose association of these bacteria within the phycosphere. In addition, the fluorescent signal of the probes was bright and easily distinguishable from autofluorescent bacteria and the dinoflagellates.

The allogamous raphid diatom Achnanthes longipes C. A. Agardh possesses a complex breeding system involving interactions between three types of clone: monoecious, unisexual and bisexual. Previous studies showed that these three types can be crossed with each other, with a tendency for sexual characteristics to be inherited: inbred monoecious lineages gave rise to monoecious or, very rarely, to bisexual clones, while inbred unisexual lineages yielded unisexual and bisexual clones. The current paper reports on the progeny of crosses between monoecious and unisexual clones and their inbred offspring. All three types of clone appeared in the F1 and F2, although unisexual clones of opposite sex to the parental clone were not found. Inbreeding depression was observed and also a tendency for ‘normal’ auxosporulation (producing two auxospores per pair of gametangia) to be replaced by ‘reduced’ or ‘intermediate’ auxosporulation (producing one auxospore per pair). In addition, patterns of incompatibility were observed that were not seen during earlier studies of clones isolated directly from nature. These included the inability of some F1 clones to mate with each other, in spite of compatibility with all other clones examined (unisexual, bisexual and monoecious).

The organization of F-actin in vegetative cells of the brown alga Sphacelaria rigidula was studied after staining with a modified rhodamine-phalloidin (Rh-Ph) protocol. The interphase vegetative cells display a well-organized F-actin cytoskeleton, consisting of cortical, endoplasmic and perinuclear arrays of actin filaments (AFs). The organization of these AFs changes slightly during mitosis, while they almost disappear at cytokinesis. The perinuclear AF population becomes more obvious during prophase, especially at the poles. At metaphase, an actin spindle is organized, co-localized with the microtubule spindle, while at anaphase an interzonal AF population appears, which persists at early telophase. At advanced telophase the image changes to a rather diffuse actin meshwork in the mid-area between the daughter nuclei. During post-telophase-early cytokinesis this AF system becomes gradually disassembled and a conspicuous actin plate is formed on the cytokinetic plane. This plate becomes more compact with the progress of cytokinesis, and seems to overcoat the patches of the forming diaphragm, and finally the daughter plasmalemmata. Treatment with cytochalasin B disturbs mitosis, not allowing the cells to proceed to anaphase, and prevents cytokinesis. These observations show that AFs are ubiquitous cytoskeletal elements of the vegetative cells of brown algae. The probable role of AFs during the cell cycle and particularly the unique actin configuration involved in cytokinesis are discussed.

The chemical composition, biosynthesis and ultrastructural organization of the cell wall of cultures of Koliella antarctica (Klebsormidiales, Chlorophyta), a green microalga obtained from samples of sea water taken near the Italian Station of Terra Nova (Ross Sea) during the austral summer 1989–1990, have been studied. Purified cell walls of the microalga were sequentially treated with chemicals which, in higher plants, solubilize matrix polysaccharides (pectins and hemicelluloses) and leave an insoluble residue considered to be α-cellulose. CDTA plus Na2CO3- and KOH-solubilized polysaccharides were made up of glucosyl residues (more than 90 mol%), as well as some minor sugars (mannose and rhamnose). Linkage analysis and enzymic hydrolysis of the matrix polysaccharides indicated the presence of β-1,4-linked glucans with minor amounts of other uncharacterized polysaccharides. The insoluble residue was composed of a small amount of crystalline α-cellulose associated with mannan or glucomannan chains. The biosynthesis of cell wall polysaccharides was studied by incubating the microalgae in the presence of D-[U-14C]glucose or myo-[U-14C]inositol. The radioactive glycosyl residues incorporated into the polysaccharides solubilized from purified cell walls of the cultured microalga corresponded to those detected in the chemical analysis of the cell wall. The myo-inositol oxidation pathway was demonstrated to be functional in the microalga. Cell wall ultrastructural observations showed a loose network of cellulose microfibrils in an extended matrix.

Eight Cocconeis Ehrenberg taxa (species and varieties) epiphytic on the leaves of Posidonia oceanica (L.) Delile were studied by scanning and transmission electron microscopy. Samples of Posidonia oceanica were collected throughout the year from seagrass meadows around the island of Ischia (Gulf of Naples, Italy). On the basis of some peculiar ultrastructural characteristics of the valve areolation, a new species, Cocconeis multiperforata sp. nov., and two new varieties, C. neothumensis var. marina var. nov., and C. scutellum var. posidoniae var. nov., are described. Ultrastructural analysis also revealed new morphological characteristics in C. britannica Naegeli in Kützing and C. maxima (Grunow) Peragallo & Peragallo, and confirmed observations of other authors for C. molesta var. crucifera Grunow, C. pellucida Grunow in Rabenhorst and C. stauroneiformis (Rabenhorst) Okuno. Our observations on the abundance of the different species provide new information on their distribution and ecology.

Distribution and phenology of some filamentous species of Rhodomelaceae in the low littoral and shallow sublittoral zones of an exposed, western Mediterranean rocky shore were studied for 1 year. The spatial distribution of many species shows a pronounced small-scale (metres or tens of metres) variability. Boergeseniella fruticulosa was most abundant in spring and fertile (tetrasporangia only) in spring and summer. Polysiphonia flocculosa was most abundant in spring and summer and reproductive throughout the year. Polysiphonia opaca was most abundant in summer and fertile in spring–summer. Maximal vegetative development and reproduction of Lophosiphonia cristata were observed in summer; conversely, Polysiphonia ceramiaeformis and Polysiphonia subulata were best developed and mostly reproductive in winter and spring. Womersleyella setacea was the least abundant of the studied species and apparently reproduced only by vegetative growth and fragmentation. The reproductive phenology of the species investigated is generally in agreement with reports for other Mediterranean regions. The life history traits of most species are typical of seaweeds in which environmental factors directly govern the onset of the main activities of the organism by meeting the primary physiological needs (‘season responders’).

This work attempts to identify growth conditions for maximal productivity of cell mass and of eicosapentaenoic acid (EPA) in ultrahigh cell density cultures of Nannochloropsis sp. Using flat plate reactors with a narrow (1–2 cm) light path and rigorous stirring exposed to high photon flux densities (1000–3000 μmol photons m−2 s−1), record population densities (1·2–1·4×1010 cells ml−1) were obtained. Consequently, the EPA content of the culture (mg l−1) was higher by some two orders of magnitude than reported hitherto for cultures of much lower cell concentrations. In continuous cultures, highest culture EPA yield coincided with maximal output rate of cell mass. The very high population densities and output rates of cell mass and of culture EPA were possible provided culture medium was replaced at least every 48 h. Inhibitory activity, for which a bioassay was developed, was thereby removed. When nutrients were added frequently in order to prevent nutrient limitation without removing the inhibitory activity in the cultures, cell proliferation ceased after reaching some 30% of the attainable maximal cell number, and the culture gradually deteriorated. Inhibitor-induced culture deterioration was fully reversible when the growth medium was replaced with fresh medium.

Adenocystis and Utriculidium share many morphological characters. They both possess a hollow, bladder-like thallus, made of a solid cortex and a fluid-filled medulla penetrated by a network of longitudinal filaments, a trichothallic apical meristem responsible for their multiaxial structure, and cryptostomata. There are two somatic phases in their life history: a haplostichous macrothallus and a polystichous microthallus. However, Adenocystis has unilocular sporangia associated with unicellular paraphyses resembling those of the Laminariales, whereas Utriculidium has plurilocular sporangia resembling those of the Scytosiphonales. Adenocystis has been classified either in the Laminariales or in different families of the Ectocarpales sensu lato, or suggested to be close to the Scytothamnales. Utriculidium has also been classified in different groups within the Ectocarpales sensu lato, depending on the authors. The two genera were either considered as closely related and even to be phases of the same complex life history, or placed in separate orders. The life histories obtained in culture have suggested that Adenocystis and Utriculidium are independent taxa. The taxonomic positions of the two genera Adenocystis and Utriculidium, which are currently unresolved, were explored in the present study, using phylogenetic analysis of combined partial LSU and SSU rDNA sequences. A data set of 20 species included Chorda, Chordaria, Dictyosiphon, Laminaria, Punctaria, Scytosiphon and Scytothamnus, the type genera of the families in which Adenocystis and/or Utriculidium were sometimes previously placed. Adenocystis and Utriculidium grouped together in all analyses within Ectocarpales sensu lato but failed to group with the ectocarpalean genera Chordaria, Dictyosiphon, Punctaria or Scytosiphon, as previously hypothesized. In fact they did not show any clear affinity with any other ectocarpalean taxa. On the basis of this result, and morphological data, we propose a new family, Adenocystaceae, to include these two genera. It is hypothesized that their current life cycles derive from an ancestral isomorphic life cycle.

The first spindle formation in karyogamy-blocked zygotes in the isogamous brown alga Scytosiphon lomentaria was studied by electron and immunofluorescence microscopy using anti-centrin and anti-β-tubulin antibodies. Zygotes blocked in karyogamy by incubation at high temperature (22 °C) were compared with normal zygotes incubated at 14 °C. In normal zygotes, after the disappearance of female gamete centrioles, centrioles from the male gamete duplicated and migrated to both poles in the metaphase, and a bipolar spindle was formed. The nuclear envelope was almost intact except at the poles. In karyogamy-blocked zygotes of S. lomentaria, both DNA synthesis and chromosomal condensation occurred in the male and female nuclei. A bipolar mitotic spindle with a pair of centrioles at each of two poles could be observed in the male nucleus, but not in the female nucleus. In some cases, after a pair of centrioles derived from the male gamete duplicated and separated, one of the two pairs migrated towards the female nucleus. In this case, a monopolar spindle was formed independently in each of the female and male nuclei. In addition to the monopolar spindle, a bipolar spindle was formed lying across the unfused male and female nuclei. Male and female chromosomes were intermixed and some of them were incorporated into the spindle. After cytokinesis of the normal and karyogamy-blocked zygotes, two daughter cells, each with one nucleus and one chloroplast, were formed.

The physiological adaptation of the phosphate uptake behaviour of the cyanobacterium Anacystis nidulans (Synechococcus leopoliensis) has been studied under phosphate-deficient conditions. We show that, during additions of pulses of phosphate, the individual cells of a population potentially develop a coherent behaviour with respect to the kinetic and energetic properties of the high-affinity phosphate uptake system. These adaptive responses are independent of the amount of stored phosphate and the absolute external phosphate concentration, but depend on the exposure time to elevated phosphate concentrations. We demonstrate that the response to additions of pulses of phosphate triggers further metabolic alterations to CO2 fixation, and the flow of fixed carbon into the pool of low-molecular weight-compounds or glycogen. These alterations may also affect subsequent growth. The ecological relevance of the adaptive phosphate uptake behaviour is discussed with respect to the establishment and stability of natural communities.

Inorganic carbon acquisition under emerged conditions was studied in the red macroalga Bostrychia scorpioides by investigating the effects of changes in the pH of the water film surrounding the thalli when emerged and of inhibitors of carbonic anhydrase (CA; EC 4.2.1.1). The CO2 uptake rate increased by approximately 150% when the pH of the water film was changed from 8·1 to 10·5. Acetazolamide (AZ), an inhibitor of the external CA, inhibited CO2 uptake by 50% at pH values higher than 8·7. The effect of ethoxyzolamide (EZ), an inhibitor of both external and internal CA, was more pronounced than that of AZ, inhibiting the CO2 uptake rates by 80% at all the pH values assayed. At pH 10·5, CO2 flux into the cells exceeded by several orders of magnitude the flux supported by the spontaneous dehydration of HCO3− to CO2 in the water film. From these results it was concluded that the main source of inorganic carbon was atmospheric CO2. The driving force for the flux of atmospheric CO2 into the cell must be the CO2 concentration gradient. Therefore, low internal CO2 concentration might be necessary to account for the CO2 fluxes measured. The function of the internal CA could be to speed up the transformation of CO2 into HCO3− inside the cell. The function of the external CA could not be fully described from the experiments although it was demonstrated that the external enzyme enhanced the CO2 flux from air to cell surface.

The phosphate uptake behaviour of phytoplankton communities in two eutrophic Uruguayan lakes has been studied using [32p]phosphate. In order to measure net incorporation of this tracer, lake samples were prelabelled with successive pulses of radioactive phosphate prior to the uptake experiments proper. The data were analysed using a proportional flow-force relationship which allows the evaluation of two characteristic parameters: a conductivity coefficient LP that here reflects the overall activity of the uptake systems of the community, and a threshold concentration [Pe]A at which net incorporation of phosphate ceases for energetic reasons. Between December 1997 and July 1998 in Lago Rodo phosphate supply by inflowing groundwater was periodically interrupted resulting in distinct states of phosphate deficiency. This showed that the phytoplankton responded to alterations in the phosphorus status of the lake by characteristic changes in LP and [Pe]A. The uptake behaviour of the phytoplankton community thus obeyed the linear flow-force relationship over a wide concentration range, in contrast to expectations of non-equilibrium thermodynamics. The observed uptake characteristics exhibited by multi-species phytoplankton ensembles are discussed with respect to a possible coherent behaviour of the constituents of these communities.

When generating kelp hybrid crosses in vitro, small blades are frequently seen in single-sex cultures of microscopic gametophytes as well as in putative hybrid crosses. We demonstrate a polymerase chain reaction (PCR)-based method that quickly determines the presence/absence of possible parental genomes in the observed blades. ITS1 nrDNA fragments that varied in length in a species-specific way were amplified. Amplification products from possible hybrid blades, apogamic blades and parental gametophytes were compared on agarose gels. Using these methods, we were able to identify a number of apogamic blades (i.e. formed directly from gametophytic cells) as well as a true hybrid sporophyte blade in crosses between Alaria marginata (Alariaceae) and Lessoniopsis littoralis (Lessoniaceae), members of two separate families of the Laminariales. To our knowledge this is the first genotypic confirmation of a hybrid kelp.

The coenocytic green alga Caulerpa racemosa colonized the Mediterranean some time after the opening of the Suez Canal in 1869. For most of the past century it has remained confined to the southeastern Mediterranean, but over the past several years the species has been reported in abundance along the western coasts of Italy, Sardinia and the Marseille area of southern France. Whether this constitutes a natural range expansion or a possible new introduction is under discussion. The purpose of this study was to establish the ITS signatures of populations inside and outside the Mediterranean. ITS1 sequences were compared among 78 clones from 21 individuals representing 11 populations of C. racemosa, collected from the Western Mediterranean, the Canary Islands, Panama and Western Australia. Intra-individual polymorphism of ITS1 was found to be as high or higher than inter-individual polymorphism across all samples tested. Polymorphism parsimony analysis, which takes into account intra-individual variation, revealed strong separation between individuals from Panama, Fuerteventura (Canary Is.) and the Mediterranean. In contrast, none of the six Mediterranean populations could be distinguished from one another. In addition, individuals and their clones collected from Perth (Australia) and Gran Canaria were spread throughout the Mediterranean clade. Possible reasons for the lack of homogenization of ITS arrays in C. racemosa under the mechanisms of concerted evolution and implications for phylogeographic interpretation are discussed and compared with Caulerpa taxifolia.