A continuous culture experiment was conducted with the marine diatom Ditylum brightwellii to assess the photo-oxidative effects of transition from dim light to high light at three successive levels. The focus was on temporary stress by ambient UVA at the water surface, via a simulation of the periodic ascent of diatoms caused by vertical mixing. Within a 14 h dim-light period, cells were exposed daily to 4 h of: (A) moderate PAR (100 μmol photon m-2 s-1), (B) high PAR (400 μmol photon m-2 s-1) and (C) high PAR (400–700 nm) plus 6·7 μmol photon m-2 s-1 (2·2 W m-2) UVA. Cell shape, life cycle and sinking rates were not affected by high PAR per se (mode B). The daily 4 h high-irradiance mode C, with additional UVA (UVA: PAR = 0·016), caused an increase in cell diameter, a decrease in length axes, and a production of vegetative resting stages. Superoxide dismutase (SOD) activities, oxidized glutathione (GSSG) pools and malondialdehyde (MDA) contents increased as well, which indicates that this biologically effective UVA dose (0·78 W m-2) promoted active oxygen production, oxidative stress and lipid peroxidation. Sinking rates in UVA-exposed D. brightwellii were higher than in those grown without UVA. A close correlation between sinking rates and MDA contents suggests that UVA-induced lipid peroxidation has altered the membrane functions which regulate buoyancy in D. brightwellii. Although UVA-induced formation of resting cells will contribute to accelerated sinking, it is not evident that this can be considered as a stress avoidance mechanism under adverse light (UVA) conditions. D. brightwellii proved to be sensitive to transitions from shade to natural surface irradiance conditions. In coastal waters, such large diatom species may experience oxidative stress after a few hours' residence near the water surface at ambient UVA levels.

The red algae generally are considered to have no motile stages. We have confirmed sporadic reports of motile spores in a few red algae by recording freshly released live spores with time-lapse videomicroscopy. Of the 25+ species investigated, only a few had spores that appeared to be immobile. Cells of unicellular species often displayed active movement although were otherwise indistinguishable from nearby immobile cells, and these may be equivalent to the motile spores released after differentiation in more complex multicellular species. There was considerable variation among members of the Bangiophycidae. Cells of Porphyridium often moved at around 0·66 μm s-1 and had conspicuous mucilage tails. Flintiella and Rhodospora showed no cellular movement. Rhodosorus sometimes moved a little and all four strains displayed continuous cytoplasmic rotation within the wall. Glaucosphaera was inert but displayed formation and transport of vesicles from the perinuclear region to the surface where they were discharged. Spores (whether monospores, tetraspores or carpospores) of almost all other species or strains available displayed various forms of motility. Some (e.g. Batrachospermum-Chantransia monospores) showed smooth, directional and continuous gliding (c. 2·2 μm s-1) for shorter or longer distances, clearly detectable to the naked eye through the microscope. In others, movement could be almost as fast or slower, non-continuous and undirectional. Certain spores (e.g. monospores of the filamentous Audouinella sp.) were amoeboid, and in some cases (e.g. Sahlingia) cells actively squeezed through the irregular interstices of a crustose colony. The mechanism of movement is unknown; polysaccharide secretion may be involved in some cases. These forms of motility may be significant as dispersal mechanisms, by moving spores out of the stagnant boundary layer, and in settling processes, by allowing spores limited ability to optimize their site of germination.

Morphology, sexual reproduction and phylogeny of a colonial green alga collected from Germany were studied in culture. Light and electron microscopy of the gelatinous (extracellular) matrix of vegetative colonies, the absence of obligately somatic cells, and the anisogamous sexual reproduction with sperm packets in this alga indicated that it is assignable to the genus Eudorina. This German alga was similar to E. elegans Ehrenberg in its multiple pyrenoids of nearly identical size and almost identical sized vegetative cells in the colony, but differed from it in having a prominent tubular structure (flagellar sheath) surrounding each flagellum in the gelatinous matrix of the vegetative colonies. Sexual reproduction was homothallic and dioecious. On the other hand, the vegetative morphology agreed well with that of Pandorina minodii Chodat with regard to multiple pyrenoids and the prominent flagellar sheaths. Thus, a new combination, Eudorina minodii (Chodat) Nozaki et Krienitz, is proposed. Molecular phylogenetic analyses of the rbcL-atpB gene sequences from the colonial Volvocales resolved that E. minodii and several heterothallic strains of E. elegans constituted a robust clade. Therefore, prominent flagellar sheaths and homothallic sexual reproduction in E. minodii may be derived characters that evolved recently within the clade.

Comparison of karyology, restriction patterns and DNA methylation levels (as gender separation could be related to some epigenetic mechanism, e.g. DNA methylation) has been performed between male and female individuals of the dioecious species, Chara tomentosa. In contrast to angiosperms, sex determination systems in Charophyceae have not been studied. High-performance liquid chromatography (HPLC) showed that the cytosine methylation level in female plants of Chara tomentosa reached 5·9 %, while in male plants it reached 9·1 %. Karyological analysis did not show any differences in metaphase chromosome number or morphology between sexes. Cytophotometric measurements of DNA content revealed differences in 1C DNA content: 7·0 pg in females and 7·4 pg in males (p < 0·001). Computer image analysis showed that in the interphase nuclei condensed chromatin consists of 34·3 % of the total chromatin in female plants, while in males it was 29·6 % of total chromatin (p < 0·001). Digestion of isolated DNAs with 12 enzymes showed poor enzymatic cleavage (for example with SacI) or restriction patterns which differed between the sexes (TaqI, Bst0I). The restriction endonucleases RsaI and BamHI revealed repetitive DNA families in males. BamHI generated bands of over 3000 bp while RsaI revealed populations of fragments 650–2000 bp long. DNA isolated from male individuals of C. tomentosa was less susceptible than DNA isolated from female plants to digestion with enzymes that are inhibited by methylation of dC residues (HpaII, HhaI).

Alaria (Alariaceae, Phaeophyceae) is a common genus of kelps in the northern hemisphere. Fourteen species are currently recognized, of which three, Alaria esculenta (L.) Greville, A. pylaii (Bory de Saint-Vincent) Greville and A. grandifolia J. Agardh, are reported for the cold-temperate North Atlantic Ocean. Alaria esculenta, the type species described originally from the North Atlantic, exhibits a range of biogeographically correlated morphotypes suggesting the possibility of multiple species, subspecies or hybrids. In Ireland we discovered an A. esculenta population with unusually long stipes resembling the type specimen of A. grandifolia described from Spitsbergen by J. Agardh in 1872. These and other plants of A. esculenta from Ireland were compared with plants from Spitsbergen fitting the description of A. grandifolia, using sexual hybridization, relative growth rate measurements and DNA sequence comparisons. Complete interfertility was observed between the different isolates. Three nucleotide substitutions (0·37 %) were found in the rbcL and RuBisCo spacer of A. grandifolia, and two in the partial 18S rRNA gene and ITS1 sequences. The relative growth rate at 10 °C of an Irish self-cross was significantly lower than those of all the other crosses. Comparison of RuBisCo spacer sequences of the Spitsbergen A. grandifolia and six A. esculenta isolates showed that A. grandifolia was identical to A. esculenta from Halifax, Canada. The partial 18S rRNA gene and ITS1 sequence of A. grandifolia was identical to that of A. praelonga from Japan and differed by a single substitution from A. esculenta from Scotland and by two nucleotide substitutions from the isolate from Ireland. The intraspecific differences in A. esculenta, together with the hybridization and morphometric results, suggest that A. grandifolia is to be considered conspecific with A. esculenta, and that A. grandifolia is most probably a large deep-water morphological variant, subspecies or ecotype of A. esculenta.

Phenotypic variation of Fucus spiralis was examined in terms of morphology and chemical composition, as determined by pyrolysis mass spectrometry (PyMS). After discriminant analysis both techniques revealed the existence of two morphotypes which were subsequently designated as F. spiralis and F. spiralis forma nanus. The nanus plants differed from the nominate spiralis in that they were shorter, narrower and had fewer branches, but had more units of growth from a single holdfast. F. spiralis f. nanus was consistently higher on the shore than F. spiralis, but the two morphotypes overlapped in terms of shore exposure level. We suggest that the formae exist within a mosaic of stable phenotypes representing populations specifically adapted to the local environment. PyMS data were in agreement with morphological descriptions showing that gross morphology can be correlated with chemical composition.

Lagynion delicatulum Skuja is a first record for New Zealand freshwaters. Individual vegetative cells in nature and culture were sessile, aflagellate, and surrounded by a hyaline lorica lacking mineral deposits. Cells were uninucleate, had one or two chloroplasts with girdle lamellae and ring nucleoids, several mitochondria, and two to four basal-body-like centrioles. Cell division was followed by the release of one or both progeny from the parent lorica as biflagellate zoospores without eyespots. The anterior flagellum bore a tomentum of fine fibrils and two balanced rows of tripartite tubular hairs. The posterior flagellum was short, scarcely visible with light microscopy. A paraflagellar rod and paraflagellar vanes were associated with its axoneme. The flagellar apparatus consisted of two basal bodies and three microtubular roots. The coplanar basal bodies diverged from each other at an angle of approximately 130°. Simple transitional helices were present. No root that could be identified with the R3 root of other stramenopiles was present. Settling zoospores produced a few long pseudopods, and lost flagella by abscission. Cells forming loricas had a circumferential array of numerous short pseudopodia. Similarities in morphology and reproduction suggest that at least those Stylococcaceae with apparently uniflagellate swarmers form a monophyletic group. Flagellar apparatus similarities between Lagynion and Hibberdia suggest a relationship, but this is not supported by previously-published phylogenies reconstructed from molecular sequences. Convergent evolution of flagellar apparatus architecture is a possibility.

The hypothesis that carbon acquisition in phaeophytes of lower organization is controlled by a blue-light-regulated C4 mechanism was tested by various approaches with the filamentous species Ectocarpus siliculosus. Here we report analyses at the enzymatic level. Enzymes potentially participating in a C4 cycle were assayed in extracts, and activities of a phosphoenolpyruvate carboxykinase (PEPCK), of NAD- and NADP-malate dehydrogenases, and of a NADP-malic enzyme were detected. No activity could be measured of phosphoenolpyruvate carboxylase or of pyruvate-Pi-dikinase, which is essential to complete the cycle by regenerating phosphoenolpyruvate from pyruvate. The kinetic properties of all enzymes were determined. Malic enzyme and PEPCK were purified to homogeneity, as tested by gel filtration. The molecular mass of native malic enzyme was determined to be 440 kDa. In SDS gel electrophoreses it produced a single band with a relative molecular mass of 53 kDa, which suggested that the native molecule might be a homo-octamer. The molecular mass of PEPCK was 90 kDa and separation on SDS gels produced two major bands, one of 56 kDa and one of 18 kDa, which appeared not to be a degradation product of the larger polypeptide. The composition of the native enzyme is unclear. In extracts, the activities of malic enzyme and PEPCK followed a circadian rhythm, but none of the enzymes tested was found to be blue-light-dependent. The above results neither prove nor dismiss the possibility that a C4 mechanism operates in Ectocarpus.

Blue light stimulates photosynthesis in brown algae by activating a mechanism for carbon acquisition. As this is connected with liberation of CO2 from an internal pool, it had previously been suggested that a C4 pathway might operate, with CO2 mobilization from an intermediate mediated by the action of blue light. The possibility that such a C4 mechanism functions was tested in Ectocarpus siliculosus by investigating metabolite pools and carbon fixation products. Extracts were analysed by HPLC with an ion exchange column. With the exception of citrate/isocitrate all metabolite levels were far too low to serve as the intermediate pool. No changes in the concentrations of any of the metabolites were found after activation by blue light following red light pre-irradiation. During short-term incubations with 14C inorganic carbon, the major label was found in phosphoglycerate. There was also clear labelling of peaks containing malate plus pyruvate and in citrate/isocitrate. In addition, glycerate contained high levels of radioactivity in some culture strains of Ectocarpus siliculosus. In chase experiments, the radioactivity of all metabolites continued to increase, indicating the presence of considerable amounts of intracellular inorganic carbon. Transient labelling was not observed for any of the fixation products. When Ectocarpus was pre-irradiated with red light and then stimulated by blue light, fixation rates increased with a time course that correlated with the activation of oxygen evolution. Photosynthetic oxygen evolution was investigated in vivo after the addition of possible intermediates to carbon-free seawater. Most of the exogenous applied substrates did not enhance photosynthesis, either in red light or after blue light stimulation. A strong enhancement seen after the addition of oxaloacetate was most likely due to release of CO2 during the breakdown of the metabolite in the medium. Taken together, the data argue strongly against the operation of a C4 pathway in Ectocarpus. It is suggested that the internal pool mobilized by blue light contains inorganic carbon.

A mechanism of bicarbonate uptake with a high sensitivity to the putative anion-exchange inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS) has been previously reported in green algae. In this study, DIDS inhibited net oxygen evolution by Dunaliella tertiolecta by up to 22 %, but internal pH regulation, intracellular CO2 accumulation, carbon fixation and affinity for dissolved inorganic carbon (DIC) in Dunaliella tertiolecta showed low or insignificant sensitivity to DIDS. However, in cells grown and tested at pH 9·5, treatment with DIDS elevated the k0·5(HCO3-), suggesting there may be a minor role for a DIDS-sensitive anion-exchange-type HCO3- transporter in DIC acquisition by D. tertiolecta at high pH. In contrast, significant external carbonic anhydrase (CAext) activity and up to 70 % inhibition of DIC-dependent O2 evolution by acetozolamide (AZ) suggest that CAext has an important role in DIC acquisition in D. tertiolecta, in normal seawater conditions and at elevated pH. Furthermore, the rate of DIC-dependent photosynthesis at high pH, in the presence of AZ, was 12 times higher than the calculated uncatalysed rate of CO2 supply from HCO3-. This requires some system for direct HCO3- uptake by D. tertiolecta, which may include a DIDS-insensitive mechanism. The effects of DIDS upon indirect measures of DIC acquisition should be interpreted cautiously as DIDS may have non-specific effects upon whole cell function, and affect ion transport processes not directly related to HCO3- uptake.

Turbidostat cultures of Dunaliella salina (Chlorophyceae) and Thalassiosira weissflogii (Bacillariophyceae) were grown at fluctuating concentrations of nitrate, phosphate and silicate. In-line measurements of PAM fluorescence were used to monitor the effects of fluctuating nutrient supply on the photochemical efficiency of photosystem II reaction centres of light-adapted cells (ΔF/F'm). Besides the maximal photochemical efficiency of photosystem II reaction centres of dark-adapted cells (Fv/Fm), chlorophyll a, particulate organic carbon, nitrogen and phosphorus, and the cell number were measured frequently during the experiments. Following nutrient-replete growth, the cells were supplied with medium from which either nitrate, phosphate or silicate was omitted. When significant effects of nutrient starvation were indicated by the fluorescence parameters, a pulse of the deficient nutrient was added to the cultures. Our experimental set-up for in-line fluorescence measurements provided sensitive and reproducible detection of the various fluorescence signals, revealing strong influences of nutrient supply on the photochemical efficiency of photosystem II. In general the fluorescence values changed substantially within 1–30 min after re-addition of the deficient nutrient. Addition of phosphate and silicate induced an immediate characteristic decrease in fluorescence, whereas nitrate addition was characterized by a strong, delayed increase in fluorescence. Complete recovery to pre-starvation fluorescence values took about 48 h in all experiments. The physiological background of nutrient uptake is used to explain the observed tight couplings between fluorescence responses and nutrient re-addition. Our study clearly demonstrates that in-line fluorescence measurements provide a new tool for the investigation of phytoplankton reactions to fluctuating nutrients and offer the possibility to detect nutrient starvation in the field.