This paper continues a series of studies of the allogamous raphid diatom Achnanthes longipes, which has a complex reproductive system combining unisexual, bisexual and monoecious behaviour. Following earlier work on the effects of inbreeding in the progeny of crosses between two unisexual clones, we studied the progeny of clones that are capable of a high degree of selfing in monoclonal culture (‘monoecious clones’). Three generations of selfed progeny were examined. In addition, we investigated the F1 generation obtained after crossing two different monoecious clones. Monoecious clones produced monoecious or, more rarely, bisexual progeny, but did not give rise to unisexual progeny. As in inbred lineages made by crossing closely related unisexual clones, inbreeding in monoecious lineages leads to a reduction in the number of gametes formed by the gametangia, from two to one. Inbred clones exhibit marked inbreeding depression and only three inbred generations were possible in monoecious lineages. In the third, final inbred generation, monoecious sexual reproduction was initiated in monoclonal cultures but gametes rarely fused and none of the auxospores and initial cells that were formed were viable; this also occurred when the inbred clones were crossed with any of the other clones available. The significance of inbreeding and other aspects of the breeding system in A. longipes is discussed.

The affinity for HCO3− and carbonic anhydrase activity (CA; EC 4.2.1.1) were determined in the red macroalga Bostrychia scorpioides. The affinity for HCO3− was assessed by using three independent methods: the effect of a high pH on photosynthetic rates, photosynthetic conductance for CO2 estimated from the initial slope of photosynthesis rate versus inorganic carbon concentration curves, and the CO2 compensation point. The results obtained from the three methods were consistent in demonstrating that B. scorpioides has a very low affinity for HCO3−, although a certain capacity for its use must not be ruled out. High activities of both external and internal CA were found. Internal CA activity was 10-fold higher than external. External and internal CA were necessary for photosynthesis at pH values higher than 7·0 because O2 evolution was inhibited after adding acetazolamide and ethoxyzolamide (inhibitors of CA). The effect of changing O2 concentration on photosynthesis was also examined. Bostrychia scorpioides exhibited a high sensitivity to O2, demonstrating a C3-like photosynthetic gas exchange physiology. Therefore, the high level of CA activity in B. scorpioides was not enough to support a high affinity for HCO3− and to prevent photorespiration.

The nucleotides and nucleoside diphosphate monosaccharides (NDP-sugars), UDP-galactose, UDP-glucose, UDP, ATP, ADP, ADP-glucose, AMP and NAD, some of which are involved in carrageenan biosynthesis, were identified in the two carrageenophytes Calliblepharis jubata and Solieria chordalis and quantified by high-performance liquid chromatography (HPLC). Of several extraction procedures evaluated, a freeze-thaw step followed by formic acid (1 M) extraction was the preferred method. The pool size of nucleotides and NDP-sugars differed between the two algal species, which may be related to their growth state. Calliblepharis, which was not growing actively, had larger pools than Solieria, which was in an active growth phase. Using an HPLC method UDP-D-glucose-4-epimerase activity was measured in cell-free extracts of both algae, for the first time in a carrageenophyte.

Immunofluorescence assays using polyclonal and preadsorbed antibodies as well as FITC-labelled lectins were employed to characterize two morphospecies of the cyanophytes Pseudanabaena catenata and P. planctonica from laboratory cultures isolated from two reservoirs in Spain and one in Denmark. Binding of both antibodies and lectins was unaffected by phase of the cell division cycle, growth phase and environmental factors such as culture medium, light or temperature. Good-quality polyclonal antibodies were obtained for several strains of both morphospecies. As expected, different morphospecies from the same location do not share common antigens while strains of the same morphospecies grown in different reservoirs do share most of their surface antigens. However, P. catenata grown at La Pinilla (Spain) does not share common antigens with P. catenata from Bastrup (Denmark). Geographical barriers, in this case, seem to play an important role in determining surface antigen diversity. Similar results are obtained when lectins are used, and some lectins are found to be useful tools for differentiating clones of the same morphospecies. From a genetic point of view there may be no clear species boundary for some morphospecies of cyanobacteria. A ‘fuzzy species’ concept, i.e. a species that belongs in part to more than one species or has characteristics of more than one species, is speculatively considered to explain genospecies boundaries of cyanobacteria.

Two brown algae, Pelvetia canaliculata and Laminaria saccharina, from the higher and lower mediolittoral belts respectively, have been tested for their capacity to overcome high-light stress in water and in air (in both fully hydrated and desiccated states). When exposed to supersaturating light irradiance in water, the two species developed non-photochemical quenching of fluorescence (NPQ) which was correlated with an increase in the de-epoxidation ratio (DR) of the xanthophyll cycle carotenoids (violaxanthin, antheraxanthin and zeaxanthin) and was followed by a slower decrease in oxygen evolution. NPQ reached values of up to 9 in P. canaliculata but only 4·5 in L. saccharina, at DRs of 0·65 and 0·5, respectively. In air, the xanthophyll cycle was also operative but the efficiency of de-epoxidation decreased linearly with the degree of hydration of the thallus. Photoprotection capacities in air also appeared higher in P. canaliculata than in L. saccharina, probably due to the higher molar content of the xanthophyll cycle pool size relative to chlorophyll a (Chl a) in the former (nearly double of that L. saccharina at 19 carotenoid molecules per 100 Chl a), which may be associated with a higher DR at the same level of desiccation. The concomitant higher accumulation of zeaxanthin in P. canaliculata might divert a higher percentage of the incident energy from the reaction centres, as demonstrated by the levels of NPQ, with steady-state fluorescence reduced to below the initial F0 level. Such differences, together with the unequal resistance to desiccation of the operation of the xanthophyll cycle, should be considered as possible factors responsible for the distribution for these two species on the shore.

Cryopreserved Chlamydomonas reinhardtii cultures remained viable when frozen by cooling slowly to −55°C, then plunging into liquid nitrogen for at least 1 day of storage. High viability (>40%) was retained when cultures contained 2–10% (v/v) methanol as a cryoprotective agent prior to freezing, while dimethyl sulphoxide was ineffective. However, methanol was lethal to cells in the presence of light. Frozen cultures became non-viable within 24 h when stored at −80°C, whereas those stored below −130°C remained viable for at least several months. Highest viability was attained in cultures that were frozen and stored at a low cell density. High viability also required that frozen cultures be warmed rapidly and the cryoprotective agent removed immediately thereafter in preparation for culturing in liquid or on solid medium. Individual cell viability was determined by measurements of colony counts after cell plating and by the penetration of Evans blue dye into non-viable cells. Viability in bulk culture was conveniently measured by comparing the rates of photosynthetic oxygen evolution, corrected for dark respiration, in previously frozen cultures with the corresponding rates in unfrozen controls. Cultures that had lost viability as measured by a decline in whole-cell photosynthetic oxygen evolution after freezing and thawing retained functional Photosystem II activity for some time thereafter, indicating that the loss in photosynthetic activity was due to some process other than photoinhibition of Photosystem II.

Partial sequences of the large subunit (LSU) and small subunit (SSU) of the ribosomal DNA of fucalean algae (Phaeophyceae) from the northern and the southern hemispheres, as well as from other brown algal genera such as Durvillaea, were analysed and compared with available morphological data. Combined SSU+LSU sequence data appear to be suitable for inferring phylogenetic relationships within the Fucales. Results suggest that Durvillaea and Notheia should be included in the Fucales and that the paraphyletic Cystoseiraceae be merged into a large family, Sargassaceae Kützing emend. De Toni. The genus Axillariella, the taxonomic position of which was uncertain, clearly belongs to the Sargassaceae. At present, at least seven families are recognized in the order Fucales: Durvillaeaceae, Seirococcaceae, Notheiaceae, Sargassaceae, Himanthaliaceae, Hormosiraceae and Fucaceae. More investigations are needed to assess whether Xiphophora should be placed in its own family.

The zygote wall in developing Durvillaea potatorum embryos is secreted soon after fertilization. Mucilage covers the surface of the zygote wall ensuring rapid adhesion of the newly fertilized egg. The zygote wall is replaced by new cell walls secreted by growing, dividing embryonic cells. Separation of the zygote wall from the embryo is evident 4 days after fertilization and is visible at the edge of the cap flanking the side of the developing embryo and/or at the curved part of the cap where it lifts away from the top of the embryo. At this stage the embryo has undergone an average of 8 cell divisions, increased 3 times in length, and started to increase in width. The zygote wall is eventually shed as it cannot accommodate embryo expansion. The growing embryo flicks out of the rigid zygote wall, which usually remains connected to the developing rhizoid by a mucilaginous strand. By day 5 the zygote wall has detached from the embryo in 22% of individuals and this increases to nearly 70% of individuals by day 10. Removal of alginate-gelling cations from the artificial sea medium indicates that the zygote wall has different properties from the underlying embryonic cell walls. Shedding of the zygote wall allows continued embryo growth, frees the embryonic thallus from the substratum and cleans the embryo surface of phenolic bodies and mucilage that are secreted soon after fertilization; it also removes micro-organisms colonizing the mucilage.

In summer, the concentrations of dissolved organic nitrogen compounds are often higher than that of inorganic nitrogen. Under such conditions it would be advantageous if phytoplankton species could utilize organic nitrogen sources, including free or combined amino acids, in addition to inorganic nitrogen. This study focused on histidine, the degradation of which potentially yields three nitrogen atoms for each molecule of histidine. In this work, histidase from Dunaliella tertiolecta, a deaminating enzyme catalysing the first steps of histidine degradation, was purified 4000-fold and partially characterized. The molecular weight of the native enzyme was estimated to be 155 kDa, corresponding to four subunits of 38 kDa. D. tertiolecta histidase is stable in the presence of dithiothreitol and is inactivated by cyanide. Histidinol phosphate, histidine and Mn2+ are effective protectors against cyanide inactivation. The enzyme did not exhibit classical Michaelis-Menten kinetics but showed a relationship between the rate of catalysis (V) and the concentration of substrate (S) that was characteristic of negative allosteric behaviour. A Hill coefficient of 4 was measured for histidine concentrations higher than 22.5 mM. Guanine, xanthine and cytosine nucleotides are inhibitors of D. tertiolecta histidase.

Several macroalgae from the Swedish West Coast (11 green, 5 red and 11 brown algal species) and two Ulva spp. from the Mediterranean were tested for mechanisms of HCO3− utilization. In all except one (Palmaria palmata), photosynthesis supported by inorganic carbon (Ci) at pH 8.7 was rapidly and substantially (>50%) inhibited by acetazolamide (AZ), an inhibitor of extracellular carbonic anhydrase activity. This suggests that extracellular dehydration followed by uptake of the CO2 formed is an important component of the HCO3− utilization mechanism(s) of these algae. In representatives of most of the green algal genera, e.g. Enteromorpha, Ulva, Chaetomorpha and Monostroma, a specific inhibitor response could be induced. This response, a sensitivity to the anion exchange inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonate (DIDS) in the presence of AZ, is characteristic of an additional mechanism of HCO3− utilization, a direct import of HCO3− across the plasma membrane, which has been previously described only for some Ulva spp. and Enteromorpha intestinalis. The DIDS sensitivity could be induced in several ways, all involving a potential shortage of the supply of CO2 for photosynthesis: through photosynthesis at high pH, through growth in strong light and nutrient-rich seawater, or through photosynthesis in the presence of AZ. Induction of photosynthesis at high pH (which is a specific feature of green macroalgae) was not always accompanied by a development of this specific DIDS sensitivity; it occurred in Chaetomorpha melagonium but not in C. linum. For Ulva lactuca cultivated in natural seawater (in continuous moderate irradiance), induction of DIDS sensitivity by high pH treatment was successful all year round, except during a period in late August. The results thus suggest that green macroalgae capable of developing photosynthesis at high pH are also capable of developing a DIDS-sensitive mechanism for HCO3− uptake, but that some additional conditions (other than low CO2 availability) must be fulfilled. The ability to dehydrate extracellular HCO3− still remained after induction of the direct HCO3− uptake, and the two mechanisms for HCO3− utilization may operate independently of one another. In fact the response to DIDS was more pronounced after the addition of AZ. The possibility that the results are related to a plasticity of the HCO3−-utilizing mechanisms of green macroalgae is discussed. Neither red nor brown macroalgae showed any sensitivity to DIDS in the presence of AZ.

Molecular phylogenetic relationships among sexually isolated subpopulations (syngens) of the morphologically defined volvocacean flagellate Pandorina morum (O. F. Müller) Bory are inferred from sequences of the internal transcribed spacer regions (ITS-1 and ITS-2) of the nuclear ribosomal DNA repeats. The resolved phylogenetic lines are composed of strains with very similar sets of traditional characters, such as zygote aggregation pattern, chromosome number and cell division timing, and are correlated with the distribution pattern of the xanthophyll loroxanthin [(3R,3′R,6′R)-β,ε-carotene-3,19,3′-triol]. Thus, loroxanthin, a phylogenetic character within the Volvocales, is also of value at the infraspecific level in P. morum, a morphologically uniform species complex, within the Volvocales.

In the paper:

Studies on the Dasyaceae. 3. Towards a phylogeny of the Dasyaceae (Ceramiales, Rhodophyta), based on comparative rbcL gene sequences and morphology, by Y. S. D. M. de Jong, A. W. G. van der Wurff, W. T. Stam & J. L. Olsen (Eur. J. Phycol., 33: 187–201, 1998), the cladogram in Fig. 2 was inadvertently printed with mixed bold and narrow lines. The bold lines have no significance.