Parasexual fusion products in green algae: Enteromorpha and Ulvaria (Ulvales,Chlorophyta)

Enteromorpha linza and Ulvaria oxysperma in North Carolina reproduce exclusively by asexual zoospores. Calcofluor white staining indicated that newly released zoospores lack significant cellulose cell wall material, making them suitable for treatment as protoplasts in a parasexual fusion process using high pH-Ca²⁺, PEG and centrifugation. Presumptive fusion products were identified by their larger size, twin chloroplasts and eyespots, and presence of fluorescence labelled and unlabelled portions. Parasexual fusion and karyogamy were confirmed by elevated levels of nuclear DNA in fusion cell germlings. In addition, aceto-orcein staining of fusion cell products revealed a diploid chromosome complement of 2N = 20 in Enteromorpha linza. Fusion cells were isolated by killing the more numerous adjacent unfused zoospores with 2-3 min exposure to blue light (410–490 nm). Unexposed fusion cells could be readily distinguished and recovered by micropipette at the 10-day stage.Center for Marine Science Research UNC-W Contribution No. 008.     

Cation Exchange Properties of the Cell Walls of Enteromorpha intestinalis (L.) Link. (Ulvales, Chlorophyta

The cell wall of Enteromorpha intestinalis (a marine alga) hasbeen found to behave as a weakly cross-linked cation exchangerin NaCl solutions from 0.1–1020 mMolal (0.1–1000mMolar). Anion adsorption could be described by Freundlich isothermsover this concentration range. The large anion, inulin carboxylate,was found to be a tracer of the anion free space of plant tissuesonly in salt solutions above 10 mMolal. The cell wall of Enteromorphahas a cation exchange capacity of about 2500 µ mol g^–1dry wt. (Na+ form). The cell wallvolume is a complex functionof p^H and the NaCl concentration. As a result, the cation exchangecapacity is only predictable on a dry weight basis. The fixednegative charges of the cell wall have a pK_a of2 in situ and1.75 in vitro, and seem to be a mixture of sulphate and carboxylsugar esters. The applicability of the Donnan equation to plant cell wallsis discussed. Interpretation of the cell wall as a single thermodynamicphase is shown to be inappropriate. A large proportion of thecell wall solution is unaffected by the fixed anions.     

Ion Exchange Fluxes of the Cell Walls of Enteromorpha intestinalis (L. ) Link (Ulvales, Chlorophyta)

The ²²Na⁺ and ³⁶CI^– exchange properties of the cell wallsof Enteromorpha intestinalis (L. ) Link in simple monovalentsalt systems have been shown to be similar to a ‘leaky’cation exchange membrane rather than a homogeneous membrane.The ion exchange properties of the cation and anion cell wallcontents are what would be expected of a cation exchange membranei. e. anion exchange is strongly dependent on the bathing electrolyteconcentration and becomes very slow in dilute salt. This wouldlead to the cell wall becoming a barrier to anions in dilutesalt. However, measurements of the anion flux across cell wallsin living and dead tissues show that anion exchange across cellwalls is facilitated by pores. The exchange kinetics of thebulk of the cell wall anions does not limit the anion flux acrosscell walls of this plant. It is concluded that the cell wallis not a critical limitation to plasmalemma fluxes of the livingplant and that unstirred layers are more important than cellwalls in the measurement of anion flux rates.     

Regeneration of plantlets fromEnteromorpha (Ulvales,Chlorophyta) protoplasts in axenic culture

High yields of protoplasts have been obtained from vegetative thalli of three species ofEnteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85–95%) occurred in the protoplasts cultured at a density greater than 1.72 × 10³ cells cm⁻² at 20 and 25°C. Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (exceptE. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (> 0.4 M) inhibited cell division and further differentiation in all species. author for correspondence     

Diluted seawater promoted the green tide of Ulva prolifera (Chlorophyta,Ulvales)

Ulva prolifera (Müller) J. Agardh is the main causative species of the 2008 Yellow Sea green tide incident. We investigated the influences of diluted seawater on the vegetative growth and reproductive cell formation of the alga. The thalli that were cultivated under low salinities (10‰ and 20‰) and low pH values (pH around 7.0) showed obvious and steady biomass gain, while those cultivated under high salinities (40‰) and relatively higher pH conditions (pH around 8.0) manifested significant biomass loss. The trend was, however, completely the opposite for reproductive cell formation and there were indications that enrichment, over very wide concentration ranges of both nitrogen and phosphate, could significantly promote vegetative growth. Results also indicated that relatively low salinity and low pH regimes boosted vegetative growth but were unfavorable for reproductive cell formation and vice versa. Based on these results, the possible origin and development mechanisms of the green tide event are discussed. Eutrophication in the Changjiang River estuary and adjacent sea areas, as well as extremely high freshwater inflows before, and during, the flood of 2007 – due to the full operation of large‐scale water facilities in the area – may have both played an important role in the formation and development of the green tide event.     

Production of bioflavor by regeneration from protoplasts of Ulva pertusa (Ulvales,Chlorophyta)

Protoplasts were isolated from thalli of Ulva pertusa using a mixed enzyme solution of 2.0% Cellulase Onozuka R-10, 2.0% Macerozyme R-10, and 2.0% Driselase. Isolated protoplasts regenerated cell walls, developed into thalli, and propagated in large numbers under aeration in the preparative scale-culture system. Typical bioflavor compounds produced from the regenerated plants, as well as from field-collected plants, were found to be long chain aldehydes, which gave a typical seaweed odor. The long chain aldehydes were formed enzymatically from unsaturated fatty acids and released into the culture fluid. A Percoll/mannitol discontinuous density gradient separation of the heterogeneous protoplasts led to a selection of cell lines with high production of bioflavor. The cells that regenerated from protoplasts were immobilized by polymer matrices such as alginate, -carrageenan, agarose, and agar. Living cells entrapped in alginate beads in aerated cultures survived best. However, the beads started to breakdown after two months. The immobilized cells demonstrated a higher bioflavor production than did the cultured cells.     

Molecular and morphological diversity of Narragansett Bay (RI,USA) Ulva (Ulvales,Chlorophyta) populations

Macroalgal bloom‐forming species occur in coastal systems worldwide. However, due to overlapping morphologies in some taxa, accurate taxonomic assessment and classification of these species can be quite challenging. We investigated the molecular and morphological characteristics of 153 specimens of bloom‐forming Ulva located in and around Narragansett Bay, RI, USA. We analyzed sequences of the nuclear internal transcribed spacer 1 region (ITS1) and the chloroplast‐encoded rbcL; based on the ITS1 data, we grouped the specimens into nine operational taxonomic units (OTUs). Eight of these OTUs have been previously reported to exist, while one is novel. Of the eight OTUs, all shared sequence identity with previously published sequences or differed by less than 1.5% sequence divergence for two molecular markers. Previously, 10 species names were reported for Ulva in Rhode Island (one blade and nine tube‐forming species) based upon morphological classification alone. Of our nine OTUs, three contained blade‐forming specimens (U. lactuca, U. compressa, U. rigida), one OTU had a blade with a tubular stipe, and six contained unbranched and/or branched tubular morphologies (one of these six, U. compressa, had both a blade and a tube morphology). While the three blade‐forming OTUs in Narragansett Bay can frequently be distinguished by careful observations of morphological characteristics, and spatial/temporal distribution, it is much more difficult to distinguish among the tube‐forming specimens based upon morphology or distribution alone. Our data support the molecular species concept for Ulva, and indicate that molecular‐based classifications of Ulva species are critical for proper species identification, and subsequent ecological assessment or mitigation of Ulva blooms.     

Cytological Investigations on Porphyra umbilicalis(L.) Kutz. var. laciniata(Lightf.) J.Ag.

1. Vegetative mitosis of Porphyra umbilicalis var. laciniatais normal, the chromosome number as seen in late prophase beingfive. Special features of this mitosis are the appearance ofseveral stained chromatin segments in early pro-phase and theformation of a crescent-shaped group of chromosomes during metaphaseand anaphase. 2. Genuine monospores have not been seen in the material investigated. 3. Both spores and spermatia arise by repeated division of amother-cell and in identical ways. Evidence is presented toshow that the first division in the mother-cell forming sporesis longitudinal. There is no evidence of reduction divisionin this division, and the chromosome number in the mother-cellis five. 4. The first division in the germinating spore is mitotic andshows five chromosomes. 5. The Conchocelis-phase is haploid throughout and produces‘fertile cell rows’ and ‘plantlets’,but spore liberation has not been observed. It is suggestedthat the ‘plantlets’ may grow out directly intothe leafy phase or may give rise to spores according to prevailingenvironmental conditions. 6. No evidence of sexual reproduction has been obtained in thepresent work and the role of the spermatium in the life-cycleis not clear. It is suggested that further work may profitablybe concerned with investigating the nature of the spermatium.     

Growth and photosynthetic responses of Ulva lactuca (Ulvales,Chlorophyta) germlings to different pH levels

In ocean ecosystems, fluctuations in seawater pH affect CO₂ fluxes, fundamentally influencing the metabolism of marine algae, especially during the early stages of macroalgal development. In this study, short-term exposure tests (minutes) and prolonged culture experiments (eight days) were performed at different pH levels to investigate the growth and photosynthetic responses of Ulva lactuca (Ulvales, Chlorophyta) germlings. Both acidified and alkalized seawater significantly depressed algal photosynthesis during short-term exposure tests. Prolonged culture in acidified or alkalized seawater also notably decreased photosynthesis rates and growth rates of U. lactuca germlings, but increased energy consumption and lipid peroxidation, indicating damage to the germlings. Our results suggested that both lowered and increased pH levels of seawater exert significant physiological stress on U. lactuca germlings.     

Genetic diversity and biogeography of native and introduced populations of Ulva pertusa (Ulvales,Chlorophyta)

Genetic diversity of native and introduced populations of Ulva pertusa (Ulvales, Chlorophyta) was examined using genetic markers of chloroplast, mitochondria and nuclear non‐coding region sequences. In the preliminary investigations to genetically identify the species for further analyses, U. pertusa was found only from temperate coasts of the more extensive collection sites including tropical coasts suggesting that it is a temperate species and basically not distributed in tropical regions. For chloroplast and mitochondrial sequences, repeating patterns of short tandem repeat sequences and nucleotide substitutions were used to recognize the haplotypes (genetic types). A total of 48 haplotypes based on combinations of chloroplast and mitochondrial haplotypes were recognized in the 244 specimens collected in the presumptive native range (Northeast Asia) and introduced populations (North America, Australia, New Zealand, Chile and Europe). Among them, 46 haplotypes (H1–H8 and H11–H48) were recognized in Northeast Asia, whereas only 1–5 haplotypes were recognized in the other areas. Nuclear microsatellite sequences were also analyzed. The lengths of the PCR products including the nuclear microsatellite region of 234 specimens were determined, and a total of 17 genotypes were recognized. Among them, 14 genotypes were found in Northeast Asia, whereas 1–7 genotypes were recognized in the other areas. Based on the results, the hypothesis that the native range of the species is in Northeast Asia was supported, and the populations outside this range were concluded to be non‐indigenous populations.     

Chemical composition and 13C NMR spectroscopic characterisation of ulvans from Ulva (Ulvales, Chlorophyta)

The chemical composition and structures of several ulvan extracts isolated from various Ulva species were studied. They were all composed mainly of rhamnose, glucuronic acid, xylose, glucose and sulphate with smaller amounts of iduronic acid and traces of galactose. Proteins were also present, most likely as contaminants. Precise quantification of the uronic acid content by chemical-enzymatic hydrolysis coupled to HPAEC-PAD analysis and by colorimetry was not achieved, most likely due to the incomplete hydrolysis of glucuronan segments, inadequate HPAEC-pulsed-amperometric response factor for iduronic acid and to a possible differential colorimetric response of the two uronic acids. ¹³C NMR spectroscopic investigation of different ulvans demonstrated that they were all based on ulvanobiuronic acid 3-sulphate A and B repeating units [β-D-Glc pA-(1->4)-α-L-Rhap3S and α-L-IdopA-(1->4)-α-L-Rha p3S, respectively] as well as contiguous β 1->4 linked D-glucuronic acids possibly occurring either in ulvan or as a separate glucuronan. Marked variations in the content of the repeating structures were seen among the different samples. However, due to the limited number of samples studied, no conclusion was reached concerning the effects of species and ecophysiological conditions on the chemistry of ulvan. This revised version was published online in June 2006 with corrections to the Cover Date.     

DNA content of Ulva compressa (Ulvales, Chlorophyta) nuclei determined with laser scanning cytometry

The green macroalgal genus Ulva (incl. Entemmorpha) contains economically valuable species, is of relevance for coastal management (green tides), and certain taxa serve as experimental organisms for fundamental research in green algae. The nuclear genome size of Ulva (Entemmorpha) compressa Linnaeus was measured in propidium iodide stained nuclei using laser scanning cytometry. Nuclei of fixed gametes yielded reproducible values, whereas nuclei extracted from multicellular gametophytes were unsuitable. With nuclei of Arabidopsis thaliana (L.) Heynh and Saccharomyces cerevisiae Hansen as references, the haploid nuclear genome size of U. compressa was calculated as 135 ± 7 Mbp. This is the smallest genome so far known from any species of Ulva.     

Molecular assessment of Ulva (Ulvales,Chlorophyta) diversity in Vietnam including the new species U. vietnamensis

Species diversity of Ulva in Vietnam was investigated using three commonly used genetic markers, the nuclear encoded rDNA ITS region and the plastid encoded rbcL and tufA genes. Single locus species delimitation methods, complemented with morphological and ecological information resulted in the delimitation of 19 species. This diversity is largely incongruent with the traditional understanding of Ulva diversity in Vietnam. Only four species identified in this study, U. lactuca, U. reticulata, U. spinulosa, and U. flexuosa, have been previously reported, and seven species, U. ohnoi, U. tepida, U. chaugulii, U. kraftiorum, U. meridionalis, U. limnetica, and U. aragoënsis, are recorded for the first time from Vietnam. Seven genetic clusters could not be associated with species names with certainty. A new species, U. vietnamensis, is described from marine to brackish coastal areas from southern Vietnam based on its morphological and molecular distinctiveness from the currently known Ulva species. A comparison with recent molecular-based studies of Ulva diversity showed that species composition in Vietnam is similar to that of adjacent countries, including Japan, China, as well as Australia. Our study emphasizes the importance of molecular data in the assessment of Ulva diversity, and indicates that a lot of diversity may still remain to be discovered, especially in tropical regions.     

Blidingia minima var. stolonifera var. nov. (Ulvales, Chlorophyta) from British Columbia: systematics, life history and morphogenesis

Blidingia minima var. stolonifera var. nov. is described from Vancouver, British Columbia. In previous literature this variety may have been confused with B. chadefaudii and B. minima var. minima. The new variety is part of the B. minima species complex in which spores germinate by evacuating the original spore and forming a germ tube. B. minima var. stolonifera is characterized by the development of marginal filaments on the basal disc that have one to five colourless cells and terminate in a cell from which a new disc grows. Colourless cells are devoid of chloroplasts and nuclei, and contain only small remnants of cytoplasm. The runner system is considered a mechanism of vegetative growth and propagation that enables a disc to cover a large amount of substratum before producing erect, unbranched thalli. In field and in culture, B. minima var. stolonifera typically reproduces by means of quadriflagellate zoospores; however, three plants from Vancouver formed biflagellate spores. These germinated poorly and developed into highly irregular, branched thalli.     

Acrochaetium botryocarpum (Harv.) J. Ag. (Rhodophyta) in Southern Australia

The morphology, ecology, cytology and systematics of Acrochaetium botryocarpum (Harv.) J. Ag. have been studied. This species occurs throughout the year in southern Australia and grows on a variety of hosts. The variable appearance of the prostrate system results from the effect of the substratum upon its morphology. Plants reach a height of 6 mm; cells of the erect filaments contain a single chromoplast with a variable number of pyrenoids. Tetrasporangial plants occur mainly in winter and sexual plants occur mainly in spring and early summer; the two generations are isomorphic. Stages of fertilisation have been observed and photographed. Acrochaetium polyrhizum (Harv.) J. Ag. is referred to the synonomy of A. botryocarpum, and A. codicolum Brg., A. grande (Levr.) De Toni fil. and A. rhizoideum (Drew) Sm. are regarded as probable synonyms. This study of A. botryocarpum indicates that host specificity, substrate relations, form of spermatangia and immediate post-fertilisation activity may not be as reliable as formerly thought for making taxonomic distinctions within the Acrochaetium-Rhodochorton complex.     

Taxonomic and ecological profile of `green tide' species of Ulva (Ulvales,Chlorophyta) in central Philippines

Hydrobiologia - Ulva spp. are common in the intertidal zones of the Philippines, but, at certain times, could over-proliferate producing blooms or `green tide' in some protected bays. In Mactan...