MOLECULAR SYSTEMATICS OF ECTOCARPUS AND KUCKUCKIA (ECTOCARPALES, PHAEOPHYCEAE) INFERRED FROM PHYLOGENETIC ANALYSIS OF NUCLEAR- AND PIASTTD-ENCODED DNA SEQUENCES

The phylogeny of Ectocarpus and Kuckuckia strains representing widely separated populations from both hemispheres was inferred from sequence analysis of the internal transcribed spacers of the nuclear ribosomal DNA (ITS 1—5.8S-ITS 2) and the spacer region in the plastid-encoded ribulose- bis -phosphate-carboxylase (RUBISCO) cistron (partial rbc L-spacer-partial rbc S ). Both sequences resulted in matching phylogenies, with the RUBISCO spacer region being most informative at the level of genera and species and the internal transcribed spacer sequences at the level of species and populations. Three major clades were formed by strains previously described by morphology and physiology as Kuckuckia, E. fasciculatus, and E. siliculosus, confirming the validity of these taxa . Ectocarpus and Kuckuckia are regarded as sibling taxa with respect to the outgroup species Feldmannia simplex, Hincksia mitchelliae, and Pilayella littoralis. The clade formed by sexual E. siliculosus strains and most asexual Ectocarpus strains was subdivided into several clades that are consistent with geographical races within E. siliculosus. The inferred phylogeny of Ectocarpus corresponds generally with results from cross-fertilization experiments, morphology, and lipid analysis. A hypothesis on the origin and dispersal of E. siliculosus suggests several natural dispersal events during periods of global cooling as well as recent and possibly anthropogenic dispersal events .     

Development and physiology of the brown alga Ectocarpus siliculosus: two centuries of research

Brown algae share several important features with land plants, such as their photoautotrophic nature and their cellulose-containing wall, but the two groups are distantly related from an evolutionary point of view. The heterokont phylum, to which the brown algae belong, is a eukaryotic crown group that is phylogenetically distinct not only from the green lineage, but also from the red algae and the opisthokont phylum (fungi and animals). As a result of this independent evolutionary history, the brown algae exhibit many novel features and, moreover, have evolved complex multicellular development independently of the other major groups already mentioned. In 2004, a consortium of laboratories, including the Station Biologique in Roscoff and Genoscope, initiated a project to sequence the genome of Ectocarpus siliculosus, a small filamentous brown alga that is found in temperate, coastal environments throughout the globe. The E. siliculosus genome, which is currently being annotated, is expected to be the first completely characterized genome of a multicellular alga. In this review we look back over two centuries of work on this brown alga and highlight the advances that have led to the choice of E. siliculosus as a genomic and genetic model organism for the brown algae.     

INHERITANCE AND MEIOTIC ELIMINATION OF A VIRUS GENOME IN THE HOST ECTOCARPUS SZLZCULOSUS (PHAEOPHYCEAE)1

The marine brown alga Ectocarpus siliculosus (Dillwyn) Lyngbye is frequently infected by a latent DNA virus that multiplies in modified sporangia and gametangia of the host. We describe a polymerase chain reaction (PCR) procedure for the amplification and detection of viral DNA in Ectocarpus. PCR analysis of parents and progeny plants confirmed that virus DNA passes through meiosis like a Mendelian trait. An infected sporophyte produced equal numbers of gametophytes with and without the viral genome. Thus, meiosis in sexual populations of the host acts as a mechanism for the creation of virus-free progeny.     

NUTRIENTS DO NOT INFLUENCE SWIMMING BEHAVIOR OR SETTLEMENT RATES OF ECTOCARPUS SILICULOSUS (PHAEOPHYCEAE) SPORES

Spores newly released from plurilocular sporangia of Ectocarpus siliculosus (Dillw.) Lyngb. sporophytes were assayed for chemotaxis to nutrients and for settlement stimulation by nutrients. To enable these measurements with relatively small volumes and numbers of released spores, we developed a computer-assisted motion-analysis assay for spore chemotaxis and verified the results with a more standard, capillary tube chemotaxis assay. The presence of a nutrient gradient did not influence the swimming behavior of E. siliculosus spores in the motion-analysis assay, and likewise no chemotactic effect was measured in the capillary tube assay. Microplate settlement assays previously utilized with bacteria and invertebrates were adapted for use with algal spores. E. siliculosus spores settled at higher rates on a hydrophobic plastic surface than on surfaces with either positively or negatively charged hydrophilic coatings. Nutrient mixtures had no effect on the rate of spore settlement on hydrophobic surfaces.     

Taxonomic value of a betaine lipid in Ectocarpus (Phaeophyceae) and the first record of a sexual population of E. fasciculatus for the Pacific Ocean

The betaine lipid DGTA differentiates between two species of Ectocarpus: it is present in E. fasciculatus Harvey, and lacking in E. siliculosus (Dillwyn) Lyngbye, Two ectocarpoid isolates from the coast of Chile, which could not be identified to species level, were found to belong to opposite DGTA types. Culture experiments showed that these plants were sporophytes. Their meiospores produced gametophytes of the species predicted by the lipid analysis. Promoted by this new approach, a sexual population of Ectocarpus fasciculatus has been detected for the first time in the Pacific Ocean.     

中国水云科Ectocarpaceae新记录Ⅱ

继续报道中国水云科新记录7种。毛果水云Ectocarpus dasycarpus,束生水云E. fasciculatus,小水云E. parvus,笔头水云E. penicillatus,群居水云E. socialis,丝状费氏藻Feldmannia filifera,壳状扭线Streblonema evagatnm。     

Cytoskeleton organisation during the infection of three brown algal species,Ectocarpus siliculosus,Ectocarpus crouaniorum and Pylaiella littoralis,by the intracellular marine oomycete Eurychasma dicksonii

Oomycete diseases in seaweeds are probably widespread and of significant ecological and economic impact, but overall still poorly understood. This study investigates the organisation of the cytoskeleton during infection of three brown algal species, Pylaiella littoralis, Ectocarpus siliculosus, and Ectocarpus crouaniorum, by the basal marine oomycete Eurychasma dicksonii. Immunofluorescence staining of tubulin revealed how the development of this intracellular biotrophic pathogen impacts on microtubule (MT) organisation of its algal host. The host MT cytoskeleton remains normal and organised by the centrosome until very late stages of the infection. Additionally, the organisation of the parasite's cytoskeleton was examined. During mitosis of the E. dicksonii nucleus the MT focal point (microtubule organisation centre, MTOC, putative centrosome) duplicates and each daughter MTOC migrates to opposite poles of the nucleus. This similarity in MT organisation between the host and pathogen reflects the relatively close phylogenetic relationship between oomycetes and brown algae. Moreover, actin labelling with rhodamine‐phalloidin in E. dicksonii revealed typical images of actin dots connected by fine actin filament bundles in the cortical cytoplasm. The functional and phylogenetic implications of our observations are discussed.     

Marine Brown Algae Requiring Vitamin B12

The brown algae Lithosiphon pusillus, Ectocarpus fasciculatus and Pylaiella litoralis were cultivated in bacteria-free cultures in artificial sea water, ASP 6F. The growth was tested with different additions of vitamins and other metabolites. Lithosiphon pusillus and Ectocarpus fasciculatus were found to require vitamin B₁₂ for optimal growth. The zoospores of Pylaiella litoralis, when cultured in medium Asp 6F with kinetin added, had an absolute requirement for vitamin B₁₂.     

Qualitative and quantitative determination of pheromone secretion in female gametes of Ectocarpus siliculosus (Phaeophyceae)

Pheromone secretion in living female gametes of Ectocarpus siliculosus was examined by closed-loop extraction and gas chromatography. The pheromone (ectocarpene) recovery efficiency was calibrated with synthetic ectocarpene and was found to depend strongly on the volume of the closed-loop system. Maximum extraction efficiency of ectocarpene (17.4%) was obtained with a small-volume system. In addition to the main compound ectocarpene, the pheromone bouquet of E. siliculosus contained minor amounts of hormosirene, multifidene and dictyotene, which until now are known only as sperm-attractants in other brown algae. The pheromone bouquet was identical in 4 clones of different geographic origins. Settled female gametes of Ectocarpus siliculosus continued to secrete pheromone and to attract male gametes for up to 7 h after their release with an ectocarpene secretion rate of 1 x 10(5) molecules s-1 cell-1.     

Reinstatement of Ectocarpus crouaniorum Thuret in Le Jolis as a third common species of Ectocarpus (Ectocarpales,Phaeophyceae) in Western Europe,and its phenology at Roscoff,Brittany

Based on morphological characters, cross‐fertility and molecular systematics, two species are currently recognized in the ubiquitous temperate brown algal genus Ectocarpus: the type species E. siliculosus (Dillwyn) Lyngbye and E. fasciculatus Harvey. We studied diversity, cross‐fertility and ecology of Ectocarpus in megatidal areas in northwest France (Western Europe) and propose to reinstate a third species, E. crouaniorum Thuret in Le Jolis. Genotyping of 67 individuals from five localities, including the type locality of E. crouaniorum, using internal transcribed spacer 1 (ITS1) length as a marker, showed that the three species co‐occurred whenever the habitat was suitable. Our survey also revealed a single putative field hybrid between E. crouaniorum and E. siliculosus, and a single individual of a further Ectocarpus genotype. In laboratory experiments, E. crouaniorum was crossed with E. siliculosus and E. fasciculatus. In 12 of 13 crosses, the zygotes did not develop (postzygotic sterility); in one experiment a viable hybrid was produced after crossing a female E. crouaniorum with a male E. siliculosus, but this hybrid was unable to form meiospores. Phylogenetic analysis of five molecular markers from the nuclear, mitochondrial and plastid genomes (in total 1818 bp) confirmed genetic separation of the three species. Ecologically, E. crouaniorum was confined to high intertidal pools and run‐offs, where the gametophyte was common from spring to summer. Another characteristic was that it usually occurred as an epiphyte of up to 12 cm in length on erect thalli of Scytosiphon lomentaria. Sporophytes of E. crouaniorum were found all year long; they were <3 cm in size or microscopic and were epilithic in the same habitat. The presence of a third species of Ectocarpus in Western Europe suggests that species diversity in this genus is larger than recognized during the last 40 years.     

SEXUAL ISOLATION BETWEEN A EUROPEAN AND AN AMERICAN POPULATION OF ECTOCARPUS SILICULOSUS (PHAEOPHYTA)1

Sexual plants of Northeastern American Ectocarpus siliculosus (Dilhw.) Lyngb. have been isolated and cultured. Female gametes produce a volatile sex hormone acting on male gametes. Combination of American and European gametes shows that the initial step of the sexual reaction (attraction of male gametes) works normally, whereas interaction of cell surfaces and fusion of gametes is prevented.     

Inhibition of Ectocarpus siliculosus infestations with copper chloride in tank cultures of Gracilaria gracilis

This study investigated copper chloride as an inhibitor of infestations of Ectocarpus siliculosus in Gracilariagracilis (Stackhouse) Steentoft, Irvine and Farnham grown in outdoor tank cultures. Copper at concentrations of 400 and 800 μg L^-1 Cu²⁺ inhibited E. siliculosusin one experiment. A reduction in the specific growth rates of G. gracilis was found for both concentrations of copper chloride in two experiments. G. gracilis recovered completely with in three weeks in one experiment, while no recovery was found in the other. Three and 5 weeks after treatment with low and high concentrations of copper chloride respectively the specific growth rates of G. gracilis was higher than that of the control in one experiment. A feeding experiment was conducted to determine the effect of copper-treated G. gracilis, used as fodder, on the growth rates of Haliotes midae (abalone) of two size classes. An isolated occurrence of growth rate inhibition of H. midae could be demonstrated in both size classes 3 months after the startof the experiment. This revised version was published online in August 2006 with corrections to the Cover Date.     

IMMUNOLOCALIZATION OF CENTER IN THE FLAGELLAR APPARATUS OF MALE GAMETES OF ECTOCARPUS SILICULOSUS (PHAEOPHYCEAE) AND OTHER BROWN ALGAL MOTILE CELLS1

Centrin or a centrin homologue was localized using immunofluorescence in the flagellar basal body region in zoids of five brown algal species: Ectocarpus siliculosus (Dillw.) Lyngb., Scytosiphon lomentaria (Lyngb.) Link, Laminaria digitata (Huds.) Lamour., Sphacelaria rigidula (Kütz.) Prud'homme van Reine, and Fucus serratus L. The antigen is restricted to short rods extending along the basal body(ies) and towards the nucleus, which always remains firmly linked to the flagellar apparatus in isolated cytoskeletons. To identify these antigenic sites, pre- and postembedding immunogold electron microscopy was applied to male gametes of E. siliculosus. At least three different structures associated with the basal bodies were antigenic: a fibrous structure connecting the proximal end of the posterior basal body to the nucleus (nucleus-basal body connector), a striated band that links the two basal bodies to each other and is located in the angel formed by them, and amorphous material interconnecting the basal bodies in their most proximal regions. In addiction, specific labeling occurs along the external surface and within the lumen of both basal bodies and in the flagellar transitional region. The possible function of these centrin-containing structures is discussed.     

Sphondylothamnion multifidum (Huds.) Näg. in western Europe

The lipid composition of 140 clonal isolates of brown algae, covering 16 species of the genera Ectocarpus, Feldmannia and Hincksia, was analysed. All taxa contained the glycolipids monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerol, and the phospholipids phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and an unidentified PX. Presence of the betaine lipid diacylglycerylhydroxymethyltrimethyl-β-alanine (DGTA) was strongly correlated with certain species in the genera Hincksia and Ectocarpus. While seven species of Hincksia contained DGTA, this lipid was absent in H. granulosa and in an unidentified Hincksia species. Due to heteromorphy of generations, species assignments in the genus Ectocarpus were made only for isolates for which the complete life history was known. We found that E. fasciculatus contains DGTA, while this lipid is absent in E. siliculosus. A collection of 40 non-sexual Ectocarpus strains included representatives with and without DGTA. The value of DGTA as a taxonomic marker in Ectocarpus and Hincksia is discussed.     

PASSAGE OF A MARINE BROWN ALGAL DNA VIRUS FROM ECTOCARPUS FASCICULATUS (ECTOCARPALES,PHAEOPHYCEAE) TO MYRIOTRICHIA CLAVAEFORMIS (DICTYOSIPHONALES,PHAEOPHYCEAE): INFECTION SYMPTOMS AND RECOVERY1

A dsDNA virus (EfasV-1) isolated from Ectocarpus fasciculatus Harvey infected Myriotrichia clavaeformis Harvey, a species belonging to a different brown algal order. The virus did not complete its infection cycle in the foreign host but caused infertility due to malformed reproductive structures. After some time in culture, the host's reproductive capacity was sometimes restored with concomitant loss of at least part of the viral genome. This incidence of interordinal virus transfer is discussed in relation to possibilities for virus-mediated horizontal gene transfer in brown algae.     

A Virus Infection in the Marine Brown Alga Ectocarpus siliculosus (Phaeophyceae)

Laboratory cultures of Ectocarpus siliculosus originating from New Zealand showed a defect in gametangium formation. Nuclear divisions in gametangium initials are not followed by cell wall formation. In the resulting multinucleate cells nuclear DNA increases dramatically, and nuclear membranes disintegrate. Eventually, the entire structure is filled with hexagonal particles of approximately 130 nm diameter. These were isolated and shown by EM to consist of a dense core surrounded by a 3-layered shell. They are released into the culture medium when the host cells burst. Ectocarpus gametes from healthy cultures could be infected by these particles. The resulting partheno-sporophytes developed pathological symptoms, suggesting that the particles are viruses. The expression of the defect is temperature dependent. At 10°C all gametangia are abnormal, while between 15 and 20 °C defective and normal gametangia and gametes are formed on the same plant. Partheno-sporophytes developing from such gametes carry the viral particles expressed in deformed unilocular and plurilocular sporangia.     

中国水云科藻类的新记录

本文报道了中国分布的水云科(褐藻门)藻类3个新记录种,即束枝水云弯枝变种;笔头状水云;石鳖费氏藻。     

The performance of aminoalkyl/fluorocarbon/hydrocarbon-modified xerogel coatings against the marine alga Ectocarpus crouaniorum: relative roles of surface energy and charge

The effect of a series of xerogel coatings modified with aminoalkyl/fluorocarbon/hydrocarbon groups on the adhesion of a new test species, the filamentous brown alga Ectocarpus crouaniorum, has been explored, and compared with the green alga Ulva linza. The results showed that E. crouaniorum adhered weakly to the less polar, low wettability coatings in the series, but stronger adhesion was shown on polar, higher surface energy coatings containing aminoalkyl groups. The results from a separate series of coatings tuned to have similar surface energies and polarities after immersion in artificial seawater (ASW), but widely different surface charges, demonstrated that surface charge was more important than surface energy and polarity in determining the adhesion strength of both E. crouaniorum and U. linza on xerogel coatings. No correlation was found between adhesion and contact angle hysteresis. X-ray photoelectron spectroscopy analysis of samples after immersion in ASW confirmed the presence of charged ammonium groups on the surface of the aminoalkylated coatings.     

Immunofluorescence Study of Microtubule Organization in Some Polarized Cell Types of Selected Brown Algae

The microtubule (Mt) organization in apical cells of Sphacelaria rigidula. as well as in branch initials of S. rigidula and Ectocarpus siliculosus, was studied by immunofluorescence. The apical interphase cells of S. rigidula show an impressive cytoskeleton of Mts, converging on the centrosome(s). A number of Mt bundles are perinuclear, but most of them run in axial orientation from the centrosomes to the cell cortex. The anterior Mt system consists of numerous thin Mt bundles, whereas the posterior system contains fewer and thicker bundles. In cells entering prophase, the cytoplasmic Mts gradually disappear. This process is somewhat faster at the posterior than at the anterior pole of the premitotic nucleus. After mitosis, the cytoplasmic Mts of the apical region appear to be re-organized more rapidly than those of the basal part of the cell. The apical daughter nucleus retains a lobed shape and condensed chromatin for a longer time, and increases considerably in size between telophase and cytokinesis, compared to the basal one. Duplication of the centrosomes proceeds more rapidly in the anterior region of apical cells than in the basal part. During branch formation in S. rigidula and E. siliculosus, a new polarity axis is established, and the Mts extend towards the protrusion into which the nucleus migrates before mitosis. After nuclear division, one of the daughter nuclei is positioned at the tip of the branch, where the apical Mt focussing point is localized.     

The ecology of some free-living ectocarpaceae

Summary 1. The work concerning the ecology of free-living marine algae is reviewed briefly.2. The species examined in this investigation are the PhaeophyceaeEctocarpus fasciculatus, E. siliculosus andPilayella littoralis.3. From culture experiments using excised filament fragments as inoculum it is shown that these species can regenerate new tissue from detached pieces.4. The longevity of the free-living plants in nature is assessed using the apparatus illustrated in Figures 1 and 2 and found to be in the region of two months.5. Free-living plants may propagate vegetatively by fragmentation but they also continue to produce unilocular and plurilocular sporangia while in this condition. The fertility of the free-living plants is usually lower than in attached plants.6. The main morphological effects of the free-living state are increase in angle of branch emergence, reduction in length of lateral branches and loss of branch pattern. This is accompanied by reduction in size of reproductive bodies. It is shown thatE. distortus andE. landsburgii constitute free-living ecads ofE. fasciculatus.7. The role of free-livingEctocarpus andPilayella in the entire species populations is thought to be minor but it is suggested that they might form a sublittoral reservoir of material in summer when intertidal conditions may be exacting.

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Die Ökologie einiger freilebender Ectocarpaceae

Kurzfassung Die BraunalgenEctocarpus fasciculatus, E. siliculosus undPilayella littoralis sind auch nach Loslösung von ihrem Substrat wachstumsfähig. Im Experiment wurden abgeschnittene Fadenstücke in einer Vorrichtung weiterkultiviert, die in Seewasser untergetaucht und so beschaffen war, daß die Lebensbedingungen so weit als möglich denen freilebender Algen entsprachen. Unter diesen Bedingungen bleiben alle drei Arten fertil und bilden uni- und plurilokuläre Sporangien aus. Diese sind jedoch kleiner, so daß weniger Schwärmer erzeugt werden als bei den festsitzenden Algen. Die losgelösten Pflanzen können sich auch vegetativ durch Fragmentation vermehren. Die Morphologie wird durch den freilebenden Zustand in folgender Weise verändert: Die Seitenzweige sind kürzer als bei den festgewachsenen Pflanzen und stehen in einem größeren Winkel zur Hauptachse. Das typische Verzweigungsmuster geht mehr oder weniger verloren; dies prägt sich beiE. fasciculatus am stärksten aus. Es wurde festgestellt, daßE. distortus undE. landsburgii freilebenden Modifikationen vonE. fasciculatus sind. Morphologische Zwischenformen entstehen, wenn freilebende Pflanzen sich mit ständig sublitoral lebenden Algen verflechten. Die interkalaren Sporangien vonP. littoralis werden terminal oder subterminal in einer ähnlichen Stellung wie beiP. varia angelegt. Die Lebensdauer der freilebenden Algen dürfte zwei Monate erreichen, doch werden die Pflanzen unter natürlichen Verhältnissen offensichtlich schon früher an den Strand gespült.