Inter-specific differences in responses to copper by natural populations of Ectocarpus

Two species of Ectocarpus, E. siliculosus (Dillw.) Lyngb. and E. fasciculatus Harv., occupying the same habitat at Port St Mary, Isle of Man, and apparently subject to the same environmental conditions, exhibit markedly different responses to copper.

The greater tolerance shown by E. siliculosus is unlikely to be attributable to direct local selection, but may result from immigration of copper-tolerant individuals from highly selective habitats nearby. E. siliculosus would appear to be a more variable species than E. fasciculatus, thereby being more readily affected by the forces of natural selection.     

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.     

Evolution and maintenance of haploid–diploid life cycles in natural populations: The case of the marine brown alga Ectocarpus

The evolutionary stability of haploid–diploid life cycles is still controversial. Mathematical models indicate that niche differences between ploidy phases may be a necessary condition for the evolution and maintenance of these life cycles. Nevertheless, experimental support for this prediction remains elusive. In the present work, we explored this hypothesis in natural populations of the brown alga Ectocarpus. Consistent with the life cycle described in culture, Ectocarpus crouaniorum in NW France and E. siliculosus in SW Italy exhibited an alternation between haploid gametophytes and diploid sporophytes. Our field data invalidated, however, the long‐standing view of an isomorphic alternation of generations. Gametophytes and sporophytes displayed marked differences in size and, conforming to theoretical predictions, occupied different spatiotemporal niches. Gametophytes were found almost exclusively on the alga Scytosiphon lomentaria during spring whereas sporophytes were present year‐round on abiotic substrata. Paradoxically, E. siliculosus in NW France exhibited similar habitat usage despite the absence of alternation of ploidy phases. Diploid sporophytes grew both epilithically and epiphytically, and this mainly asexual population gained the same ecological advantage postulated for haploid–diploid populations. Consequently, an ecological interpretation of the niche differences between haploid and diploid individuals does not seem to satisfactorily explain the evolution of the Ectocarpus life cycle.     

Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales,Phaeophyceae)

The genus Ectocarpus (Ectocarpales, Phaeophyceae) contains filamentous algae widely distributed in marine and estuarine habitats of temperate regions in both hemispheres. While E. siliculosus has become a model organism for genomics and genetics of the brown macroalgae, accurate species delineation, distribution patterns and diversity for the genus Ectocarpus remain problematic. In this study, we used three independent species delimitation approaches to generate a robust species hypothesis for 729 Ectocarpus specimens collected mainly along the European and Chilean coasts. These approaches comprised phylogenetic reconstructions and two bioinformatics tools developed to objectively define species boundaries (General Mixed Yule Coalescence Method and Automatic Barcode Gap Discovery). Our analyses were based on DNA sequences of two loci: the mitochondrial cytochrome oxidase subunit 1 and the nuclear internal transcribed spacer 1 of the ribosomal DNA. Our analyses showed the presence of at least 15 cryptic species and suggest the existence of incomplete lineage sorting or introgression between five of them. These results suggested the possible existence of different levels of reproductive barriers within this species complex. We also detected differences among species in their phylogeographic patterns, range and depth distributions, which may suggest different biogeographic histories (e.g., endemic species or recent introductions).     

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.     

Worldwide occurrence of virus-infections in filamentous marine brown algae

Virus infections were detected inEctocarpus siliculosus andEctocarpus fasciculatus on the coasts of Ireland, California, Peru, southern South America, Australia and New Zealand; in threeFeldmannia species on the coasts of Ireland, continental Chile and Archipelago Juan Fernandez (Chile); and inLeptonematella from Antarctica. Natural populations on the Irish coast contained 3% infected plants inE. fasciculatus, and less than 1% inFeldmannia simplex. On the Californian coast, 15 to 25% ofEctocarpus isolates were infected. Virus symptoms were absent inE. siliculosus from Peru, but appeared after meiosis in laboratory cultures. The virus particles inE. fasciculatus are identical in size and capsid structure to those reported forE. siliculosus, while the virus inF. simplex is smaller and has a different envelope. Our findings suggest that virus infections are a common and worldwide phenomenon in filamentous brown algae.     

Hybridization between two cryptic filamentous brown seaweeds along the shore: analysing pre‐ and postzygotic barriers in populations of individuals with varying ploidy levels

We aimed to study the importance of hybridization between two cryptic species of the genus Ectocarpus, a group of filamentous algae with haploid–diploid life cycles that include the principal genetic model organism for the brown algae. In haploid–diploid species, the genetic structure of the two phases of the life cycle can be analysed separately in natural populations. Such life cycles provide a unique opportunity to estimate the frequency of hybrid genotypes in diploid sporophytes and meiotic recombinant genotypes in haploid gametophytes allowing the effects of reproductive barriers preventing fertilization or preventing meiosis to be untangle. The level of hybridization between E. siliculosus and E. crouaniorum was quantified along the European coast. Clonal cultures (568 diploid, 336 haploid) isolated from field samples were genotyped using cytoplasmic and nuclear markers to estimate the frequency of hybrid genotypes in diploids and recombinant haploids. We identified admixed individuals using microsatellite loci, classical assignment methods and a newly developed Bayesian method (XPloidAssignment), which allows the analysis of populations that exhibit variations in ploidy level. Over all populations, the level of hybridization was estimated at 8.7%. Hybrids were exclusively observed in sympatric populations. More than 98% of hybrids were diploids (40% of which showed signs of aneuploidy) with a high frequency of rare alleles. The near absence of haploid recombinant hybrids demonstrates that the reproductive barriers are mostly postzygotic and suggests that abnormal chromosome segregation during meiosis following hybridization of species with different genome sizes could be a major cause of interspecific incompatibility in this system.     

Sexual reproduction in British Ectocarpus Siliculosus (Phaeophyta)

One male and one female gametophyte of Ectocarpus siliculosus have been detected in a sample of 19 plants taken from the Isle of Man. Fertilization takes place between gametes formed in plurilocular gametangia. Clear cross-fertility exists with Mediterranean strains. This result confirms the notion that the concept of the gametic character of meiospores in Ectocarpus should be abandoned.     

A myrionemoid variant of Ectocarpus fasciculatus Harv.

Culture experiments on Ectocarpus fasciculatus Harv. have been carried out using material from three separate localities. Under certain conditions, which have yet to be exactly defined, sporelings develop into plants quite unlike ‘normal’ Ectocarpus but resembling Myrionema corunnae Sauv. Reference is made to a field observation of a similar phenomenon by Jaasund (1965) and the status of M. corunnae is discussed.     

The occurrence of variant forms in cultures of species of Ectocarpus and Giffordia

Apparent variants, resembling Hecatonema sp., have been isolated from cultures of Ectocarpus fasciculatus Harv., Ectocarpus siliculosus (Dillw.) Lyngb., Giffordia irregularis (Kütz.) Le Jol. and Giffordia mitchellae (Harv.) Hamel. The origin and life history of the hecatonemoid forms is described. Their possible roles are discussed and an argument is presented in support of the hypothesis that they are contaminants.     

Revisiting Australian Ectocarpus subulatus (Phaeophyceae) From the Hopkins River: Distribution,Abiotic Environment,and Associated Microbiota

In 1995 a strain of Ectocarpus was isolated from Hopkins River Falls, Victoria, Australia, constituting one of few available freshwater or nearly freshwater brown algae, and the only one belonging to the genus Ectocarpus. It has since been used as a model to study acclimation and adaptation to low salinities and the role of its microbiota in these processes. To provide more background information on this model, we assessed if Ectocarpus was still present in the Hopkins river 22 years after the original finding, estimated its present distribution, described its abiotic environment, and determined its in situ microbial composition. We sampled for Ectocarpus at 15 sites along the Hopkins River as well as 10 neighboring sites and found individuals with ITS and cox1 sequences identical to the original isolate at three sites upstream of Hopkins River Falls. The salinity of the water at these sites ranged from 3.1 to 6.9, and it was rich in sulfate (1–5 mM). The diversity of bacteria associated with the algae in situ (1312 operational taxonomic units) was one order of magnitude higher than in previous studies of the original laboratory culture, and 95 alga-associated bacterial strains were isolated from algal filaments on site. In particular, species of Planctomycetes were abundant in situ but rare in laboratory cultures. Our results confirmed that Ectocarpus was still present in the Hopkins River, and the newly isolated algal and bacterial strains offer new possibilities to study the adaptation of Ectocarpus to low salinity and its interactions with its microbiome.     

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.     

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.

     

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.     

Copper stress proteomics highlights local adaptation of two strains of the model brown alga Ectocarpus siliculosus

Ectocarpus siliculosus is a cosmopolitan brown alga with capacity to thrive in copper enriched environments. Analysis of copper toxicity was conducted in two strains of E. siliculosus isolated from (i) an uncontaminated coast in southern Peru (Es32) and (ii) a copper polluted rocky beach in northern Chile (Es524). Es32 was more sensitive than Es524, with toxicity detected at 50 μg/L Cu, whereas Es524 displayed negative effects only when exposed to 250 μg/L Cu. Differential soluble proteome profiling for each strain exposed to sub‐lethal copper levels allowed to identify the induction of proteins related to processes such as energy production, glutathione metabolism as well as accumulation of HSPs. In addition, the inter‐strain comparison of stress‐related proteomes led to identify features related to copper tolerance in Es524, such as striking expression of a PSII Mn‐stabilizing protein and a Fucoxanthine chlorophyll a–c binding protein. Es524 also expressed specific stress‐related enzymes such as RNA helicases from the DEAD box families and a vanadium‐dependent bromoperoxidase. These observations were supported by RT‐qPCR for some of the identified genes and an enzyme activity assay for vanadium‐dependent bromoperoxidase. Therefore, the occurrence of two different phenotypes within two distinct E. siliculosus strains studied at the physiological and proteomic levels strongly suggest that persistent copper stress may represent a selective force leading to the development of strains genetically adapted to copper contaminated sites.     

The genome‐scale metabolic network of Ectocarpus siliculosus (EctoGEM): a resource to study brown algal physiology and beyond

Brown algae (stramenopiles) are key players in intertidal ecosystems, and represent a source of biomass with several industrial applications. Ectocarpus siliculosus is a model to study the biology of these organisms. Its genome has been sequenced and a number of post‐genomic tools have been implemented. Based on this knowledge, we report the reconstruction and analysis of a genome‐scale metabolic network for E. siliculosus, EctoGEM ( http://ectogem.irisa.fr ). This atlas of metabolic pathways consists of 1866 reactions and 2020 metabolites, and its construction was performed by means of an integrative computational approach for identifying metabolic pathways, gap filling and manual refinement. The capability of the network to produce biomass was validated by flux balance analysis. EctoGEM enabled the reannotation of 56 genes within the E. siliculosus genome, and shed light on the evolution of metabolic processes. For example, E. siliculosus has the potential to produce phenylalanine and tyrosine from prephenate and arogenate, but does not possess a phenylalanine hydroxylase, as is found in other stramenopiles. It also possesses the complete eukaryote molybdenum co‐factor biosynthesis pathway, as well as a second molybdopterin synthase that was most likely acquired via horizontal gene transfer from cyanobacteria by a common ancestor of stramenopiles. EctoGEM represents an evolving community resource to gain deeper understanding of the biology of brown algae and the diversification of physiological processes. The integrative computational method applied for its reconstruction will be valuable to set up similar approaches for other organisms distant from biological benchmark models.     

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.