Urban Tillmann

Azadinium spinosum gen. et sp. nov. (Dinophyceae) identified as a primary producer of azaspiracid toxins.

Azaspiracids (AZAs) are a group of lipophilic marine biotoxins associated with human incidents of shellfish poisoning. During a research cruise to the North Sea, we analysed size-fractionated plankton for AZA by mass spectrometry and successfully isolated an AZA-producing dinoflagellate from the east coast of Scotland. As shown previously, an axenic culture of this dinoflagellate produces AZA 1, AZA 2 and an isomer of AZA 2. Here we give a taxonomic description of this new taxon Azadinium spinosum gen. et sp. nov., as a de novo producer of AZAs. Azadinium spinosum is a small (12–16 µm length and 7–11 µm width) peridinin-containing photosynthetic dinoflagellate with a superficial resemblance under light microscopy to gymnodinioids, but with a thin theca. The large nucleus is spherical and located posteriorly, whereas the single chloroplast is parietal, lobed, and typically extends into both the epi- and hyposome. The Kofoidian thecal tabulation is APC, 4′, 3a, 6″, 6C, 5?S, 6′″, 2″″. This plate pattern has an epithecal affinity to the Peridiniales and a hypothecal affinity to the Gonyaulacales, but is distinctly different from described dinoflagellate genera. The assignment of A. spinosum to the dinoflagellates is supported by molecular phylogenetic analysis of four genes, SSU rDNA, LSU rDNA (D1/D2 region), ITS and cytochrome oxidase (sub-unit 1) (COI). In agreement with the morphological description, phylogenetic analysis did not show any particularly close affiliation to the Peridiniales or Gonyaulacales, nor to any other dinoflagellate order represented in molecular databases. Consequently, we erected a new genus, Azadinium, for this taxon. However the ordinal affiliation of the genus is uncertain. This study represents the first description and confirmation of a new dinoflagellate species capable of producing AZA and is thus an important advance in surveillance programmes for toxigenic microalgae and toxins of human health significance.

PHENOTYPIC VARIATION AND GENOTYPIC DIVERSITY IN A PLANKTONIC POPULATION OF THE TOXIGENIC MARINE DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE)1

Multiple clonal isolates from a geographic population of Alexandrium tamarense (M. Lebour) Balech from the North Sea exhibited high genotypic and phenotypic variation. Genetic heterogeneity was such that no clonal lineage was repeatedly sampled according to genotypic markers specified by amplified fragment length polymorphism (AFLP) and microsatellites. Subsampling of genotypic data from both markers showed that ordination of individuals by pair‐wise genetic dissimilarity indices was more reliable by AFLP (482 biallelic loci) than by microsatellites (18 loci). However, resulting patterns of pair‐wise genetic similarities from both markers were significantly correlated (Mantel test P < 0.005). The composition of neurotoxins associated with paralytic shellfish poisoning (PSP) was also highly diverse among these isolates and allowed clustering of toxin phenotypes based on prevalence of individual toxins. Correlation analysis of pair‐wise relatedness of individual clones according to PSP‐toxin profiles and both genotypic characters failed to yield close associations. The expression of allelochemical properties against the cryptophyte Rhodomonas salina (Wisłouch) D. R. A. Hill et Wetherbee and the predatory dinoflagellate Oxyrrhis marina Dujard. manifested population‐wide variation of responses in the target species, from no visible effect to complete lysis of target cells. Whereas the high genotypic variation indicates high potential for adaptability of the population, we interpret the wide phenotypic variation as evidence for lack of strong selective pressure on respective phenotypic traits at the time the population was sampled. Population markers as applied here may elucidate the ecological significance of respective traits when followed under variable environmental conditions, thereby revealing how variation is maintained within populations.

Implications of life‐history transitions on the population genetic structure of the toxigenic marine dinoflagellate Alexandrium tamarense

Genotypic or phenotypic markers for characterization of natural populations of marine microalgae have typically addressed questions regarding differentiation among populations, usually with reference to a single or few clonal isolates. Based upon a large number of contemporaneous isolates from the same geographical population of the toxigenic species Alexandrium tamarense from the North Sea, we uncovered significant genetic substructure and low but significant multilocus linkage disequilibrium (LD) within the planktonic population. Between the alternative molecular genotyping approaches, only amplified fragment length polymorphism (AFLP) revealed cryptic genetic population substructure by Bayesian clustering, whereas microsatellite markers failed to yield concordant patterns. Both markers, however, gave evidence for genetic differentiation of population subgroups as defined by AFLP. A considerable portion of multilocus LD could be attributed to population subdivision. The remaining LD within population subgroups is interpreted as an indicator of frequency shifts of clonal lineages during vegetative growth of planktonic populations. Phenotypic characters such as cellular content and composition of neurotoxins associated with paralytic shellfish poisoning (PSP) and allelochemical properties may contribute to intra‐ or inter‐annual differentiation of planktonic populations, if clonal lineages that express these characters are selectively favoured. Nevertheless, significant phenotypic differentiation for these characters among the genetically differentiated subgroups was only detected for PSP toxin content in two of the four population subgroups. By integrating the analysis of phenotypic and genotypic characteristics, we developed a conceptual population genetic model to explain the importance of life‐cycle dynamics and transitions in the evolutionary ecology of these dinoflagellates.

The Relevance of Marine Chemical Ecology to Plankton and Ecosystem Function: An Emerging Field

Marine chemical ecology comprises the study of the production and interaction of bioactive molecules affecting organism behavior and function. Here we focus on bioactive compounds and interactions associated with phytoplankton, particularly bloom-forming diatoms, prymnesiophytes and dinoflagellates. Planktonic bioactive metabolites are structurally and functionally diverse and some may have multiple simultaneous functions including roles in chemical defense (antipredator, allelopathic and antibacterial compounds), and/or cell-to-cell signaling (e.g., polyunsaturated aldehydes (PUAs) of diatoms). Among inducible chemical defenses in response to grazing, there is high species-specific variability in the effects on grazers, ranging from severe physical incapacitation and/or death to no apparent physiological response, depending on predator susceptibility and detoxification capability. Most bioactive compounds are present in very low concentrations, in both the producing organism and the surrounding aqueous medium. Furthermore, bioactivity may be subject to synergistic interactions with other natural and anthropogenic environmental toxicants. Most, if not all phycotoxins are classic secondary metabolites, but many other bioactive metabolites are simple molecules derived from primary metabolism (e.g., PUAs in diatoms, dimethylsulfoniopropionate (DMSP) in prymnesiophytes). Producing cells do not seem to suffer physiological impact due to their synthesis. Functional genome sequence data and gene expression analysis will provide insights into regulatory and metabolic pathways in producer organisms, as well as identification of mechanisms of action in target organisms. Understanding chemical ecological responses to environmental triggers and chemically-mediated species interactions will help define crucial chemical and molecular processes that help maintain biodiversity and ecosystem functionality.

The importance of sediments in the transformation and turnover of nutrients and organic matter in the Wadden Sea and German Bight

From 1994 through 1996 transformation processes in the water column of the German Bight and the adjacent Wadden Sea were investigated in the projects TRANSWATT and KUSTOS. On the basis of a review of carbon and nutrient budgets we examine the role of processes in the sediment for overall carbon and nutrient cycling in the Wadden Sea and adjacent German Bight. We distinguish two aspects: the sediment as the site where organic matter is rapidly turned over and the sediment as the site where organic matter and nutrients are immobilized. The relative importance of the sediment for the remineralisation of organic matter depends on the water depth: The review of carbon budgets suggests that in the Wadden Sea (2 - 3 m) about 50% of the remineralisation occurs in the sediment. In the German Bight (20 m), 10 - 20% of the primary production is remineralised in the sediment. The budgets further show that the Wadden Sea is heterotrophic. About 100 gC m-2 y1 is imported from the coastal zone. This implies a net autotrophy of the coastal zone, which is in line with the results from the projects TRANSWATT and KUSTOS. Within the Wadden Sea, organic matter has to be turned over two to three times and in the German Bight three to four times to explain the annual primary production. This is lower than in the offshore North Sea where annual turnover rates up to five have been found. Several processes remove nutrients on longer time-scales from the biogeochemical cycle. The importance of the local formation of phosphorus containing minerals like apatite as a phosphorus sink is shown. A discussion of several denitrification estimates concludes that in the German Bight and adjacent Wadden Sea on average about 8 -16% of the total nitrogen influx (from the coastal zone, from rivers and via the atmosphere) is lost to the atmosphere.

Azadinium obesum (Dinophyceae), a new nontoxic species in the genus that can produce azaspiracid toxins

Tillmann U., Elbrächter M., John U., Krock B. and Cembella A. 2010. Azadinium obesum (Dinophyceae), a new nontoxic species in the genus that can produce azaspiracid toxins. Phycologia 49: 169–182. DOI: 10.2216/09-35.1 The novel dinoflagellate taxon we describe here as Azadinium obesum sp. nov. was isolated as clone 2E10 from the North Sea along the Scottish east coast, the same locality as for Azadinium spinosum Elbrächter et Tillmann, the type and formerly only species for this genus. In contrast to A. spinosum, a known producer of azaspiracid (AZA) toxins, the isolate of A. obesum produces no known AZA analogues detectable by liquid chromatography coupled with tandem mass spectrometry. Azadinium obesum is a small (13–18 µm length; 10–14 µm width) photosynthetic dinoflagellate with a thin theca exhibiting the Kofoidean plate tabulation: Po, cp, X, 4′, 3a, 6″, 6C, 5?S, 6′″, 2″″. This species is morphologically distinguished from A. spinosum by slightly larger mean cell size, consistent absence of an antapical spine, the lack of a stalked pyrenoid and several details of the plate configuration. Among these thecal features, the first precingular (1″) plate of A. obesum does not touch the first epithecal intercalary plate and is four sided rather than five sided as in A. spinosum. Furthermore, in A. obesum the lower half of the first apical (1′) plate is very narrow and tongue-like, and precingular plates 1″ and 6″ are very close together, whereas these diverge in A. spinosum. DNA sequence and phylogenetic analysis elucidates and supports the separation (but close affinity) of A. obesum and A. spinosum, as well as the description of the former as a distinct species. Phylogenetic interpretation of the four genes analysed – internal transcribed spacer, 18S rDNA, 28S rDNA (D1/D2) and cytochrome oxidase I – further validates the recently erected genus Azadinium Elbrächer et Tillmann but does not clarify the position of the genus with respect to higher taxonomic levels within the subclass Peridiniphycidae.

Preliminary Characterization of Extracellular Allelochemicals of the Toxic Marine Dinoflagellate Alexandrium tamarense Using a Rhodomonas salina Bioassay

Members of the marine dinoflagellate genus Alexandrium are known to exude allelochemicals, unrelated to well-known neurotoxins (PSP-toxins, spirolides), with negative effects on other phytoplankton and marine grazers. Physico/chemical characterization of extracellular lytic compounds of A. tamarense, quantified by Rhodomonas salina bioassay, showed that the lytic activity, and hence presumably the compounds were stable over wide ranges of temperatures and pH and were refractory to bacterial degradation. Two distinct lytic fractions were collected by reversed-phase solid-phase extraction. The more hydrophilic fraction accounted for about 2% of the whole lytic activity of the A. tamarense culture supernatant, while the less hydrophilic one accounted for about 98% of activity. Although temporal stability of the compounds is high, substantial losses were evident during purification. Lytic activity was best removed from aqueous phase with chloroform-methanol (3:1). A “pseudo-loss” of lytic activity in undisturbed and low-concentrated samples and high activity of an emulsion between aqueous and n-hexane phase after liquid-liquid partition are strong evidence for the presence of amphipathic compounds. Lytic activity in the early fraction of gel permeation chromatography and lack of activity after 5 kD ultrafiltration indicate that the lytic agents form large aggregates or macromolecular complexes.

A new non-toxic species in the dinoflagellate genus Azadinium: A. poporum sp. nov.

A new dinoflagellate species, Azadinium poporum sp. nov., was isolated as three clones from the southern North Sea off the Danish coast. In contrast to the type species A. spinosum, a known producer of azaspiracid (AZA) toxins, the isolates of A. poporum produce no known AZA analogues detectable by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Azadinium poporum is a small (11–16 µm length; 8–12 µm width) photosynthetic dinoflagellate with a thin theca exhibiting the Kofoidean plate tabulation: Po, cp, X, 4′, 3a, 6″. 6C, 5?S, 6′″, 2″″. This species is morphologically distinguished from the type A. spinosum by a slightly lower mean cell length/width ratio, consistent absence of an antapical spine, the presence of more than one stalked pyrenoid, and the conspicuous arrangement of the ventral pore, located at the junction of the pore plate and the first two apical plates. This latter feature also distinguishes A. poporum from A. obesum. As in A. spinosum, but differing from A. obesum, the first precingular (1″) plate of A. poporum touches the first epithecal intercalary plate 1a. DNA sequences and phylogenetic analysis of four nuclear-encoded genes, namely the internal transcribed spacer (ITS), 18S rDNA, 28S rDNA (D1/D2) and cytochrome oxidase I (COI), support the separation of A. poporum from (but with a close affinity to) other Azadinium species (A. obesum and A. spinosum), as well as its description as a distinct species within the recently erected genus Azadinium Elbrächter & Tillmann. Nevertheless, in spite of the phylogenetic consistency at the generic level, the position of the genus with respect to higher taxonomic levels within the subclass Peridiniphycidae could not be further clarified.

Amphidoma languida sp. nov. (Dinophyceae) reveals a close relationship between Amphidoma and Azadinium

Toxic algae such as Alexandrium and Azadinium have an important ecological impact and have originated several times independently within the dinophytes. Their closest relatives, however, are mostly unknown at present. A new dinophyte species, Amphidoma languida sp. nov., was isolated from Bantry Bay (Ireland) during a period of elevated azaspiracid toxicity in mussels. The new species was described in detail, and its phylogenetic position was analysed, by using a combination of light and electron microscopy, chemical detection methods, and sequence comparison of concatenated ribosomal RNA sequence data. Morphological similarities, such as cingular and hypothecal plates, the number and arrangement of sulcal plates, and the characteristic apical pore complex with a small X-plate centrally invading the first apical plate, indicated a close relationship between Amphidoma and Azadinium. However, no known azaspiracid analogues were detected in A. languida by liquid chromatography coupled with tandem mass-spectrometry. In a molecular phylogeny, the Amphidomataceae including Amphidoma and Azadinium were an independent lineage among other monophyletic major groups of the dinophytes such as the Suessiales, Prorocentrales, Gonyaulacales, and Peridiniales. Thus, the taxonomic affiliation of Azadinium is clarified, and our data may prove helpful in the development of specific and reliable molecular detection methods of toxic Azadinium.

First Report of the Photosynthetic Dinoflagellate Genus Azadinium in the Pacific Ocean: Morphology and Molecular Characterization of Azadinium cf. poporum

A strain of a dinoflagellate belonging to the genus Azadinium was obtained by the incubation of sediments collected from Shiwha Bay, Korea. This report of the genus Azadinium is the first outside of northern Europe and furthermore from the Pacific Ocean. The diagnostic morphological features of the isolate very closely resemble the recently described species Azadinium poporum isolated from the North Sea. However, the shape of the 3′ apical plate and the occasional morphological variations unreported from A. poporum bring minor distinctions between strains from different locations. The DNA sequences of small subunit, ITS, and large subunit (LSU) rDNA differed by 0.2%, 2.6%, and 3.6%, respectively, from those of A. poporum, whereas the COI gene was identical to those found in all strains of Azadinium. Phylogenetic analyses of the ribosomal DNA regions generally positioned the Korean strain as a sister taxon of A. poporum. However, the Korean isolate tends to occupy a basal position within Azadinium species with ITS rDNA and LSU rDNA. Using liquid chromatography coupled with tandem mass spectrometry, no known azaspiracids were detected. The slight but discernible morphological differences, the distinct rDNA sequences, and the tendency of the Korean strain to diverge phylogenetically based on ITS rDNA and LSU rDNA from A. poporum do not enable us to clearly assign the isolate to A. poporum. However, these characteristics do not allow us to classify it as a distinct species, and it is therefore designated as Azadinium cf. poporum. The examination of more strains to find more diagnostic characteristics might enable the attribution of this material to a well‐defined taxonomic position.