Living in a cloud: intraspecific facilitation by allelochemical mediated grazing protection 1 2

Abstract 23 Dinoflagellates are a major cause of harmful algal blooms, with consequences for coastal marine 24 ecosystem functioning and services. Alexandrium tamarense is one of the most abundant and 25 widespread toxigenic species in the temperate northern and southern hemisphere, and produces 26 paralytic shellfish poisoning toxins as well as lytic allelochemical substances. These bioactive 27 compounds may support the success of A. tamarense and its ability to form blooms. Here we 28 investigate grazing of highly (Alex2) and moderately (Alex4) allelochemical active A. tamarense 29 strains and a non-allelochemical active conspecific (Alex5) by the heterotrophic dinoflagellate 30 Polykrikos kofoidii in monoclonal and mixed experimental set-ups. While Alex4 and particularly 31 Alex5 were strongly grazed by P. kofoidii in monoclonal set-ups, both strains did grow well in 32 the mixed assemblages (Alex4+Alex5 and Alex2+Alex5). Hence, the allelochemical active 33 strains facilitate the non-active strain by protecting the population against grazing. This 34 allelochemical mediated facilitation resembles associational resistance observed in various 35 macroalgal and plant communities. Occurring intraspecifically it may partly explain the high 36 genotypic and phenotypic diversity of Alexandrium populations. It is furthermore conceivable 37 that these diverse populations comprise multiple cooperative traits that may support mutual 38 intraspecific facilitation, which in turn will promote the success of this notorious harmful algal 39 bloom species. 40 4

High Genetic Diversity and Fine-Scale Spatial Structure in the Marine Flagellate Oxyrrhis marina (Dinophyceae) Uncovered by Microsatellite Loci

Free-living marine protists are often assumed to be broadly distributed and genetically homogeneous on large spatial scales. However, an increasing application of highly polymorphic genetic markers (e.g., microsatellites) has provided evidence for high genetic diversity and population structuring on small spatial scales in many free-living protists. Here we characterise a panel of new microsatellite markers for the common marine flagellate Oxyrrhis marina. Nine microsatellite loci were used to assess genotypic diversity at two spatial scales by genotyping 200 isolates of O. marina from 6 broad geographic regions around Great Britain and Ireland; in one region, a single 2 km shore line was sampled intensively to assess fine-scale genetic diversity. Microsatellite loci resolved between 1–6 and 7–23 distinct alleles per region in the least and most variable loci respectively, with corresponding variation in expected heterozygosities (He) of 0.00–0.30 and 0.81–0.93. Across the dataset, genotypic diversity was high with 183 genotypes detected from 200 isolates. Bayesian analysis of population structure supported two model populations. One population was distributed across all sampled regions; the other was confined to the intensively sampled shore, and thus two distinct populations co-occurred at this site. Whilst model-based analysis inferred a single UK-wide population, pairwise regional FST values indicated weak to moderate population sub-division (0.01–0.12), but no clear correlation between spatial and genetic distance was evident. Data presented in this study highlight extensive genetic diversity for O. marina; however, it remains a substantial challenge to uncover the mechanisms that drive genetic diversity in free-living microorganisms

Patterns of post-glacial genetic differentiation in marginal populations of a marine microalga

Peer reviewe

A Multi-Year Comparison of Spirolide Profiles in Planktonic Field Samples from the North Sea and Adjacent Waters

Alexandrium ostenfeldii isolates from distinct geographical locations showed almost identical profiles, primarily consisting of 20-methyl spirolide G (20-meG). Whereas the Scottish isolate produces only this variant, the Irish isolate additionally yields slight amounts of 13-desmethyl spirolide C (13-desmeC). These profiles were also reflected in the field data, where 20-meG was the most abundant spirolide throughout all samples and years.This work was financed by the HGF through the research programmes MARCOLPOLI and PACES and is part of the GEOHAB CORE research project on HABs in Fjords and Coastal Embayments

Evolutionary ecology of Alexandrium(Dinophyceae)with special emphasis on genotypic and phenotypic variation in the toxigenic species A. tamarense

In this thesis, broad phenotypic variation in the genus Alexandrium with respect to allelochemical properties targeting a wide range of other planktonic protists was observed and indications of co-evolutionary processes shaping the respective allelopathic phenotype were found. At the intra- and inter-population level in the species A. tamarense large genotypic variation was observed. A conceptual population genetic model on how this variation is preserved in the long run - despite evidence of clonal selection and frequency shifts of clonal lineages during vegetative planktonic growth - is presented. Phenotypic characters such as allelochemical properties and content and relative composition of paralytic shellfish poisoning toxins (PSP) may be such characters that are under directional selection during planktonic population growth and harmful algal bloom (HAB) development. However, isolates from the populations studied varied widely with respect to these quantitative genetic characters

Evolutionsbiologie der Gattung Alexandrium (Dinophyceae) mit besonderem Schwerpunkt auf genotypischer und phänotypischer Variation in der toxischen Art A. tamarense

In this thesis, broad phenotypic variation in the genus Alexandrium with respect to allelochemical properties targeting a wide range of other planktonic protists was observed and indications of co-evolutionary processes shaping the respective allelopathic phenotype were found. At the intra- and inter-population level in the species A. tamarense large genotypic variation was observed. A conceptual population genetic model on how this variation is preserved in the long run - despite evidence of clonal selection and frequency shifts of clonal lineages during vegetative planktonic growth - is presented. Phenotypic characters such as allelochemical properties and content and relative composition of paralytic shellfish poisoning toxins (PSP) may be such characters that are under directional selection during planktonic population growth and harmful algal bloom (HAB) development. However, isolates from the populations studied varied widely with respect to these quantitative genetic characters

Bioactive compounds of marine dinoflagellate isolates from western Greenland and their phylogenetic association within the genus Alexandrium

The diversity and biogeography of populations of the toxigenic marine dinoflagellate genus Alexandrium, a major global cause of paralytic shellfish poisoning (PSP), are represented by only a few studies based upon a low number of cultured isolates and remain poorly described in Arctic and sub-Arctic waters. Multiple clonal isolates (n = 22) of the Alexandrium tamarense species complex, and a single isolate of A. tamutum, were collected from the water column while on board an oceanographic expedition to the west coast of Greenland. After culturing of these isolates under controlled conditions, their phylogenetic affinities within the genus Alexandrium were characterized by sequence analysis of nuclear large subunit (LSU) rDNA. Based upon morphological and molecular genetic criteria, all isolates of the A. tamarense species complex were consistent with membership in the Group I ribotype (previously known as the North American ribotype). Phenotypic signatures were also analyzed based upon their respective profiles of paralytic shellfish toxins (PST) and allelochemical interactions against a target cryptophyte Rhodomonas, as determined by lytic potency. All isolates conforming to the A. tamarense Group I produced PST, but no toxins were detected in A. tamutum P2E2. Unusually, only carbamoyl toxins were produced among the A. tamarense Group I isolates from Greenland; sulfocarbamoyl derivatives, generally present in A. tamarense population from other locations, including the Arctic, North Pacific and North Atlantic, were absent from all isolates. Allelochemical activity, causing cell lysis of Rhodomonas, but generally being unrelated to cellular PST, was expressed by all A. tamarense isolates and also by A. tamutum, but varied widely in potency. Comparison of the genotypic (rDNA) and phenotypic (PST profile, allelochemical activity) characteristics of Greenland isolates with those of other Arctic populations reveals a complex pattern of intra-specific diversity. Estimation of diversity relationships is problematic because of the distinct patterns of divergence and lack of evidence of linkage among the alternative biomarkers and morphology. Nevertheless, such studies are necessary as the basis for constructing hindcasting scenarios and predicting changes in Alexandrium species distribution in the Arctic from the regional to the global scale

Growth and bioactive secondary metabolites of arctic Protoceratium reticulatum (Dinophyceae)

Harmful algal blooms are mainly caused by marine dinoflagellates and are known to produce potent toxins that may affect the ecosystem, human activities and health. Such events have increased in frequency and intensity worldwide in the past decades. Numerous processes involved in Global Change are amplified in the Arctic, but little is known about species specific responses of arctic dinoflagellates. The aim of this work was to perform an exhaustive morphological, phylogenetical and toxinological characterization of Greenland Protoceratium reticulatum and, in addition, to test the effect of temperature on growth and production of bioactive secondary metabolites. Seven clonal isolates, the first isolates of P. reticulatum available from arctic waters, were phylogenetically characterized by analysis of the LSU rDNA. Six isolates were further characterized morphologically and were shown to produce both yessotoxins (YTX) and lytic compounds, representing the first report of allelochemical activity in P. reticulatum. As shown for one of the isolates, growth was strongly affected by temperature with a maximum growth rate at 15 8C, a significant but slow growth at 1 8C, and cell death at 25 8C, suggesting an adaptation of P. reticulatum to temperate waters. Temperature had no major effect on total YTX cell quota or lytic activity but both were affected by the growth phase with a significant increase at stationary phase. A comparison of six isolates at a fixed temperature of 10 8C showed high intraspecific variability for all three physiological parameters tested. Growth rate varied from 0.06 to 0.19 d�1, and total YTX concentration ranged from 0.3 to 15.0 pg YTX cell�1 and from 0.5 to 31.0 pg YTX cell�1 at exponential and stationary phase, respectively. All six isolates performed lytic activity; however, for two isolates lytic activity was only detectable at higher cell densities in stationary phase

Genetic differentiation and phenotypic characteristics of geographically separated populations of the Alexandrium tamarense North American ribotype

The Alexandrium tamarense species complex contains prominent paralytic shellfish poisoning (PSP) toxin producers that can be further discriminated either on the basis of morphological characteristics or by DNA sequence divergence. The North American clade, as defined by its LSU ribosomal DNA sequence, is the most widely distributed representative of the PSP toxin-producing A. tamarense clades. Populations of this clade cause recurrent blooms in many regions of the world. Natural populations from North America, Northern Europe and Japan exhibit notable genetic differentiation that can be detected by molecular markers with different resolution properties. Whereas molecular sequence analysis of ribosomal DNA yields only a coarse resolution pattern of regional subclades, mitochondrial DNA sequences and microsatellites, as well as Amplified Fragment Length Polymorphism (AFLP) analysis, allow the estimation of genetic differentiation between contiguous populations. No congruence of any of the genetic markers were found between the expression of PSP toxin phenotypes or allelochemical properties that can affect grazers or competing algal species, but inter-population differences in PSP toxin profile were apparent on a broad geographical scale. The variable expression of the allelopathic phenotype within a population from Northern Europe was used to experimentally test the protective benefit of allelochemical properties on bloom formation