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

Molecular ecological study of the toxic marine dinoflagellate Alexandrium tamarense

In this thesis double digest restriction-site associated DNA sequencing was established for the marine microalgae Alexandrium tamarense and microsatellite marker analysis as well as 28S analysis of four A. tamarense populations was performed. A. tamarense belongs to the phylum dinoflagellata, a large group of flagellate protists with unique features in respect to their genome structure and cell organization. Furthermore this microalga is responsible for a lot of HABs. A group of three morphologically very similar species form the A. tamarense” species complex”, which is further subdivided into five distinct ribotype groups. Questions like the biogeography of this species complex as well as the question how to bring molecular data about separation of clades and morphological characteristics of defined species in line remain partly unsolved by now. Special emphasis in this thesis lays on the biogeography of ribotype group I of the A. tamarense species complex. Previous studies addressing the above mentioned questions used molecular markers which cover just a tiny fraction of the genome. ddRADSeq produces a lot more markers leading to a higher coverage of the genome and thus a much higher resolution for molecular ecological studies. In order to provide results for comparison with ddRADSeq, analysis were performed with an already established marker system. Hence microsatellite analysis with 10 microsatellite loci was performed. The four tested populations were from 3 different geographic origins: Alaska, Greenland and Northsea. Principle Coordinate and Structure analysis were performed to reveal inter- and intrapopulation structuring. A Mantel´s test was conducted to test if geographic distance correlates with genetic distance. The ddRADSeq approach could be established successfully. Results from microsatellite analysis revealed separation of Alaska´s population into two distinct clusters in Structure as well as in PCoA analysis. These findings support previously proposed models of biogeographic distribution of the group I ribotye from the A. tamarense species complex

Intraspecific facilitation by allelochemical mediated grazing protection within a toxigenic dinoflagellate population

Dinoflagellates are a major cause of harmful algal blooms (HABs), with consequences for coastal marine ecosystem functioning and services. Alexandrium fundyense (previously Alexandrium tamarense) is one of the most abundant and widespread toxigenic species in the temperate Northern and Southern Hemisphere and produces paralytic shellfish poisoning toxins as well as lytic allelochemical substances. These bioactive compounds may support the success of A. fundyense and its ability to form blooms. Herewe investigate the impact of grazing on monoclonal and mixed set-ups of highly (Alex2) and moderately (Alex4) allelochemically active A. fundyense strains and a non-allelochemically active conspecific (Alex5) by the heterotrophic dinoflagellate Polykrikos kofoidii. While Alex4 and particularly Alex5 were strongly grazed by P. kofoidii when offered alone, both strains grew well in the mixed assemblages (Alex4 þ Alex5 and Alex2 þ Alex5). Hence, the allelochemical active strains facilitated growth of the non-active strain by protecting the population as awhole against grazing. Based on our results, we argue that facilitation among clonal lineages within a species may partly explain the high genotypic and phenotypic diversity of Alexandrium populations. Populations of Alexandrium may comprise multiple cooperative traits that act in concert with intraspecific facilitation, and hence promote the success of this notorious HAB species

The ecological and evolutionary concept behind allelochemical interactions for Alexandrium populations

Alexandrium tamarense is a prominent harmful algal bloom forming species known to produce paralytic shellfish poisoning toxins (PSTs), as well as bioactive substances acting as allelochemicals. These specific allelochemical substances have not yet been structurally elucidated, but they have strong negative effects on a broad array of species, including many auto- and heterotrophic microplankton. The impact, however, of allelochemicals on actual bloom formation is currently under debate. Here we show the potential effect of allelochemicals on reducing grazing pressure on A. tamarense, while additionally demonstrating positive effects on the intra-population level. We established an allele-specific quantitative PCR (asqPCR) assay based on two A. tamarense microsatellite markers. The application of asqPCR distinguishes among three different strains of A. tamarense (A2, A4 and A5) in a mixed assemblage. Strain A2 expressed strong allelochemical activity, while A4 was only moderately lytic against targets and A5 was not lytic at all. All strains were grown individually, mixed either pairwise with each other, and/or pairwise together with a common protistan grazer, the dinoflagellate Polykrikos koifidii. When offered individual Alexandrium strains, the predator clearly grazed upon A5, moderate on A4 yet not upon A2. In pairwise mixtures, however, no prey strain-selection was observed. Finally, we show that the ecological concept of facilitation, invoking positive species interactions (here intra-specific), is a plausible explanation for the role of allelochemicals for natural populations of Alexandrium and their success in bloom formation

Intraspecific facilitation by allelochemical mediated grazing protection within a toxigenic dinoflagellate population, link to supplementary material

Dinoflagellates are a major cause of harmful algal blooms, with consequences for coastal marine ecosystem functioning and services. Alexandrium tamarense is one of the most abundant and widespread toxigenic species in the temperate northern and southern hemisphere, and produces paralytic shellfish poisoning toxins as well as lytic allelochemical substances. These bioactive compounds may support the success of A. tamarense and its ability to form blooms. Here we investigate the impact of grazing on monoclonal and mixed set-ups of highly (Alex2) and moderately (Alex4) allelochemically active A. tamarense strains and on a non-allelochemically active conspecific (Alex5) by the heterotrophic dinoflagellate Polykrikos kofoidii. While Alex4 and particularly Alex5 were strongly grazed by P. kofoidii when offered alone, both strains grew well in the mixed assemblages (Alex4+Alex5 and Alex2+Alex5). Hence, the allelochemical active strains facilitated growth of the non-active strain by protecting the population as a whole against grazing. Based on our results, we argue that facilitation among clonal lineages within a species may partly explain the high genotypic and phenotypic diversity of Alexandrium populations. Populations of Alexandrium may comprise multiple cooperative traits that act in concert with intraspecific facilitation, and hence promote the success of this notorious harmful algal bloom species

Data from: Low genetic diversity despite multiple introductions of the invasive plant species Impatiens glandulifera in Europe

Background: Invasive species can be a major threat to native biodiversity and the number of invasive plant species is increasing across the globe. Population genetic studies of invasive species can provide key insights into their invasion history and ensuing evolution, but also for their control. Here we genetically characterise populations of Impatiens glandulifera, an invasive plant in Europe that can have a major impact on native plant communities. We compared populations from the species’ native range in Kashmir, India, to those in its invaded range, along a latitudinal gradient in Europe. For comparison, the results from 39 other studies of genetic diversity in invasive species were collated. Results: Our results suggest that I. glandulifera was established in the wild in Europe at least twice, from an area outside of our Kashmir study area. Our results further revealed that the genetic diversity in invasive populations of I. glandulifera is unusually low compared to native populations, in particular when compared to other invasive species. Genetic drift rather than mutation seems to have played a role in differentiating populations in Europe. We find evidence of limitations to local gene flow after introduction to Europe, but somewhat less restrictions in the native range. I. glandulifera populations with significant inbreeding were only found in the species’ native range and invasive species in general showed no increase in inbreeding upon leaving their native ranges. In Europe we detect cases of migration between distantly located populations. Human activities therefore seem to, at least partially, have facilitated not only introductions, but also further spread of I. glandulifera across Europe. Conclusions: Although multiple introductions will facilitate the retention of genetic diversity in invasive ranges, widespread invasive species can remain genetically relatively invariant also after multiple introductions. Phenotypic plasticity may therefore be an important component of the successful spread of Impatiens glandulifera across Europe

Low genetic diversity despite multipleintroductions of the invasive plant species Impatiens glandulifera in Europe

Background: Invasive species can be a major threat to native biodiversity and the number of invasive plant speciesis increasing across the globe. Population genetic studies of invasive species can provide key insights into theirinvasion history and ensuing evolution, but also for their control. Here we genetically characterise populations ofImpatiens glandulifera, an invasive plant in Europe that can have a major impact on native plant communities. Wecompared populations from the species’ native range in Kashmir, India, to those in its invaded range, along alatitudinal gradient in Europe. For comparison, the results from 39 other studies of genetic diversity in invasivespecies were collated. Results: Our results suggest that I. glandulifera was established in the wild in Europe at least twice, from an areaoutside of our Kashmir study area. Our results further revealed that the genetic diversity in invasive populations ofI. glandulifera is unusually low compared to native populations, in particular when compared to other invasivespecies. Genetic drift rather than mutation seems to have played a role in differentiating populations in Europe. Wefind evidence of limitations to local gene flow after introduction to Europe, but somewhat less restrictions in thenative range. I. glandulifera populations with significant inbreeding were only found in the species’ native rangeand invasive species in general showed no increase in inbreeding upon leaving their native ranges. In Europe wedetect cases of migration between distantly located populations. Human activities therefore seem to, at leastpartially, have facilitated not only introductions, but also further spread of I. glandulifera across Europe. Conclusions: Although multiple introductions will facilitate the retention of genetic diversity in invasive ranges,widespread invasive species can remain genetically relatively invariant also after multiple introductions. Phenotypicplasticity may therefore be an important component of the successful spread of Impatiens glandulifera across Europe.Funders:  Research Foundation - Flanders (FWO); CBD (Center for Biodiversity Dynamics), NTNU</p