Final Report Summary - IDA (Intraspecific diversity and adaptability of Fucus vesiculosus at range limits) Intraspecific genetic diversity is the 'raw material' that allows species to adjust to a changing world. For predictions of the effects of global change, it is crucial to know if low genetic diversity is a result of local adaptation or of other processes. The Baltic Sea is an extreme marine environment and its extreme temperature and salinity gradients are likely to be strong selective forces. A recent review revealed that for many Baltic Sea species, intraspecific genetic diversity was lower than in the North Sea or Atlantic. It remains unknown, however, if genetic diversity declines with increases in environmental stress and/or if marginal populations (assumed to experience highest selection) show the lowest genetic diversity. Conversely, the question if populations with high genetic diversity are more resistant against a particular stessor than those with low diversity is unanswered in many cases, too. One focus of the project lies on the selective forces behind diversity loss in bladder-wrack, Fucus vesiculosus, a canopy forming seaweed and keystone species in the Baltic Sea. To explore population genetic diversity and structure of F. vesiculosus along the environmental gradients in the Baltic Sea, we are collaborating with the lab of Kerstin Johannesson at the Sven Lovén Centre for Marine Sciences in Tjärnö, Sweden. However, microsatellite data turned out to provide too low resolution to compile a detailed genetic structure for Baltic Sea F. vesiculosus. Thus, restriction associated deoxyribonucleic acid sequencing (RAD-seq) markers are being developed in Tjärnö and as soon as these are available we can continue working on this objective. To determine the variation of fitness of different genotypes we challenged germlings of F. vesiculosus from the Kiel Fjord area, Germany, with temperature and salinity stress in laboratory experiments. Temperature and salinity affected the germination success of F. vesiculosus and sensitivity towards thermal stress varied significantly amongst genotypes. The optimal conditions for germination of fucoid zygotes were found to be at 15 degrees of Celsius and 17 psu. At 5 degrees of Celsius germination of F. vesiculosus was significantly higher than at 25 degrees of Celsius, implying that hot summer seasons impair germination success more than cold winter temperatures. All surviving germlings and their parents of two fully replicated experiments were frozen to further investigate the genetic basis of thermal tolerance. Microsatellite loci, which are located close to genes under potential thermal selection and are expected to co-vary with these genes will be analysed on these samples. With this tool as well as with RAD-seq markers we may be able to discern genotypes in individuals or populations, which are tolerant or sensitive against thermal stress. We further developed expressed sequence tags (ESTs) to search for genetic polymorphisms that are directly linked to genes with putative function, mainly to explore the genetic basis of inducible anti-herbivore defence in Fucus vesiculosus using microarrays. During inducible defence, the brown alga produces metabolites that act against herbivores 'on demand'. However, the triggers and the nature of these metabolites are largely unknown. For design of the arrays, we employed next-generation sequencing (454) resulting in an EST library with 25 498 gene models spotted on the array. For tests on the arrays, RNA from three fully replicated defence induction experiments was harvested on different points in time. In one experiment, isopods grazed on the algae directly, the other two employed oligoalginate and methyl-jasmonate as triggers. Feeding assays proofed that all treatments in all three experiments induced a significantly reduced palatability of F. vesiculosus to isopods. Treatments in all three experiments modified genome expression substantially demonstrating for the first time that the ecofunctional genomics approach allows to gain new insights into the underlying processes of defence in rockweeds. Since our exploration of the genetic basis of inducible anti-herbivore defence in Fucus vesiculosus is pioneering work on a non-model organism, our EST library has a low annotation rate (i.e. only 20 % of our sequences can be annotated to sequences or gene models with known function of other organisms). To increase annotation rate and to further identify putative candidate genes for inducible anti-herbivore defence we are currently comparing our EST libraries of Fucus vesiculosus with all unigenes compiled by 454 transcriptome sequencing of the other brown algae Lessonia nigrescens and Laminaria digitata (grazed and ungrazed) in Roscoff, France. Comparisons of all three species upregulation of genes under herbivore grazing may additionally result in better knowledge on the genetic basis and cellular pathways of inducible defence. Since it has been shown that herbivore defence may fail under elevated temperatures, our research may help to interpret possible breakdowns in populations of these foundational seaweed species under climates change and may help to identify applicable bioactive compounds. The microarray developed for exploring the genetic basis of inducible anti-herbivore defence is currently also used for further exploring transcriptomic and genomic differences between Fucus vesiculosus and Fucus radicans. F. radicans, a recently described species of brown seaweed from the northern Baltic Sea is an unparalleled example of rapid speciation in a species-poor open marine ecosystem. F. radicans is endemic to the Baltic and is most closely related to F. vesiculosus. Neutral DNA markers have shown significant genotypic differences between both species, but cannot provide a satisfying phylogeographic pattern. Cross genomic hybridisation (CGH) is planned to fill this gap and is currently performed in this project collaboration with the labs of Kerstin Johannesson in Tjärnö, Sweden, and Uwe John in Bremerhaven, Germany. Transcriptomic differences may additionally identify functionally important genes which may have played central roles in this speciation process. Determination of optimal living conditions of the two Fucus species will additionally help us to identify those abiotic parameters that may be important in the reproductive isolation of both species. We expect to deliver critical information on expression patterns under different environmental parameters that will provide a fundamental basis for management and monitoring of keystone seaweed species.