Despite their clear importance and potential to broadly influence host populations, little is known about pathogen interactions within a broader ecosystem. This project will use the well-studied Daphnia magna-Pasteuria ramosa as a model host-parasite system to investigate how various factors (temperature, route of transmission and selection for host resistance) interact to influence the repeatable seasonal epidemic dynamics observed in nature in order to further understand the ecological and evolutionary drivers of epidemics. The novelty offered by this project is that it will be using a combination of field observations, mesocosm and laboratory experiments along side with mathematical modelling, aiming to quantify and measure the relative importance of factors that are often recognised to be important for epidemic dynamics. These factors are not well understood, and provide a comprehensive understanding of host-parasite-community dynamics and the ecological, evolutionary and epidemiological processes that govern them. The novelty offered by this project is that it demonstrates interactions between parasites, hosts and other species in the ecosystem, as well as the long-term impact of temperature on host-parasite communities. The combination of different approaches proposed in this project allow us to disentangle the complex relationships between hosts, parasites, the broader ecological community, and the abiotic environment and gain further insight to the rules that govern epidemic cycles and ecological feedback loops. By doing so, it allows to estimate the effect of climate change (in particular warmer summers) on the future dynamics of this host-parasite system. Understanding the drivers of infectious disease dynamics and the complex mechanisms of transmission and their relationship to the biotic and abiotic environment are more important than ever as we face the dual and linked challenges of global change and emerging infectious diseases.

Studies in diverse biological systems have led us to believe that host-parasite coevolution is responsible for the extraordinary genetic diversity seen in some genomic regions, such as MHC genes in jawed vertebrates and R-genes in plants. What is still missing, however, is the functional link between genetic variants for host resistance and parasite infectivity on the level of individual interactions and genomic signatures that are predicted to result from these interactions. In this research proposal-a continuation of my previous SNF grant-I address this topic, focusing on a well-established host-parasite system: the water flea Daphnia magna and its virulent bacterial parasite Pasteuria ramosa. This model system has the potential to reveal the mechanistic connection between resistance and infectivity on the individual level and genomic variation at the species level. Our work may thus serve as a case study for demonstrating how selection on specific host- parasite interactions creates genomic patterns of long-term balancing selection and trans-species polymorphism. The proposed research has three main aims, each with two subprojects (SPs): Aim 1: Identify resistance and infectivity genes: We will use genomic approaches to screen hosts (SP1) and parasites (SP2) for loci related to their interactions, with the overarching aim of understanding how these genes interact to produce phenotypic variation. Specifically, we will map genes for polymorphisms in host resistance and parasite infection using stratified genome-wide association studies (GWAS). Aim 2: Long-term consequences of coevolution: Here we will analyse genomes of D. magna and two related species, D. similis and D. sinensis, for long-term balancing selection and trans-species polymorphism (TSP). SP3 will focus on a known host supergene for resistance, a gene so diverse that it requires a haplotype approach, using long-read sequencing. SP4 will employ candidate-free genome scans to search for signatures of balancing selection and TSP in genotypes of the three species collected over a broad geographic scale. Aim 3: Short-term dynamics of coevolution: Genomes of naturally infected hosts will be sequenced together with those of the infecting parasite. These data will be used to test directly for genetic interactions between host and parasite loci, i.e. interspecies linkage disequilibrium (SP5). Variation at the resulting host and parasite candidate loci will then be tracked over two years in longitudinal samples from a natural population and also in sediment cores taken from the same population (SP6). This will allow us to follow coevolution at the interacting genes over time spans from years to decades. SP6 depends on results from SP5. These subprojects complement each other, promising to create a comprehensive picture of the nature and dynamics of the genes that underlay coevolutionary interactions of this system over both the short-term (via patterns of parasitism and selection) and the long-term (via genomic signatures). The proposed research will close the gap between theory, individual level patterns of disease and genomic signatures at disease loci. This is important not only for this system, but for many other host-parasite systems undergoing antagonistic evolution, including humans and their parasites.

Testing predictions of Red Queen coevolution, by Dieter Ebert, Universität Basel SUMMARY It is believed that many biological phenomena (e.g. the evolution of sex, Batesian mimicry, and immune response) have evolved as a consequence of host-parasite coevolution. The leading hypothesis for such coevolution is based on time-lagged negative frequency dependent selection (NFDS), also known as Red Queen coevolution. Evidence from population genetics (e.g. high genetic diversity and balancing selection at disease loci) is consistent with the idea that NFDS drives coevolution in host-parasite systems, but other models of coevolution (e.g., selective sweeps) cannot be easily excluded. Here I propose to test the predictions of the Red Queen coevolution hypothesis by analysis of hosts and parasite dynamics in one well-characterized population. In this Daphnia magna population we observe yearly strong epidemics of the bacterial parasite Pasteuria ramosa. I propose to test the hypothesis that coevolution in this system is driven by NFDS. Subproject A aims to identify the host genes that prevent parasite attachment in one particular population ("The Swisspond"), which has yearly strong epidemics of Pasteuria ramosa. We plan to compare different host-genotypes by sequencing from each resistotype multiple genomes and conduct genome-wide association mapping. In a next step we aim to pinpoint the exact genes responsible for the resistance polymorphism by employing molecular tools (CRISPR/Cas9) to knock down candidate genes in the regions of interest. The cloned offspring of the knock down genotypes will be tested for phenotypic effects in response to multiple parasite clones. Subproject B continues our efforts to find the infectivity genes in the bacterial pathogen Pasteuria. This parasite has now been fully sequenced and genomes from different isolates with known infection characteristics will be compared. As a further step we plan to do a proteomics approach, to identify the proteins which are expressed on the spore surface and which are responsible for the attachment of the parasite to the host. Subproject C aims to uncover the genetic interaction matrix between host and parasite genotypes. Host and parasite isolates from the field will be cloned in the laboratory and the mode of inheritance of resistance will be worked out by conducting genetic crosses among hosts. At the same time, infected hosts will be collected across the course of an epidemic in the field and will be genotypes for candidate genes at host resistance loci and parasite infectivity loci. This "co-genotyping" will allow estimating the strength of genetic host-parasite interactions during natural epidemics and enable us to estimate the strength of selection acting on different genotypes. Expected value of the research: This research aims to provide a convincing case study on the validity and predictions of the Red Queen coevolution hypothesis, offering urgently needed genetic data for theoretical and empirical research in evolution, epidemiology and disease biology. It has implications for our understanding of how coevolution shapes genetic diversity and genomes.

Testing genetic assumptions and predictions of Red Queen coevolution Grant proposal for the Swiss Nationalfonds by Dieter Ebert, Universität Basel Background : It is believed that many biological phenomena (e.g. the evolution of sex, Batesian mimicry, and immune response) have evolved as a consequence of host-parasite coevolution. The leading hypothesis for such coevolution is based on time-lagged negative frequency dependent selection (NFDS), also known as Red Queen coevolution. Evidence from population genetics (e.g. high genetic diversity and balancing selection at disease loci) is consistent with the idea that NFDS drives coevolution in host-parasite systems, but other models of coevolution (e.g., selective sweeps) cannot be easily excluded. Here I propose to test the specific predictions of Red Queen coevolution by undertaking a genetic analysis of the genes under selection and their evolutionary dynamics, focusing on the genetic interactions of Daphnia magna and its bacterial parasite Pasteuria ramosa . I will test my main hypothesis-that coevolution in this system is driven by NFDS-by exploring the genetic interaction matrix of host and parasite, characterizing the genes involved in these interactions, and tracing their dynamics over a 50-year period in pond sediments that contain layered archives of host and parasite populations. Subproject A aims to uncover the genetic interaction matrix between host and parasite genotypes. We focus on the attachment step of Pasteuria, because polymorphism in attachment genes explains most of Daphnia's variation in resistance. Subproject B aims to identify the host genes that prevent parasite attachment. We have already mapped the location of these genes to a small genomic region. I suggest using association mapping in natural populations to further narrow down this region. Next, we will employ molecular evolution tools to test the prediction that allelic variants at these loci are old and under balancing selection. Furthermore, we will trace temporal changes at the candidate loci and of resistance phenotypes in layered pond sediments that harbour resting stages of host and parasite. This will enable us to observe if there is a signature of fluctuating selection on resistance genes. Subproject C will focus on identifying the infectivity genes in Pasteuria. This parasite shows frequent recombination, allowing us to use a genome-wide approach to analyse genetic diversity in several Pasteuria genomes from different infectotypes (carrying different attachment genes). The candidate regions in the genome will then be tested for associations with the parasite's attachment phenotypes using material from natural populations. Next, the prediction of rapid allele frequency changes will be tested in sediment cores. Finally, we will test the prediction that parasite infectotypes track host resistotypes by combining the sediment core results of Subprojects B and C. Expected value of the research: This research aims to provide a convincing case study on the validity and predictions of the Red Queen coevolution hypothesis, offering urgently needed genetic data for theoretical and empirical research in evolution, epidemiology and disease control. It has implications for our understanding of how coevolution shapes the phenomena related to host-parasite interactions.

Loss of Heterozygosity during Asexual Reproduction Summary Sexual reproduction is a widespread phenomenon in nature. Numerous reviews and theoretical studies have discussed this as a paradoxical situation because everything else being equal, sexual reproduction has high costs. However, the widespread existence and persistence of sexual reproduction combined with the observation that asexual lineages are usually young indicates that sexual reproduction has evolutionary advantages that outweigh its costs. A number of potential advantages have been suggested, among them A) that sexual lineages can purge deleterious mutations more effectively, B) that sexual lineages have a higher potential to evolve in a changing environment, and C) that asexual lines have a lower fitness than commonly expected, because they are faced with an ongoing loss of heterozygosity due to ameiotic recombination. This can occur as either reciprocal (crossover) or nonreciprocal (gene conversion) exchange in apomictic germ-line cells. This latter idea is at the centre of this proposal. Asexual lines often emerge from sexual ancestors, raising the question of why asexuals do not out-compete their sexual relatives. It has been suggested that the loss of heterozygosity (LOH) and its associated loss of fitness may reduce the long-term advantages of asexual reproduction. Recently, LOH has been discovered in asexually reproducing Daphnia. Building on this finding, we here propose two sub-projects to quantify LOH and its fitness consequences during the asexual reproduction of D. magna, taking advantage of recently developed genomic tools. Sub-project A will focus on the estimation of LOH in single generations. Using a high density SNP and INDEL array, we will quantify LOH in several mother-daughter pairs. This approach will not entirely exclude selection: selection during early development against offspring (embryos) with LOH would remain unnoticed. Therefore, we will compliment it with a genome-wide analysis of Mendelian ratios of the codominant markers. Selection against recessive deleterious alleles would distort these ratios and would, in combination with a genetic map, allow us to estimate their number and distribution in the genome. From this one can estimate how much LOH is underestimated due to early selection. Sub-project B will examine the fitness consequences of LOH in asexually propagating lines with and without competition. Furthermore, this project attempts to assess the effect of inbreeding load on LOH under competitive conditions. These experiments will enable us to estimate the longer-term consequences of LOH in asexual populations. This proposed research will provide further insights into the evolution of asexuality, the maintenance of sexual recombination and the genetic mechanisms at work during reproduction. It also promises to increase our understanding of the effects and distributions of deleterious alleles in the genome and of the evolution of genome structure in asexuals.

STRESSFLEA will develop and use genomics tools to unravel patterns and mechanisms of adaptation to anthropogenic and natural stressors in natural populations of the water flea Daphnia magna. STRESSFLEA has three objectives: 1. Obtaining insight into the functional genomic underpinning of genetic adaptation to specific stressors. Combining genome scans, candidate gene approaches and QTL mapping, we will identify genes underlying specific adaptations along environmental gradients. 2. Obtaining insight into the mechanisms by which natural Daphnia populations respond to multiple stressors, using genomics tools to identify processes responsible for correlated genetic responses (trade-offs, pleiotropy, linkage). 3. Reconstructing evolutionary processes at large spatial and temporal scales through the use of genomic markers linking variation at specific genes to trait values and fitness. STRESSFLEA will invest strongly in the development of genomics tools and thus contribute to the development of Daphnia magna as a key model system in ecological and functional genomics of stress responses. Daphnia magna is one of the best studied species in ecology, evolution and ecotoxicology. Combining this knowledge with functional genomics provides unique opportunities to understand the mechanistic underpinning of local adaptation to complex selection gradients in nature.

The research I am proposing continues the work of my group on the outgoing grant " Processes and mechanisms of antagonistic host-parasite coevolution" (Nr. 31003A-116029). For the parasite-host systems my group has been focussing on we now have good evidence that rapid coevolution is taking place: parasites track the locally abundant host genotypes and hosts evolve resistance. What we lack is an understanding of the underlying genetic mechanism and with it, the best model to explain the processes at work. Here we focus on discovering the genetic mechanisms underlying the host-parasite interactions because this knowledge will allow us to identify the model to best explain coevolution. The two most often discussed models are time lacked negative frequency dependent selection (the Red Queen hypothesis) and coevolution by selective sweeps. Knowing the genetics would allow us to pinpoint the appropriate coevolutionary model. The Pasteuria-Daphnia model Pasteruia ramosa is an endoparasitic bacterium infecting several Daphnia species as well as some other Cladocera. Despite of Pasteuria's seemingly low degree of specificity on the host species level, this bacterium shows a remarkable degree of host genotype specificity. The same clone of Pasteuria shows high specificity in infection for only certain clones of D. magna, but is also specific to certain clones of other species, e.g. D. longispina or D. pulex. I am proposing that one Ph.D. student project continues to work on the genetic interactions between Daphnia and parasite clones. The aims of this work would be 1) Conduct multiple crosses between Daphnia clones and elucidate the segregation patterns of resistance. 2) Test for linkage among resistance loci. 3) To determine whether resistance loci show epistasis, i.e., if combinations of alleles result in different phenotypes than their effects alone would suggest. The postdoc project proposed here focuses on the population genetic structure of Pasteuria ramosa. For this we plan to sample Pasteuria from different spatial and temporal scales and obtain sequence data on putative resistance loci and house-keeping genes. This project has three parts: 1) Global scale: With Pasteuria samples across Europe and North America. 2) Intermediate temporal scale: Using Pasteuria samples from the layered sediments of lakes (sediment cores), we plan to reconstruct the temporal changes of the Pasteuria populations over periods of about 30 years. 3) Short temporal scale: To link the changes in allele frequencies expected from the sediment cores with events during natural selection, we will collect time series of Pasteuria-infected Daphnia during epidemics in natural populations. These data will allow us to follow the microevolutionary changes during coevolution and to quantify the strength of selection acting on candidate genes. The Octosporea-Daphnia model Octosporea bayeri is a microsporidian parasite with both vertical and horizontal transmission. Preliminary data suggest, that O. bayeri adapts by means of epigenetic genome modification. The second Ph.D. project for which I seek funding with this proposal is to verify or reject the epigenetics hypothesis for O. bayeri. First we will carry out detailed experiments to test for epigenetic adaptation. If these experiments suggest that epigenetic effects are likely to play a role for parasite adaptation, we will use "bisulfite sequencing" to obtain methylation patterns of O. bayeri kept in different host environments. This would allow us to find candidate genes responsible for parasite adaptation to its host clones. The topic of parasite adaptation by means of epigenetics has received little attention by evolutionary biologists, but promises an exciting field for understanding host-parasite coevolution.

Daphnia Functional Genomics Resource Understanding the interaction of the genome and the environment is an important public health consideration since many human disorders with complex genetic architectures are highly influenced by interactions between genetic and environmental factors. A critical need is the development of study systems with well advanced genomic infrastructures that also have tractable and well understood ecological contexts. Daphnia is a logical candidate study system for further development to fill this gap. Daphnia has long been considered one of the premiere systems for ecological study and recent advances have lead to a rapidly expanding genomic infrastructure including a complete genome sequence and gene expression arrays. To advance Daphnia as a model for understanding the interplay between genome tructure/function and environmental factors in the development of complex phenotypes, we propose to establish mapped QTL panels and a SNP database as a shared resource for the research community. Since unique genotypes of Daphnia can be maintained indefinitely through clonal reproduction, mapped QTL panels will provide a sustainable resource enabling multiple researchers to capitalize on the rapidly advancing genomic infrastructure of Daphnia. Increasingly, research on complex phenotypes utilizes a combination of QTL analysis and microarray expression profiles (eQTLs). The application of these two methods, referred to as genetical genomics, in a model system in which environmental conditions can be accurately and systematically manipulated will significantly advance our understanding of the relationship between the phenotype and the underlying genotypic and environmental effects. Specifically we will: (1) Establish QTL mapping panels of F2 recombinant lines from relevant strains of Daphnia. (2) Develop a SNP database to facilitate fine-scale mapping. (3) Generate a high-density genetic map for each panel using a combination of microsatellite loci and SNP markers. (4) Establish the bioinformatics infrastructure to integrate genetic and phenotypic data from these panels with existing genomic data. (5) Maintain and distribute live cultures of the recombinant lines to enable QTL studies relevant to a wide range of research areas and provide bioinformatics support for this resource.

Processes and mechanisms of antagonistic coevolution The research I am proposing addresses basic aspects of the coevolution between hosts and their parasites. Many biological and medical phenomena have been explained to be a consequence of reciprocal host-parasite coevolution. Some of these explanations require specific and rapid antagonistic coevolution to take place. Experimental coevolution of viruses in bacteria or cell cultures gave evidence for coevolution by selective sweeps, but we have little, and mostly indirect evidence for coevolution with plant and animal hosts. However, population genetic consideration suggests that rapid antagonistic coevolution in plant and animal host systems should be dominated by negative frequency dependent selection. In this proposal I ask for funds to carry out experiments with populations of the waterfleas Daphnia magna and its microparasites to deepen our understanding of the genetic processes and mechanisms of coevolution. D. magna reproduce sexually and clonally, the later with a generation time of only 10 days. Two parasites, the microsporidium, Octosporea bayeri, and the bacterium, Pasteuria ramosa, will be used in the experiments. I propose a project with 3 sub-projects to elucidate the mechanisms and patterns of host-parasite coevolution. Sub-Project A aims to find direct experimental evidence for rapid and specific coevolution with Daphnia and a microsporidian parasite under natural conditions. This will include time-shift cross-infection experiments using hosts and parasites stored at different times of the coevolution. Sub-Project B is about finding the infectivity genes in the bacterial parasite, Pasteuria. Sub-Project C proposes experiments to elucidate the mechanisms at work shaping the genetic epidemiology and coevolution of Pasteuria with its waterflea host. With my research I hope to establish a case study, which would provide urgently needed data to test assumptions and to estimate parameters for epidemiological and (co-)evolutionary models of infectious diseases. It would allow streamlining treatments against pests and parasites and to make more accurate predictions about infectious diseases evolution. It will further provide insight into natural phenomena, which are suggested to be a consequence of rapid antagonistic coevolution.

Host-Parasite Evolution The main focus of this project are question related to the evolution, genetics and ecology of host-parasite interactions. The research includes questions about the adaptive significance of parasite virulence (Why do hosts get sick? Is virulence adaptive for the parasite?) and the adaptive significance of genetic variation and sexual recombination (What is sex good for?). Our work also includes the study of inbreeding and inbreeding depression and its relationship to parasitism. We use experimental epidemiology and experiemental evolution to approach these questions. Our main study organisms are microparasites (bacteria, fungi, protozoans) and their hosts, waterfleas of the genus Daphnia . This system allows us to estimate fitness components of hosts and parasites, which is essential for the quantification of costs and benefits in both antagonists. The research includes field work in natural Daphnia populations and experimental setups in the laboratory. Some of our field work is done in a Daphnia rock pool metapopulation in Southern Finland. We also use genoic approaches to find genes involved in host-parasite interactions