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Genomic regions that play a role in parasite defense are often found to be highly variable, with the major histocompatibility complex serving as an iconic example. Single nucleotide polymorphisms may represent only a small portion of this variability, with Indel polymorphisms and copy number variation further contributing. In extreme cases, haplotypes may no longer be recognized as orthologous. Understanding the evolution of such highly divergent regions is challenging because the most extreme variation is not visible using reference-assisted genomic approaches. Here we analyze the case of the Pasteuria Resistance Complex in the crustacean Daphnia magna , a defense complex in the host against the common and virulent bacterium Pasteuria ramosa. Two haplotypes of this region have been previously described, with parts of it being nonhomologous, and the region has been shown to be under balancing selection. Using pan-genome analysis and tree reconciliation methods to explore the evolution of the Pasteuria Resistance Complex and its characteristics within and between species of Daphnia and other Cladoceran species, our analysis revealed a remarkable diversity in this region even among host species, with many nonhomologous hyper-divergent haplotypes. The Pasteuria Resistance Complex is characterized by extensive duplication and losses of Fucosyltransferase (FuT) and Galactosyltransferase (GalT) genes that are believed to play a role in parasite defense. The Pasteuria Resistance Complex region can be traced back to common ancestors over 250 million years. The unique combination of an ancient resistance complex and a dynamic, hyper-divergent genomic environment presents a fascinating opportunity to investigate the role of such regions in the evolution and long-term maintenance of resistance polymorphisms. Our findings offer valuable insights into the evolutionary forces shaping disease resistance and adaptation, not only in the genus Daphnia , but potentially across the entire Cladocera class. [ABSTRACT FROM AUTHOR]

Balancing selection is an evolutionary process that maintains genetic polymorphisms at selected loci and strongly reduces the likelihood of allele fixation. When allelic polymorphisms that predate speciation events are maintained independently in the resulting lineages, a pattern of trans-species polymorphisms may occur. Trans-species polymorphisms have been identified for loci related to mating systems and the MHC, but they are generally rare. Trans-species polymorphisms in disease loci are believed to be a consequence of long-term host-parasite coevolution by balancing selection, the so-called Red Queen dynamics. Here we scan the genomes of three crustaceans with a divergence of over 15 million years and identify 11 genes containing identical-by-descent trans-species polymorphisms with the same polymorphisms in all three species. Four of these genes display molecular footprints of balancing selection and have a function related to immunity. Three of them are located in or close to loci involved in resistance to a virulent bacterial pathogen, Pasteuria, with which the Daphnia host is known to coevolve. This provides rare evidence of trans-species polymorphisms for loci known to be functionally relevant in interactions with a widespread and highly specific parasite. These findings support the theory that specific antagonistic coevolution is able to maintain genetic diversity over millions of years. Trans-species polymorphisms (TSP) in disease loci are thought to be caused by long-term host-parasite coevolution. Here, the authors identify consistent TSPs across three species of Daphnia and find several genes related to immunity function for resistance to a virulent bacterial pathogen. [ABSTRACT FROM AUTHOR]

Understanding the genomic basis of infectious disease is a fundamental objective in co-evolutionary theory with relevance to healthcare, agriculture, and epidemiology. Models of host-parasite co-evolution often assume that infection requires specific combinations of host and parasite genotypes. Co-evolving host and parasite loci are, therefore, expected to show associations that reflect an underlying infection/resistance allele matrix, yet little evidence for such genome-to-genome interactions has been observed among natural populations. We conducted a study to search for this genomic signature across 258 linked host (Daphnia magna) and parasite (Pasteuria ramosa) genomes. Our results show a clear signal of genomic association between multiple epistatically interacting loci in the host genome, and a family of genes encoding for collagen-like protein in the parasite genome. These findings are supported by laboratory-based infection trials, which show strong correspondence between phenotype and genotype at the identified loci. Our study provides clear genomic evidence of antagonistic co-evolution among wild populations. [ABSTRACT FROM AUTHOR]

The dynamics of extinction and (re)colonization in habitat patches are characterizing features of dynamic metapopulations, causing them to evolve differently than large, stable populations. The propagule model, which assumes genetic bottlenecks during colonization, posits that newly founded subpopulations have low genetic diversity and are genetically highly differentiated from each other. Immigration may then increase diversity and decrease differentiation between subpopulations. Thus, older and/or less isolated subpopulations are expected to have higher genetic diversity and less genetic differentiation. We tested this theory using whole-genome pool-sequencing to characterize nucleotide diversity and differentiation in 60 subpopulations of a natural metapopulation of the cyclical parthenogen Daphnia magna. For comparison, we characterized diversity in a single, large, and stable D. magna population. We found reduced (synonymous) genomic diversity, a proxy for effective population size, weak purifying selection, and low rates of adaptive evolution in the metapopulation compared with the large, stable population. These differences suggest that genetic bottlenecks during colonization reduce effective population sizes, which leads to strong genetic drift and reduced selection efficacy in the metapopulation. Consistent with the propagule model, we found lower diversity and increased differentiation in younger and also in more isolated subpopulations. Our study sheds light on the genomic consequences of extinction–(re)colonization dynamics to an unprecedented degree, giving strong support for the propagule model. We demonstrate that the metapopulation evolves differently from a large, stable population and that evolution is largely driven by genetic drift. [ABSTRACT FROM AUTHOR]

Analysing variation in a species' genomic diversity can provide insights into its historical demography, biogeography and population structure, and thus its ecology and evolution. Although such studies are rarely undertaken for parasites, they can be highly revealing because of the parasite's co‐evolutionary relationships with hosts. Modes of reproduction and transmission are thought to be strong determinants of genomic diversity for parasites and vary widely among microsporidia (fungal‐related intracellular parasites), which are known to have high intraspecific genetic diversity and interspecific variation in genome architecture. Here we explore genomic variation in the microsporidium Hamiltosporidium, a parasite of the freshwater crustacean Daphnia magna, looking especially at which factors contribute to nucleotide variation. Genomic samples from 18 Eurasian populations and a new, long‐read‐based reference genome were used to determine the roles that reproduction mode, transmission mode and geography play in determining population structure and demographic history. We demonstrate two main Hamiltosporidium tvaerminnensis lineages and a pattern of isolation‐by‐distance, but note an absence of congruence between these two parasite lineages and the two Eurasian host lineages. We suggest a comparatively recent parasite spread through Northern Eurasian host populations after a change from vertical to mixed‐mode transmission and the loss of sexual reproduction. While gaining knowledge about the ecology and evolution of this focal parasite, we also identify common features that shape variation in genomic diversity for many parasites, such as distinct modes of reproduction and the intertwining of host–parasite demographies. [ABSTRACT FROM AUTHOR]

The link between long-term host–parasite coevolution and genetic diversity is key to understanding genetic epidemiology and the evolution of resistance. The model of Red Queen host–parasite coevolution posits that high genetic diversity is maintained when rare host resistance variants have a selective advantage, which is believed to be the mechanistic basis for the extraordinarily high levels of diversity at disease-related genes such as the major histocompatibility complex in jawed vertebrates and R-genes in plants. The parasites that drive long-term coevolution are, however, often elusive. Here we present evidence for long-term balancing selection at the phenotypic (variation in resistance) and genomic (resistance locus) level in a particular host–parasite system: the planktonic crustacean Daphnia magna and the bacterium Pasteuria ramosa. The host shows widespread polymorphisms for pathogen resistance regardless of geographic distance, even though there is a clear genome-wide pattern of isolation by distance at other sites. In the genomic region of a previously identified resistance supergene, we observed consistent molecular signals of balancing selection, including higher genetic diversity, older coalescence times, and lower differentiation between populations, which set this region apart from the rest of the genome. We propose that specific long-term coevolution by negative-frequency-dependent selection drives this elevated diversity at the host's resistance loci on an intercontinental scale and provide an example of a direct link between the host's resistance to a virulent pathogen and the large-scale diversity of its underlying genes. [ABSTRACT FROM AUTHOR]

Understanding how diversity is maintained in natural populations is a major goal of evolutionary biology. In coevolving hosts and parasites, negative frequency‐dependent selection is one mechanism predicted to maintain genetic variation. While much is known about host diversity, parasite diversity remains understudied in coevolutionary research. Here, we survey natural diversity in a bacterial parasite by characterizing infection phenotypes for over 50 isolates in relation to 12 genotypes of their host, Daphnia magna. We find striking phenotypic variation among parasite isolates, and we discover the parasite can infect its host through at least five different attachment sites. Variation in attachment success at each site is explained to varying degrees by host and parasite genotypes. A spatial correlation analysis showed that infectivity of different isolates does not correlate with geographic distance, meaning isolates from widespread populations are equally able to infect the host. Overall, our results reveal that infection phenotypes of this parasite are highly diverse. Our results are consistent with the prediction that under Red Queen coevolutionary dynamics both the host and the parasite should show high genetic diversity for traits of functional importance in their interactions. [ABSTRACT FROM AUTHOR]

Inbreeding refers to the fusion of related individuals' gametes, with self‐fertilization (selfing) being an extreme form of inbreeding—involving gametes produced by the same individual. Selfing is expected to reduce heterozygosity by an average of 50% in one generation; however, little is known about the empirical variation on a genome level surrounding this figure and the factors that affect variation. We selfed genotypes of the cyclic parthenogen Daphnia magna and analysed whole genomes of mothers and selfed offspring, observing the predicted 50% heterozygosity reduction on average. We also saw substantial variation around this value and significant differences among mother–offspring pairs. Crossover analysis confirmed the known trend of recombination occurring more often towards the telomeres. This effect was shown, through simulations, to increase the variance of heterozygosity reduction compared to when a uniform distribution of crossovers was used. Similarly, we simulated inbred line production after several generations of selfing and we observed higher variance in achieved homozygosity when we consider a higher recombination rate towards the telomeres. Our empirical and simulation study highlights that the expected mean values of heterozygosity reduction show remarkable variation, which can help understand, for example, differences among inbred individuals. [ABSTRACT FROM AUTHOR]

Knowledge of the genetic architecture of pathogen infectivity and host resistance is essential for a mechanistic understanding of coevolutionary processes, yet the genetic basis of these interacting traits remains unknown for most host–pathogen systems. We used a comparative genomic approach to explore the genetic basis of infectivity in Pasteuria ramosa , a Gram-positive bacterial pathogen of planktonic crustaceans that has been established as a model for studies of Red Queen host–pathogen coevolution. We sequenced the genomes of a geographically, phenotypically, and genetically diverse collection of P. ramosa strains and performed a genome-wide association study to identify genetic correlates of infection phenotype. We found multiple polymorphisms within a single gene, Pcl7 , that correlate perfectly with one common and widespread infection phenotype. We then confirmed this perfect association via Sanger sequencing in a large and diverse sample set of P. ramosa clones. Pcl7 codes for a collagen-like protein, a class of adhesion proteins known or suspected to be involved in the infection mechanisms of a number of important bacterial pathogens. Consistent with expectations under Red Queen coevolution, sequence variation of Pcl7 shows evidence of balancing selection, including extraordinarily high diversity and absence of geographic structure. Based on structural homology with a collagen-like protein of Bacillus anthracis , we propose a hypothesis for the structure of Pcl7 and the physical location of the phenotype-associated polymorphisms. Our results offer strong evidence for a gene governing infectivity and provide a molecular basis for further study of Red Queen dynamics in this model host–pathogen system. [ABSTRACT FROM AUTHOR]

Parasite‐mediated selection varying across time and space in metapopulations is expected to result in host local adaptation and the maintenance of genetic diversity in disease‐related traits. However, nonadaptive processes like migration and extinction‐(re)colonization dynamics might interfere with adaptive evolution. Understanding how adaptive and nonadaptive processes interact to shape genetic variability in life‐history and disease‐related traits can provide important insights into their evolution in subdivided populations. Here we investigate signatures of spatially fluctuating, parasite‐mediated selection in a natural metapopulation of Daphnia magna. Host genotypes from infected and uninfected populations were genotyped at microsatellite markers, and phenotyped for life‐history and disease traits in common garden experiments. Combining phenotypic and genotypic data a QST–FST‐like analysis was conducted to test for signatures of parasite mediated selection. We observed high variation within and among populations for phenotypic traits, but neither an indication of host local adaptation nor a cost of resistance. Infected populations have a higher gene diversity (Hs) than uninfected populations and Hs is strongly positively correlated with fitness. These results suggest a strong parasite effect on reducing population level inbreeding. We discuss how stochastic processes related to frequent extinction‐(re)colonization dynamics as well as host and parasite migration impede the evolution of resistance in the infected populations. We suggest that the genetic and phenotypic patterns of variation are a product of dynamic changes in the host gene pool caused by the interaction of colonization bottlenecks, inbreeding, immigration, hybrid vigor, rare host genotype advantage and parasitism. Our study highlights the effect of the parasite in ameliorating the negative fitness consequences caused by the high drift load in this metapopulation. [ABSTRACT FROM AUTHOR]

• All the five genera of the Daphniidae were included in this study. • A highly supported phylogenies of the Daphniidae was obtained using 636 nuclear single-copy genes. • Some discrepancies between nuclear and mtDNA based trees were observed at deeper nodes. • A new candidate sister lineage for Daphnia magna was identified. • We dated the Daphniidae phylogeny using both fossil records and substitution rates. In the post-genomic era, much of phylogenetic analyses still relies on mitochondrial DNA, either alone or in combination with few nuclear genes. Although this approach often makes it possible to construct well-supported trees, it is limited because mtDNA describes the history of a single locus, and nuclear phylogenies based on a few loci may be biased, leading to inaccurate tree topologies and biased estimations of species divergence time. In this study, we perform a phylogenomic analysis of the Daphniidae family (Crustacea: Branchiopoda: Anomopoda) including some of the most frequently studied model organisms (Daphnia magna and D. pulex) whose phylogenetic relationships have been based primarily on an assessment of a few mtDNA genes. Using high-throughput sequencing, we were able to assemble 38 whole mitochondrial genomes and draft nuclear genomes for 18 species, including at least one species for each known genus of the family Daphniidae. Here we present phylogenies based on 636 nuclear single-copy genes shared among all sampled taxa and based on whole mtDNA genomes. The phylogenies we obtained were highly supported and showed some discrepancies between nuclear and mtDNA based trees at deeper nodes. We also identified a new candidate sister lineage of Daphnia magna. Our time-calibrated genomic trees, which we constructed using both fossil records and substitution rates, yielded very different estimates of branching event times compared to those based on mtDNA. By providing multi-locus, fossil-calibrated trees of the Daphniidae, our study contributes to an improved phylogenetic framework for ecological and evolutionary studies that use water fleas as a model system. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • The average nucleotide identity within D. magna ranged between 98.9% and 95.9%. • π showed a decreasing cline in moving from the Middle East to Northern Europe. • The HPDI of divergence between West and East populations had a median of ∼290 kBP. • The value of ω ranged between 0.04 and 0.17. • ω of genomes sampled from non-rock pools was 0.0798 and rock pools was 0.1603. Abstract Phylogeography places population genetics in an explicitly spatial context, and in doing so attempts to reconstruct the historical and contemporary evolutionary processes acting across a species range through space and time. Here we present the phylogeographical structure of Daphnia magna as determined for full mitochondrial genomes from samples of 60 populations throughout much of the species known range, including Europe, the Middle East, and Asia. Contrary to previous analyses, the present analysis of the mitochondrial genome reveals coarse-grained (continental scale) evidence for spatial structure, and in particular a deep split between Western Eurasia and East Asian D. magna lineages. In contrast to previous analyses with nuclear genetic markers, our mitogenomic analysis reveals much less structure within lineages. We quantify divergence between species using the full mitochondrial genome sequence of a closely related species, D. similis. The distribution of European and Middle Eastern genetic diversity is consistent with a rapid demographic expansion following the end of the most recent ice age about 10,000 years before present. By estimating species wide distributions of d N /d S in mtDNA, we provide evidence that the effectiveness of purifying selection on protein coding genes in the mitochondrial genome of coastal rock pool populations, which have pronounced extinction-colonization dynamics, is reduced compared to larger and more stable non-rock pool populations. The present study adds important insights into the evolutionary history of a widely used model organism in ecology, evolution and ecotoxicology, and highlights the utility of phylogeographic analysis of organellar genomes to understand evolutionary processes. [ABSTRACT FROM AUTHOR]

Identifying the presence and magnitude of population genetic structure remains a major consideration in evolutionary biology as doing so allows one to understand the demographic history of a species as well as make predictions of how the evolutionary process will proceed. Next-generation sequencing methods allow us to reconsider previous ideas and conclusions concerning the distribution of genetic variation, and what this distribution implies about a given species evolutionary history. A previous phylogeographic study of the crustacean Daphnia magna suggested that, despite strong genetic differentiation among populations at a local scale, the species shows only moderate genetic structure across its European range, with a spatially patchy occurrence of individual lineages. We apply RAD sequencing to a sample of D. magna collected across a wide swath of the species' Eurasian range and analyse the data using principle component analysis ( PCA) of genetic variation and Procrustes analytical approaches, to quantify spatial genetic structure. We find remarkable consistency between the first two PCA axes and the geographic coordinates of individual sampling points, suggesting that, on a continent-wide scale, genetic differentiation is driven to a large extent by geographic distance. The observed pattern is consistent with unimpeded (i.e. no barriers, landscape or otherwise) migration at large spatial scales, despite the fragmented and patchy nature of favourable habitats at local scales. With high-resolution genetic data similar patterns may be uncovered for other species with wide geographic distributions, allowing an increased understanding of how genetic drift and selection have shaped their evolutionary history. [ABSTRACT FROM AUTHOR]

The determinants of variation in a species' genome-wide nucleotide diversity include historical, environmental, and stochastic aspects. This diversity can inform us about the species' past and present evolutionary dynamics. In parasites, the mode of transmission and the interactions with the host might supersede the effects of these aspects in shaping parasite genomic diversity. We used genomic samples from 10 populations of the microsporidian parasite Ordospora colligata to investigate present genomic diversity and how it was shaped by evolutionary processes, specifically, the role of phylogeography, co-phylogeography (with the host), natural selection, and transmission mode. Although very closely related microsporidia cause diseases in humans, O. colligata is specific to the freshwater crustacean Daphnia magna and has one of the smallest known eukaryotic genomes. We found an overlapping phylogeography between O. colligata and its host highlighting the long-term, intimate relationship between them. The observed geographic distribution reflects previous findings that O. colligata exhibits adaptations to colder habitats, which differentiates it from other microsporidian gut parasites of D. magna predominantly found in warmer areas. The co-phylogeography allowed us to calibrate the O. colligata phylogeny and thus estimate its mutation rate. We identified several genetic regions under potential selection. Our whole-genome study provides insights into the evolution of one of the most reduced eukaryotic genomes and shows how different processes shape genomic diversity of an obligate parasite. [ABSTRACT FROM AUTHOR]

Salinization poses an increasing problem worldwide, threatening freshwater organisms and raising questions about their ability to adapt. We explored the mechanisms enabling a planktonic crustacean to tolerate elevated salinity. By gradually raising water salinity in clonal cultures from 185 Daphnia magna populations, we showed that salt tolerance strongly correlates with native habitat salinity, indicating local adaptation. A genome-wide association study (GWAS) further revealed a major effect of the Alpha,alpha-trehalose-phosphate synthase (TPS) gene, suggesting that trehalose production facilitates salinity tolerance. Salinity-tolerant animals showed a positive correlation between water salinity and trehalose concentrations, while intolerant animals failed to produce trehalose. Animals with a non-functional TPS gene, generated through CRISPR-Cas9, supported the trehalose role in salinity stress. Our study highlights how a keystone freshwater animal adapts to salinity stress using an evolutionary mechanism known in bacteria, plants, and arthropods. [Display omitted] • Salinity tolerance is a locally adapted trait in Daphnia magna • The trehalose TPS gene shows a strong association with salinity tolerance • Trehalose content increases with salinity, allowing survival of tolerant genotypes • Disabling the TPS gene reduces salinity tolerance by preventing trehalose synthesis Santos et al. show that tolerance to elevated salinity in D. magna is mediated by trehalose. Genomic analysis and CRISPR-Cas9 technique provide evidence for the crucial role of the TPS gene in trehalose production and local adaptation. These findings reveal how this species uses a mechanism known in certain bacteria, plants, and other invertebrates. [ABSTRACT FROM AUTHOR]