Long-term balancing selection for pathogen resistance maintains trans-species polymorphisms in a planktonic crustacean.

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.

Acclimation to warmer temperatures can protect host populations from both further heat stress and the potential invasion of pathogens.

Thermal acclimation can provide an essential buffer against heat stress for host populations, while acting simultaneously on various life-history traits that determine population growth. In turn, the ability of a pathogen to invade a host population is intimately linked to these changes via the supply of new susceptible hosts, as well as the impact of warming on its immediate infection dynamics. Acclimation therefore has consequences for hosts and pathogens that extend beyond simply coping with heat stress-governing both population growth trajectories and, as a result, an inherent propensity for a disease outbreak to occur. The impact of thermal acclimation on heat tolerances, however, is rarely considered simultaneously with metrics of both host and pathogen population growth, and ultimately fitness. Using the host Daphnia magna and its bacterial pathogen, we investigated how thermal acclimation impacts host and pathogen performance at both the individual and population scales. We first tested the effect of maternal and direct thermal acclimation on the life-history traits of infected and uninfected individuals, such as heat tolerance, fecundity, and lifespan, as well as pathogen infection success and spore production. We then predicted the effects of each acclimation treatment on rates of host and pathogen population increase by deriving a host's intrinsic growth rate (rm) and a pathogen's basic reproductive number (R0). We found that direct acclimation to warming enhanced a host's heat tolerance and rate of population growth, despite a decline in life-history traits such as lifetime fecundity and lifespan. In contrast, pathogen performance was consistently worse under warming, with within-host pathogen success, and ultimately the potential for disease spread, severely hampered at higher temperatures. Our results suggest that hosts could benefit more from warming than their pathogens, but only by linking multiple individual traits to population processes can the full impact of higher temperatures on host and pathogen population dynamics be realised.

Coinfection frequency in water flea populations is a mere reflection of parasite diversity.

In nature, parasite species often coinfect the same host. Yet, it is not clear what drives the natural dynamics of coinfection prevalence. The prevalence of coinfections might be affected by interactions among coinfecting species, or simply derive from parasite diversity. Identifying the relative impact of these parameters is crucial for understanding patterns of coinfections. We studied the occurrence and likelihood of coinfections in natural populations of water fleas (Daphnia magna). Coinfection prevalence was within the bounds expected by chance and parasite diversity had a strong positive effect on the likelihood of coinfections. Additionally, coinfection prevalence increased over the season and became as common as a single infection. Our results demonstrate how patterns of coinfection, and particularly their temporal variation, are affected by overlapping epidemics of different parasites. We suggest that monitoring parasite diversity can help predict where and when coinfection prevalence will be high, potentially leading to increased health risks to their hosts.

Phylogeny, morphology, virulence, ecology, and host range of Ordospora pajunii (Ordosporidae), a microsporidian symbiont of Daphnia spp.

The impacts of microsporidia on host individuals are frequently subtle and can be context dependent. A key example of the latter comes from a recently discovered microsporidian symbiont of Daphnia, the net impact of which was found to shift from negative to positive based on environmental context. Given this, we hypothesized low baseline virulence of the microsporidian; here, we investigated the impact of infection on hosts in controlled conditions and the absence of other stressors. We also investigated its phylogenetic position, ecology, and host range. The genetic data indicate that the symbiont is Ordospora pajunii, a newly described microsporidian parasite of Daphnia. We show that O. pajunii infection damages the gut, causing infected epithelial cells to lose microvilli and then rupture. The prevalence of this microsporidian could be high (up to 100% in the lab and 77% of adults in the field). Its overall virulence was low in most cases, but some genotypes suffered reduced survival and/or reproduction. Susceptibility and virulence were strongly host-genotype dependent. We found that North American O. pajunii were able to infect multiple Daphnia species, including the European species Daphnia longispina, as well as Ceriodaphnia spp. Given the low, often undetectable virulence of this microsporidian and potentially far-reaching consequences of infections for the host when interacting with other pathogens or food, this Daphnia-O. pajunii symbiosis emerges as a valuable system for studying the mechanisms of context-dependent shifts between mutualism and parasitism, as well as for understanding how symbionts might alter host interactions with resources. IMPORTANCE: The net outcome of symbiosis depends on the costs and benefits to each partner. Those can be context dependent, driving the potential for an interaction to change between parasitism and mutualism. Understanding the baseline fitness impact in an interaction can help us understand those shifts; for an organism that is generally parasitic, it should be easier for it to become a mutualist if its baseline virulence is relatively low. Recently, a microsporidian was found to become beneficial to its Daphnia hosts in certain ecological contexts, but little was known about the symbiont (including its species identity). Here, we identify it as the microsporidium Ordospora pajunii. Despite the parasitic nature of microsporidia, we found O. pajunii to be, at most, mildly virulent; this helps explain why it can shift toward mutualism in certain ecological contexts and helps establish O. pajunii is a valuable model for investigating shifts along the mutualism-parasitism continuum.

Divergence of the Host-Associated Microbiota with the Genetic Distance of Host Individuals Within a Parthenogenetic Daphnia Species.

The taxonomic composition of the microbiota in the gut and epidermis of animals is known to vary among genetically and physiologically different host individuals within the same species. However, it is not clear whether the taxonomic composition diverges with increasing genetic distance of the host individuals. To unveil this uncertainty, we compared the host-associated microbiota among the genotypes within and between genetically distant lineages of parthenogenetic Daphnia cf. pulex across different physiological states, namely, well-fed, starved, and dead. Metagenomic analysis with 16S rRNA showed that, regardless of the host genotypes, diversity of the host-associated microbiota was high when the host individuals were fed food and gradually decreased when they were starved until they died. However, the difference in the host-associated microbiota, that is, ß-diversity, was significant among the genotypes within and between the host lineages when they were fed. Although some bacteria in the microbiota, such as Limnohabitans, Rhodococcus, and Aeromicrobium, were found abundantly and commonly in all host genotypes; others, such as those of Holosoporacea, were found only in the genotypes of a specific lineage. Accordingly, the ß-diversity tended to increase with increasing genetic distance of the host individuals. These results support an idea that the host-associated microbiota diverged with genetic divergence in the host species and that at least some bacteria are highly dependent on the genetically specific metabolites produced by the host individuals.

The timings of host diapause and epidemic progression mediate host genetic diversity and future epidemic size in Daphnia-parasite populations.

Epidemics commonly exert parasite-mediated selection and cause declines in host population genetic diversity. This can lead to evolution of resistance in the long term and smaller subsequent epidemics. Alternatively, the loss of genetic diversity can increase host vulnerability to future disease spread and larger future epidemics. Matters are made more complex by the fact that a great many host organisms produce diapausing life stages in response to environmental change (often as a result of sexual reproduction; e.g. plant seeds and invertebrate resting eggs). These diapausing stages can disrupt the relationship between past epidemics, host genetic diversity and future epidemics because they allow host dispersal through time. Specifically, temporally dispersing hosts avoid infection and thus selection from contemporary parasites, and also archive genetic variation for the future. We studied 80 epidemics in 20 semi-natural populations of the temporally dispersing crustacean Daphnia magna and its sterilizing bacterial parasite Pasteuria ramosa, and half of these populations experienced a simulated environmental disturbance treatment. We found that early initiation of diapause relative to the timing of the epidemic led to greater host genetic diversity and reduced epidemic size in the subsequent year, but this was unaffected by environmental disturbance.

Polystyrene nanoplastics differentially influence the outcome of infection by two microparasites of the host Daphnia magna.

The accumulation of micro- and nanoplastic particles in freshwater bodies has given rise to much concern regarding their potential adverse effects on aquatic biota. Beyond their known effects on single species, recent experimental evidence suggests that host-parasite interactions can also be affected by environmental concentrations of micro- and nanoplastics. However, investigating the effects of contaminants in simplified infection settings (i.e. one host, one parasite) may understate their ecological relevance, considering that co-infections are common in nature. We exposed the cladoceran Daphnia magna to a fungal parasite of the haemolymph (Metschnikowia bicuspidata) and a gut microsporidium (Ordospora colligata), either in single or co-infection. In addition, Daphnia were raised individually in culture media containing 0, 5 or 50 mg l-1 of polystyrene nanoplastic beads (100 nm). Only few infections were successful at the higher nanoplastic concentration, due to increased mortality of the host. While no significant effect of the low concentration was detected on the microsporidium, the proportion of hosts infected by the fungal parasite increased dramatically, leading to more frequent co-infections under nanoplastic exposure. These results indicate that nanoplastics can affect the performance of distinct pathogens in diverging ways, with the potential to favour parasite coexistence in a common zooplanktonic host. This article is part of the theme issue 'Infectious disease ecology and evolution in a changing world'.

Out of the 'host' box: extreme off-host conditions alter the infectivity and virulence of a parasitic bacterium.

Disease agents play an important role in the ecology and life history of wild and cultivated populations and communities. While most studies focus on the adaptation of parasites to their hosts, the adaptation of free-living parasite stages to their external (off-host) environment may tell us a lot about the factors that shape the distribution of parasites. Pasteuria ramosa is an endoparasitic bacterium of the water flea Daphnia with a wide geographical distribution. Its transmission stages rest outside of the host and thus experience varying environmental regimes. We examined the life history of P. ramosa populations from four environmental conditions (i.e. groups of habitats): the factorial combinations of summer-dry water bodies or not, and winter-freeze water bodies or not. Our goal was to examine how the combination of winter temperature and summer dryness affects the parasite's ability to attach to its host and to infect it. We subjected samples of the four groups of habitats to temperatures of 20, 33, 46 and 60°C in dry and wet conditions, and exposed a susceptible clone of Daphnia magna to the treated spores. We found that spores which had undergone desiccation endured higher temperatures better than spores kept wet, both regarding attachment and subsequent infection. Furthermore, spores treated with heightened temperatures were much less infective and virulent. Even under high temperatures (60°C), exposed spores from all populations were able to attach to the host cuticle, albeit they were unable to establish infection. Our work highlights the sensitivity of a host-free resting stage of a bacterial parasite to the external environment. Long heatwaves and harsh summers, which are becoming more frequent owing to recent climate changes, may therefore pose a problem for parasite survival. This article is part of the theme issue 'Infectious disease ecology and evolution in a changing world'.

Temperature modifies trait-mediated infection outcomes in a Daphnia-fungal parasite system.

One major concern related to climate change is that elevated temperatures will drive increases in parasite outbreaks. Increasing temperature is known to alter host traits and host-parasite interactions, but we know relatively little about how these are connected mechanistically-that is, about how warmer temperatures impact the relationship between epidemiologically relevant host traits and infection outcomes. Here, we used a zooplankton-fungus (Daphnia dentifera-Metschnikowia bicuspidata) disease system to experimentally investigate how temperature impacted physical barriers to infection and cellular immune responses. We found that Daphnia reared at warmer temperatures had more robust physical barriers to infection but decreased cellular immune responses during the initial infection process. Infected hosts at warmer temperatures also suffered greater reductions in fecundity and lifespan. Furthermore, the relationship between a key trait-gut epithelium thickness, a physical barrier-and the likelihood of terminal infection reversed at warmer temperatures. Together, our results highlight the complex ways that temperatures can modulate host-parasite interactions and show that different defense components can have qualitatively different responses to warmer temperatures, highlighting the importance of considering key host traits when predicting disease dynamics in a warmer world. This article is part of the theme issue 'Infectious disease ecology and evolution in a changing world'.

QTL study reveals candidate genes underlying host resistance in a Red Queen model system.

Specific interactions of host and parasite genotypes can lead to balancing selection, maintaining genetic diversity within populations. In order to understand the drivers of such specific coevolution, it is necessary to identify the molecular underpinnings of these genotypic interactions. Here, we investigate the genetic basis of resistance in the crustacean host, Daphnia magna, to attachment and subsequent infection by the bacterial parasite, Pasteuria ramosa. We discover a single locus with Mendelian segregation (3:1 ratio) with resistance being dominant, which we call the F locus. We use QTL analysis and fine mapping to localize the F locus to a 28.8-kb region in the host genome, adjacent to a known resistance supergene. We compare the 28.8-kb region in the two QTL parents to identify differences between host genotypes that are resistant versus susceptible to attachment and infection by the parasite. We identify 13 genes in the region, from which we highlight eight biological candidates for the F locus, based on presence/absence polymorphisms and differential gene expression. The top candidates include a fucosyltransferase gene that is only present in one of the two QTL parents, as well as several Cladoceran-specific genes belonging to a large family that is represented in multiple locations of the host genome. Fucosyltransferases have been linked to resistance in previous studies of Daphnia-Pasteuria and other host-parasite systems, suggesting that P. ramosa spore attachment could be mediated by changes in glycan structures on D. magna cuticle proteins. The Cladoceran-specific candidate genes suggest a resistance strategy that relies on gene duplication. Our results add a new locus to a growing genetic model of resistance in the D. magna-P. ramosa system. The identified candidate genes will be used in future functional genetic studies, with the ultimate aim to test for cycles of allele frequencies in natural populations.

Host-Associated Bacterial Communities Vary Between Daphnia galeata Genotypes but Not by Host Genetic Distance.

Host genotype may shape host-associated bacterial communities (commonly referred to as microbiomes). We sought to determine (a) whether bacterial communities vary among host genotypes in the water flea Daphnia galeata and (b) if this difference is driven by the genetic distance between host genotypes, by using D. galeata genotypes hatched from sediments of different time periods. We used 16S amplicon sequencing to profile the gut and body bacterial communities of eight D. galeata genotypes hatched from resting eggs; these were isolated from two distinct sediment layers (dating to 1989 and 2009) of a single sediment core of the lake Greifensee, and maintained in a common garden in laboratory cultures for 5 years. In general, bacterial community composition varied in both the Daphnia guts and bodies; but not between genotypes from different sediment layers. Specifically, genetic distances between host genotypes did not correlate with beta diversity of bacterial communities in Daphnia guts and bodies. Our results indicate that Daphnia bacterial community structure is to some extent determined by a host genetic component, but that genetic distances between hosts do not correlate with diverging bacterial communities.

Genetic slippage after sex maintains diversity for parasite resistance in a natural host population.

Although parasite-mediated selection is a major driver of host evolution, its influence on genetic variation for parasite resistance is not yet well understood. We monitored resistance in a large population of the planktonic crustacean Daphnia magna over 8 years, as it underwent yearly epidemics of the bacterial pathogen Pasteuria ramosa. We observed cyclic dynamics of resistance: Resistance increased throughout the epidemics, but susceptibility was restored each spring when hosts hatched from sexual resting stages. Host resting stages collected across the year showed that largely resistant host populations can produce susceptible sexual offspring. A genetic model of resistance developed for this host-parasite system, based on multiple loci and strong epistasis, is in partial agreement with our findings. Our results reveal that, despite strong selection for resistance in a natural host population, genetic slippage after sexual reproduction can be a strong factor for the maintenance of genetic diversity of host resistance.

Stochastic variation in gut bacterial community affects reproductive rates in the water flea Daphnia magna.

It is well-documented that perturbation of the gut bacterial community can influence the reproductive rates of the host. Less is known about how natural ecological processes can change the bacterial composition in the gut and how such changes influence the reproductive rate of the host. Here, we provide novel experimental insights into such processes using the clonally reproducing water flea, Daphnia magna. A total of 20 replicate cultures were reared for 5 weeks (Phase 1) to allow for divergence of bacterial communities through stochastic processes (i.e. drift, founder effects, and/or colonization). Duplicate cultures created from each of these were reared for 21 days (Phase 2) while recording reproductive rates. There was a significant repeatability in reproductive rates between these duplicates, suggesting that divergence of the bacterial communities during Phase 1 translated into reproductive rate effects during Phase 2. This was further supported by significant differences in the relative abundance of gut bacteria (investigated by amplicon sequencing of a part of the 16S rRNA gene) between cultures with high and low reproductive rate in Phase 2. These results are consistent with the hypothesis that stochastic processes can cause natural variation in the bacterial composition in the gut, which in turn affect host reproductive rates.

Infection by a eukaryotic gut parasite in wild Daphnia sp. associates with a distinct bacterial community.

Host-associated bacterial communities play an important role in host fitness and resistance to diseases. Yet, few studies have investigated tripartite interaction between a host, parasite and host-associated bacterial communities in natural settings. Here, we use 16S rRNA gene amplicon sequencing to compare gut- and body- bacterial communities of wild water fleas belonging to the Daphnia longispina complex, between uninfected hosts and those infected with the common and virulent eukaryotic gut parasite Caullerya mesnili (Family: Ichthyosporea). We report community-level changes in host-associated bacteria with the presence of the parasite infection; namely decreased alpha diversity and increased beta diversity at the site of infection, i.e. host gut (but not host body). We also report decreased abundance of bacterial taxa proposed elsewhere to be beneficial for the host, and an appearance of taxa specifically associated with infected hosts. Our study highlights the host-microbiota-infection link in a natural system and raises questions about the role of host-associated microbiota in natural disease epidemics as well as the functional roles of bacteria specifically associated with infected hosts.

Transgenerational plasticity alters parasite fitness in changing environments.

Transgenerational plasticity can help organisms respond rapidly to changing environments. Most prior studies of transgenerational plasticity in host­parasite interactions have focused on the host, leaving us with a limited understanding of transgenerational plasticity of parasites. We tested whether exposure to elevated temperatures while spores are developing can modify the ability of those spores to infect new hosts, as well as the growth and virulence of the next generation of parasites in the new host. We exposed Daphnia dentifera to its naturally co-occurring fungal parasite Metschnikowia bicuspidata, rearing the parasite at cooler (20°C) or warmer (24°C) temperatures and then, factorially, using those spores to infect at 20 and 24°C. Infections by parasites reared at warmer past temperatures produced more mature spores, but only when the current infections were at cooler temperatures. Moreover, the percentage of mature spores was impacted by both rearing and current temperatures, and was highest for infections with spores reared in a warmer environment that infected hosts in a cooler environment. In contrast, virulence was influenced only by current temperatures. These results demonstrate transgenerational plasticity of parasites in response to temperature changes, with fitness impacts that are dependent on both past and current environments.

Host Sex Modulates the Energetics of Pathogen Proliferation and Its Dependence on Environmental Resources.

AbstractSex differences in immunity are predicted to underlie much of the frequently observed sex differences in the prevalence or severity of infection. We propose the additional hypothesis that differences in the ability of males and females to acquire and use resources will also affect how readily a pathogen can convert host energy into transmission stages, thereby contributing to sex differences in infection dynamics. To test this we manipulated the resource environment of male and female Daphnia magna by altering the availability of food and then exposed hosts to a bacterial pathogen. We measured the production of transmission spores and virulence via the reduction in life span, together with feeding rates and changes in mass-independent metabolic rate, as a measure of the intake and expenditure of energy during infection. When raised in the presence of high resource levels, females more readily allowed for resources in the environment to be translated to pathogen exploitation, as represented by increased spore production, greater virulence, and higher energy use. In contrast, the traits of infected males were robust to changes in resource availability. High food availability thus exaggerated the degree of sexual dimorphism observed between the sexes. It also modified the relationship between host energy use, virulence, and pathogen spore production for each sex. These results suggest that a host's resource environment can affect how a male or female is exploited by a pathogen and may thus be an additional factor driving sex-specific patterns of disease susceptibility or severity.

Zooplankton as a Transitional Host for Escherichia coli in Freshwater.

This study shows that Escherichia coli can be temporarily enriched in zooplankton under natural conditions and that these bacteria can belong to different phylogroups and sequence types (STs), including environmental, clinical, and animal isolates. We isolated 10 E. coli strains and sequenced the genomes of two of them. Phylogenetically, the two isolates were closer to strains isolated from poultry meat than to freshwater E. coli, albeit their genomes were smaller than those of the poultry isolates. After isolation and fluorescent protein tagging of strains ED1 and ED157, we show that Daphnia sp. can take up these strains and release them alive again, thus becoming a temporary host for E. coli. In a chemostat experiment, we show that this association does not prolong bacterial long-term survival, but at low abundances it also does not significantly reduce bacterial numbers. We demonstrate that E. coli does not belong to the core microbiota of Daphnia, suffers from competition by the natural Daphnia microbiota, but can profit from its carapax to survive in water. All in all, this study suggests that the association of E. coli with Daphnia is only temporary, but the cells are viable therein, and this might allow encounters with other bacteria for genetic exchange and potential genomic adaptation to the freshwater environment. IMPORTANCE The contamination of freshwater with feces-derived bacteria is a major concern regarding drinking water acquisition and recreational activities. Ecological interactions promoting their persistence are still very scarcely studied. This study, which analyses the survival of E. coli in the presence of zooplankton, is thus of ecological and water safety relevance.

Daily feeding rhythm linked to microbiome composition in two zooplankton species.

Host-associated microbial communities are impacted by external and within-host factors, i.e., diet and feeding behavior. For organisms known to have a circadian rhythm in feeding behavior, microbiome composition is likely impacted by the different rates of microbe introduction and removal across a daily cycle, in addition to any diet-induced changes in microbial interactions. Here, we measured feeding behavior and used 16S rRNA sequencing to compare the microbial community across a diel cycle in two distantly related species of Daphnia, that differ in their life history traits, to assess how daily feeding patterns impact microbiome composition. We find that Daphnia species reared under similar laboratory conditions have significantly different microbial communities. Additionally, we reveal that Daphnia have daily differences in their microbial composition that correspond with feeding behavior, such that there is greater microbiome diversity at night during the host's active feeding phase. These results highlight that zooplankton microbiomes are relatively distinct and are likely influenced by host phylogeny.

Disentangling Diet- and Medium-Associated Microbes in Shaping Daphnia Gut Microbiome.

Host genotype and environment are considered crucial factors in shaping Daphnia gut microbiome composition. Among the environmental factors, diet is an important factor that regulates Daphnia microbiome. Most of the studies only focused on the use of axenic diet and non-sterile medium to investigate their effects on Daphnia microbiome. However, in natural environment, Daphnia diets such as phytoplankton are associated with microbes and could affect Daphnia microbiome composition and fitness, but remain relatively poorly understood compared to that of axenic diet. To test this, we cultured two Daphnia magna genotypes (genotype-1 and genotype-2) in sterile medium and fed with axenic diet. To check the effects of algal diet-associated microbes versus free water-related microbes, Daphnia were respectively inoculated with three different inoculums: medium microbial inoculum, diet-associated microbial inoculum, and medium and diet-mixed microbial inoculum. Daphnia were cultured for 3 weeks and their gut microbiome and life history traits were recorded. Results showed that Daphnia inoculated with medium microbial inoculum were dominated by Comamonadaceae in both genotypes. In Daphnia inoculated with mixed inoculum, genotype-1 microbiome was highly changed, whereas genotype-2 microbiome was slightly altered. Daphnia inoculated with diet microbial inoculum has almost the same microbiome in both genotypes. The total number of neonates and body size were significantly reduced in Daphnia inoculated with diet microbial inoculum regardless of genotype compared to all other treatments. Overall, this study shows that the microbiome of Daphnia is flexible and varies with genotype and diet- and medium-associated microbes, but not every bacteria is beneficial to Daphnia, and only symbionts can increase Daphnia performance.

Balancing Selection for Pathogen Resistance Reveals an Intercontinental Signature of Red Queen Coevolution.

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.