Host phenotype and microbiome vary with infection status, parasite genotype, and parasite microbiome composition.

A growing literature demonstrates the impact of helminths on their host gut microbiome. We investigated whether the stickleback host microbiome depends on ecoevolutionary variables by testing the impact of exposure to the cestode parasite Schistocephalus solidus with respect to infection success, host genotype, parasite genotype, and parasite microbiome composition. We observed constitutive differences in the microbiome of sticklebacks of different origin, and those differences increased when sticklebacks exposed to the parasite resisted infection. In contrast, the microbiome of successfully infected sticklebacks varied with parasite genotype. More specifically, we revealed that the association between microbiome and immune gene expression increased in infected individuals and varied with parasite genotype. In addition, we showed that S. solidus hosts a complex endomicrobiome and that bacterial abundance in the parasite correlates with expression of host immune genes. Within this comprehensive analysis we demonstrated that (i) parasites contribute to modulating the host microbiome through both successful and unsuccessful infection, (ii) when infection is successful, the host microbiome varies with parasite genotype due to genotype-dependent variation in parasite immunomodulation, and (iii) the parasite-associated microbiome is distinct from its host and impacts the host immune response to infection.

Cross-continental experimental infections reveal distinct defence mechanisms in populations of the three-spined stickleback Gasterosteus aculeatus.

Epidemiological traits of host-parasite associations depend on the effects of the host, the parasite and their interaction. Parasites evolve mechanisms to infect and exploit their hosts, whereas hosts evolve mechanisms to prevent infection and limit detrimental effects. The reasons why and how these traits differ across populations still remain unclear. Using experimental cross-infection of three-spined stickleback Gasterosteus aculeatus and their species-specific cestode parasites Schistocephalus solidus from Alaskan and European populations, we disentangled host, parasite and interaction effects on epidemiological traits at different geographical scales. We hypothesized that host and parasite main effects would dominate both within and across continents, although interaction effects would show geographical variation of natural selection within and across continents. We found that mechanisms preventing infection (qualitative resistance) occurred only in a combination of hosts and parasites from different continents, while mechanisms limiting parasite burden (quantitative resistance) and reducing detrimental effects of infection (tolerance) were host-population specific. We conclude that evolution favours distinct defence mechanisms on different geographical scales and that it is important to distinguish concepts of qualitative resistance, quantitative resistance and tolerance in studies of macroparasite infections.

Repeatability of Adaptive Radiation Depends on Spatial Scale: Regional Versus Global Replicates of Stickleback in Lake Versus Stream Habitats.

The repeatability of adaptive radiation is expected to be scale-dependent, with determinism decreasing as greater spatial separation among "replicates" leads to their increased genetic and ecological independence. Threespine stickleback (Gasterosteus aculeatus) provide an opportunity to test whether this expectation holds for the early stages of adaptive radiation-their diversification in freshwater ecosystems has been replicated many times. To better understand the repeatability of that adaptive radiation, we examined the influence of geographic scale on levels of parallel evolution by quantifying phenotypic and genetic divergence between lake and stream stickleback pairs sampled at regional (Vancouver Island) and global (North America and Europe) scales. We measured phenotypes known to show lake-stream divergence and used reduced representation genome-wide sequencing to estimate genetic divergence. We assessed the scale dependence of parallel evolution by comparing effect sizes from multivariate models and also the direction and magnitude of lake-stream divergence vectors. At the phenotypic level, parallelism was greater at the regional than the global scale. At the genetic level, putative selected loci showed greater lake-stream parallelism at the regional than the global scale. Generally, the level of parallel evolution was low at both scales, except for some key univariate traits. Divergence vectors were often orthogonal, highlighting possible ecological and genetic constraints on parallel evolution at both scales. Overall, our results confirm that the repeatability of adaptive radiation decreases at increasing spatial scales. We suggest that greater environmental heterogeneity at larger scales imposes different selection regimes, thus generating lower repeatability of adaptive radiation at larger spatial scales.

Examining the role of parasites in limiting unidirectional gene flow between lake and river sticklebacks.

Parasites are important selective agents with the potential to limit gene flow between host populations by shaping local host immunocompetence. We report on a contact zone between lake and river three-spined sticklebacks (Gasterosteus aculeatus) that offers the ideal biogeographic setting to explore the role of parasite-mediated selection on reproductive isolation. A waterfall acts as a natural barrier and enforces unidirectional migration from the upstream river stickleback population to the downstream river and lake populations. We assessed population genetic structure and parasite communities over four years. In a set of controlled experimental infections, we compared parasite susceptibility of upstream and downstream fish by exposing laboratory-bred upstream river and lake fish, as well as hybrids, to two common lake parasite species: a generalist trematode parasite, Diplostomum pseudospathaceum, and a host-specific cestode, Schistocephalus solidus. We found consistent genetic differentiation between upstream and downstream populations across four sampling years, even though the downstream river consisted of ~10% first-generation migrants from the upstream population as detected by parentage analysis. Fish in the upstream population had lower genetic diversity and were strikingly devoid of macroparasites. Through experimental infections, we demonstrated that upstream fish and their hybrids had higher susceptibility to parasite infections than downstream fish. Despite this, naturally sampled upstream migrants were less infected than downstream residents. Thus, migrants coming from a parasite-free environment may enjoy an initial fitness advantage, but their descendants seem likely to suffer from higher parasite loads. Our results suggest that adaptation to distinct parasite communities can influence stickleback invasion success and may represent a barrier to gene flow, even between close and connected populations.

Correction to: Specificity of resistance and geographic patterns of virulence in a vertebrate hostparasite system.

After publication of the original article [1], the authors have notified us that the incorrect version of Fig. 4 was used. Below you can find the both incorrect and correct versions of the figure.

The right response at the right time: Exploring helminth immune modulation in sticklebacks by experimental coinfection.

Parasites are one of the strongest selective agents in nature. They select for hosts that evolve counter-adaptive strategies to cope with infection. Helminth parasites are special because they can modulate their hosts' immune responses. This phenomenon is important in epidemiological contexts, where coinfections may be affected. How different types of hosts and helminths interact with each other is insufficiently investigated. We used the three-spined stickleback (Gasterosteus aculeatus) - Schistocephalus solidus model to study mechanisms and temporal components of helminth immune modulation. Sticklebacks from two contrasting populations with either high resistance (HR) or low resistance (LR) against S. solidus, were individually exposed to S. solidus strains with characteristically high growth (HG) or low growth (LG) in G. aculeatus. We determined the susceptibility to another parasite, the eye fluke Diplostomum pseudospathaceum, and the expression of 23 key immune genes at three time points after S. solidus infection. D. pseudospathaceum infection rates and the gene expression responses depended on host and S. solidus type and changed over time. Whereas the effect of S. solidus type was not significant after three weeks, T regulatory responses and complement components were upregulated at later time points if hosts were infected with HG S. solidus. HR hosts showed a well orchestrated immune response, which was absent in LR hosts. Our results emphasize the role of regulatory T cells and the timing of specific immune responses during helminth infections. This study elucidates the importance to consider different coevolutionary trajectories and ecologies when studying host-parasite interactions.

Specificity of resistance and geographic patterns of virulence in a vertebrate host-parasite system.

BACKGROUND: Host genotype - parasite genotype co-evolutionary dynamics are influenced by local biotic and abiotic environmental conditions. This results in spatially heterogeneous selection among host populations. How such heterogeneous selection influences host resistance, parasite infectivity and virulence remains largely unknown. We hypothesized that different co-evolutionary trajectories of a vertebrate host-parasite association result in specific virulence patterns when assessed on a large geographic scale. We used two reference host populations of three-spined sticklebacks and nine strains of their specific cestode parasite Schistocephalus solidus from across the Northern Hemisphere for controlled infection experiments. Host and parasite effects on infection phenotypes including host immune gene expression were determined. RESULTS: S. solidus strains grew generally larger in hosts coming from a population with high parasite diversity and low S. solidus prevalence (DE hosts). Hosts from a population with low parasite diversity and high S. solidus prevalence (NO hosts) were better able to control the parasite's growth, regardless of the origin of the parasite. Host condition and immunological parameters converged upon infection and parasite growth showed the same geographic pattern in both host types. CONCLUSION: Our results suggest that NO sticklebacks evolved resistance against a variety of S. solidus strains, whereas DE sticklebacks are less resistant against S. solidus. Our data provide evidence that differences in parasite prevalence can cause immunological heterogeneity and that parasite size, a proxy for virulence and resistance, is, on a geographic scale, determined by main effects of the host and the parasite and less by an interaction of both genotypes.

Virulence in the three-spined stickleback specific parasite Schistocephalus solidus is inherited additively.

Parasite virulence is a key trait in host-parasite interactions and plays a crucial role in infection dynamics. Our study system offers the rare opportunity to study the virulence of an individual macroparasite (Schistocephalus solidus) in its vertebrate fish host (Gasterosteus aculeatus). The size of the tapeworm in the fish can be regarded as a good proxy for individual parasite virulence, as parasite size correlates negatively with fitness traits of the stickleback host (i.e. the bigger the parasite, the lower the host's reproductive success) as well as directly with the number of parasite offspring to be expected. To investigate how virulence is inherited, laboratory bred, parasite-naïve stickleback were infected with a cross of two S. solidus populations of either high or low virulence, as well as one hybrid cross between the two. The relative weight of the parasite as expressed in the parasite index served as a measure of virulence. Furthermore, we measured several condition and immune related traits in the fish host to assess parasite impact on the stickleback. We hypothesized that parasite virulence is to a large extent genetically determined and correlated with several fitness traits in the stickleback host. We found that virulence is inherited additively in S. solidus, with hybrids of high and low virulence parasites displaying intermediate levels. However, contrary to expectation, infection rate of S. solidus in three-spined stickleback is not related to virulence. Even though the presence of the parasite caused differences in host condition, these were indistinguishable between the different levels of virulence in this experiment. Fish immune traits also showed a response to infection but had no correlation with level of parasite virulence. With this experiment we have taken the first step towards understanding how virulence is inherited and how it is driven in the Schistocephalus-stickleback system, even though virulence, as measured here, does not directly translate into cost for the host. A better understanding of the costs inflicted on the host by S. solidus infection is needed to understand this interaction in greater detail.

Resist Globally, Infect Locally: A Transcontinental Test of Adaptation by Stickleback and Their Tapeworm Parasite.

Parasite infections are a product of both ecological processes affecting host-parasite encounter rates and evolutionary dynamics affecting host susceptibility. However, few studies examine natural infection variation from both ecological and evolutionary perspectives. Here, we describe the ecological and evolutionary factors generating variation in infection rates by a tapeworm (Schistocephalus solidus) in a vertebrate host, the threespine stickleback (Gasterosteus aculeatus). To explore ecological aspects of infection, we measured tapeworm prevalence in Canadian stickleback inhabiting two distinct environments: marine and freshwater. Consistent with ecological control of infection, the tapeworm is very rare in marine environments, even though marine fish are highly susceptible. Conversely, commonly infected freshwater stickleback exhibit substantial resistance in controlled laboratory trials, suggesting that high exposure risk overwhelms their recently evolved resistance. We also tested for parasite adaptation to its host by performing transcontinental reciprocal infections, using stickleback and tapeworm populations from Europe and western Canada. More infections occurred in same-continent host-parasite combinations, indicating parasite "local" adaptation, at least on the scale of continents. However, the recently evolved immunity of freshwater hosts applies to both local and foreign parasites. The pattern of adaptation described here is not wholly compatible with either of the common models of host-parasite coevolution (i.e., matching infection or targeted recognition). Instead, we propose a hybrid, eco-evolutionary model to explain the remarkable pattern of global host resistance and local parasite infectivity.

The role of prezygotic isolation mechanisms in the divergence of two parasite species.

BACKGROUND: The formation of reproductive barriers in diverging lineages is a prerequisite to complete speciation according to the biological species concept. In parasites with complex life cycles, speciation may be driven by adaptation to different intermediate hosts, yet diverging lineages can still share the same definitive host where reproduction takes place. In these cases, prezygotic isolation mechanisms should evolve very early and be particularly strong, preventing costly unfavourable matings. In this study, we investigated the importance of prezygotic barriers to reproduction in two cestode species that diverged 20-25mya and show an extraordinary degree of specificity to different intermediate hosts. Both species share the same definitive hosts and hybridize in the laboratory. Yet, natural hybrids have so far not been detected. METHODS: We used a combination of different experiments to investigate the role of prezygotic barriers to reproduction in the speciation of these parasites. First, we investigated whether hybridization is possible under natural conditions by exposing lab-reared herring gulls (Larus argentatus, the definitive hosts) to both parasites of either sympatric or allopatric combinations. In a second experiment, we tested whether the parasites prefer conspecifics over parasites from a different species in dichotomous mate choice trials. RESULTS: Our results show that the two species hybridize under natural conditions with parasites originating either from sympatric or allopatric populations producing hybrid offspring. Surprisingly, the mate choice experiment indicated that both parasite species prefer mates of the different species to conspecifics. CONCLUSIONS: Neither fundamental constraints against hybridization in a natural host nor assortative mate choice sufficiently explain the persistent segregation of the two tapeworm species in nature. Hence, postzygotic ecological selection against hybrids is presumably the more important driving force limiting gene flow between the two parasite sister species.

Comparative transcriptomics of stickleback immune gene responses upon infection by two helminth parasites, Diplostomum pseudospathaceum and Schistocephalus solidus.

Immune systems of vertebrates are much more diverse than previously thought, in particular at the base of the vertebrate clade. RNA-seq was used to describe in detail the transcriptomic response of stickleback hosts to infection by two helminth parasites, the trematode Diplostomum pseudospathaceum (2 genotypes plus a genotype mix) and the cestode Schistocephalus solidus. Based on a global transcription profiling, we present immune genes that are active during chronic or multiple repeated infection. We found that the transcription profiles of D. pseudospathaceum genotypes were as divergent as those of the two parasite species. When comparing the host immune response, only 5 immune genes were consistently upregulated upon infection by both species. These genes indicated a role for enhanced toll like receptor (TLR) activity (CTSK, CYP27B1) and an associated positive regulation of macrophages (CYP27B1, THBS1) for general helminth defense. We interpret the largely differentiated gene expression response among parasite species as general redundancy of the vertebrate immune system, which was also visible in genotype-specific responses among the different D. pseudospathaceum infections. The present study provides the first evidence that IL4-mediated activation of T-helper lymphocyte cells is also important in anti-helminthic immune responses of teleost fish.

Effects of environmental variation on host-parasite interaction in three-spined sticklebacks (Gasterosteus aculeatus).

Recent research provides accumulating evidence that the evolutionary dynamics of host-parasite adaptations strongly depend on environmental variation. In this context, the three-spined stickleback (Gasterosteus aculeatus) has become an important research model since it is distributed all over the northern hemisphere and lives in very different habitat types, ranging from marine to freshwater, were it is exposed to a huge diversity of parasites. While a majority of studies start from explorations of sticklebacks in the wild, only relatively few investigations have continued under laboratory conditions. Accordingly, it has often been described that sticklebacks differ in parasite burden between habitats, but the underlying co-evolutionary trajectories are often not well understood. With the present review, we give an overview of the most striking examples of stickleback-parasite-environment interactions discovered in the wild and discuss two model parasites which have received some attention in laboratory studies: the eye fluke Diplostomum pseudospathacaeum, for which host fish show habitat-specific levels of resistance, and the tapeworm Schistocephalus solidus, which manipulates immunity and behavior of its stickleback host to its advantage. Finally, we will concentrate on an important environmental variable, namely temperature, which has prominent effects on the activity of the immune system of ectothermic hosts and on parasite growth rates.

Experimental parasite community ecology: intraspecific variation in a large tapeworm affects community assembly.

Non-random species associations occur in naturally sampled parasite communities. The processes resulting in predictable community structure (e.g. particular host behaviours, cross-immunity, interspecific competition) could be affected by traits that vary within a parasite species, like growth or antigenicity. We experimentally infected three-spined sticklebacks with a large tapeworm (Schistocephalus solidus) that impacts the energy needs, foraging behaviour and immune reactions of its host. The tapeworms came from two populations, characterized by high or low growth in sticklebacks. Our goal was to evaluate how this parasite, and variation in its growth, affects the acquisition of other parasites. Fish infected with S. solidus were placed into cages in a lake to expose them to the natural parasite community. We also performed a laboratory experiment in which infected fish were exposed to a fixed dose of a common trematode parasite. In the field experiment, infection with S. solidus affected the abundance of four parasite species, relative to controls. For two of the four species, changes occurred only in fish harbouring the high-growth S. solidus; one species increased in abundance and the other decreased. These changes did not appear to be directly linked to S. solidus growth though. The parasite exhibiting elevated abundance was the same trematode used in the laboratory infection. In that experiment, we found a similar infection pattern, suggesting that S. solidus affects the physiological susceptibility of fish to this trematode. Associations between S. solidus and other parasites occur and vary in direction. However, some of these associations were contingent on the S. solidus population, suggesting that intraspecific variability can affect the assembly of parasite communities.

Reciprocal cross infection of sticklebacks with the diphyllobothriidean cestode Schistocephalus solidus reveals consistent population differences in parasite growth and host resistance.

BACKGROUND: In host-parasite evolutionary arms races, parasites are generally expected to adapt more rapidly, due to their large population sizes and short generation times. There exist systems, though, where parasites cannot outpace their hosts because of similar generation times in both antagonists. In those cases concomitant adaptation is expected. METHODS: We tested this hypothesis in the three-spined stickleback-Schistocephalus solidus tapeworm system, where generation times are comparable in both organisms. We chose two populations of sticklebacks which differ prominently in the prevalence of S. solidus and consequently in its level of selective pressure. We performed a full factorial common garden experiment. Particularly, Norwegian (NO) and German (DE) sticklebacks, as well as hybrids between both stickleback populations and in both parental combinations, were exposed each to a single S. solidus originating from the same two host populations. RESULTS: We found the infection phenotype to depend on the host population, the parasite population, but not their interaction. NO-parasites showed higher infectivity than DE-parasites, with NO-sticklebacks also being more resistant to DE-parasites than to the sympatric NO-parasite. Reciprocally, DE-hosts were more susceptible to the allopatric NO-parasite while DE-parasites grew less than NO-parasites in all stickleback groups. Despite this asymmetry, the ratio of worm to host weight, an indicator of parasite virulence, was identical in both sympatric combinations, suggesting an optimal virulence as a common outcome of parallel coevolved systems. In hybrid sticklebacks, intermediate infection rates and growth of S. solidus from either origin suggests a simple genetic basis of resistance. However, comparison of infection phenotypes in NO-maternal and DE-maternal hybrid sticklebacks indicates local adaptation to the sympatric counterpart in both the host and the parasite. CONCLUSIONS: Host-parasite systems with similar generation time show evidence for concomitant reciprocal adaptation resulting in parasite optimal virulence and host parasite specific resistance.

Transcriptome profiling of immune tissues reveals habitat-specific gene expression between lake and river sticklebacks.

The observation of habitat-specific phenotypes suggests the action of natural selection. The three-spined stickleback (Gasterosteus aculeatus) has repeatedly colonized and adapted to diverse freshwater habitats across the northern hemisphere since the last glaciation, while giving rise to recurring phenotypes associated with specific habitats. Parapatric lake and river populations of sticklebacks harbour distinct parasite communities, a factor proposed to contribute to adaptive differentiation between these ecotypes. However, little is known about the transcriptional response to the distinct parasite pressure of those fish in a natural setting. Here, we sampled wild-caught sticklebacks across four geographical locations from lake and river habitats differing in their parasite load. We compared gene expression profiles between lake and river populations using 77 whole-transcriptome libraries from two immune-relevant tissues, the head kidney and the spleen. Differential expression analyses revealed 139 genes with habitat-specific expression patterns across the sampled population pairs. Among the 139 differentially expressed genes, eight are annotated with an immune function and 42 have been identified as differentially expressed in previous experimental studies in which fish have been immune challenged. Together, these findings reinforce the hypothesis that parasites contribute to adaptation of sticklebacks in lake and river habitats.

Immunity comes first: the effect of parasite genotypes on adaptive immunity and immunization in three-spined sticklebacks.

Adaptive immunity in vertebrates can confer increased resistance against invading pathogens upon re-infection. But how specific parasite genotypes affect the temporal transition from innate to adaptive immunity under continual exposure to parasites is poorly understood. Here, we investigated the effects of homologous and heterologous exposures of genetically distinct parasite lineages of the eye fluke Diplostomum pseudospathaceum on gene expression patterns of adaptive immunity in sticklebacks (Gasterosteus aculeatus). Observable differences in gene expression were largely attributable to final exposures while there was no transcription pattern characteristic for a general response to repeated infections with D. pseudospathaceum. None of the final exposure treatments was able to erase the distinct expression patterns resulting from a heterologous pre-exposed fish. Interestingly, heterologous final exposures showed similarities between different treatment groups subjected to homologous pre-exposure. The observed pattern was supported by parasite infection rates and suggests that host immunization was optimized towards an adaptive immune response that favored effectiveness against parasite diversity over specificity.

Diversity, specificity and speciation in larval Diplostomidae (Platyhelminthes: Digenea) in the eyes of freshwater fish, as revealed by DNA barcodes.

Larvae (metacercariae) in some species of Diplostomidae (Platyhelminthes: Digenea) inhabit fish eyes and are difficult to identify to species based on morphology. DNA barcoding has clarified the diversity and life cycles of diplostomids in North America, Europe and Africa, but has seldom been used in parasites sampled in large numbers or at large spatial scales. Here, distance-based analysis of cytochrome c oxidase 1 barcodes and, in some specimens, internal transcribed spacer (ITS-1, 5.8S, ITS-2) sequences was performed for over 2000 diplostomids from Africa, the Middle East, Europe, Asia and the Americas. Fifty-two species of Diplostomum, Tylodelphys and Austrodiplostomum (Digenea: Diplostomidae) were distinguished. The 52 species comprise 12 identified species, six species in two species complexes and 34 putative species, and 33/52 had been delineated in previous studies. Most (23/40) of the unidentified, putative species distinguished by cytochrome c oxidase 1 distances were supported by at least one additional line of evidence. As the intensity of sampling of the 52 species increased, variation in cytochrome c oxidase 1 decreased between and increased within species, while the spatial scale at which species were sampled had no effect. Nonetheless, variation between species always exceeded variation within species. New life-cycle linkages, geographic and host records, and genetic data were recorded in several species, including Tylodelphys jenynsiae, Tylodelphys immer and Diplostomum ardeae. Species of Diplostomum inhabiting the lens are less host-specific and less numerous than those infecting other tissues, suggesting that reduced immune activity in the lens has influenced rates of speciation.

Identification of candidate mimicry proteins involved in parasite-driven phenotypic changes.

BACKGROUND: Endoparasites with complex life cycles are faced with several biological challenges, as they need to occupy various ecological niches throughout their development. Host phenotypes that increase the parasite's transmission rate to the next host have been extensively described, but few mechanistic explanations have been proposed to describe their proximate causes. In this study we explore the possibility that host phenotypic changes are triggered by the production of mimicry proteins from the parasite by using an ecological model system consisting of the infection of the threespine stickleback (Gasterosteus aculeatus) by the cestode Schistocephalus solidus. METHOD: Using RNA-seq data, we assembled 9,093 protein-coding genes from which ORFs were predicted to generate a reference proteome. Based on a previously published method, we built two complementary analysis pipelines to i) establish a general classification of protein similarity among various species (pipeline A) and ii) identify candidate mimicry proteins showing specific host-parasite similarities (pipeline B), a key feature underlying the possibility of molecular mimicry. RESULTS: Ninety-four tapeworm proteins showed high local sequence homology with stickleback proteins. Four of these candidates correspond to secreted or membrane proteins that could be produced by the parasite and eventually be released in or be in contact with the host to modulate physiological pathways involved in various phenotypes (e.g. behaviors). One of these candidates belongs to the Wnt family, a large group of signaling molecules involved in cell-to-cell interactions and various developmental pathways. The three other candidates are involved in ion transport and post-translational protein modifications. We further confirmed that these four candidates are expressed in three different developmental stages of the cestode by RT-PCR, including the stages found in the host. CONCLUSION: In this study, we identified mimicry candidate peptides from a behavior-altering cestode showing specific sequence similarity with host proteins. Despite their potential role in modulating host pathways that could lead to parasite-induced phenotypic changes and despite our confirmation that they are expressed in the developmental stage corresponding to the altered host behavior, further investigations will be needed to confirm their mechanistic role in the molecular cross-talk taking place between S. solidus and the threespine stickleback.

Genomics of divergence along a continuum of parapatric population differentiation.

The patterns of genomic divergence during ecological speciation are shaped by a combination of evolutionary forces. Processes such as genetic drift, local reduction of gene flow around genes causing reproductive isolation, hitchhiking around selected variants, variation in recombination and mutation rates are all factors that can contribute to the heterogeneity of genomic divergence. On the basis of 60 fully sequenced three-spined stickleback genomes, we explore these different mechanisms explaining the heterogeneity of genomic divergence across five parapatric lake and river population pairs varying in their degree of genetic differentiation. We find that divergent regions of the genome are mostly specific for each population pair, while their size and abundance are not correlated with the extent of genome-wide population differentiation. In each pair-wise comparison, an analysis of allele frequency spectra reveals that 25-55% of the divergent regions are consistent with a local restriction of gene flow. Another large proportion of divergent regions (38-75%) appears to be mainly shaped by hitchhiking effects around positively selected variants. We provide empirical evidence that alternative mechanisms determining the evolution of genomic patterns of divergence are not mutually exclusive, but rather act in concert to shape the genome during population differentiation, a first necessary step towards ecological speciation.