Stepwise recombination suppression around the mating-type locus in an ascomycete fungus with self-fertile spores.

Recombination is often suppressed at sex-determining loci in plants and animals, and at self-incompatibility or mating-type loci in plants and fungi. In fungal ascomycetes, recombination suppression around the mating-type locus is associated with pseudo-homothallism, i.e. the production of self-fertile dikaryotic sexual spores carrying the two opposite mating types. This has been well studied in two species complexes from different families of Sordariales: Podospora anserina and Neurospora tetrasperma. However, it is unclear whether this intriguing association holds in other species. We show here that Schizothecium tetrasporum, a fungus from a third family in the order Sordariales, also produces mostly self-fertile dikaryotic spores carrying the two opposite mating types. This was due to a high frequency of second meiotic division segregation at the mating-type locus, indicating the occurrence of a single and systematic crossing-over event between the mating-type locus and the centromere, as in P. anserina. The mating-type locus has the typical Sordariales organization, plus a MAT1-1-1 pseudogene in the MAT1-2 haplotype. High-quality genome assemblies of opposite mating types and segregation analyses revealed a suppression of recombination in a region of 1.47 Mb around the mating-type locus. We detected three evolutionary strata, indicating a stepwise extension of recombination suppression. The three strata displayed no rearrangement or transposable element accumulation but gene losses and gene disruptions were present, and precisely at the strata margins. Our findings indicate a convergent evolution of self-fertile dikaryotic sexual spores across multiple ascomycete fungi. The particular pattern of meiotic segregation at the mating-type locus was associated with recombination suppression around this locus, that had extended stepwise. This association between pseudo-homothallism and recombination suppression across lineages and the presence of gene disruption at the strata limits are consistent with a recently proposed mechanism of sheltering deleterious alleles to explain stepwise recombination suppression.

Size Variation of the Nonrecombining Region on the Mating-Type Chromosomes in the Fungal Podospora anserina Species Complex.

Sex chromosomes often carry large nonrecombining regions that can extend progressively over time, generating evolutionary strata of sequence divergence. However, some sex chromosomes display an incomplete suppression of recombination. Large genomic regions without recombination and evolutionary strata have also been documented around fungal mating-type loci, but have been studied in only a few fungal systems. In the model fungus Podospora anserina (Ascomycota, Sordariomycetes), the reference S strain lacks recombination across a 0.8-Mb region around the mating-type locus. The lack of recombination in this region ensures that nuclei of opposite mating types are packaged into a single ascospore (pseudohomothallic lifecycle). We found evidence for a lack of recombination around the mating-type locus in the genomes of ten P. anserina strains and six closely related pseudohomothallic Podospora species. Importantly, the size of the nonrecombining region differed between strains and species, as indicated by the heterozygosity levels around the mating-type locus and experimental selfing. The nonrecombining region is probably labile and polymorphic, differing in size and precise location within and between species, resulting in occasional, but infrequent, recombination at a given base pair. This view is also supported by the low divergence between mating types, and the lack of strong linkage disequilibrium, chromosomal rearrangements, transspecific polymorphism and genomic degeneration. We found a pattern suggestive of evolutionary strata in P. pseudocomata. The observed heterozygosity levels indicate low but nonnull outcrossing rates in nature in these pseudohomothallic fungi. This study adds to our understanding of mating-type chromosome evolution and its relationship to mating systems.

Convergent recombination cessation between mating-type genes and centromeres in selfing anther-smut fungi.

The degree of selfing has major impacts on adaptability and is often controlled by molecular mechanisms determining mating compatibility. Changes in compatibility systems are therefore important evolutionary events, but their underlying genomic mechanisms are often poorly understood. Fungi display frequent shifts in compatibility systems, and their small genomes facilitate elucidation of the mechanisms involved. In particular, linkage between the pre- and postmating compatibility loci has evolved repeatedly, increasing the odds of gamete compatibility under selfing. Here, we studied the mating-type chromosomes of two anther-smut fungi with unlinked mating-type loci despite a self-fertilization mating system. Segregation analyses and comparisons of high-quality genome assemblies revealed that these two species displayed linkage between mating-type loci and their respective centromeres. This arrangement renders the same improved odds of gamete compatibility as direct linkage of the two mating-type loci under the automictic mating (intratetrad selfing) of anther-smut fungi. Recombination cessation was found associated with a large inversion in only one of the four linkage events. The lack of trans-specific polymorphism at genes located in nonrecombining regions and linkage date estimates indicated that the events of recombination cessation occurred independently in the two sister species. Our study shows that natural selection can repeatedly lead to similar genomic patterns and phenotypes, and that different evolutionary paths can lead to distinct yet equally beneficial responses to selection. Our study further highlights that automixis and gene linkage to centromeres have important genetic and evolutionary consequences, while being poorly recognized despite being present in a broad range of taxa.

Congruent population genetic structures and divergence histories in anther-smut fungi and their host plants Silene italica and the Silene nutans species complex.

Study of the congruence of population genetic structure between hosts and pathogens gives important insights into their shared phylogeographical and coevolutionary histories. We studied the population genetic structure of castrating anther-smut fungi (genus Microbotryum) and of their host plants, the Silene nutans species complex, and the morphologically and genetically closely related Silene italica, which can be found in sympatry. Phylogeographical population genetic structure related to persistence in separate glacial refugia has been recently revealed in the S. nutans plant species complex across Western Europe, identifying several distinct lineages. We genotyped 171 associated plant-pathogen pairs of anther-smut fungi and their host plant individuals using microsatellite markers and plant chloroplastic single nucleotide polymorphisms. We found clear differentiation between fungal populations parasitizing S. nutans and S. italica plants. The population genetic structure of fungal strains parasitizing the S. nutans plant species complex mirrored the host plant genetic structure, suggesting that the pathogen was isolated in glacial refugia together with its host and/or that it has specialized on the plant genetic lineages. Using random forest approximate Bayesian computation (ABC-RF), we found that the divergence history of the fungal lineages on S. nutans was congruent with that previously inferred for the host plant and probably occurred with ancient but no recent gene flow. Genome sequences confirmed the genetic structure and the absence of recent gene flow between fungal genetic lineages. Our analyses of individual host-pathogen pairs contribute to a better understanding of co-evolutionary histories between hosts and pathogens in natural ecosystems, in which such studies remain scarce.

Independent domestication events in the blue-cheese fungus Penicillium roqueforti.

Domestication provides an excellent framework for studying adaptive divergence. Using population genomics and phenotypic assays, we reconstructed the domestication history of the blue cheese mould Penicillium roqueforti. We showed that this fungus was domesticated twice independently. The population used in Roquefort originated from an old domestication event associated with weak bottlenecks and exhibited traits beneficial for pre-industrial cheese production (slower growth in cheese and greater spore production on bread, the traditional multiplication medium). The other cheese population originated more recently from the selection of a single clonal lineage, was associated with all types of blue cheese worldwide except Roquefort, and displayed phenotypes more suited for industrial cheese production (high lipolytic activity, efficient cheese cavity colonization ability and salt tolerance). We detected genomic regions affected by recent positive selection and putative horizontal gene transfers. This study sheds light on the processes of rapid adaptation and raises questions about genetic resource conservation.

Multiple infections, relatedness and virulence in the anther-smut fungus castrating Saponaria plants.

Multiple infections (co-occurrence of multiple pathogen genotypes within an individual host) can have important impacts on diseases. Relatedness among pathogens can affect the likelihood of multiple infections and their consequences through kin selection. Previous studies on the castrating anther-smut fungus Microbotryum lychnidis-dioicae have shown that multiple infections occur in its host plant Silene latifolia. Relatedness was high among fungal genotypes within plants, which could result from competitive exclusion between unrelated fungal genotypes, from population structure or from interactions between plant and fungal genotypes for infection ability. Here, we aimed at disentangling these hypotheses using M. saponariae and its host Saponaria officinalis, both experimentally tractable for these questions. By analysing populations using microsatellite markers, we also found frequent occurrence of multiple infections and high relatedness among strains within host plants. Infections resulting from experimental inoculations in the greenhouse also revealed high relatedness among strains co-infecting host plants, even in clonally replicated plant genotypes, indicating that high relatedness within plants did not result merely from plant x fungus interactions or population structure. Furthermore, hyphal growth in vitro was affected by the presence of a competitor growing nearby and by its genetic similarity, although this latter effect was strain-dependent. Altogether, our results support the hypothesis that relatedness-dependent competitive exclusion occurs in Microbotryum fungi within plants. These microorganisms can thus respond to competitors and to their level of relatedness.

Co-occurrence among three divergent plant-castrating fungi in the same Silene host species.

The competitive exclusion principle postulates that different species can only coexist in sympatry if they occupy distinct ecological niches. The goal of this study was to understand the geographical distribution of three species of Microbotryum anther-smut fungi that are distantly related but infect the same host plants, the sister species Silene vulgaris and S. uniflora, in Western Europe. We used microsatellite markers to investigate pathogen distribution in relation to host specialization and ecological factors. Microbotryum violaceo-irregulare was only found on S. vulgaris at high elevations in the Alps. Microbotryum lagerheimii could be subdivided into two genetically differentiated clusters, one on S. uniflora in the UK and the second on S. vulgaris in the Alps and Pyrenees. The most abundant pathogen species, M. silenes-inflatae, could be subdivided into four genetic clusters, co-occurring in the Alps, the UK and the Pyrenees, and was found on both S. vulgaris and S. uniflora. All three fungal species had high levels of homozygosity, in agreement with the selfing mating system generally observed in anther-smut fungi. The three pathogen species and genetic clusters had large range overlaps, but occurred at sites with different elevations, temperatures and precipitation levels. The three Microbotryum species thus do not appear to be maintained by host specialization or geographic allopatry, but instead may occupy different ecological niches in terms of environmental conditions.

A microRNA allele that emerged prior to apple domestication may underlie fruit size evolution.

The molecular genetic mechanisms underlying fruit size remain poorly understood in perennial crops, despite size being an important agronomic trait. Here we show that the expression level of a microRNA gene (miRNA172) influences fruit size in apple. A transposon insertional allele of miRNA172 showing reduced expression associates with large fruit in an apple breeding population, whereas over-expression of miRNA172 in transgenic apple significantly reduces fruit size. The transposon insertional allele was found to be co-located with a major fruit size quantitative trait locus, fixed in cultivated apples and their wild progenitor species with relatively large fruit. This finding supports the view that the selection for large size in apple fruit was initiated prior to apple domestication, likely by large mammals, before being subsequently strengthened by humans, and also helps to explain why signatures of genetic bottlenecks and selective sweeps are normally weaker in perennial crops than in annual crops.

Strong phylogeographic co-structure between the anther-smut fungus and its white campion host.

Although congruence between host and pathogen phylogenies has been extensively investigated, the congruence between host and pathogen genetic structures at the within-species level has received little attention. Using an unprecedented and comprehensive collection of associated plant-pathogen samples, we investigated the degree of congruence between the genetic structures across Europe of two evolutionary and ecological model organisms, the anther-smut pathogen Microbotryum lychnidis-dioicae and its host plant Silene latifolia. We demonstrated a significant and particularly strong level of host-pathogen co-structure, with three main genetic clusters displaying highly similar spatial ranges in Western Europe, Eastern Europe and Italy, respectively. Correcting for the geographical component of genetic variation, significant correlations were still found between the genetic distances of anther-smut and host populations. Inoculation experiments suggested plant local adaptation, at the cluster level, for resistance to pathogens. These findings indicate that the pathogen remained isolated in the same fragmented southern refugia as its host plant during the last glaciation, and that little long-distance dispersal has occurred since the recolonization of Europe for either the plant or the pathogen, despite their known ability to travel across continents. This, together with the inoculation results, suggests that coevolutionary and competitive processes may be drivers of host-pathogen co-structure.

Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant.

Cold-adapted organisms with current arctic-alpine distributions have persisted during the last glaciation in multiple ice-free refugia, leaving footprints in their population structure that contrast with temperate plants and animals. However, pathogens that live within hosts having arctic-alpine distributions have been little studied. Here, we therefore investigated the geographical range and population structure of a fungus parasitizing an arctic-alpine plant. A total of 1437 herbarium specimens of the plant Silene acaulis were examined, and the anther smut pathogen Microbotryum silenes-acaulis was present throughout the host's geographical range. There was significantly greater incidence of anther smut disease in more northern latitudes and where the host locations were less dense, indicating a major influence of environmental factors and/or host demographic structure on the pathogen distribution. Genetic analyses with seven microsatellite markers on recent collections of 195 M. silenes-acaulis individuals revealed three main genetic clusters, in North America, northern Europe and southern Europe, likely corresponding to differentiation in distinct refugia during the last glaciation. The lower genetic diversity in northern Europe indicates postglacial recolonization northwards from southern refugia. This study combining herbarium surveys and population genetics thus uniquely reveals the effects of climate and environmental factors on a plant pathogen species with an arctic-alpine distribution.

Lower prevalence but similar fitness in a parasitic fungus at higher radiation levels near Chernobyl.

Nuclear disasters at Chernobyl and Fukushima provide examples of effects of acute ionizing radiation on mutations that can affect the fitness and distribution of species. Here, we investigated the prevalence of Microbotryum lychnidis-dioicae, a pollinator-transmitted fungal pathogen of plants causing anther-smut disease in Chernobyl, its viability, fertility and karyotype variation, and the accumulation of nonsynonymous mutations in its genome. We collected diseased flowers of Silene latifolia from locations ranging by more than two orders of magnitude in background radiation, from 0.05 to 21.03 µGy/h. Disease prevalence decreased significantly with increasing radiation level, possibly due to lower pollinator abundance and altered pollinator behaviour. Viability and fertility, measured as the budding rate of haploid sporidia following meiosis from the diploid teliospores, did not vary with increasing radiation levels and neither did karyotype overall structure and level of chromosomal size heterozygosity. We sequenced the genomes of twelve samples from Chernobyl and of four samples collected from uncontaminated areas and analysed alignments of 6068 predicted genes, corresponding to 1.04 × 10(7)  base pairs. We found no dose-dependent differences in substitution rates (neither dN, dS, nor dN/dS). Thus, we found no significant evidence of increased deleterious mutation rates at higher levels of background radiation in this plant pathogen. We even found lower levels of nonsynonymous substitution rates in contaminated areas compared to control regions, suggesting that purifying selection was stronger in contaminated than uncontaminated areas. We briefly discuss the possibilities for a mechanistic basis of radio resistance in this nonmelanized fungus.

History of expansion and anthropogenic collapse in a top marine predator of the Black Sea estimated from genetic data.

Two major ecological transitions marked the history of the Black Sea after the last Ice Age. The first was the postglacial transition from a brackish-water to a marine ecosystem dominated by porpoises and dolphins once this basin was reconnected back to the Mediterranean Sea (ca. 8,000 y B.P.). The second occurred during the past decades, when overfishing and hunting activities brought these predators close to extinction, having a deep impact on the structure and dynamics of the ecosystem. Estimating the extent of this decimation is essential for characterizing this ecosystem's dynamics and for formulating restoration plans. However, this extent is poorly documented in historical records. We addressed this issue for one of the main Black Sea predators, the harbor porpoise, using a population genetics approach. Analyzing its genetic diversity using an approximate Bayesian computation approach, we show that only a demographic expansion (at most 5,000 y ago) followed by a contemporaneous population collapse can explain the observed genetic data. We demonstrate that both the postglacial settlement of harbor porpoises in the Black Sea and the recent anthropogenic activities have left a clear footprint on their genetic diversity. Specifically, we infer a strong population reduction (~90%) that occurred within the past 5 decades, which can therefore clearly be related to the recent massive killing of small cetaceans and to the continuing incidental catches in commercial fisheries. Our study thus provides a quantitative assessment of these demographically catastrophic events, also showing that two separate historical events can be inferred from contemporary genetic data.

Repeated loss of function at HD mating-type genes and of recombination suppression without mating-type locus linkage in anther-smut fungi.

A wide diversity of mating systems occur in nature, with frequent evolutionary transitions in mating-compatibility mechanisms. Basidiomycete fungi typically have two mating-type loci controlling mating compatibility, HD and PR, usually residing on different chromosomes. In Microbotryum anther-smut fungi, there have been repeated events of linkage between the two mating-type loci through chromosome fusions, leading to large non-recombining regions. By generating high-quality genome assemblies, we found that two sister Microbotryum species parasitizing Dianthus plants, M. superbum and M. shykoffianum, as well as the distantly related M. scorzonarae, have their HD and PR mating-type loci on different chromosomes, but with the PR mating-type chromosome fused with part of the ancestral HD chromosome. Furthermore, progressive extensions of recombination suppression have generated evolutionary strata. In all three species, rearrangements suggest the existence of a transient stage of HD-PR linkage by whole chromosome fusion, and, unexpectedly, the HD genes lost their function. In M. superbum, multiple natural diploid strains were homozygous, and the disrupted HD2 gene was hardly expressed. Mating tests confirmed that a single genetic factor controlled mating compatibility (i.e. PR) and that haploid strains with identical HD alleles could mate and produce infectious hyphae. The HD genes have therefore lost their function in the control of mating compatibility in these Microbotryum species. While the loss of function of PR genes in mating compatibility has been reported in a few basidiomycete fungi, these are the first documented cases for the loss of mating-type determination by HD genes in heterothallic fungi. The control of mating compatibility by a single genetic factor is beneficial under selfing and can thus be achieved repeatedly, through evolutionary convergence in distant lineages, involving different genomic or similar pathways.

A new cheese population in Penicillium roqueforti and adaptation of the five populations to their ecological niche.

Domestication is an excellent case study for understanding adaptation and multiple fungal lineages have been domesticated for fermenting food products. Studying domestication in fungi has thus both fundamental and applied interest. Genomic studies have revealed the existence of four populations within the blue-cheese-making fungus Penicillium roqueforti. The two cheese populations show footprints of domestication, but the adaptation of the two non-cheese populations to their ecological niches (i.e., silage/spoiled food and lumber/spoiled food) has not been investigated yet. Here, we reveal the existence of a new P. roqueforti population, specific to French Termignon cheeses, produced using small-scale traditional practices, with spontaneous blue mould colonisation. This Termignon population is genetically differentiated from the four previously identified populations, providing a novel source of genetic diversity for cheese making. The Termignon population indeed displayed substantial genetic diversity, both mating types, horizontally transferred regions previously detected in the non-Roquefort population, and intermediate phenotypes between cheese and non-cheese populations. Phenotypically, the non-Roquefort cheese population was the most differentiated, with specific traits beneficial for cheese making, in particular higher tolerance to salt, to acidic pH and to lactic acid. Our results support the view that this clonal population, used for many cheese types in multiple countries, is a domesticated lineage on which humans exerted strong selection. The lumber/spoiled food and silage/spoiled food populations were not more tolerant to crop fungicides but showed faster growth in various carbon sources (e.g., dextrose, pectin, sucrose, xylose and/or lactose), which can be beneficial in their ecological niches. Such contrasted phenotypes between P. roqueforti populations, with beneficial traits for cheese-making in the cheese populations and enhanced ability to metabolise sugars in the lumber/spoiled food population, support the inference of domestication in cheese fungi and more generally of adaptation to anthropized environments.

Domestication in dry-cured meat Penicillium fungi: Convergent specific phenotypes and horizontal gene transfers without strong genetic subdivision.

Some fungi have been domesticated for food production, with genetic differentiation between populations from food and wild environments, and food populations often acquiring beneficial traits through horizontal gene transfers (HGTs). Studying their adaptation to human-made substrates is of fundamental and applied importance for understanding adaptation processes and for further strain improvement. We studied here the population structures and phenotypes of two distantly related Penicillium species used for dry-cured meat production, P. nalgiovense, the most common species in the dry-cured meat food industry, and P. salamii, used locally by farms. Both species displayed low genetic diversity, lacking differentiation between strains isolated from dry-cured meat and those from other environments. Nevertheless, the strains collected from dry-cured meat within each species displayed slower proteolysis and lipolysis than their wild conspecifics, and those of P. nalgiovense were whiter. Phenotypically, the non-dry-cured meat strains were more similar to their sister species than to their conspecific dry-cured meat strains, indicating an evolution of specific phenotypes in dry-cured meat strains. A comparison of available Penicillium genomes from various environments revealed HGTs, particularly between P. nalgiovense and P. salamii (representing almost 1.5 Mb of cumulative length). HGTs additionally involved P. biforme, also found in dry-cured meat products. We further detected positive selection based on amino acid changes. Our findings suggest that selection by humans has shaped the P. salamii and P. nalgiovense populations used for dry-cured meat production, which constitutes domestication. Several genetic and phenotypic changes were similar in P. salamii, P. nalgiovense and P. biforme, indicating convergent adaptation to the same human-made environment. Our findings have implications for fundamental knowledge on adaptation and for the food industry: the discovery of different phenotypes and of two mating types paves the way for strain improvement by conventional breeding, to elucidate the genomic bases of beneficial phenotypes and to generate diversity.

Domestication of the Emblematic White Cheese-Making Fungus Penicillium camemberti and Its Diversification into Two Varieties.

Domestication involves recent adaptation under strong human selection and rapid diversification and therefore constitutes a good model for studies of these processes. We studied the domestication of the emblematic white mold Penicillium camemberti, used for the maturation of soft cheeses, such as Camembert and Brie, about which surprisingly little was known, despite its economic and cultural importance. Whole-genome-based analyses of genetic relationships and diversity revealed that an ancient domestication event led to the emergence of the gray-green P. biforme mold used in cheese making, by divergence from the blue-green wild P. fuscoglaucum fungus. Another much more recent domestication event led to the generation of the P. camemberti clonal lineage as a sister group to P. biforme. Penicillium biforme displayed signs of phenotypic adaptation to cheese making relative to P. fuscoglaucum, in terms of whiter color, faster growth on cheese medium under cave conditions, lower amounts of toxin production, and greater ability to prevent the growth of other fungi. The P. camemberti lineage displayed even stronger signs of domestication for all these phenotypic features. We also identified two differentiated P. camemberti varieties, apparently associated with different kinds of cheeses and with contrasted phenotypic features in terms of color, growth, toxin production, and competitive ability. We have thus identified footprints of domestication in these fungi, with genetic differentiation between cheese and wild populations, bottlenecks, and specific phenotypic traits beneficial for cheese making. This study has not only fundamental implications for our understanding of domestication but can also have important effects on cheese making.

Multiple convergent supergene evolution events in mating-type chromosomes.

Convergent adaptation provides unique insights into the predictability of evolution and ultimately into processes of biological diversification. Supergenes (beneficial gene linkage) are striking examples of adaptation, but little is known about their prevalence or evolution. A recent study on anther-smut fungi documented supergene formation by rearrangements linking two key mating-type loci, controlling pre- and post-mating compatibility. Here further high-quality genome assemblies reveal four additional independent cases of chromosomal rearrangements leading to regions of suppressed recombination linking these mating-type loci in closely related species. Such convergent transitions in genomic architecture of mating-type determination indicate strong selection favoring linkage of mating-type loci into cosegregating supergenes. We find independent evolutionary strata (stepwise recombination suppression) in several species, with extensive rearrangements, gene losses, and transposable element accumulation. We thus show remarkable convergence in mating-type chromosome evolution, recurrent supergene formation, and repeated evolution of similar phenotypes through different genomic changes.

Blue cheese-making has shaped the population genetic structure of the mould Penicillium roqueforti.

BACKGROUND: Penicillium roqueforti is a filamentous fungus used for making blue cheeses worldwide. It also occurs as a food spoiler and in silage and wood. Previous studies have revealed a strong population genetic structure, with specific traits associated with the different populations. Here, we used a large strain collection from worldwide cheeses published recently to investigate the genetic structure of P. roqueforti. PRINCIPAL FINDINGS: We found a genetic population structure in P. roqueforti that was consistent with previous studies, with two main genetic clusters (W+C+ and W-C-, i.e., with and without horizontal gene transferred regions CheesyTer and Wallaby). In addition, we detected a finer genetic subdivision that corresponded to the environment and to protected designation of origin (PDO), namely the Roquefort PDO. We indeed found evidence for eight genetic clusters, one of the cluster including only strains from other environments than cheeses, and another cluster encompassing only strains from the Roquefort PDO. The W-C- and W+C+ cheese clusters were not the most closely related ones, suggesting that there may have been two independent domestication events of P. roqueforti for making blue cheeses. SIGNIFICANCE: The additional population structure revealed here may be relevant for cheese-makers and for understanding the history of domestication in P. roqueforti.

Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: Towards a comprehensive conservation strategy of the wild apple in Europe.

Crop-to-wild gene flow can reduce the fitness and genetic integrity of wild species. Malus sylvestris, the European crab-apple fruit tree in particular, is threatened by the disappearance of its habitat and by gene flow from its domesticated relative, Malus domestica. With the aims of evaluating threats for M. sylvestris and of formulating recommendations for its conservation, we studied here, using microsatellite markers and growth experiments: (i) hybridization rates in seeds and trees from a French forest and in seeds used for replanting crab apples in agrosystems and in forests, (ii) the impact of the level of M. domestica ancestry on individual tree fitness and (iii) pollen dispersal abilities in relation to crop-to-wild gene flow. We found substantial contemporary crop-to-wild gene flow in crab-apple tree populations and superior fitness of hybrids compared to wild seeds and seedlings. Using paternity analyses, we showed that pollen dispersal could occur up to 4 km and decreased with tree density. The seed network furnishing the wild apple reintroduction agroforestry programmes was found to suffer from poor genetic diversity, introgressions and species misidentification. Overall, our findings indicate supported threats for the European wild apple steering us to provide precise recommendations for its conservation.