Fanny E Hartmann
email: fanny.hartmann@u-psud.fr.

Ricardo Rodriguez

Fantin La Vega

E Carpentier

Pierre Gladieux

Amandine Cornille

Michael E Hood

Tatiana Giraud

Ricardo C Rodríguez De La Vega

Fantin Carpentier

Understanding Adaptation

Keywords: Coevolution, Host Specialization, and Mating System in Castrating Anther-Smut Fungi by Combining Population and Comparative Genomics comparative genomics, population genomics, transcriptomics, adaptation, positive selection, selective sweeps, divergence, gene flow, rearrangements, suppressed recombination

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Introduction

Pathogens thrive using living organisms as nutritional resources, which reduces their host fitness. This leads to coevolutionary arms races, in which pathogens are selected for increased abilities of host infection and exploitation, while hosts are selected for mechanisms of resistance to particular diseases. Such coevolution occurs on short evolutionary scales, as a never-ending process of adaptation and counter-adaptation (113). Across macro-evolutionary scales, some pathogens also may undergo host shifts, forming new species by specialization in combination with new hosts [START_REF] De Vienne | Cospeciation vs host-shift speciation: methods for testing, evidence from natural associations and relation to coevolution[END_REF]. Coevolution is a very different evolutionary process from host specialization, despite the terms often being used interchangeably, and may involve different genomic mechanisms and/or molecular interactions that have yet to be well resolved [START_REF] De Vienne | Cospeciation vs host-shift speciation: methods for testing, evidence from natural associations and relation to coevolution[END_REF].

An integrated understanding of the ecological and genetic/genomic mechanisms underlying both coevolution and host specialization by pathogens is of fundamental importance. These phenomena indeed represent cases of rapid adaptation, diversification and long-term species interactions, shedding light on the processes generating and maintaining biodiversity and ecosystem dynamics. Furthermore, knowledge on the genomic mechanisms involved in coevolution and host specialization in fungal pathogens is important for controlling crop and animal diseases and preventing emerging diseases that are a rising threat in domestic and wild populations [START_REF] Fisher | Emerging fungal threats to animal, plant and ecosystem health[END_REF][START_REF] Gladieux | The population biology of fungal invasions[END_REF]. Fungi are the most important plant pathogens, causing dramatic crop diseases, including many devastating diseased that are newly emergent following host shifts [START_REF] Anderson | Emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers[END_REF][START_REF] Desprez-Loustau | The fungal dimension of biological invasions[END_REF][START_REF] Fisher | Worldwide emergence of resistance to antifungal drugs challenges human health and food security[END_REF].

Fungal pathogens also have to cope with their abiotic environment, such as temperature and humidity [START_REF] Abbate | Cooccurrence among three divergent plant-castrating fungi in the same Silene host species[END_REF][START_REF] Cordier | The composition of phyllosphere fungal assemblages of European beech (Fagus sylvatica) varies significantly along an elevation gradient[END_REF][START_REF] Desprez-Loustau | Relating germination dynamics of Melampsora pinitorqua teliospores to temperature and rainfall during overwintering[END_REF][START_REF] Enjalbert | Genetic evidence of local adaptation of wheat yellow rust (Puccinia striiformis f. sp. tritici) within France[END_REF]129). Understanding the mechanisms of adaptation to climatic variables is thus similarly of fundamental and applied interest. Adaptation ability is however impacted by genetic diversity and gene flow, which are themselves influenced by dispersal rates and mating systems, that are therefore important life history traits to study for an integrated understanding of evolution, adaptation, population subdivision and speciation [START_REF] Barrett | Life history determines genetic structure and evolutionary potential of host-parasite interactions[END_REF][START_REF] Gandon | Local adaptation, evolutionary potential and hostparasite coevolution: interactions between migration, mutation, population size and generation time[END_REF][START_REF] Gibson | Sibling competition arena: selfing and a competition arena can combine to constitute a barrier to gene flow in sympatry[END_REF].

From the advent of modern genetics a century ago, anther-smut fungi (Microbotryum violaceum species complex, previously Ustilago violacea) have served as useful models for the molecular controls of mating and adaptations to abiotic conditions [START_REF] Abbate | Elevational disease distribution in a natural plantpathogen system: insights from changes across host populations and climate[END_REF][START_REF] Baker | Infection of species of Melandrium by Ustilago violacea (Pers.) Fuckel and the transmission of the resultant disease[END_REF][START_REF] Biere | Anther smut transmission in Silene latifolia and Silene dioica: impact of host traits, disease frequency, and host density[END_REF][START_REF] Carlsson | Epidemiology of anther-smut disease (Microbotryum violaceum) and numeric regulation of populations of Silene dioica[END_REF][START_REF] Garber | Genetic mapping of a phytopathogenic Basidiomycete, Ustilago violacea[END_REF][START_REF] Garber | Transmission genetics of Microbotryum violaceum (Ustilago violacea): a case study[END_REF][START_REF] Goldschmidt | Vererbungsversuche mit den biologischen arten den Antherenbrandes (Ustilago violacea Pers.)[END_REF][START_REF] Kneip | Untersuchungen uber den Antherenbrand (Ustilago violacea Pers.): Ein Beitrag zum Sexualitätproblem[END_REF]123,124). With advances in population genetics and genomics, emphasis has grown with regard to the natural diversity within this pathogen group, the dynamics of diseases, the mating systems and genetic differentiation in relation to host plants in natural ecosystems [START_REF] Abbate | Cooccurrence among three divergent plant-castrating fungi in the same Silene host species[END_REF][START_REF] Bueker | Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant[END_REF][START_REF] Cattrall | Genetics of Ustilago violacea. III. Crossing over and nondisjunction[END_REF][START_REF] Day | 30 -Ustilago violacea, anther smut of the Caryophyllaceae[END_REF][START_REF] Day | The production and characteristics of diploids in Ustilago violacea[END_REF][START_REF] Freeman | Isolates of Microbotryum violaceum from North American host species are phylogenetically distinct from their European host-derived counterparts[END_REF][START_REF] Giraud | Patterns of within population dispersal and mating of the fungus Microbotryum violaceum parasitising the plant Silene latifolia[END_REF][START_REF] Giraud | Selfing propensity under choice conditions in a parasitic fungus, Microbotryum violaceum, and parameters influencing infection success in artificial inoculations[END_REF][START_REF] Gac | Phylogenetic evidence of hostspecific cryptic species in the anther smut fungus[END_REF]105,111,131,132). The anther-smut fungi belong to the Microbotryum genus (basidiomycetes), which castrate plants of the Caryophyllaceae family, replacing the pollen with their spores and aborting ovaries (Figure 1B). They constitute an excellent model pathosystem, with hundreds of closely related fungal species specialized on different host plants, resulting from numerous host shifts, with conspicuous symptoms, a rich scientific history and occurring in natural ecosystems (Figure 1A) [START_REF] Hood | Distribution of the anther-smut pathogen Microbotryum on species of the Caryophyllaceae[END_REF][START_REF] Kemler | Implications of molecular characters for the phylogeny of the Microbotryaceae (Basidiomycota: Urediniomycetes)[END_REF][START_REF] Gac | Phylogenetic evidence of hostspecific cryptic species in the anther smut fungus[END_REF]111,118). Furthermore, they are phylogenetically close to the rust fungi as damaging crop pathogens (127). Most Microbotryum species are highly host-specific, but a few are more generalist, parasitizing closely related host species (Figure 1A) [START_REF] Gac | Evolution of reproductive isolation within a parasitic fungal species complex[END_REF]105). Other Microbotryum species, while distantly related, co-occur on the same host species, representing cases of convergence (Figure 1A) [START_REF] Abbate | Cooccurrence among three divergent plant-castrating fungi in the same Silene host species[END_REF][START_REF] Gac | Phylogenetic evidence of hostspecific cryptic species in the anther smut fungus[END_REF].

Host-pathogen coevolution in the Microbotryum model systems has been suggested based on patterns of plant local adaptation [START_REF] Delmotte | Host and parasite population structure in a natural plant-pathogen system[END_REF][START_REF] Feurtey | Strong phylogeographic co-structure between the anther-smut fungus and its white campion host[END_REF] and congruent plant-pathogen genetic structure [START_REF] Feurtey | Strong phylogeographic co-structure between the anther-smut fungus and its white campion host[END_REF].

Microbotryum species show little pre-zygotic isolation and increasing post-zygotic isolation strength with phylogenetic distance [START_REF] Büker | Experimental hybridization and backcrossing reveal forces of reproductive isolation in Microbotryum[END_REF][START_REF] De Vienne | Hybrid sterility and inviability in the parasitic fungal species complex Microbotryum[END_REF][START_REF] Gac | Evolution of reproductive isolation within a parasitic fungal species complex[END_REF], which may allow gene flow among closely related species. Moreover, abiotic factors have been shown to play a role with the disease interactions in important ways [START_REF] Abbate | Cooccurrence among three divergent plant-castrating fungi in the same Silene host species[END_REF], and Microbotryum fungi display an interesting mating system, with predominant automixis (i.e., intra-tetrad selfing), which has fostered multiple chromosomal rearrangements across the genus linking the mating-type loci controlling gamete compatibility [START_REF] Branco | Evolutionary strata on young mating-type chromosomes despite the lack of sexual antagonism[END_REF][START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF]. A consideration of these features altogether allows the studying adaptation, coevolution, host specialization, differentiation and mating systems with unique power.

For tackling this complex suite of questions, comparative genomics and population genomics constitute highly relevant and complementary approaches, addressing different time scales of evolution. In contrast to life history traits, ecology and population structure, which have been extensively studied [START_REF] Alexander | Population dynamics and genetics of plant disease: a case study of anther-smut disease[END_REF][START_REF] Antonovics | Epidemiology of anther-smut infection of Silene alba (= S. latifolia) caused by Ustilago violacea : patterns of spore deposition in experimental populations[END_REF][START_REF] Bernasconi | Silene as a model system in ecology and evolution[END_REF][START_REF] Kaltz | Male and female Silene latifolia plants differ in per-contact risk of infection by a sexually transmitted disease[END_REF], the genetic basis of interactions between Microbotryum fungi and their hosts is still little known; genomic approaches can elucidate the mechanisms and the functions involved in adaptation, coevolution, host specialization and speciation in this pathosystem. Analyses of gene expression between different stages of the life cycle can also inform on these processes. In particular, the pathogen's mating systems also influence adaptation, coevolution and host specialization [START_REF] Gibson | Sibling competition arena: selfing and a competition arena can combine to constitute a barrier to gene flow in sympatry[END_REF][START_REF] Giraud | Linking the emergence of fungal plant diseases with ecological speciation[END_REF], especially in anther-smut fungi that are obligately completing the sexual cycle upon every disease transmission. Genomics can further help to understand the evolution of mating systems by studying the changes at the mating-type loci.

In this review, we discuss the recent insights into our understanding of adaptation, coevolution and host specialization in anther-smut fungi gained from gene expression data and comparative genomics (part 1) and from population genomics (parts 2 & 3). We then discuss insights gained from genomics on mating system evolution (part 4). We illustrate that the combination of multiple genomic approaches is needed for a full understanding of evolution, as comparative genomics, population genomics and transcriptomics address different timescales and have power for detecting different footprints of adaptive events.

Finally, future challenges to be addressed using genomics tools are discussed (part 5).

1-COMPARATIVE GENOMICS AND TRANSCRIPTOMICS APPROACHES TO UNDERSTAND ADAPTATION AND HOST SPECIALIZATION IN ANTHER-SMUT

FUNGI

The sequencing of genomes and transcriptomes of Microbotryum species sheds light on pathogenicity, adaptation and specialization mechanisms across long evolutionary timescales; speciation events in castrating Microbotryum fungi have been dated from 0.4 to 11 MYA (26, 72) (Figure 2A). Phylogenomics enables obtaining an accurate understanding of the lineage histories, and comparative genomics is highly suitable to identify genetic changes associated with diversification at such large evolutionary scales.

Genome architecture and identification of candidate genes involved in pathogenicity using expression data

One of the best studied anther-smut species is M. lychnidis-dioicae, parasitizing the white campion Silene latifolia (Figure 1B). The diploid genomes of the Lamole M. lychnidis-dioicae strain was the first eukaryote genome to be assembled with new sequencing technologies [START_REF] Badouin | Chaos of rearrangements in the mating-type chromosomes of the anther-smut fungus Microbotryum lychnidis-dioicae[END_REF].

Comparative analysis of the Lamole strain of M. lychnidis-dioicae with other basidiomycetes genomes revealed specific gene content features such as the absence of plant cell wall degrading enzymes and expanded repertoires of major facilitator superfamily transporters, secretory lipases, glycosyltransferases and enzymes that could manipulate host development (104). Such features are likely related to the castrating and biotrophic lifestyle of anther-smut fungi (104), where the fungus takes up a largely symptomless residence between the host cells in the plant's growing points/meristems until the host initiates flower development.

Additionally, this pathogen has a remarkable ability to developmentally transform female host plants to take on a male-like floral structure, with the growth of stamens that then bare spores in place of pollen and the abortion of the ovary early in its development [START_REF] Audran | Microsporogenesis and pollen grains in Silene dioica (L.) Cl. and alterations in its anthers parasitised by Ustilago violacea (Pers.) Rouss[END_REF]. Apart from the accumulation of transposable elements (TEs) in the non-recombining regions of the matingtype chromosomes, there was no genome compartmentalization into more or less repeat-rich regions on autosomes [START_REF] Badouin | Chaos of rearrangements in the mating-type chromosomes of the anther-smut fungus Microbotryum lychnidis-dioicae[END_REF]104), in contrast to some other fungal pathogens with isochore genomic architecture and localization of effector genes in repeat-rich regions [START_REF] Gout | Lost in the middle of nowhere: the AvrLm1 avirulence gene of the Dothideomycete Leptosphaeria maculans[END_REF].

Nevertheless, transposable elements were locally associated across the Lamole M. lychnidisdioicae genome with gene clusters of small secreted proteins and genes affected by within species presence-absence polymorphism, suggesting a role of transposable elements in genome rearrangements and duplications of genes putatively involved in host adaptation [START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeographic structure[END_REF]104). Although footprints typical of genome defense mechanisms against TEs, similar to repeat-induced point mutation (RIP), were identified in anther-smut genomes, a massive burst-like expansion of Gypsy-like retrotransposons in a Microbotryum strain suggested that persistent transposable elements activity and expansion can occur [START_REF] Horns | Massive expansion of Gypsy-Like retrotransposons in Microbotryum fungi[END_REF][START_REF] Horns | Patterns of repeat-induced point mutation in transposable elements of Basidiomycete fungi[END_REF].

Transcriptomics conducted at several in vitro stages allow detecting genes upregulated in certain conditions and thus likely involved in important functions at a given life stage.

Transcriptomic analyses using the Lamole M. lychnidis-dioicae strain enabled identifying genes likely associated with nutrient uptake, the mating program and the dikaryotic switch [START_REF] Fontanillas | Degeneration of the non-recombining regions in the mating-type chromosomes of the anther-smut fungi[END_REF]104,125,126,136). In silico effector gene prediction combining in planta expression data, sequence conservation and predicted localization, allowed identifying small secreted proteins genes as candidate effectors, i.e. involved in pathogenicity, in M. lychnidis-dioicae, M. silenes-dioicae and M. violaceum var paradoxa [START_REF] Beckerson | Cause and effectors: Secretome comparison of members from the anther-smut pathogen species complex, Microbotryum violaceum[END_REF][START_REF] Kuppireddy | Identification and initial characterization of the effectors of an anther smut fungus and potential host target proteins[END_REF]. Eight genes in M. silenes-dioicae and three genes in M. violaceum var paradoxa predicted to encode secreted proteins were further confirmed to be secreted using yeast secretion trap [START_REF] Beckerson | Cause and effectors: Secretome comparison of members from the anther-smut pathogen species complex, Microbotryum violaceum[END_REF][START_REF] Kuppireddy | Identification and initial characterization of the effectors of an anther smut fungus and potential host target proteins[END_REF]. Compared expression data in male and female S. latifolia individuals during fungal infection revealed pathogen-mediated changes in sex-biased gene expression and altered sexual dimorphism in the host (137).

Another transcriptome analysis of the early development stages of infected flowers detailed changes in gene expression in M. lychnidis-dioicae, identifying gene categories likely to manipulate the host development and reproductive system, such as potential effectors and virulence factors (125). Further coupling experiments of host and pathogen gene expression changes, and in further paired host-Microbotryum fungi, should help deciphering the major components of the tight host-pathogen interactions described in the system.

Comparative genomics studies within the Microbotryum genus

Comparative genomics among Microbotryum fungi, and with other plant pathogens, has provided insights into the specificity of castrating biotrophic pathogens growing intracellularly (115) relative to other forms of parasitic nutritional ecology. Comparative genomics among anther-smut fungi specialized on different hosts can help unravel the genomic determinants of host specificity as well as the shared pathogenicity mechanisms. Indeed, while substantial insights has been gained by the study of individual genomes of Microbotryum species, whether features such as the conspicuous lack of cell-wall degrading enzymes in the M. lychnidis-dioicae Lamole genome are common to the genus cannot be known without a comparative genomics analysis that addresses both distantly and closelyrelated species [START_REF] Hardison | Comparative Genomics[END_REF].

Early comparative studies focused on orthologous genes across single pass Sanger-sequenced cDNA libraries, i.e. expressed sequence tags, from four Microbotryum species. The primary focus was looking for signals of positive selection in terms of frequent amino-acid changes [START_REF] Aguileta | Finding candidate genes under positive selection in non-model species: examples of genes involved in host specialization in pathogens[END_REF]. A subset of the genes evolving under positive selection between species was further shown to be under strong purifying selection within two closely-related Microbotryum species, M. lychnidis-dioicae and M. silenes-dioicae, suggesting that adaptive changes concomitant with host shifts can be later fixed due to strong functional constraints within species [START_REF] Gladieux | Purifying selection after episodes of recurrent adaptive diversification in fungal pathogens[END_REF]. Although the inferred function of some of the orthologous groups with signals of positive selection could be associated with aspects of virulence or speciation, none of these displayed features of effectors (such as secretory signals), likely because the expressed sequence tags did not exhaustively cover the genomes. Indeed, only 53 clusters of orthologs shared by at least three species and at least 300 nucleotides long could be retrieved [START_REF] Aguileta | Finding candidate genes under positive selection in non-model species: examples of genes involved in host specialization in pathogens[END_REF].

Therefore, even though these analyses demonstrated the utility of comparative genomics to identify candidate genes for diversifying selection in non-model organisms, the lack of whole genome sequences prevented any insight about presence-absence polymorphisms or substitutions both known to be important for adaptation to new hosts.

The number of high-quality genomes assemblies or shotgun sequencing from Microbotryum species/strains has exploded recently, reaching nearly a hundred as by late 2018 (Table 1; Figure 1A; [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF][START_REF] Badouin | Chaos of rearrangements in the mating-type chromosomes of the anther-smut fungus Microbotryum lychnidis-dioicae[END_REF][START_REF] Branco | Evolutionary strata on young mating-type chromosomes despite the lack of sexual antagonism[END_REF][START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF][START_REF] Carpentier | Convergent recombination cessation between mating-type genes and centromeres in selfing anther-smut fungi[END_REF][START_REF] Fortuna | Polymorphic microsatellite markers for the tetrapolar anther-smut fungus Microbotryum saponariae based on genome sequencing[END_REF]112,134)). In comparative genomics, near-complete gene lists can be clustered to obtain groups of homologous sequences that can be then used to build phylogenetic profiles of gene content. Such comparisons allow the identification of gene families that are species-specific and those that have been expanded or reduced in particular lineages [START_REF] Ames | Determining the evolutionary history of gene families[END_REF][START_REF] Han | Estimating gene gain and loss rates in the presence of error in genome assembly and annotation using CAFE 3[END_REF]. Species-or population-specific genes are either derived from within-group innovation, a rather uncommon phenomenon [START_REF] Casola | From de novo to "de nono": The majority of novel protein coding genes identified with phylostratigraphy are old genes or recent duplicates[END_REF], the result of differential losses or gene duplications [START_REF] Hartmann | Distinct trajectories of massive recent gene gains and losses in populations of a microbial eukaryotic pathogen[END_REF], or due to the non-vertical acquisition of gene-coding genome fragments, for instance horizontal gene transfer [START_REF] Feurtey | Interspecific gene exchange as a driver of adaptive evolution in fungi[END_REF]. Expanded gene families require the escape from the rampant pseudogenization (non-functionalization) of duplicated genes (99), whereas reduced or complete losses of gene families is often related to ecological shifts (117), rendering the product of those genes no longer needed for survival [START_REF] Albalat | Evolution by gene loss[END_REF]. Understanding these processes is fundamental to the study of evolutionary ecology as they help to explain the genomic architecture underlying the phenomenon of adaptive divergence. In silico annotations and comparative analyses have identified hundreds of candidate effectors across multiple Microbotryum species, enriched in gene families showing presence-absence polymorphism across species (Figure 2B) (112), along with orthologous genes with landmarks of positive selection between species and purifying selection within species [START_REF] Beckerson | Cause and effectors: Secretome comparison of members from the anther-smut pathogen species complex, Microbotryum violaceum[END_REF], thus generalizing and expanding previous findings. High-quality genome assemblies revealed little genomic rearrangements in autosomes [START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF].

Studies of positive selection based on the comparisons of non-synonymous and synonymous

substitution rates (dN/dS; (135)) and on the comparisons of the proportions of nonsynonymous and synonymous polymorphisms within species and differences between species (McDonald and Kreitman test; (100)) revealed no signature of diversifying selection between sister Microbotryum species specialized on two closely related host species [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF], but detected a dozen of genes encoding secreted proteins with signs of positive selection between more distantly related Microbotryum species specialized on more distant host species (Figure 2C) [START_REF] Beckerson | Cause and effectors: Secretome comparison of members from the anther-smut pathogen species complex, Microbotryum violaceum[END_REF]. Future comparative genomics studies encompassing all currently sequenced genomes will likely have high power to detect genes involved in host specialization by allowing further disentangling the effects of pathogen and host phylogenetic distances. In particular, combining population and comparative analyses should be very powerful to identify genes under diversifying selection between species and purifying selection within species as well as species-specific gene gains and losses. Building gene genealogies based on whole genomes also allowed to resolve previously ambiguous relationships among some anther-smut species (Figure 1A). The comparison of repeat contents and genomic rearrangements between genomes will be a further key step to understand the role of genome dynamics in adaptive processes in anther-smut fungi.

2-POPULATION GENOMICS TO IDENTIFY ADAPTIVE GENETIC VARIATION IN NATURAL PATHOGEN POPULATIONS

Population genomics is a complementary approach to comparative genomics for understanding adaptation in pathogen populations. Population genomics indeed address more recent adaptive events, and on a broader range of evolutionary genetic phenomena, not only gene gains/losses and recurrent changes in amino-acids. Selective sweeps can be detected using population genomics, which can reveal positive selection on a single amino-acid change or basepair substitutions in non-coding regions. Furthermore, population genomics can address the questions of the genomic bases of host-pathogen coevolution and local adaptation, that constitute more recent selection compared to the long-term selection underlying host specialization, and possibly differential selection among geographically distant populations (Figure 2A) [START_REF] Croll | The genetic basis of local adaptation for pathogenic fungi in agricultural ecosystems[END_REF][START_REF] Grünwald | Population genomics of fungal and oomycete pathogens[END_REF]107). In contrast to major fungal-plant pathosystems, no gene-for-gene relationship has been reported for anther-smut fungi. Instead, the probability of infection shows quantitative variation [START_REF] Alexander | Spread of anther-smut disease (Ustilago violacea) and character correlations in a genetically variable experimental population of Silene alba[END_REF][START_REF] Alexander | Population dynamics and genetics of plant disease: a case study of anther-smut disease[END_REF][START_REF] Chung | Variation in resistance to multiple pathogen species: anther smuts of Silene uniflora[END_REF], which suggests a rather complex genetic basis of co-evolution and host local adaptation. Genome-wide population genomics approaches in anthersmut fungi allowed identification of the complex genetic basis of recent adaptive events through genome scans of selective sweeps and gene-presence absence polymorphism [START_REF] Aguileta | Lower prevalence but similar fitness in a parasitic fungus at higher radiation levels near Chernobyl[END_REF][START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF][START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeographic structure[END_REF]. Selective sweep analyses allow one to identify loci that have recently been under positive selection within populations and thus likely underlying coevolution and local adaptation, whereas genes involved in host specialization are likely under purifying selection within species after the initial adaptive events following host shifts. Analyses of whole genome sequences of 53 genomes of the anther-smut sister species M. lychnidis-dioicae and M. silenes-dioicae identified selective sweeps (Figure 2D) [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF], likely resulting from dynamic co-evolutionary arm race of the fungus with its hosts. The overlap between genes differentially expressed in planta and in vitro and those lying within selective sweeps, together with functional annotations, provided clues to genes and functions involved in plantpathogen interaction in the Microbotryum-Silene system. Candidate genes included glycoside hydrolases, pectin lyases and an extracellular membrane protein with CFEM domain [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF]. The pectin lyase function seems relevant in that Microbotryum fungi grow between cells of the meristem (115), which is a pectin-occupied space. Extracellular membrane proteins with a cysteine-rich CFEM domain are present in effectors in several fungal pathogens [START_REF] Kulkarni | Novel G-protein-coupled receptorlike proteins in the plant pathogenic fungus Magnaporthe grisea[END_REF]. This study was also an opportunity to test for differences in intensity of coevolution between anther smut fungi on different hosts. Interestingly, differences in the number and the location of the selective sweeps were found between sister species. Footprints of positive selection affected 17 % of the genome in M. lychnidis-dioicae and 1 % of the genome in M. silenesdioicae [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF]. Selective sweeps were scattered throughout the genomes. Linkage disequilibrium was found to decay relatively slowly with physical distance along chromosomes, as expected for selfing species, but still indicated effective recombination.

Polymorphism in each fungal species was negatively correlated with the recombination rates along chromosomes, consistent with recurrent positive and/or background selection erasing diversity on larger genomic regions when recombination is less frequent [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF].

Population genomics can also contribute to our understanding of the impact of recent anthopogenic factors on the genome and subsequent adaptation. Analyses of M. lychnidisdioicae genomes along a gradient of ionizing radiation levels around Chernobyl showed no evidence of deleterious mutation accumulation in the form of non-synonymous substitutions [START_REF] Aguileta | Lower prevalence but similar fitness in a parasitic fungus at higher radiation levels near Chernobyl[END_REF]. Lower mean values of dN/dS were even found in Chernobyl compared to other areas of the same eastern genetic cluster (3), which may be due to stronger selection in contaminated areas against individuals bearing mildly deleterious mutations, i.e. stronger purifying selection.

In addition to genome scans looking at signatures of positive selection, other population genomic approaches make use of the genetic variation in pathogen populations to identify the genomic architecture of local adaptation [START_REF] Bartoli | Genome-wide association studies in plant pathosystems: toward an ecological genomics approach[END_REF][START_REF] Croll | The genetic basis of local adaptation for pathogenic fungi in agricultural ecosystems[END_REF]107,114). Population genomics enables the unravelling the genomic bases of adaptation to abiotic conditions by searching for correlations between local population allele frequencies and local environments (genetic-environment association methods) [START_REF] Hoban | Finding the genomic basis of local adaptation: Pitfalls, practical solutions, and future directions[END_REF]. Such approaches can be used in anther-smut fungi along altitudinal clines in Alpine populations on Dianthus or Silene hosts. Studies on the three species parasitizing S. vulgaris in particular could be interesting as elevation and climate has been shown to impact these anther-smut fungi [START_REF] Abbate | Elevational disease distribution in a natural plantpathogen system: insights from changes across host populations and climate[END_REF][START_REF] Abbate | Cooccurrence among three divergent plant-castrating fungi in the same Silene host species[END_REF]. Strong population structure as found in many Microbotryum species at European scale [START_REF] Abbate | Cooccurrence among three divergent plant-castrating fungi in the same Silene host species[END_REF][START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF][START_REF] Bueker | Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant[END_REF][START_REF] Fortuna | Multiple infections, relatedness and virulence in the anther-smut fungus castrating Saponaria plants[END_REF]105) might be a challenge to the use of such methods in particular, but these methods can be utilized at small geographical scales and/or in species with less population subdivision.

Gene copy number variation segregating within species is also a widespread and an important source of genetic variation and several examples of adaptive evolution through gene loss or gene gain have been identified in agricultural fungal plant pathogens [START_REF] Fouché | The birth and death of effectors in rapidly evolving filamentous pathogen genomes[END_REF]. Population genomics allow to explore the extent and adaptive potential of such within-species variation.

Gene presence/absence polymorphism was found to contribute to the genetic variation in populations of the two closely related species of castrating anther-smut fungi, M. lychnidisdioicae and M. silenes-dioicae [START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeographic structure[END_REF]. Genes displaying presence/absence polymorphism were mostly recently acquired, in a single species, through duplications in multiple-gene families and few genes predicted to encode secreted proteins were affected, suggesting defense against host recognition by other genetic changes than gene loss or gain. Although most gene presence/absence polymorphisms were likely neutral, the putative functions of some genes affected by presence-absence polymorphism (e.g., secreted proteins) or their localization within previously identified selective sweeps suggested that some gene loss or gain events may be adaptive [START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeographic structure[END_REF].

3-INSIGHTS INTO THE DYNAMICS OF DIVERGENCE AND GENE FLOW FROM POPULATION GENOMICS

By providing a glimpse into intra-specific genetic diversity and its variation across the genome, population genomics analyses are also highly useful to understand processes underlying species divergence and phylogeography, quantifying rates of gene flow and its heterogeneity along genomes, and providing accurate estimation of population size variations.

The occurrence of multiple Microbotryum sister species pairs in sympatry makes the system a perfect model to study the dynamics of divergence and gene flow in fungal pathogen populations.

Contrasted patterns of interspecific gene flow in the Microbotryum genus: a speciation continuum?

The two pathogens M. lychnidis-dioicae and M. silenes-dioicae and their respective sister host plants, Silene latifolia and S. dioica, are ubiquitous in Europe and their geographic distributions are largely overlapping, providing an ideal system for research on the formation and maintenance of species in sympatry. Microsatellite data from samples across Europe revealed rare disease transmission events between the host species and rare pathogen hybrids [START_REF] Gladieux | Maintenance of fungal pathogen species that are specialized to different hosts: allopatric divergence and introgression through secondary contact[END_REF]132). However, these approaches using a dozen microsatellite markers may lack power.

Analyses of whole genome sequences of many pathogen samples that appeared of pure ancestry based upon the microsatellite data then revealed no evidence for admixture, indicating that introgression does not persist beyond one or two generations [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF]. In the laboratory, both fungal species can infect both host plants [START_REF] De Vienne | Phylogenetic determinants of potential host shifts in fungal pathogens[END_REF][START_REF] Gibson | Performance of a hybrid fungal pathogen on pure-species and hybrid host plants[END_REF][START_REF] Gac | Evolution of reproductive isolation within a parasitic fungal species complex[END_REF]. Experimental crosses showed little premating isolation by assortative mating between the two pathogen species [START_REF] Büker | Experimental hybridization and backcrossing reveal forces of reproductive isolation in Microbotryum[END_REF][START_REF] Gac | Evolution of reproductive isolation within a parasitic fungal species complex[END_REF]132), even at sympatric sites (110), and a lack of post-mating barriers [START_REF] De Vienne | Hybrid sterility and inviability in the parasitic fungal species complex Microbotryum[END_REF][START_REF] Gac | Evolution of reproductive isolation within a parasitic fungal species complex[END_REF]. Hybrids were viable and fertile at least through the F2 generation in the greenhouse [START_REF] De Vienne | Phylogenetic determinants of potential host shifts in fungal pathogens[END_REF][START_REF] Gac | Evolution of reproductive isolation within a parasitic fungal species complex[END_REF]131). F2 hybrids produced by selfed F1s had mostly returned to homozygosity, suggesting that genomic content derived from one of the two parental species had already begun to be purged [START_REF] Büker | Experimental hybridization and backcrossing reveal forces of reproductive isolation in Microbotryum[END_REF][START_REF] De Vienne | Phylogenetic determinants of potential host shifts in fungal pathogens[END_REF]. This latter finding, combined with the fact that introgression does not appear to persist in nature, is consistent with strong genome-wide selection by the host plant and the scattering of genes involved in host specialization across the genome, as revealed in genome scans of selective sweeps [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF]. FST values were found near their maximum all along their genomes (Figure 3A).

Whereas strict host specialization is often the rule on Silene species [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF][START_REF] Gac | Phylogenetic evidence of hostspecific cryptic species in the anther smut fungus[END_REF]133), on Dianthus hosts in contrast population genetics approaches revealed four Microbotryum lineages with broader and overlapping host specificities (Figure 1A) [START_REF] Gac | Phylogenetic evidence of hostspecific cryptic species in the anther smut fungus[END_REF]105). One Microbotryum lineage was found only on D. pavonius while the others occurred spread across several host species, some of them being shared among Microbotryum lineages. The sympatry of Microbotryum lineages within populations, in particular in the Alps, led to hybridization (105). The individuals with mixed ancestry based on clustering analyses of microsatellite data suffered from significant meiotic sterility, which confirmed they were hybrids between species (105). The larger host ranges of Microbotryum lineages on Dianthus hosts may be explained by the recent divergence of their host plants. The Dianthus genus has indeed undergone a recent radiation in Europe with morphologically diverse European Dianthus species restricted to small geographically restricted ranges (130). The full extent and evolutionary consequences of the hybridization on pathogen dynamics and evolution remains to be explored. Along this line, the Dianthus-Microbotryum system may become, in the coming years, a tractable model to investigate the impact of gene flow during divergence, and whether selection due to local/host adaptation can make some genomic regions more or less permeable to gene flow, which represents a current debate in evolutionary biology [START_REF] Cruickshank | Reanalysis suggests that genomic islands of speciation are due to reduced diversity, not reduced gene flow[END_REF]. These questions could not be addressed so far based on the population genomics analyses of M. lychnidis-dioicae and M. silenes-dioicae as no genomic introgression could be detected in natural populations [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF]. In contrast, the hybrids detected in natural populations on Dianthus hosts with significant sterility suggest the occurrence of introgressions (105). Other pairs of Microbotryum species might also be suitable to address these questions of the impact and heterogeneity of gene flow along the genome. For example, anther-smut fungi on the closely related and sympatric native American species S. virginica and S. caroliniana [START_REF] Antonovics | Herbarium studies on the distribution of anther-smut fungus (Microbotryum violaceum) and Silene species (Caryophyllaceae) in the eastern United States[END_REF][START_REF] Antonovics | An anther-smut disease (Ustilago violacea) of fire-pink (Silene virginica): Its biology and relationship to the anther-smut disease of white campion (Silene alba)[END_REF] could not be separated into host-specialized species based on a few gene genealogies [START_REF] Freeman | Isolates of Microbotryum violaceum from North American host species are phylogenetically distinct from their European host-derived counterparts[END_REF][START_REF] Kemler | Implications of molecular characters for the phylogeny of the Microbotryaceae (Basidiomycota: Urediniomycetes)[END_REF]111).

In this system, population genomics should allow elucidating whether anther-smut fungi on these American Silene species show host differentiation or genome-wide gene flow, or introgression only in genomic regions not involved in host specialization. Based on the few genomes available so far [START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF], we find FST values between Microbotryum populations on the two hosts, S. virginica and S. caroliniana, that are lower and more heterogeneous along the genomes than between M. lychnidis-dioicae and M. silenes-dioicae referenced above (Figure 3B). These initial results suggests the occurrence of gene flow in some genomic regions. The situation of anther-smut fungi on S. vulgaris, with three distant lineages with convergent specialization on this same host species (2), would also be worth exploring using population genomics to determine the extent of introgression and its genomic localization, and whether the interspecific exchange of alleles has been deleterious or adaptive.

Another promising approach in anther-smut fungi for identifying genomic regions involved in host adaptation will be to perform genome scans of differentiation between closely related species or host races, if possible to avoid the potential pitfalls of such approaches [START_REF] Cruickshank | Reanalysis suggests that genomic islands of speciation are due to reduced diversity, not reduced gene flow[END_REF]. This could contribute to our understanding of the role of gene flow in the early stages of divergence and to identifying genomic regions less permeable to gene flow because of selection for host adaptation and/or genetic incompatibilities between lineages [START_REF] Bierne | The coupling hypothesis: why genome scans may fail to map local adaptation genes[END_REF][START_REF] Cruickshank | Reanalysis suggests that genomic islands of speciation are due to reduced diversity, not reduced gene flow[END_REF]. More generally, such population genomics approaches would be valuable to use in plant pathogen fungi.

Phylogeography and demographic history inferences

Microbotryum lychnidis-dioicae and M. silenes-dioicae also constituted case studies in providing one of the most clear-cut examples of phylogeographic structure in pathogens, thanks to a collection of samples whose density and geographical scale was unprecedented for a disease association in natural populations. In M. lychnidis-dioicae, clustering analyses based on microsatellite markers (133), as well as nuclear gene sequences [START_REF] Gladieux | Purifying selection after episodes of recurrent adaptive diversification in fungal pathogens[END_REF][START_REF] Gladieux | Maintenance of fungal pathogen species that are specialized to different hosts: allopatric divergence and introgression through secondary contact[END_REF], revealed the existence of three genetically distinct clusters, reflecting recolonization from well-recognized southern refugia after glaciation. Little admixture has been found between clusters based on microsatellites [START_REF] Feurtey | Strong phylogeographic co-structure between the anther-smut fungus and its white campion host[END_REF]133), and this has later been confirmed by whole genome sequences [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF]. Indeed, SNPs (single nucleotide polymorphisms) revealed few shared polymorphisms and many fixed differences among the clusters, and pairwise FST values between them were high (0.56-0.74; Figures 3C andD), supporting low levels of inter-cluster gene flow [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF]. Whole genome sequences provided further insights into the age of divergence between the three M. lychnidis-dioicae lineages (Southern, Western and Eastern clusters), sequential size changes in the population size of derived lineages and also supported low levels of gene flow [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF].

Most notably, the pathogen genetic structure closely matched with the genetic structure of the host species S. latifolia with the same regionally defined Southern, Western and Eastern clusters, indicating that the anther-smut pathogen remained during the last glaciation in the same three distinct refugia as its host (i.e. in the Iberian, Italian and Balkan peninsulas) [START_REF] Feurtey | Strong phylogeographic co-structure between the anther-smut fungus and its white campion host[END_REF].

The congruence of population structures between M. lychnidis-dioicae and its host appeared even stronger than what could be expected because of isolation by distance alone, suggesting that coevolution has played a significant role in the congruence of the population structures [START_REF] Feurtey | Strong phylogeographic co-structure between the anther-smut fungus and its white campion host[END_REF]. Genome-wide gene presence-absence polymorphism recovered the same population structure [START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeographic structure[END_REF]. Inoculation experiments, indicating plant local adaptation for resistance to pathogens [START_REF] Feurtey | Strong phylogeographic co-structure between the anther-smut fungus and its white campion host[END_REF][START_REF] Kaltz | Local maladaptation in the anther-smut fungus Microbotryum violaceum to its host plant Silene latifolia: evidence from a cross-inoculation experiment[END_REF][START_REF] Kaltz | Within-and among-population variation in infectivity, latency and spore production in a host-pathogen system[END_REF], were consistent with a contribution of adaptive factors to the observed congruence between pathogen and host population structures.

Microsatellite markers and genome-wide SNPs indicated that M. silenes-dioicae also exhibited a genetic structure, albeit with biogeographic patterns more difficult to interpret [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF]133) and very low FST values genome-wide (Figure 3E). Genome-wide gene presence/absence polymorphism revealed two different clusters with a more obvious east/west separation [START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeographic structure[END_REF], that may correspond to local adaptation of S. dioica clusters [START_REF] Hathaway | Geographically congruent large-scale patterns of plastid haplotype variation in the European herbs Silene dioica and S. latifolia (Caryophyllaceae)[END_REF]109), although this remains to be assessed. This case study shows the power of various kinds of population genomic studies to unravel weak and/or adaptive population subdivision.

4-UNRAVELLING MATING SYSTEM AND GAMETE COMPATIBILITY SYSTEMS USING BOTH COMPARATIVE GENOMICS AND POPULATION

GENOMICS

The combination of comparative genomics and population genomics also can reveal remarkable transitions in mating systems by elucidating the changes in genomic mechanisms controlling mating compatibility. For the broad group of basidiomycete fungi, gamete compatibility is controlled by two loci acting at the haploid stage, mating being successful only between haploid cells carrying different alleles at both mating-type loci [START_REF] Coelho | Fungal sex: The Basidiomycota[END_REF]. The two mating-type loci are i) the PR locus which encodes pheromone genes and a pheromone receptor gene implicated in gamete recognition and fusion, and ii) the HD locus which encodes homeodomain protein-coding genes allowing, after fusion, for the maintenance of the dikaryon and hyphal growth [START_REF] Feldbrügge | Regulation of mating and pathogenic development in Ustilago maydis[END_REF][START_REF] Kües | Life history and developmental processes in the basidiomycete Coprinus cinereus[END_REF]. Most basidiomycetes are outcrossing and have these two loci unlinked, although some fungi in this group have linked mating-type loci (103).

Linkage of the two mating type loci is considered to be favored due to increased odds of gamete compatibility under selfing when mating-type loci are linked (103). Interestingly, most Microbotryum species are highly selfing and were long known to segregate only two mating type phenotypes, but it remained uncertain whether this was due to mating-type loci linkage or to the loss of role in mating-type determinism for one of the two mating-type loci, as both cases occurred in basidiomycetes [START_REF] Hood | Breaking linkage between mating compatibility factors: Tetrapolarity in Microbotryum[END_REF]103). Comparative genomics of well-assembled genomes allowed to resolve the complex genome architecture and long-term evolutionary history of the repeat-rich and rearranged mating type chromosomes in anther-smut fungi, and population genomics datasets were essential for identifying young events of recombination suppression.

Population genomics confirmed high rates of selfing in all studied Microbotryum species, by showing high levels of genome-wide homozygosity [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF][START_REF] Branco | Evolutionary strata on young mating-type chromosomes despite the lack of sexual antagonism[END_REF][START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF] and confirmed massive recombination suppression on mating-type chromosomes [START_REF] Hood | Extensive divergence between mating-type chromosomes of the anther-smut fungus[END_REF]134). High-quality genomes assemblies allowed reconstructing the history of genomic events underlying the shift in gamete compatibility system [START_REF] Branco | Evolutionary strata on young mating-type chromosomes despite the lack of sexual antagonism[END_REF][START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF]. The long-read sequencing technology allowed assembling the two repeat-rich mating-type chromosomes of the Lamole M. lychnidis-dioicae strain, which confirmed linkage between the two mating-type loci HD and PR (Figure 4A) [START_REF] Badouin | Chaos of rearrangements in the mating-type chromosomes of the anther-smut fungus Microbotryum lychnidis-dioicae[END_REF]. Genome comparisons between multiple Microbotryum species showed that the ancestral state had unlinked mating-type loci on two distinct chromosomes, and that independent rearrangements and chromosome fusions occurred in multiple species, convergently linking the two mating-type loci by large regions without recombination (Figure 4B) [START_REF] Branco | Evolutionary strata on young mating-type chromosomes despite the lack of sexual antagonism[END_REF][START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF]. This shows that natural selection can repeatedly lead to similar phenotypes through multiple different evolutionary pathways.

Following recombination suppression, a chaos of rearrangements occurred on mating-type chromosomes [START_REF] Badouin | Chaos of rearrangements in the mating-type chromosomes of the anther-smut fungus Microbotryum lychnidis-dioicae[END_REF], as well as TE and non-synonymous substitution accumulation [START_REF] Badouin | Chaos of rearrangements in the mating-type chromosomes of the anther-smut fungus Microbotryum lychnidis-dioicae[END_REF][START_REF] Fontanillas | Degeneration of the non-recombining regions in the mating-type chromosomes of the anther-smut fungi[END_REF], as is typical in non-recombining regions, and in particular on sex chromosomes [START_REF] Bachtrog | Y-chromosome evolution: emerging insights into processes of Ychromosome degeneration[END_REF]. The highquality assemblies allowed the detailed characterization of extensive rearrangements and repeat accumulations on the two mating-type chromosomes [START_REF] Badouin | Chaos of rearrangements in the mating-type chromosomes of the anther-smut fungus Microbotryum lychnidis-dioicae[END_REF][START_REF] Branco | Evolutionary strata on young mating-type chromosomes despite the lack of sexual antagonism[END_REF][START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF]. Another important characteristic feature of sex chromosomes was observed on the mating-type chromosomes of multiple Microbotryum species, i.e., the stepwise extension of the regions with recombination suppression. The progressive extension of the regions without recombination revealed a pattern of clear "evolutionary strata", i.e., decreasing divergence between alleles on the alternative mating-type chromosomes farther from the mating-type loci (Figure 4C).

Population genomics was essential for providing evidence of early events of recombination suppression in several species, by showing the segregation of alleles according to their associated mating-type, decreased levels of diversity as expected under lower population effective sizes and that high divergence between alleles associated with the alternative mating types was due to balancing selection on mating types rather than elevated substitution rates [START_REF] Branco | Evolutionary strata on young mating-type chromosomes despite the lack of sexual antagonism[END_REF][START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF]. Indeed, as soon as recombination ceased, alleles on the two mating-type chromosomes diverged gradually with time (Figure 4D). Finding such evolutionary strata in fungi, which lack male and female roles, challenged the classical view for the evolution of sex chromosomes. Indeed stepwise recombination suppression in sex chromosomes was thought to be due primarily to sexual antagonism, i.e., the selection to link genes with alleles beneficial in one sex, and deleterious in the other, to the sex determining gene [START_REF] Bergero | The evolution of restricted recombination in sex chromosomes[END_REF]. The finding of evolutionary strata in fungi without sexual antagonism indicates that alternative hypotheses should be explored to explain the progressive spread of recombination suppression, such as overdominance, epigenetic modifications associated with transposable elements or neutral rearrangements [START_REF] Ironside | No amicable divorce? Challenging the notion that sexual antagonism drives sex chromosome evolution[END_REF]108).

5-CONCLUDING REMARKS AND PERSPECTIVES

A thorough understanding of the major roles played by pathogens requires the integrative study of both ongoing processes of coevolution and dynamics of specialization that impact the emergence new diseases. Investigations of the anther-smut fungi utilizing comparative approaches to genomics and gene expression profiles, combined with population-level studies, illustrate the strength of combining different genomic approaches addressing different scales of evolution (Figure 2). The availability of genomic data for multiple sister species and multiple populations within species makes the anther-smut system quite exceptional for identifying the genetic mechanisms involved in adaptation, coevolution, host specialization and mating system at different evolutionary times (Table 1; Figure 1). Comparative genomics has long been the predominant approach for studying adaptation in plant fungal pathogens [START_REF] Duplessis | Obligate biotrophy features unraveled by the genomic analysis of rust fungi[END_REF][START_REF] Floudas | The paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes[END_REF][START_REF] Friesen | Emergence of a new disease as a result of interspecific virulence gene transfer[END_REF]102,107) and has provided important insights into the mechanisms of adaptation, e.g., through horizontal gene transfers, gene gains/losses, hybridization or recurrent aminoacid changes [START_REF] Gladieux | Fungal evolutionary genomics provides insight into the mechanisms of adaptive divergence in eukaryotes[END_REF]. Comparative genomics by definition does not consider population-level variation, such that population genomics is a complementary approach for insights into evolutionary processes acting at the local and regional scales. For example, several recent studies have revealed gene presence/absence polymorphism within species [START_REF] Hartmann | Distinct trajectories of massive recent gene gains and losses in populations of a microbial eukaryotic pathogen[END_REF][START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeographic structure[END_REF]119,120).

In addition, comparative genomics can only detect a specific type of positive selection, involving frequent changes of amino-acids. Positive selection of a single amino-acid change or of regulatory regions can only be detected by looking for selective sweeps using population genomics. Some recent studies based on population genomics have in fact revealed important aspects of adaptation in fungal plant pathogens, showing footprints of introgression, selective sweeps and amino acid-changes [START_REF] Gladieux | Gene flow between divergent cereal-and grass-specific lineages of the rice blast fungus Magnaporthe oryzae[END_REF][START_REF] Hartmann | Genome-wide evidence for divergent selection between populations of a major agricultural pathogen[END_REF]101,116,121,122). Furthermore, cross referencing candidate genes that are highlighted by multiple indications of being subject to natural selection during parasitism as outlined here (e.g. genes found within a selective sweep, upregulated in the plant and having experienced gene family expansion compared to other fungal pathogens) can strengthen their putative roles as pathogen effector that are central in the specificity of fungal-plant combinations. Functional studies can help understanding the role of the candidate genes. Promising transformation protocol have been developed in M. lychnidis-dioicae (128) and will likely facilitate the characterization of key genes involved in the interaction between the anther-smut fungi and their Caryophyllaceae host plants, an important challenge for the coming years. Transcriptomes and epigenomes of multiple Microbotryum species and multiple strains within species will likely be complementary to the current available genomic ressources to identify the role of regulatory and epigenetic mechanisms in the adaptation of anther-smut fungi to their hosts and environment, their divergence and their mating-type chromosome organisation, contributing to further understanding the mechanisms involved in adaptive processes in fungal pathogen populations. It will also be interesting to investigate similar levels of among and within species sampling and genome sequencing using pathogens of different levels of obligate parasitism, including facultative and opportunistic pathogens, as well as hemibiotrophy and necrotrophy. 120. Steenwyk JL, Soghigian JS, Perfect JR, Gibbons JG. 2016. Copy number variation contributes to cryptic genetic variation in outbreak lineages of Cryptococcus gattii from the North American Pacific Northwest. BMC Genomics. 17 Caryophyllaceae), that are a consensus from previous phylogenetic analyses [START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF][START_REF] Carpentier | Convergent recombination cessation between mating-type genes and centromeres in selfing anther-smut fungi[END_REF]111).

Tables and Figures

Availability of short reads or long reads-based genome assemblies or population genomic data for the species of anther-smut fungi as presented in Table 1 is for FST distributions are shown as squares); pictures of infected hosts by each host-specialized species (Photo credits: Michael E. Hood). For each pairwise comparison, FST values were calculated per gene for all genes present on autosomal contigs (i.e. not belonging to matingtype chromosomes) and carrying at least 1 SNP using the PopGenome R package (106). Red dashed lines correspond to median FST values. Genomic data were described in [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF][START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF]134).

Mapping, SNP calling and FST calculations were described in [START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF][START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeographic structure[END_REF]. lychnidis-dioicae mating-type chromosome evolution from the two ancestral mating-type chromosomes through a centromere-to-telomere fusion event, which brought the PR and HD loci onto the same chromosome and allowed their linkage through a recombination suppression (dashed lines). (C) Demonstration of stepwise recombination suppression using per-gene synonymous divergence and their respective standard error (dS ± SE) between alleles from M. lychnidis-dioicae associated to the a1 versus a2 mating types along the matingtype chromosome gene order of M. lagerheimii, as a proxy for ancestral gene order. Evolutionary strata of different divergence levels (colored differently) shows that

  (1):700 121. Stukenbrock EH, Christiansen FB, Hansen TT, Dutheil JY, Schierup MH. 2012. Fusion of two divergent fungal individuals led to the recent emergence of a unique widespread pathogen species. Proc. Natl. Acad. Sci. 109(27):10954-59 122. Taylor JW, Branco S, Gao C, Hann-Soden C, Montoya L, et al. 2017. Sources of fungal genetic variation and associating It with phenotypic diversity. Microbiol. Spectr. 5(5): 123. Thrall PH, Biere A, Antonovics J. 1993. Plant life-history and disease susceptibility--The occurrence of Ustilago violacea on different species within the Caryophyllaceae. J. Ecol. 81(3):489-98 124. Thrall PH, Burdon JJ. 2004. Host-pathogen life history interactions affect biological control success. Weed Technol. 18(sp1):1269-74 125. Toh SS, Chen Z, Rouchka EC, Schultz DJ, Cuomo CA, Perlin MH. 2018. Pas de deux: an intricate dance of anther smut and Its host. G3 Genes Genomes Genet. g3.300318.2017 126. Toh SS, Chen Z, Schultz DJ, Cuomo CA, Perlin MH. 2017. Transcriptional analysis of mating and pre-infection stages of the anther smut, Microbotryum lychnidis-dioicae. Microbiology. 163(3):410-20 127. Toh SS, Perlin MH. 2016. Resurgence of less-studied smut fungi as models of phytopathogenesis in the omics age. Phytopathology. 106(11):1244-54 128. Toh SS, Treves DS, Barati MT, Perlin MH. 2016. Reliable transformation system for Microbotryum lychnidis dioicae informed by genome and transcriptome project. Arch. Microbiol. 198(8):813-25

Figure 1 :

 1 Figure 1: Specialization and co-phylogenies of anther-smut fungi and their

  indicated with a black square near the fungal cladogram leaves. Dashed lines indicate specialization of a fungal species on a host species, with pink lines for fungal species infecting different hosts, and orange links for fungal species infecting the same host. The sequenced strain M. intermedium was sampled on Salvia pratensis, although this fungal species is usually found on Scabiosa hosts. (B) Infected host plants by anther-smut fungi. Numbers refer to host species as in panel A. Spores of anther-smut fungi are visible in the anthers of the flowers (Photo credits: Michael E. Hood, Tatiana Giraud, Maarteen Strack van Schijndel).

Figure 2 :

 2 Figure 2: Evolutionary processes in anther-smut fungi studied by comparative genomics

( 2 )

 2 Identification of genes under positive selection using polymorphism in one species and divergence with an outgroup using McDonald-Kreitman tests. An excess of the ratio between non-synonymous (DN) and synonymous (DS) substitutions between species compared to the ratio between synonymous (PS) and non-synonymous (PN) polymorphisms within species is indicative of positive selection within the focal species indicated by an asterisk. Examples are shown for the orthologous group ORTHAg06728 and ORTHAg05587 (20). (3) Genome scan to identify selective sweeps based on allele frequency spectrum in M. lychnidis-dioicae. A selective sweep is characterized by an excess of rare variants. Composite likelihood ratio (CLR) tests estimate the probability of the presence of a selective sweep taking into account demographic history and genome-wide allele frequency spectrum (15).

Figure 3 :

 3 Figure 3: Distribution of divergence along genomes between species of host-specialized

Figure 4 :

 4 Figure 4: Genomic rearrangements and evolutionary strata on mating-type

Table 1 : Whole genome public resources in anther-smut fungi (Microbotryum genus).

 1

ACKNOWLEDGMENTS

This work was supported by a Marie Curie European grant (PRESTIGE-2016-4-0013 to FEH). FEH received the Young Biological Researcher Prize from the Fondation des Treilles, created by Anne Gruner Schlumberger, which supports research in Science and Art (http://www.les-treilles.com). A B Salvia pratensis Knautia arvensis Myosoton aquaticum Saponaria officinalis Dianthus superbus Dianthus pavonius Dianthus seguieri Silene caroliniana Silene virginica Silene lemmonii Silene dioica Silene latifolia Silene vulgaris Silene paradoxa Silene italica Silene nutans Silene acaulis Silene otites Lychnis flos-cuculi Microbotryum Hosts M. intermedium M. scabiosae M. v. caroliniana M. stellariae M. lagerheimii M. saponariae M. carthusianorum M. superbum M. v. paradoxa M. v. italica M. silenes-acaulis M. major M. v. lemmonii M. silenes-dioicae M. lychnidis-dioicae M. violaceo-irregulare M. violaceum s. str. M. silenes-inflatae M. coronariae

recombination suppression extended stepwise from the HR and PR mating-type loci. (D)

Examples of two gene genealogies showing contrasted clustering levels of alleles at nonmating-type genes associated with the a1 versus a2 mating types (dark grey and light grey squares, respectively, at the tips of the gene genealogy). The left panel shows the gene genealogy of a gene belonging to the pseudo-autosomal region (or PAR), with no transspecific polymorphism, i.e., intermingled alleles associated with a1 and a2 mating types. The right panel shows the gene genealogy of a gene belonging to the non-recombining region, with completely separated alleles associated with a1 versus a2 mating types of both M. lychnidis-dioicae and M. silenes-dioicae, and thus trans-specific polymorphism. The branch length scale is indicated at the bottom of each gene genealogy. Long-reads Short-reads

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(3) (4)

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Figure 1

Evolutionary events