Philippe Darriet

Vittorio Capozzi

Mariette Hugo

Lucie

Marie Geoffrey

Maroula Julie Marion

Paul Henschke

Simon Schmidt

Thanks Radka

Caroline Kate

Simon Dillon

Charly, Bill, Ben, Lizzy Marcell Lyubo

Joël, Perrine Agathe Federico

Marta Avramova
email: avramova.marta@gmail.com

Emilien Alice Cibrario

Emilien Peltier

Monika Coton

Emmanuel Coton

Joseph Schacherer

Giuseppe Spano

Giuseppe Blaiotta

Franck Salin

Marguerite Dols-Lafargue

Paul Grbin

Chris Curtin

Warren Albertin

Isabelle Masneuf-Pomarede

Isabelle Masneuf-Pomarède

Hany Abdo

Margaux Paulin

Patricia Ballestra

Marguerite Dols- Lafargue

" Brettanomyces

" Tolérance

Isabelle Masneuf- Pomarede

Anthony Borneman

Cristian Varela

"Brettanomyces bruxellensis population survey reveals a diploid-triploid complex structured according to substrate of isolation and geographical distribution", submitted in Scientific Reports Poster presentations

Keywords: génétique des populations, Brettanomyces bruxellensis, sulfites, avantage sélectif, polyploïdie population genetics, Brettanomyces bruxellensis, sulphites, selective advantage, polyploidy

Génétique des populations et diversité de l'espèce Brettanomyces bruxellensis. Etude de la tolérance aux sulfites Résumé Brettanomyces bruxellensis est un microorganisme qui est considéré comme la cause majeure des défauts microbiologiques du vin. L'importance de cette levure à l'échelle industrielle est liée au fait qu'elle est isolée à partir de substrats différents tels que la bière, le kombucha, les molasses utilisées pour la production de bioéthanol et autres. Ce projet a pour objectif d'étudier la diversité génétique de l'espèce en se basant sur une large population d'isolats provenant de niches écologiques et géographiques variées. Pour ce faire, une méthode de génotypage robuste (analyse microsatellite) a été optimisée et appliquée sur la population, mettant en évidence la coexistence de populations diploïdes et triploïdes à l'échelle globale. Puis, la relation entre regroupement génétique et traits physiologiques a été explorée. Notamment, l'étude de la tolérance aux sulfites a été effectuée sur un sous-ensemble de souches représentatif de la population. Les résultats obtenus mettent en évidence un lien entre groupes génétiques et comportement vis-à-vis des sulfites. Des expériences de compétition en présence de dioxyde de soufre montrent un avantage sélectif des souches tolérantes aux sulfites par rapport aux souches sensibles, suggérant ainsi une adaptation spécifique au principal antiseptique utilisé en oenologie. Ce travail contribue à une meilleure connaissance de cette levure d'altération du vin en termes de diversité génétique et phénotypique et permet d'émettre des hypothèses sur les stratégies évolutives d'adaptation au milieu anthropique de cette espèce modèle non conventionnelle.

Résumé détaillé

Brettanomyces bruxellensis est un microorganisme qui est considéré comme la cause majeure des défauts microbiologiques du vin. L'importance de cette levure à l'échelle industrielle est liée au fait qu'elle est isolée à partir de substrats différents tels que la bière, le kombucha (thé fermenté), les molasses utilisées pour la production de bioéthanol, etc. où cette levure pourrait être considérée comme étant bénéfique (voire nécessaire) ou néfaste pour l'élaboration et la qualité du produit. Au cours des dernières décennies, l'étude scientifique de cette levure ubiquiste ont révélé une variabilité phénotypique importante pour l'espèce en terme de croissance, métabolisme, tolérance à des facteurs biotiques et abiotiques variées, et autres.

Particulièrement, dans le domaine de l'oenologie, cette variabilité rend difficile la prévention et l'élimination de ce microorganisme d'altération. Pour chercher à comprendre ces variations phénotypiques intraspécifiques, la diversité génétique de B. bruxellensis a été explorée, mettant en évidence une variabilité génétique importante au sein de l'espèce. Notamment, une étude des souches isolées en Australie a mis en évidence différents groupes génétiques, avec un génotype prépondérant parmi les vins australiens. De plus, les souches de ce génotype se sont avérées très tolérantes aux sulfites. Plus tard, une étude de génomique comparative de 4 souches de cette espèce a mis en évidence des souches présentant niveaux de ploïdie différents, certaines souches étant diploïdes et autres -triploïdes, avec la souche appartenant au génotype le plus répandu en Australie étant triploïde. Ces études ont souligné un possible lien entre génotype et tolérance aux sulfites pour cette population d'isolats australiens. Les sulfites étant le moyen le plus utilisé pour prévenir et éliminer le développement de B. bruxellensis dans le domaine de l'oenologie, ce lien méritait d'être exploré à plus grande échelle. De plus, à ce stade de la connaissance de cette levure d'intérêt industriel, il était intéressant d'explorer la génétique des populations de l'espèce et les facteurs qui la déterminent.

Dans ce contexte, ce projet a pour objectif d'étudier la diversité génétique de l'espèce B. bruxellensis en se basant sur une large population d'isolats provenant de niches écologiques et géographiques variées. Pour ce faire une collection riche composée de 1488 isolats de substrats et origines géographiques a été utilisée. Ensuite, une méthode de génotypage robuste (analyse microsatellite) a été optimisée et appliquée sur la population, confirmant la diversité génotypique de l'espèce et mettant en évidence la coexistence de populations diploïdes et triploïdes à l'échelle globale. Par des analyses statistiques, il est démontré que la population est structurée en fonction du niveau de ploïdie, le type de substrat et l'origine géographique des isolats, suggérant une influence anthropique sur la biodiversité spatiale de B. bruxellensis.

Dans un deuxième temps, la relation entre regroupement génétique et traits physiologiques a été explorée. Notamment, l'étude de la tolérance aux sulfites a été effectuée sur un sous-ensemble de souches représentatif de la population (39 souches de substrats et origines géographiques différents). Des fermentations en petite échelle (fermenteurs de 3 mL) ont été effectuées dans un milieu modèle et à des différentes concentrations en sulfites variant de 0 à 0.6 mg/L de SO 2 moléculaire.

Les paramètres de croissance phase de latence, vitesse de croissance et population maximale ont été suivis et la base de données ainsi obtenue a été traitée par des analyses statistiques. Les résultats confirment le lien entre regroupement génétique et comportement vis-à-vis des sulfites pour ce sous-ensemble d'isolats représentatifs de l'espèce.

En effet, le lien entre configuration génétique et tolérance aux sulfites, combiné à la dissémination des souches triploïdes tolérantes au SO 2 mènent à l'hypothèse que cette configuration génétique pourrait apporter un avantage sélectif dans les conditions oenologiques, notamment en présence de SO 2 . Pour vérifier cette hypothèse, des expériences de compétition entre souches ont été menées en présence de dioxyde de soufre. Les isolats représentatifs des trois génotypes majeurs rencontrés en milieu oenologique ont été marqués avec des gènes de résistance aux antibiotiques en utilisant un protocole de transformation basé sur le mécanisme de jonction des extrémités non homologues. Les résultats montrent un avantage sélectif des souches tolérantes aux sulfites par rapport aux souches sensibles, suggérant ainsi une adaptation spécifique au principal antiseptique utilisé en oenologie.

Dans un dernier temps, l'étude du lien groupe génétique/tolérance aux sulfites a été approfondi en élargissant le panel de souches phénotypes à 145 souches représentatives de la population B. bruxellensis étudiée. Cette démarche a permis de confirmer ce lien ainsi que d'explorer la possibilité d'utiliser des marqueurs moléculaires pour discriminer les souches sensibles des tolérantes. De plus, des différents types de comportements en terme de croissance ont été observés parmi les souches de B. bruxellensis qui survivent à des fortes de doses de SO 2 soulignant des phénotypes résistants et tolérants. Humans consume and produce fermented beverages since millennia. It was through empirical experience that ancient people discovered the pleasant properties of the liquids resulting from fruit crushing and subsequent fermentation. Vessels containing traces of fermented beverage based on rice, honey and fruit (hawthorn or grape, elucidated via the presence of calcium salt of tartaric acid residues) were discovered in China and were dated back to 7000 BC [START_REF] Mcgovern | Fermented beverages of pre-and proto-historic China[END_REF]. The oldest chemical evidence of grape-wine mixed with Pistacia atlantica resin was found in Iran and was evaluated to 5400-5000 BC [START_REF] Mcgovern | Neolithic resinated wine[END_REF] which coincides with the period when the first human population settlements and plant and animal domestication took place [START_REF] Mcgovern | Neolithic resinated wine[END_REF]. Thus, by inducing, managing, and favouring fermentation of fruit and other materials, human societies have (possibly unconsciously) interacted with microbial populations responsible of the fermentation process.

Non-exhaustive list of fermented products made by humans and yeasts would include the most popular ones like wine, beer, and bread, but also yoghourt, chocolate, cheese, etc. This list is further broadened by fermented plants that are followed by distillation processes (tequila, whiskey, and others). Even if unconscious of the existence of yeasts performing the fermentation, people probably noticed that for example bread was lighter and better for consumption after addition of small quantity of wine (that was putatively a first form of leavened bread) (Mortimer, 2000a). Thus, unconsciously, since ancient time humans were in constant interaction with microorganisms. Nowadays, we know that fermented beverages present various types of ecological environments for the development of multiple genera, species, and strains of microorganismsmoulds, yeast, bacteria, and viruses. Indeed, it was not until the 1870s, following the work of Louis Pasteur, that it was demonstrated that small unicellular organisms (yeasts) were responsible for the transformation of sugars contained in fruit to alcohol and carbon dioxide in the reaction called fermentation. Central role in the knowledge of yeasts and their metabolism is occupied by the species Saccharomyces cerevisiae (the so-called baker's yeast) which is in the origin of various fermented beverages.

Focus on wine yeast

Wine is probably the most ancient fermented beverage consumed by people and this is partially due to fact that wine would occur "naturally" without addition of leaven. The provenance of yeast performing grape fermentation is still a controversy. Some authors suggest that the yeasts are already present on the grapes' surface and perform the fermentation once the grapes are crushed and their sugars released [START_REF] Mortimer | On the origins of wine yeast[END_REF]. It was believed for long time that S. cerevisiae cells were found only in association with human environments. Some authors suggested that wine yeasts were present on the grapes surface and could also be transported in the winery through insects and other vectors, subsequently residing on the winemaking equipment [START_REF] Naumov | Genetic identification of biological species in theSaccharomyces sensu stricto complex[END_REF]. Interestingly, the main yeast performing grape fermentation -S. cerevisiae, is detected on grape surface but at very low frequencies compared to other species [START_REF] Goddard | Saccharomyces cerevisiae: a nomadic yeast with no niche?[END_REF][START_REF] Mortimer | On the origins of wine yeast[END_REF]. This observation led to another model suggesting that S. cerevisiae was a "nomad yeast" without particular niche [START_REF] Goddard | Saccharomyces cerevisiae: a nomadic yeast with no niche?[END_REF]. Eventually, S. cerevisiae is present in probably all grape fermentations and it plays a central role in winemaking for several reasons: i) it is the only non-spoilage yeast species related to winemaking environment that is able to produce and survive to high concentrations of ethanol, ii) it is able the consume all sugars present in the grape must, thus reducing the risk of sluggish fermentation and spoilage during wine storage, iii) it is associated with enzymatic activities implied in the transformation of aromatic precursors contained in the grape. Thus, over the last decades, people have adapted their winemaking practices in order to favour the development of this particular species, often avoiding the occurrence of other microorganisms. The most straightforward example for this is the selection and production of the first active dried yeast designed for controlled inoculation of grape must by winemakers in the 1970s (see [START_REF] Chambers | Fermenting knowledge: the history of winemaking, science and yeast research[END_REF]. At present, the majority of conventional winemakers use this technique in order to avoid sluggish fermentations and generally to obtain better control of wine fermentation. Subsequently, multiple scientific articles focused on the selection and development of S. cerevisiae strains to obtain adapted sugar/ethanol yield, aromatic characteristics, and metabolic features corresponding to the winemaking environment (SO 2 , copper, and ethanol tolerance, flocculation capacity, and others) (see [START_REF] Chambers | Fermenting knowledge: the history of winemaking, science and yeast research[END_REF].

Nowadays, there is a high and rising interest for the so-called non-Saccharomyces yeast (or species other than the ones form Saccharomyces genus). Those species are generally developed in the early stages of grape fermentation process. Later, their population declines and is displaced by S. cerevisiae. Thus, their presence in the beginning of the fermentation is acting on wine properties [START_REF] Fleet | Yeast interactions and wine flavour[END_REF]. From oenological point of view, the impact of non-Saccharomyces yeast could be related to the volatile metabolites production involved in wine aroma [START_REF] Fleet | Yeast interactions and wine flavour[END_REF]. From a biological point of view, non-Saccharomyces yeasts are modifying the physical environment for S. cerevisiae (and other microorganisms). Also, some non-Saccharomyces yeast species are described as spoilage microorganisms in wine, leading to altered wine taste, aroma, and/or mouthfeel. Thus, nowadays the importance of non-Saccharomyces yeast in winemaking is undisputable (both as beneficial and spoilage microorganisms). The attempt to produce more complex and diverse wines has led to high and rising interest for various non-Saccharomyces yeast species for the controlled must inoculation.

Among the commercialised species available for co-inoculation with S. cerevisiae, are Torulaspora delbrueckii, Metschnikowia pulcherrima, Lachancea kluyveri, Lachancea thermotolerans [START_REF] Jolly | Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered[END_REF][START_REF] Masneuf-Pomarede | The Genetics of Nonconventional Wine Yeasts: Current Knowledge and Future Challenges[END_REF].

Yeast as human commensals and pathogens

Different yeast species are part of human body as commensals. However, in some cases strains can become pathogenic, especially in the case of immunocompromised patients (ex. HIV patients). Among others, popular human-pathogen yeast are several species from the Candida genus (ex. C. albicans, C. glabrata), as well as Cryptococcus neoformans [START_REF] Hazen | New and emerging yeast pathogens[END_REF][START_REF] Wertheimer | Ploidy dynamics and evolvability in fungi[END_REF]. Interestingly, Saccharomyces cerevisiae is sometimes encountered as human pathogen [START_REF] Wertheimer | Ploidy dynamics and evolvability in fungi[END_REF][START_REF] Zhu | Whole Genome Analysis of 132 Clinical Saccharomyces cerevisiae Strains Reveals Extensive Ploidy Variation[END_REF].

Yeast as model organisms

From a fundamental point of view, yeasts are model organisms for the study of higher eukaryote cell metabolism, human-cell ageing, and cancer [START_REF] Botstein | Yeast as a Model Organism[END_REF][START_REF] Denoth Lippuner | Budding yeast as a model organism to study the effects of age[END_REF][START_REF] Wassmann | Mitotic checkpoints: from yeast to cancer[END_REF], especially after the first genome sequence S. cerevisiae was published [START_REF] Goffeau | Life with 6000 Genes[END_REF]. This is mainly due to the genetic similarity with multicellular eukaryotes combined with the easiness of manipulating those unicellular organisms in laboratory. The ability to manipulate S. cerevisiae in the laboratory in a highly controlled manner allows studying of genome instability, which is a typical trait of cancer cells [START_REF] Wassmann | Mitotic checkpoints: from yeast to cancer[END_REF]. The short generation time of yeasts in the laboratory combined with the availability of genetic transformation tools (see [START_REF] Chambers | Fermenting knowledge: the history of winemaking, science and yeast research[END_REF] for review), makes of yeasts irreplaceable tools for revealing gene functions and evolution mechanisms for eukaryotes.

Yeast as "cell factories"

The importance of yeasts is enhanced by the use of those unicellular microorganisms as "cell factories" for the production of different molecules of industrial and pharmaceutical interest with among others vanillin, insulin, and hepatitis B vaccine [START_REF] Hou | Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae[END_REF][START_REF] Joan | Microbial factories for recombinant pharmaceuticals[END_REF][START_REF] Jørgen | Improved vanillin production in baker's yeast through <it>in silico </it>design[END_REF]. Particularly interesting industrial application of yeasts from environmental point of view is the production of ethanol, butanol, and isoprenoids that can be used as biofuel [START_REF] Gírio | Hemicelluloses for fuel ethanol: A review[END_REF][START_REF] Hong | Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries[END_REF].

Overall, the use of yeasts in beverage production and other industrial branches led to a gradual control of the fermentation processes on the behalf of people. On one hand, people are providing and modifying the environment in a way that would favour or limit the development of certain types of microorganisms. On the other hand, people would actively breed, modify, and select for organisms of interest. In industry, this is mostly driven by the idea of maintaining quality product as well as preventing production of inconsumable (and therefore non-sellable) products, thus guaranteeing optimal process efficiency. In this context, it is essential to i) know the biological material that is developing in fermentation conditions and ii) explore its behaviour in different conditions, in order to iii) be able to predict its impact on the product, and eventually adapt the used techniques according to that.

Yeast population genetics -an approach to study yeast evolution

The qualitative analysis of microbial diversity of an ecosystem can be done on multiple levelstype of microorganisms (yeast, mould, bacteria), species level (for example, identify the yeast species present in a wine sample), and intraspecies level (identify the variability of strains present among the species). If we take the example of wine, studies of microbiome related to winemaking often explore the species diversity. However, many characteristics of those organisms could present high level of intraspecies variability with subsequent variable repercussions on wine qualities. In this chapter, different methods for the assessment of genetic intraspecies diversity among strains are going to be discussed with an accent on wine-related species (Table 1.1). Few of those methods have furthermore the advantage to elucidate genetic relations between strains (AFLP, MLST, WGS and SNPs analysis). In recent years, the advancements in the fields of both high throughput techniques (Next Generation Sequencing) and bioinformatics, made possible the analysis of large amount of genetic data. Further, population genetics is the scientific enquiry that puts phenotypic variation in the context of genetic evolution [START_REF] Chakravarti | Population genetics-making sense out of sequence[END_REF]. Comparison between individuals and populations with their specific genetic characteristics, allows exploring the genetic bases behind phenotypic variation. Thus, population genetics brings insights into the evolutionary history of a species, and the factors playing major role in shaping its population structure [START_REF] Mcdonald | The Population Genetics of Fungi: Tools and Techniques[END_REF].

Genetic relations between different populations are essential for the study of the evolutionary success of a species and the putative relation with human activity. Population genetics in microbiology can be studied through various methods that should have the ability to i) be discriminant, ii) reproducible, iii) and to highlight genetic relations between different genotype (or genome) profiles observed. Even if genome sequencing is the most complete and accurate method for this type of analysis, other methods have the advantage to be easier to apply and analyse, by still remaining accurate. Microsatellite markers (or SSR standing for single sequence repeats) have few very strong advantages, among others: codominance (meaning that if there are two or more different alleles present for a locus, they would be visible), neutrality (related to the fact that they are generally not subject of selective pressure), low risk of homoplasy (they are generally specific and unique to the species), high variability among strains [START_REF] Clark | Resolving microsatellite genotype ambiguity in populations of allopolyploid and diploidized autopolyploid organisms using negative correlations between allelic variables[END_REF][START_REF] Guichoux | Current trends in microsatellite genotyping[END_REF][START_REF] Vieira | Microsatellite markers: what they mean and why they are so useful[END_REF]. Furthermore, microsatellite analyses allow the screening of a high number of isolates and establishment of genetic relations between individuals, and give indications on the ploidy level of the studied organism. Thus, the use of microsatellite analysis for population genetics studies allows covering a wide range of genetic diversity at intraspecies level. (Albertin et al., 2014a, 2014b, 2016[START_REF] Almeida | A Gondwanan Imprint on Global Diversity and Domestication of Wine and Cider Yeast Saccharomyces uvarum[END_REF][START_REF] Sam Crauwels | Brettanomyces Bruxellensis, Essential Contributor in Spontaneous Beer Fermentations Providing Novel Opportunities for the Brewing Industry[END_REF][START_REF] Sam Crauwels | Brettanomyces Bruxellensis, Essential Contributor in Spontaneous Beer Fermentations Providing Novel Opportunities for the Brewing Industry[END_REF][START_REF] Ayoub | Application of Multi Locus Sequence Typing to the analysis of the biodiversity of indigenous Saccharomyces cerevisiae wine yeasts from Lebanon[END_REF][START_REF] Barquet | Tandem repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-Saccharomyces subspecies[END_REF][START_REF] Börlin | Cellar-Associated Saccharomyces cerevisiae Population Structure Revealed High-Level Diversity and Perennial Persistence at Sauternes Wine Estates[END_REF][START_REF] Cadez | Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera[END_REF][START_REF] Cubillos | Self-fertilization is the main sexual reproduction mechanism in native wine yeast populations[END_REF]C. Curtin et al., 2012b;[START_REF] Dubourdieu | Identification des souches de levures isolées de vins par l'analyse de leur ADN mitochondrial[END_REF][START_REF] Dunn | Microarray karyotyping of commercial wine yeast strains reveals shared, as well as unique, genomic signatures[END_REF][START_REF] Erny | Ecological Success of a Group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii Hybrids in the Northern European Wine-Making Environment[END_REF][START_REF] Fay | Evidence for Domesticated and Wild Populations of Saccharomyces cerevisiae[END_REF][START_REF] Gallone | Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts[END_REF][START_REF] Grangeteau | Diversity of yeast strains of the genus Hanseniaspora in the winery environment: What is their involvement in grape must fermentation?[END_REF][START_REF] Guijo | Determination of the relative ploidy in different Saccharomyces cerevisiae strains used for fermentation and "flor" film ageing of dry sherry-type wines[END_REF][START_REF] Hranilovic | The evolution of Lachancea thermotolerans is driven by geographical determination, anthropisation and flux between different ecosystems[END_REF]Ibeas et al., 1996;[START_REF] Legras | Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history[END_REF][START_REF] Legras | Optimisation of interdelta analysis for Saccharomyces cerevisiae strain characterisation[END_REF][START_REF] Liti | Population genomics of domestic and wild yeasts[END_REF][START_REF] Lopandic | Genetically different wine yeasts isolated from Austrian vine-growing regions influence wine aroma differently and contain putative hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii[END_REF][START_REF] Lopes | Spoilage yeasts in Patagonian winemaking: molecular and physiological features of Pichia guilliermondii indigenous isolates[END_REF][START_REF] Martorell | Molecular typing of the yeast species Dekkera bruxellensis and Pichia guilliermondii recovered from wine related sources[END_REF][START_REF] Masneuf-Pomarede | The yeast Starmerella bacillaris (synonym Candida zemplinina) shows high genetic diversity in winemaking environments[END_REF][START_REF] Miot-Sertier | Development of a molecular method for the typing of Brettanomyces bruxellensis (Dekkera bruxellensis) at the strain level[END_REF][START_REF] Muñoz | Multilocus sequence typing of oenological Saccharomyces cerevisiae strains[END_REF][START_REF] Querol | A Comparative Study of Different Methods of Yeast Strain Characterization[END_REF][START_REF] Richards | A database of microsatellite genotypes for Saccharomyces cerevisiae[END_REF][START_REF] Salinas | Genomic and phenotypic comparison between similar wine yeast strains of Saccharomyces cerevisiae from different geographic origins[END_REF][START_REF] Schacherer | Comprehensive polymorphism survey elucidates population structure of Saccharomyces cerevisiae[END_REF][START_REF] Schuller | Survey of molecular methods for the typing of wine yeast strains[END_REF][START_REF] Schuller | Survey of molecular methods for the typing of wine yeast strains[END_REF][START_REF] Sturm | Influence of grape treatment on the wine yeast populations isolated from spontaneous fermentations[END_REF][START_REF] Tofalo | Diversity of Candida zemplinina strains from grapes and Italian wines[END_REF][START_REF] Urso | Yeast biodiversity and dynamics during sweet wine production as determined by molecular methods[END_REF] Among unicellular eukaryotes, S. cerevisiae is the most broadly studied model species and this mirrors its importance as industrial yeast with high contribution to fundamental knowledge of micro-and macro-organisms. Population genetics of the species highlighted great genetic diversity and grouping according to type of industrial fermentation environment [START_REF] Aa | Population structure and gene evolution in Saccharomyces cerevisiae[END_REF][START_REF] Fay | Evidence for Domesticated and Wild Populations of Saccharomyces cerevisiae[END_REF][START_REF] Gallone | Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts[END_REF][START_REF] Legras | Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history[END_REF][START_REF] Liti | Population genomics of domestic and wild yeasts[END_REF][START_REF] Schacherer | Comprehensive polymorphism survey elucidates population structure of Saccharomyces cerevisiae[END_REF]. Industrial fermentations are directly related to human activity; therefore this correlation is a strong indicator of domestication of S. cerevisiae. Indeed, population genomics studies led to the hypothesis that at least two lineages of S. cerevisiae population (namely, European/Wine and Sake group) were a subject of domestication [START_REF] Liti | Population genomics of domestic and wild yeasts[END_REF][START_REF] Schacherer | Comprehensive polymorphism survey elucidates population structure of Saccharomyces cerevisiae[END_REF]. Precisely, wine strains were demonstrated to form a specific cluster, first by study of polymorphic sites at five unlinked loci [START_REF] Fay | Evidence for Domesticated and Wild Populations of Saccharomyces cerevisiae[END_REF], and then, by microsatellite analysis [START_REF] Legras | Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history[END_REF] and genome sequencing [START_REF] Liti | Population genomics of domestic and wild yeasts[END_REF][START_REF] Schacherer | Comprehensive polymorphism survey elucidates population structure of Saccharomyces cerevisiae[END_REF]. These findings were also supported by certain genetic signatures of domestication, related to human activity such as tolerance to sulphur dioxide, copper, and other chemical agents [START_REF] Pérez-Ortín | Molecular characterization of a chromosomal rearrangement involved in the adaptive evolution of yeast strains[END_REF][START_REF] Warringer | Trait Variation in Yeast Is Defined by Population History[END_REF]. Recently, the domestication of beer S. cerevisiae strains was also elucidated [START_REF] Gallone | Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts[END_REF]. At the beginning of the era of S. cerevisiae population structure analysis, natural isolates were missing. However, it was recently discovered that wild S. cerevisiae populations were present on oak barks and associated soil from the Mediterranean region [START_REF] Almeida | A population genomics insight into the Mediterranean origins of wine yeast domestication[END_REF]. This made it possible to compare the "wild" populations to the human-associated ones and highlighted a group of grape wine fermentation-related genes that were present among wine strains and absent among the wild oak ones [START_REF] Almeida | A population genomics insight into the Mediterranean origins of wine yeast domestication[END_REF]. This analysis confirmed the predictions of population diversification related to domestication. [START_REF] Almeida | A population genomics insight into the Mediterranean origins of wine yeast domestication[END_REF][START_REF] Almeida | A Gondwanan Imprint on Global Diversity and Domestication of Wine and Cider Yeast Saccharomyces uvarum[END_REF][START_REF] Baker | The Genome Sequence of Saccharomyces eubayanus and the Domestication of Lager-Brewing Yeasts[END_REF][START_REF] Gallone | Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts[END_REF][START_REF] Libkind | Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast[END_REF][START_REF] Liti | Population genomics of domestic and wild yeasts[END_REF][START_REF] Schacherer | Comprehensive polymorphism survey elucidates population structure of Saccharomyces cerevisiae[END_REF][START_REF] Sicard | Bread, beer and wine: Yeast domestication in the Saccharomyces sensu stricto complex[END_REF] Several population genetics studies were performed on different wine yeast species that are also present in other beverages [START_REF] Albertin | Hanseniaspora uvarum from Winemaking Environments Show Spatial and Temporal Genetic Clustering[END_REF](Albertin et al., , 2014b(Albertin et al., , 2014a;;[START_REF] Hranilovic | The evolution of Lachancea thermotolerans is driven by geographical determination, anthropisation and flux between different ecosystems[END_REF][START_REF] Masneuf-Pomarede | The yeast Starmerella bacillaris (synonym Candida zemplinina) shows high genetic diversity in winemaking environments[END_REF]. The use of microsatellite analysis for those studies makes it possible to deduce ploidy level of the species, thus leading to hypotheses on their life cycle.

Interestingly, not all studied wine-related yeast populations clustered according to the same factors as S. cerevisiae. For some species, populations cluster according to geographical origin (S. bacillaris [START_REF] Masneuf-Pomarede | The yeast Starmerella bacillaris (synonym Candida zemplinina) shows high genetic diversity in winemaking environments[END_REF], H. uvarum [START_REF] Albertin | Hanseniaspora uvarum from Winemaking Environments Show Spatial and Temporal Genetic Clustering[END_REF]). This would suggest that the factors related to the geographical localisation are more important for those populations than factors related to the industrial fermentation environment. In other cases, niche type was demonstrated to be the determining factor for population structure. This was the Can a microorganism be "domesticated"?

Domestication is a term generally used for the relationship between man and plants or animals. However, several yeast species from the Saccharomyces genus are also considered as domesticated by humans (see [START_REF] Liti | Population genomics of domestic and wild yeasts[END_REF], Shacherer, 2009[START_REF] Libkind | Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast[END_REF][START_REF] Gallone | Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts[END_REF][START_REF] Baker | The Genome Sequence of Saccharomyces eubayanus and the Domestication of Lager-Brewing Yeasts[END_REF][START_REF] Almeida | A population genomics insight into the Mediterranean origins of wine yeast domestication[END_REF][START_REF] Almeida | A Gondwanan Imprint on Global Diversity and Domestication of Wine and Cider Yeast Saccharomyces uvarum[END_REF][START_REF] Sicard | Bread, beer and wine: Yeast domestication in the Saccharomyces sensu stricto complex[END_REF]. Based on the studies cited above, there are few major characteristics that should be present for defining a species or sub-population of strains as "domesticated": i) genetic fingerprint of domestication whose presence and expression contributes to adaptation to human-related environment; ii) phenotypic characteristics related to human activity; iii) niche specialisation or adaptation to artificial man-made environment which is related to the other two points; iv) presence of traits that are desirable for humans (e.g. production of aroma compounds of interest, fermentation efficiency, etc.); v) genetic distinction between human-related and natural isolates. A relevant example is a sub-group of beer S. cerevisiae industrial strains which are characterised by a decay of sexual reproduction, convergent evolution towards industrially favourable traits like maltotriose utilisation, stress resistance and non-production of offflavours [START_REF] Gallone | Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts[END_REF].

case for T. delbrueckii (Albertin et al., 2014a) and L. thermotolerans [START_REF] Hranilovic | The evolution of Lachancea thermotolerans is driven by geographical determination, anthropisation and flux between different ecosystems[END_REF] (both commercialised for must inoculation in combination with S. cerevisiae), for which a genetic differentiation between natural and industrial isolates was observed. Microsatellite genotype analysis was also applied for Brettanomyces bruxellensis (Albertin et al., 2014b) wine spoilage yeast which is also isolated from other fermented products. For this species, clonal populations were shown to persist over decades in the same winery (Albertin et al., 2014b). The same study also highlighted the high dissemination of the species, as genetically close strains were detected at distant geographical locations (Albertin et al., 2014b). The significance of those population genetics studies is related to the contribution to a more holistic picture of the species, their adaptation and evolution in human-related environments.

Ploidy level among wine (and other) yeast of interest

"One of the most striking features of genome structure is its lability." [START_REF] Otto | The Evolutionary Consequences of Polyploidy[END_REF] Genetic variability is in the origin of genetic diversity and the subsequent adaptation capacity of a species. Yeast genomes can gain variability through different mechanisms including sexual reproduction (mix and shuffle of two parent genomes, single point mutations (ex. changes in single nucleotides), InDels (insertions or deletions events of relatively short pieces of DNA), transposons (mobile genetic elements that can cause mutations by insertion), genetic recombination (reorganisation of parts of the genome), or acquisition of exogenic DNA pieces by horizontal gene transfer (reviewd by [START_REF] Dequin | The genomes of fermentative Saccharomyces[END_REF][START_REF] Steensels | Improving industrial yeast strains: exploiting natural and artificial diversity[END_REF] (Figure 1.1). In the following section a particular attention will be attributed to ploidy variation as a source of genomic plasticity for few species of industrial and clinical importance. Polyploidy is the state of having more than one (for haploid organisms) or two (for diploid organisms) sets of chromosomes. To further discuss the incidence and impact of polyploidy, it is important to define different types of polyploidy as described in the relevant literature (see the box below).

according to [START_REF] Chen | Molecular mechanisms of polyploidy and hybrid vigor[END_REF][START_REF] Soltis | Polyploidy and genome evolution in plants[END_REF] 1.

How does polyploidy occur?

Generally, polyploidy is linked to impaired chromosome segregation that can be due to various genetic and environmental factors [START_REF] Otto | The Evolutionary Consequences of Polyploidy[END_REF]. Polyploidy can be achieved by inhibition of some or all aspects of mitosis in variant cell cycles [START_REF] Frawley | Polyploidy[END_REF]. Thus, polyploids arise when a rare mitotic or meiotic catastrophe causes the formation of gametes with more than one set of chromosomes. Further, diploid gametes can fuse with haploid ones, and produce triploids that can either be sterile, or further give polyploid gametes [START_REF] Ramsey | Pathways, Mechanisms, and Rates of Polyploid Formation in Flowering Plants[END_REF]. Also, the fusion of diploid zygotes would result in tetraploid daughter cells.

Different types of polyploidy

Diploidy: The state of being diploid; that is, containing two complete sets of chromosomes (or genomes).

Aneuploidy: the state of having chromosome number that is not the exact multiple of the typical haploid set for a species.

Polyploidy: the state of having more than one (for haploid organisms) or two (for diploid organisms) complete sets of chromosomes Autopolyploidy: the state of polyploidy resulting from genome doubling that arises within a species; it may involve a single individual or crossing between individuals from genetically distinct lineages within a species.

Allopolyploidy: polyploidy formed through the combined processes of interspecific hybridisation and mutation of chromosome number.

Thus, a whole genome duplication (WGD) caused by "abnormal" cell division could lead to autopolyploidy [START_REF] Peer | The evolutionary significance of polyploidy[END_REF]. Whereas autopolyploids formation results "simply" from mutation of chromosome number, allopolyploidy is the result of concurrent hybridisation and mutation in chromosome number [START_REF] Comai | The advantages and disadvantages of being polyploid[END_REF]. Furthermore, polyploidy is prevalent among hybrid taxa which is possibly related to meiotic pairing [START_REF] Otto | The Evolutionary Consequences of Polyploidy[END_REF]. Diploid hybrids are prone to form unreduced gametes, which have the same number of chromosomes as the somatic cells and thus the rate of polyploids is often increased in hybrid lineages [START_REF] Otto | The Evolutionary Consequences of Polyploidy[END_REF].

These phenomena generally have a fitness cost on the respective organisms due to the difficulty to maintain imbalanced chromosome number during cell division, the propensity of polyploid mitosis and meiosis to produce aneuploid cells and the associated epigenetic instability, as well as other effects related to nuclear and cell enlargement [START_REF] Comai | The advantages and disadvantages of being polyploid[END_REF]. From a structural and regulatory point of view, increasing genomic content of the cell can lead to increased nucleus and cell volume (especially in the case of somatic polyploidy which concerns multicellular organisms and is not discussed in details in this work) [START_REF] Melaragno | Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis[END_REF].

Consequently, the surface to volume ratio of the cell is modified, and could lead to dosage imbalance, regulatory repercussions [START_REF] Comai | The advantages and disadvantages of being polyploid[END_REF], and/or lower growth rate depending on the environment [START_REF] Mable | Ploidy evolution in the yeast Saccharomyces cerevisiae: a test of the nutrient limitation hypothesis[END_REF].

Because of those disadvantages related to polyploidy, it is generally accepted that the polyploid state is maintained only if it confers selective advantage to the cell and/or respective population in a particular environment condition [START_REF] Wertheimer | Ploidy dynamics and evolvability in fungi[END_REF]. Immediate advantages of polyploidy are related to increased genetic variation and possible changes in gene expression (especially in the case of allopolyploids resultant from diverged lineages [START_REF] Otto | The Evolutionary Consequences of Polyploidy[END_REF] and epigenetic remodelling [START_REF] Peer | The evolutionary significance of polyploidy[END_REF] (Figure 1.2, from [START_REF] Rancati | Karyotypic changes as drivers and catalyzers of cellular evolvability: A perspective from non-pathogenic yeasts[END_REF].

Genome plasticity of polyploids could be a result of gene redundancy (presence of the same gene in multiple copies). This phenomenon has masking effect on mutations that could otherwise be deleterious, but also provides field for evolutionary experimentation through neofunctionalisation of the respective genes [START_REF] Comai | The advantages and disadvantages of being polyploid[END_REF][START_REF] Peer | The evolutionary significance of polyploidy[END_REF]. In allopolyploids, successful genetic combinations could be favoured through heterosis [START_REF] Otto | The Evolutionary Consequences of Polyploidy[END_REF]. Thus, by changing the genomic context of certain genetic features (or genome repatterning), polyploidy can lead to increased variability. This variable genetic background is especially important for small populations which are result from bottleneck phenomena. In those cases, it would be the balance between fitness cost and survival novelty of the newly formed genetic configuration that would determine the prosperity of the polyploid lineage.

Several famous polyploids

Actually, most of the crops that sustain humanity are polyploids (Paterson and Wendel, 2015) and this highlights the industrial importance of polyploidy, especially for plants.

In many cases, characteristics of polyploids were of interest for the production managed [START_REF] Chen | Molecular mechanisms of polyploidy and hybrid vigor[END_REF]Leitch and Leitch, 2008).

Sample of polyploid agricultural crops.

Showing oil from oilseed rape, bread from bread wheat, rope from sisal, coffee beans, banana, cotton, potatoes, and maize. (Leitch and Leitch, 2008) For this state to remain stable, it is required that the conditions wouldn't allow displacement by the polyploids' diploid relatives [START_REF] Otto | The Evolutionary Consequences of Polyploidy[END_REF]. If this condition is satisfied, the polyploid population can eventually establish leading to adaptation. Higher polyploid states are often associated with reproductive incompatibility with parent diploid cells and therefore postzygotic reproductive isolation. Generally, adapted polyploids further enter an evolutionary path of diploidisation during which duplicated genes can be lost, retained or undergo sub-or neofunctionalisation [START_REF] Comai | The advantages and disadvantages of being polyploid[END_REF]. The increased number of gene copies can fuel new beneficial mutations and lead to specific adaptations. Thus, polyploidisation may favour long-term diversification, evolutionary success, and possibly speciation.

Polyploidy in yeast

In fungi, aneuploidy and polyploidy have been shown to confer selective advantage in extreme conditions, such as high osmotic pressure, presence of drugs, low temperature, and others [START_REF] Albertin | Evidence for autotetraploidy associated with reproductive isolation in Saccharomyces cerevisiae: towards a new domesticated species[END_REF][START_REF] Albertin | Polyploidy in fungi: evolution after whole-genome duplication[END_REF][START_REF] Mulla | Yeast: a simple model system to study complex phenomena of aneuploidy[END_REF][START_REF] Todd | Ploidy Variation in Fungi -Polyploidy, Aneuploidy, and Genome Evolution[END_REF][START_REF] Wertheimer | Ploidy dynamics and evolvability in fungi[END_REF]. Polyploids are often observed among yeast species in particular.

Many yeast species are commensals related to humans but can become pathogenic in some occasions, especially in the case of immunocompromised patients [START_REF] Odds | Candida and candidosis: a review and bibliography. 2nd edition. Candida and candidosis: a review and bibliography[END_REF][START_REF] Todd | Ploidy Variation in Fungi -Polyploidy, Aneuploidy, and Genome Evolution[END_REF][START_REF] Wertheimer | Ploidy dynamics and evolvability in fungi[END_REF][START_REF] Zhu | Whole Genome Analysis of 132 Clinical Saccharomyces cerevisiae Strains Reveals Extensive Ploidy Variation[END_REF]. The most prevalent yeast related to fungal infections is Candida albicans [START_REF] Wertheimer | Ploidy dynamics and evolvability in fungi[END_REF]. This opportunistic yeast is known to have a complex life cycle with alternation of haploid and diploid populations [START_REF] Hickman | The /`obligate diploid/' Candida albicans forms mating-competent haploids[END_REF]. Antibiotics from the azole family are the most popular method to treat candidiasis but cases of fluconazole resistance have often been reported among isolates [START_REF] White | Clinical, cellular, and molecular factors that contribute to antifungal drug resistance[END_REF].

The latter antibiotic triggers an enzyme involved in the ergosterol pathway which is coded by the gene ERG1 (see White et al., 1998 for review). It was experimentally demonstrated that resistant strains were aneuploid for a region of the left arm of chromosome 5 that contained the drug-trigger gene, as well as other genes related to drug efflux and transcription factors that positively regulate a subset of efflux pump genes [START_REF] Selmecki | Aneuploidy and isochromosome formation in drug-resistant Candida albicans[END_REF]. Therefore, this aneuploidy contributes to resistant phenotype by both i) higher synthesis rate of the fluconazole trigger and ii) higher drug efflux rate. Another fungal species -Cryptococcus neoformans, which is generally an environmental saprophyte, is also reported to lead to meningoencephalitis in humans, especially in the late 1980s when the incidence of HIV patients increased (May et al., 2016). C. neoformans is most often found in haploid state and can reproduce both sexually and asexually. Strikingly, during infections, C. neoformans cells can gain virulence through the formation of "titan cells" with ploidy ranging from 4n to >64n [START_REF] Feldmesser | Dynamic changes in the morphology of Cryptococcus neoformans during murine pulmonary infection[END_REF][START_REF] Okagaki | Cryptococcal Cell Morphology Affects Host Cell Interactions and Pathogenicity[END_REF] (reviewed by [START_REF] Todd | Ploidy Variation in Fungi -Polyploidy, Aneuploidy, and Genome Evolution[END_REF]. Higher ploidy of those cells is associated to volume increase and subsequent protection from phagocytosis by immune cells [START_REF] Okagaki | Cryptococcal Cell Morphology Affects Host Cell Interactions and Pathogenicity[END_REF]. Furthermore, when treated with fluconazole, polyploid strains gave rise to different aneuploid daughter cells [START_REF] Gerstein | Polyploid Titan Cells Produce Haploid and Aneuploid Progeny To Promote Stress Adaptation[END_REF]. Thus, aneuploid formation contributes to rapid generation of diversity to C. neoformans. As a consequence, the population is more prone to rapidly correspond to the changes in host-related environment, leading to successful virulence.

Strikingly, recent study has demonstrated that 70% among 132 S. cerevisiae clinical isolates were aneu-or polyploid [START_REF] Zhu | Whole Genome Analysis of 132 Clinical Saccharomyces cerevisiae Strains Reveals Extensive Ploidy Variation[END_REF]. It was suggested that the higher ploidy level of those strains contributes to the transition from industrial to human pathogen lifestyle [START_REF] Zhu | Whole Genome Analysis of 132 Clinical Saccharomyces cerevisiae Strains Reveals Extensive Ploidy Variation[END_REF]. Indeed, previous population genomics studies suggested that S. cerevisiae clinical isolates originate from industry-related strains that have gained the ability to colonise human tissues [START_REF] Schacherer | Comprehensive polymorphism survey elucidates population structure of Saccharomyces cerevisiae[END_REF]. In a clinical context, genetic flexibility following polyploidisation can promote tumorigenesis in mammalian cells [START_REF] Fujiwara | Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells[END_REF]. Thus, S. cerevisiae is an important model for polyploid behaviour and evolution. Indeed, in vitro evolution experiments with isogenic haploid, diploid, and tetraploid S. cerevisiae strains highlighted that polyploidy can, not only promote, but also accelerate adaptation [START_REF] Selmecki | Polyploidy can drive rapid adaptation in yeast[END_REF]. Actually, yeast polyploidy is far from being an exceptional event, as polyploidy is in the origin of the whole Saccharomyces genus which was the subject of whole genome duplication (WGD) [START_REF] Wolfe | Molecular evidence for an ancient duplication of the entire yeast genome[END_REF]) that occurred 100 million years ago. Among other features, WGD implied duplication of glycolytic genes [START_REF] Conant | Increased glycolytic flux as an outcome of whole-genome duplication in yeast[END_REF] and subsequent enhanced sugar metabolism and ethanol make-accumulate-consume strategy [START_REF] Rozpędowska | Parallel evolution of the make-accumulateconsume strategy in Saccharomyces and Dekkera yeasts[END_REF][START_REF] Tessonnière | Design and performance testing of a real-time PCR assay for sensitive and reliable direct quantification of Brettanomyces in wine[END_REF]. This event coincides with the period when fleshy fruit colonised the Earth surface thus providing sugar-rich environment. Therefore, rapid sugar consumption associated with production of ethanol (which is toxic for microorganisms concurrent in the same niche), associated with ethanol accumulation and subsequent consumption, possibly gave selective advantage to Saccharomyces yeast [START_REF] Tessonnière | Design and performance testing of a real-time PCR assay for sensitive and reliable direct quantification of Brettanomyces in wine[END_REF]. This ancient WGD event is considered a key event in Saccharomyces clade leading to adaptation of the species to environmental changes. Polyploid S. cerevisiae strains are also related to industrial environments. For instance, autotetraploid S. cerevisiae strains are shown to be specifically related to baking environment [START_REF] Albertin | Evidence for autotetraploidy associated with reproductive isolation in Saccharomyces cerevisiae: towards a new domesticated species[END_REF]. It was suggested that the autotetraploid state of the respective populations conferred adaptation to baking-specific conditions through high osmotic pressure tolerance and high metabolic efflux [START_REF] Albertin | Evidence for autotetraploidy associated with reproductive isolation in Saccharomyces cerevisiae: towards a new domesticated species[END_REF]. Stable S. cerevisiae autopolyploid populations were also isolated from millet beer [START_REF] Safadi | A polyploid population of Saccharomyces cerevisiae with separate sexes (dioecy)[END_REF] and sherry-type wines [START_REF] Guijo | Determination of the relative ploidy in different Saccharomyces cerevisiae strains used for fermentation and "flor" film ageing of dry sherry-type wines[END_REF][START_REF] Naumov | Natural Polyploidization of Some Cultured Yeast Saccharomyces Sensu Stricto: Auto-and Allotetraploidy[END_REF]. The occurrence of autopolyploid S. cerevisiae in those specific environments suggests the putative industrial interest of this genomic state, which would be related to their high metabolic flux leading to high process efficiency.

Apart from autopolyploids resulting from WGD, allopolyploid populations, and precisely hybrid species, are also encountered in human-related industrial environments. Populations resulting from hybridisation phenomena were often reported among the Saccharomyces genus and are wittingly or unwittingly utilised by people for the production of fermented products (Table 1.2, reviewed by [START_REF] Marsit | Diversity and adaptive evolution of Saccharomyces wine yeast: a review[END_REF][START_REF] Morales | Evolutionary Role of Interspecies Hybridization and Genetic Exchanges in Yeasts[END_REF]. [START_REF] Bradbury | A homozygous diploid subset of commercial wine yeast strains[END_REF][START_REF] Gangl | Exceptional fermentation characteristics of natural hybrids from Saccharomyces cerevisiae and S. kudriavzevii[END_REF]Gonzalez et al., 2007;[START_REF] Lopandic | Genetically different wine yeasts isolated from Austrian vine-growing regions influence wine aroma differently and contain putative hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii[END_REF][START_REF] Masneuf | New Hybrids between Saccharomyces Sensu Stricto Yeast Species Found among Wine and Cider Production Strains[END_REF][START_REF] Borneman | The genome sequence of the wine yeast VIN7 reveals an allotriploid hybrid genome with Saccharomyces cerevisiae and Saccharomyces kudriavzevii origins[END_REF][START_REF] Erny | Ecological Success of a Group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii Hybrids in the Northern European Wine-Making Environment[END_REF]Schutz et al., 1994;[START_REF] Arroyo-López | Effects of temperature, pH and sugar concentration on the growth parameters of Saccharomyces cerevisiae, S. kudriavzevii and their interspecific hybrid[END_REF] [START_REF] Arroyo-López | Effects of temperature, pH and sugar concentration on the growth parameters of Saccharomyces cerevisiae, S. kudriavzevii and their interspecific hybrid[END_REF][START_REF] Belloch | Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex[END_REF][START_REF] Borneman | The genome sequence of the wine yeast VIN7 reveals an allotriploid hybrid genome with Saccharomyces cerevisiae and Saccharomyces kudriavzevii origins[END_REF][START_REF] Bradbury | A homozygous diploid subset of commercial wine yeast strains[END_REF][START_REF] De Barros Lopes | Evidence for multiple interspecific hybridization in Saccharomyces sensu stricto species[END_REF][START_REF] Erny | Ecological Success of a Group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii Hybrids in the Northern European Wine-Making Environment[END_REF][START_REF] Gangl | Exceptional fermentation characteristics of natural hybrids from Saccharomyces cerevisiae and S. kudriavzevii[END_REF][START_REF] González | Molecular Characterization of New Natural Hybrids of Saccharomyces cerevisiae and S. kudriavzevii in Brewing[END_REF][START_REF] González | Enological characterization of natural hybrids from Saccharomyces cerevisiae and S. kudriavzevii[END_REF][START_REF] Goldstein | Natural hybrids from Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations[END_REF][START_REF] Libkind | Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast[END_REF][START_REF] Lopandic | Genetically different wine yeasts isolated from Austrian vine-growing regions influence wine aroma differently and contain putative hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii[END_REF][START_REF] Masneuf | New Hybrids between Saccharomyces Sensu Stricto Yeast Species Found among Wine and Cider Production Strains[END_REF][START_REF] Schütz | Dynamics of the yeast strain population during spontaneous alcoholic fermentation determined by CHEF gel electrophoresis[END_REF] Popular example is allotetraploid S. pastorianus (used for the elaboration of lager-style beers) which is the result of hybridisation between S. cerevisiae parent and cryotolerant species that was recently elucidated to be S. eubayanus, and was reported to be present in natural environments in Patagonia [START_REF] Libkind | Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast[END_REF], Tibet [START_REF] Bing | Evidence for a Far East Asian origin of lager beer yeast[END_REF], North America [START_REF] Peris | Population structure and reticulate evolution of Saccharomyces eubayanus and its lager-brewing hybrids[END_REF] and New Zealand [START_REF] Gayevskiy | Saccharomyces eubayanus and Saccharomyces arboricola reside in North Island native New Zealand forests[END_REF]. Through the combination of ecological studies and comparative genomics, it was demonstrated that S. eubayanus genome sequence was 99.5% identical to the non-S. cerevisiae portion of S. pastorianus' genome and suggested related changes in sulphite and sugar metabolism that are important for lager-beer related environment [START_REF] Libkind | Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast[END_REF] (Figure 1.3). Thus, the resultant S. pastorianus is well adapted to lager beer styles which are characterised by bottom fermentation at cold temperatures. Other hybrids were also elucidated in the brewing environments, and some of the ale strains also appear to be hybrids [START_REF] Rainieri | The inheritance of mtDNA in lager brewing strains[END_REF].

Another type of hybrid between S. cerevisiae x S. kudriavzevii was elucidated for Trappist beer isolates [START_REF] González | Molecular Characterization of New Natural Hybrids of Saccharomyces cerevisiae and S. kudriavzevii in Brewing[END_REF] (Table 1.2). Hybrids resulting from the latter combination of parent species (S. cerevisiae x S. kurdiavzevii) were also isolated from wine-related environments [START_REF] Belloch | Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex[END_REF][START_REF] Goldstein | Natural hybrids from Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations[END_REF][START_REF] Lopandic | Genetically different wine yeasts isolated from Austrian vine-growing regions influence wine aroma differently and contain putative hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii[END_REF] where they were well adapted to low-temperature fermentation [START_REF] Belloch | Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex[END_REF] and were associated with interesting ester production profile [START_REF] Lopandic | Genetically different wine yeasts isolated from Austrian vine-growing regions influence wine aroma differently and contain putative hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii[END_REF]. In wine, hybrids between S. cerevisiae x S. bayanus are also encountered [START_REF] Belloch | Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex[END_REF][START_REF] Goldstein | Natural hybrids from Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations[END_REF][START_REF] Lopandic | Genetically different wine yeasts isolated from Austrian vine-growing regions influence wine aroma differently and contain putative hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii[END_REF][START_REF] Masneuf | New Hybrids between Saccharomyces Sensu Stricto Yeast Species Found among Wine and Cider Production Strains[END_REF]. Strikingly, triple hybrids between the species S. bayanus x S. kudriavzevii x S. cerevisiae were also described in both cider [START_REF] Masneuf | New Hybrids between Saccharomyces Sensu Stricto Yeast Species Found among Wine and Cider Production Strains[END_REF], and wine [START_REF] Goldstein | Natural hybrids from Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations[END_REF]. Those cases taken together suggested that establishment of hybrid populations is a common phenomenon among Saccharomyces yeast related to beer, wine, and cider fermentations. Interestingly, S. bayanus itself is considered a hybrid species with contributions from S. uvarum, S. eubayanus and S. cerevisiae [START_REF] Libkind | Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast[END_REF][START_REF] Nguyen | Molecular typing demonstrates homogeneity of Saccharomyces uvarum strains and reveals the existence of hybrids between S. uvarum and S. cerevisiae, including the S. bayanus type strain CBS 380[END_REF]. Furthermore, even inter-family hybrid between Hanseniaspora vinae and S. cerevisiae [START_REF] Cappello | Molecular and physiological characteristics of a grape yeast strain containing atypical genetic material[END_REF] , and Zygosaccharomyces bailii and S. cerevisiae [START_REF] Novo | Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118[END_REF] were formed through horizontal gene transfer in grape environment. The evolutionary success of hybrids originates in bringing together characteristics of two (or more) divergent species, thus leading to beneficial combination of metabolic, morphological, and genetic features, which would allow survival in specific conditions and occupation of related environments.

Successful polyploid state and hybridisation related to industrial fermentation environments is not limited to the Saccharomyces genus. An example is the species Brettanomyces bruxellensis, for which triploids resulting from least two independent hybridisation events were reported (Borneman et al., 2014). Allopolyploid strains of this wine-spoilage species happen to correspond to a highly disseminated genotype among wine B. bruxellensis isolates from Australia, representing 92% of the total population (Curtin et al., 2007). For this species, the putative advantages of the allopolyploid state are still to be elucidated.

Brettanomyces bruxellensis

History of the species Brettanomyces bruxellensis

Brettanomyces bruxellensis is a yeast species isolated from various fermented beverages and is often associated with its wine spoilage capacity and contribution to some specialty beer aroma profile. The etymology Brettanomyces comes from the Greek words Brettano [British brewer] [START_REF] Libkind | Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast[END_REF] A model of the formation of S. pastorianus and the hybrid strains of S. bayanus. First, wild S. eubayanus and ale-type S. cerevisiae hybridized to form an allotetraploid that gave rise to S. pastorianus. Second, domestication imposed strong selective pressure for strains with the most desirable brewing properties. Third, in the brewing vats with high densities of S. pastorianus, cell lysis releases large DNA fragments that occasionally transform, fourth, contaminating wild strains of S. eubayanus because of the lack of pure culture techniques. Fifth, multiple hybridization events with wild strains of S. uvarum gave rise to CBS 380T and NBRC 1948. This model does not exclude prior or parallel involvement of S. uvarum in brewing or contamination.

and Myces [fungus]. This name appeared for the first time in scientific literature in the year 1904 [START_REF] Claussen | On a Method for the Application of Hansen's Pure Yeast System in the Manufacturing of Well-Conditioned English Stock Beers[END_REF]. It was Claussen, who first used this name to describe the species, which he attributed to the Torula genus and considered an essential contributor to typical aroma profile of English Ales [START_REF] Claussen | On a Method for the Application of Hansen's Pure Yeast System in the Manufacturing of Well-Conditioned English Stock Beers[END_REF] (and thus, the word "Brettano" in the name that he has given to the species). The industrial importance of the species was underscored since its first description, as B. bruxellensis was the first microorganism ever to be patented (UK patent number GB190328184). Indeed, in his work Claussen insisted that "…the action of Brettanomyces is absolutely necessary to bring English stock beers into proper cask and bottle condition, and to impart to them that peculiar and remarkably fine flavour which in a great measure determines their value." [START_REF] Claussen | On a Method for the Application of Hansen's Pure Yeast System in the Manufacturing of Well-Conditioned English Stock Beers[END_REF]. Actually, following Claussen's report, other scientists declared that they had previously isolated yeast with similar morphology and aromatic characteristics in Kalinkin brewery in Russia and Guinness' Chemist Laboratory in Ireland in the years 1889 and 1899 respectively (see [START_REF] Gilliland | Brettanomyces. I. Occurrence, Characteristics, and Effects on Beer Flavour[END_REF]). Further, in 1921 Kufferath and Van Laer isolated Brettanomyces from Belgian Lambic beer and named it Brettanomyces bruxellensis (Kluyver and Custers, 1921). Lambic is the result of complex spontaneous open-tank fermentation which lasts for one to three years. During that time, multiple families, genera and strains of microorganisms act sequentially to contribute to the peculiar organoleptic characteristics of the final beverage. Indeed, the most characteristic property of Lambic beer is its aroma profile and mouthfeel that are believed to be directly related to the microorganisms involved in the fermentation, with B. bruxellensis being detected at the end of the process [START_REF] Spitaels | The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer[END_REF]. Later on, B. bruxellensis was also isolated from wine [START_REF] Cocolin | Molecular detection and identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in spoiled wines[END_REF]Curtin et al., 2007;[START_REF] Esteve-Zarzoso | Yeast Population Dynamics during the Fermentation and Biological Aging of Sherry Wines[END_REF][START_REF] Krumbholz | Pilot scale repeated fed-batch fermentation processes of the wine yeast Dekkera bruxellensis for mass production of resveratrol from Polygonum cuspidatum[END_REF][START_REF] Peynaud | Sur les brettanomyces isolés de raisins et de vins[END_REF][START_REF] Walt | The wine yeasts of the Cape[END_REF][START_REF] Wright | Brettanomyces in the New Zealand wine industry[END_REF]. There, this microorganism was described as a spoilage factor related to high acidity and unpleasant aromas [START_REF] Peynaud | Sur les brettanomyces isolés de raisins et de vins[END_REF]). In the 1990s, B. bruxellensis was shown to be responsible for the production of ethyl-phenols in wine [START_REF] Heresztyn | Metabolism of volatile phenolic compounds from hydroxycinnamic acids byBrettanomyces yeast[END_REF][START_REF] Chatonnet | Incidence des conditions de fermentation et d'élevage des vins blancs secs en barriques sur leur composition en substances cédées par le bois de chêne[END_REF].

Those molecules are associated to aromatic descriptors like horse leather, barnyard, and medicinal [START_REF] Chatonnet | Incidence des conditions de fermentation et d'élevage des vins blancs secs en barriques sur leur composition en substances cédées par le bois de chêne[END_REF]. This characteristic is the main property that defines B. bruxellensis as wine spoilage yeast. Additionally, it is also associated to mousiness and high acidity [START_REF] Grbin | Mousy off-flavour production in grape juice and wine by Dekkera and Brettanomyces yeasts[END_REF][START_REF] Peynaud | Sur les brettanomyces isolés de raisins et de vins[END_REF]. Intense research on the species B. bruxellensis related to wine followed after the 90s (possibly due to the fact that it was demonstrated to be involved in ethyl phenols production) with rising interest over the last 15 years (the role of B. bruxellensis in wine will be detailed in the next sections). Over the decades, Brettanomyces genus enlarged and other species were included and excluded from the yeast taxonomy books [START_REF] Sam Crauwels | Brettanomyces Bruxellensis, Essential Contributor in Spontaneous Beer Fermentations Providing Novel Opportunities for the Brewing Industry[END_REF][START_REF] Steensels | Brettanomyces yeasts -From spoilage organisms to valuable contributors to industrial fermentations[END_REF]. Up to date, there are five species -B. bruxellensis, B. anomalus, B. custerianus, B. naardenensis, and B. nanus. The name Dekkera bruxellensis was also introduced for the teleomorph form of B. bruxellensis following the observation of spore formation reported by Walt and Kerken back in 1960. Therefore the designation Brettanomyces/Dekkera bruxellensis was often used in scientific publications. However, to our knowledge no further prove of sporulation was reported. This, combined with the fact that by the rules of the Melbourne code species should be designated with only one valid name, as well as the tendency to end dual nomenclature for fungi [START_REF] Hibbett | Fungal systematics: is a new age of enlightenment at hand?[END_REF] leads to the prioritisation of the name Brettanomyces bruxellensis for the species over the last years, even if Dekkera is still used by some authors. Interestingly, microbiome descriptive studies on multiple fermented food and beverages highlighted presence of B. bruxellensis. Isolates belonging to the species were found on grapes [START_REF] Renouf | Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries[END_REF], in cider [START_REF] Coton | Yeast ecology in French cider and black olive natural fermentations[END_REF][START_REF] Morrissey | The role of indigenous yeasts in traditional Irish cider fermentations[END_REF], kombucha tea [START_REF] Coton | Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods[END_REF][START_REF] Teoh | Yeast ecology of Kombucha fermentation[END_REF], kefir [START_REF] Laureys | Microbial Species Diversity, Community Dynamics, and Metabolite Kinetics of Water Kefir Fermentation[END_REF], olives [START_REF] Coton | Yeast ecology in French cider and black olive natural fermentations[END_REF], bioethanol production plants [START_REF] Basílio | Detection and identification of wild yeast contaminants of the industrial fuel ethanol fermentation process[END_REF][START_REF] Beckner | Microbial contamination of fuel ethanol fermentations[END_REF][START_REF] De Souza Liberal | Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation[END_REF][START_REF] Passoth | Dekkera bruxellensis and Lactobacillus vini Form a Stable Ethanol-Producing Consortium in a Commercial Alcohol Production Process[END_REF][START_REF] Souza | The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation[END_REF], agave fermentation for tequila production [START_REF] Lachance | Yeast communities in a natural tequila fermentation[END_REF][START_REF] Lappe-Oliveras | Yeasts associated with the production of Mexican alcoholic nondistilled and distilled Agave beverages[END_REF], soft drinks [START_REF] Deak | Modified indirect conductimetric technique for detecting low populations of yeasts in beverage concentrates and carbonated beverages[END_REF][START_REF] Put | Heat resistance studies on yeast spp. causing spoilage in soft drinks[END_REF][START_REF] Yarrow | Brettanomyces abstinens sp.n[END_REF], sourdough [START_REF] Hammes | Microbial ecology of cereal fermentations[END_REF][START_REF] Meroth | Identification and Population Dynamics of Yeasts in Sourdough Fermentation Processes by PCR-Denaturing Gradient Gel Electrophoresis[END_REF]), yoghourt (Kosse et al., 1997), etc. Particularly interesting case is the one of B. bruxellensis occurrence in bioethanol production plants [START_REF] Basílio | Detection and identification of wild yeast contaminants of the industrial fuel ethanol fermentation process[END_REF][START_REF] Beckner | Microbial contamination of fuel ethanol fermentations[END_REF][START_REF] De Souza Liberal | Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation[END_REF][START_REF] Passoth | Dekkera bruxellensis and Lactobacillus vini Form a Stable Ethanol-Producing Consortium in a Commercial Alcohol Production Process[END_REF][START_REF] Souza | The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation[END_REF]. Bioethanol production through microorganism fermentation allows ethanol synthesis from organic matter, thus presenting an eco-friendly process for the production of ethanol which can be used as fuel. This process involves highly controlled large-scale fermentations mainly held by selected S. cerevisiae strains. The fermentation conditions are characterised by low pH, presence of inhibitor factors, abundance of complex sugars, etc. Even in those harsh conditions, contaminant B. bruxellensis strains were isolated [START_REF] Basílio | Detection and identification of wild yeast contaminants of the industrial fuel ethanol fermentation process[END_REF][START_REF] Beckner | Microbial contamination of fuel ethanol fermentations[END_REF][START_REF] De Souza Liberal | Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation[END_REF][START_REF] Souza | The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation[END_REF]. Furthermore, they were able to displace S. cerevisiae population during controlled fermentation [START_REF] Souza | The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation[END_REF]. Thus, even if mainly considered spoilage yeast in bioethanol production [START_REF] Basílio | Detection and identification of wild yeast contaminants of the industrial fuel ethanol fermentation process[END_REF][START_REF] Beckner | Microbial contamination of fuel ethanol fermentations[END_REF][START_REF] De Souza Liberal | Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation[END_REF], B. bruxellensis is also seen as potential microorganism for the fermentation of molasses for bioethanol production [START_REF] Blomqvist | Dekkera bruxellensis-spoilage yeast with biotechnological potential, and a model for yeast evolution, physiology and competitiveness[END_REF][START_REF] Passoth | Dekkera bruxellensis and Lactobacillus vini Form a Stable Ethanol-Producing Consortium in a Commercial Alcohol Production Process[END_REF]. Still in the industrial context, B. bruxellensis was suggested as the most efficient organism among five other species for the synthesis of resveratrol (Kuo et al., 2017). Those aspects highlight the industrial impact of the yeast B. bruxellensis.

Genetics of Brettanomyces bruxellensis

Since the first pioneer scientific articles on Brettanomyces bruxellensis, the words "diversity" and "variability" often accompanied the description of the species. Indeed, still in the 1960s and specifically in the wine context, Peynaud and Domercq spoke about the variability of different strains in means of sugar consumption [START_REF] Peynaud | Sur les brettanomyces isolés de raisins et de vins[END_REF]. Further, other phenotypic aspects were highlighted as variable among strains, such as growth capacity [START_REF] Agnolucci | Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines[END_REF][START_REF] Barbin | Diversity in spoilage yeast dekkera/Brettanomyces bruxellensis isolated from French red wine. Assessment during fermentation of synthetic wine medium[END_REF][START_REF] Fugelsang | Population Dynamics and Effects of Brettanomyces bruxellensis Strains on Pinot noir (Vitis vinifera L .) Wines[END_REF][START_REF] Oelofse | Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production[END_REF][START_REF] Romano | Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine[END_REF]Vigentini et al., 2008a), sugar metabolism (Conterno et al., 2006;[START_REF] Crauwels | Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains[END_REF][START_REF] Crauwels | Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains[END_REF][START_REF] Galafassi | Dekkera/Brettanomyces yeasts for ethanol production from renewable sources under oxygen-limited and low-pH conditions[END_REF], nitrogen source utilisation (Borneman et al., 2014;[START_REF] Crauwels | Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains[END_REF], ethyl phenols production [START_REF] Agnolucci | Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines[END_REF]Conterno et al., 2006;[START_REF] Crauwels | Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains[END_REF][START_REF] Toro | Intraspecific biodiversity and "spoilage potential" of Brettanomyces bruxellensis in Apulian wines[END_REF][START_REF] Martorell | Molecular typing of the yeast species Dekkera bruxellensis and Pichia guilliermondii recovered from wine related sources[END_REF][START_REF] Renouf | Evidence for differences between B. bruxellensis strains originating from an enological environment[END_REF]Vigentini et al., 2008a), behaviour in viable but not cultivable state [START_REF] Capozzi | Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach[END_REF]Longin et al., 2016a), and response to abiotic factors like temperature [START_REF] Barata | The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine[END_REF]Conterno et al., 2006), pH [START_REF] Blomqvist | Fermentation characteristics of Dekkera bruxellensis strains[END_REF]Conterno et al., 2006), oxygen availability [START_REF] Capusoni | Effects of oxygen availability on acetic acid tolerance and intracellular pH in Dekkera bruxellensis[END_REF]Du Toit et al., 2005a;[START_REF] Uscanga | Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production[END_REF] and sulfur dioxide (SO 2 ) [START_REF] Agnolucci | Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis[END_REF]A. Barata et al., 2008;Conterno et al., 2006;[START_REF] Crauwels | Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains[END_REF]C. Curtin et al., 2012b;[START_REF] Vigentini | Assessment of the Brettanomyces bruxellensis metabolome during sulphur dioxide exposure[END_REF]. To seek explanation for these variations, different scientific teams have explored the genetic diversity of the species. Despite several studies on the genetic diversity of this species using fingerprinting techniques such as Random Amplified Polymorphism DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), pulsed field electrophoresis (REA-PFGE), and mtDNA restriction analysis [START_REF] Agnolucci | Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines[END_REF][START_REF] Campolongo | Prevalence and Biodiversity of Brettanomyces bruxellensis in Wine from Northwestern Italy[END_REF]Conterno et al., 2006;Curtin et al., 2012a;Curtin et al., 2007;[START_REF] Toro | Intraspecific biodiversity and "spoilage potential" of Brettanomyces bruxellensis in Apulian wines[END_REF][START_REF] Joseph | Production of Volatile Compounds by Wine Strains of Brettanomyces bruxellensis Grown in the Presence of Different Precursor Substrates[END_REF][START_REF] Martorell | Molecular typing of the yeast species Dekkera bruxellensis and Pichia guilliermondii recovered from wine related sources[END_REF][START_REF] Oelofse | Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production[END_REF][START_REF] Vigentini | Intraspecific variations of Dekkera/Brettanomyces bruxellensis genome studied by capillary electrophoresis separation of the intron splice site profiles[END_REF], our understanding of the B. bruxellensis global population structure and the factors that drive it remains limited. Some of those genotypic studies suggested a correlation with geographical origin of the isolates (Conterno et al., 2006). Others highlighted correlations between genotypic profile and phenotypic characteristics (e.g. SO 2 tolerance and ethyl phenols production (Conterno et al., 2006)). For example, Conterno et al., 2006 highlighted a particularly "dangerous" genotypic group correlated with high ethyl phenol production and SO 2 tolerance (Conterno et al., 2006;Curtin et al., 2012a). Meanwhile, other scientific teams were also interested in the genetic specificities of B. bruxellensis. [START_REF] Woolfit | Genome Survey Sequencing of the Wine Spoilage Yeast Dekkera (Brettanomyces) bruxellensis[END_REF] made a first attempt to obtain partial genome sequence of the strain CBS 2499, and highlighted a proteome enriched in transporters and genes involved in nitrogen and lipid metabolism [START_REF] Woolfit | Genome Survey Sequencing of the Wine Spoilage Yeast Dekkera (Brettanomyces) bruxellensis[END_REF]. In this work, it was suggested that those characteristics could be related to the environment from which the isolate was obtained (namely wine) and could confer survival in this medium characterised by low nutrient availability and high ethanol content. In this first genomic work on B. bruxellensis, it was suggested that this strain was haploid following the observation that compared to Saccharomyces cerevisiae there was a small number of lineagespecific duplicated genes [START_REF] Woolfit | Genome Survey Sequencing of the Wine Spoilage Yeast Dekkera (Brettanomyces) bruxellensis[END_REF]. Therefore, at this stage, it was assumed that B. bruxellensis was a haploid species. However, very quickly this assumption was changed by another study which was published two years later by the same scientific team. Indeed, [START_REF] Hellborg | Complex Nature of the Genome in a Wine Spoilage Yeast, Dekkera bruxellensis[END_REF] discussed the high karyotype variability among 30 B. bruxellensis strains with different geographical origin [START_REF] Hellborg | Complex Nature of the Genome in a Wine Spoilage Yeast, Dekkera bruxellensis[END_REF]. By applying PFGE analysis (Pulsed Field Gel Electrophoresis), the authors underscored a remarkable karyotype variability (ranging from 4 to 9 chromosomes), a characteristics that is not common for eukaryotes [START_REF] Hellborg | Complex Nature of the Genome in a Wine Spoilage Yeast, Dekkera bruxellensis[END_REF]. Thus, the simple haploid organisation was excluded, and new hypothesis of polyploid state of the species arose [START_REF] Hellborg | Complex Nature of the Genome in a Wine Spoilage Yeast, Dekkera bruxellensis[END_REF]. Further confirmation of the polyploid state of the species was provided by the partial sequence analysis of five genes that showed heterozygosity and presence of different haplotypes for the same strain [START_REF] Hellborg | Complex Nature of the Genome in a Wine Spoilage Yeast, Dekkera bruxellensis[END_REF]. Those haplotypes were virtually re-grouped by their sequence similarities, and it was noticed that there was more resemblances between haplotypes from the same group but different strains, than in between haplotypes from the same strain [START_REF] Hellborg | Complex Nature of the Genome in a Wine Spoilage Yeast, Dekkera bruxellensis[END_REF]. This led to the hypothesis that hybridisation events occurred during the evolutionary history of the species [START_REF] Hellborg | Complex Nature of the Genome in a Wine Spoilage Yeast, Dekkera bruxellensis[END_REF]. This information, taken together with the high karyotype variability led to the assumption that the species was possibly asexual. Based on those findings, two major hypotheses were made on the evolutionary history of the species: i) hybridisation with closely related species led to asexuality of the progenitors and mutations accumulation in the resultant descendant population, ii) a diploid progenitor existed, that became asexual and accumulated mutations that led to high intraspecies diversity. Hints on the plausibility of those hypotheses were provided by the whole genome sequencing of few B. bruxellensis isolates that was published during the following years [START_REF] Piškur | The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties[END_REF]. Actually, up to date, the sequences of ten isolates from different fermentation and geographic regions are available (Borneman et al., 2014;[START_REF] Crauwels | Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing[END_REF]Curtin et al., 2012;[START_REF] Fournier | High-quality de novo genome assembly of the Dekkera bruxellensis UMY321 yeast isolate using Nanopore MinION sequencing[END_REF][START_REF] Olsen | De novo assembly of Dekkera bruxellensis: a multi technology approach using short and long-read sequencing and optical mapping[END_REF][START_REF] Piškur | The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties[END_REF][START_REF] Valdes | Draft genome sequence and transcriptome analysis of the wine spoilage yeast Dekkera bruxellensis LAMAP2480 provides insights into genetic diversity, metabolism and survival[END_REF]. Intriguingly, the first genomic sequences of strains belonging to the species B. bruxellensis highlighted diploid [START_REF] Piškur | The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties[END_REF] and triploid (Curtin et al., 2012) strains. Also, B. bruxellensis was actually genetically distant from other food-related yeast species (Figure 1.4). Indeed, previous work highlighted that B. bruxellensis and S. cerevisie (baker's yeast) evolved separately and their lineages separated 200 million years ago [START_REF] Rozpędowska | Parallel evolution of the make-accumulateconsume strategy in Saccharomyces and Dekkera yeasts[END_REF] (or 100 million years before the whole genome duplication [START_REF] Woolfit | Genome Survey Sequencing of the Wine Spoilage Yeast Dekkera (Brettanomyces) bruxellensis[END_REF] that occurred in S. cerevisiae lineage). However, both lineages developed, independently, similar survival strategies based on make-accumulate-consume metabolism. For S. cerevisiae, whole genome duplication (WGD) led to duplication of genes related to the adaptation of high-sugar environment possibly in response to the environmental changes related to the concomitant abundance of flowering plants. The fact that B. bruxellensis has gained the same adaptation mechanism based on make-accumulate-consume strategy without WGD makes of those two species an excellent model for convergent evolution.

Indeed, the genome sequence of the strain CBS 2499 provided insights to the mechanisms of its high ethanol production and tolerance which lays in the lineage-specific duplication of ADH gene-family [START_REF] Piškur | The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties[END_REF] (a trait also typical of S. cerevisiae but which evolved independently in B. bruxellensis). Those genes are also related to the synthesis of higher alcohols and aromatic esters precursors and it was suggested that this could partially be the cause of B. bruxellensis' peculiar aromatic profile. Another striking feature of B. bruxellensis' [START_REF] Curtin | Harnessing improved understanding of Brettanomyces bruxellensis biology to mitigate the risk of wine spoilage[END_REF] genome was revealed by the de-novo sequence assembly of the wine strain AWRI1499, which presented a triploid genome consisting of moderately heterozygous diploid genome and associated haploid genome from another closely related species (Curtin et al., 2012).

Strikingly, the triploid strain was a representative of a highly dispersed and SO 2 tolerant genotype among Australian isolates. This led to the first hypothesis that this polyploid state could actually confer selective advantage in winemaking conditions to strains of this group (Borneman et al., 2014). It seems that this phenomenon is not only present in Australia, as strains with similar microsatellite profile were isolated from wines from France and South Africa (Albertin et al., 2014b). In the next years, few other genomes became available to the scientific community (Borneman et al., 2014;[START_REF] Crauwels | Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing[END_REF][START_REF] Fournier | High-quality de novo genome assembly of the Dekkera bruxellensis UMY321 yeast isolate using Nanopore MinION sequencing[END_REF][START_REF] Olsen | De novo assembly of Dekkera bruxellensis: a multi technology approach using short and long-read sequencing and optical mapping[END_REF][START_REF] Valdes | Draft genome sequence and transcriptome analysis of the wine spoilage yeast Dekkera bruxellensis LAMAP2480 provides insights into genetic diversity, metabolism and survival[END_REF]. Comparative genomics study revealed that there are at least two independent hybridisation events leading to two divergent triploid populations (Borneman et al., 2014) (Figure 1.5). This scenario is similar to the one of Saccharomyces genus where interspecific hybrids are often formed and combine characteristics of two parent species to confront environmental changes. Indeed, the parallel between those two lineages was underscored at the time of the first partial genome analysis published by Hellborg and Piškur [START_REF] Hellborg | Complex Nature of the Genome in a Wine Spoilage Yeast, Dekkera bruxellensis[END_REF]. Eventually, it was even suggested that it is possible that B. bruxellensis, rather than being one species, is actually a consortium of species [START_REF] Curtin | Harnessing improved understanding of Brettanomyces bruxellensis biology to mitigate the risk of wine spoilage[END_REF]. However, this hypothesis remains to be discussed among the scientific community.

Another sequence of a strain isolated from beer became available in 2014 [START_REF] Crauwels | Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing[END_REF]. This diploid beer strain was compared with the two available sequences of diploid wine strain (CBS 2499) and triploid wine strain (AWRI1499). Analysis of SNPs (Single Nucleotide Polymorphisms), CNVs (Copy Number Variations) and indels (insertions and/or deletions of genomic fragments) highlighted at least two regions that were present in the wine strains but missing in the beer isolate [START_REF] Crauwels | Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing[END_REF]. Those regions were related to nitrogen and carbon metabolism and, in this work, it was suggested that they could be useful for wine-type environment. Hypothetically, presence of those regions could be associated with fitness cost in beer environment and were therefore eliminated by selective pressure in the latter [START_REF] Crauwels | Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing[END_REF]. In this study, another intriguing observation was made. LSU rRNA analysis allowed the establishment of genetic relations between strains and it was interesting to notice that soft drinks isolates clustered separately from wine and beer strains [START_REF] Crauwels | Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing[END_REF]. The authors therefore suggested that this was a hint for possible niche adaptation among the species B. bruxellensis [START_REF] Crauwels | Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing[END_REF]. Indeed, this hypothesis was partially supported by phenotypic test performed with eight strains from wine, beer, and soft drink grown in different types of beverages [START_REF] Crauwels | Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains[END_REF]. The results of this study reported that only wine bruxellensis strains is predicted to contain a conserved diploid set of chromosomes. In addition, AWRI1499 and AWRI1608 are predicted to both contain a third full set of chromosomes that have been inherited from more distantly related strains or a closely related species that is unique to each strain (Dekkera x and Dekkera y). From (Borneman et al., 2014) strains were able to grow in wine medium suggesting niche adaptation for those isolates [START_REF] Crauwels | Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains[END_REF].

Recently, another remarkable feature of B. bruxellensis' genome was elucidated. Apart from the differences in ploidy level between strains (Borneman et al., 2014), it was also demonstrated that the diploid strain CBS 2499 possesses "atypical" centromere loci that are prone to induce ploidy changes [START_REF] Ishchuk | Novel Centromeric Loci of the Wine and Beer Yeast Dekkera bruxellensis CEN1 and CEN2[END_REF]. Insertions of the centromeric regions CEN1 and CEN2 led to ploidy shifts and phenotypic switchdevelopment of fluffy colonies with 3 times more biofilm production [START_REF] Ishchuk | Novel Centromeric Loci of the Wine and Beer Yeast Dekkera bruxellensis CEN1 and CEN2[END_REF]. This led to the hypothesis that this genetic feature can be related to adaptation to low-nutrient environment [START_REF] Ishchuk | Novel Centromeric Loci of the Wine and Beer Yeast Dekkera bruxellensis CEN1 and CEN2[END_REF] (such as wine). Furthermore, from a fundamental point of view this study demonstrated for the first time the presence of miniature inverted repeat transposable element (MITE)genetic feature that is encountered among animals and plant species but was described for the first time in yeast. This characteristic enhances the importance of B. bruxellensis as model yeast species for the study of genome plasticity in eukaryote organisms. The availability of high-quality sequences that is on the path of intense development [START_REF] Fournier | High-quality de novo genome assembly of the Dekkera bruxellensis UMY321 yeast isolate using Nanopore MinION sequencing[END_REF][START_REF] Olsen | De novo assembly of Dekkera bruxellensis: a multi technology approach using short and long-read sequencing and optical mapping[END_REF] will pave the way for future population genomics studies that would possibly elucidate more remarkable adaptation features of the species B. bruxellensis.

At present, there are some answers to the questions that were raised by the first genomic study performed back in 2007 [START_REF] Woolfit | Genome Survey Sequencing of the Wine Spoilage Yeast Dekkera (Brettanomyces) bruxellensis[END_REF]: i) the remarkable karyotype variation could be explained by the incidence of polyploid strains and high genome plasticity related to specific centromeric loci structure leading to ploidy shifts, ii) hybridisation events were highlighted for the species and this gives indications for the putative evolutionary strategy of the species.

However, the incidence of polyploid state remains to be elucidated among strains from various substrates and geographic origins, and the sexuality of the species remains an open question.

Occurrence of Brettanomyces bruxellensis in wine

Brettanomyces bruxellensis was first isolated from winemaking-related environment in the 1930s from a French wine [START_REF] Krumbholz | Pilot scale repeated fed-batch fermentation processes of the wine yeast Dekkera bruxellensis for mass production of resveratrol from Polygonum cuspidatum[END_REF]. In this work, the species was defined as Mycotorula intermedia but it was later re-classified as B. bruxellensis [START_REF] Peynaud | Sur les brettanomyces isolés de raisins et de vins[END_REF]. This case is a good example of change of species name that has occurred multiple times for Brettanomyces species (see [START_REF] Steensels | Brettanomyces yeasts -From spoilage organisms to valuable contributors to industrial fermentations[END_REF]. Here, the name Brettanomyces bruxellensis will be used for all species that were firstly published with other names but are re-defined as B. bruxellensis to date. Further report of B. bruxellensis isolation from grape must in France dates back to 1956, when Peynaud et Domercq analysed different B. bruxellensis strains from Bordeaux region for their morphology, sugar consumption, and impact on wine organoleptic properties [START_REF] Peynaud | Sur les brettanomyces isolés de raisins et de vins[END_REF]. These authors also mentioned that previous B. bruxellensis isolations occurred in the 1950s from highly acid wine from Italy, from Jura wine associated with yeast film developed on the surface, and a sparkling wine from Germany (see [START_REF] Peynaud | Sur les brettanomyces isolés de raisins et de vins[END_REF]. They described the cells as slow growing, elongated, and not forming spores. They were associated with peculiar aromatic profile described as fruity/aldehyde-like, but also sour, acetamide-like repugnant aroma (the latter was described as "mousiness") [START_REF] Peynaud | Sur les brettanomyces isolés de raisins et de vins[END_REF]. Four years after this work, B. bruxellensis was also described as being part of the wine yeasts of the Cape, South Africa [START_REF] Walt | The wine yeasts of the Cape[END_REF]. Later on, B. bruxellensis was treated in relation to New Zealand wine industry [START_REF] Wright | Brettanomyces in the New Zealand wine industry[END_REF], where Brett contamination was widespread for the vintage 1971, and interestingly it was of higher incidence among fortifying spirit production sites [START_REF] Wright | Brettanomyces in the New Zealand wine industry[END_REF]. Over the last decades, B. bruxellensis presence was further reported in Spain, Australia, USA, Chile, etc. [START_REF] Agnolucci | Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines[END_REF]Curtin et al., 2007;[START_REF] Ganga | Effect of wine yeast monoculture practice on the biodiversity of non-Saccharomyces yeasts[END_REF]Ibeas et al., 1996). The importance of this yeast for the winemaking industry is mostly related to the production of ethyl phenols, associated with unpleasant aromas, provoking consumers' rejection and subsequent economic loss for the producers [START_REF] Loureiro | Spoilage yeasts in the wine industry[END_REF][START_REF] Wedral | The challenge of Brettanomyces in wine[END_REF]. B. bruxellensis' spoilage potential is further enhanced by its variability, scavenger metabolism (low nutrient requirements), and tolerance to sulfur dioxide, which will be discussed in the following sections.

Impact of Brettanomyces bruxellensis on organoleptic wine qualities

In the 1990s, it was demonstrated that B. bruxellensis was able to convert hydroxycinnamic acids (HCAs) in ethyl phenols (EPs) (namely 4-ethylphenol and 4-ethylguaiacol, 4-EP and 4-EG) [START_REF] Heresztyn | Metabolism of volatile phenolic compounds from hydroxycinnamic acids byBrettanomyces yeast[END_REF][START_REF] Chatonnet | Incidence des conditions de fermentation et d'élevage des vins blancs secs en barriques sur leur composition en substances cédées par le bois de chêne[END_REF]. The latter molecules are associated to descriptors like horse sweat, medicinal, barnyard, leather (Chatonnet et al., 1995a[START_REF] Chatonnet | Incidence des conditions de fermentation et d'élevage des vins blancs secs en barriques sur leur composition en substances cédées par le bois de chêne[END_REF]. At that time, the perception threshold for those molecules was fixed at 425 µg.L -1 , and it was generally accepted that Brett character was not preferred by consumers (Chatonnet et al., 1995a[START_REF] Chatonnet | Incidence des conditions de fermentation et d'élevage des vins blancs secs en barriques sur leur composition en substances cédées par le bois de chêne[END_REF][START_REF] Curtin | Harnessing improved understanding of Brettanomyces bruxellensis biology to mitigate the risk of wine spoilage[END_REF]. It was demonstrated that those molecules can also affect wine taste associated to metallic characters [START_REF] Lattey | Consumer acceptability, sensory properties and expert quality judgements of Australian Cabernet Sauvignon and Shiraz wines[END_REF]. Interestingly, previous studies showed that Beaujolais wines with ethyl phenol concentration well above the sensory threshold (around 2000 µg.L -1 ) were preferred by consumers [START_REF] Etievant | Sensory impact of volatile phenols on red wine aroma: influence of carbonic maceration and time of storage[END_REF]. This result, however, remains and exception from the general trend, and wine with high ethyl phenols are usually not appreciated by consumers [START_REF] Curtin | Harnessing improved understanding of Brettanomyces bruxellensis biology to mitigate the risk of wine spoilage[END_REF]. Even if EPs seem to have an important role for wine perception, the role of those molecules for the cell remains unknown. For now, the main hypothesis is that 4-EP and 4-EG synthesis is involved in i) maintaining of redox balance (Du Toit et al., 2005a;[START_REF] Fugelsang | Population Dynamics and Effects of Brettanomyces bruxellensis Strains on Pinot noir (Vitis vinifera L .) Wines[END_REF][START_REF] Liti | Population genomics of domestic and wild yeasts[END_REF], ii) detoxification through hydroxycinnamic acids conversion [START_REF] Carmona | Comparative proteome analysis of Brettanomyces bruxellensis under hydroxycinnamic acid growth[END_REF][START_REF] Harris | Dekkera and Brettanomyces growth and utilisation of hydroxycinnamic acids in synthetic media[END_REF], iii) attraction of insects which act as dissemination vectors [START_REF] Dweck | Olfactory Proxy Detection of Dietary Antioxidants in Drosophila[END_REF]. Despite those hypotheses, the clear role of EPs synthesis for the cell and related population remains to be elucidated. There is also a controversy concerning the intraspecies variability for 4-EP and 4-EG production, some authors reporting that the production is the same among isolates [START_REF] Curtin | Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma[END_REF]Joseph et al., 2013) and others claiming that there is certain degree of variability (Conterno et al., 2006;Vigentini et al., 2008b). It is neither clear whether cells which are not actively proliferating are able to synthesise vinyl phenols and/or ethyl phenols [START_REF] Agnolucci | Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis[END_REF][START_REF] Serpaggi | Characterization of the "viable but nonculturable" (VBNC) state in the wine spoilage yeast Brettanomyces[END_REF] or not (André [START_REF] Barata | The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine[END_REF][START_REF] Dias | Factors affecting the production of 4-ethylphenol by the yeast Dekkera bruxellensis in enological conditions[END_REF]Longin et al., 2016a;[START_REF] Romano | Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine[END_REF]Vigentini et al., 2008b), with different studies claiming controversial observations on the subject.

Additional to ethyl phenols production, B. bruxellensis has other incidence on wine organoleptic qualities. Namely, mousiness (or "goût de souris") was firstly reported by [START_REF] Peynaud | Sur les brettanomyces isolés de raisins et de vins[END_REF]) and explored more thoroughly by [START_REF] Grbin | Mousy off-flavour production in grape juice and wine by Dekkera and Brettanomyces yeasts[END_REF].

Furthermore, isovaleric acids related to rancid aromas [START_REF] Curtin | Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma[END_REF] and high acidity [START_REF] Peynaud | Sur les brettanomyces isolés de raisins et de vins[END_REF][START_REF] Romano | Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine[END_REF]Vigentini et al., 2008b) were associated to B. bruxellensis. Interestingly, isobutyric and isovaleric were shown to have masking effect on the detection of EPs [START_REF] Romano | Sensory and analytical re-evaluation of "Brett character[END_REF]. Other authors suggest that the differences related to the latter characteristics are not perceptible if EPs are present above their sensory threshold [START_REF] Curtin | Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma[END_REF], which defines EPs as the major factor related to B. bruxellensis wine spoilage. The negative impact on the organoleptic qualities of wine is intensified by the masking effect of those molecules of the fruity aromas of wine [START_REF] Tempere | The olfactory masking effect of ethylphenols: Characterization and elucidation of its origin[END_REF].

Brettanomyces bruxellensis spoilage prevention and elimination

Prediction and prevention methods in wine

For a long time Brettanomyces bruxellensis spoilage was thought to be result of inefficient winery sanitation. Even if the correlation between wine hygiene and B. bruxellensis spoilage is still a controversy, it is generally recommended to keep good hygiene of the winery equipment.

Nowadays, different types of software allow the prediction of putative B. bruxellensis spoilage.

They often take into account wine physicochemical properties (ethanol content, pH, etc.), winemaking practices (SO 2 addition dose and frequency, filtration, etc.), and winery-related environmental factors (e.g. temperature). Thus, winemakers can evaluate a risk for B. bruxellensis spoilage (high, low, or inexistent) and can adapt winemaking practices according to the risk of spoilage.

Prevention techniques often include adapted SO 2 addition (discussed below), and in some cases control of the quantity of EP precursors [START_REF] Benito | Minimization of ethylphenol precursors in red wines via the formation of pyranoanthocyanins by selected yeasts[END_REF] (that is expected to lower the risk of EPs formation by B. bruxellensis). However, the latter technique does not lead to B. bruxellensis elimination from wine if it is already present.

Detection of Brettanomyces bruxellensis in wine

Probably the most straightforward method for Brettanomyces bruxellensis detection is to smell Brett character related to the wine. However, more precise and efficient detection methods can be used both in the winery and with the assistance of specialised laboratories.

Microscope observation is sometimes used as simple way for detecting B. bruxellensis but is of controversial reliability. Direct observation of wine sample by optical microscopy was believed to allow B. bruxellensis cells identification thanks to their specific elongated shape and the susceptibility to form pseudohyphal structures [START_REF] Peynaud | Sur les brettanomyces isolés de raisins et de vins[END_REF]. However, now it is known that B. bruxellensis cell morphology is of heterogeneous nature and depends on multiple factors. Thus, this type of observations should be interpreted with care. Also, if B. bruxellensis cells are not observed by microscope when analysing wine sample, this doesn't mean that they are not present at all for the reasons described above. In this case, it is advised to apply other detection techniques. Flow cytometry is a culture-independent method that allows quality-and quantity-analysis of resident population. Coupled with fluorescence in situ hybridisation (FCM-FISH) this technique becomes a valuable tool for the specific counting of B. bruxellensis population present in wine sample and evaluating of cell physiological state and viability (Longin et al., 2016a;[START_REF] Serpaggi | Specific identification and quantification of the spoilage microorganism Brettanomyces in wine by flow cytometry: A useful tool for winemakers[END_REF]. However, this method is difficult to apply in the winery and demands highly specialised equipment and manipulators.

"Classic" method for B. bruxellensis detection consists in plating on selective medium and enumeration of viable and cultivable population (culture-dependant method). Different types of media were developed for the selective isolation of B. bruxellensis containing antibiotics eliminating other microorganisms (moulds, bacteria, Saccharomyces, and non-Saccharomyces yeasts) and/or containing p-coumaric acid as EP precursor and which would help identification by aroma detection by the person performing the analysis. This method has the advantage to be relatively cheap, and easy to apply by untrained personnel. However, B. bruxellensis being a slow-growing organism, the response is obtained after 5-10 days and thus does not allow immediate reaction to the contamination. Another recurrent problem of culture-dependant detection of B. bruxellensis is the fact that this species can enter into a viable but non cultivable (VBNC) [START_REF] Agnolucci | Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis[END_REF][START_REF] Capozzi | Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach[END_REF]Longin et al., 2016a;[START_REF] Serpaggi | Characterization of the "viable but nonculturable" (VBNC) state in the wine spoilage yeast Brettanomyces[END_REF], and therefore cells could be present without being detected on selective medium. The spoilage potential of VBNC cells will be discussed further. Culture independent methods are possible alternative to enumeration on selective medium. These techniques are based on the amplification of B. bruxellensis species specific DNA fragments by PCR reaction. Speciesspecific PCR can be only qualitative (showing B. bruxellensis presence or absence in the sample), or quantitative (qPCR) giving an idea of the population level in the sample (Longin et al., 2016b;[START_REF] Phister | Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine[END_REF][START_REF] Tessonnière | Design and performance testing of a real-time PCR assay for sensitive and reliable direct quantification of Brettanomyces in wine[END_REF]. Another technique consists in cells detection through plasmon resonance biosensors (Manzano et al., n.d.). The advantage of those methods is mainly the time efficiencya sample can be taken and the result can be obtained in few hours. However, special equipment and trained personnel are needed for the test application. Furthermore, those techniques have the disadvantage to sometimes detect DNA of cells that are inactive or dead, possibly leading to false positives.

Elimination methods for Brettanomyces bruxellensis

When prevention methods weren't applied or were inefficient, it is possible to act against the already present B. bruxellensis population. Elimination methods can be roughly grouped in chemical and physical methods. Further, physical methods can act by directly removing the cells present in the wine, or by altering their physiological state.

The most broadly used chemical method is sulfur dioxide (SO 2 ) addition. Efficient SO 2 utilisation includes both efficient dose and frequency of SO 2 application to must and wine. The major advantage of this method is the fact that SO 2 also has other beneficial properties on wine quality as an antioxidant and antioxidasic agent (meaning that it inhibits the action of oxidation enzymes like laccase) [START_REF] Ribéreau-Gayon | Traité d'oenologie[END_REF]. Furthermore, it is a popular and cheap method that is easy to apply. However, sulfur dioxide overdose could have repercussions on wine organoleptic qualities and its concentration in final wine is a subject of regulation. Thus, it is important to determine the optimal SO 2 dose in order to obtain efficacy without interfering with wine quality. When deciding SO 2 adjustment, pH and ethanol content of the wine should be taken into account. An additional challenge is the variability among strains in means of SO 2 sensitivity. The latter two aspects will be discussed in details in further sections. Other additives used in winemaking are chitosana hydrophilic biopolymer with antimicrobial properties [START_REF] Petrova | Effectiveness of chitosan preparations against Brettanomyces bruxellensis grown in culture media and red wines[END_REF][START_REF] Taillandier | Effect of a fungal chitosan preparation on Brettanomyces bruxellensis, a wine contaminant[END_REF], and DMDC (dimethyl dicarbonate) [START_REF] Delfini | Fermentability of grape must after inhibition with dimethyl dicarbonate (DMDC)[END_REF]. Killer toxins produced by non-Saccharomyces yeast were also studied over the last few years and their effect on B. bruxellensis and possible application in winemaking remains to be investigated [START_REF] Comitini | Kluyveromyces wickerhamii killer toxin: purification and activity towards Brettanomyces/Dekkera yeasts in grape must[END_REF][START_REF] Mehlomakulu | Characterization of novel killer toxins secreted by wine-related non-Saccharomyces yeasts and their action on Brettanomyces spp[END_REF][START_REF] Mehlomakulu | Non-Saccharomyces killer toxins: Possible biocontrol agents against Brettanomyces in wine?[END_REF] Physical removal of B. bruxellensis cells can be done by filtering [START_REF] Duarte | Filter Media Comparison for the Removal of Brettanomyces bruxellensis from Wine[END_REF][START_REF] Renouf | Evidence for differences between B. bruxellensis strains originating from an enological environment[END_REF][START_REF] Umiker | Removal of Brettanomyces Bruxellensis from Red Wine Using Membrane Filtration[END_REF]. The choice of filter can be done according to the material of the membrane and its porosity -often 1 or 0.45 µm (and more rarely 0.22 µm) of diameter. This method has the advantage to be easy to apply and the possibility to be coupled with chemical techniques like SO 2 addition. However, it should be taken into account that B. bruxellensis cell size has been shown to be variable depending on the physiological state [START_REF] Serpaggi | Characterization of the "viable but nonculturable" (VBNC) state in the wine spoilage yeast Brettanomyces[END_REF]. Thus, it is possible that some filters would still allow the passage of some cells that could give rise to higher population level later on. Apart from filtration, other physical methods can be used, such as pulsed electric field [START_REF] Delsart | Impact of pulsed-electric field and high-voltage electrical discharges on red wine microbial stabilization and quality characteristics[END_REF], low electric current [START_REF] Lustrato | Inactivation of wine spoilage yeasts Dekkera bruxellensis using low electric current treatment (LEC)[END_REF], high hydrostatic pressure [START_REF] González-Arenzana | Inactivation of Brettanomyces bruxellensis by High Hydrostatic Pressure technology[END_REF], high temperature treatment [START_REF] Fabrizio | Heat inactivation of wine spoilage yeast Dekkera bruxellensis by hot water treatment[END_REF], high power ultrasonics [START_REF] Luo | Viability of common wine spoilage organisms after exposure to high power ultrasonics[END_REF], and others. All these methods have the advantage to avoid chemicals addition to wine. However, their efficacy, price, and application mode should be further studied in order to expand their use in the wine industry.

Post-spoilage curative methods

One of the biggest problems concerning B. bruxellensis is the fact that often winemakers become aware of the contamination when EPs are already produced above the olfactory detection threshold. Unfortunately, in those cases, even if the contaminant population is removed by one of the methods cited above, the Brett aroma character remains present in the wine. To address this problem, a few techniques were developed for the removal of EPs from wine. Those methods are based on reverse osmosis for the removal of EPs from wine [START_REF] Ugarte | Reduction of 4-Ethylphenol and 4-Ethylguaiacol Concentration in Red Wines Using Reverse Osmosis and Adsorption[END_REF] or addition of polymers that bind selectively to EPs [START_REF] Carrasco-Sánchez | Removal of 4-Ethylphenol and 4-Ethylguaiacol with Polyaniline-Based Compounds in Wine-Like Model Solutions and Red Wine[END_REF][START_REF] Garcia | Molecularly imprinted polymers as a tool for the study of the 4-ethylphenol metabolic pathway in red wines[END_REF][START_REF] Teixeira | Volatile phenols depletion in red wine using molecular imprinted polymers[END_REF][START_REF] Larcher | 4-Ethylphenol and 4-ethylguaiacol depletion in wine using esterified cellulose[END_REF]. Some authors even suggested that there was a post-bottling solution to the problem of EPs in wine, showing that suberin in cork closure has an EP-binding capacity [START_REF] Gallardo-Chacón | Sorption of 4-ethylphenol and 4-ethylguaiacol by suberin from cork[END_REF].

However, those methods need further validation and do not solve the problem with the contamination itself, which means that B. bruxellensis could develop in the winery during the next vintages.

To sum up, different methods for prevention, detection, and elimination of B. bruxellensis are available on the market. However, sulfur dioxide addition remains undoubtedly the most broadly used antimicrobial agent in winemaking.

Focus on sulfur dioxide use in wine

Use of sulfur dioxide in wine

Sulfur dioxide is the most broadly used antimicrobial agent in wine. Its success is related to multiple beneficial physicochemical properties when it comes to wine quality. Sulfur dioxide has not only antimicrobial, but also antioxidant, and antoxidasic effect on wine (Pascal [START_REF] Ribéreau-Gayon | Traité d'oenologie[END_REF]. The reactive molecule of SO 2 actually binds to some oxidases like tyrosinase and laccase that could otherwise be detrimental for wine quality. Despite the beneficial properties of SO 2 , its use should be done with care, as excessive SO 2 addition could have negative effect on wine aroma and colour intensity [START_REF] Bakker | Effect of sulphur dioxide and must extraction on colour, phenolic composition and sensory quality of red table wine[END_REF][START_REF] Ribéreau-Gayon | Traité d'oenologie[END_REF]. As a chemical additive, SO 2 (often added in wine as potassium metabisulphite solution or sulfur tablets that are burnt in the barrels) is a subject of regulation.

Thus, the final doses of total sulfur dioxide in wine for most European countries are 150 mg.L -1 for red wines, 200 mg.L -1 for white and rosé wines, 300 mg.L -1 for red, white, and rosé wines with more than 4 g.L -1 of reducing substances, and 400 mg.L -1 for sweet wines (OIV, Office Internationale de la Vigne et du Vin, 2015 http://www.oiv.int/public/medias/4086/e-coei-1soudio.pdf). The legislation often refers to total form of sulfur dioxide due to the variability of free and bound forms according to the physicochemical properties of the wine (described below).

The most popular shapes of sulfur dioxide addition are the burning of sulfur tablets for barrel sanitation and the addition of potassium metabisulfite solution (K 2 S 2 O 5 or PBS). In aqueous solution, sulfur dioxide is present in three different chemical species depending on the pH of the solutionbisulphite ion (HSO 3 -), molecular SO 2 .H 2 O, and sulphite ion SO 3 -(Figure 1. [START_REF] Mcdonald | The Population Genetics of Fungi: Tools and Techniques[END_REF].

The chemical equilibrium between those species is dependent on pH and at the moderately acidic pH of wine (around pH 3.5) the most abundant SO 2 form is HSO 3 -(Figure 1.6). In wine, the balance between those two forms is further complicated by the fact that SO 2 can bind different molecules present in the medium like acetaldehyde, glutathione, glucose, anthocyanins, etc. Thus, in the winemaking context, several different forms should be differentiated: i) bound form of SO 2the one comprising SO 2 linked to other molecules, ii) free SO 2the fraction of SO 2 that is not bound to other molecules, and iii) molecular SO 2 that is part of the free SO 2 and represents the fraction of free SO 2 that is in neutral SO 2 .H 2 O form.

The combination of bound and free SO 2 is referred to as total SO 2 . From a microbiological point of view, the molecular SO 2 (mSO 2 ) is the one that has antimicrobial action (Du Toit et al., 2005a;[START_REF] Macris | Transport and toxicity of sulphur dioxide in Saccharomyces cerevisiae var ellipsoideus[END_REF]. In practice, the free SO 2 can be measured via titration methods, and further the mSO 2 fraction can be deduced from the measured free SO 2 concentration according to the wine's pH, temperature, and alcohol content (mSO 2 fraction decreases with higher pH, lower ethanol content, and lower temperature). The antimicrobial action of mSO 2 is considered to occur on multiple levels. Molecular SO 2 is a small molecule with no charge that can enter the cell passively [START_REF] Stratford | Sulphur Dioxide Resistance in Saccharomyces cerevisiae and Saccharomycodes ludwigii[END_REF], or via specific membrane transporters [START_REF] Macris | Transport and toxicity of sulphur dioxide in Saccharomyces cerevisiae var ellipsoideus[END_REF][START_REF] Park | Evidence for Sulfite Proton Symport in Saccharomyces cerevisiae[END_REF][START_REF] Pilkington | Reactions of Saccharomyces cerevisiae and Zygosaccharomyces bailii to Sulphite[END_REF]. Since the first contact with the cell, it could lead to morphological and physiological changes linked to the binding to cell wall and membrane components [START_REF] Anacleto | Kinetics and activation energetics of death in Saccharomyces cerevisiae induced by sulfur dioxide[END_REF]. Another morphological effect on the cell, induced by SO 2 is cell disruption and subsequent leakage of metabolites, as well as possible binding and inactivating of the membrane ATPases leading to decrease in intracellular ATP concentration (reviewed by [START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF]. Once inside the cell, the molecule of SO 2 faces a change in pH (the intracellular pH being around 6.5). Therefore, the fraction of mSO 2 lowers, whereas HSO 3 -and SO 3 -concentrations increase (Figure 1. [START_REF] Mcdonald | The Population Genetics of Fungi: Tools and Techniques[END_REF]. Subsequently, to attain osmotic balance between different SO 2 species, more mSO 2 molecules can enter the cell from the extracellular environment. Inside the cell, bisulphite ion (HSO 3 -) can block essential metabolic pathways through binding with various cell metabolites. Among others, SO 2 can bind to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) a key enzyme of the glycolysis [START_REF] Hinze | Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite[END_REF], leading to lower ATP generation and a decrease of the energy available for the cell. Sulfur dioxide can also bind to acetaldehyde [START_REF] Rankine | Influence of yeast strain on binding of sulphur dioxide in wines, and on its formation during fermentation[END_REF], consequently blocking the synthesis of ethanol and therefore decrease of NADH regeneration rate.

Furthermore, SO 2 can bind to metabolism substrates like glucose, leading to lower nutrients available for the cell [START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF]. Finally, SO 2 has been reported to act on genetic level by causing point mutations, thus altering cell function [START_REF] Shapiro | Genetic effects of bisulfite (sulfur dioxide)[END_REF].

To sum up, SO 2 is an essential contributor quality in conventional winemaking with beneficial effects on multiple levels. Its antimicrobial activity is undisputable but complex phenomenon.

However, some yeast species have managed to develop tolerance to SO 2 . The related mechanisms have been thoroughly studied for Saccharomyces cerevisiae and will be reviewed in the following section.

Sulfur dioxide tolerance in Saccharomyces cerevisiae

Nowadays, Saccharomyces cerevisiae is often inoculated to wine must because of its high fermentation efficiency (sugar to ethanol yield), and capacity to consume all the sugars present in the must without residuals. This property is essential in winemaking because even low quantities of residual sugars can present source of nutrients for microorganisms during wine storage, and lead to subsequent wine spoilage. Thus, the capacity to tolerate SO 2 without repercussions on cell metabolism is an essential element for the selection of commercial S.

cerevisiae strains for wine production. Subsequently, this characteristic has been thoroughly studied for this species.

As described in the previous section, SO 2 acts on multiple physiological, morphological, and genetic levels. Therefore, it is expected that SO 2 tolerance would be the result of different cell properties combined together. Indeed, there are various mechanisms that have been described

in the literature for being related to SO 2 tolerance. Briefly they can be grouped in i) sulfur reduction mechanisms, ii) sulfur oxidation, iii) sulphutolysis, and iv) active efflux of SO 2 molecule (reviewed by [START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF] and represented on Figure 1.7). Reduction mechanisms for coping with high concentrations of SO 2 involve utilisation of HSO 3 -ions in the sulphur amino acid biosynthesis (SAAB) pathway. As shown on Figure 1.7, HSO 3 -ions are intermediaries in this pathway; therefore increased SAAB can be a way to utilise HSO 3 -ions and make them beneficial for the cell metabolism. SAAB pathway is downregulated by the concentration of the final products of the reaction, like methionine, and it was indeed demonstrated that higher concentration of methionine was associated to lower SO 2 resistance of yeast cells [START_REF] Aranda | Sulfur and Adenine Metabolisms Are Linked, and Both Modulate Sulfite Resistance in Wine Yeast[END_REF]. Sulfur dioxide oxidation is a mechanism that has been described in higher eukaryotes but for now has not been elucidated in yeast. Similarly, sulphitolysis of bisulphite anions through chemical reaction with the oxidised form of glutathione is a controversy and has not been clearly demonstrated for unicellular eukaryotes [START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF]. However, yeast cells can have another "trick" to cope with the excess of intracellular SO 2acetaldehyde (ethanal) synthesis. Acetaldehyde production is particularly interesting in the context of winemaking, as this molecule has strong impact on wine organoleptic qualities being associated with bruised-apple off-flavour when present at high concentrations. Acetaldehyde is strongly reactive molecule and can therefore bind SO 2 (as well as other molecules present in wine). However, demonstrating whether acetaldehyde is a response to or consequence of SO 2 intoxication, is a challenge [START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF].

The most studied (and probably most efficient) trick of the cell for coping with SO 2 intoxication is the active efflux through a specific plasma membrane pump -Ssu1p [START_REF] Park | Evidence for Sulfite Proton Symport in Saccharomyces cerevisiae[END_REF]. This mechanism was thoroughly explored over the last 15 years, and some evolutionary pathways of its acquisition were highlighted. Overall, the SSU1 gene was shown to exist in at least two alleles, one of which is (SSU1-R) related to resistance and is subject of high heterozygosity between strains [START_REF] Aa | Population structure and gene evolution in Saccharomyces cerevisiae[END_REF]. Actually, this allele was shown to be the result of specific translocation between chromosomes VIII-XVI [START_REF] Nardi | A sulphite-inducible form of the sulphite efflux gene SSU1 in a Saccharomyces cerevisiae wine yeast[END_REF][START_REF] Pérez-Ortín | Molecular characterization of a chromosomal rearrangement involved in the adaptive evolution of yeast strains[END_REF]. Furthermore, this translocation has been shown to have adaptive importance to winemaking conditions (where SO 2 is broadly used), as it was very common for wine strains and missing in S. cerevisiae strains from other industrial fermentation environments [START_REF] Pérez-Ortín | Molecular characterization of a chromosomal rearrangement involved in the adaptive evolution of yeast strains[END_REF]. Surprisingly, another translocation event of similar nature was also highlighted between chromosomes XV and XVI [START_REF] Zimmer | QTL Dissection of Lag Phase in Wine Fermentation Reveals a New Translocation Responsible for Saccharomyces cerevisiae Adaptation to Sulfite[END_REF]. Again, this translocation was specific to wine strains and showed to confer selective advantage in presence of SO 2 via shorter lag phase compared to other strains. The latter study highlighted that genome plasticity and namely translocation events are important evolutionary element for the SO 2 tolerance of S. cerevisiae wine strains.

Several authors highlighted a general cell response to SO 2 , described as viable but not cultivable (VBNC) state. VBNC is a common term used for bacteria but it was only recently that it started to be used for unicellular eukaryote organisms as well. VBNC describes a state for which cells are viable but don't grow on culture medium as a result of stress-response to environmental factor. Particular trait of VBNC is the reversibility of this state meaning that the cells are able to "resuscitate" if the stress-factor is removed from the medium. Here, the word "reversible" is important, as VBNC state is demonstrated via experimental inducing of this state but should also involve resurrection when the stress agent is removed. Indeed, it was suggested that S. cerevisiae enters into a VBNC state after SO 2 addition and was able to re-gain cultivability when mSO 2 fraction was lowered in the medium through pH increase. The existence of the VBNC state is important in oenological context as it could lead to false negatives when microbiological analysis is performed through culture-dependant methods.

Particularly, the importance of VBNC state was highlighted for B. bruxellensisa species that is characterised by variable SO 2 tolerance.

a [START_REF] Rex | Resistance of Candida species to fluconazole[END_REF], b [START_REF] Brauner | Distinguishing between resistance, tolerance and persistence to antibiotic treatment[END_REF] 1.4.5.

Sulfur dioxide tolerance in Brettanomyces bruxellensis

Generally, non-Saccharomyces yeast species are considered as SO 2 sensitive, meaning that they are not viable after SO 2 addition in winemaking conditions. However, some cases of SO 2 tolerant species have been recently discussed namely for Zygosaccharomyces balii and Brettanomyces bruxellensis (Zuehlke et al., 2013). B. bruxellensis presents a major problem for

Resistance/Tolerance

When referring to survival strategies of microorganisms in presence of SO 2 , both the terms "tolerance" and "resistance" are often used without clearly defining the meaning behind them. The same ambiguity is encountered among clinical scientific studies a , where usually, tolerance is related to survival, whereas resistance refers to the capacity to actively grow at the presence of an antibiotic (which is often associated to specific molecular mechanisms and is inherited). Typical method for defining efficient drug dose for the eradication of resistant microorganisms is MIC (minimum inhibitory concentration) a,b . However, this method is not well adapted to tolerant microorganisms, as they are able to survive (even if not actively growing) at transient drug concentrations higher than the MIC. Recently, MDK (minimum duration for killing) was suggested as quantitative indicator for tolerance b . In clinical context, those differences are important as they can lead to a misclassification of tolerant pathogens as resistant or vice versa, subsequently leading to inefficient treatment. In the context of winemaking and the use of SO 2 as antimicrobial agent in particular, these terms are even more difficult to define, as the kinetics of different forms of SO 2 is complex (Figure 1.6) and highly variable in the wine matrix. The molecular form of SO 2 (mSO 2 ) constantly varies in wine due to SO 2 additions and readjustments, wine practices related to contact of wine with oxygen, binding of SO 2 to various molecules present in wine, and other physicochemical variations. Therefore, the definition of MIC or MDK is difficult in these conditions, because they are variable according to wine characteristics and wine storage conditions. However, it is important to keep in mind that different mechanisms at the cellular and population level could be in the origin of the survival and growth of spoilage of microorganism in the presence of SO 2 . Subsequently, related microorganisms should potentially be treated with different concentration and frequencies of antimicrobial addition.

winemakers all over the world and is broadly discussed, especially over the last two decades.

Subsequently, intense research was done on those properties of B. bruxellensis that are directly related to winemaking spoilage on one hand, and survival of this yeast in winemaking conditions, on the other. Interestingly for scientists, but sadly for winemakers, B. bruxellensis' tolerance to SO 2 seems highly variable. Indeed, different studies report concentrations ranging from 0.2 to 1.1 mg.L -1 for preventing B. bruxellensis' growth [START_REF] Curtin | Harnessing improved understanding of Brettanomyces bruxellensis biology to mitigate the risk of wine spoilage[END_REF]. Further, those variations were defined as strain-dependant, and in some cases were linked to genotype. Conterno et al., 2006 highlighted that among several groups defined by 26S rDNA analysis, one was with particularly high spoilage potential because of its survival rate in presence of SO 2 and ethyl phenols (EPs) production profile. Another striking example is the study of Curtin et al., 2012a which demonstrated that the most prevalent genotype among Australian isolates (as defined by AFLP analysis), was also associated with high tolerance to SO 2 (>0.6 mg.L -1 mSO 2 ). It was further shown that a strain from this genotypic group was actually an allopolyploid (triploid to be precise) resulting from interspecific hybridisation event with a species closely related to B. bruxellensis. This led to a hypothesis for revealing the mechanisms behind B. bruxellensis' variable tolerance to SO 2 , as strains that were SO 2 sensitive had different genetic configuration. Indeed, it was suggested that the triploid state of AWRI1499 strain could be involved in the tolerance to SO 2 . Furthermore, it was recently demonstrated via microsatellite analysis, that triploid strains from the same genotypic group as AWRI1499 are also present in France and South Africa (Albertin et al., 2014b) highlighting that the occurrence of this genotype is not limited to Australia. To support the hypothesis of SO 2 tolerance for the isolates that are genetically close to AWRI1499 it would be interesting to correlate genotype and phenotype for more strains from various origins.

Overall, variability of SO 2 tolerance for the species B. bruxellensis presents a problem for winemakers, as doses which are efficient and at the same time not excessive are difficult to define. The issue is even more complex than that, as initial population level was also demonstrated to have a significant effect on SO 2 efficacy with higher initial population levels leading to a decrease of SO 2 efficacy (Longin et al., 2016a).

Interestingly [START_REF] Serpaggi | Characterization of the "viable but nonculturable" (VBNC) state in the wine spoilage yeast Brettanomyces[END_REF]. Proteomics analysis of the VBNC state was performed in the context of this study, and the difference in proteins related to glycolytic flux and redox balance was shown to be modified as a result from SO 2 exposure [START_REF] Serpaggi | Characterization of the "viable but nonculturable" (VBNC) state in the wine spoilage yeast Brettanomyces[END_REF]. The main role of redox balance-and carbon catabolismrelated genes expression associated with VBNC state in B. bruxellensis was further confirmed by transcriptomics analysis [START_REF] Capozzi | Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach[END_REF]. This approach also demonstrated a true "resuscitation" of the cells [START_REF] Capozzi | Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach[END_REF] after stress removal induced by pH increase.

The resuscitation rather than re-growth of small part of the population was supported by the observed repression of DNA replication genes during VBNC state [START_REF] Capozzi | Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach[END_REF]. This study furthermore reported the intraspecies variability among strains concerning the entry in VBNC. Whereas EP production by cells in VBNC was observed by Serpaggi et al, another study claimed the lack of EP synthesis during this state (Longin et al., 2016a). The latter also evaluated the state of the cell membrane after SO 2 exposure and highlighted increase cell membrane permeability related to this stress factor (Longin et al., 2016a).

Overall, the current knowledge on B. bruxellensis SO 2 tolerance phenomenon reveals great variability among strains. This characteristic renders the definition of the optimal and efficient SO 2 dose for B. bruxellensis spoilage prevention a challenge. Promising link between SO 2 tolerance and genotype was underscored by the screening of 41 B. bruxellensis isolates from Australia, highlighting a widely spread SO 2 tolerant genotype among the continent (Curtin et al., 2012a). The presence of this genotypic family was further shown to have specific genetic configurationtriploid state resultant from hybridisation event, and isolates from this genetic group were furthermore detected in France and South Africa (Albertin et al., 2014b). The link between genotype and phenotype is therefore a potential way to evaluate SO 2 tolerance among B. bruxellensis and merits further exploration at finer scale.

Abstract

Brettanomyces bruxellensis is a unicellular fungus of increasing industrial and scientific interest over the past 15 years. Previous studies revealed high genotypic diversity among B.

bruxellensis strains as well as strain-dependent phenotypic characteristics. Genomic assemblies revealed that some strains harbour triploid genomes and based upon prior genotyping it was inferred that a triploid population was widely dispersed across Australian wine regions. We performed an intraspecific diversity genotypic survey of 1488 B. bruxellensis isolates from 29 countries, 5 continents and 9 different fermentation niches. Using microsatellite analysis in combination with different statistical approaches, we demonstrate that the studied population is structured according to ploidy level, substrate of isolation and geographical origin of the strains, underlying the relative importance of each factor. We found that geographical origin has a different contribution to the population structure according to the substrate of origin, suggesting an anthropic influence on the spatial biodiversity of this microorganism of industrial interest. The observed clustering was correlated to variable stress response, as strains from different groups displayed variation in tolerance to the wine preservative sulfur dioxide (SO 2 ).

The potential contribution of the triploid state for adaptation to industrial fermentations and dissemination of the species B. bruxellensis is discussed.

Introduction

Grape derived wine is one of the most popular alcoholic beverages and has been produced by humans since ancient times. It is the result of grape juice fermentation by yeasts which consume the fruit sugars and mainly release ethanol and carbon dioxide. Even though microorganisms are an essential part of the winemaking process, they must cope with a very hostile and variable environment, characterised by high initial sugar content and subsequent high ethanol content, low pH, presence of antimicrobial agents, and lack of nutrients. Despite these stressful conditions, some opportunistic microorganisms manage to survive and multiply during and after alcoholic fermentation. A striking example is the wine spoilage yeast

Brettanomyces bruxellensis (teleomorph Dekkera bruxellensis) that is typically detected during wine aging but also at lower frequency during the early stages of the winemaking process (grapes and must) [START_REF] Renouf | Evidence for differences between B. bruxellensis strains originating from an enological environment[END_REF][START_REF] Renouf | Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries[END_REF].When it grows in wine, B. bruxellensis produces odorant molecules (namely volatile phenols), which are associated with unpleasant aromas described as barnyard, horse sweat, Band-aid ® (Chatonnet et al., 1995a[START_REF] Chatonnet | Incidence des conditions de fermentation et d'élevage des vins blancs secs en barriques sur leur composition en substances cédées par le bois de chêne[END_REF][START_REF] Heresztyn | Metabolism of volatile phenolic compounds from hydroxycinnamic acids byBrettanomyces yeast[END_REF]. Therefore, the presence of B. bruxellensis in wine often provokes rejection by consumers and serious economic losses for winemakers [START_REF] Wedral | The challenge of Brettanomyces in wine[END_REF].

The wider industrial relevance of this yeast is highlighted by the fact that it is isolated from various fermented beverages and products. For example, B. bruxellensis is an essential contributor to the elaboration of some specialty Belgian and American beers, which are the result of complex spontaneous fermentations performed by various genera of bacteria and yeasts [START_REF] Bokulich | Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale[END_REF][START_REF] Steensels | Brettanomyces yeasts -From spoilage organisms to valuable contributors to industrial fermentations[END_REF]. Indeed, B. bruxellensis was the first microorganism to be patented for its contribution to English 'stock' ales [START_REF] Claussen | On a Method for the Application of Hansen's Pure Yeast System in the Manufacturing of Well-Conditioned English Stock Beers[END_REF][START_REF] Claussen | On a Method for the Application of Hansen's Pure Yeast System in the Manufacturing of Well-Conditioned English Stock Beers[END_REF]. This yeast has also been isolated from other fermented beverages and food like kombucha, kefir, cider, and olives [START_REF] Coton | Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods[END_REF][START_REF] Schifferdecker | The wine and beer yeast Dekkera bruxellensis[END_REF][START_REF] Steensels | Brettanomyces yeasts -From spoilage organisms to valuable contributors to industrial fermentations[END_REF]. Interestingly, B. bruxellensis was reported to be a common contaminant in bioethanol production plants [START_REF] Passoth | Dekkera bruxellensis and Lactobacillus vini Form a Stable Ethanol-Producing Consortium in a Commercial Alcohol Production Process[END_REF][START_REF] Souza | The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation[END_REF], and under the right conditions can take the place of the industrial Saccharomyces cerevisiae strains and perform molasses fermentation [START_REF] Souza | The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation[END_REF].

The recurrent problem of B. bruxellensis in wine and its potential use for beer and bioethanol industrial fermentations has led to high and rising interest in this yeast species. Various studies highlighted great phenotypic diversity of B. bruxellensis regarding growth capacity [START_REF] Agnolucci | Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines[END_REF][START_REF] Barbin | Diversity in spoilage yeast dekkera/Brettanomyces bruxellensis isolated from French red wine. Assessment during fermentation of synthetic wine medium[END_REF][START_REF] Fugelsang | Population Dynamics and Effects of Brettanomyces bruxellensis Strains on Pinot noir (Vitis vinifera L .) Wines[END_REF][START_REF] Oelofse | Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production[END_REF][START_REF] Romano | Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine[END_REF]Vigentini et al., 2008a), sugar metabolism (Conterno et al., 2006;[START_REF] Crauwels | Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains[END_REF][START_REF] Crauwels | Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains[END_REF][START_REF] Galafassi | Dekkera/Brettanomyces yeasts for ethanol production from renewable sources under oxygen-limited and low-pH conditions[END_REF], nitrogen source utilisation (Borneman et al., 2014;[START_REF] Crauwels | Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains[END_REF], volatile phenols production [START_REF] Agnolucci | Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines[END_REF]Conterno et al., 2006;[START_REF] Crauwels | Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains[END_REF][START_REF] Toro | Intraspecific biodiversity and "spoilage potential" of Brettanomyces bruxellensis in Apulian wines[END_REF][START_REF] Martorell | Molecular typing of the yeast species Dekkera bruxellensis and Pichia guilliermondii recovered from wine related sources[END_REF][START_REF] Renouf | Evidence for differences between B. bruxellensis strains originating from an enological environment[END_REF][START_REF] Romano | Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine[END_REF], behaviour in viable but not cultivable state [START_REF] Capozzi | Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach[END_REF], and response to abiotic factors like temperature [START_REF] Barata | The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine[END_REF]Conterno et al., 2006), pH [START_REF] Blomqvist | Fermentation characteristics of Dekkera bruxellensis strains[END_REF]Conterno et al., 2006), oxygen availability [START_REF] Capusoni | Effects of oxygen availability on acetic acid tolerance and intracellular pH in Dekkera bruxellensis[END_REF]Du Toit et al., 2005a;[START_REF] Uscanga | Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production[END_REF] and sulfur dioxide (SO 2 ) [START_REF] Agnolucci | Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis[END_REF][START_REF] Barata | The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine[END_REF]Conterno et al., 2006;[START_REF] Crauwels | Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains[END_REF]Curtin et al., 2012a;[START_REF] Vigentini | Assessment of the Brettanomyces bruxellensis metabolome during sulphur dioxide exposure[END_REF].

This phenotypic variation makes it difficult to predict the spoilage potential of B. bruxellensis and is therefore a major concern for winemakers. For example, across several studies the concentration of molecular SO 2 (mSO 2 ) required to stop B. bruxellensis' growth ranged from 0.2 to 1.1 mg.L -1 [START_REF] Curtin | Harnessing improved understanding of Brettanomyces bruxellensis biology to mitigate the risk of wine spoilage[END_REF]. This observed variability was at least partly due to the use of different strains. However, only a few studies have attempted to correlate SO 2 tolerance to a genotypic profile (Conterno et al., 2006;Curtin et al., 2012a). A striking example is the study of 41 B. bruxellensis wine isolates from Australia showing that the most common genotype (92% of studied isolates) was correlated with SO 2 tolerance, thus suggesting that SO 2 usage patterns may have created a selective pressure on this population (Curtin et al., 2012a).

Despite several studies that have explored genetic diversity of this species using fingerprinting techniques such as Random Amplified Polymorphism DNA (RAPD), Amplified Fragment

Length Polymorphism (AFLP), pulsed field electrophoresis (REA-PFGE), and mtDNA restriction analysis [START_REF] Agnolucci | Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines[END_REF][START_REF] Campolongo | Prevalence and Biodiversity of Brettanomyces bruxellensis in Wine from Northwestern Italy[END_REF]Conterno et al., 2006;Curtin et al., 2012a;Curtin et al., 2007;[START_REF] Toro | Intraspecific biodiversity and "spoilage potential" of Brettanomyces bruxellensis in Apulian wines[END_REF][START_REF] Joseph | Production of Volatile Compounds by Wine Strains of Brettanomyces bruxellensis Grown in the Presence of Different Precursor Substrates[END_REF][START_REF] Martorell | Molecular typing of the yeast species Dekkera bruxellensis and Pichia guilliermondii recovered from wine related sources[END_REF][START_REF] Oelofse | Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production[END_REF][START_REF] Vigentini | Intraspecific variations of Dekkera/Brettanomyces bruxellensis genome studied by capillary electrophoresis separation of the intron splice site profiles[END_REF], our understanding of the B. bruxellensis global population structure and the factors that drive it remains limited. Several studies highlight an important intraspecific diversity of B. bruxellensis [START_REF] Agnolucci | Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines[END_REF]Conterno et al., 2006;Curtin et al., 2007;[START_REF] Vigentini | Intraspecific variations of Dekkera/Brettanomyces bruxellensis genome studied by capillary electrophoresis separation of the intron splice site profiles[END_REF] which makes the prediction of its occurrence and behaviour in industrial fermentations difficult. Further, recent genetic studies on a limited number of strains (Albertin et al., 2014b;Borneman et al., 2014;Curtin et al., 2012) have suggested that polyploidy and hybridisation may play a significant role in microevolution of the species, along with plasticity in chromosomal structure due to "untraditional" centromeres [START_REF] Ishchuk | Novel Centromeric Loci of the Wine and Beer Yeast Dekkera bruxellensis CEN1 and CEN2[END_REF]. The role of polyploidy in adaptive changes to suit environment and/or lifestyle has been observed in other organisms [START_REF] Albertin | Polyploidy in fungi: evolution after whole-genome duplication[END_REF][START_REF] Comai | The advantages and disadvantages of being polyploid[END_REF][START_REF] Selmecki | Polyploidy can drive rapid adaptation in yeast[END_REF][START_REF] Wertheimer | Ploidy dynamics and evolvability in fungi[END_REF], notably for S.

cerevisiae which shares similar fermentation niches to those occupied by B. bruxellensis.

To enhance our knowledge of the global B. bruxellensis population, here we used a recently developed microsatellite profiling method (Albertin et al., 2014b) to genotype 1488 isolates from various fermentation niches across five continents. Typing based on microsatellite markers is a rapid, reliable and discriminant genotyping approach that has been successfully used to decipher complex population structures [START_REF] Guichoux | Current trends in microsatellite genotyping[END_REF][START_REF] Vieira | Microsatellite markers: what they mean and why they are so useful[END_REF] and provide insight into the ploidy-state (Albertin et al., 2014b). The performed research work aimed to determine the population structure of a large B. bruxellensis collection and test for a link between the identified subpopulations and their adaptive ability, with a focus on tolerance to sulfur dioxide. Strain isolation from contaminated wines was performed by spreading 100 µL of wine sample on solid YPD medium containing 10 g.L -1 yeast extract (Difco Laboratories, Detroit M1), 10 g.L -1 bactopeptone (Difco Laboratories, Detroit M1), 20 g.L -1 D-glucose (Sigma-Aldrich) and 20 g.L -1 agar (Sigma-Aldrich). This medium was supplemented with antibiotics in order to limit the growth of bacteria (5 g.L -1 chloramphenicol Sigma-Aldrich), moulds (7.5 g.L -1 biphenyl, Sigma-Aldrich), and yeast of the Saccharomyces genus (50 g.L -1 cycloheximide, Sigma-Aldrich). The samples were then incubated at 30 °C for 5 to 10 days. Ten colonies were then picked randomly and analysed by PCR using the DB1/DB2 primers (Ibeas et al., 1996) (Eurofins MWG Operon, Les Ulis, France) for species identity confirmation (DNA extraction was performed as described below for the microsatellite analysis). Putative B. bruxellensis colonies were streaked and grown on selective YPD medium twice consecutively in order to insure the strain purity. Colonies that gave a positive result by PCR DB1/DB2 were stored at -80 °C in 50% YPD/glycerol medium.

Materials

Genotyping by microsatellite analysis

DNA extraction

For DNA extraction, strains were grown on YPD solid medium at 30 °C for 5 to 7 days and fresh colonies were lysed in 30 µL of 20 mM NaOH solution heated at 99 °C for 10 minutes using iCycler thermal cycler (Biorad, Hercules, CA, USA).

Microsatellite loci identification and primers design

Twelve pairs of primers were designed on the basis of the de-novo genome assembly of the triploid B. bruxellensis strain AWRI1499 ( Curtin et al., 2012) as previously described by (Albertin et al., 2014b). Four pairs of primers were added to the eight that were previously described in order to improve the discriminative power of the test and to insure its robustness (Supplementary Table S-2.1 online and in Appendix).

Microsatellites amplification

In order to reduce the time and cost of analysis, some of the PCR reactions were multiplexed as shown in the Tm column in Supplementary Table S-2.1 online and in Appendix. By this procedure the number of PCR reactions per sample was reduced from 12 to 9.

PCR reactions were performed in a final volume of 15 µL containing 1 µL of DNA extract (extraction performed as described above), 0.05 µM of forward primer, 0.5 µM of reverse primer and labelled primer (or 1 µL in the case of duplex PCR reactions), 1x Taq-&GO (MP Biomedicals, Illkirch, France). The forward primers were tailed on their 5' end with M13 sequence as described by [START_REF] Schuelke | An economic method for the fluorescent labeling of PCR fragments[END_REF]. Universal M13 primers were labelled with FAM-, HEX-, AT565-(equivalent to PET) or AT550-(equivalent to NED) fluorescent dies (Eurofins MWG Operon, Les Ulis, France). This method allows labelling of several microsatellite marker primers with the same fluorochrome marked primer (M13) instead of marking each of the 12 forward primers and thus reduces significantly the analysis cost.

Touch-down PCR was carried out using an iCycler thermal cycler (Biorad, Hercules, CA, USA). The program consisted of an initial denaturation step of 1 min at 94 °C followed by 10 cycles of 30 s at 94 °C, 30 s at Tm+10 °C (followed by a 1 °C decrease per cycle until Tm is reached) and 30 s at 72 °C, then 20 cycles of 30 s at 94 °C, 30 s at Tm and 30 s at 72 °C, and a final extension step of 2 min at 72 °C.

Amplicons were first analysed by a microchip electrophoresis system (MultiNA, Shimadzu)

and the optimal conditions for PCR amplifications were assessed. Then, the exact sizes of the amplified fragments were determined using the ABI3730 DNA analyser (Applied Biosystems) (a core facility of INRA, UMR Biodiversité Gènes et Ecosystèmes, PlateForme Génomique, 33610 Cestas, France). Prior to the ABI3730 analysis, PCR amplicons were diluted (1800-fold for FAM, 600-fold for HEX, 1200-fold for AT565 and 1800-fold for AT550) and multiplexed in formamide. The LIZ 600 molecular marker (ABI GeneScan 600 LIZ Size Standard, Applied Biosystems) was diluted 100-fold and added to each multiplex. Before loading, diluted amplicons were heated 4 min at 94 °C. Allele size was recorded manually using GeneMarker Demo software V2.2.0 (SoftGenetics).

Microsatellite data analysis

To investigate the genetic relationships between strains, the microsatellite dataset was analysed using the Poppr package (Kamvar et al., 2014) in R (3.1.3 version, https://www.r-project.org).

A dendrogram was established using Bruvo's distance (Bruvo et al., 2004) and Neighbour Joining (NJ) clustering (Paradis et al., 2004). Bruvo's distance takes into account the mutational process of microsatellite loci and is well adapted to populations with mixed ploidy levels and is therefore suitable for the study of the B. bruxellensis strain collection used in this work. Supplementary tests were applied to the same dataset in order to confirm the clusters obtained by Neighbour Joining. First, an UPGMA (Unweighted Pair Group Method with Arithmetic Mean) analysis was compared with NJ. Then, the partition method [START_REF] Prosperi | A novel methodology for large-scale phylogeny partition[END_REF] was applied in order to confirm the reliability of the nodes obtained by NJ. Also, a principal component analysis (PCA) by the ade4 package in R (https://www.r-project.org) was applied to the same dataset and finally, the function 'find.clusters' available in the adegenet R package was used to identify clusters by successive K-means [START_REF] Jombart | adegenet: a R package for the multivariate analysis of genetic markers[END_REF]. Further, AMOVA (analysis of molecular variance) was used to assess the relative importance of geographical localisation and substrate origin regarding B. bruxellensis genetic diversity. To confirm the results obtained by the AMOVA analysis, the link between genetic divergence and geographic distance was further evaluated by MANTEL test.

Core genotype analysis

Among the 124 alleles included in the initial dataset, 70 were found to be significantly associated with the triploid isolates (χ² test, p<0.01) and were excluded to create a new dataset comprising alleles common to all groups and representative of the core genotype (i.e. the genotype common to all groups).

For the inference of population structure with this dataset, LEA package was used [START_REF] Frichot | LEA: An R package for landscape and ecological association studies[END_REF] in combination with the TESS tool to map the geographical cluster assignments of the ancestral populations as defined by [START_REF] Höhna | TESS: an R package for efficiently simulating phylogenetic trees and performing Bayesian inference of lineage diversification rates[END_REF] [START_REF] Höhna | TESS: an R package for efficiently simulating phylogenetic trees and performing Bayesian inference of lineage diversification rates[END_REF].

Further, a differentiation test analysis was performed by calculating the fixation index (F ST ) for the core diploid genotype.

Sulphite tolerance assessment

The assay was performed in liquid medium containing 6.7 g.L -1 of YNB (Difco TM Yeast Nitrogen Base, Beckton, Dickinson and Company), 2.5 g.L -1 D-glucose, 2.5 g.L -1 D-Fructose, 5% (v/v) ethanol and increasing concentrations of potassium metabisulphite (PMB, K 2 S 2 O 5 )(Thermo Fischer Scientific) in order to obtain 0, 0.2, 0.4 and 0.6 mg.L -1 mSO 2 final concentrations. For the calculation of mSO 2 it was considered that K 2 S 2 0 5 corresponds to about 50% of total SO 2 (therefore a solution of 10 g.L -1 K 2 S 2 0 5 corresponds to approximately 5 g.L -1 total SO 2 ). In order to deduce the final mSO 2 concentration, the free SO 2 concentration was assessed by aspiration/titration method. Then, the mSO 2 was estimated by taking into account the final pH, temperature and alcohol content of the medium (resource available at http://www.vignevin-sudouest.com/services-professionnels/formulaires-calcul/so2-actif.php).

Final pH was adjusted to 3.5 with phosphoric acid (1M H 3 PO 4 ) and the four media (corresponding to the 4 different concentrations of SO 2 ) were filtered separately with 0.22 µm pore filter (Millipore).

Small-scale fermentations were performed in sterile 4 ml spectrophotometer cuvettes containing a sterile magnet stirrer (Dutscher, France). The cells were grown on YPD agar and inoculated into the YNB-based medium without SO 2 . After 96 h of pre-culture (the point at which all strains reached stationary phase), the cells were inoculated at OD 600 0.1 in a final volume of 3 ml. The inoculated medium was then covered with 300 µL of sterile silicone oil (Sigma-Aldrich) to avoid oxidation of the medium which could favour the free SO 2 consumption. Then, the cuvette was capped with a plastic cap (Dutscher) and sealed with parafilm. A sterile needle was added by piercing the cap to allow CO 2 release. The "nanofermenters" were then placed in a spectrophotometer cuvettes container box and on a 15 multipositions magnetic stirrer plate at 25 °C (the final temperature in the "nano-fermenters" was therefore 29 °C due to the stirrer heating). Optical density (OD 600 ) was measured every 24h during at least 300h to follow cell population growth until stationary phase was reached.

For each growth curve, the following three parameters were calculated: maximal OD was the maximal OD reached at 600 nm, the lag phase (in hours) was the time between inoculation and the beginning of cell growth (5% maximal OD increase), and finally, the maximal growth rate was calculated (maximal number of division per hour based on the OD measurement divided by time). A non-parametric Kruskal-Wallis test was used at α=5% to identify the means that were significantly different.

Results

B. bruxellensis genotyping analysis and population structure

The B. bruxellensis collection used in this study comprised 1488 isolates from 29 countries and 9 different substrates, the majority of strains (87%) originating from wine (Supplementary All isolates were shown to be heterozygous for at least one locus. Many isolates were shown to have more than 2 alleles per locus. About half of the isolates had up to 3 alleles per locus (792 isolates) and some had up to 4 and 5 alleles per locus (67 and 1 isolates, respectively). The high number of isolates with up to 3 alleles per locus suggests the existence of triploidy in the studied population. The same observation was reported previously by (Curtin et al., 2012) and (Borneman et al., 2014) who performed de-novo sequencing and comparative genomics respectively, highlighting two triploid strains having different triploidisation origins.

The raw data obtained by the microsatellite analysis corresponds to the alleles (i.e. the size of the amplified microsatellite sequences) per locus and per strain (Supplementary Table online).

This data was further used for the construction of a dendrogram reflecting the genetic proximity between strains (Figure 2.1 A). The method was based on Bruvo's distance and Neighbour Joining (NJ) and was chosen for being reliable and suitable for populations with mixed ploidy levels. The population clusters in 3 main genetic groups (Figure 2.1 A).

Additional methods, including complementary tests and Bayesian approaches were applied to verify the reliability of the clustering obtained by NJ (Figure 2.1). The NJ tree showed three main branches that were almost perfectly conserved with UPGMA method (Figure 2.1 A and B). Then, a principal component analysis (PCA) was performed using R ade4 package (Figure 2.1 C). The PCA analysis showed that the three main groups were almost identical to the clusters previously defined. Furthermore, the partition method [START_REF] Prosperi | A novel methodology for large-scale phylogeny partition[END_REF] was applied on the same dataset. This algorithm identifies monophyletic clusters for which the individuals are more closely related than randomly selected individuals. The reliability of the node is then computed and nodes with reliability higher than 90% are considered (Figure 2.1 D). The partition method also confirmed the three main clusters obtained with NJ as reliable.

Finally, clusters were identified using successive K-means (adegenet package, function 'find.clusters'). This function implements the clustering procedure used in Discriminant Analysis of Principal Components (DAPC) [START_REF] Jombart | Discriminant analysis of principal components: a new method for the analysis of genetically structured populations[END_REF], where successive K-means are run with an increasing number of clusters (k), associated with a statistical measure of goodness of fit. This approach identified 3 clusters, once again very similar to those obtained by NJ (Figure 2.1 E). Overall, the five approaches taken together confirmed the reliability of the three main clusters observed in the studied B. bruxellensis population. The PCA was computed using the ade4 package in R. For isolates with incomplete genotyping, the missing data was inferred from the closest neighbour using Bruvo's distance. Isolates are shown with the same colours as in A. D-Node reliability using the partition method [START_REF] Prosperi | A novel methodology for large-scale phylogeny partition[END_REF]. Only the nodes with reliability >90% are shown on the NJ tree. E-Cluster identification using successive K-means. The find.cluster function from the adegenet package in R was applied, using within-groups sum of squares (WSS) statistics and the default criterion diffNgroup. This tool identifies an optimal number of 3 clusters, represented on the NJ tree using different arbitrary colours. F-Inferred ploidy. The maximum number of alleles per locus was computed. Isolates with up to 2 alleles/locus were considered as diploid (2n).

Isolates with up to 3 alleles/locus were considered as triploid (3n), and the number of loci showing up to 3 alleles was recorded (1-2 loci, or more than 2 loci showing up to three alleles). Finally, isolates with up to 4 or 5 alleles/locus were noted as 4n/5n. The inferred ploidy is represented on the NJ tree.

Since B. bruxellensis is known to exhibit different ploidy levels (Borneman et al., 2014;Curtin et al., 2012), we inferred putative ploidy level based on the microsatellite genotyping. Isolates with up to 2 alleles per locus were considered diploid and noted 2n (Figure 2.1 F). Isolates with up to 3 alleles/locus were considered triploid (3n). Finally, isolates with up to 4-5 alleles/locus were noted as 4n/5n. The ploidy level coincided clearly with the three main branches of the dendrogram, the red and orange groups being mostly triploid and the blue-green mostly diploid. Within this last cluster, two triploid sub-groups based on the substrate origin and ploidy level of the strains were defined, marked with blue and cyan colours. Finally, the combination of different methods and factors defined of 3 main groups, the 'diploid' one being further divided into 3 subgroups (Table 2.1 and Figure 2.2). To assess the relative importance of geographical localisation, substrate origin and ploidy level on B. bruxellensis' population structure, an analysis of molecular variance (AMOVA) was performed. The three factors were shown to be significant (p-value<0.0001). Ploidy level explained 46.9% of the variance, whereas the geographical origin and substrate factors explained only small proportions of the total variation (around 5% for each) (Table 2.2).

However, when considering non-wine isolates, the geographical origin explains 54.8% of the total variance, suggesting that wine genotypes are highly disseminated across the regions studied in comparison with other substrates. The correlation between genetic and geographic distance matrix (MANTEL test) was also significant (p-value=0.0009), confirming that the genetic variation of the total population is significantly related to geographical localisation. The MANTEL test, performed only on the wine strains (p-value=0.0040), also confirmed the results obtained with AMOVA, suggesting a different population structure amongst wine strains compared to those from the other niches. Most classical population genetic analyses cannot be performed using our initial microsatellite dataset since B. bruxellensis population include diploid and polyploid isolates, and most traditional analyses are not available for mixed ploidy levels. To overcome such difficulties, we excluded the alleles identified as specific to the isolates showing more than 3 alleles for at least one locus. Among the 124 alleles included in the initial dataset, 70 were found to be significantly associated with the triploid isolates (χ² test, p-value<0.01), and were excluded to create a new dataset comprising alleles representative of the core genotype (i.e. the genotype common to all groups). This approach is justified as previous comparative genomics studies showed that B. bruxellensis isolates shared a core diploid genome (Borneman et al., 2014).

The obtained core genotype dataset showed up to 2 alleles per locus for most individuals (1350 out of 1488) and only 138 remaining individuals had loci with 3 or 4 alleles. This indicates that the removal of specific triploid alleles allowed us to have access to the core diploid genome common to all B. bruxellensis isolates. Loci with more than 2 alleles were considered as missing data and only concerned 138 individuals, of which 130 only had one locus with 3 alleles.

Ancestral populations and inference of population structure

LEA package and the snmf function in R were used to infer population structure for the 'core were associated with only one ancestral population. Finally, only the tequila/ethanol group (CBS 5512-like) seemed to be associated with more than one ancestry. Altogether, the population structure analysis on the core diploid genotype confirmed the previous clustering and suggested the existence of only one ancestral population for each current population.

Population differentiation analysis

A population differentiation analysis was performed by calculating the fixation index (F ST ) on the core diploid genotype dataset (Figure 2.4). The wine AWRI1499-like population is highly differentiated from beer AWRI1608-like and wine CBS 2499-like groups (with F ST 0.36 and 0.39 respectively). This confirms the grouping obtained by the previous analyses. In addition, the pairwise F ST values showed high differentiation between beer AWRI1608-like and wine CBS 2499-like populations (F ST 0.28). The L14165-like kombucha population seems to be mostly differentiated from the 1608-like beer population and is closer to CBS 5512-like tequila/ethanol group. Finally, it is interesting to point out that the CBS 5512-like group is not highly differentiated from all other groups, which is congruent with the fact that population structure analysis inferred multiple ancestries populations for that group.

Sulphite tolerance

Sulfur dioxide tolerance was assayed for a subset of B. bruxellensis (a total of 39 strains). The chosen strains were selected according to their various geographical origins, substrates and different genetic groups. Some isolates showing identical microsatellite genotypes were included to evaluate possible sulfur tolerance variation between strains with undifferentiated genotypic patterns (13-EN11C11=L0417=L0424; UWOPS 92-244.4=UWOPS 92-262.3;L0469=L14186). Each strain was grown in medium with increasing SO 2 concentration (ranging from 0 to 0.6 mg.L -1 molecular SO 2 ) in biological triplicates, so that more than 480 fermentations were monitored.

Three growth parameters (lag phase, maximum growth rate, maximal OD) in the presence of four different concentrations of mSO 2 were followed until stationary phase was reached or for a maximum of 300h when growth was slow or absent. The isolates presented different behaviour according to mSO 2 concentration (Figure 2.5). Based on the growth parameters of the strains when exposed to increased concentrations of mSO 2 , two main groups were identified: 1) sensitive strains (S) characterised by an altered growth with i) a significant lag phase prolongation, ii) a significant decrease in maximum growth rate, and/or iii) significant decrease in maximum OD 600 (e.g. the sensitive strain L0422 had a lag phase of 17.2 h, 40.7 h, 255.8 h and growth absence, growth rate values were 0.11, 0.07, 0.02 divisions/h and growth absence for and OD 600 2, 1.9, 0.8 and no growth at 0, 0.2, 0.4 and 0.6 mg.L -1 mSO 2 respectively); 2) tolerant strains (T) that showed unmodified growth rate and maximum OD 600 but sometimes a significant prolongation of lag phase was observed (e.g. the tolerant strain AWRI1499 had a maximal growth rate of 0.07, 0.09, 0.08 and 0.07 divisions/h, OD 600 1.9, 2.0, 1.9 and 1.9, lag phase of 75, 56.5, 91.5 and 110.3 h at 0, 0.2, 0.4 and 0.6 mg.L -1 mSO 2 respectively for the same strain) (mean values of those parameters for each strain are shown in Supplementary Table S-2.4 online in Appendix). A clear relation between genetic group and SO 2 tolerance was highlighted (Figure 2.5). The isolates from groups AWRI1608-like, CBS 5512-like, CBS 2499-like and L14165-like were mostly identified as sensitive (S), whereas the triploid AWRI1499-like and triploid L0308-like groups were mostly classified as tolerant (T).

Furthermore, the isolates with an identical microsatellite profile presented similar behaviour in means of growth parameters in the different conditions studied here (Figure 2.5 and Supplementary Table S-2.4 online in Appendix).

Discussion

The yeast B. bruxellensis has gained importance for its impact not only in wine industry, but also in beer-and bioethanol-associated fermentation processes. Subsequently, many independent studies were held and results were obtained on different B. bruxellensis collections but without leading to a holistic picture of the B. bruxellensis species. In this study, a large collection of B. bruxellensis strains (1488 isolates) from various substrates (9, the majority of strains (87%) being isolated from wine) and geographic origins (5 continents) was genotyped.

The use of a reliable and robust method (microsatellite analysis) determined a general picture of the species' genetic diversity and population structure. The analysis of the complete genotype dataset highlighted 3 main genetic clusters in the B. bruxellensis population represented by the AWRI1499-like group, AWRI1608-like and CBS 2499-like group correlating with ploidy level and substrate of isolation. Three sub-clusters were also defined for their ploidy level and substrate of isolation, namely tequila/ethanol CBS 5512-like group, wine L0308-like, and kombucha L14165-like group. Our results are consistent with comparative genomics analysis showing that the AWRI1499, AWRI1608 and AWRI1613 (genetically close to the strain CBS 2499) strains are genetically distant and that the AWRI1499 and AWRI1608 strains are triploid while AWRI1613 is diploid (Borneman et al., 2014).

Heterozygosity for at least one out of the 12 microsatellite loci was shown for all B. bruxellensis isolates. This observation supports the assumption that a simple haploid organisation of the genome is excluded, which is congruent with previous results based on the Southern analysis of single gene probes of 30 B. bruxellensis strains from different geographical origins [START_REF] Hellborg | Complex Nature of the Genome in a Wine Spoilage Yeast, Dekkera bruxellensis[END_REF]. In comparison, using microsatellite analysis, [START_REF] Legras | Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history[END_REF] reported 102 out of 410 S. cerevisiae isolates (about 25%) and 75% of Saccharomyces uvarum strains (among 108 isolates from various geographical and substrates origins) to be homozygous [START_REF] Legras | Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history[END_REF]. In general, highly homozygous strains are associated with sporulation and selfing phenomena [START_REF] Mortimer | Genome renewal: A new phenomenon revealed from a genetic study of 43 strains of Saccharomyces cerevisiae derived from natural fermentation of grape musts[END_REF]. So, this could suggest that in the case of B. bruxellensis these mechanisms are non-existent or very rare amongst isolates from industrial fermentation environments. Indeed, there is only one study to our knowledge [START_REF] Walt | The wine yeasts of the Cape[END_REF], which reports spore formation for B. bruxellensis (and therefore its teleomorph form Dekkera bruxellensis). In the scenario of rare or nonexistent sexual reproduction, a large proportion of heterozygous strains would promote higher phenotypic diversity and therefore colonisation of new niches and adaptation to new environments [START_REF] Magwene | Revisiting Mortimer's Genome Renewal Hypothesis: Heterozygosity, Homothallism, and the Potential for Adaptation in Yeast[END_REF].

Our results confirm on a large scale the assumption that the B. bruxellensis population is composed of strains with different ploidy level (Albertin et al., 2014b;Borneman et al., 2014;Curtin et al., 2012;[START_REF] Hellborg | Complex Nature of the Genome in a Wine Spoilage Yeast, Dekkera bruxellensis[END_REF], as 57.8% of the isolates were shown to have more than 2 alleles for at least one locus. Moreover, polyploid strains were associated with various fermentation niches and geographical regions. A strong correlation between genetic clustering and ploidy level was highlighted, with some clusters predicted to be diploid (CBS 2499-like) while others were composed of mainly triploid isolates (e.g. AWRI1499-and AWRI1608-like). The latter two clusters derive from distinct ancestral populations and thus, presumably from different triploidisation events. The polyploid state typically has a high fitness cost on the eukaryote cell due to the difficulty to maintain imbalanced number of chromosomes during cell division as well as other effects caused by nucleus and cell enlargement [START_REF] Comai | The advantages and disadvantages of being polyploid[END_REF]. Thus, it is presumed that a stable polyploid or aneuploid state is maintained when it confers advantage for the survival of the cell in particular conditions [START_REF] Wertheimer | Ploidy dynamics and evolvability in fungi[END_REF]. Indeed, aneuploidy and polyploidy contribute to genome plasticity and have been shown to confer selective and fitness advantages to fungi in extreme conditions, such as the presence of high concentrations of drugs, high osmotic pressure, low temperature, and others (see [START_REF] Albertin | Polyploidy in fungi: evolution after whole-genome duplication[END_REF][START_REF] Mulla | Yeast: a simple model system to study complex phenomena of aneuploidy[END_REF]Wertheimer et al., 2016 for review). Similar observations have been made in clinical microbiology, for example, 70% of 132 completely sequenced S. cerevisiae clinical isolates with different geographic origins were shown to be poly-or aneuploid [START_REF] Zhu | Whole Genome Analysis of 132 Clinical Saccharomyces cerevisiae Strains Reveals Extensive Ploidy Variation[END_REF]. It has been suggested that the aneuploid state contributes to the transition from commercial (industrial fermentations) to clinical (human pathogen lifestyle) environments. Aneuploidy was also reported for another human pathogen -C. albicans, for which an aneuploidy of an isochromosome [i(5L)] is shown to confer resistance to fluconazole [START_REF] Selmecki | Aneuploidy and isochromosome formation in drug-resistant Candida albicans[END_REF]. In the industry, stable autotetraploid S. cerevisiae strains have been described among isolates from a bakery environment and it was suggested that their prevalence in sour dough fermentation could be the result of human selection for tolerance to high osmotic pressure and high metabolic fluxhighly favourable characteristics for baking [START_REF] Albertin | Evidence for autotetraploidy associated with reproductive isolation in Saccharomyces cerevisiae: towards a new domesticated species[END_REF]. In the case of B. bruxellensis, however, polyploidy seems to be not only due to a "simple" duplication of chromosomes and/or regions of chromosomes but is the result of independent hybridisation events with closely or distantly related unknown species (Borneman et al., 2014), which result in allotriploid strains. Efficient hybrid species are not rare in human related fermentations [START_REF] Albertin | Polyploidy in fungi: evolution after whole-genome duplication[END_REF][START_REF] Querol | The complex and dynamic genomes of industrial yeasts[END_REF][START_REF] Steensels | Improving industrial yeast strains: exploiting natural and artificial diversity[END_REF] and often the hybridisation with a genetically close species is believed to confer tolerance to specific stress factor in a given environment. This is the case of S. pastorianus, used for lager beer fermentations characterised with low temperatures. This yeast has recently been shown to be a hybrid between S. cerevisiae and S. eubayanusa cryotolerant species isolated from forests in Patagonia [START_REF] Libkind | Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast[END_REF], Tibet [START_REF] Bing | Evidence for a Far East Asian origin of lager beer yeast[END_REF] and recently from New Zealand [START_REF] Gayevskiy | Saccharomyces eubayanus and Saccharomyces arboricola reside in North Island native New Zealand forests[END_REF]. Thus, presumably sterile hybrids were naturally generated and they multiplied clonally, accumulating mutations which enhanced the adaptability of the new "species" [START_REF] Libkind | Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast[END_REF]. Hybrids are also a widespread state among wine yeast, where natural or laboratory obtained combinations between two species could have interesting technological properties [START_REF] Le Jeune | Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum[END_REF][START_REF] Masneuf | New Hybrids between Saccharomyces Sensu Stricto Yeast Species Found among Wine and Cider Production Strains[END_REF][START_REF] Naumov | Natural Polyploidization of Some Cultured Yeast Saccharomyces Sensu Stricto: Auto-and Allotetraploidy[END_REF][START_REF] Sipiczki | Interspecies hybridization and recombination in Saccharomyces wine yeasts[END_REF][START_REF] Steensels | Improving industrial yeast strains: exploiting natural and artificial diversity[END_REF]. Other form of genome dynamics was also highlighted for the diploid CBS 2499 strain possessing specific centromeric loci configuration that enables genome rearrangements and ploidy shifts [START_REF] Ishchuk | Novel Centromeric Loci of the Wine and Beer Yeast Dekkera bruxellensis CEN1 and CEN2[END_REF]. Based on the body of knowledge concerning other polyploid micro-and macro-organisms and the prevalence of polyploid strains highlighted in this study, we assume that B. bruxellensis has adapted to environmental stress factors by the means of genome plasticity, namely polyploidy.

Our study showed that at least one group, the AWRI1499-like triploid wine group, is composed of wine isolates that are highly tolerant to SO 2 and that are clearly divergent from other B. bruxellensis clusters (F ST higher than 0.35 when compared with AWRI1608-like and CBS 2499-like groups). Nevertheless, for some wine samples, isolates from both AWRI1499-like triploid group and the CBS 2499-like diploid group were identified. Coexistence of diploid and polyploid (auto-and allopolyploid) "microspecies" has often been reported for plants, in which the polyploids are widely distributed as opposed to the diploids that have a more restricted distribution [START_REF] Stebbins | The Significance of Polyploidy in Plant Evolution[END_REF]. Babcock and Stebbins were the first to name this coexistence of populations a diploid-polyploid complex [START_REF] Babcock | American species of Crepis[END_REF] for a Crepis species defined as a group of interrelated and interbreeding species that also have different levels of ploidy. These authors claimed that such polyploid complex can arise when there are at least two genetically isolated diploid populations and auto-and allopolyploid derivatives that coexist and interbreed.

In the case of B. bruxellensis, the sexual cycle of this yeast is not yet elucidated and interbreeding remains to be evidenced. However, we propose that B. bruxellensis could be described as a diploid-triploid complex, in which sub-populations with different ploidy levels coexist.

To obtain a deeper understanding of the factors shaping B. bruxellensis population structure, we explored the impact of geographical localisation and industrial fermentation environment of origin on the total genetic variance. Contribution of the "geographic origin" factor to the population structure was shown to be significant yet only explained a relatively small proportion of variation. However, the variance proportion explained by this factor is much higher when considering non-wine isolates, suggesting that wine strains are highly dispersed worldwide. This dispersal could easily reflect exchange of material and human transport associated with winemaking, followed by adaptation to local winemaking practices (Curtin et al., 2007). Exchange of material also happens between different industries, which would facilitate local transfer of microorganisms between beverages. For example, some beers are aged in oak barrels previously used for winemaking [START_REF] Sanna | Effect of wine barrel ageing or sapa addition on total polyphenol content and antioxidant activities of some Italian craft beers[END_REF]. Also, in the past, beer fermentation is thought to have been initiated by the addition of a small amount of wine (Mortimer, 2000b). Such exchanges could be a possible explanation for the low (but significant) contribution of the "substrate of isolation" factor to the total genetic variance in the studied population (5.93%, p-value<0.0001). Substrate of isolation and geographic origin contributed to a similar extent to the total genetic variance of the population. However, this percentage remained low (5%) compared to S. cerevisiae for which geographic origin was shown to contribute to 28% of the genetic variance [START_REF] Legras | Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history[END_REF], and Candida albicans for which 39% were reported [START_REF] Fundyga | Population structure of Candida albicans, a member of the human flora, as determined by microsatellite loci[END_REF]. For S. cerevisiae, a significant contribution of geographic origin to the genetic variance is often perceived as a sign of local domestication [START_REF] Almeida | A population genomics insight into the Mediterranean origins of wine yeast domestication[END_REF][START_REF] Legras | Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history[END_REF]. Like S. cerevisiae, B. bruxellensis is isolated from human-conducted fermentations including beer and wine. However, until now there are no B. bruxellensis isolates from "natural" non-human related habitats contrary to the case of S. cerevisiae [START_REF] Sampaio | Natural Populations of Saccharomyces kudriavzevii in Portugal Are Associated with Oak Bark and Are Sympatric with S. cerevisiae and S. paradoxus[END_REF][START_REF] Sniegowski | Saccharomyces cerevisiae and Saccharomyces paradoxus coexist in a natural woodland site in North America and display different levels of reproductive isolation from European conspecifics[END_REF][START_REF] Wang | Surprisingly diverged populations of Saccharomyces cerevisiae in natural environments remote from human activity[END_REF]. A recent comparative study of strains with different industrial origins and their growth capacities in various type of media (wine, beer, and soft drink) suggests adaptation of B. bruxellensis strains to different fermented beverages [START_REF] Crauwels | Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains[END_REF]. In our study, a low but significant contribution of substrate of isolation to the total genetic variance of the species was highlighted (5.93 %, p-value<0.0001), which is an indicator for the adaptation of certain sub-groups to different human-related niches (e.g. winemaking conditions, kombucha fermentation, and others). This structuration is further accompanied by a specific genetic configuration, some groups being mostly diploid and others polyploid.

The hypothesis that the triploid state of B. bruxellensis is maintained for some genetic groups because of its contribution to adaptation to a certain type of environment or stress factors is strongly supported by the sulphite tolerance assay performed in our study. This indicated that strains representative of the globally dispersed wine triploid AWRI1499-like group are highly tolerant to SO 2 . Sulfur dioxide is the most common antimicrobial agent used in winemaking.

However, very tolerant B. bruxellensis strains have been reported [START_REF] Curtin | Harnessing improved understanding of Brettanomyces bruxellensis biology to mitigate the risk of wine spoilage[END_REF].

Particularly, in Australia 92% of the isolates are genetically close to a strain that has be shown to be triploid by genome sequencing and highly tolerant to SO 2 (normal growth at more than 0.6 mg.L -1 mSO 2 ) (Curtin et al., 2012a). Here, we show that isolates from this genetic group are highly represented worldwide, namely in France, Italy, Portugal, Southern Argentina and Chile. Furthermore, we confirmed on a larger scale (39 strains from different geographical and fermentation niches) that even high SO 2 doses could not guarantee the absence of growth of these strains and therefore their potential to spoil wine. In this context, it is worth noting that isolates from substrates other than wine, were all sensitive to SO 2 which suggests a direct link between SO 2 exposure in wine and tolerance to this compound. Survival in the presence of SO 2 has been broadly studied in S. cerevisiae but is still not fully elucidated. SO 2 is a small and reactive molecule that could enter the cell passively or via selective transport [START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF]. Once inside the cell, SO 2 can interact with different enzymes and molecules thus having an impact on the basic metabolic pathways of the cell, such as glycolysis. Strategies to tolerate SO 2 are also numerous, like its action on the cell: through the production of molecules that bind SO 2 (acetaldehyde, pyruvate, and others), SO 2 oxidation and SO 2 active efflux by sulphite pump (SSU1) [START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF]. Even if in B. bruxellensis these mechanisms are not elucidated, SO 2 tolerance could be linked to different aspectspresence of gene(s) coding for a sulphite transporter or presence of this gene (or genes) in multiple copies and therefore overexpression, differences in the gene regulation leading to more efficient response to SO 2 toxicity, or morphological and physiological state of the cell that would give it the ability to tolerate this antimicrobial agent (cell membrane structure, growth, etc.). The fact that all the highly tolerant B.bruxellensis strains are triploid indicates that this genetic configuration could contribute to SO 2 tolerance. As mentioned in the previous paragraphs, polyploid states are maintained when they confer a selective advantage. In this case, we can hypothesise that the allotriploid AWRI1499-like strains combine genetic and physiological characteristics from the parent genomes that confer to them the ability to survive in the presence of SO 2 .

A possible strategy to cope with the issue of highly tolerant strains would be the increase of SO 2 concentration added to the must and wine. However, the strong legislation and consumer pressure to reduce any kind of wine additives makes it undesirable to produce wines with high concentrations of SO 2 which would be needed for the prevention of AWRI1499-like strains growth. Therefore, the genetic content of B. bruxellensis has to be considered when choosing spoilage prevention and treatment methods in the winery in order to obtain optimal effect with minimum intervention. Overall, our results show that polyploid strains are widely disseminated and suggest that B. bruxellensis is a diploid-triploid complex whose population structure has been influenced by the use of sulfur dioxide as a preservative in winemaking. Thus, we highlight the importance of B. bruxellensis species as a non-conventional model microorganism for the study of polyploidy as an adaptation mechanism to human-related environments.

Data availability

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

Introduction

Brettanomyces bruxellensis is a microorganism that is gaining high and rising industrial importance over the last years. Comparative genomics studies have revealed a complex genetic structure of the species, as some strains are diploid (e. g. CBS 2499), whereas others are allotriploid (e. g. AWRI1499 and AWRI1608), resulting from independent hybridisation events (Borneman et al., 2014). In a recent study, we demonstrated that whole genetic clusters (namely CBS 2499-like, AWRI1499-like and AWRI1608-like) correlate with ploidy level of the strains and substrate of isolation (Avramova et al., submitted). Indeed, this yeast species is isolated from various human-related fermentation environments (see Avramova et al., submitted) and was suggested to have strong niche adaptation corresponding to different types of fermented beverages [START_REF] Crauwels | Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains[END_REF]Avramova et al., submitted), especially for the wine strains [START_REF] Crauwels | Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains[END_REF]Avramova et al., submitted). In wine, B. bruxellensis is considered a spoilage species, mainly due to the production of ethyl phenols [START_REF] Chatonnet | Incidence des conditions de fermentation et d'élevage des vins blancs secs en barriques sur leur composition en substances cédées par le bois de chêne[END_REF], which confer unpleasant aromas described as barnyard, horse sweat, and medicinal (Chatonnet et al., 1995b;[START_REF] Heresztyn | Metabolism of volatile phenolic compounds from hydroxycinnamic acids byBrettanomyces yeast[END_REF]. The spoilage potential of this yeast is further enhanced by its tolerance to sulfur dioxide (SO 2 ) [START_REF] Agnolucci | Impact of sulphur dioxide on the viability, culturability, and volatile phenol production of Dekkera bruxellensis in wine[END_REF][START_REF] Agnolucci | Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis[END_REF]Curtin et al., 2012a) the most broadly used antimicrobial agent in winemaking. Several scientific works have treated this issue over the last decade [START_REF] Agnolucci | Impact of sulphur dioxide on the viability, culturability, and volatile phenol production of Dekkera bruxellensis in wine[END_REF]A. Barata et al., 2008;[START_REF] Capozzi | Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach[END_REF]Curtin et al., 2012a;[START_REF] Vigentini | Assessment of the Brettanomyces bruxellensis metabolome during sulphur dioxide exposure[END_REF]Zuehlke and Edwards, 2013;Avramova et al., submitted), and have underscored variable tolerance to SO 2 that correlates with genetic grouping of B. bruxellensis [START_REF] Agnolucci | Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines[END_REF]C. Curtin et al., 2012b). Strikingly, 92% of the Australian isolates (C. Curtin et al., 2012b) and high percentage of the Bordeaux isolates (Cibrario et al, in preparation) were reported to have genotypes previously correlated with high SO 2 tolerance (C. Curtin et al., 2012b) , Avramova et al., submitted. Ironically, it was suggested that SO 2 use applied constant selective pressure on B. bruxellensis wine population, thus leading to the establishment of tolerant genotypes (C. Curtin et al., 2012b) and rendering common doses of SO 2 inefficient against this spoilage microorganism. Sulfur dioxide tolerance is accepted to be a hallmark of domestication for Saccharomyces cerevisiae wine strains [START_REF] Legras | Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history[END_REF] and thus it was recently suggested that B. bruxellensis was a species closely related to human activity (Avramova et al., submitted). The problem becomes even more complex, as the most tolerant strains have a allopolyploid configuration (Avramova et al., submitted;Borneman et al., 2014;Curtin et al., 2012) and co-exist with diploid populations in a diploid-triploid complex (Avramova et al., submitted). These characteristics make of B. bruxellensis an excellent model organism for the study of polyploidy as an adaptation strategy in eukaryotes. Indeed, the striking correlation between genetic configuration (namely, allotriploid state corresponding to AWRI1499-like group) and SO 2 tolerance, taken together with the high dispersal of triploid genotypes (C. Curtin et al., 2012b), Avramova et al., submitted, Cibrario et al., in preparation) led to the hypothesis that the genetic configuration of AWR1499-like strains is conferring selective advantage in winemaking conditions (Borneman et al., 2014) and exposure to SO 2 (Avramova et al., submitted). The aim of the present study is to verify this hypothesis. For that purpose, competition experiments were designed to evaluate the relative fitness of an allotriploid AWRI1499 strain versus a reference diploid strain (CBS 2499) and a divergent allotriploid (AWRI1608) in means of growth capacity. In order to differentiate the competitor and reference strain in mixed culture, competition trials with yeast at intraspecies level often involve constructing strains with auxotrophic or antibiotic resistance markers through genetic transformation. However, genetic manipulation of B. bruxellensis is still not a common laboratory practice, and directed genetic transformation presents a challenge due to the putative asexuality of this organism [START_REF] Curtin | Genomic insights into the evolution of industrial yeast species Brettanomyces bruxellensis[END_REF]Avramova et al., submitted) and its mixed ploidy level (Borneman et al., 2014;[START_REF] Schifferdecker | Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast[END_REF]Avramova et al., submitted). To address this problem, non-directed methods were made available to the scientific community over the last years, based on both auxotrophic [START_REF] Schifferdecker | Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast[END_REF] and antibiotic resistance [START_REF] Miklenić | Improved electroporation procedure for genetic transformation of Dekkera/Brettanomyces bruxellensis[END_REF][START_REF] Miklenić | Genetic transformation of the yeast Dekkera/Brettanomyces bruxellensis with non-homologous DNA[END_REF] markers.

Here, we used an optimised version of the protocol published by (Miklenić et al., 2013) (Varela et al., in preparation) and we constructed strains with antibiotic resistance cassettes, as those markers were shown to be better adapted for competition experiments [START_REF] Baganz | Suitability of replacement markers for functional analysis studies inSaccharomyces cerevisiae[END_REF].

The strains AWRI1499, AWRI1608 and AWRI1626 (=CBS 2499) were chosen as representatives of the three main B. bruxellensis genetic groups, associated with different genetic background, ploidy level, and SO 2 tolerance (Avramova et al., submitted). Thus, we tested the hypothesis of selective advantage of AWRI1499-like strains over AWRI1608-like or CBS 2499-like strains in presence of SO 2 through competition experiments. Additionally, our experimental design brings new insights on B. bruxellensis' interactions at intraspecies level and the impact of polyploidy on the adaptation capacities of the species.

Materials and methods

Brettanomyces bruxellensis strains

Three B. bruxellensis wine strains -AWRI1499 (allotriploid, SO 2 tolerant), AWRI1608 (allotriploid, SO 2 sensitive) and AWRI1626 (equivalent to CBS 2499, diploid, SO 2 sensitive) were chosen as representatives of the three major genetic groups of the B. bruxellensis population (Albertin et al., 2014b;Borneman et al., 2014) (see Avramova et al., submitted) and their different tolerance to sulfur dioxide (SO 2 ) (see Avramova et al., submitted) (strain details in Table 3.1, and growth profiles in Supplementary Figure S-3.1 in Appenndix).

Strains construction and selection of transformants

To differentiate AWRI1499, AWRI1608 and AWRI1626 in a mixed culture, strains were transformed with genes conferring resistance to either G418 (geneticin) or ClonNat (nourseothricin). Plasmids pMK-T-TDH1pr-KanMX and pMK-T-TDH1pr-NatMX were used to amplify transformation cassettes. In both plasmids the gene conferring antibiotic resistance is under the control of the strong B. bruxellensis promoter TDH1. B. bruxellensis strains were transformed by electroporation following the protocol suggested by [START_REF] Miklenić | Genetic transformation of the yeast Dekkera/Brettanomyces bruxellensis with non-homologous DNA[END_REF].

Briefly, strains were grown over night in liquid YPD medium (10 g.L -1 yeast extract (Difco Laboratories, Detroit M1), 10 g.L -1 bactopeptone (Difco Laboratories, Detroit M1), and 20 g.L - 1 D-glucose (Sigma-Aldrich)). Then, 1 mL of culture was inoculated in 200 mL of fresh medium and incubated on orbital shaker (180 rpm, 28 °C) until a concentration of 5.10 7 cells.mL -1 was achieved. Cells were then centrifuged at (3000 rpm, 4 min, room temperature), supernatant was carefully decanted and cells were washed with 50 mL deionised water (this step was repeated three times). Then, cells were re-suspended in 20 mL of a freshly made solution containing 35mM of dithiothreitol) and 100 mM lithium acetate, and incubated for 45 minutes at 28 °C with gentle shaking (140 rpm). After, cells were centrifuged (3000 rpm, 4 minutes, 4 °C) and pellet was washed with ice-cold sterile deionised water. From this step on, until the end of the electroporation procedure, the cells were maintained on ice. The washing procedure was then repeated two more times but with 20 mL of ice-cold 1M sorbitol solution.

After, the cells were re-suspended in 1M sorbitol (ice-cold), so the total volume of suspension was 500 µL, 50 µL aliquots were distributed in micro-centrifuge tubes (1.5 mL, Eppendorf). A volume of 1 µL containing the DNA cassettes was then introduced into the samples, mixed thoroughly with a micropipette and incubated on ice for 5 minutes. Further, the samples were placed in separate 0.2 cm-gap electroporation cuvettes (Bio-Rad) previously kept on ice, and pulsed (1.8 kV, 5 ms) with Bio-Rad Gene Pulser Electroporator (600 Ω, 25 µF). Immediately after the pulse, 1 mL of 1M sorbitol:YPD (1:1) ice-cold solution was added, and the samples were incubated at room temperature for 20 minutes without shaking. After the incubation step, the cells were placed in 10 mL sterile tubes and 1 mL of YPD was added. The samples were incubated overnight at 28 °C, 140 rpm and spread on YPD-G418 plates (G418, 200 mg.L -1 ) or YPD-Cln plates (Cln, 50 mg.L -1 ) depending on the transformation; each sample was inoculated on 2 plates. Plates were then incubated at 30 °C and transformants were visible after 5-10 days.

Transformation validation

To validate the successful transformation of the three B. bruxellensis strains, 8 random transformants for each strain were re-streaked on solid YPD medium containing the antibiotic to which the transformant was expected to be resistant (G418, 200 mg.L -1 for KanMX transformants and ClonNat, 50 mg.L -1 for NatMX transformants) (see Figure 3.1). Isolates were then tested by TYPEBrett PCR (method patent number PCT/FR2016/052701) to confirm the identity of the species and transformants' genetic group (Figure 3.1, Step 1.). Isolates were then streaked on plates containing the reciprocal antibiotic (e.g. G418 for isolates transformed with the NatMX cassette and ClonNat for isolates transformed with the KanMX cassette) (Figure 3.1, Step 2.); transformants which did not grow on reciprocal antibiotics were retained for further validation. DNA insertion in B. bruxellensis strains during transformation is nonhomologous and random [START_REF] Miklenić | Genetic transformation of the yeast Dekkera/Brettanomyces bruxellensis with non-homologous DNA[END_REF], and therefore phenotypical neutrality of the obtained transformants was evaluated. This was done by following cell growth in 96-well plates (Greiner, Frickenhausen, Germany) containing YNB medium (YNB, 6.7 g.l -1 , D-Glucose, 2.5 g.l -1 , and D-Fructose, 2.5 g.l -1 , pH was adjusted to 3.5 with HCl 1M and the medium filtered with 0.22 µm pore filter) (Figure 3.1, Step 3.). Briefly, strains were first grown in YNB medium for 4 days and then, inoculated at OD 600 0.1 in a final volume of 200 µL medium. Plates were covered with Breathe-Easy membranes (Diversified Biotech, Dedham, MA) and incubated at 28 °C. Inoculations were performed in triplicate and using the wild type (WT) strains as a reference. Cell growth for each well was measured for 4 days with a microplate reader spectrophotometer (Spectramax M2, Molecular Devices). Transformants which showed similar growth curves to the corresponding wild-type strains were conserved in glycerol stock at -80 C (Figure 3.1., Step 4.) and named according to strain, antibiotic cassette and isolate number, thus transformant name 1499_K_3, 1499 stands for wild type strain AWRI1499, K for the KanMX cassette inserted and 3 for the number of isolate from the eight that were chosen for transformation validation.

Competition experiments -media and growth conditions

Only transformants that had same growth profile as the wild type strains (Figure 3.2) were considered phenotypically neutral and were used for the rest of the experiment. 3.2) were inoculated in equivalent proportions in order to obtain an OD 600 0.1 (i.e. 0.05 OD 600 for each strain) (Figure 3.3, Step 2.). Competition experiments were performed in synthetic media as described in Avramova et al., submitted, containing YNB, 6.7 g.L -1 , D-Glucose, 2.5 g.L -1 , D-Fructose, 2.5 g.L -1 , ethanol, 5%, pH adjusted to 3.5 with HCl 1M and filtered with 0.22 µm pore filter. Potassium metabisulphite K 2 S 2 O 5 was added at different concentrations from freshly made stock solution at 10 g.L -1 . The final concentration was adjusted to obtain 0, 0.2, 0.4, and 0.6 mg.L -1 molecular SO 2 (mSO 2 ). Free SO 2 was measured by the aspiration/titration method (http://www.moundtop.com/so2/SO2-Aspiration-Procedure2.pdf) and molecular mSO 2 was estimated considering final temperature, pH and DNA insertion in B. bruxellensis strains during transformation is non-homologous and random [START_REF] Miklenić | Genetic transformation of the yeast Dekkera/Brettanomyces bruxellensis with non-homologous DNA[END_REF], and therefore controls were included to evaluate the possible impact of the transformation on the growth of the strains and their tolerance to SO 2 . To assess the possible impact of the introduced DNA cassette strains containing either antibiotic marker were evaluated, for instance strain A-KanMX vs strain B-NatMX and strain A-NatMX vs strain B-KanMX). Additionally, controls strain A-KanMX vs strain A-NatMX and strain B-KanMX vs strain B-NatMX were also included for all competing pairs at every mSO 2 concentrations (Supplementary Figure S-3.2 in Appendix). To avoid the potential bias introduced by using strains resulting from random insertion, different transformants (different transformed clones)

were used as 'biological triplicates'. To test the repeatability of the competition experiments the same AWRI1499 transformants were used in both competition sets, AWRI1499 vs AWRI1608 and AWRI1499 vs AWRI1626.

Colony enumeration

Strain enumeration in mixed culture was assessed by plating on agar plates containing either G418, 200 mg.L -1 or ClonNat, 50 mg.L -1 . At each transfer step, samples were spread on agar plates (Cln and G418 separately) at two concentrations (no dilution and dilution 10 -3 ; dilutions were done in PBS solution containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 , pH adjusted to 7.4) using an automated spiral plater (Whitly). The spiral plater is dispensing a constant sample volume from the centre to the periphery of the plate, thus creating gradual decrease in cell concentration. Thus, the enumeration detection range covered 5 orders of magnitude (from 10 2 to 10 7 ). Briefly, samples were thoroughly mixed with vortex machine ). Further, both strains are inoculated in ratio 1:1 and total OD 600 0.1 in fresh media (with different concentrations of SO 2 ) (2.). The mixed culture is grown until stationary phase. At that point, sample is taken for OD 600 measure (3.); spreading on agar plates containing antibiotics (4.) which are later incubated and colonies are counter after 10 days; and sample is used for re-inoculation in fresh medium with the same concentration of SO 2 (5.). The cycles are repeated until approximate 20 generations, corresponding to 4 cycles.

OD 600 0.05 and 50 µL were spread with spiral plater on plates containing 25 mL of medium. Plates were then incubated at 28 C for 10 days and at least two enumerations per sample were performed using Protocol 3 Colony Counter (Synbiosis) using the "Two sector" option of the included software at maximum sensitivity and option "Split touching colonies". The counts were manually adjusted to eliminate false positives and add colonies that were not taken into account by the software. Final colony counts were used to obtain log 2 ratios for each competing pair, log 2 (AWRI1499/AWRI1626) and log 2 (AWRI1499/AWRI1608). Log 2 ratios were then used in a heat map using heatmap.2 plot in gplots R package (http://www.r-project.org/).

Wild type control

In order to test if the introduction of DNA affected strain growth and competition ability, wildtype strains were subjected to competition experiments. Thus, AWRI1499 and AWRI1608 strains were inoculated following the procedure indicated above. Fermentations were performed in triplicate for each mSO 2 concentration (0, 0.2, 0.4, and 0.6 mg.L -1 mSO 2 ). Based on results obtained with transformed strains, samples from 0, 8 and 12 generations were chosen for colony enumeration. For each sample 30 random colonies were picked and lysed in 30 µL of 20mM NaOH and then heated for 10 minutes at 99°C with iCycler thermal cycler (Biorad, Hercules, CA, USA). Then, TYPEBrett PCR (patent number PCT/FR2016/052701) was used to identify the genetic group of each colony.

Results

Briefly, transformants were validated by testing their phenotypical neutrality in means of growth behaviour and cross antibiotic resistance. Validated transformants were used in pairwise competitiondiploid AWRI1626 vs triploid AWRI1499, and triploid AWRI1608 vs triploid AWRI1608. The transformants are results from random DNA insertion of antibiotic resistance cassette via non-homologous recombination. Thus, controls comprising strains with same genetic background but different antibiotic resistance markers were included in the experiment.

Validation of transformants

A total of 48 transformant colonies (corresponding to 2 antibiotic resistance marker cassettes per genetic background and 8 colonies per combination 'genetic background and antibiotic resistance marker') were validated confirming yeast species, genetic group identity, as well as antibiotic resistance (Table 3.3). This ensured that no contaminations occurred during transformation (as transformations of several strains were performed simultaneously) and that the DNA cassette insertion did not affect regions used for species and genetic group identification. Only one strain however, showed resistance to the reciprocal antibiotic (Table 3.3). Most transformants showed a similar growth profile to the corresponding wild-type strain (Figure 3.2), although some differences were observed for 6 of the AWRI1608 transformants.

Therefore, AWRI1608 KanMX 2, 5, 6 and AWRI1608NatMX 1, 3, and 8 (Table 3.3) were not included in the competition experiments.

Competition experiments

As controls, transformants with the same genetic background but different selective markers were subjected to competition experiments under different mSO 2 concentrations. Figure 3.4

shows the log 2 of the ratio between KanMX over NatMX transformants. No patterns were observed regarding antibiotic cassettes or mSO 2 concentration between competing pairs. This suggested that the dominance observed for some individual isolates was random and not related to the nature of the DNA cassette. In fact, differences in log 2 ratios for repeated AWRI1499 competing pairs were related to the initial ratio at inoculation time (G0).

In competitions experiments between AWRI1499 and AWRI1608, transformants of this latter strain outcompeted AWRI1499 isolates at 0, 0.2, and 0.4 mg.L -1 mSO 2 , whereas at 0.6 mg.L -1 AWRI1499 transformants outcompeted AWRI1608 isolates. For these strains, the proportions of AWRI1499 and AWRI1608 in the initial inoculum were not always equal (see column G0

on Figure 3.6), but this did not affect the subsequent behaviour of the strains during the experiment, as pairs with higher initial inoculum of AWRI1608 than AWRI1499 behaved in the same way as competing pairs where at inoculation there were higher numbers of AWRI1499 or equal proportions between strains (e. g. couples 1 and 2 at 0 mSO 2 ).

Interestingly, at low mSO 2 concentrations AWRI1499 transformants were not detected by colony counting (<400 UFC.mL -1 ). This finding differs from the results obtained in competition experiments between AWRI1499 and AWRI1626 where both strains coexisted up to G20 stage (Supplementary Table S-3.1). At 0.6 mg.L -1 mSO 2 , two out of the six competing pairs (pairs 10 and 12, Figure 3.5) showed a dominance of the AWRI1608 transformant over the AWRI1499 isolate. This indicated that although transformants were carefully selected, the random nature of the transformation process can still affect the ability of certain isolates to compete.

Competition experiments performed with the wild-type strains AWRI1499 and AWRI1608 (Figure 3.6) validated the results obtained with the transformants and confirmed the reliability of the proposed experimental design. Wild-type competition experiments were particularly useful to confirm the dominance of AWRI1499 over AWRI1608 at 0.6 mg.L -1 mSO 2 .

Discussion

Previous studies highlighted contrasted tolerance to sulfur dioxide among Brettanomyces bruxellensis strains, which was correlated with differences in the strains' genetic background (C. Curtin et al., 2012b). It was further hypothesised that strains from AWRI1499-like group were specifically adapted to medium with SO 2a characteristic in relation with human activity (Avramova et al., submitted). However, to confirm the latter hypothesis, the relative selective advantage of these strains should be evaluated through competition experiments in presence or absence of SO 2 . It was previously demonstrated that, antibiotic resistance markers were suited for competition trials due to their negligible effect on transformants' growth rate when compared to wild type [START_REF] Baganz | Suitability of replacement markers for functional analysis studies inSaccharomyces cerevisiae[END_REF]. Different cassette types were developed [START_REF] Goldstein | Natural hybrids from Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations[END_REF], allowing the broad application of this method for yeast relative fitness evaluation in industrial and clinical context. However, genetic transformation is still not ordinary part of laboratory work with B. bruxellensis. Despite the growing industrial beneficial mutations [START_REF] Selmecki | Polyploidy can drive rapid adaptation in yeast[END_REF], changes in cell physiology [START_REF] Hegreness | An Equivalence Principle for the Incorporation of Favorable Mutations in Asexual Populations[END_REF], or compensation of deleterious mutations. Other cases of adaptation to industrial environments through polyploidisation have already been described, such as the case of autotetraploid S. cerevisiae strains used in in baking for which the tetraploid state is assumed to confer fitness in the presence of high osmotic pressure [START_REF] Albertin | Evidence for autotetraploidy associated with reproductive isolation in Saccharomyces cerevisiae: towards a new domesticated species[END_REF]. The allopolyploid state of the strains AWRI1499 and AWRI1608 originate from independent hybridisation events between a diploid parent strain and unknown donors of haploid additional genome. Brettanomyces bruxellensis being supposedly an asexual organism (Avramova et al., submitted;[START_REF] Curtin | Genomic insights into the evolution of industrial yeast species Brettanomyces bruxellensis[END_REF], allopolyploidy is a plausible evolution strategy for expanding environmental tolerance. Similar cases are reported within Saccharomyces genus where triploid hybrids exist and often combine beneficial traits from two (or more) parents [START_REF] Blein-Nicolas | A systems approach to elucidate heterosis of protein abundances in yeast[END_REF][START_REF] Libkind | Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast[END_REF][START_REF] Marsit | Diversity and adaptive evolution of Saccharomyces wine yeast: a review[END_REF][START_REF] Masneuf | New Hybrids between Saccharomyces Sensu Stricto Yeast Species Found among Wine and Cider Production Strains[END_REF][START_REF] Querol | The complex and dynamic genomes of industrial yeasts[END_REF]. Thus, the question if the evolutionary success of allotriploid strains (AWRI1499-like and AWRI1608-like) would be related to characteristics other than SO 2 tolerance remains open and could be explored with the experimental approach presented here.

The contrasted relative fitness of the strain AWRI1499 at different SO 2 concentration confirms the previously stated hypothesis that AWRI1499-like strains could have a relative selective advantage in the presence of SO 2 (Borneman et al., 2014;Curtin et al., 2012). Similar situation of selective advantage related to SO 2 tolerance was reported when studying competition between S. cerevisiae strains [START_REF] Pérez-Torrado | Ecological interactions among Saccharomyces cerevisiae strains: insight into the dominance phenomenon[END_REF][START_REF] Zimmer | QTL Dissection of Lag Phase in Wine Fermentation Reveals a New Translocation Responsible for Saccharomyces cerevisiae Adaptation to Sulfite[END_REF]. Indeed, it was pointed out that sulphite tolerance, particularly SSU1 overexpression [START_REF] Pérez-Torrado | Ecological interactions among Saccharomyces cerevisiae strains: insight into the dominance phenomenon[END_REF] and shorter lag phase in presence of SO 2 [START_REF] Zimmer | QTL Dissection of Lag Phase in Wine Fermentation Reveals a New Translocation Responsible for Saccharomyces cerevisiae Adaptation to Sulfite[END_REF], take part in the factors leading to ecological advantage of dominant S. cerevisiae strains. Taking into account that AWRI1499 is specifically adapted to high concentrations of SO 2 , it can be assumed that, the spoilage prevention practice of SO 2 addition, also leads to unwitting selection for a SO 2 tolerant genotype. This hypothesis is further confirmed by previous ecology studies on the species, reporting a high proportion of AWRI1499-like isolates which presented 92% of Australian isolates (C. Curtin et al., 2012b;Chris D. Curtin et al., 2007). This was also recently confirmed for the Bordeaux region, where AWRI1499-like strains represented 50% out of 731 isolates together with diploid CBS 2499-like isolates (Cibrario et al, in preparation). Moreover, an emergence of the AWRI1499-like group over the last 25 years (after the year 1990) was suggested (Cibrario et al, in preparation), which also strikingly correlates with the period when B. bruxellensis was associated for the first time with the presence of ethyl phenols in wine [START_REF] Heresztyn | Metabolism of volatile phenolic compounds from hydroxycinnamic acids byBrettanomyces yeast[END_REF][START_REF] Chatonnet | Incidence des conditions de fermentation et d'élevage des vins blancs secs en barriques sur leur composition en substances cédées par le bois de chêne[END_REF]. This scientific discovery possibly drew an increased attention on this spoilage microorganism in the wine industry and was followed by rise of SO 2 use that acted as selection pressure on B. bruxellensis wine population. In S. cerevisiae, the translocations responsible for adaptation to sulphite [START_REF] Pérez-Ortín | Molecular characterization of a chromosomal rearrangement involved in the adaptive evolution of yeast strains[END_REF][START_REF] Zimmer | QTL Dissection of Lag Phase in Wine Fermentation Reveals a New Translocation Responsible for Saccharomyces cerevisiae Adaptation to Sulfite[END_REF], were demonstrated to be prevalent among wine strains [START_REF] Zimmer | QTL Dissection of Lag Phase in Wine Fermentation Reveals a New Translocation Responsible for Saccharomyces cerevisiae Adaptation to Sulfite[END_REF]. It was suggested that these strains may have been selected by human activity for their rapid colonisation of medium containing SO 2 [START_REF] Zimmer | QTL Dissection of Lag Phase in Wine Fermentation Reveals a New Translocation Responsible for Saccharomyces cerevisiae Adaptation to Sulfite[END_REF]. In B. bruxellensis, the existence of a major allotriploid group tolerant to sulphites is another example of human related selection process of wine microorganisms through SO 2 addition. However, for this species the molecular mechanisms behind SO 2 tolerance remain to be revealed.

Human-conducted fermentations provide complex environments where multiple genera, species, and strains of microorganisms coexist in the presence of various stress factors.

Microbial interactions therefore take place and are important elements influencing wine quality [START_REF] Fleet | Yeast interactions and wine flavour[END_REF]. Even if the interactions between wine yeast have been broadly studied at the interspecies level, the relations between strains of the same species have often been neglected [START_REF] Fleet | Yeast interactions and wine flavour[END_REF]. In ecology, interactions are generally classified as cooperation (a behaviour which provides a benefit to another individual, and which is selected for because of its beneficial effect on the recipient [START_REF] West | Social semantics: altruism, cooperation, mutualism, strong reciprocity and group selection[END_REF], and competition (the negative effects which one organism has upon another by consuming, or controlling access to, a resource that is limited in availability). More specifically, competition can be symmetrical (where the two populations coexist) and asymmetrical (where one population excludes the other) [START_REF] Shearer | Fungal competition[END_REF]. The competition trial performed here, highlighted coexistence of both AWRI1499 and AWRI1626 strains at 0, 0.2, and 0.4 mg.L -1 mSO 2 (even if there was always dominance of one population over the other). This suggests populations of both genetic groups would be able to share the same niche space. The coexistence of AWRI1499 and AWRI1626 strains demonstrated in this experiment also supports the idea of a diploid-triploid complex suggested previously, where diploid and triploid B. bruxellensis populations coexist (see Avramova et al., submitted). It is interesting to notice that in the case of AWRI1499 (allotriploid) and AWRI1608 (divergent allotriploid), the relation is different, as AWRI1608 completely displaces AWRI1499 and at G20 AWRI1499 population is under the detection limit (<400 UFC.mL -1 , Supplementary Table S-3.1 in Appendix). These results suggest that, at low SO 2 concentration, an actual competition with exclusion [START_REF] Hibbing | Bacterial competition: surviving and thriving in the microbial jungle[END_REF] occurs between the triploid strains AWRI1499 and AWRI1608. Thus, the comparison between the two competition couples demonstrates different interaction profiles and highlights the complexity of intraspecies relations for B. bruxellensis.

The industrially relevant yeast B. bruxellensis is an exceptional model of adaptation to diverse human-related environments via genome plasticity and acquisition of stable polyploid populations. The proposed competition protocol will allow taking into account the interactions between B. bruxellensis strains when studying this yeast's striking adaptation capacity.

Introduction

Yeast metabolism is one of the multiple factors shaping wine aromatic and flavour properties [START_REF] Fleet | Yeast interactions and wine flavour[END_REF]. Through various processes, winemakers manage the transformation of must into wine, aiming to obtain high quality product according to their wants and the expectations of their customers. However, wine chemical and microbiological properties being in constant evolution throughout the winemaking process, there are always parameters that are difficult to control. One example of such phenomenon is spoilage by Brettanomyces bruxellensisyeast related to production of off-aromas with descriptors "barnyard", "horse sweat", "medicinal" [START_REF] Heresztyn | Metabolism of volatile phenolic compounds from hydroxycinnamic acids byBrettanomyces yeast[END_REF][START_REF] Chatonnet | Incidence des conditions de fermentation et d'élevage des vins blancs secs en barriques sur leur composition en substances cédées par le bois de chêne[END_REF]. The most common method to prevent and/or control B.

bruxellensis spoilage is the addition of sulfur dioxide into must and wine. Sulphites are used in winemaking at least since the 18 th century and are introduced by both burning of sulphur tablets in barrels and in liquid form (mainly potassium bisulphite solution addition to must and wine) [START_REF] Ribéreau-Gayon | Traité d'oenologie[END_REF]. Sulphur dioxide is broadly used in winemaking not only for its antiseptic action, but also for its antioxidant, and antioxidasic properties [START_REF] Ribéreau-Gayon | Traité d'oenologie[END_REF]. Thus, SO 2 addition is the preferred choice when it comes to B. bruxellensis spoilage prevention. Unfortunately, over the last years, B. bruxellensis was reported to be tolerant to commonly used doses of SO 2 . Furthermore, its survival in presence of SO 2 was shown to be variable among isolates [START_REF] Agnolucci | Impact of sulphur dioxide on the viability, culturability, and volatile phenol production of Dekkera bruxellensis in wine[END_REF][START_REF] Barata | The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine[END_REF]Curtin et al., 2012a). This variability makes the prediction of B. bruxellensis spoilage potential and the choice of adequate antimicrobial agent a challenge for winemakers. Recently, it was shown that B. bruxellensis SO 2 sensitivity correlates with genotype defined by AFLP (Curtin et al., 2012a) and microsatellite markers (Avramova et al., submitted). Among the six main clusters of B.

bruxellensis population (Avramova et al., submitted), the AWRI1499-like genetic cluster was highlighted to comprise isolates with high SO 2 tolerance (Avramova et al., submitted).

Furthermore, the strain AWRI1499 was demonstrated to have a selective advantage in presence of SO 2 when compared to other wine strains, thus suggesting a specific adaptation of isolates from AWRI1499-like genetic cluster to this antimicrobial and higher wine spoilage potential (Avramova et al., in preparation). The aims of this study were i) to extend the screening of SO 2 sensitivity to 106 additional isolates, ii) to confirm the correlation between genetic clusters and SO 2 sensitivity to a larger collection representative of the global B. bruxellensis population and iii) to validate the applicability of a method for B. bruxellensis SO 2 tolerance prediction through genetic markers analysis by using microsatellites.

Material and methods

Strains

In this study, 106 strains from different geographical and industrial fermentation origins were used based on their microsatellite profile as defined in the previous chapter. Those strains were evaluated for their tolerance to SO 2 using the same protocol as in Avramova et al., submitted which made possible the combination of both datasets together to give a total of 145 strains (Table 4.1).

Sulphite tolerance assessment

The assay was performed in liquid medium containing 6.7 g.L -1 of YNB (Difco TM Yeast Nitrogen Base, Beckton, Dickinson and Company), 2.5 g.L -1 D-glucose, 2.5 g.L -1 D-Fructose, 5% (v/v) ethanol and increasing concentrations of potassium metabisulphite (PMB, K 2 S 2 O 5 ) (Thermo Fischer Scientific) in order to obtain 0, 0.2, 0.4 and 0.6 mg.L -1 mSO 2 final concentrations. For the calculation of mSO 2 it was considered that K 2 S 2 0 5 corresponds to about 50% of total SO 2 (therefore a solution of 10 g.L -1 K 2 S 2 0 5 corresponds to approximately 5 g.L -1 total SO 2 ). In order to deduce the final mSO 2 concentration, the free SO 2 concentration was assessed by aspiration/titration method. Then, the mSO 2 was estimated by taking into account the final pH, temperature and alcohol content of the medium (resource available at http://www.vignevin-sudouest.com/services-professionnels/formulaires-calcul/so2-actif.php).

Final pH was adjusted to 3.5 with phosphoric acid (1M H 3 PO 4 ) and the four media (corresponding to the 4 different concentrations of SO 2 ) were filtered separately with 0.22 µm pore filter (Millipore).

Small-scale fermentations were performed in sterile 4 ml spectrophotometer cuvettes containing a sterile magnet stirrer (Dutscher, France). The cells were grown on YPD agar and inoculated into the YNB-based medium without SO 2 . After 96 h of pre-culture (the point at which all strains reached stationary phase), the cells were inoculated at OD 600 0.1 in a final volume of 3 ml. The inoculated medium was then covered with 300 µL of sterile silicone oil (Sigma-Aldrich) to avoid oxidation of the medium which could favour the free SO 2 consumption. Then, the cuvette was capped with a plastic cap (Dutscher) and sealed with parafilm. A sterile needle was added by piercing the cap to allow CO 2 release. The "nanofermenters" were then placed in a spectrophotometer cuvettes container box and on a 15 multipositions magnetic stirrer plate at 25 °C (the final temperature in the "nano-fermenters" was therefore 29 °C due to the stirrer heating). Optical density (OD 600 ) was measured every 24 hours during at least 300h to follow cell population growth until stationary phase was reached.

For each growth curve, the following three parameters were calculated: maximal OD was the maximal OD reached at 600 nm, the lag phase (in hours) was the time between inoculation and the beginning of cell growth (5% maximal OD increase), and finally, the maximal growth rate was calculated (maximal number of division per hour based on the OD measurement divided by time). A non-parametric Kruskal-Wallis test was used at α=5% to identify the means that were significantly different.

Results

Growth behaviour in presence of SO2

A total of more than 2050 small-scale fermentations were performed corresponding to each strain tested at increasing concentrations of mSO 2 (0, 0.2, 0.4 and 0.6 mg.L - worldwide. The groups AWRI1499-like, and L0308-like presented 91% (40 out of 44 isolates) tolerant/resistant phenotypes, whereas the groups CBS 2499-like, L14165-like, AWRI1608like and CBS 5512-like -88% (89 out of 101 isolates) sensitive strains. For wine strains, the proportions were 95% and 87% respectively.

Discussion

Sulphur dioxide is usually used by winemakers as preventive or curative treatment for Brettanomyces bruxellensis contamination. Concentrations of 0.2 to 0.5 mg.L -1 molecular SO 2 are typically reported to inhibit growth in wine [START_REF] Barata | The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine[END_REF]Conterno et al., 2006).

However, B. bruxellensis was considered to be rather sulphite tolerant [START_REF] Agnolucci | Impact of sulphur dioxide on the viability, culturability, and volatile phenol production of Dekkera bruxellensis in wine[END_REF] A. [START_REF] Barata | The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine[END_REF]C. Curtin et al., 2012b). Moreover, the tolerance to sulphite is strain dependant [START_REF] Agnolucci | Impact of sulphur dioxide on the viability, culturability, and volatile phenol production of Dekkera bruxellensis in wine[END_REF]Avramova et al., submitted;[START_REF] Barata | The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine[END_REF]Curtin et al., 2012a;Vigentini et al., 2008b) and sulphite efficiency is population level dependant (Longin et al., 2016a). Previous studies highlighted genotype dependant tolerance to sulfur dioxide for B.

bruxellensis among Australian isolates (C. Curtin et al., 2012a), that was recently confirmed with 39 isolates representative of the global B. bruxellensis population (Avramova et al., submitted). Taking into account the high intra-species genetic diversity of B. bruxellensis, 106 additional isolates from various origins were included to the previous phenotypic test to confirm the link between genotype and SO 2 tolerance at larger and finer scale. The final aim of this study was to explore the potential use of genetic markers to assess the genetic group and then, to deduce SO 2 tolerance of B. bruxellensis strains.

In our previous study, it was noticed that representatives of the triploid L0308-like group exhibited a peculiar profile characterised by unmodified growth parameters at all tested SO 2 concentrations. However, these observations were based on only two isolates with similar origin (Avramova et al., submitted). To complement these results, we analysed 9 additional L0308-like strains from different origins and confirmed their "resistant" phenotype. Here, a resistant phenotype corresponds to behaviour, for which there were no significant differences for all studied growth parameters at increasing SO 2 concentration. On the other hand, "tolerant" strains were those for which lag phase was modified when SO 2 was increasing. Those two terms are used in clinical microbiology, where they serve to describe microbial pathogenicity [START_REF] Anderson | Evolution of antifungal-drug resistance: mechanisms and pathogen fitness[END_REF][START_REF] Brauner | Distinguishing between resistance, tolerance and persistence to antibiotic treatment[END_REF]. Often, tolerance is related to the capacity of the organism to survive under inhibition by an agent, whereas resistance is linked to the capacity to actively proliferate in presence of antibiotic, and is measured as minimum inhibitory concentration or fitness [START_REF] Anderson | Evolution of antifungal-drug resistance: mechanisms and pathogen fitness[END_REF]. The peculiarity of SO 2 application, however, is that the active antimicrobial fraction (mSO 2 ) of this agent is variable (depending on environmental parameters such as temperature, alcohol content and pH) and decreases over time due to free SO 2 combination. Furthermore, B. bruxellensis is able to enter a VBNC (viable but not cultivable) state after sulphites addition [START_REF] Agnolucci | Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis[END_REF][START_REF] Capozzi | Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach[END_REF]Du Toit et al., 2005b;Longin et al., 2016a;[START_REF] Serpaggi | Characterization of the "viable but nonculturable" (VBNC) state in the wine spoilage yeast Brettanomyces[END_REF], followed by growth recovery with sulphites decrease over time. In winemaking, suphite levels are often re-adjusted at different time intervals, thus creating seasonality in SO 2 administration during the winemaking process.

In these conditions, the actual survival of B. bruxellensis in wine could be related to i) survival and growth besides initial "hit" with SO 2 , that could be related to resistant-type mechanism and ii) survival at the initial SO 2 "hit" and until a stage when mSO 2 concentration is lower in the medium, followed by growth recovery that could be described as tolerance mechanism. Indeed, resistant and tolerant phenotypes are often interconnected and related to different types of metabolism and cell structure differences. In clinical microbiology, it is suggested that tolerant and resistant strains would be treated differently (resistant being treated with higher doses and shorter treatment, whereas tolerant strains would be treated with lower doses but extended treatment duration [START_REF] Brauner | Distinguishing between resistance, tolerance and persistence to antibiotic treatment[END_REF]. The detection of both resistant and tolerant growth profiles in the present dataset suggests that B. bruxellensis strains have developed not one, but multiple strategies to cope with SO 2 present in wine.

Here, the majority of tolerant or resistant strains were isolated from wine (46 out of 52). This suggests a strong link between SO 2 exposure and recent emergence of tolerant/resistant phenotypes that was discussed in previous studies (Previous chapters, Cibrario et al., in increased yeast cell permeability, which probably leads to cell death. The ability of cells to restore functional cell permeability could constitute another sulphite adaptation mechanism for B. bruxellensis. The molecule of SO 2 having various effects on the cell structure, metabolism and genome [START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF], those mechanisms can be numerous including synthesis of binding molecules (like acetaldehyde), specific membrane structure, etc. [START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF].

The sensitivity/survival phenotype in presence of SO 2 correlates with genotypic profiles defined by microsatellite analysis of 39 strains (Avramova et al., submitted) and this was validated here with the additional set of 106 strains. The groups CBS 2499-like (diploid), L14165-like (diploid), AWRI1608-like (triploid), and CBS 5512-like (triploid) are all susceptible to SO 2 presence in synthetic medium. On the contrary, AWRI1499-like (triploid) and L0308-like (triploid) survived in presence of high concentrations of SO 2 . This behaviour was confirmed by independent study performed in wine medium, where the strain L0417 (AWRI1499-like) was demonstrated to be more tolerant than L02E2 (CBS 2499-like). The use of microsatellites as selection markers was previously proposed for S. cerevisiae wine strains [START_REF] Franco-Duarte | Computational models reveal genotype-phenotype associations in Saccharomyces cerevisiae[END_REF][START_REF] Renouf | Evidence for differences between B. bruxellensis strains originating from an enological environment[END_REF]. In the latter work, 30 different phenotype analyses were performed, and SO 2 tolerance was highlighted to be one of the factors that correlate the most with microsatellite patterns. In the winemaking context, SO 2 tolerance is a positive trait for the selection of S. cerevisiae, whereas it is the opposite for B. bruxellensis strains, for which it is directly related to spoilage potential. Defining SO 2 tolerance through genetic markers can therefore be used as an efficient tool to adapt antimicrobial treatment in the winery. Similar methods are used for resistance prediction for pathogenic fungi [START_REF] Irinyi | International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database--the quality controlled standard tool for routine identification of human and animal pathogenic fungi[END_REF][START_REF] Park | Establishing Surrogate Markers for Fluconazole Resistance in Candida albicans[END_REF]. Namely, in the case of C. albicans, PCR-based methods were proposed for the detection of mutations related to fluconazole resistance [START_REF] Park | Establishing Surrogate Markers for Fluconazole Resistance in Candida albicans[END_REF]. This method allows the adoption of alternative techniques to cope with this microorganism. Contrary to fluconazole which targets lanosterol 14-demethylase (the product of ERG1) [START_REF] White | Resistance Mechanisms in Clinical Isolates of Candida albicans[END_REF], SO 2 has a very broad range of activities on the cell on structural, genetic and metabolic level [START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF], and detection method of specific mutation responsible for resistance would be a challenge. Therefore, the strong correlation between genotype and SO 2 tolerance presents a reliable alternative for the prediction of this phenotype through microsatellite analysis. Indeed, resistant/tolerant genotypes were predicted at 95% (of 44 strains) based on the genotype profile. For comparison, this percentage was 91% for C. albicans (based on 32 isolates) when using targeted PCR [START_REF] Park | Establishing Surrogate Markers for Fluconazole Resistance in Candida albicans[END_REF]. Combined with the fact that clonal populations of B. bruxellensis strains were isolated over a long period of time in the same winery (Albertin et al., 2014b), the use of microsatellite markers is also applicable as a prediction method based on spoilage populations from previous vintages. Here, we suggest the use of microsatellite markers as reliable genetic markers for predicting spoilage potential in means of SO 2 tolerance for B. bruxellensis populations. This method could allow application of adequate antimicrobial techniques according to the survival mechanism in presence of SO 2 of the contaminating B. bruxellensis population, and therefore assure efficient spoilage prevention with minimal intervention.

countries, ii) represented a high proportion of the overall wine isolates (>40%), and iii) were almost exclusively comprised of wine isolates. Thus, a strong support for the hypothesis of link between genotype and isolation environment was provided. This observation was extended to other niches than wine, as we found a correlation between genotypic profile and related industrial fermentation environment for beer, bioethanol/tequila, and kombucha. To further study the specific adaptation of the species to various fermentation environments, it would be interesting to optimise and apply the competition protocol developed for this study and explore other parameters than SO 2 tolerance. In particular, it would be of high interest to investigate the selective advantage of strains with different genetic background in other variable conditions relevant to winemakingethanol concentration, pH, temperature, etc. Also, to confirm the correlation between genotype (and associated ploidy level) and substrate of isolation, it would be interesting to perform competition experiments between strains isolated from different beverages and in media corresponding to the physicochemical characteristics of the latter (e.g. beer, bioethanol). Thus, it could be tested if strains from the different genetic groups have indeed a specific advantage in the media that they were isolated from.

Wine presents peculiar conditions for the development of microorganisms and one of the specificities related to winemaking is the use of sulfur dioxide. Indeed, it was previously demonstrated that isolates from the group AWRI1499 were highly tolerant to SO 2 and it was suggested that this was an important piece of the puzzle of the adaptation of this genetic group to wine. By this work, we highlight that AWRI1499-like triploid strains are tolerant to sulfur dioxide, whereas other genotypes were sensitive to this antimicrobial agent. The specific adaptation to SO 2 of the strain AWRI1499 was further tested and confirmed in this work. For this aim, B. bruxellensis isolates were transformed and put in pairwise competition at different SO 2 concentrations. Genetic transformation of triploid B. bruxellensis strains was successfully performed for the first time (to our knowledge). The results demonstrated the selective advantage of AWRI1499 in presence of SO 2 thus supporting the hypothesis that this phenotypic characteristic can be a major component for the adaptation of this genetic group to winemaking and subsequently its high dissemination worldwide. It would be of interest to further verify those results in wine.

In fermentation environments different species and strains of microorganisms co-exist in the same space. Thus, the interactions that occur between them have impact on the final product and are interesting subject for future research. In particular, interspecies interaction between S.

cerevisiae and B. bruxellensis can be interesting especially in the case of bioethanol production where both species were detected together. Also, the nature of intraspecific interactions for B.

bruxellensis could be explored with the proposed competition protocol in winemaking and brewery context.

This strong link between genotype and sulphites tolerance presents an opportunity to diagnose SO 2 sensitivity for B. bruxellensis. Variable SO 2 tolerance of B. bruxellensis is a major concern for winemakers who aim to use optimal doses of this antimicrobial to attain a compromise between its beneficial and detrimental effects on wine quality. With the aim to design molecular markers that can be used in practice to deduce SO 2 sensitivity of spoilage-associated B. bruxellensis population, we have explored the SO 2 tolerance of a total of 145 isolates from various origins and substrates. Intriguingly, it was confirmed that only wine strains exhibited SO 2 tolerance. Furthermore, different degrees of SO 2 tolerance/resistance were highlighted, some phenotypes were characterised by unmodified growth even at 0.6 mg.L -1 mSO 2 . These observations suggest that there are probably not only one, but multiple strategies for coping with this antimicrobial for B. bruxellensis. To complement these results, it would be interesting Overall this work presents industrial interest for the winemaking community, as it demonstrates the dissemination of highly SO 2 tolerant strains worldwide. In perspective it would be of interest to explore growth behaviour and viability of "tolerant" and "resistant" B. bruxellensis

strains

Introduction

Brettanomyces bruxellensis (B. bruxellensis) is a spoilage yeast associated with major wine aroma defects which are present in about 25% of red wines [START_REF] Gerbaux | Étude des phénols volatils dans les vins de Pinot noir en Bourgogne[END_REF]Conterno et al. 2006). The 'Brett' character often leads to consumers' rejection and therefore economic loss. Even if numerous prevention and elimination methods are available for winemakers, the problem often persists from one year to another. Thus, controlling B. bruxellensis contamination risk is often a priority when vinification and wine ageing management decisions are made. The importance of the B. bruxellensis issue is underscored by the fact that this species is detected worldwide and in substrates other than wine.

Sulfur dioxide (SO 2 ) addition is the most common technique to prevent B. bruxellensis development during the winemaking process. However, high SO 2 levels are not always associated with premium wines styles and many wine consumers are seeking wines with lower SO 2 levels. Furthermore, it was recently shown that some B. bruxellensis strains are tolerant to SO 2 . The use of SO 2 as a prevention method against B. bruxellensis contamination might not prove a long-term strategy.

Here, we present our results concerning the intraspecific diversity of B. bruxellensis -on both genotypic and phenotypic level -and we focus particularly on B. bruxellensis' sensitivity to SO 2 .

Brettanomyces bruxellensis and its genetic diversity

Numerous tools allow the detection and quantification of the species B. bruxellensis, although few were developed for genotypic analysis at intra-species level. Therefore, little evidence is available on the species' ecology and genetic diversity. Even though the population level can be quantified in a wine or must sample in a reliable way, it was, until now, relatively challenging to assess the nature of the strains present in the sample and their contamination and spoilage activity.

A collection of 1280 B. bruxellensis isolates from 29 countries was assembled and considered in this study (Figure 1). microsatellite sequence repeats (Albertin et al. 2014) (a scheme representing the main steps of the method is provided in Figure 2). The strains used in the study were isolated from wine, sourced from other laboratories or were already present in Centre Ressources Biologiques (CRB) oenologie strain collection. DNA extraction was performed from a single fresh colony by treatment with 30 µl of 20 mM NaOH and 99 °C heat for 10 minutes. Microsatellite analysis was done by amplifying Simple Sequence Repeat (or SSR) regions as described by Albertin et al. (2014). Amplicon sizes were measured by ABI3730 DNA analyser and GeneMarker® software. Raw data was treated on R software using Poppr Package (Kamvar et al. 2014).

Figure 2. Schematic of experimental approach

This method allows the establishment of the genetic links between strains, revealing significant genetic diversity within the species. In agreement with the first genome studies of the species (Curtin et al. 2012b;Borneman et al. 2014), we confirm the existence of triploid strains possessing every gene in three copies instead of the common two. These triploid strains possess an additional genome whose origin remains unknown at present. Remarkably, a high proportion of genotyped strains are triploid. The hypothesis is that the triploid state could confer specific traits to B. bruxellensis which would be advantageous for the adaptation to wine-type environment.

The 1280 B. bruxellensis isolates were genotyped by microsatellite analysis highlighting 617 genetic profiles clustered in three main genetic groups (A, B, and C). Group A consists of strains that are triploid and isolated from wine, group B consists of a second type of triploid strains isolated from beer and wine, and group C consists of mostly diploid strains isolated from wine and other substrates (kombucha, tequila, bioethanol, etc.). This significant genetic diversity may help to explain the considerable phenotypic variation of the strains shown in previous studies, particularly growth capacity, ethyl phenol production and/or SO 2 tolerance. Interestingly, different genetic groups have been shown to co-exist in the same winery or wine sample and from one sample to another taken from the same source.

Brettanomyces bruxellensis and use of sulfur dioxide

The active form of SO 2 (i.e. molecular SO 2 ) is the most common method to fight against B. bruxellensis. The efficiency of SO 2 depends on the dose applied, the pH, ethanol content and temperature of the medium. It is generally considered that 0.5 mg/l molecular SO 2 is sufficient to inhibit B. bruxellensis 1280 isolates from diverse substrates and geographic origins SSR analysis (Simple sequence repeat) 12 microsatellite sequences (Albertin et al. 2014) Genetic relationship between strains Poppr R Package (Kamvar et al. 2014) Bruvo's distance (Bruvo et al. 2004) Neugbour Joining (Paradis 2004) growth and that 0.7-0.8 mg/l of molecular SO 2 is a lethal dose [START_REF] Chatonnet | Brettanomyces: mythes et réalités[END_REF], although the levels change with wine pH: 30 mg/l of free SO 2 at pH 3.6 equates to 60 mg/l at pH 3.9.

The existence of strains that are tolerant to SO 2 was recently highlighted. In Australia, a strategy to control B. bruxellensis based on the use of SO 2 was applied over the last ten years or more. B. bruxellensis isolates were collected from different wines treated with SO 2 and the studied population clustered in eight different genetic profiles as estimated by Amplified Fragment Length Polymorphism (Curtin 2007). A group comprising the majority among these strains (85 % of the isolates) was shown to be highly tolerant to SO 2 and cells could grow at 0.6 mg/l of molecular SO 2 (Curtin et al. 2012a). These findings suggest that using SO 2 to manage B. bruxellensis may apply a selective pressure to the population and ultimately lead to the emergence of highly resistant strains. Thus, it is important to understand any links between the genetic linkage of the strains and their resistance to SO 2 .

To spread these observations to other winemaking regions, we performed at laboratory scale a phenotypic test to evaluate the SO 2 tolerance of 33 strains representative of the genetic diversity of the species (strains varied in their geographical region, substrate of isolation and genetic group A, B or C as previously defined by microsatellite analysis). Strain growth was characterised in a synthetic laboratory medium in triplicate and under anaerobic conditions.

It was observed that growth in the presence of increasing concentrations of SO 2 is significantly different for each genetic group (Figure 3). For the strains from group A, the lag phase is slightly but significantly longer with an increasing SO 2 concentration. However, once the growth has started, the SO 2 concentration doesn't have any significant effect on the other growth parameters (i.e. growth rate, maximum population attained and time taken to attain the maximum population; only maximum population is shown) and this observation was valid even for the concentration of 0.6 mg/l of molecular SO 2 . Thus, these strains are considered tolerant to SO 2 : apart from the longer lag phase, they have a 'normal' growth from 0.2 to 0.6 mg/l molecular SO 2 . On the contrary, the growth of the strains belonging to strains B and C is strongly affected by the concentration of molecular SO 2 and this is valid for doses higher than 0.4 mg/l (and even 0.2 for several strains). These strains are considered sensitive to SO 2 .

Consequently, the adjustment of the molecular SO 2 even at 0.6 mg/l could be insufficient when SO 2 tolerant strains are present. A tool for diagnosing Brettanomyces bruxellensis' SO 2 sensitivity

In our laboratory, we have developed and filed a patent for a simple molecular test which highlights the genetic group of a given B. bruxellensis isolate (A, B or C) and therefore predicts its SO 2 sensitivity. This test is based on simple Polymerase Chain Reaction (PCR) analysis on colonies isolated on selective solid medium. The analysis relies on the size of the amplification fragment produced at the end of the PCR, which varies with the genetic group of the strain. An example of a result after the test is performed on three different colonies is shown in Figure 4. As a next step, we aim to develop a quantitative PCR-tool that would eliminate the cultivation step of the analysis and allow faster quantification.

Conclusion

The study of B. bruxellensis' genetic diversity revealed an unexpected genomic complexity. Various B. bruxellensis groups exist which differ in terms of sulfite tolerance. In the collection of isolates studied by microsatellite analysis, 34% of the strains are potentially very tolerant to SO 2 , illustrating the fast adaptation capacity of the species. The phenomenon is widespread -the sulfite-tolerant isolates were detected not only in different French winemaking regions but also in other countries such as Australia.

In the actual context of chemical input reduction in the wine industry (particularly the use of SO 2 ), it is now possible to assess the SO 2 sensitivity of B. bruxellensis contaminating a wine sample. This may help the winemaker to select a strategy to prevent and control spoilage, and avoid the use SO 2 when it is not likely to be effective -winemakers should utilise SO 2 addition only on wines that are not contaminated by tolerant strains.

  Globalement, ce travail contribue à une meilleure connaissance de B. bruxellensis, levure d'altération du vin, en termes de diversité génétique et phénotypique et permet d'émettre des hypothèses sur les stratégies évolutives d'adaptation au milieu anthropique de cette espèce modèle non conventionnelle. Mots clefs : génétique des populations, Brettanomyces bruxellensis,

Figure1. 1 .

 1 Figure1.1. Origins of genetic variation in yeast. Genetic variation can be caused by several different mechanisms. For sake of simplicity, only one chromosome per yeast cell is displayed (green or purple). Different color shades represent homologous chromosomes. In (e), a second chromosome is represented in red. (a) Sexual reproduction: after sporulation and concomitant meiotic cross-over events in the parental strains (2n), genomes of two haploid (n) segregants can hybridize, a process called mating. (b) Point mutations: changes in single nucleotides. These mutations can be synonymous or nonsynonymous: synonymous mutations do not change the amino acid sequence, while nonsynonymous mutations do. Nonsynonymous mutations are therefore more likely to alter the phenotype. (c) InDels: insertion and deletion events of relatively short pieces of DNA. (d) Transposons: insertion of transposable elements in the genome. (e) Changes in ploidy level: the whole genome, or large parts, is duplicated or lost, which can result in poly-or aneuploidies. (f) Horizontal gene transfer: transfer of genes by means other than regular sexual reproduction. (g) Genetic recombination: reorganization of parts of the genome. It can act on both homologous (cross-over and gene conversion) and nonhomologous loci (ectopic recombination). Homologous recombination such as gene conversion (nonreciprocal transfer of genetic material between highly homologous genes) occurs relatively frequently and can sometimes give rise to novel or modified traits. Ectopic recombination events such as TY-promoted chromosomal translocations are rarer, but can drastically rearrange the genome, and even generate novel genes.[START_REF] Steensels | Improving industrial yeast strains: exploiting natural and artificial diversity[END_REF]

  by people and they selected for them consciously or unconsciously. Some popular examples are the potato (Solanum tuberosum; 2n=4x=48), bread wheat (Triticum aestivum, 2n=6x=42), maize (Zea mays; 2n=4x=20), bread wheat (Triricum aestivum, 2n=6x=42), upland cotton (Gossypium hirsutum; 2n=4x=52), oilseed rape (Brassica napus; 2n=4x=38), sisal (Agave sisalana; 2n=5x= 80), banana (Musa; 2n=3x=33), coffee (Coffea arabica; 2n=4x=44)

Figure 1 . 2 .

 12 Figure1.2. Karyotypic changes as catalysers of genetic variation. Graphical representation of the potential snowballing effects of karyotype changes (underlined in the blue, Events section) on genome alterations. For instance, whole-genome duplication events leading to polyploidy result in geometric imbalances underlying chromosome instability. The ensuing chromosome missegregation events lead to loss of heterozygosity and/or aneuploidy, which in turn, through alteration of gene function and/or imbalanced gene expression, elevate all forms of genome instability, further perpetuating the vicious cycle of ever-increasing accumulation of various types of mutations. For completeness, the figure illustrates also the role of genetic instability and sequence mutations and how the various types of mutations and genome instability are intertwined with each other. Solid arrows: documented links. Dashed arrows indicate hypothetical links.[START_REF] Rancati | Karyotypic changes as drivers and catalyzers of cellular evolvability: A perspective from non-pathogenic yeasts[END_REF]

Figure 1 .

 1 Figure 1.3. A model of the formation of S. pastorianus and the hybrid strains of S. bayanus.[START_REF] Libkind | Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast[END_REF] A model of the formation of S. pastorianus and the hybrid strains of S. bayanus. First, wild S. eubayanus and ale-type S. cerevisiae hybridized to form an allotetraploid that gave rise to S. pastorianus. Second, domestication imposed strong selective pressure for strains with the most desirable brewing properties. Third, in the brewing vats with high densities of S. pastorianus, cell lysis releases large DNA fragments that occasionally transform, fourth, contaminating wild strains of S. eubayanus because of the lack of pure culture techniques. Fifth, multiple hybridization events with wild strains of S. uvarum gave rise to CBS 380T and NBRC 1948. This model does not exclude prior or parallel involvement of S. uvarum in brewing or contamination.

Figure 1 . 4 .

 14 Figure 1.4. Schematic overview of Brettanomyces bruxellensis phylogenetic relationship to other yeast species. 'Whole-genome'-based phylogenies place B. bruxellensis in an intermediate evolutionary group with methylotropic species Komagetalla pastoris, Kuraishia capsulata and Ogataea angusta/O. polymorpha (Left). A multi-gene phylogeny expands upon the relationship between these species and places K. pastoris outside of the B. bruxellensis containing clade (Right, red branches). The relative positions of other Brettanomyces species have been estimated based upon a separate multi-gene phylogeny (Right, purple branches). From[START_REF] Curtin | Harnessing improved understanding of Brettanomyces bruxellensis biology to mitigate the risk of wine spoilage[END_REF]

Figure 1 . 5 .

 15 Figure 1.5. Schematic representation of four D. bruxellensis strain genomes. Each of the D.bruxellensis strains is predicted to contain a conserved diploid set of chromosomes. In addition, AWRI1499 and AWRI1608 are predicted to both contain a third full set of chromosomes that have been inherited from more distantly related strains or a closely related species that is unique to each strain (Dekkera x and Dekkera y). From(Borneman et al., 2014)

Figure1. 6 .

 6 Figure1.6. SO 2 species in aqueous solution and their representation in percentage of total SO2 throughout the pH range. pK1 is the dissociation constant of the chemical reaction SO2•H2O↔HSO3-+H+ and pK2 that of the reaction HSO3-↔SO32-+H+. The effective pH range of wine is highlighted. From[START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF]

Figure 1 .

 1 Figure 1.7. A summary of the sulphate assimilation pathway and the cellular and molecular responses of S. cerevisiae to the presence of SO 2 . SAAB sulphur amino acid biosynthesis, SR sulphur reduction[START_REF] Divol | Surviving in the presence of sulphur dioxide: Strategies developed by wine yeasts[END_REF]

Figure 2 . 1 .

 21 Figure 2.1. B. bruxellensis population clusters identification by combining different tools and parameters. A-Dendrogram using Bruvo's distance and NJ clustering. The figure was produced using the poppr package in R. B-Dendrogram using Bruvo's distance and UPGMA clustering. The figure was produced using poppr. Isolates are shown in the same colours as in A. C-Principal component analysis (PCA) based on microsatellite genotyping.The PCA was computed using the ade4 package in R. For isolates with incomplete genotyping, the missing data was inferred from the closest neighbour using Bruvo's distance. Isolates are shown with the same colours as in A. D-Node reliability using the partition method[START_REF] Prosperi | A novel methodology for large-scale phylogeny partition[END_REF]. Only the nodes with reliability >90% are shown on the NJ tree. E-Cluster identification using successive K-means. The find.cluster function from the adegenet package in R was applied, using within-groups sum of squares (WSS) statistics and the default criterion diffNgroup. This tool identifies an optimal number of 3 clusters, represented on the NJ tree using different arbitrary colours. F-Inferred ploidy. The maximum number of alleles per locus was computed. Isolates with up to 2 alleles/locus were considered as diploid (2n). Isolates with up to 3 alleles/locus were considered as triploid (3n), and the number of loci showing up to 3 alleles was recorded (1-2 loci, or more than 2 loci showing up to three alleles). Finally, isolates with up to 4 or 5 alleles/locus were noted as 4n/5n. The inferred ploidy is represented on the NJ tree.

Figure 2 . 2 .

 22 Figure 2.2. Dendrogram of 1488 isolates of B. bruxellensis using 12 microsatellite markers. The dendrogram was drawn via the poppr package, using Bruvo's distance and NJ clustering. Five clusters were considered and are represented by different colours. Isolates displaying identical genotypes are represented by a unique tip whose size is proportional to the number of isolates. Inferred ploidy was made as described in Figure 2.1 F. The histograms represent the distribution of isolates depending on the substrate and the five considered clusters. The pie chart illustrates the proportion of the strains originating from different types of sources.

  diploid' dataset. The number of ancestral populations tested ranged from K=1 to K = 15 (100 repetitions), and entropy criterion was computed to choose the number of ancestral populations explaining the genotypic data in the best way (Supplementary Figure S-2.1 online and in Appendix). Entropy was minimal for K=5 ancestral populations (K=3, 4, 5, 6 shown on Supplementary Figure S-2.2 online and in Appendix). Such Bayesian analysis shows that these 5 ancestral populations are congruent with previous analyses that considered the complete dataset (Figure2.3): the AWRI1499-like (wine, red) and AWRI1608-like (beer, orange) groups were associated with only one ancestral population. Likewise, most of the blue-green subgroups (wine CBS 2499-like, wine L0308-like, kombucha L14165-like) previously defined

Figure 2 . 3 .

 23 Figure 2.3. Ancestral populations of 1488 B. bruxellensis strains. STRUCTURE plots for K=5 (the number of ancestral population with lowest entropy, see Supplementary Fig. S1 online). Each bar represents an isolate and the colour of the bar represents the estimated ancestry proportion of each of the K clusters. The same colour code is kept as in Figure 2.1 and 2.2.

Figure 2 . 4 .

 24 Figure 2.4. Population differentiation represented by fixation index (F ST ) of B. bruxellensis genetic groups between each other. The range of F ST is from 0 to 1, 1 meaning that the two populations do not share any genetic diversity.

Figure 2 . 5 .

 25 Figure 2.5. Growth parameters of B. bruxellensis strains at different concentrations of SO 2 . 39 strains belonging to the 6 genetic groups defined previously were tested in small scale fermentations and growth (OD 600 ) was measured in media containing different concentrations of sulfur dioxide (0, 0.2, 0.4, and 0.6 mg.L -1 mSO 2 ) and in biological triplicates. Three parameters were considered: lag phase (h): end of lag phase considered when OD above initial OD*5%; maximal growth rate (r) = number of cellular divisions per hour; maximal OD; S and T stand for sensitive and tolerant (Kruskal-Wallis test, α=5%). Genetic groups are represented in the same colours as on Figure 2.2.

Figure 3 . 1 .

 31 Figure 3.1. Transformation validation steps. After transformation, the culture containing the transformants is spread on YPD plates supplemented with antibiotic. Eight colonies are randomly picked and re-streaked on fresh plates. After 5-10 days of incubation, the transformants' species and genetic group are confirmed by PCR analysis (Step 1.). If the result corresponds to the expected one, biomass is re-streaked on another antibiotic to assess putative cross-resistance which is undesirable (Step 2.). If there is no growth, transformants' growth is assessed in YNB medium and compared to the wild type (WT) (Step 3.). If the transformants cover those parameters, they are conserved in glycerol stock at -80 °C for further use.

Figure 3 . 2 .

 32 Figure 3.2. Growth profiles of transformed strains in chemically defined medium. A-AWRI1499 and 16 transformed isolates, B-AWRI1608 and 16 transformed isolates, C-AWRI1626 and 14 transformed isolates. Strains were grown in YNB medium until stable stationary phase was reached and growth profile was compared with the one of the wild-type parent strain.

  medium (calculations available at https://www.vignevin-sudouest.com/servicesprofessionnels/formulaires-calcul/so2-actif.php). Competition experiments were performed in 50 mL centrifuge tubes (Grenier Bio-One) with 25 mL of medium at 28 C and 140 rpm rotation shaking. Cultures were grown until an approximate OD 600 of 1.5-2 (roughly 4 generations)(Figure 3.3, Step 3.). At this point, samples were taken and spread on YPD plates containing antibiotics as indicated below (Figure3.3, Step 4.), simultaneously cells were reinoculated in fresh medium at OD 600 0.1 (Figure3.3, Step 5). These transfers were repeated until 20 generations of the total population were attained (or total of 4 transfer steps).

Figure 3 . 3 .

 33 Figure 3.3. Transfer cycles. Pre-cultures are grown for each transformant from a competition couple until stationary phase (1.). Further, both strains are inoculated in ratio 1:1 and total OD 600 0.1 in fresh media (with different concentrations of SO 2 ) (2.). The mixed culture is grown until stationary phase. At that point, sample is taken for OD 600 measure (3.); spreading on agar plates containing antibiotics (4.) which are later incubated and colonies are counter after 10 days; and sample is used for re-inoculation in fresh medium with the same concentration of SO 2 (5.). The cycles are repeated until approximate 20 generations, corresponding to 4 cycles.

Figure 3 . 5 .

 35 Figure 3.5. Competition between transformants with different genetic backgrounds. Transformants with different genetic background were put in competition. Each square represents the log 2 of the ratio between strains (1499/1626) on the left and (1499/1608) on the right.

Figure 3 . 6 .

 36 Figure 3.6. Wild-type controls of the competition experiments. WT strains AWRI1499 and AWR1608 were put in competition as controls.

Figure 4 . 1 .

 41 Figure 4.1. Examples of three different growth profiles at four mSO 2 concentrations.

Figure 4 . 2 .

 42 Figure 4.2. Growth parameters of 145 B. bruxellensis isolates grown at different SO 2 concentrations. Growth parameters lag phase (h), maximum growth rate (division per hour), and maximum OD are presented for each isolates. Isolates are grouped by genetic group as defined by Avramova et al., submitted: in order CBS 2499-like group (cyan), L14165-like (lawn green), AWRI1608-like (orange), AWRI1499like (red), dark blue (CBS 3025-like), light blue (L0308-like). Vertical traits present standard deviations.

  to extend the study by performing the experiments in wine and follow the cell viability and physiological state of the cells by methods others than the spectrophotometry (e.g. flow cytometry). Microsatellite markers can therefore present a tool for molecular diagnosis of SO 2 sensitivity in B. bruxellensis. In the future, the combination of population genetics and in-depth genome sequencing, would allow the elucidation of the molecular mechanisms underlying SO 2 tolerance in B. bruxellensis. Different strategies to cope with this toxic compound have been highlighted for the species S. cerevisiae. In particular, active efflux of SO 2 is related to translocations for the latter species. From an evolutionary point of view, it would be intriguing to verify if B. bruxellensis has acquired SO 2 tolerance through other genetic mechanisms, like polyploidy in particular and genomic plasticity in general. By the combination of high-quality genome sequencing and directed genetic transformation, candidate genes could be designated and tested for their contribution to SO 2 tolerance. Better understanding of the mechanism of SO 2 tolerance in B. bruxellensis will possibly give indications for the better management of this key issue in the winemaking context.From a fundamental point of view, we provide a holistic picture of the population genetics of the species B. bruxellensis. Structuring according to substrate of isolation is highlighted, suggesting an influence of human activity on B. bruxellensis population structure. Furthermore, a coexistence of diploid and triploid populations is underscored and we propose that B. bruxellensis could be described as a diploid-triploid complex. These characteristics make of B. bruxellensis a non-conventional model microorganism for the study of polyploid populations and the impact of polyploidy on the adaptation capacities of an organisma concept of industrial and clinical importance. Comparative genomics would allow the construction of more complete picture of the genome dynamics of the species. In particular, comparison between strains from different industrial environments could highlight genomic features associated with adaptation to specific niche-types which would lead to a better understanding of the evolution of the species and its putative relation with human activity. In perspective, a fortuitous isolation of Brettanomyces wild strains would allow the comparison between industrial and natural isolates. Thus, the relation between human activity and B. bruxellensis evolution could be explored and verified. The availability of high-quality sequences could lead to the "discovery" of the mysterious donors of the additional genomes of allopolyploid B. bruxellensis strains.

Figure 1 .

 1 Figure 1. Geographic origin of B. bruxellensis strains used in this study. The substrate of origin is indicated by the colour as follows: red -wine, grape and wine equipment; orange -beer; bluetequila; green -kombucha; violet -bioethanol; yellow -others (cider and other fermented beverages).

Figure 3 .

 3 Figure 3. Growth parameters of different B. bruxellensis strains belonging to three major genetic groups (A -8 strains, B -8 strains and C -17 strains) in media with increasing molecular SO 2 concentrations. Kruskal-Wallis test was performed for every parameter and every group of strains, different letters (a, b, c, d, ab) indicate significantly different mean values at 5% threshold.

Figure 4 .

 4 Figure 4. Result from the PCR to determine if a B. bruxellensis isolate belongs to the group A, B, or C. The 470 bp band is specific to the B. bruxellensis species(Ibeas at al. 1996), a 281 band is specific to strains from group A, a 356 band is specific to strains from group B and there is no band specific to group C. Thus, a single analysis permits (i) to confirm that the isolate belongs to B. bruxellensis species and (ii) to predict its sensitivity to SO 2 . Of the 1280 isolates studied, 435 belong to group A, 206 to group B and 639 to group C. The group A isolates come from various wine regions in France (Bordeaux, Bourgogne, Jura, Languedoc, Côtes du Rhône) but also from Italy and Australia. No link was established between the genetic group and the geographical origin of the strains.

Table 1 .1. Techniques for yeast differentiation at intraspecies level Method a Species References

 1

	b

Table 1 .2. Few examples of hybrid species related to wine, beer, and cider fermentations Parental species Industrial fermentation evironment Industrial relevance References a S. cerevisiae x S. kudravzevii Beer

 1

		Low-temperature	Gonzàlez et al., 2008;
		fermentation;	Belloch et al., 2008
		Adaptation to
		fluctuating conditions;
		Production of glycerol
		and aroma compounds
	Wine	Efficient glucose and
		fructose fermentation;
		Ethanol production;
		Aromatic profile/Ester
		production; Low
		temperature
		fermentation

; S. cerevisiae x S. eubayanus Beer

		(Lager)	Low-temperature	Libkind et al., 2011
			fermentation
	S. cerevisiae x S. bayanus	Beer		Gonzàlez et al., 2008
		Wine		Naumov et al., 2000;
	S. cerevisiae x S. uvarum	Wine		Le Jeune et al., 2007;
	S

. cerevisiae x S. kudravzevii x S. bayanus

	WIne	Borneman et al., 2012;
		Gonzalez et al., 2006
	Cider	Masneuf et al., 1998;
		de Barros Lopes et al.,
		2002;
	a

  , similarly to S. cerevisiae, VBNC state was reported for B. bruxellensis. It was first suggested by du Toit et al. who observed an initial loss of cultivability after SO 2 addition, followed by growth recovery after oxygen administration(Du Toit et al., 2005a). Intrigued by those observations, and the recurrent problem of SO 2 tolerance of B. bruxellensis, other teams studied the phenomena of cultivability loss and growth recovery associated to SO 2 . Agnolucci et al. measured both viability via trypan blue cell staining, and cultivability on agar plates after SO 2 exposure for seven different strains[START_REF] Agnolucci | Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis[END_REF]. Based on those experiments, they claimed that different concentrations of sulfur dioxide were necessary for the induction of VBNC state among strains, and reported the production of vinyl phenols during this putative

metabolically inactive state

[START_REF] Agnolucci | Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis[END_REF]

. Later, Serpaggi et al. reported ethyl phenol production during VBNC state in B. bruxellensis

and methods 2.3.1. Yeast strains B

  . bruxellensis strains used in this study were collected from different origins: i) from CRB Oenologie collection (Centre de Ressources Biologiques Oenologie, Institut des Sciences de la Vigne et du Vin, France), ii) sent from other laboratories, and iii) isolated from wines for the purpose of this work. Overall, the collection of B. bruxellensis used in this study contained 1488 isolates (Supplementary Table online and available upon request) which were further analysed by genotyping.

  Table online and available upon request). The 1488 isolates were genotyped with 12 primer

pairs amplifying microsatellite regions, including four new loci in addition to the eight previously published

(Albertin et al., 2014b)

. Characteristics of the different loci and number of alleles are given in Supplementary Table online and available upon request. One locus out of the four additional loci (D1) displayed a high allelic diversity, presenting 18 different alleles.

Table 2 .1. Clusters considered as a result of the microsatellite analysis and cluster validation witn five different clustering methods

 2

	Group name	Number of	Number of	Putative ploidy (for the	Substrate
		isolates	genotypes	majority of the isolates in the
				group)
	AWRI1499-like	548	197	Triploid	Mostly from wine
	AWRI1608-like	210	127	Triploid	Beer and Wine
	CBS 2499-like	573	208	Diploid	Wine
	L0308-like	37	26	Triploid	Wine
	CBS 5512-like	18	16	Triploid	Bioethanol and tequila
	L14165-like	108	58	Diploid	Kombucha

Table 2 .2. Impact of geographical localisation, substrate origin and ploidy on the population variance (AMOVA test)

 2

	Factor	%Variance p-value
	Country	4.89	<0.0001
	Country (wine isolates)	3.7	<0.0001
	Country (non-wine isolates)	54.8	<0.0001
	Substrate	5.93	<0.0001
	Ploidy	46.9	<0.0001
	2.4.2. Core genotype analysis
	2.4.2.

1 Core diploid data subset

Table 3 .1. Strains used in this study Strain name Source of isolation

 3

			Country of isolation	Ploidy level	Sequence reference SO 2 tolerance
	AWRI1499	Wine	Australia	3n	Curtin et al., 2012 Tolerant
	AWRI1608	Wine	Australia	3n	Borneman et al.,	Sensitive
					2014
	AWRI1626 (=CBS 2499) Wine	France	2n	Piškur et al., 2012 Sensitive

Table 3 .2. Competition couples

 3

	Set 1	Set 2
	AWRI1626 vs AWRI1499 AWRI1608 vs AWRI1499
	1626_N_2 vs 1499_K_3 1608_N_4 vs 1499_K_3
	1626_K_3 vs 1499_N_2 1608_K_1 vs 1499_N_2
	1626_N_6 vs 1499_K_4 1608_N_5 vs 1499_K_4
	1626_K_4 vs 1499_N_4 1608_K_4 vs 1499_N_4
	1626_N_7 vs 1499_K_7 1608_N_7 vs 1499_K_7
	1626_K_6 vs 1499_N_5 1608_K_7 vs 1499_N_5

Table 3 .3. Transformation validation -validated (+) and non-validated (-) parameters

 3

	Transformant	TYPEBrett	Antibiotic	Growth on	Growth YNB
			resistance	reciprocal antibiotic
	1499_K_1	+	+	+	+
	1499_K_2	+	+	+	+
	1499_K_3	+	+	+	+
	1499_K_4	+	+	+	+
	1499_K_5	+	+	+	+
	1499_K_6	+	+	+	+
	1499_K_7	+	+	+	+
	1499_K_8	+	+	+	+
	1499_N_1	+	+	+	+
	1499_N_2	+	+	+	+
	1499_N_3	+	+	+	+
	1499_N_4	+	+	+	+
	1499_N_5	+	+	+	+
	1499_N_6	+	+	+	+
	1499_N_7	+	+	+	+
	1499_N_8	+	+	+	+
	1626_K_1	+	+	+	+
	1626_K_2	+	+	+	+
	1626_K_3	+	+	+	+
	1626_K_4	+	+	+	+
	1626_K_5	+	+	+	+
	1626_K_6	+	+	+	+
	1626_K_7	+	+	+	+
	1626_K_8	+	+	+	+
	1626_N_1	+	+	+	+
	1626_N_2	+	+	+	+
	1626_N_3	+	+	+	+
	1626_N_4	+	+	+	+
	1626_N_5	+	+	+	+
	1626_N_6	+	+	+	+
	1626_N_7	+	+	+	+
	1626_N_8	+	+	-	+
	1608_K_1	+	+	+	+
	1608_K_2	+	+	+	-
	1608_K_3	+	+	+	+
	1608_K_4	+	+	+	+
	1608_K_5	+	+	+	-
	1608_K_6	+	+	+	-
	1608_K_7	+	+	+	+
	1608_K_8	+	+	+	+
	1608_N_1	+	+	+	-
	1608_N_2	+	+	+	+
	1608_N_3	+	+	+	-
	1608_N_4	+	+	+	+
	1608_N_5	+	+	+	+
	1608_N_6	+	+	+	+
	1608_N_7	+	+	+	+
	1608_N_8	+	+	+	-

Table 4 .1. Strains used for sulfur dioxide tolerance assay Strain Substrate Country Vintage Genetic group Phenotype analysis reference

 4

	13EN11C5	Wine	France	2013 CBS 2499-like	Avramova et al., submitted
	13E5A6	Wine	France	2013 CBS 2499-like	This study
	12AVB1	Wine	France	1912 CBS 2499-like	This study
	26AVB2	Wine	France	1926 CBS 2499-like	This study
	59AVB3	Wine	France	1959 CBS 2499-like	This study
	19b/19	Wine	Germany NA	CBS 2499-like	This study
	AWRI 2915	Wine	Australia 2014 CBS 2499-like	This study
	AWRI1615	Wine	Australia 2003 CBS 2499-like	This study
	B001-14 T28 7	Wine	France	2014 CBS 2499-like	This study
	B002-14 T14 7	Wine	France	2014 CBS 2499-like	This study
	CBS 2499	Wine	France	1990 CBS 2499-like	Avramova et al., submitted
	DEN6_12_10	Wine	Denmark 2012 CBS 2499-like	This study
	DEN6_13_2	Wine	Denmark 2012 CBS 2499-like	This study
	DEN612_9	Wine	Denmark 2012 CBS 2499-like	Avramova et al., submitted
	GB64	Wine	Italy	2014 CBS 2499-like	This study
	GB70	Wine	Italy	2014 CBS 2499-like	This study
	ISA2150	Wine	Portugal	2002 CBS 2499-like	This study
	KOM14106	Kombucha France	NA	CBS 2499-like	Avramova et al., submitted
	L02/E2 AZ	Wine	France	NA	CBS 2499-like	Avramova et al., submitted

.2. SO2 tolerance/resistance and relation to genetic group

  Supplementary Table S-4.1 in Appendix), three growth curve types were defined for the different concentrations of SO 2 : i) sensitive strains with significantly longer lag phase and slower growth rate and/or lower maximum OD in presence of increasing concentrations of SO 2 , ii) tolerant strains for which lag phase was longer when SO 2 was present but the others parameters remained unchanged, and iii) resistant strains for which all parameters are not significantly impacted whatever the concentrations of SO 2 considered (examples of each growth profile are shown on Figure 4.1 with the respective values for different growth parameters). Differences in growth parameters were presented per genetic group (as defined by microsatellite analysis) (Figure 4.2, Supplementary Figure S-4.1 in Appendix, Table4.2). It can be noted that strains that belong to the same genetic cluster have similar response to SO 2 in

	4.3

1

) at least in triplicate. The 145 strains had different response to sulphur dioxide concentrations in means of lag phase, growth rate, and maximum OD. According to those growth parameters' variation, (means of growth profile. Also, 48 out of 52 tolerant or resistant strains (Table

4

.2) are isolated from wine

(Table 4.1)

. All tolerant and resistant strains were isolated after 1990, and from

Table 4 .2. Number of isolates by genetic group and phenotype

 4

	Genetic group	Sensitive Tolerant Resistant	Total
	CBS 2499-like	38	1	3	42
	L14165-like	14	3	1	18
	AWRI1608-like	27	2	1	30
	AWRI1499-like	4	7	21	32
	CBS 5512-like	10	0	1	11
	L308-like	0	1	11	12
	Total	93	14	38	145

  at a finer scale. Phenotypic experiments combined with deeper knowledge of the genetics of the species would be useful for revealing the mechanisms behind those different phenotypes. The striking correlation between SO 2 tolerance and specific genotypic configuration presents a great tool for the evaluation of SO 2 sensitivity of resident B. bruxellensis strains. In the future, a finer diagnosis of B. bruxellensis spoilage population in means of genetic group, related SO 2 survival behaviour, and population level, would contribute to the use of optimal SO 2 concentrations at adequate time intervals, thus leading to more efficient SO 2 management. between genotype and sulphite sensitivity was demonstrated for a large collection of isolates representative of the global B. bruxellensis population.iii)Wine triploid AWRI1499-like strains are tolerant to SO 2 concentrations as high as 0.6 mg.L -1 mSO 2 and are highly disseminated across wine producing regions worldwide.iv)Competition experiments confirmed that tolerance to SO 2 confers a selective advantage compared to sensitive strains in medium containing SO 2 ; this indicates specific adaptation of SO 2 tolerant B. bruxellensis populations through a human-

	Chapter 6. Conclusions
	The present work led to several key findings: Supplementary Table S-3.1. (continued) AWRI1499 vs AWRI1608
				RAW
	SO2	Couple	Doublings	0	4	8	12	16	20
	i)	Polyploidy is a highly represented state among B. bruxellensis species revealing a 0 1499 vs 1499_K_3 4.60E+05 7.48E+06 2.38E+06 8.52E+04 1.65E+04 1.60E+03
		0 1499 vs 1608_N_4	3.80E+05 1.44E+07 8.13E+06 5.08E+06 1.46E+07 1.25E+07
		0 1499 vs 1499_N_2 diploid-triploid complex structured according to substrate of isolation and 5.66E+04 1.32E+06 3.60E+05 2.21E+04 1.40E+03 3.00E+02 0 1499 vs 1608_K_1 1.56E+05 1.39E+07 1.23E+07 8.31E+06 1.18E+07 8.64E+06
		0 1499 vs 1499_K_4 geographic origin. 0 1499 vs 1608_N_5	2.99E+04 7.69E+04 1.93E+04 1.94E+04 2.50E+04 1.16E+03 2.02E+05 2.62E+06 8.15E+06 2.00E+07 4.53E+07 7.40E+06
	ii)	0 1499 vs 1499_N_4 0 1499 vs 1608_K_4 0 1499 vs 1499_K_7 0 1499 vs 1608_N_7 0 1499 vs 1499_N_5 0 1499 vs 1608_K_7 0.2 1499 vs 1499_K_3 0.2 1499 vs 1608_N_4 0.2 1499 vs 1499_N_2 0.2 1499 vs 1608_K_1 0.2 1499 vs 1499_K_4 0.2 1499 vs 1608_N_5 0.2 1499 vs 1499_N_4 0.2 1499 vs 1608_K_4 0.2 1499 vs 1499_K_7 0.2 1499 vs 1608_N_7 0.2 1499 vs 1499_N_5 0.2 1499 vs 1608_K_7 0.4 1499 vs 1499_K_3 A strong link related selection process. 7.20E+05 2.14E+05 1.66E+03 6.60E+02 3.99E+02 3.99E+02 6.60E+05 2.72E+06 6.40E+06 4.93E+07 2.05E+07 9.47E+06 6.17E+04 2.78E+06 4.60E+05 4.27E+04 2.85E+03 8.00E+02 2.10E+05 2.10E+07 1.01E+07 1.03E+07 1.20E+07 2.27E+07 1.10E+06 2.37E+05 3.54E+04 2.36E+03 3.99E+02 3.99E+02 1.14E+04 1.91E+07 1.02E+07 7.17E+06 8.32E+06 9.52E+06 4.60E+05 1.97E+05 4.80E+05 1.42E+04 5.00E+02 3.99E+02 3.80E+05 1.52E+06 1.80E+06 5.28E+06 2.76E+06 2.70E+06 5.66E+04 1.04E+05 1.66E+03 3.99E+02 3.99E+02 3.99E+02 1.56E+05 2.72E+06 1.00E+06 6.47E+06 2.30E+06 5.20E+06 2.99E+04 5.66E+04 6.20E+02 3.99E+02 NA NA 2.02E+05 3.12E+06 9.80E+05 1.96E+06 NA NA 7.20E+05 6.60E+05 5.40E+04 7.80E+02 NA NA 6.60E+05 3.12E+06 8.80E+05 3.88E+06 NA NA 6.17E+04 2.15E+05 5.95E+04 5.00E+02 3.99E+02 3.99E+02 2.10E+05 2.38E+06 7.60E+05 1.60E+06 4.04E+06 5.53E+06 1.10E+06 4.34E+04 5.40E+02 3.99E+02 3.99E+02 3.99E+02 1.14E+04 2.58E+06 1.44E+06 8.80E+05 5.03E+06 1.50E+06 4.60E+05 4.20E+05 1.11E+05 3.99E+02 3.99E+02 3.99E+02 0.4 1499 vs 1608_N_4 3.80E+05 3.40E+05 5.00E+05 1.62E+06 2.00E+06 2.12E+06
	v)	0.4 1499 vs 1499_N_2 Three B. bruxellensis strains with different ploidy levels and genetic backgrounds 5.66E+04 2.42E+06 1.04E+03 4.67E+03 3.99E+02 3.99E+02 0.4 1499 vs 1608_K_1 1.56E+05 9.60E+05 1.12E+06 6.20E+06 1.32E+06 2.02E+06
		0.4 1499 vs 1499_K_4 were successfully transformed. 2.99E+04 1.53E+05 1.28E+06 3.56E+06 3.56E+06 7.00E+05 0.4 1499 vs 1608_N_5 2.02E+05 2.30E+06 1.31E+06 3.92E+06 3.92E+06 1.63E+07
		0.4 1499 vs 1499_N_4	7.20E+05 1.48E+06 1.08E+07 1.15E+07 1.09E+05 7.00E+05
	vi)	Microsatellite analysis can be used for molecular diagnosis of SO 2 sensitivity of B. 0.4 1499 vs 1608_K_4 6.60E+05 1.38E+06 1.31E+07 4.52E+06 5.06E+06 3.99E+02
		0.4 1499 vs 1499_K_7	6.17E+04 1.14E+05 3.60E+05 3.99E+02 3.99E+02 3.99E+02
		bruxellensis strains. 0.4 1499 vs 1608_N_7	2.10E+05 1.81E+05 2.22E+06 2.57E+06 3.96E+06 1.42E+06
		0.4 1499 vs 1499_N_5	1.10E+06 3.60E+05 1.95E+07 6.61E+04 4.60E+05 6.03E+06
		0.4 1499 vs 1608_K_7	1.14E+04 7.80E+05 1.01E+07 1.20E+06 8.60E+05 3.04E+06
		0.6 1499 vs 1499_K_3	4.60E+05 5.29E+07 2.72E+06 8.71E+06 5.95E+07 3.60E+06
		0.6 1499 vs 1608_N_4	3.80E+05 7.20E+05 1.33E+04 1.72E+05 4.80E+05 2.18E+04
		0.6 1499 vs 1499_N_2	5.66E+04 1.50E+06 2.14E+05 2.28E+06 4.00E+06 1.93E+04
		0.6 1499 vs 1608_K_1	1.56E+05 1.04E+05 1.10E+06 2.46E+06 5.19E+06 5.80E+05
		0.6 1499 vs 1499_K_4	2.99E+04 5.07E+06 4.78E+07 3.83E+07 7.95E+07 7.03E+07
		0.6 1499 vs 1608_N_5	2.02E+05 1.86E+05 1.98E+05 2.23E+05 1.93E+04 4.57E+03
		0.6 1499 vs 1499_N_4	7.20E+05 6.28E+07 2.25E+07 2.16E+07 1.63E+05 NA
		0.6 1499 vs 1608_K_4	6.60E+05 1.61E+04 3.99E+02 3.99E+02 3.99E+02 NA
		0.6 1499 vs 1499_K_7	6.17E+04 2.10E+06 3.03E+07 5.17E+06 2.84E+06 5.28E+06
		0.6 1499 vs 1608_N_7	2.10E+05 3.99E+02 3.99E+02 3.99E+02 3.99E+02 3.99E+02
		0.6 1499 vs 1499_N_5	1.10E+06 3.92E+06 1.08E+03 3.99E+02 NA	NA
		0.6 1499 vs 1608_K_7	1.14E+04 1.84E+06 2.20E+05 7.00E+05 NA	NA

Scientific teams from all over the world have focused their work on the genome sequencing of different strains of B. bruxellensis. Thanks to this recent knowledge, and particularly the sequences provided by the work ofCurtin et al. (2012b), our team developed 12 genetic markers based on

Acknowledgements

Acknowledgments

The authors would like to thank the CIVB (Conseil Interprofessionnel du Vin de Bordeaux) and Région Aquitaine for the financial support. Also, the authors thank Lucile Pic (ICV, France), Viriginie Serpaggi (Inter Rhône, France), Manuel Ferreira (Technical University of Lisbon, Portugal), Marcos Morais (Universidade Federal de Pernambuco, Brazil), Morvan Koarer (IFV, France), Microflora (France), Marc-André Lachance (University of Western Ontario, Canada), Marina Bely (ISVV, France), Cécile Thibon (ISVV, France), Hervé Alexandre (Université de Bourgogne, France), Cédric Longin (Université de Bourgogne, France), Iavor Tchobanov (University of Food Technology, Plovdiv, Bulgaria), and all organisations, researchers and winemakers who provided isolates and contaminated wine samples for the purpose of this study.

construction of the project and for yours ideas, excitement and guidance throughout the project. Paul, thank

References 145

Transformants for the three strains, AWRI1499, AWRI1608 and AWRI1626 were assessed by pairwise competition experiments (AWRI1499 vs AWRI1626, and AWRI1499 vs AWRI1608). For AWRI1499 vs AWRI1626, two growth patterns were observed (Figure 3.5). At low mSO 2 concentrations (0 and 0.2 mg.L -1 ), AWRI1626 transformants outcompeted AWRI1499 transformants and this phenomenon is maintained and/or intensified over time depending on the competing pair. Although AWRI1626 transformants dominated over AWRI1499 transformants, AWRI1499 isolates showed moderate population numbers (in the order of 10 5 -10 6 UFC.mL -1 , raw count numbers are shown in Supplementary Table S-3.1 in Appendix). On the contrary, at high mSO 2 concentrations (0.4 and 0.6 mg.L -1 ), AWRI1499 transformants outcompeted AWRI1626 isolates and this dominance seemed more intense for 0.6 mg.L -1 of mSO 2 than for 0.4 mg.L -1 . No differences were observed between the pairs 1499_K vs 1626_N and 1499_N vs 1626_K, suggesting that there is no effect of the type of cassette on the competition behaviour of the transformants. importance of this non-conventional microorganism, there is no protocol for its directed genetic transformation. Protocols for undirected genetic transformation of B. bruxellensis strains with both auxotrophic [START_REF] Schifferdecker | Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast[END_REF] and antibiotic resistance markers [START_REF] Miklenić | Improved electroporation procedure for genetic transformation of Dekkera/Brettanomyces bruxellensis[END_REF][START_REF] Miklenić | Genetic transformation of the yeast Dekkera/Brettanomyces bruxellensis with non-homologous DNA[END_REF] were made available for the scientific community, but are still not broadly applied. Here, we used an optimised protocol for transformation with antibiotic resistance markers method based on non-homologous end joining for three previously sequenced B.

bruxellensis strains (Borneman et al., 2014;Curtin et al., 2012;[START_REF] Piškur | The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties[END_REF]. We report the first successful genetic transformation of triploid B. bruxellensis strains to our knowledge -AWRI1499 and AWRI1608. The transformation protocol used in this study led to the construction of phenotypically neutral strains in means of cross-resistance and growth behaviour and opens the way for further genetic studies on this non-conventional yeast.

Previous studies highlighted major genetic and phenotypic differences between the strains AWRI1499, AWRI1608 and CBS 2499 (=AWRI1626) underscoring differences in ploidy level and genetic configuration. Here, we use those three strains as representatives of the major genetic clusters of the global B. bruxellensis population (Avramova et al., submitted), characterised by different ploidy level (diploid and triploid) and sulphite sensitivity (tolerant and sensitive). Pairwise competitions showed that, at low SO 2 concentrations, the diploid AWRI1626 strain has a relative selective advantage compared to the allotriploid AWRI1499 until a switching point (>0.2 mg.L -1 mSO 2 ), when AWRI1499 takes over AWRI1626. Similar situation was observed for AWRI1608 vs AWRI1499 (both triploid) but with a switching point at 0.4 mg.L -1 mSO 2 . Strikingly, in the case of B. bruxellensis, all highly SO 2 tolerant wine strains are shown to be triploid (see Avramova et al., submitted). Stable polyploid state is thought to be maintained only if it confers selective advantage to the cell [START_REF] Wertheimer | Ploidy dynamics and evolvability in fungi[END_REF], despite the possible slowing down of growth rate and cell metabolism [START_REF] Otto | The Evolutionary Consequences of Polyploidy[END_REF].

Polyploidy was shown to confer adaptive capacity to yeast cells through higher rate of

Chapter 4. Molecular diagnosis of Brettanomyces bruxellensis' sulfur dioxide sensitivity through genotype specific method

Marta Avramova 1,2 , Amélie Vallet-Courbin 3 , Julie Maupeu 3 , Isabelle Masneuf-Pomarède 1,4 , Warren Albertin Curtin et al., 2012). This hypothesis is also phenotypically supported as all resistant and tolerant strains were isolated from wines from recent vintages (after 1990) (Table 4.1). Furthermore, the strain AWRI1499 (a representative of allotriploid AWRI1499-like group) was shown to have a specific selective advantage in presence of SO 2 when compared to two other wine strains representatives of diploid CBS 2499like and triploid AWRI1608-like groups (Avramova et al., in preparation). This data taken together highlights the role of SO 2 , and therefore human activity, in shaping B. bruxellensis population structure, which was discussed in the previous chapters.

Sulphur dioxide resistance is broadly studied in S. cerevisiae and the main mechanisms related to this phenotype is efflux through Ssu1p active pump [START_REF] Nardi | A sulphite-inducible form of the sulphite efflux gene SSU1 in a Saccharomyces cerevisiae wine yeast[END_REF][START_REF] Park | SSU1 mediates sulphite efflux in Saccharomyces cerevisiae[END_REF][START_REF] Pérez-Ortín | Molecular characterization of a chromosomal rearrangement involved in the adaptive evolution of yeast strains[END_REF]. It was demonstrated that SSU1-R allele which is involved in SO 2 resistance, is the product of reciprocal translocation between chromosomes VII and XVI, thus highlighting the importance of gross chromosomal rearrangements in the adaptive evolution of S. cerevisiae [START_REF] Pérez-Ortín | Molecular characterization of a chromosomal rearrangement involved in the adaptive evolution of yeast strains[END_REF]. Later, another translocation involved in SO 2 tolerance (XV-t-XVI) was shown to shorten lag phase in presence of SO 2 , thus conferring relative selective advantage compared to nt-XVI strains [START_REF] Zimmer | QTL Dissection of Lag Phase in Wine Fermentation Reveals a New Translocation Responsible for Saccharomyces cerevisiae Adaptation to Sulfite[END_REF]. Following those studies, it was suggested that those translocations were empirically selected by humans [START_REF] Pérez-Ortín | Molecular characterization of a chromosomal rearrangement involved in the adaptive evolution of yeast strains[END_REF][START_REF] Zimmer | QTL Dissection of Lag Phase in Wine Fermentation Reveals a New Translocation Responsible for Saccharomyces cerevisiae Adaptation to Sulfite[END_REF]. The lack of effect of SO 2 on lag phase observed for the resistant B. bruxellensis strains could be related to similar mechanisms. Indeed, allele specific expression of efflux pump BbSSU1 was detected by comparative transcriptomics (Curtin et al., ISSY2015 abstract). The mechanisms related to the resistant phenotype in B. bruxellensis remain to be elucidated. As for the tolerant strains, the longer lag phase would reflect the time needed for the adaptation through complex mechanisms or the survival until a lower mSO 2 concentration is attained in the medium. Using staining with propidium iodide detection by flow cytometer analysis (Longin et al., 2016a), Longin et al. showed that sulphite induces

Chapter 5. Discussion and perspectives

Brettanomyces bruxellensis is a yeast species that was known for more than a century and is associated to a peculiar aromatic profile in various fermented beverages. First described in beer, B. bruxellensis was revealed as an essential contributor to the organoleptic qualities of some specialty beer-types. In wine however, this same yeast is undesirable due to the production of ethyl phenols related to unpleasant aromas. Spoilage by this microorganism leads to consumers' rejection and subsequent economic losses for winemakers. Strains of this species were isolated from other fermented products like kombucha, cider, olives, etc. In particular, B. bruxellensis is of great industrial interest in the context of bioethanol production plants where it was initially considered a contamination organism but could be potentially used for the molasses fermentation. Thus, B. bruxellensis develops in multiple industrial fermentation environments where its presence (in some cases essential, whereas in othersundesirable) rarely remains unnoticed. It was mainly the wine spoilage potential of B. bruxellensis that drew great attention on this species on behalf of the scientific community over the last twenty years. Subsequently, high diversity was underscored for B. bruxellensisboth at the phenotypic and genetic levels. However, those studies remained marginal and didn't lead to a holistic picture of the species. To address this problem, we performed a population genetics study on a large number of B. bruxellensis strains through microsatellite markers analysis. This genotyping method has the advantage to give indication on the genetic relations between strains and the ploidy level of a given organism. Indeed, ploidy is an essential parameter to take into account when studying B. bruxellensis, as strains with different ploidy level were highlighted following whole genome sequencing. The results presented here confirm on a large scale the assumption that B. bruxellensis population is composed of strains with different ploidy level, as 57.8% of the isolates were shown to have more than 2 alleles for at least one locus. The studied population is structured according to ploidy level, substrate of isolation and geographical origin of the strains, underlying the relative importance of each factor.

By the use of microsatellite analysis, we were able to confirm the triploid state of AWRI1499like and AWRI1608-like strains, and we suggest the presence of other triploid groups, namely L0308-like wine group and L14165-like tequila/bioethanol group. Those results lead to the assumption that multiple polyploidisation events occurred among B. bruxellensis population and that polyploidy could present and adaptation mechanism to various environment types.

Another interesting observation concerning the ploidy level of the species was that among the numerous and various isolates analysed, there was high heterozygosity level and no putative haploids, supporting the general view that B. bruxellensis could be an asexual organism. In perspective, the developed microsatellite genotyping method and the knowledge of the overall population structure of the species will allow to take into account the genetic diversity of B. bruxellensis when evaluating its phenotypic properties by choosing relevant representatives of the population for the experiments. Multiple morphological and physiological aspects on cell and population level remain to be explored. Namely, other parameters like pseudohyphal growth or tolerance to antimicrobial agents could be studied in the context of winemaking and other industrial fermentation environments. Subsequently, this would lead to better knowledge of the adaptation mechanisms of this yeast, as well as the factors that impact its survival, growth, and spoilage potential. Furthermore, the putative role of the triploid state of beer and bioethanol strains for the adaptation to their respective fermentation environments could be explored.

Intriguingly, the triploid AWRI1499-like strains presented the majority of Australian B. bruxellensis isolates from wine. Thus, a possible link between the polyploid state of those strains and the adaptation to winemaking environment was suggested. Here, we demonstrated that isolates from the AWRI1499-like genetic group: i) were highly disseminated in different

Appendix

Supplementary

Written communications

Why is the genetic diversity of Brettanomyces bruxellensis important for winemakers and is it related to sulfur dioxide tolerance?

M. Avramova 1,6 , A. Cibrario 1 , M. Coton 2 , E. Coton 2 , F. Salin 3 , W. Albertin 1,4 , C. Curtin 6 , I. Masneuf-Pomarède 1,5

Abstract

The environmental conditions of wine are unfavourable for growth of many microorganisms; however, Brettanomyces bruxellensis is highly adapted to the winemaking process, which implies resistance to sulfur dioxide, high ethanol tolerance, growth on limited nitrogen sources and tolerance of low pH. This yeast's metabolism results in an alteration of the wine's flavour profile (unpleasant 'leathery' and/or 'mousy' characters), thus leading to economic losses. B. bruxellensis is also associated with other industrial fermentations such as beer, cider, kombucha (fermented tea), kefir and bioethanol. In these products, the desirability/undesirability of this yeast is unclear and still debated.

The industrial importance of B. bruxellensis has led to the study of its genome and population structure. Previous studies revealed a high genotypic diversity at intra-species level and that phenotypic characteristics are strain-dependent. Furthermore, a comparison of genome assemblies revealed the coexistence of diploid and triploid populations and high dissemination of a triploid population in wine fermentations in Australia. We have conducted a genotyping study of a large population of B. bruxellensis isolates from five continents and different substrates using microsatellite markers. The results suggest that B. bruxellensis species is structured according to ploidy level and substrate. The potential contribution of the triploid state to the adaptation to industrial fermentations and to the dissemination of B. bruxellensis is discussed. This works focuses on the ability of different strains of B. bruxellensis to survive in the presence of sulfur dioxide.