Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non-Saccharomyces origin. We identified Zygosaccharomyces bailii, a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and nonSaccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations. adaptive evolution ͉ comparative genomics ͉ horizontal gene transfer ͉ introgression ͉ Zygosaccharomyces bailii

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1–D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5–6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

Polyploidization is an important process in the evolution of eukaryotic genomes, but ensuing molecular mechanisms remain to be clarified. Autopolyploidization or whole-genome duplication events frequently are resolved in resulting lineages by the loss of single genes from most duplicated pairs, causing transient gene dosage imbalance and accelerating speciation through meiotic infertility. Allopolyploidization or formation of interspecies hybrids raises the problem of genetic incompatibility (Bateson-Dobzhansky-Muller effect) and may be resolved by the accumulation of mutational changes in resulting lineages. In this article, we show that an osmotolerant yeast species, Pichia sorbitophila, recently isolated in a concentrated sorbitol solution in industry, illustrates this last situation. Its genome is a mosaic of homologous and homeologous chromosomes, or parts thereof, that corresponds to a recently formed hybrid in the process of evolution. The respective parental contributions to this genome were characterized using existing variations in GC content. The genomic changes that occurred during the short period since hybrid formation were identified (e.g., loss of heterozygosity, unilateral loss of rDNA, reciprocal exchange) and distinguished from those undergone by the two parental genomes after separation from their common ancestor (i.e., NUMT (NUclear sequences of MiTochondrial origin) insertions, gene acquisitions, gene location movements, reciprocal translocation). We found that the physiological characteristics of this new yeast species are determined by specific but unequal contributions of its two parents, one of which could be identified as very closely related to an extant Pichia farinosa strain.

The evolutionary history of the characters underlying the adaptation of microorganisms to food and biotechnological uses is poorly understood. We undertook comparative genomics to investigate evolutionary relationships of the dairy yeast Geotrichum candidum within Saccharomycotina. Surprisingly, a remarkable proportion of genes showed discordant phylogenies, clustering with the filamentous fungus subphylum (Pezizomycotina), rather than the yeast subphylum (Saccharomycotina), of the Ascomycota. These genes appear not to be the result of Horizontal Gene Transfer (HGT), but to have been specifically retained by G. candidum after the filamentous fungi–yeasts split concomitant with the yeasts’ genome contraction. We refer to these genes as SRAGs (Specifically Retained Ancestral Genes), having been lost by all or nearly all other yeasts, and thus contributing to the phenotypic specificity of lineages. SRAG functions include lipases consistent with a role in cheese making and novel endoglucanases associated with degradation of plant material. Similar gene retention was observed in three other distantly related yeasts representative of this ecologically diverse subphylum. The phenomenon thus appears to be widespread in the Saccharomycotina and argues that, alongside neo-functionalization following gene duplication and HGT, specific gene retention must be recognized as an important mechanism for generation of biodiversity and adaptation in yeasts.

The molecular and evolutionary processes underlying fungal domestication remain largely unknown despite the importance of fungi to bioindustry and for comparative adaptation genomics in eukaryotes. Wine fermentation and biological ageing are performed by strains of S. cerevisiae with, respectively, pelagic fermentative growth on glucose and biofilm aerobic growth utilizing ethanol. Here, we use environmental samples of wine and flor yeasts to investigate the genomic basis of yeast adaptation to contrasted anthropogenic environments. Phylogenetic inference and population structure analysis based on single nucleotide polymorphisms revealed a group of flor yeasts separated from wine yeasts. A combination of methods revealed several highly differentiated regions between wine and flor yeasts, and analyses using codon-substitution models for detecting molecular adaptation identified sites under positive selection in the high-affinity transporter gene ZRT1. The cross-population composite likelihood ratio revealed selective sweeps at three regions, including in the hexose transporter gene HXT7, the yapsin gene YPS6 and the membrane protein coding gene MTS27. Our analyses also revealed that the biological ageing environment has led to the accumulation of numerous mutations in proteins from several networks, including Flo11 regulation and divalent metal transport. Together, our findings suggest that the tuning of FLO11 expression and zinc transport networks are a distinctive feature of the genetic changes underlying the domestication of flor yeasts. Our study highlights the multiplicity of genomic changes underlying yeast adaptation to man-made habitats and reveals that flor/wine yeast lineage can serve as a useful model for studying the genomics of adaptive divergence.

The delineation of species among strains assigned to Debaryomyces hansenii was examined using a gene genealogies-based approach in order to compare spliceosomal intron sequences found in four housekeeping genes (ACT1, TUB2, RPL31 and RPL33). This revealed four distinct groups of strains containing, respectively, D. hansenii var. hansenii CBS 767 T , D. hansenii var. fabryi CBS 789 T , Candida famata var. flareri CBS 1796 T (the anamorph of D. hansenii var. fabryi CBS 789 T ) and Debaryomyces tyrocola CBS 766 T , whose species status was no longer accepted. The sequence divergence between these groups, reaching in some cases over 20 %, unambiguously isolated the groups as separate taxa, leading to a proposal for the reinstatement of the originally described species D. hansenii CBS 767 T and D. tyrocola CBS 766 T . The variety D. hansenii var. fabryi was further subdivided into two taxa, Debaryomyces fabryi CBS 789 T and Candida flareri CBS 1796 T (previously C. famata var. flareri and Blastodendrion flareri). The comparison of intron sequences therefore exposed cryptic species whose phenotypic traits are not distinguishable from known species, but which have significantly diverged from the genetic point of view. Hence, we describe the new taxon Debaryomyces macquariensis sp. nov. CBS 5571 T is related to, but clearly distinct from, the Debaryomyces species mentioned above. The approach used in this work has also revealed the existence of populations within the newly delineated species D. hansenii and genetic exchanges between these populations, indicating an unexpected genetic diversity within this part of the genus Debaryomyces.

Geotrichum candidum is a ubiquitous yeast and an essential component in the production of many soft cheeses. We developed a multilocus sequence typing (MLST) scheme with five retained loci (NUP116, URA1, URA3, SAPT4 and PLB3) which were sufficiently divergent to distinguish 40 sequence types (STs) among the 67 G. candidum strains tested. Phylogenetic analyses defined five main clades; one clade was restricted to environmental isolates, three other clades included distinct environmental isolates and dairy strains, while the fifth clade comprised 34 strains (13 STs), among which all but two were isolated from milk, cheese or the dairy environment. These findings suggest an adaptation to the dairy ecosystems by a group of specialized European G. candidum strains. In addition, we developed a polymerase chain reaction inter-long terminal repeat scheme, a fast and reproducible random amplification of polymorphic DNA-like method for G. candidum, to type the closely related dairy strains, which could not be distinguished by MLST. Overall, our findings distinguished two types of dairy strains, one forming a homogeneous group with little genetic diversity, and the other more closely related to environmental isolates. Neither regional nor cheese specificity was observed in the dairy G. candidum strains analysed. This present study sheds light on the genetic diversity of both dairy and environmental strains of G. candidum and thus extends previous characterizations that have focused on the cheese isolates of this species. Copyright © 2016 John Wiley & Sons, Ltd.

Among ascomycetous yeasts, the CTG clade is so-called because its constituent species translate CTG as serine instead of leucine. Though the biology of certain pathogenic species such as Candida albicans has been much studied, little is known about the life cycles of non-pathogen species of the CTG clade. Taking advantage of the recently obtained sequence of the biotechnological Millerozyma (Pichiasorbitophila) farinosa strain CBS 7064, we used MLST to better define phylogenic relationships between most of the Millerozyma farinosa strains available in public collections. This led to the constitution of four phylogenetic clades diverging from 8% to 15% at the DNA level and possibly constituting a species complex (M. farinosa) and to the proposal of two new species:Millerozyma miso sp. nov. CBS 2004T ( = CLIB 1230T) and Candida pseudofarinosa sp. nov.NCYC 386T( = CLIB 1231T).Further analysis showed that M. farinosa isolates exist as haploid and inter-clade hybrids. Despite the sequence divergence between the clades, secondary contacts after reproductive isolation were evidenced, as revealed by both introgression and mitochondria transfer between clades. We also showed that the inter-clade hybrids do sporulate to generate mainly viable vegetative diploid spores that are not the result of meiosis, and very rarely aneuploid spores possibly through the loss of heterozygosity during sporulation. Taken together, these results show that in this part of the CTG clade, non-Mendelian genetic exchanges occur at high rates through hybridization between divergent strainsfrom distinct clades and subsequent massive loss of heterozygosity. This combination of mechanisms could constitute an alternative sexuality leading to an unsuspected biodiversity.