Tempo and Mode of Genome Evolution in the Budding Yeast Subphylum.

Budding yeasts (subphylum Saccharomycotina) are found in every biome and are as genetically diverse as plants or animals. To understand budding yeast evolution, we analyzed the genomes of 332 yeast species, including 220 newly sequenced ones, which represent nearly one-third of all known budding yeast diversity. Here, we establish a robust genus-level phylogeny comprising 12 major clades, infer the timescale of diversification from the Devonian period to the present, quantify horizontal gene transfer (HGT), and reconstruct the evolution of 45 metabolic traits and the metabolic toolkit of the budding yeast common ancestor (BYCA). We infer that BYCA was metabolically complex and chronicle the tempo and mode of genomic and phenotypic evolution across the subphylum, which is characterized by very low HGT levels and widespread losses of traits and the genes that control them. More generally, our results argue that reductive evolution is a major mode of evolutionary diversification.

Taxogenomic placement of Rasporella oleae and Rasporella dianae gen. and spp. nov., two insect associated yeast species.

During the course of independent studies in Europe, North America, and Africa, seven yeast strains were isolated from insect frass, decaying wood, tree flux, and olive oil sediment. Phylogenetic analysis of two barcoding DNA regions (internal transcribed spacer and the D1/D2 domain of the LSU rRNA gene) revealed that they belong to two closely related undescribed species distinct from all genera in the family Debaryomycetaceae. For reliable taxonomic placement the genomes of four strains of the two novel species and six type strains of closely related species were sequenced. Orthologous genes from 54 genomes of representatives of the Pichiomycetes and 23 outgroup taxa were concatenated to construct a fully supported phylogenetic tree. Consistent with the assumptions, we found that the two new species belong to a novel genus. In addition, the delimitation of the novel species was supported by genetic distance calculations from average nucleotide identity (ANI) and digital DNA:DNA hybridization (dDDH) values. The physiological characterization of the novel species was generally consistent with their genomic content. All strains had two alleles encoding secretory lipase in either two or three copies depending on the species. However, lipolytic activity was detected only in strains with three copies of the secretory lipase gene. Nevertheless, lipolytic activity might be related to their association with the insect gut. Based on these results, formal descriptions of the new genus Rasporella gen. nov. and of two new species Rasporella dianae sp. nov. (holotype UCDFST 68-643T , MycoBank no.: 850238) and Rasporella oleae sp. nov. (holotype ZIM 2471T , MycoBank no.: 850126) are provided.

Yeasts from temperate forests.

Yeasts are ubiquitous in temperate forests. While this broad habitat is well-defined, the yeasts inhabiting it and their life cycles, niches, and contributions to ecosystem functioning are less understood. Yeasts are present on nearly all sampled substrates in temperate forests worldwide. They associate with soils, macroorganisms, and other habitats and no doubt contribute to broader ecosystem-wide processes. Researchers have gathered information leading to hypotheses about yeasts' niches and their life cycles based on physiological observations in the laboratory as well as genomic analyses, but the challenge remains to test these hypotheses in the forests themselves. Here, we summarize the habitat and global patterns of yeast diversity, give some information on a handful of well-studied temperate forest yeast genera, discuss the various strategies to isolate forest yeasts, and explain temperate forest yeasts' contributions to biotechnology. We close with a summary of the many future directions and outstanding questions facing researchers in temperate forest yeast ecology. Yeasts present an exciting opportunity to better understand the hidden world of microbial ecology in this threatened and global habitat.

Extensive loss of cell-cycle and DNA repair genes in an ancient lineage of bipolar budding yeasts.

Cell-cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low Guanine-Cytosine (GC) content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18/21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, a faster-evolving lineage (FEL), which began diversifying approximately 87 million years ago (mya), and a slower-evolving lineage (SEL), which began diversifying approximately 54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. E.g., all species lost the cell-cycle regulator WHIskey 5 (WHI5), and the FEL lost components of the spindle checkpoint pathway (e.g., Mitotic Arrest-Deficient 1 [MAD1], Mitotic Arrest-Deficient 2 [MAD2]) and DNA-damage-checkpoint pathway (e.g., Mitosis Entry Checkpoint 3 [MEC3], RADiation sensitive 9 [RAD9]). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene 3-MethylAdenine DNA Glycosylase 1 (MAG1), which is part of the base-excision repair pathway, and the DNA photolyase gene PHotoreactivation Repair deficient 1 (PHR1), which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POLymerase 4 [POL4] and POL32) and telomere-associated genes (e.g., Repressor/activator site binding protein-Interacting Factor 1 [RIF1], Replication Factor A 3 [RFA3], Cell Division Cycle 13 [CDC13], Pbp1p Binding Protein [PBP2]). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell-cycle and genome integrity genes and pathways, and may represent a novel, to our knowledge, system for studying cellular life without them.

Clavispora santaluciae f.a., sp. nov., a novel ascomycetous yeast species isolated from grapes.

During a study of yeast diversity in Azorean vineyards, four strains were isolated which were found to represent a novel yeast species based on the sequences of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and of the D1/D2 domain of the large subunit (LSU) rRNA gene, together with their physiological characteristics. An additional strain isolated from Drosophila suzukii in Italy had identical D1/D2 sequences and very similar ITS regions (five nucleotide substitutions) to the Azorean strains. Phylogenetic analysis using sequences of the ITS region and D1/D2 domain showed that the five strains are closely related to Clavispora lusitaniae, although with 56 nucleotide differences in the D2 domain. Intraspecies variation revealed between two and five nucleotide differences, considering the five strains of Clavispora santaluciae. Some phenotypic discrepancies support the separation of the new species from their closely related ones, such as the inability to grow at temperatures above 35 °C, to produce acetic acid and the capacity to assimilate starch. Neither conjugations nor ascospore formation were observed in any of the strains. The name Clavispora santaluciae f.a., sp. nov., is proposed to accommodate the above noted five strains (holotype, CBS 16465T; MycoBank no., MB 835794).

Starmerella vitis f.a., sp. nov., a yeast species isolated from flowers and grapes.

A novel yeast species of Starmerella vitis f.a. sp. nov. is proposed to accommodate five strains isolated from flowers, grapes and an insect in the Azores, Canada, Hungary, Palau and Taiwan. As the strains were genetically distinct, we used parsimony network analysis based on ITS-D1/D2 sequences to delineate the species in a statistically objective manner. According to sequence comparisons and phylogenetic analysis, the novel species is most closely related to Starmerella lactis-condensi. The two species cannot be distinguished by conventional physiological tests. The type strain of Starmerella vitis f.a., sp. nov. is CBS 16418T; Mycobank number MB 835251.

Isolation and Identification of Indigenous Wine Yeasts and Their Use in Alcoholic Fermentation.

RESEARCH BACKGROUND: In our study, spontaneous alcoholic fermentations were carried out to isolate non-Saccharomyces and Saccharomyces yeasts from grape must from different vine-growing regions in Slovenia. Additionally, the diversity of native Saccharomyces cerevisiae strains was evaluated during the process. EXPERIMENTAL APPROACH: During spontaneous alcoholic fermentations the yeast population of non-Saccharomyces and Saccharomyces yeasts was sampled. We used eleven microsatellite markers to determine the genetic diversity of S. cerevisiae strains. In addition, different ratios of the indigenous strains of S. cerevisiae, Hanseniaspora uvarum and Starmerella bacillaris were tested for their possible use in alcoholic fermentation with inoculated yeasts by monitoring its course and measuring the concentration of aroma compounds in wine. RESULTS AND CONCLUSIONS: Sequencing of the internal transcribed spacer (ITS) regions of ribosomal DNA showed that of 64 isolates, 46 strains represent S. cerevisiae and 18 strains belong to non-Saccharomyces yeasts. The identified non-Saccharomyces yeast species were H. uvarum, Pichia kudriavzevii, Saturnispora diversa and S. bacillaris. The dendrogram grouped S. cerevisiae strains into 14 groups. The number of S. cervisiae strains isolated from the musts was 10 (Posavje), 11 (Podravje) and 25 (Primorska vine-growing region). On the other hand, the alcoholic fermentation with inoculated yeasts, in which the native S. cerevisiae strain predominated over H. uvarum and S. bacillaris, gave the most promising result due to the highest alcoholvolume fraction, the lowest acetic acid concentration and significantly higher concentrations of volatile thiols 3-mercaptohexyl acetate (3MHA) and 3-mercaptohexan-1-ol (3MH), 2-methylpropanol, 2-methylbutanol, 3-methylbutanol and 2-phenylethanol) in the produced wine. NOVELTY AND SCIENTIFIC CONTRIBUTION: We confirmed the potential use of indigenous S. cerevisiae and non-Saccharomyces yeasts in alcoholic fermentation with inoculated yeasts, which allows the positive properties of the yeast strains to be expressed and good quality wines to be produced. Thus, the results are encouraging for winemakers to create different wine styles associated with a particular terroir using indigenous yeasts.

The influence of Dekkera bruxellensis on the transcriptome of Saccharomyces cerevisiae and on the aromatic profile of synthetic wine must.

A double compartment membrane system was constructed in order to systematically study possible microbial interactions between yeasts Saccharomyces cerevisiae and Dekkera bruxellensis and their impact on wine aroma. The presence of D. bruxellensis induced 77 transcripts of S. cerevisiae. These were mostly of unknown function; however, some were involved in thiamine biosynthesis and in amino acid and polyamine transport, suggesting a competitive relationship between the two yeast species. Among the transcripts with no biological function, 14 of them were found to be the members of the PAU gene family that is associated with response to anaerobiosis stress. In separated cultures, S. cerevisiae produced glycerol which was subsequently consumed by D. bruxellensis. The concentration of ethylphenols was reduced and we assume that they were absorbed onto the surfaces of S. cerevisiae yeast walls. Also in separated cultures, D. bruxellensis formed a typical profile of aromatic esters with decreased levels of acetate esters and increased level of ethyl esters.

In Saccharomyces cerevisiae gene targeting fidelity depends on a transformation method and proportion of the overall length of the transforming and targeted DNA.

Gene replacement is one of the most essential approaches in construction of the genetically modified yeast strains. However, the fidelity of gene targeting and the effort needed for construction of a particular strain can vary significantly. We investigated the influence of two important factors-the choice of the transformation method and the design of the transforming DNA fragment, which can vary in overall length (including flanking regions and selectable marker) compared to the length of the targeted region in the genome. Gene replacement fidelity was determined in several assays using electroporation and spheroplast transformation, and compared with our previous results obtained by lithium acetate. We have demonstrated clearly that gene targeting fidelity depends on the transformation protocol, being highest for lithium acetate method. In contrast, lower fidelity was observed with electroporation and spheroplast transformation. Additionally, the fidelity also depends on a design of the transformation assay, since a higher overall length ratio of the transforming DNA and targeted region results in higher fidelity. Moreover, the karyotype analysis of the aberrant transformants by qPCR demonstrates that gene targeting can result in diploidisation of haploid strains, most likely via targeted chromosome duplication followed by subsequent duplication of other chromosomes.

Kuraishia mediterranea sp. nov., a methanol-assimilating yeast species from olive oil and its sediment.

Six yeast strains isolated from olive oil sediments and spoiled olive oils originating from Slovenia and Portugal, respectively, proved to represent an undescribed yeast species based on DNA sequence comparisons. The analysis of gene sequences for internal transcribed spacer regions and the large subunit rRNA gene D1/D2 domain placed the novel species in the genus Kuraishia in a subclade containing Kuraishiacapsulata, the type species of the genus. Although the novel species is well separated genetically from the recognized species of the genus, only a minor phenotypic difference differentiating it from Kuraishia capsulata and K. molischiana was observed. Relevant to its isolation source, no lipolytic activity was detected in the strains of the novel species. To accommodate the above-noted strains, Kuraishia mediterranea sp. nov. (holotype: ZIM 2473T; isotype: CBS 15107T; MycoBank no.: MB 822817) is proposed.

Brettanomyces acidodurans sp. nov., a new acetic acid producing yeast species from olive oil.

Two yeast strains representing a hitherto undescribed yeast species were isolated from olive oil and spoiled olive oil originating from Spain and Israel, respectively. Both strains are strong acetic acid producers, equipped with considerable tolerance to acetic acid. The cultures are not short-lived. Cellobiose is fermented as well as several other sugars. The sequences of their large subunit (LSU) rRNA gene D1/D2 domain are very divergent from the sequences available in the GenBank. They differ from the closest hit, Brettanomyces naardenensis by about 27%, mainly substitutions. Sequence analyses of the concatenated dataset from genes of the small subunit (SSU) rRNA, LSU rRNA and translation elongation factor-1α (EF-1α) placed the two strains as an early diverging member of the Brettanomyces/Dekkera clade with high bootstrap support. Sexual reproduction was not observed. The name Brettanomyces acidodurans sp. nov. (holotype: NCAIM Y.02178T; isotypes: CBS 14519T = NRRL Y-63865T = ZIM 2626T, MycoBank no.: MB 819608) is proposed for this highly divergent new yeast species.

Zygosaccharomyces favi sp. nov., an obligate osmophilic yeast species from bee bread and honey.

Five yeast strains representing a hitherto undescribed yeast species were isolated from bee bread and honey in Hungary. They are obligate osmophilic, i.e. they are unable to grow in/on high water activity culture media. Following isogamous conjugation, they form 1-4 spheroid or subspheroid ascospores in persistent asci. The analysis of the sequences of their large subunit rRNA gene D1/D2 domain placed the new species in the Zygosaccharomyces clade. In terms of pairwise sequence similarity, Zygosaccharomyces gambellarensis is the most closely related species. Comparisons of D1/D2, internal transcribed spacer and translation elongation factor-1α (EF-1α) gene sequences of the five strains with that of the type strain of Z. gambellarensis revealed that they represent a new yeast species. The name Zygosaccharomyces favi sp. nov. (type strain: NCAIM Y.01994(T) = CBS 13653(T) = NRRL Y-63719(T) = ZIM 2551(T)) is proposed for this new yeast species, which based on phenotype can be distinguished from related Zygosaccharomyces species by its obligate osmophilic nature. Some intragenomic sequence variability, mainly indels, was detected among the ITS copies of the strains of the new species.

Hanseniaspora nectarophila sp. nov., a yeast species isolated from ephemeral flowers.

Seven apiculate yeast strains that were isolated from the flowers of Syphocampylus corymbiferus Pohl in Brazil are genetically, morphologically and phenotypically distinct from recognized species of the genera Hanseniaspora and Kloeckera. Genetic discontinuities between the novel strains and their closest relatives were found using a networking approach based on the concatenated sequences of the rRNA gene (internal transcribed spacer and D1/D2 of the LSU), and the protein-coding genes for actin and translation elongation factor-1α. Phylogenetic analysis based on the rRNA and the actin gene placed the novel species represented by the strains in close relationship to Hanseniaspora meyeri and Hanseniaspora clermontiae. PCR fingerprinting with microsatellite primers confirmed the genetic heterogeneity of the novel species. The name Hanseniaspora nectarophila sp. nov. is proposed, with UFMG POG a.1(T) ( = ZIM 2311(T)  = CBS 13383(T)) as the type strain; MycoBank no. MB807210. As the current description of the genus does not allow the presence of multilateral budding, an emended diagnosis of the genus Hanseniaspora Zikes is proposed.

High-throughput screening of non-conventional yeasts for conversion of organic waste to microbial oils via carboxylate platform.

Converting waste into high-value products promotes sustainability by reducing waste and creating new revenue streams. This study investigates the potential of diverse yeasts for microbial oil production by utilizing short-chain fatty acids (SCFAs) that can be produced from organic waste and focuses on identifying strains with the best SCFA utilisation, tolerance and lipid production. A collection of 1434 yeast strains was cultivated with SCFAs as the sole carbon source. Eleven strains emerged as candidates with promising growth rates and high lipid accumulation. Subsequent fermentation experiments in liquid SCFA-rich media, which focused on optimizing lipid accumulation by adjusting the carbon to nitrogen (C/N) ratio, showed an increase in lipid content at a C/N ratio of 200:1, but with a concurrent reduction in biomass. Two strains were characterized by their superior ability to produce lipids compared to the reference strain Yarrowia lipolytica CECT124: Y. lipolytica EXF-17398 and Pichia manshurica EXF-7849. Characterization of these two strains indicated that they exhibit a biotechnologically relevant balance between maximizing lipid yield and maintaining growth at high SCFA concentrations. These results emphasize the potential of using SCFAs as a sustainable feedstock for oleochemical production, offering a dual benefit of waste valorisation and microbial oil production.

Ogataea kolombanensis sp. nov., Ogataea histrianica sp. nov. and Ogataea deakii sp. nov., three novel yeast species from plant sources.

Nine methanol-assimilating yeast strains isolated from olive oil sediments in Slovenia, extra virgin olive oil from Italy and rotten wood collected in Hungary were found to form three genetically separated groups, distinct from the currently recognized yeast species. Sequence analysis from genes of the small subunit (SSU) rRNA, internal transcribed spacer region/5.8S rRNA, large subunit (LSU) rRNA D1/D2 domains and translational elongation factor-1α (EF-1α) revealed that the three closely related groups represent three different undescribed yeast species. Sequence analysis of the LSU rRNA gene D1/D2 domains placed the novel species in the Ogataea clade. The three novel species are designated as Ogataea kolombanensis sp. nov. (type strain: ZIM 2322(T) = CBS 12778(T) = NRRL Y-63657(T)), Ogataea histrianica sp. nov. (type strain: ZIM 2463(T) = CBS 12779(T) = NRRL Y-63658(T)) and Ogataea deakii sp. nov. (type strain: NCAIM Y.01896(T) = CBS 12735(T) = NRRL Y-63656(T)).

Genomic and ecological factors shaping specialism and generalism across an entire subphylum.

Organisms exhibit extensive variation in ecological niche breadth, from very narrow (specialists) to very broad (generalists). Paradigms proposed to explain this variation either invoke trade-offs between performance efficiency and breadth or underlying intrinsic or extrinsic factors. We assembled genomic (1,154 yeast strains from 1,049 species), metabolic (quantitative measures of growth of 843 species in 24 conditions), and ecological (environmental ontology of 1,088 species) data from nearly all known species of the ancient fungal subphylum Saccharomycotina to examine niche breadth evolution. We found large interspecific differences in carbon breadth stem from intrinsic differences in genes encoding specific metabolic pathways but no evidence of trade-offs and a limited role of extrinsic ecological factors. These comprehensive data argue that intrinsic factors driving microbial niche breadth variation.

Candida adriatica sp. nov. and Candida molendinolei sp. nov., two yeast species isolated from olive oil and its by-products.

Thirteen strains isolated from virgin olive oil or its by-products in several Mediterranean countries were found to be phenotypically and genetically divergent from currently recognized yeast species. Sequence analysis of the large subunit (LSU) rDNA D1/D2 domain and internal transcribed spacer regions/5.8S rDNA revealed that the strains represented two novel species described as Candida adriatica sp. nov. (type strain ZIM 2334(T) = CBS 12504(T) = NCAIM Y.02001(T)) and Candida molendinolei sp. nov. (type strain DBVPG 5508(T) = CBS 12508(T) = NCAIM Y.02000(T)). Phylogenetic analysis based on concatenated sequences of the small subunit rRNA gene, the D1/D2 region of the LSU rDNA and the translation elongation factor-1α gene suggested that C. adriatica sp. nov. and C. molendinolei sp. nov. should be placed within the Lindnera and Nakazawaea clades, respectively.

Whole-Genome Sequencing and Annotation of the Yeast Clavispora santaluciae Reveals Important Insights about Its Adaptation to the Vineyard Environment.

Clavispora santaluciae was recently described as a novel non-Saccharomyces yeast species, isolated from grapes of Azores vineyards, a Portuguese archipelago with particular environmental conditions, and from Italian grapes infected with Drosophila suzukii. In the present work, the genome of five Clavispora santaluciae strains was sequenced, assembled, and annotated for the first time, using robust pipelines, and a combination of both long- and short-read sequencing platforms. Genome comparisons revealed specific differences between strains of Clavispora santaluciae reflecting their isolation in two separate ecological niches-Azorean and Italian vineyards-as well as mechanisms of adaptation to the intricate and arduous environmental features of the geographical location from which they were isolated. In particular, relevant differences were detected in the number of coding genes (shared and unique) and transposable elements, the amount and diversity of non-coding RNAs, and the enzymatic potential of each strain through the analysis of their CAZyome. A comparative study was also conducted between the Clavispora santaluciae genome and those of the remaining species of the Metschnikowiaceae family. Our phylogenetic and genomic analysis, comprising 126 yeast strains (alignment of 2362 common proteins) allowed the establishment of a robust phylogram of Metschnikowiaceae and detailed incongruencies to be clarified in the future.

Novakomyces olei sp. nov., the First Member of a Novel Taphrinomycotina Lineage.

Taphrinomycotina is the smallest subphylum of the phylum Ascomycota. It is an assemblage of distantly related early diverging lineages of the phylum, comprising organisms with divergent morphology and ecology; however, phylogenomic analyses support its monophyly. In this study, we report the isolation of a yeast strain, which could not be assigned to any of the currently recognised five classes of Taphrinomycotina. The strain of the novel budding species was recovered from extra virgin olive oil and characterised phenotypically by standard methods. The ultrastructure of the cell wall was investigated by transmission electron microscopy. Comparisons of barcoding DNA sequences indicated that the investigated strain is not closely related to any known organism. Tentative phylogenetic placement was achieved by maximum-likelihood analysis of the D1/D2 domain of the nuclear LSU rRNA gene. The genome of the investigated strain was sequenced, assembled, and annotated. Phylogenomic analyses placed it next to the fission Schizosaccharomyces species. To accommodate the novel species, Novakomyces olei, a novel genus Novakomyces, a novel family Novakomycetaceae, a novel order Novakomycetales, and a novel class Novakomycetes is proposed as well. Functional analysis of genes missing in N. olei in comparison to Schizosaccharomyces pombe revealed that they are biased towards biosynthesis of complex organic molecules, regulation of mRNA, and the electron transport chain. Correlating the genome content and physiology among species of Taphrinomycotina revealed some discordance between pheno- and genotype. N. olei produced ascospores in axenic culture preceded by conjugation between two cells. We confirmed that N. olei is a primary homothallic species lacking genes for different mating types.

Hanseniaspora smithiae sp. nov., a Novel Apiculate Yeast Species From Patagonian Forests That Lacks the Typical Genomic Domestication Signatures for Fermentative Environments.

During a survey of Nothofagus trees and their parasitic fungi in Andean Patagonia (Argentina), genetically distinct strains of Hanseniaspora were obtained from the sugar-containing stromata of parasitic Cyttaria spp. Phylogenetic analyses based on the single-gene sequences (encoding rRNA and actin) or on conserved, single-copy, orthologous genes from genome sequence assemblies revealed that these strains represent a new species closely related to Hanseniaspora valbyensis. Additionally, delimitation of this novel species was supported by genetic distance calculations using overall genome relatedness indices (OGRI) between the novel taxon and its closest relatives. To better understand the mode of speciation in Hanseniaspora, we examined genes that were retained or lost in the novel species in comparison to its closest relatives. These analyses show that, during diversification, this novel species and its closest relatives, H. valbyensis and Hanseniaspora jakobsenii, lost mitochondrial and other genes involved in the generation of precursor metabolites and energy, which could explain their slower growth and higher ethanol yields under aerobic conditions. Similarly, Hanseniaspora mollemarum lost the ability to sporulate, along with genes that are involved in meiosis and mating. Based on these findings, a formal description of the novel yeast species Hanseniaspora smithiae sp. nov. is proposed, with CRUB 1602 H as the holotype.