A genome-informed higher rank classification of the biotechnologically important fungal subphylum Saccharomycotina.

The subphylum Saccharomycotina is a lineage in the fungal phylum Ascomycota that exhibits levels of genomic diversity similar to those of plants and animals. The Saccharomycotina consist of more than 1 200 known species currently divided into 16 families, one order, and one class. Species in this subphylum are ecologically and metabolically diverse and include important opportunistic human pathogens, as well as species important in biotechnological applications. Many traits of biotechnological interest are found in closely related species and often restricted to single phylogenetic clades. However, the biotechnological potential of most yeast species remains unexplored. Although the subphylum Saccharomycotina has much higher rates of genome sequence evolution than its sister subphylum, Pezizomycotina, it contains only one class compared to the 16 classes in Pezizomycotina. The third subphylum of Ascomycota, the Taphrinomycotina, consists of six classes and has approximately 10 times fewer species than the Saccharomycotina. These data indicate that the current classification of all these yeasts into a single class and a single order is an underappreciation of their diversity. Our previous genome-scale phylogenetic analyses showed that the Saccharomycotina contains 12 major and robustly supported phylogenetic clades; seven of these are current families (Lipomycetaceae, Trigonopsidaceae, Alloascoideaceae, Pichiaceae, Phaffomycetaceae, Saccharomycodaceae, and Saccharomycetaceae), one comprises two current families (Dipodascaceae and Trichomonascaceae), one represents the genus Sporopachydermia, and three represent lineages that differ in their translation of the CUG codon (CUG-Ala, CUG-Ser1, and CUG-Ser2). Using these analyses in combination with relative evolutionary divergence and genome content analyses, we propose an updated classification for the Saccharomycotina, including seven classes and 12 orders that can be diagnosed by genome content. This updated classification is consistent with the high levels of genomic diversity within this subphylum and is necessary to make the higher rank classification of the Saccharomycotina more comparable to that of other fungi, as well as to communicate efficiently on lineages that are not yet formally named. Taxonomic novelties: New classes: Alloascoideomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Dipodascomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Lipomycetes M. Groenew., Hittinger, Opulente, A. Rokas, Pichiomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Sporopachydermiomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Trigonopsidomycetes M. Groenew., Hittinger, Opulente & A. Rokas. New orders: Alloascoideomycetes: Alloascoideales M. Groenew., Hittinger, Opulente & A. Rokas; Dipodascomycetes: Dipodascales M. Groenew., Hittinger, Opulente & A. Rokas; Lipomycetes: Lipomycetales M. Groenew., Hittinger, Opulente & A. Rokas; Pichiomycetes: Alaninales M. Groenew., Hittinger, Opulente & A. Rokas, Pichiales M. Groenew., Hittinger, Opulente & A. Rokas, Serinales M. Groenew., Hittinger, Opulente & A. Rokas; Saccharomycetes: Phaffomycetales M. Groenew., Hittinger, Opulente & A. Rokas, Saccharomycodales M. Groenew., Hittinger, Opulente & A. Rokas; Sporopachydermiomycetes: Sporopachydermiales M. Groenew., Hittinger, Opulente & A. Rokas; Trigonopsidomycetes: Trigonopsidales M. Groenew., Hittinger, Opulente & A. Rokas. New families: Alaninales: Pachysolenaceae M. Groenew., Hittinger, Opulente & A. Rokas; Pichiales: Pichiaceae M. Groenew., Hittinger, Opulente & A. Rokas; Sporopachydermiales: Sporopachydermiaceae M. Groenew., Hittinger, Opulente & A. Rokas. Citation: Groenewald M, Hittinger CT, Bensch K, Opulente DA, Shen X-X, Li Y, Liu C, LaBella AL, Zhou X, Limtong S, Jindamorakot S, Gonçalves P, Robert V, Wolfe KH, Rosa CA, Boekhout T, Cadez N, Péter G, Sampaio JP, Lachance M-A, Yurkov AM, Daniel H-M, Takashima M, Boundy-Mills K, Libkind D, Aoki K, Sugita T, Rokas A (2023). A genome-informed higher rank classification of the biotechnologically important fungal subphylum Saccharomycotina. Studies in Mycology 105: 1-22. doi: 10.3114/sim.2023.105.01 This study is dedicated to the memory of Cletus P. Kurtzman (1938-2017), a pioneer of yeast taxonomy.

Towards yeast taxogenomics: lessons from novel species descriptions based on complete genome sequences.

In recent years, 'multi-omic' sciences have affected all aspects of fundamental and applied biological research. Yeast taxonomists, though somewhat timidly, have begun to incorporate complete genomic sequences into the description of novel taxa, taking advantage of these powerful data to calculate more reliable genetic distances, construct more robust phylogenies, correlate genotype with phenotype and even reveal cryptic sexual behaviors. However, the use of genomic data in formal yeast species descriptions is far from widespread. The present review examines published examples of genome-based species descriptions of yeasts, highlights relevant bioinformatic approaches, provides recommendations for new users and discusses some of the challenges facing the genome-based systematics of yeasts.

The yeast genus Tortispora gen. nov., description of Tortispora ganteri sp. nov., Tortispora mauiana f.a., sp. nov., Tortispora agaves f.a., sp. nov., Tortispora sangerardonensis f.a., sp. nov., Tortispora cuajiniquilana f.a., sp. nov., Tortispora starmeri f.a., sp. nov. and Tortispora phaffii f.a., sp. nov., reassignment of Candida caseinolytica to Tortispora caseinolytica f.a., comb. nov., emendation of Botryozyma, and assignment of Botryozyma, Tortispora gen. nov. and Trigonopsis to the family Trigonopsidaceae fam. nov.

We describe the yeast genus Tortispora gen. nov., an early-diverging lineage in the Saccharomycetales that displays the formation of helical ascospores. The genus is based on 16 strains resembling Candida caseinolytica that were isolated from necrotic plant tissue in warm regions of the New World. Based on sequences of the D1/D2 domains of the nuclear large subunit rRNA gene, as well as other data, the strains are assigned to eight distinct species. The species are nutritionally specialized and share the unusual ability to hydrolyse casein and to grow on 1-butanol as sole carbon source. One species of the proposed new genus produces a simple ascus with a helical ascospore, whereas other species of the clade have failed to form ascospores. All species in the clade, including C. caseinolytica, are assigned to Tortispora gen. nov. The new binomials are Tortispora ganteri sp. nov., type species of the genus (SUB 86-469.5(T) = CBS 12581(T) = NRRL Y-17035(T)), Tortispora caseinolytica f.a., comb. nov. (UCD-FST 83-438.3(T) = CBS 7781(T) = NRRL Y-17796(T)), Tortispora mauiana f.a., sp. nov. (UWOPS 87-2430.3(T) = CBS 12803(T) = NRRL Y-48832(T)), Tortispora agaves f.a., sp. nov. (UWOPS 94-257.6(T) = CBS 12794(T) = NRRL Y-63662(T)), Tortispora sangerardonensis f.a., sp. nov. (UWOPS 00-157.1(T) = CBS 12795(T) = NRRL Y-63663(T)), Tortispora cuajiniquilana f.a., sp. nov. (UWOPS 99-344.4(T) = CBS 12796(T) = NRRL Y-63664(T)), Tortispora starmeri f.a., sp. nov. (G 91-702.5(T) = CBS 12793(T) = NRRL Y-63665(T)) and Tortispora phaffii f.a., sp. nov. (UWOPS 91-445.1(T) = CBS 12804(T) = NRRL Y-48833(T)). In addition, species formerly assigned to the genus Ascobotryozyma are reassigned to the genus Botryozyma. The genera Trigonopsis, Botryozyma and Tortispora are assigned to the family Trigonopsidaceae fam. nov.

Proliferating larval cestodiasis: report of a case.

A 72-year-old Filipino man who had emigrated to Canada had an abdominal infection with a proliferating acephalic larval cestode. Small, irregularly shaped parasites were within the substance of a lymph node, where they provoked a fibrogranulomatous and eosinophilic reaction. The parasite is described and is identified as an undifferentiated sparganum or tetrahyridium based upon comparison with two similar parasites from patients in Taiwan and Paraguay.

Lachancea nothofagi sp. nov., a yeast associated with Nothofagus species in Patagonia, Argentina.

Six strains of a novel yeast species were isolated from Nothofagus species trees in native forests in Patagonia, Argentina. The strains were isolated from bark, fluxes and the ectomycorrhizospheric soil fraction of Nothofagus antarctica, Nothofagus nervosa and Nothofagus pumilio. Analysis of the D1/D2 large-subunit rDNA sequences indicated that the novel species belonged to the genus Lachancea and is closely related to Lachancea meyersii. The name Lachancea nothofagi sp. nov. is proposed to accommodate these strains. The type strain is UWOPS 99-807.3(T) (=CBS 11611(T)=NRRL Y-48670(T)).

Kluyveromyces: systematics since 1970.

The taxonomy of Kluyveromyces has been the object of intense study since van der Walt's (1970) monograph. This is an account of the major developments and the classification to be adopted in the 4th edition of The Yeasts, a Taxonomic Study. The guiding principles that will be followed in eventual revisions of the genus are presented.

Restriction mapping of rDNA and the taxonomy of Kluyveromyces van der Walt emend. van der Walt.

Ribosomal DNA from the type strains of 13 nomenspecies of Kluyveromyces and from other strains were mapped with 11 restriction endonucleases. The length of the repeating unit ranged from ca. 8.4 kb (in K. aestuarii) to ca. 10.9 kb (in K. phaffii). The length variation resided as expected in the nontranscribed spacer. The patterns confirmed some of the inferences articulated by various students of the genus. The closely related species K. marxianus and K. lactis constituted a core to which could be linked first K. wickerhamii and K. dobzhanskii and then K. aestuarii. The presumed relatedness between K. waltii and K. thermotolerans was endorsed by rDNA mapping as well, but evidence linking these two species to the rest of the genus is wanting. The restriction patterns suggest that the multispored species together with K. delphensis form a loose assemblage acting as a bridge between the "core" species and the species K. phaffii and K. lodderi.

Yeast communities in a natural tequila fermentation.

Fresh and cooked agave, Drosophila spp., processing equipment, agave molasses, agave extract, and fermenting must at a traditional tequila distillery (Herradura, Amatitan, Jalisco, México) were studied to gain insight on the origin of yeasts involved in a natural tequila fermentations. Five yeast communities were identified. (1) Fresh agave contained a diverse mycobiota dominated by Clavispora lusitaniae and an endemic species, Metschnikowia agaveae. (2) Drosophila spp. from around or inside the distillery yielded typical fruit yeasts, in particular Hanseniaspora spp., Pichia kluyveri, and Candida krusei. (3) Schizosaccharomyces pombe prevailed in molasses. (4) Cooked agave and extract had a considerable diversity of species, but included Saccharomyces cerevisiae. (5) Fermenting juice underwent a gradual reduction in yeast heterogeneity. Torulaspora delbrueckii, Kluyveromyces marxianus, and Hanseniaspora spp. progressively ceded the way to S. cerevisiae, Zygosaccharomyces bailii, Candida milleri, and Brettanomyces spp. With the exception of Pichia membranaefaciens, which was shared by all communities, little overlap existed. That separation was even more manifest when species were divided into distinguishable biotypes based on morphology or physiology. It is concluded that crushing equipment and must holding tanks are the main source of significant inoculum for the fermentation process. Drosophila species appear to serve as internal vectors. Proximity to fruit trees probably contributes to maintaining a substantial Drosophila community, but the yeasts found in the distillery exhibit very little similarity to those found in adjacent vegetation. Interactions involving killer toxins had no apparent direct effects on the yeast community structure.

Hanseniaspora nodinigri, a new yeast species found in black knots (Dibotryon morbosum) of Prunus virginiana.

The new yeast species Hanseniaspora nodinigri is described to accommodate members of the genus Hanseniaspora that are unable to assimilate glucono-sigma-lactone and isolated from stromatal tissue of black knots (Dobotryon morbosum) of chokecherry, Prunus virginiana. The newly described taxon shows much resemblance, by other criteria, to H. vineae van der Walt et Tscheuschner and H. osmophila (Niehaus) Phaff, Miller et Shifrine.

Current views on the yeast species.

The application of the biological species concept to the delineation of yeast species is becoming a reality. Studies centred on heterothallic Pichia and related species, and on homothallic species of Kluyveromyces have facilitated a better understanding of the concept of 'yeast species'.

Phylogenetic study of ribosomal DNA of cactophilic Pichia species by restriction mapping.

The rDNAs of strains of the cactophilic Pichia species P. amethionina, P. antillensis, P. barkeri, P. cactophila, P. caribaea, P. deserticola, P. heedii, P. kluyveri, P. norvegenesis, P. opuntiae, P. pseudocactophila, P. thermotolerans and their varieties and anamorphs were mapped with 15 restriction endonucleases, and compared to P. membranaefaciens and P. salictaria as possible non-cactophilic relatives. The existence of species complexes among those taxa was confirmed. P. membranaefaciens was a plausible ancestral species, and its closest relative in the cactophilic group was P. deserticola. These two species appeared to be moderately related to P. heedii and to P. barkeri, but the latter was shown clearly to belong to the P. kluyveri complex, in spite of a 6 mol% G+C difference in their nuclear DNAs. P. cactophila and P. pseudocactophila ostensibly emerged from P. norvegensis, a facultatively cactophilic yeast. The P. amethionina, P. cactophila and P. opuntiae species complexes appeared independent from one another and from all other species studied. P. salictaria did not appear to be related to P. amethionina.

The use of N,N,N',N'-tetramethylphenylenediamine to detect peroxidase activity on polyacrylamide electrophoresis gels.

N,N,N',N'-Tetramethylphenylenediamine (TMPD) acts as an effective indicator of peroxidase activity on polyacrylamide electrophoresis gels. The test is easy to perform, rapid, sensitive, and reliable. The procedure produces vivid bright blue bands (Wursters blue) on a clear background. TMPD and Wursters blue did not interfere with a number of other electrophoresis stains subsequently applied. These included total protein staining with Coomassie blue, and a number of pigment producing electrophoresis stains used to investigate melanogenesis-related enzymes in the black yeast Phaeococcomyces sp.

The yeast genus Starmerella gen. nov. and Starmerella bombicola sp. nov., the teleomorph of Candida bombicola (Spencer, Gorin & Tullock) Meyer & Yarrow.

Seven strains of a heterothallic haploid yeast species were isolated from flowers of Calystegia sepium (hedge bindweed, Convolvulaceae) and associated sap beetles of the genus Conotelus. Conjugation was observed between some of the isolates and the type strain of Candida bombicola, resulting in evanescent asci with one ascospore with a convoluted surface. The sequences of the D1/D2 variable domain of the large subunit of the rDNAs of three strains differed by only one or two bases from that of the type strain. The new genus Starmerella, with the single species Starmerella bombicola, is proposed to accommodate the teleomorph of C. bombicola. The designated isotype is strain UWO(PS)97-118I (H-; CBS 8451I).

Predacious yeasts.

Haustorium-mediated predation was observed in seven yeast species. Arthroascus javanensis, Botryoascus synnaedendrus, Guilliermondella selenospora, Saccharomycopsis fibuligera, and three hitherto unknown species penetrate and kill other yeasts. These yeasts share an unusual requirement for organic sulphur. One isolate recovered from Australian Hibiscus was studied in detail and found to attack a broad range of prey species, including ascomycetous and basidiomycetous yeasts as well as moulds. Predation was most effective when growth was on a solid surface and the medium was poor in complex nutrients. Organic sulphur (exemplified by methionine) was identified as a key factor. It serves as a nutritional benefit to the predator and, depending on the concentration, acts as either an inhibitor of predation or possibly a signal for detection of prey. Sampling of a yeast habitat with a medium selective for selenium-resistant yeasts indicated that auxotrophic and predacious yeasts might be more widespread than anticipated.

Mating in the heterothallic haploid yeast Clavispora opuntiae, with special reference to mating type imbalances in local populations.

Mating was studied in the haploid, heterothallic yeast Clavispora opuntiae to assess the importance of nutritional, genetic, and other factors that may favour mating and recombination. Local populations of this yeast generally exhibit dramatic inequalities in mating type distributions, suggesting that mating is rare in nature even though most isolates mate freely in the laboratory. The absence of assimilable nitrogen is prerequisite to mating competence, presumably by causing G1 arrest. Maximum mating competence is found in cells entering stationary phase in nitrogen-limited media. Unlike the vast majority of mating yeasts, C. opuntiae does not appear to produce diffusible mating factors (sex pheromones), and mating-competent cells do not undergo sexual agglutination. Pairwise cell contact appears to be the only signal that triggers the sexual process in this case. In order to determine if mating type imbalances in nature are caused by reduced fertility of 'consanguine' crosses, meiotic recombination was measured in pairs of strains that varied in their genetic distances as indicated by restriction mapping. That hypothesis was rejected, as recombination efficiency decreased with increasing genetic distance. We conclude that the rarity of mating in local populations is exacerbated by the stringent physical (pairwise cell contact) and nutritional (nitrogen depletion) conditions that will allow mating to proceed. Parallels are drawn with mating patterns observed in Clavispora lusitaniae.

A comparison of yeast communities found in necrotic tissue of cladodes and fruits ofOpuntia stricta on Islands in the Caribbean Sea and where introduced into Australia.

Yeast communities growing in the decaying tissues (cladodes and fruits) ofOpuntia stricta (prickly pear cactus) and associated yeast vectors (Drosophila species) were compared in two geographic regions (Caribbean and eastern Australia). The Australian yeast community provides an interesting comparison to the Caribbean community, because the host plantO. stricta was introduced to Australia over 100 years ago. Many of the yeasts found in the Australian system also were introduced during a period of biological control (1926-1935) when they accompanied rotting prickly pear cladodes and insects shipped to Australia from the Americas. The yeast community composition (proportion of each species) is compared at several levels of organization: (1) within and between regions, (2) across seasons and years, and (3) within and between tissue types. The yeast species composition of the cladode communities are similar from locality to locality, season to season, and year to year, with the region-to-region similarity slightly less. The composition of the fruit-yeast communities are distinct from region to region and only show some overlap with the cladodes within regions when collected simultaneously in the same locality. It is suggested that the cladode-microorganism-Drosophila system is relatively closed (little extrinsic influence) whereas the fruit-microorganism-Drosophila system is open (large extrinsic influence).

Yeast communities associated with Drosophila species and related flies in an eastern oak-pine forest: a comparison with western communities.

Intestinal yeast mycobiota were studied in 14 species of Drosophila and in the drosophilid species Chymomyza amoena, captured at Pinery Provincial Park, Ontario. Over 56 yeast species, some undescribed, were isolated. These yeast communities were compared with those from two similar surveys conducted in western portions of North America. The community structures were influenced significantly by the habitat rather than phylogeny of the flies. Geographic separation was a factor affecting yeast taxa frequencies in the fly species, but it was largely overshadowed by ecological factors when the communities were described physiologically. The notion that habitats are filled by yeasts which add up to a suitable physiological potential, more or less independently of their taxonomic affinities, was thus confirmed.

Yeasts from exudates ofQuercus, Ulmus, Populus, andPseudotsuga: New isolations and elucidation of some factors affecting ecological specificity.

The yeast flora associated with exudates ofQuercus, Ulmus, Populus, andPseudotsuga was examined in the light of new isolations in geographic areas different from those in previous reports. Application of multivariate analytic methods indicated that geographic distance, although a meaningful ecological factor, is largely overshadowed by host tree specificity, provided that yeast community physiological profiles and not yeast taxa, are used as ecological descriptors. Some physiological attributes used in classifying yeasts were identified as particularly important in shaping the yeast communities of those trees. The possible divergence between these attributes and those generally considered taxonomically useful is discussed.

Purification and partial characterization of an exo-beta-glucanase from the yeast Kluyveromyces aestaurii.

The intracellular-periplasmic exo-1,3-beta-glucanase (EC 3.2.1.58) has been extracted from the yeast Kluyveromyces aestuarii and purified to immunoelectrophoretic homogeneity by ion-exchange and gel-exclusion chromatography. The kinetic constants and activation energies for laminarin, p-nitrophenyl-beta-D-glucoside, and pustulan have been determined, along with the effect of pH. Evidence is presented indicating that the enzyme is composed of a single polypeptide chain, about 24% carbohydrates, and its molecular weight was estimated to be 43 000.

Metschnikowia hawaiiensis sp. nov., a heterothallic haploid yeast from Hawaiian morning glory and associated drosophilids.

A new haploid, heterothallic yeast species was isolated repeatedly from morning glory (Ipomoea acuminata) flowers and from two associated drosophilid species, Scaptomyza calliginosa and Drosophila floricola, in a Hawaiian kipuka. Haploid strains of this organism multiply asexually by budding and, under nutrient deprivation, by the formation of long germ tubes that develop into branching true mycelia. Mating compatibility is controlled by two alleles of a single locus. Plasmogamy between compatible strains is followed by the development of very large elongate asci bearing vestiges of the zygotes and the formation in each ascus of two unusually large aciculate ascospores similar to those formed by members of the genus Metschnikowia. Membership in the genus Metschnikowia is supported by the physiological profile of the yeast, which is typical of the genus but not identical to the profile of any previously described species. The name Metschnikowia hawaiiensis is proposed to emphasize the geographic origin of the new species, not its habitat, which has not been determined precisely. The holotype strain of M. hawaiiensis is strain UWO(PS) 87-2167.2 (= ATCC 76059 = CBS 7432), and the isotype strain is strain UWO(PS) 87-2203.2 (= ATCC 76058 = CBS 7433).