Diversity of yeast species from Dutch garden soil and the description of six novel Ascomycetes.

A Citizen Science initiative by the Westerdijk Fungal Biodiversity Institute and the Utrecht University Museum gave rise to a project where fungal and yeast isolates were obtained and identified from Dutch soil samples. During the current study, 386 yeast strains were isolated from 157 different locations in the Netherlands. These strains were identified using sequence data of the large-subunit rRNA gene (D1/D2 region) and the internal transcribed spacer 1 and 2 regions. A total of 53 different yeast species were found as well as 15 potentially novel species. Six novel ascomycetous species are described during this study that include Hanseniaspora mollemarum sp. nov., Ogataea degrootiae sp. nov., Pichia gijzeniarum sp. nov., Saccharomycopsis oosterbeekiorum sp. nov., Trichomonascus vanleenenius sp. nov. and Zygoascus flipseniorum sp. nov. This study made it possible to incorporate numerous yeast isolates into the CBS collection without any restrictions, which make these isolates readily available for use by others. Many of the isolates represented species of which only a few isolates or even only a single ex-type strain were available. Therefore, it is a clear indication that such biodiversity-orientated Citizen Science projects can enrich the pool of available yeasts for future research projects.

Description of Hyphopichia buzzinii f.a., sp. nov. and Hyphopichia homilentoma comb. nov., the teleomorph of Candida homilentoma.

During studies of the yeast diversity associated with rotting wood in Brazil and fruits, plants and insects in French Guiana, three strains of a new species were isolated. Analysis of the sequences of the internal transcribed spacer (ITS)-5.8S and D1/D2 domains of the large subunit of the rRNA gene showed that this species belongs to the genus Hyphopichia and its closest relative is Candida homilentoma. These species differ by 44 nucleotide substitutions in D1/D2 sequences. A new species Hyphopichia buzzinii f. a., sp. nov., is proposed to accommodate these isolates. The type strain of Hyphopichia buzzinii sp. nov. is CLIB 1739T (=CBS 14300T = UFMG-CM-Y6121T; MycoBank number is MB 815609). In addition, we isolated 11 strains of C. homilentoma from rotting wood, leaf surfaces, and water bodies in Brazil, and these strains when crossed among one another and with the type strain (CBS 6312T) of this species, produced hat-shaped ascospores typical of the genus Hyphopichia. We describe the teleomorph of C. homilentoma as a new combination, Hyphopichia homilentoma comb. nov. (type strain CBS 6312T; MycoBank number is MB 820009). We also propose to transfer the other six Candida species of the Hyphopichia clade to this genus as new combinations. Hyphopichia homilentoma produced ethanol and xylitol from D-xylose whereas H. buzzinii was only able to convert this pentose to xylitol.

First DNA Barcoding Survey in Bulgaria Unveiled Huge Diversity of Yeasts in Insects.

In this study, we conducted a comprehensive survey aimed at assessing the diversity of yeast species inhabiting the guts of various insect species collected mainly from two Bulgarian National Parks, namely, Rila, and Pirin. The insect specimens encompass a broad taxonomic spectrum, including representatives from Coleoptera, Orthoptera, Lepidoptera, Hymenoptera, Dermaptera, Isopoda, and Collembola. Yeast strains were identified with DNA barcoding using the ribosomal markers, specifically, the D1/D2 domains of the ribosomal large subunit (LSU) and the internal transcribed spacers regions ITS 1 + 2 (ITS). The analysis unveiled the presence of 89 ascomycetous and 18 basidiomycetous yeast isolates associated with the insect specimens. Furthermore, our study identified 18 hitherto unknown yeast species.

Genome Analysis of a Newly Discovered Yeast Species, Hanseniaspora menglaensis.

Annual surveys of Irish soil samples identified three isolates, CBS 16921 (UCD88), CBS 18246 (UCD443), and CBS 18247 (UCD483), of an apiculate yeast species within the Hanseniaspora genus. The internal transcribed spacer (ITS) and D1/D2 region of the large subunit (LSU) rRNA sequences showed that these are isolates of the recently described species Hanseniaspora menglaensis, first isolated from Southwest China. No genome sequence for H. menglaensis is currently available. The genome sequences of the three Irish isolates were determined using short-read (Illumina) sequencing, and the sequence of one isolate (CBS 16921) was assembled to chromosome level using long-read sequencing (Oxford Nanopore Technologies). Phylogenomic analysis shows that H. menglaensis belongs to the fast-evolving lineage (FEL) of Hanseniaspora. Only one MAT idiomorph (encoding MATα1) was identified in all three sequenced H. menglaensis isolates, consistent with one mating type of a heterothallic species. Genome comparisons showed that there has been a rearrangement near MATα of FEL species compared to isolates from the slowly evolving lineage (SEL).

The teleomorph state of Candida deformans Langeron & Guerra and description of Yarrowia yakushimensis comb. nov.

Yarrowia lipolytica is the only known species in the teleomorph (i.e. sexual) genus Yarrowia and has its anamorph (i.e. asexual state) classified in the genus Candida Berkhout as Candida lipolytica. This species can be found readily in nature, has significant industrial value and is important to the food and medical fields. Candida deformans was first considered a variety of C. lipolytica and was later listed as a synonym of this species. More recent studies based on sequence variation in the nuclear rRNA gene sequences suggested C. deformans to be a separate species with no known teleomorph linked to it. In this study we show that C. deformans strains, obtained from South Africa, can mate with strains present in the CBS yeast collection and produce a Yarrowia teleomorph, described here as Yarrowia deformans. Strains of Candida yakushimensis nom. inval., a species also belonging to the Yarrowia phylogenetic clade, were also studied and described as a novel species of Yarrowia.

Species reassignment of Geotrichum bryndzae, Geotrichum phurueaensis, Geotrichum silvicola and Geotrichum vulgare based on phylogenetic analyses and mating compatibility.

The present classification of Galactomyces and its anamorph, Geotrichum, is based on various studies that used morphology, ecology, biochemistry, DNA-DNA reassociation comparisons and gene sequencing. In this study, the identities of strains of the Centraalbureau voor Schimmelcultures yeast culture collection, as well as seven strains from South Africa, were examined by analyses of the nucleotide divergence in the internal transcribed spacer regions of the nuclear rRNA gene (nrRNA) operon, the D1/D2 domains of the 26S rRNA gene and partial actin gene sequences as well as compatibility studies. The South African strains were assigned to species in the genus Galactomyces. The phylogenetic analyses and mating studies revealed that Geotrichum silvicola and Geotrichum bryndzae are synonyms of Galactomyces candidus and that Geotrichum vulgare is a synonym of Galactomyces pseudocandidus.

Five novel Wickerhamomyces- and Metschnikowia-related yeast species, Wickerhamomyces chaumierensis sp. nov., Candida pseudoflosculorum sp. nov., Candida danieliae sp. nov., Candida robnettiae sp. nov. and Candida eppingiae sp. nov., isolated from plants.

On the basis of nucleotide divergences in the D1/D2 domain of the 26S rRNA gene and the internal transcribed spacers (ITS) domain of the rRNA gene, five novel yeast species, Wickerhamomyces chaumierensis sp. nov. (CBS 8565(T)  = JCM 17246(T)), Candida pseudoflosculorum sp. nov. (CBS 8584(T)  = JCM 17242(T)), Candida danieliae sp. nov. (CBS 8533(T)  = JCM 17247(T)), Candida robnettiae sp. nov. (CBS 8580(T)  = JCM 17243(T)) and Candida eppingiae sp. nov. (CBS 8586(T)  = JCM 17241(T)), isolated from plants in Thailand and Guyana, are proposed in this study.

Re-examination of strains formerly assigned to Hyphopichia burtonii, the phylogeny of the genus Hyphopichia, and the description of Hyphopichia pseudoburtonii sp. nov.

On the basis of the nucleotide divergence in the internal transcribed spacer (ITS) domain of the rRNA gene and the D1/D2 domain of the 26S rRNA gene, species of the genus Hyphopichia can be divided into different phylogenetic groups. In this study, three distinct phylogenetic clusters as well as a novel species, Hyphopichia pseudoburtonii sp. nov. with type strain CBS 2455(T) (=JCM 16346(T)), are introduced. Mating studies indicate that strains belonging to Hyphopichia pseudoburtonii are heterothallic.

DNA barcoding revealed Nematodospora valgi gen. nov., sp. nov. and Candida cetoniae sp. nov. in the Lodderomyces clade.

During a yeast biodiversity survey conducted in 2009-2011 in Bulgaria (South Eastern Europe) five strains of a novel ascomycetous yeast species were isolated from the beetle Valgus hemipterus (Cetoniinae) collected from two localities, namely Osogovska Planina Mountain and Nature Park Zlatni Pyasatsi. Phylogenetic analysis using combined sequences of the D1/D2 domains of the large subunit ribosomal DNA (LSU rDNA) and the internal transcribed spacers 1 + 2 regions (ITS1+2) placed the novel species on a separate branch near the basal part of the Lodderomyces clade. The novel species has a unique ascospore morphology distinct from those of the closely related teleomorphic genus Lodderomyces. Based on phylogenetic analysis and morphology of the ascospores we propose Nematodospora valgi gen. nov., sp. nov. to accommodate these isolates (MB811804 D37S(T), MB802458). Two strains of a novel anamorphic yeast species were isolated from the beetles Cetonia aurata and Oxythyrea funesta (Cetoniinae) collected in East Rhodopies and Sofia city, respectively. DNA barcoding analysis placed the new yeast species within the Candida parapsilosis subclade. Here, we present the description of a new yeast species, Candida cetoniae sp. nov. (IMB1R2(T), MB803501) to accommodate these two strains. The ecology and biogeography of the insect-associated yeasts of the Lodderomyces clade is discussed.

Ribosomal DNA sequencing and reinstatement of the genus Arthroascus von Arx.

Sequence analysis of the D1/D2 domain of 26S rDNA was conducted upon seven Arthroascus strains from different geographic localities. The European and Asian species Arthroascus schoenii was documented from the North-American continent and from the Island of Hawaii. We discuss the heterogeneity of the genus Saccharomycopsis sensu Kurtzman and Robnett 1995. On the basis of molecular and genetic data the genus Arthroascus von Arx is reinstated.

The treasure trove of yeast genera and species described by Johannes van der Walt (1925-2011).

Yeast taxonomy and systematics have in recent years been dealt with intensively primarily by a small group of individual researchers with particular expertise. Amongst these was Johannes P. van der Walt, who had a major role in shaping our current understanding of yeast biodiversity and taxonomy. Van der Walt based his taxonomic studies not only on available cultures, but also by going into the field to isolate yeasts from various substrates. This pioneering work led to the discovery of many new genera and species, which were deposited in the Centraalbureau voor Schimmelcultures (CBS) collections for future studies in taxonomy, genomics, and industrial uses. These treasures collected during more than 60 years provide an outstanding legacy to the yeast community and will continue to exist in his absence. This contribution provides a comprehensive overview of the current nomenclatural and taxonomic status of the yeast genera and species introduced by van der Walt during his career.

Polyphasic re-examination of Yarrowia lipolytica strains and the description of three novel Candida species: Candida oslonensis sp. nov., Candida alimentaria sp. nov. and Candida hollandica sp. nov.

The type strain of Yarrowia lipolytica and 38 strains identified as Yarrowia lipolytica, four strains of Candida deformans, including the type and two subcultures of the type, two strains of Candida galli and six unidentified strains that resembled Y. lipolytica were examined by PCR fingerprints using primers M13 and (GAC)5. The same strains, together with four strains of the recently introduced Candida yakushimensis nom. inval., were sequenced for the D1/D2 domain of the 26S rRNA gene and parts of the ITS domain and also studied for their physiological properties. Of the strains identified previously as Y. lipolytica, CBS 2076 had the same fingerprint as the type of C. deformans and strain CBS 4855 was distinct from all other strains. The six strains that resembled Y. lipolytica were separated into two groups distinct from any of the other clades. A total of six groups obtained by fingerprint and sequence data were evaluated by performing DNA reassociation reactions. Mating experiments among the 35 strains of Y. lipolytica sensu stricto showed that 15 strains represented one mating type and 16 strains represented the opposite mating type, while four strains were self-sporulating. Teleomorph states were not produced by C. deformans, C. galli or any of the unidentified isolates. However, positive mating reactions were rarely observed in crosses among C. galli and some strains of Y. lipolytica and C. deformans. Consequently, sharing the same mating type system, C. deformans and C. galli could be considered anamorphs of unnamed Yarrowia species. Results from PCR fingerprints, sequencing and mating studies support the grouping of the studied strains into Y. lipolytica, C. galli, C. deformans, C. yakushimensis nom. inval. and three novel species in the Yarrowia clade: Candida oslonensis sp. nov. (type strain CBS 10146(T) =NRRL Y-48252(T); Mycobank number MB 510769), Candida alimentaria sp. nov. (type strain CBS 10151(T) =NRRL Y-48253(T); Mycobank number MB 510770) and Candida hollandica sp. nov. (type strain CBS 4855(T) =NRRL Y-48254(T); Mycobank number MB 510771).

Phylogenetic placement of Hanseniaspora-Kloeckera species using multigene sequence analysis with taxonomic implications: descriptions of Hanseniaspora pseudoguilliermondii sp. nov. and Hanseniaspora occidentalis var. citrica var. nov.

Two protein-coding genes, actin and translation elongation factor-1alpha (EF-1alpha), as well as two ribosomal gene regions, D1/D2 domains of the large subunit and both internal transcribed spacers including the 5.8S gene region, were evaluated regarding their usefulness for reconstruction of phylogenetic relationships in the Hanseniaspora-Kloeckera species group. This included analyses of sequence divergence values, heterogeneity of evolutionary rates and the reliability of the inferred trees. Both protein-coding genes showed greater capacities to resolve at the strain level and between the closely related species of Hanseniaspora-Kloeckera, compared with the ribosomal gene regions. However, to obtain a fully resolved and reliable phylogenetic tree that reflected the biological relationships it was necessary to combine three congruent sequence datasets. The novel species Hanseniaspora pseudoguilliermondii sp. nov. (type strain CBS 8772T) is described as a result of the application of various molecular approaches to delimit species. Furthermore, incongruent gene genealogies of genetically divergent strains of Hanseniaspora occidentalis, as determined by amplified fragment length polymorphism analysis and DNA-DNA reassociation measurements, indicated the presence of two novel varieties, H. occidentalis var. occidentalis (type strain CBS 2592T) and H. occidentalis var. citrica var. nov. (type strain CBS 6783T), which could be distinguished by habitat preference.

Molecular-genetic diversity of the ascomycetous yeast genus Arthroascus: Arthroascus babjevae sp. nov., Arthroascus fermentans var. arxii var. nov. and geographical populations of Arthroascus schoenii.

Using molecular and genetic analyses, 28 Arthroascus strains were analysed, isolated from widely different geographical localities in Europe, North America, Far-East Asia and Hawaii. Most of the strains have been assigned to the species Arthroascus schoenii. PCR-RAPD revealed two Japanese Arthroascus strains (UCD 67-278 and IFO 10138) to have peculiar patterns. Comparative rDNA (D1/D2 26S, ITS1 and ITS2) sequence analysis showed that the two strains respectively represent a novel species and a novel variety. Based on the results of sequence analysis, genetic hybridization and DNA-DNA reassociation, two new members of the genus Arthroascus are formally described, Arthroascus babjevae sp. nov. (type strain UCD 67-278(T)=CBS 9167(T)) and Arthroascus fermentans var. arxii var. nov. (type strain IFO 10138(T)=CBS 9168(T)). These results show that A. schoenii has a worldwide distribution, while the species Arthroascus javanensis is represented only by the type culture CBS 2555(T), isolated in Indonesia. Cluster analysis revealed a correlation between PCR-RAPD fingerprints and geographical origin of the A. schoenii strains. Despite this molecular differentiation, A. schoenii strains collected in different regions of the world formed predominantly fertile hybrids, with normal recombination of control markers.

Taxonomy and phylogeny of the ascomycetous yeast genus Zygoascus, with proposal of Zygoascus meyerae sp. nov. and related anamorphic varieties.

Physiological characters, mating compatibility, PCR-RAPD fingerprints, mol% G + C content, DNA-DNA relatedness, and large-subunit and internal transcribed spacer rRNA gene sequences of strains assigned to the genus Zygoascus were re-examined. On the basis of those data, and after phylogenetic analyses, an emendation of Zygoascus hellenicus (type material is a cross of CBS 6736(T) x CBS 5839(T)) is proposed, comprising two novel anamorphic varieties, Candida steatolytica var. steatolytica (CBS 6736(T)) and C. steatolytica var. inositophila (CBS 5839(T)). A novel teleomorphic species, Zygoascus meyerae sp. nov. (type material is a cross of CBS 4099(T) x CBS 7521(T)) is described, together with two novel anamorphic varieties corresponding to it, Candida hellenica var. hellenica (CBS 4099(T)) and C. hellenica var. acidophila (CBS 7115(T)).

Genome comparisons in the genus Dipodascus de Lagerheim.

Phenotypic characteristics of physiology and morphology of 71 strains belonging to the genus Dipodascus de Lagerheim were examined. The GC contents of genomic DNAs of 46 strains were calculated from the thermal denaturation curves using the spectrophotometric method. The first derivatives of the melting curves revealed that the DNAs of these strains are heterogeneous; four categories could be recognized. However, DNA similarity values calculated by using DNA-DNA reassociation kinetics showed that each category could be subdivided further. Two categories were separated into four subgroups each; the other two yielded five subgroups each. Strains belonging to the same subgroup exhibited high levels of DNA similarity ranging from 82 to 100%. The 18 subgroups represented 13 currently accepted Dipodascus species and five anamorphic Geotrichum species, four representing novel taxa. A phenotypic key to distinguish the taxa of Dipodascus, Galactomyces and Geotrichum is presented.

Ascobotryozyma cognata sp. nov., a new ascomycetous yeast associated with nematodes from wood-boring beetle galleries.

A new species of Ascobotryozyma, A. cognata sp. nov. (anamorph Botryozyma cognata), was isolated from beetle galleries in Idaho, USA. A. cognata was found on the surface of free-living nematodes, Panagrellus dubius, collected from galleries created by the long-horned beetle Saperda calcarata in Populus (aspen), and the weevil Cryptorhynchus lapathi in Salix (willow). A. cognata isolates were collected from similar habits and in relatively close proximity to those of A. americana, the only species described from North America. The recognition of A. cognata as a distinct species was supported by morphological and molecular data. Thallus cells of A. cognata were significantly shorter than those of A. americana. Low DNA reassociation values, notably different randomly amplified polymorphic DNA (RAPD), inter-sequence simple repeat (ISSR), and amplified fragment-length polymorphic (AFLP) fingerprints, and sequence divergence in both the D1/D2 domain of the nuc-LSU rDNA and an additional unidentified region were all consistent with the recognition of a new species.

Botryozyma mucatilis sp. nov., an anamorphic ascomycetous yeast associated with nematodes in poplar slime flux.

A new species of Botryozyma, Botryozyma mucatilis, was isolated from the surface of free-living nematodes, Panagrellus dubius, inhabiting slime flux from hybrid poplars, Populus deltoidesxtrichocarpa, in Oregon, USA. This species was discovered in relatively close proximity to the teleomorphic species Ascobotryozyma americana and Ascobotryozyma cognata, both collected from P. dubius nematodes inhabiting beetle galleries in Populus spp. and Populus and Salix spp., respectively. B. mucatilis is recognized as a distinct species based on molecular and morphological data. Sequence divergence in both the D1/D2 domain of the nuclear large-subunit rDNA and internal transcribed spacer region rDNA, low DNA reassociation values, notably different amplified fragment-length polymorphic fingerprints, and significantly longer cells all support the designation of a novel species.

Hanseniaspora meyeri sp. nov., Hanseniaspora clermontiae sp. nov., Hanseniaspora lachancei sp. nov. and Hanseniaspora opuntiae sp. nov., novel apiculate yeast species.

Fourteen apiculate yeast strains isolated from various sources in South Africa, North America and the Hawaiian islands were found to be genetically divergent from other Hanseniaspora-Kloeckera species by using randomly amplified polymorphic DNA (RAPD)-PCR. After cluster analysis of the RAPD-PCR fingerprints, five groups were recognized. DNA reassociation values among representatives of these groups and strains of Hanseniaspora-Kloeckera species revealed that the strains represent five novel species. Four are described here as novel species of HANSENIASPORA: Hanseniaspora meyeri sp. nov. (type CBS 8734(T)), Hanseniaspora clermontiae sp. nov. (type CBS 8821(T)), Hanseniaspora lachancei sp. nov. (type CBS 8818(T)) and Hanseniaspora opuntiae sp. nov. (type CBS 8733(T)). The fifth novel species, which is represented by only a single strain, CBS 8772, is not introduced as a new taxon. Phylogenetic analyses of the D1/D2 region of the 26S rDNA and internal transcribed spacer (ITS) regions with 5.8S rDNA sequences placed H. meyeri, H. clermontiae, H. lachancei, H. opuntiae and strain CBS 8772 close to Hanseniaspora uvarum and Hanseniaspora guilliermondii. The key characteristics for standard physiological identification of H. clermontiae and H. lachancei were respectively maximal growth temperature and assimilation of 2-keto-D-gluconate. However, physiological characteristics did not allow the distinction of H. opuntiae and strain CBS 8772 from H. guilliermondii or H. meyeri from H. uvarum. These three novel taxa can be identified by either ITS sequencing or PCR-RFLP of ITS regions using restriction enzymes MboII and HinfI.

Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera.

Three molecular methods, RAPD-PCR analysis, electrophoretic karyotyping and RFLP of the PCR-amplified ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), were studied for accurate identification of Hanseniaspora and Kloeckera species as well as for determining inter- and intraspecific relationships of 74 strains isolated from different sources and/or geographically distinct regions. Of these three methods, PCR-RFLP analysis of ITS regions with restriction enzymes DdeI and HinfI is proposed as a rapid identification method to discriminate unambiguously between all six Hanseniaspora species and the single non-ascospore-forming apiculate yeast species Kloeckera lindneri. Electrophoretic karyotyping produced chromosomal profiles by which the seven species could be divided into four groups sharing similar karyotypes. Although most of the 60 strains examined exhibited a common species-specific pattern, a different degree of chromosomal-length polymorphism and a variable number of chromosomal DNA fragments were observed within species. Cluster analysis of the combined RAPD-PCR fingerprints obtained with one 10-mer primer, two microsatellite primers and one minisatellite primer generated clusters which with a few exceptions are in agreement with the groups as earlier recognized in DNA-DNA homology studies.