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.

Taxonomy of the yeast genus Vanderwaltozyma and proposal of Vanderwaltozyma meishanica sp. nov., Vanderwaltozyma huisunica sp. nov., and Vanderwaltozyma molinica sp. nov.

Using electrophoretic karyotyping, RAPD fingerprinting and phylogenetic analysis of ribosomal RNA gene sequences, twenty-six Vanderwaltozyma strains were studied. Out of 19 strains isolated in mountainous areas of Taiwan, eighteen strains were isolated from soil and one strain was isolated from the fruiting body of mushroom, six were identified as V. polyspora and three as V. verrucispora. Based on the results of a multigene sequence analysis (D1/D2, ITS and mitochondrial COX II gene) and DNA-DNA reassociation, three new ascosporic members of the genus Vanderwaltozyma are formally described: V. huisunica sp. nov. (GA1S06T = CBS 12250T = BCRC 23260T), V. meishanica sp. nov. (EN4S02T = CBS 12249T = BCRC 23255T) and V. molinica sp. nov. (GJ8S05T = CBS 12251T = BCRC 23264T), and the holotypes of these novel species are assigned as BCRC 23260T, BCRC 23255T and BCRC 23264T, respectively.

Exploiting heterozygosity in industrial yeasts to create new and improved baker's yeasts.

The main aim of the work was to utilize heterozygosity of industrial yeast strains to construct new baker's yeast strains. Commercial baker's yeast strain ALKO 743, its more ethanol tolerant descendant ALKO 554 selected initially for growth over 300 generations in increasing ethanol concentrations in a glucose medium, and ALKO 3460 from an old domestic sour dough starter were used as starting strains. Isolated meiotic segregants of the strains were characterized genetically for sporulation ability and mating type, and the ploidy was determined physically. Heterozygosity of the segregant strains was estimated by a variety of molecular characterizations and fermentation and growth assays. The results showed wide heterozygosity and that the segregants were clustered into subgroups. This clustering was used for choosing distantly or closely related partners for strain construction crosses. Intrastrain hybrids made with segregants of ALKO 743 showed 16-24% hybrid vigour or heterosis. Interstrain hybrids with segregants of ALKO 743 and ALKO 3460 showed a wide variety of characteristics but also clear heterosis of 27-31% effects as assayed by lean and sugar dough raising. Distiller's yeast ALKO 554 turned out to be a diploid genetic segregant and not just a more ethanol tolerant mutant of the tetraploid parent strain ALKO 743.

Description of Komagataella mondaviorum sp. nov., a new sibling species of Komagataella (Pichia) pastoris.

Five methylotrophic strains (UCDFST 71-1024T, UCDFST 54-11.16, UCDFST 54-11.141, UCDFST 68-967.1 and UCDFST 74-1030) from the Phaff Yeast Culture Collection (University of California Davis, USA) that were originally designated as Pichia pastoris were found to represent a novel Komagataella species. Strains of Komagataella mondaviorum sp. nov. UCDFST 71-1024T(type strain) = CBS 15017, UCDFST 54-11.16, UCDFST 54-11.141, UCDFST 68-967.1, and UCDFST 74-1030 were isolated in USA, respectively, from cottonwood tree Populus deltoides in 1971 (Davis, CA), slime flux of Quercus sp. in 1954 (CA), exudate of black oak Q. kelloggii in 1954 (Central Sierra Nevada. CA), dry frass from Salix sp. in 1968 (Soleduck Road, Olympic National Park, WA) and from flux of hackberry tree Celtis sp. in 1974 (CA). The new species was differentiated from Komagataella kurtzmanii, Komagataella pastoris, Komagataella phaffii, Komagataella populi, Komagataella pseudopastoris and Komagataella ulmi by divergence in gene sequences for D1/D2 LSU rRNA, ITS1-5.8S-ITS2, RNA polymerase subunit I and translation elongation factor-1α. Komagataella mondaviorum sp. nov. is registered in MycoBank under MB 821789.

Ogataea haglerorum sp. nov., a novel member of the species complex, Ogataea (Hansenula) polymorpha.

Three strains representing a novel species of the Ogataea clade were isolated by W. T. Starmer and H. J. Phaff from rotting tissue of Opuntia phaeacantha in Arizona, USA. Analyses of the sequences of the D1/D2 LSU rRNA gene, ITS1-5.8S-ITS2, and translation elongation factor-1α (EF-1 α) showed that this novel species belongs to the Ogataea polymorpha complex formed by Ogataea angusta, Ogataea parapolymorpha and Ogataea polymorpha. The novel species differs from these species by 4-5 nucleotide substitutions in the D1/D2 domain, by 28-29 nucleotide substitutions in the EF-α gene and by 18-24 nucleotide substitutions and 2-5 indels in the ITS-5.8S region. The name Ogataea haglerorum sp. nov. is proposed for this novel species. The type strain is VKPM Y-2583T (=CBS 14645T=UCDFST 17-101T). The Mycobank number is MB 819772.

Ancient and recent origins of shared polymorphisms in yeast.

Shared genetic polymorphisms between populations and species can be ascribed to ancestral variation or to more recent gene flow. Here, we mapped shared polymorphisms in Saccharomyces cerevisiae and its sister species Saccharomyces paradoxus, which diverged 4-6 million years ago. We used a dense map of single-nucleotide diagnostic markers (mean distance 15.6 base pairs) in 1,673 sequenced S. cerevisiae isolates to catalogue 3,852 sequence blocks (≥5 consecutive markers) introgressed from S. paradoxus, with most being recent and clade-specific. The highly diverged wild Chinese S. cerevisiae lineages were depleted of introgressed blocks but retained an excess of individual ancestral polymorphisms derived from incomplete lineage sorting, perhaps due to less dramatic population bottlenecks. In the non-Chinese S. cerevisiae lineages, we inferred major hybridization events and detected cases of overlapping introgressed blocks across distinct clades due to either shared histories or convergent evolution. We experimentally engineered, in otherwise isogenic backgrounds, the introgressed PAD1-FDC1 gene pair that independently arose in two S. cerevisiae clades and revealed that it increases resistance against diverse antifungal drugs. Overall, our study retraces the histories of divergence and secondary contacts across S. cerevisiae and S. paradoxus populations and unveils a functional outcome.

Molecular genetic diversity of the Saccharomyces yeasts in Taiwan: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii.

Genetic hybridization, sequence and karyotypic analyses of natural Saccharomyces yeasts isolated in different regions of Taiwan revealed three biological species: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii. Intraspecies variability of the D1/D2 and ITS1 rDNA sequences was detected among S. cerevisiae and S. kudriavzevii isolates. According to molecular and genetic analyses, the cosmopolitan species S. cerevisiae and S. kudriavzevii contain local divergent populations in Taiwan, Malaysia and Japan. Six of the seven known Saccharomyces species are documented in East Asia: S. arboricola, S. bayanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus.

Komagataella kurtzmanii sp. nov., a new sibling species of Komagataella (Pichia) pastoris based on multigene sequence analysis.

A novel methanol assimilating yeast species Komagataella kurtzmanii is described using the type strain VKPM Y-727 (=KBP Y-2878 = UCD-FST 76-20 = Starmer #75-208.2 = CBS 12817 = NRRL Y-63667) isolated by W.T. Starmer from a fir flux in the Catalina Mountains, Southern AZ, USA. The new species is registered in MycoBank under MB 803919. The species was differentiated by divergence in gene sequences for D1/D2 LSU rRNA, ITS1-5.8S-ITS2, RNA polymerase subunit I, translation elongation factor-1α and mitochondrial small subunit rRNA. K. kurtzmanii differs from its phenotypically similar sibling species Komagataella pastoris, Komagataella pseudopastoris, Komagataella phaffii, Komagataella populi and Komagataella ulmi by absence of growth at 35 °C and inability to assimilate trehalose.

Five novel species of the anamorphic genus Candida in the Cyberlindnera clade isolated from natural substrates in Taiwan.

Twelve strains representing five novel yeast species were isolated from natural samples distributed in mountain areas in Taiwan during 2007 and 2009. Sequence analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene revealed that these species are members of the Cyberlindnera clade. These five new species have a greater than 1% difference from their closest relatives in the sequences of the D1/D2 domain of the LSU rRNA gene and were well separated from their closest relatives in terms of physiological characteristics. Moreover, a sexual state could not be found in these five novel yeast species. Therefore, the scientific names of Candida maesa sp. nov. (type strain GJ8L01(T)), Candida takata sp. nov. (type strain EN25S01(T)), Candida taoyuanica sp. nov. (type strain GY15S07(T)), Candida hungchunana sp. nov. (type strain NC3W71(T)) and Candida stauntonica sp. nov. (type strain GY13L05(T)) were proposed to accommodate these yeasts.

Genetic identification of new biological species Saccharomyces arboricolus Wang et Bai.

Direct genetic testing for hybrid sterility unambiguously showed that the newly described yeast Saccharomyces arboricolus Wang et Bai is reproductively isolated from Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces kudriavzevii, Saccharomyces mikatae and Saccharomyces paradoxus and, therefore, represents a new biological species of the genus Saccharomyces. Combined phylogenetic analysis of the rDNA repeat sequences (18S, 26S, ITS), nuclear ACT1 and mitochondrial ATP9 genes revealed that S. arboricolus, along with S. kudriavzevii and S. bayanus, is distantly related to the other four biological species.

Viral dsRNA in the wine yeast Saccharomyces bayanus var. uvarum.

The presence of viral dsRNA (L and M fractions) in the cryophilic yeast Saccharomyces bayanus var. uvarum is documented here for the first time. Sixty-eight strains of different origins were analyzed. Most of them did not carry dsRNA; the L fraction was found in seven strains, while 11 strains had both L and M fractions. The size of the L fraction was invariable (4.5 kb), as in the cultured yeast Saccharomyces cerevisiae. In contrast to L-dsRNA, the M fraction varied in size from ca. 1.2 to 1.8 kb. In total, seven different M-dsRNA types were recognized (M1-M3 and M8-M11), predominantly among French wine strains of S. bayanus var. uvarum. Phenotypic analysis revealed that the M-dsRNAs found were cryptic and may represent mutant forms of killer plasmids.

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.

Genetic diversity study of the yeast Saccharomyces bayanus var. uvarum reveals introgressed subtelomeric Saccharomyces cerevisiae genes.

Intraspecies polymorphism of the yeast Saccharomyces bayanus var. uvarum was studied using the polymerase chain reaction with a microsatellite primer (GTG)(5). Sixty-nine strains of different origins were analyzed. There existed a correlation between PCR patterns of the strains and the source of their isolation: the type of wine and the particular winemaking region. Southern hybridization analysis revealed for the first time introgression between Saccharomyces cerevisiae and S. bayanus var. uvarum. Two strains isolated from alcoholic beverages in Hungary and identified by genetic analysis as S. bayanus var. uvarum were found to harbor a number of S. cerevisiae subtelomeric sequences: Y', SUC, RTM and MAL.

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.

Molecular genetic study of introgression between Saccharomyces bayanus and S. cerevisiae.

The genomic constitution of different S. bayanus strains and natural interspecific Saccharomyces hybrids has been studied by genetic and molecular methods. Unlike S. bayanus var. uvarum, some S. bayanus var. bayanus strains (the type culture CBS 380, CBS 378, CBS 425, CBS 1548) harbour a number of S. cerevisiae subtelomeric sequences: Y', pEL50, SUC, RTM and MAL. The two varieties, having 86-100% nDNA-nDNA reassociation, are partly genetically isolated from one another but completely isolated from S. cerevisiae. Genetic and molecular data support the maintaining of var. bayanus and var. uvarum strains in the species S. bayanus. Using Southern hybridization with species-specific molecular markers, RFLP of the MET2 gene and flow cytometry analysis, we showed that the non-S. cerevisiae parents are different in lager brewing yeasts and in wine hybrid strains. Our results suggest that S. pastorianus is a hybrid between S. cerevisiae and S. bayanus var. bayanus, while S. bayanus var. uvarum contributed to the formation of the wine hybrids S6U and CID1. According to the partial sequence of ACT1 gene and flow cytometry analysis, strain CID1 is a triple hybrid between S. cerevisiae, S. kudriavzevii and S. bayanus var. uvarum.

Five new combinations in the yeast genus Zygofabospora Kudriavzev emend. G. Naumov (pro parte Kluyveromyces) based on genetic data.

The rDNA sequencing data obtained during the last 5 years in several laboratories clearly demonstrate that within the heterogeneous genus Kluyveromyces there is a group of highly related species, which we refer to as the genus Zygofabospora Kudriavzev 1960 emend. G. Naumov 2002. This genus includes four hybridizing species, Zf. marxiana, Zf. dobzhanskii, Zf. lactis, Zf. wickerhamii (Zygofabospora sensu stricto), and two taxonomically related species, Zf. aestuarii, Zf. nonfermentans (Nagahama et al.) G. Naumov comb. nov. (Zygofabospora sensu lato). We studied the relationships of the yeasts composing the Zf. lactis complex. Genetic hybridization analysis and molecular karyotyping revealed partial genetic-isolation varieties, Zf. lactis var. drosophilarum (Shehata et al.) G. Naumov comb. nov. and Zf. lactis var. phaseolospora (Shehata et al.) G. Naumov comb. nov. from North America, and Zf. lactis var. krassilnikovii (Kudriavzev) G. Naumov comb. nov. from Europe. The dairy yeast Zf. lactis var. lactis G. Naumov comb. nov. yields highly fertile hybrids with its wild ancestor Zf. lactis var. krassilnikovii and semi-sterile hybrids with North American taxa. Besides, Zf. lactis var. lactis and Zf. lactis var. krassilnikovii formed fertile hybrids with the South African yeast Zf. lactis var. vanudenii (van der Walt et Nel) G. Naumov comb. nov. The reinstatement of the latter yeast at the variety level has been done taking into account the results of the present study and the literature data on its geographic isolation, high divergence of the karyotype and mitochondrial DNA.

Molecular-genetic differentiation of the dairy yeast Kluyveromyces lactis and its closest wild relatives.

The currently accepted formal division of the species Kluyveromyces lactis into two taxonomic varieties, Kl. lactis var. lactis and Kl. lactis var. drosophilarum, is based arbitrarily on phenotypic and ecological characters. On the other hand, the genetic hybridisation analysis and molecular karyotyping of its synonyms allowed us [FEMS Yeast Res. 2 (2002) 39] to reinstate them in the genus Zygofabospora Kudriavzev emend G. Naumov (=Kluyveromyces Kurtzman et al., 2001) as the varieties Zf. lactis var. lactis, Zf. lactis var. krassilnikovii, Zf. lactis var. drosophilarum, Zf. lactis var. phaseolospora and Zf. lactis var. vanudenii. In the present work, we studied forty Kl. lactis strains of different geographic and ecological origins by means of restriction analysis of the PCR-amplified non-coding nrDNA regions encompassing the intergenic spacer 2 (IGS2) and the internal transcribed spacers (ITS1 and ITS2). The results confirmed the complex structure of Kl. lactis. Moreover, four additional genetic populations were identified: three in North America ('aquatic', 'pseudovanudenii' and 'new') and one in Far-East Asia ('oriental'). Comparative sequence analysis of the 5.8S-rRNA gene and the two internal transcribed spacers revealed that the populations 'aquatic' and 'oriental' formed distinct taxa which are phylogenetically separate from the five known populations. However, some discrepancies were observed between the restriction and sequencing data. Genetic hybridisation analysis needs to be done to further elucidate the genetic relationships between the populations of Kl. lactis.

Molecular genetic identification of Saccharomyces sensu stricto strains from African sorghum beer.

Genetic relationships of 24 phenotypically different strains isolated from sorghum beer in West Africa and the type cultures of the Saccharomyces sensu stricto species were investigated by universally primed polymerase chain reaction (PCR) analysis, microsatellite fingerprinting and PCR-restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers. The results demonstrate that internal transcribed spacer (ITS) PCR-RFLP analysis with the endonucleases HaeIII, HpaII, ScrFI and TaqI is useful for discriminating S. cerevisiae, S. kudriavzevii, S. mikatae from one another and from the S. bayanus/S. pastorianus and S. cariocanus/S. paradoxus pairs. The sorghum beer strains exhibited the same restriction patterns as the type culture of S. cerevisiae CBS 1171. PCR profiles generated with the microsatellite primer (GTG)(5) and the universal primer N21 were almost identical for all isolates and strain CBS 1171. Despite phenotypic peculiarities, the strains involved in sorghum beer production in Ghana and Burkina Faso belong to S. cerevisiae. However, based on sequencing of the rDNA ITS1 region and Southern hybridisation analysis, these strains represent a divergent population of S. cerevisiae.

High-rate evolution of Saccharomyces sensu lato chromosomes.

Forty isolates belonging to the Saccharomyces sensu lato complex were analyzed for one nuclear and two mitochondrial sequences, and for their karyotypes. These data are useful for description and definition of yeast species based on the phylogenetic species concept. The deduced phylogenetic relationships among isolates based on the nuclear and mitochondrial sequences were usually similar, suggesting that horizontal transfer/introgression has not been frequent. The highest degree of polymorphism was observed at the chromosome level. Even isolates which had identical nuclear and mitochondrial sequences often exhibited variation in the number and size of their chromosomes. Apparently, yeast chromosomes have been frequently reshaped and therefore also the position of genes has been dynamic during the evolutionary history of yeasts.