Sex in the cold: taxonomic reorganization of psychrotolerant yeasts in the order Leucosporidiales.

Species of Leucosporidiales are a group of psychrotolerant yeasts with biotechnological potential. In the present work, we studied the phenotypic, genetic and sexual characteristics of three species of this genus (Leucosporidium scottii, Leucosporidiella creatinivora and Le. yakutica) to clarify the evolutionary relationship among these closely related taxa. From the results obtained, it becomes clear that these yeasts can interbreed. Although genetic delimitation is possible for the three species, the extent of nucleotide substitutions and phenotypic differences observed between them are lower than that expected for species that have ended the speciation process. Our taxonomic conclusion is to maintain the three taxa until further genomic data are gathered. However, the concept of L. scottii species complex is proposed for this group of species. Finally, we transfer all Leucosporidiella and Mastigobasidium species to Leucosporidium (Leucosporidiales), and, in order to end the polyphyly condition of these taxa, we propose the new genus Pseudoleucosporidium gen. nov. and the new combination Peudoleucosporidium fasciculatum comb. nov.

Favored isolation and rapid identification of the astaxanthin-producing yeast Xanthophyllomyces dendrorhous(Phaffia rhodozyma) from environmental samples.

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) yeasts are biotechnologically exploited as a natural source of astaxanthin for aquaculture. Based on results of recent studies, it has become clear that this species possesses a greater genetic variability generating the necessity to uncover it and assess its potential for the astaxanthin industry. However, difficulties for the isolation of the X. dendrorhous hinder extensive environmental surveys which need to be carried out to better understand the habitat, distribution and genetic diversity of this species. We extensively searched for distinctive physiological traits of X. dendrorhours by testing phenotypic properties simultaneously with a panel of common sympatric fungi. As a result we obtained a new and innovative strategy for improving X. dendrorhous recovery rate and identification from environmental samples. This strategy involved the use of trehalose-based media, and a rapid X. dendrorhous identification method based on the simultaneous spectrophotometric detection of astaxanthin and UV-absorbing compounds (mycosporines). The proposed procedures proved effective in field trials conducted in natural environments of Patagonia (Argentina) and thus represent an important tool for the discovery of new astaxanthin-producing strains of X. dendrorhous useful for the aquaculture industry.

Phylogenetic distribution of fungal mycosporines within the Pucciniomycotina (Basidiomycota).

The synthesis of the UV Absorbing compounds named mycosporines (MYCs) has been detected in a few basidiomycetous yeast groups. Conspicuous accumulation of mycosporine-glutaminol-glucoside (MGG) in yeasts requires photo-induction and its photoprotective function has been postulated. The distribution of the ability to produce MYCs appeared to be related to the yeast taxonomic affiliation. In view of the potential significance of MYCs in yeast taxonomy, we here studied the distribution of this trait among dimorphic basidiomycetes of the Pucciniomycotina. Of the 94 fungal species (377 strains and 33 genera) tested, almost half were MYC-positive and MGG was the main compound produced. MGG synthesis was observed for representatives of five of seven Pucciniomycotina classes, indicating that this trait is widely distributed in this group. MGG detection proved useful for the differentiation of species of the polyphyletic genera, such as Rhodotorula and Sporobolomyces, that are phylogenetically separated. MGG quantification and UV tolerance studies in Cystobasidiomycetes supported the idea that the habitat of origin of each strains is important in the level of MGG synthesis and that MYCs have a photoprotective function in yeasts. The taxonomic value of this trait in fungal systematics is discussed.

Production of the UVB-absorbing compound mycosporine-glutaminol-glucoside by Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

The ability of the basidiomycetous yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) to accumulate astaxanthin is responsible for the industrial use of this yeast as a microbial source of pigments for aquaculture. It is also hypothesized that astaxanthin accounts for its ability to thrive in highly oxidative and UV-exposed habitats. Here, we assessed the ability of this species to synthesize UV-absorbing compounds generally known as mycosporines, evaluated the effect of culture media in the production of these compounds and compared its UV growth resistance and tolerance with other yeasts. The 48 wild and collection strains screened were positive for mycosporines and a unique compound identified as mycosporine-glutaminol-glucoside (MGG) was detected. Thus, the ability of X. dendrorhous to produce MGG, as described here for the first time, is so far unique among the Cystofilobasidiales. The compound was synthesized constitutively, although growth under visible light and, to a greater extent, UVA radiation stimulated its production. Strains from UV-exposed habitats produced larger quantities and oligotrophic complex media seemed to favor MGG accumulation. UV tolerance and survival of X. dendrorhous was high and comparable to that of the polyextremophilic Rhodotorula mucilaginosa. The taxonomical and ecological implications of the production of MGG by X. dendrorhous are discussed.

Description of Holtermanniella gen. nov., including Holtermanniella takashimae sp. nov. and four new combinations, and proposal of the order Holtermanniales to accommodate tremellomycetous yeasts of the Holtermannia clade.

The novel genus Holtermanniella is proposed here to accommodate four Cryptococcus species closely related to Holtermannia corniformis that are included in the Holtermannia clade (Basidiomycota, Agaricomycotina). Thus, four novel combinations are proposed: Holtermanniella nyarrowii comb. nov., Holtermanniella festucosa comb. nov., Holtermanniella mycelialis comb. nov. and Holtermanniella wattica comb. nov. In addition, a novel anamorphic yeast species was studied with 15 isolates obtained from different habitats around the world. Analysis of the sequences of the D1/D2 region of their large subunit rDNA showed that the novel species is placed phylogenetically within the Holtermannia clade of the Tremellomycetes (Agaricomycotina, Basidiomycota). PCR fingerprinting and sequencing of ITS1-5.8S-ITS2 showed genetic intraspecific variability among the strains: three groups were formed, which did not correlate with geographical origin or substrate. This novel species, designated the type species of Holtermanniella gen. nov., is described as Holtermanniella takashimae sp. nov.; the type strain is CBS 11174(T) (=HB 982(T) =DBVPG 8012(T)). The order Holtermanniales ord. nov. is proposed here to include Holtermannia (the type genus) and Holtermanniella.

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) on stromata of Cyttaria hariotii in northwestern Patagonian Nothofagus forests.

The occurrence and distribution of Xanthophyllomyces dendrorhous associated with Cyttaria hariotii parasitizing three Nothofagus species (N. dombeyi, N. antarctica and N. pumilio) in northwestern Patagonia (Argentina), as well as the factors that may affect this distribution were herein studied. Between 2000 and 2007, samples were obtained from 18 different locations. Based on physiological tests and morphological characteristics of sexual structures, 72 isolates were identified as X. dendrorhous. Representative strains were studied by MSP-PCR fingerprinting and sequence analysis of the ITS region. MSP-PCR fingerprints were similar for the newly isolated strains, and were also identical to the profiles of the strains previously found in this region. Patagonian strains appear to be a genetically uniform and distinct population, supporting the hypothesis that the association with different host species has determined genetically distinct X. dendrorhous populations worldwide. X. dendrorhous was recovered from N. dombeyi and N. antarctica. Approximately half the sampling sites and samples were positive for X. dendrorhous, but the isolation recovery rate was low. X. dendrorhous was absent in the early stages of ascostromata maturation, becoming more abundant in later stages. The present work represents a step forward in the understanding of the natural distribution and ecology of this biotechnologically relevant yeast.

The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica.

The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4°C and 20°C, indicating that they could be metabolically active in the sampled substrates.

Photoprotection by carotenoid pigments in the yeast Rhodotorula mucilaginosa: the role of torularhodin.

In this paper we report the relationship between carotenoids and ergosterol and cell UV-B resistance in different strains of the yeast Rhodotorula mucilaginosa. Cell survival was studied using a set of 13 strains; additionally, two mutants (a hyper-producing one and a colourless one) in combination with diphenylamine (DPA), a carotenogenesis inhibitor, were used. A positive correlation between total carotenoids and survival to UV-B radiation was found. However, when individual carotenoid concentrations were tested, only torularhodin was found to be significantly related to UV-B survival. On the contrary, ergosterol did not affect survival. The hyper-pigmented strain showed an enhanced survival (up to 250%) compared to the parental strain, while the survival of the albino mutant was similar to that experienced by the parental strain; however, observed changes in survival were dose dependent. The cyclobutane pyrimidine dimers (CPDs), one of the major forms of DNA damage caused by UV exposure, appears as unrelated to the accumulation of carotenoids and cell survival. These results indicate that bearing higher torularhodin concentrations enhances UV-B survival in yeasts and, thus, the accumulation of this pigment constitutes an important mechanism that improves the resistance of yeasts to UV-B.

Yeast diversity in the acidic Rio Agrio-Lake Caviahue volcanic environment (Patagonia, Argentina).

The Rio Agrio and Lake Caviahue system (RAC), in Northwestern Patagonia, is a natural acidic environment. The aims of this study were to characterize the yeast community and to provide the first ecological assessment of yeast diversity of this extreme aquatic environment. Yeast occurrence and diversity were studied at seven sites where the water pH varied between 1.8 and 6.7. Yeast CFU counts in the river ranged from 30 to 1200 CFU L(-1), but in the Lake the values were lower (30-60 CFU L(-1)). A total of 25 different yeast species were found, 11 of which belonged to undescribed taxa. Among these was an unusual strongly acidophilic Cryptococcus species. The RAC yeast community resembles that of acidic aquatic environments resulting from anthropic activities such as the São Domingos mines in Portugal and the Rio Tinto in Spain, respectively. The isolated yeast species were organized into different grades of adaptation to the RAC aquatic system. Based on the proposed grades, Rhodotorula mucilaginosa, Rhodosporidium toruloides and two novel Cryptococcus species were the most adapted species. These Cryptococcus species are apparently specialists of acidic aquatic environments, and might bear physiological features that possibly account for their ability to thrive in such extreme environments.

Biodiversity of cold-adapted yeasts from glacial meltwater rivers in Patagonia, Argentina.

The occurrence of culturable yeasts in glacial meltwater from the Frías, Castaño Overo and Río Manso glaciers, located on Mount Tronador in the Nahuel Huapi National Park (Northwestern Patagonia, Argentina) is presented. Subsurface water samples were filtered for colony counting and yeast isolation. The total yeast count ranged between 6 and 360 CFU L(-1). Physiologic and molecular methods were employed to identify 86 yeast isolates. In agreement with yeast diversity data from studies for Antarctic and Alpine glaciers, the genera Cryptococcus, Leucosporidiella, Dioszegia, Rhodotorula, Rhodosporidium, Mrakia, Sporobolomyces, Udeniomyces and Candida were found. Cryptococcus and Leucosporidiella accounted for 50% and 20% of the total number of strains, respectively. Among 21 identified yeast species, Cryptococcus sp. 1 and Leucosporidiella fragaria were the most frequent. The typically psychrophilic Mrakia yeast strain and three new yeast species, yet to be described, were also isolated. All yeast strains were able to grow at 5, 10, and 15 degrees C. Among yeast strains expressing extracellular enzymatic activity, higher proteolytic and lipolytic activities were obtained at 4 degrees C than at 20 degrees C.

Occurrence of photoprotective compounds in yeasts from freshwater ecosystems of northwestern Patagonia (Argentina).

In this paper we present the results of research on the occurrence, induction and role of photoprotective compounds (PPCs) present in native aquatic yeasts from freshwater Patagonian ecosystems. We focus on the effect of UV radiation (UVR) as a factor that controls the level of photoprotection of yeasts, and explore its potential significance in shaping yeast distributional patterns. The research presented here combines field surveys and laboratory work, including the isolation and culture of native yeasts strains, and laboratory assays under different radiation conditions. The results obtained suggest that yeasts are common dwellers of oligotrophic Patagonian water bodies, and provide the first evidence of the distribution of PPC (carotenoid and mycosporine)-producing yeasts in temperate freshwaters. A greater proportion of carotenogenic yeasts were observed in high-elevation lakes. The yeast strains isolated from these environments were found to produce higher amounts of mycosporines (MYCs), and to present higher tolerance to UVB exposure than those from piedmont lakes. Patagonian yeasts have only one type of MYC, mycosporine-glutaminol-glucoside (myc-glu-glu), which seems common to all other yeasts. By analyzing the production of myc-glu-glu in a large number of yeasts belonging to different taxonomic groups, we propose that this compound may have potential use as a chemotaxonomic marker in yeast systematics. Collectively, our work reveals that in Patagonian freshwater yeasts there is an apparent relationship between the ability to produce PPCs, their tolerance to UV exposure and their success in colonizing habitats highly exposed to UVR.

Mycosporines in carotenogenic yeasts.

The ability to produce mycosporines (MYCs) in 157 pigmented yeast strains (eight genera, 25 species) isolated from natural environments of Patagonia (Argentina) was assessed. The strains belong to four taxonomic groups: the Sporidiobolales and Erythrobasidium clade of the class Urediniomycetes, and Cystofilobasidiales and Tremellales of the class Hymenomycetes. Induction of MYCs did not occur in all yeast strains tested and appeared to be an exclusive trait of members of the Erythrobasidium clade and Tremellales. This is the first report on the production of MYCs by pigmented species from the latter group, as well as the first extensive screening of mycosporinogenic yeasts. The consistent occurrence of MYCs in some specific phylogenetic groups suggests this trait bears evolutionary significance and that the presence/absence of MYCs may have potential applications in yeast systematics.

Production of torularhodin, torulene, and ß-carotene by Rhodotorula yeasts.

Yeasts of the genera Rhodotorula are able to synthesize different pigments of high economic value like ß-carotene, torulene, and torularhodin, and therefore represent a biotechnologically interesting group of yeasts. However, the low production rate of pigment in these microorganisms limits its industrial application. Here we describe some strategies to obtain hyperpigmented mutants of Rhodotorula mucilaginosa by means of ultraviolet-B radiation, the procedures for total carotenoids extraction and quantification, and a method for identification of each pigment.

Yeasts from glacial ice of Patagonian Andes, Argentina.

Glacial ice and snow are known habitats for cold-adapted microorganisms. Research on cold-adapted yeast biodiversity from Perito Moreno and Mount Tronador glaciers (Patagonia, Argentina), and production of extracellular enzymatic activity at low temperatures (5 and 18 °C), was performed and described in this study. Ninety percent (90%) of the isolates were basidiomycetous; 16 genera and 29 species were identified. Twenty-five percent (25%) of total isolates corresponded to psychrophilic yeasts, whereas 75% were psychrotolerant yeasts. Eighty-five percent (85%) of all isolates had at least one enzymatic activity. Multiple correspondence analysis and cluster classification revealed a relationship between certain genera and some enzymatic activities. Cold-adapted yeast isolates were able to hydrolyze natural compounds (casein, lipids, starch, pectin, and carboxymethylcellulose) at low temperatures, suggesting a significant ecological role of these organisms as organic matter decomposers and nutrient cyclers. These yeasts are especially relevant for metabolic and ecological studies, as well as for yeast-based biotechnological process at low temperatures.

Cryptococcus species (Tremellales) from glacial biomes in the southern (Patagonia) and northern (Svalbard) hemispheres.

Cryptococcus species (Basidiomycota) were isolated as the predominant yeast from glacial biomes of both Patagonia (Argentina) and the Svalbard archipelago (Norway). For a selected group of Cryptococcus belonging to Tremellales, assimilative profile, production of extracellular hydrolytic enzymes and ribosomal DNA internal transcribed spacer and large subunit (D1/D2) sequences were analysed. Cryptococcus victoriae, which was originally described from Antarctica, was the most frequently found species at both locations. High variability within the species was observed and described at the genotypic and phenotypic levels, two newly described species were found in both Patagonia and Svalbard: Cryptococcus fonsecae and Cryptococcus psychrotolerans. Two other new species were found only in Patagonia: Cryptococcus frias and Cryptococcus tronadorensis. Three additional new taxa were found, but they are not named as they were only represented by single isolates.

Yeasts from an oligotrophic lake in Patagonia (Argentina): diversity, distribution and synthesis of photoprotective compounds and extracellular enzymes.

Nahuel Huapi (NH) Lake is an oligotrophic temperate lake of glacial origin with high transparency, surrounded by well-developed forests and located at San Carlos de Bariloche, Nahuel Huapi National Park, in Patagonia, Argentina. In this lake, we characterized yeast distribution and diversity along a south-to-north transect and established a relationship between the ability to produce photoprotective compounds (PPCs) (carotenoid pigments and mycosporines) and the occurrence of yeast at different collection points. Subsurface water samples were filtered for yeast isolation. Total yeast counts ranged between 22 and 141 CFU L(-1) , and the highest values corresponded to the most impacted sites. Littoral sites had a low proportion of yeast-producing PPCs and this group prevailed in pelagic sites. This is probably a result of the high transparency of the water and the increased UV exposure. The yeast community from NH Lake showed a high species richness and a uniform distribution of taxa between pelagic and border collection points. Yeasts were identified as belonging to 14 genera and 34 species. Rhodotorula mucilaginosa and Cryptococcus victoriae were the most frequently found species, representing 14.4% and 13.6% of the total yeast isolates, respectively. Most of the yeast isolates demonstrated at least one extracellular enzymatic activity (mainly cellulase and lipase activities), which suggested that these microorganisms are metabolically active in the lake.

UVB photoprotective role of mycosporines in yeast: photostability and antioxidant activity of mycosporine-glutaminol-glucoside.

Several yeast species are able to synthesize and accumulate UV-radiation-absorbing mycosporine metabolites that are of unclear physiological function. In this work we analyzed the relationship between mycosporine-glutaminol glucoside (MGG) production, cell survival after UVB irradiation, and formation of cyclobutane pyrimidine dimers (CPDs). We also assessed the photostability and singlet oxygen quenching activity of MGG. A set of nine isolates of the basidiomycetous yeast Cryptococcus steppossus cultured in both dark and light conditions was used for the studies. Survival of the UVB-irradiated isolates and MGG concentration had a linear relationship when the concentration was over 2.5 mg g(-1). CPD accumulation and MGG accumulation were inversely related. MGG in aqueous solution was photostable with a photodecomposition quantum yield of 1.16 × 10(-5). MGG quenching of singlet oxygen was also observed, and the rate constant for the process in D(2)O was 5.9 × 10(7) M(-1) s(-1). Our results support the idea that MGG plays an important role as a UVB photoprotective metabolite in yeasts by protecting against direct damage on DNA and probably against indirect damage by singlet oxygen quenching.

Cystobasidiomycetes yeasts from Patagonia (Argentina): description of Rhodotorula meli sp. nov. from glacial meltwater.

A basidiomycetous yeast, strain CRUB 1032(T), which formed salmon-pink colonies, was isolated from glacial meltwater in Patagonia, Argentina. Morphological, physiological and biochemical characterization indicated that this strain belonged to the genus Rhodotorula. Molecular taxonomic analysis based on the 26S rDNA D1/D2 domain and internal transcribed spacer region sequences showed that strain CRUB 1032(T) represents an undescribed yeast species, for which the name Rhodotorula meli sp. nov. is proposed (type strain is CRUB 1032(T)=CBS 10797(T)=JCM 15319(T)). Phylogenetic analysis showed that Rhodotorula lamellibrachii was the closest known species, which, together with R. meli, formed a separate cluster related to the Sakaguchia clade within the Cystobasidiomycetes. Additional Patagonian yeast isolates of the class Cystobasidiomycetes are also investigated in the present work.

Wickerhamomyces patagonicus sp. nov., an ascomycetous yeast species from Patagonia, Argentina.

Eight strains of a novel yeast species were isolated from tree saps of 'Coihue' (Nothofagus dombeyi, Nothofagaceae) and glacial meltwater (Castaño Overo River) in the Nahuel Huapi National Park, Patagonia, Argentina. The sequences of the D1/D2 domains of the large subunit of the rRNA gene showed that this novel yeast species belongs to the Wickerhamomyces genus (Order Saccharomycetales, Family Wickerhamomycetaceae). The closest related species were Candida ponderosae and Wickerhamomyces chambardii. Wickerhamomyces patagonicus sp. nov. is proposed to accommodate these novel strains, with the type strain CRUB 1724(T) (=CBS 11398(T) =JCM 16381(T)).

Cryptococcus spencermartinsiae sp. nov., a basidiomycetous yeast isolated from glacial waters and apple fruits.

Seven strains representing a novel yeast species belonging to the genus Cryptococcus were isolated from different substrates from Patagonia, Argentina, and The Netherlands. Three strains were isolated from a meltwater river draining from the Frias glacier at Mount Tronador situated in Nahuel Huapi National Park (Patagonia) and four were isolated from apple surfaces in Randwijk, The Netherlands. Analysis of the D1/D2 large-subunit rRNA gene and ITS region sequences indicated that these strains represent a single species that is distinct from other species of the Tremellales clade. The name Cryptococcus spencermartinsiae sp. nov. is proposed to accommodate these strains. The type strain is CRUB 1230(T) (=CBS 10760(T) =DBVPG 8010(T)).