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During a survey of floricolous yeasts in Portugal, a basidiomycetous yeast representing a novel species in the genus Hannaella was isolated in Portugal from the flower of Lantana camara , an ornamental exotic species native to Central and South America. A combination of phylogenetic analyses of DNA barcode sequences used in yeast molecular systematics, namely the D1/D2 domain and the complete internal transcribed spacer (ITS) region supported the recognition of a new species of Hannaella , that we designate Hannaella floricola sp. nov. (ex-type strain PYCC 9191 T =CBS 18097 T ). Although the assignment of the new species to the genus Hannaella was evident, the detection of its closest relatives appeared more problematic. Nevertheless, our analyses suggested that H. floricola sp. nov. belongs a clade that also includes H. coprosmae , H. oryzae and H. surugaensis , together four candidate novel species. In addition we provide the molecular identification of several unidentified strains whose D1/D2 and ITS sequences are available from GenBank.

An inventory of culturable yeasts from the soil and water of natural CO2 springs (mofettes) in northeast Slovenia is presented. In mofettes, CO2 of geological origin reaches the soil surface causing temporarily and spatially stable hypoxic environments in soil and water. In total, 142 yeast strains were isolated and identified from high CO2 and control meadow soil, meadow ground-water, forest pond and stream water. All water locations showed below-ground CO2 release. They were assigned to six basidiomycetous yeast genera (six species) and 11 ascomycetous genera (18 species). All ascomycetous yeasts, with the exception of Debaryomyces hansenii, were able to grow under elevated CO2 and fermented glucose. Candida sophiae-reginae, Pichia fermentans and Candida vartiovaarae were the dominating species in meadow and forest high CO2 exposed water. Meyerozyma guilliermondii and Wickerhamomyces anomalus predominated in high CO2 exposed soils. Using high dilution plating of a mofette soil sample, four strains of an unknown basidiomycetous species were isolated and are here newly described as Occultifur mephitis based on molecular phylogenetic and phenotypic criteria. The type strain of Occultifur mephitis is EXF-6436 T [CBS 14611=PYCC 7049, LT594852 (D1/D2), KX929055 (ITS)]. An additional three isolated strains are EXF-6437 (LT594853, KX929056), EXF-6473 (LT594863, KX929057) and EXF-6482 (LT594867, KX929054), as well as a strain reported from previous studies isolated from a leaf of Cistus albidus in Portugal (CBS 10223=PYCC 6067), EU002842 (D1/D2), KY308183 (ITS).

Yeasts are common inhabitants of different types of aquatic habitats, including marine and estuarine waters and rivers. Although numerous studies have surveyed yeast occurrence in these habitats, the identification of autochthonous populations has been problematic because several yeast species seem to be very versatile and therefore mere presence is not sufficient to establish an ecological association. In the present study we investigated the dynamics of the yeast community in the Tagus river estuary (Portugal) by combining a microbiological study involving isolation, quantification, and molecular identification of dominant yeast populations with the analysis of hydrological and hydrographical data. We set out to test the hypothesis of the multiple origins of estuarine yeast populations in a transect of the Tagus estuary and we postulate four possible sources: open sea, terrestrial, gastrointestinal and the estuary itself in the case of populations that have become resident. Candida parapsilosis and Pichia guilliermondii were correlated with Escherichia coli, which indicated an intestinal origin. Other cream-colored yeasts like Debaryomyces hansenii and Candida zeylanoides had similar dynamics, but no association with E. coli and quite distinct ecological preferences. They might represent a group of resident estuarine populations whose primary origin is diverse and can include marine, terrestrial, and gastrointestinal habitats. Another major yeast population was represented by Rhodotorula mucilaginosa. The cosmopolitan nature of that species and its moderate association with E. coli point to terrestrial sources as primary habitats.

Local adaptations within species are often governed by several interacting genes scattered throughout the genome. Single-locus models of selection cannot explain the maintenance of such complex variation because recombination separates co-adapted alleles. Here we report a previously unrecognized type of intraspecific multi-locus genetic variation that has been maintained over a vast period. The galactose (GAL) utilization gene network of Saccharomyces kudriavzevii, a relative of brewer's yeast, exists in two distinct states: a functional gene network in Portuguese strains and, in Japanese strains, a non-functional gene network of allelic pseudogenes. Genome sequencing of all available S. kudriavzevii strains revealed that none of the functional GAL genes were acquired from other species. Rather, these polymorphisms have been maintained for nearly the entire history of the species, despite more recent gene flow genome-wide. Experimental evidence suggests that inactivation of the GAL3 and GAL80 regulatory genes facilitated the origin and long-term maintenance of the two gene network states. This striking example of a balanced unlinked gene network polymorphism introduces a remarkable type of intraspecific variation that may be widespread.

In this report, we describe three novel asexual basidiomycetous yeast species, Cryptococcus aciditolerans sp. nov. (type strain CBS 10872T=SDY 081T), Cryptococcus ibericus sp. nov. (type strain CBS 10871T=SDY 022T) and Cryptococcus metallitolerans sp. nov. (type strain CBS 10873T=SDY 190T), which were isolated from acid rock drainage collected at the São Domingos mine in southern Portugal. Phylogenetic analysis of molecular sequence data indicated that the novel species belong to the order Filobasidiales of the class Tremellomycetes and form a well-separated clade, next to Cryptococcus gastricus and Cryptococcus gilvescens. Since the novel species also share a peculiar ecology, being able to thrive under extreme environmental conditions characterized by very low pH and high concentrations of heavy metals, we designate this combination of phylogenetic and ecological characteristics as an ecoclade.

Here we report the isolation of four Saccharomyces species (former Saccharomyces sensu stricto group) from tree bark. The employment of two temperatures (10 degrees C in addition to the more commonly used 30 degrees C) resulted in the isolation of S. kudriavzevii and S. uvarum, two species that grow at low temperatures, in addition to S. cerevisiae and S. paradoxus. A clear bias was found toward the bark of certain trees, particularly certain oak species. Very often, more than one Saccharomyces species was found in one locality and occasionally even in the same bark sample. Our evidence strongly suggests that (markedly) different growth temperature preferences play a fundamental role in the sympatric associations of Saccharomyces species uncovered in this survey. S. kudriavzevii was isolated at most of the sites sampled in Portugal, indicating that the geographic distribution of this species is wider than the distribution assumed thus far. However, the Portuguese S. kudriavzevii population exhibited important genetic differences compared to the type strain of the species that represents a Japanese population. In this study, S. kudriavzevii stands out as the species that copes better with low temperatures.

Here we report on the results of a survey of the yeast populations occurring on submerged leaves (alder, eucalyptus and oak) in a natural mountain stream, during different phases of their decomposition and through two consecutive years. Leaf litter mass loss, total yeast counts, Shannon-Weiner index (H'), yeast community structure and physiologic abilities were analyzed to evaluate the dynamics of yeast communities during decay. Seventy-two yeast taxa were recorded, and in all litter types, species of basidiomycetous affinity predominated over ascomycetous ones. Discriminant analysis of presence/absence data (yeast species) showed significant differences both among substrate types (P<0.0026) and with decomposition time (P<0.0001). Carbon and nitrogen source utilization by yeast strains also varied with the substrate (P<0.0001) and decomposition time (P<0.0001). Further conclusions were that: (1) all litter types have in common ubiquitous yeast species, such as Cryptococcus albidus, Debaryomyces hansenii and Rhodotorula glutinis, among the common 20 yeast species; (2) only a few species were dominant, and most species were rare, being recorded once or twice throughout decomposition; and (3) the order of yeast appearance, and their substrate assimilation patterns, strongly suggest a succession phenomenon. Finally, explanations for the distribution patterns and variations in yeast communities are discussed.

Temperature gradient gel electrophoresis (TGGE) was employed for the assessment of yeast diversity in the estuary of the Tagus river (Portugal). The molecular detection of yeasts was carried out directly from water samples and, in parallel, a cultivation approach by means of an enrichment step was employed. A nested PCR was employed to obtain a fungal amplicon containing the D2 domain of the 26S rRNA gene. For identification the TGGE bands were extracted, re-amplified, and sequenced. Fourteen fungal taxa were detected and all except one were yeasts. Most yeast sequences corresponded to members of the Ascomycota and only three belonged to the Basidiomycota. Five yeasts (four ascomycetes and one basidiomycete) could not be identified to the species level due to the uniqueness of their sequences. The number of species detected after enrichment was higher than the number of taxa found using the direct detection method. This suggests that some yeast populations are present in densities that are below the detection threshold of the method. With respect to the analysis of the yeast community structure, our results indicate that the dominant populations belong to Debaryomyces hansenii, Rhodotorula mucilaginosa, Cryptococcus longus, and to an uncultured basidiomycetous yeast phylogenetically close to Cr. longus. The combined analysis of direct detection and cultivation approaches indicates a similar community structure at the two sampled sites since nine species were present at both localities.

Seven phylloplane yeast strains that were collected in the Arrábida Natural Park, Portugal, and identified preliminarily as Bullera alba, the anamorphic stage of Bulleromyces albus, were investigated. In contrast to Bulleromyces albus, these isolates produced a brownish pigment when grown on potato dextrose agar. The pigment caused darkening of the cultures and diffused into the culture medium. Mating studies revealed that the Arrábida isolates did not react with the different mating types of Bulleromyces albus, but were sexually compatible with them and produced mycelium with clamp connections, haustoria and transversally septate basidia that ejected the basidiospores. Various taxonomic criteria that were evaluated during the present study and comparison with other sexual taxa of the Tremellales indicated that this teleomorph should be classified in a novel genus. Therefore, Auriculibuller fuscus gen. nov., sp. nov. (type strain, PYCC 5690(T)=CBS 9648(T)) is proposed. In addition, during the course of this investigation, a member of a novel Bullera species, Bullera japonica sp. nov. (type strain, PYCC 4534(T)=CBS 2013(T)), was found among collection isolates that were identified formerly as Bullera alba. In molecular phylogenetic analysis of the D1/D2 domains of the 26S rDNA and the internal transcribed spacer region, the two taxa were found to be closely related, but distinct at the species level.

The occurrence and diversity of yeasts in seawater was investigated in a study site located 20 Km off Faro, Portugal, above the Alvares Cabral Trench. A total of 43 water samples from different layers (above the permanent thermocline, under the thermocline and near the bottom) and directly from the surface, originated 234 isolates. All the isolates were identified using a molecular approach that included, in a first stage, MSP-PCR fingerprinting. A total of 31 MSP-PCR classes were formed, 8 for the pigmented yeasts and 23 for the non-pigmented yeasts. The pink coloured isolates were identified by direct comparison of the new fingerprints with those obtained for representative strains of the various species. For identification of the non-pigmented yeasts, a representative isolate of each MSP-PCR class was selected for sequence analysis and compared with reference sequences. The five most abundant yeast species were Sakaguchia dacryoidea, Pseudozyma aphidis, Rhodosporidium babjevae, R. diobovatum and Debaryomyces hansenii. The distribution of isolates and species in the major taxonomic groups indicated that the number of basidiomycetous yeasts and their diversity are prevalent in relation to their ascomycetous counterpart. Diversity indices were determined and superficial water and water near the bottom had the highest diversity. The sampling effort effectiveness was estimated, and found to correspond to approximately 60% of the species present. MSP-PCR identification proved suitable for pigmented basidiomycetous yeasts and, when used in conjunction with sequence analysis, was effective for the characterization of non-pigmented populations. Our results indicate that the MSP-PCR fingerprinting method is appropriate for the characterization of large groups of isolates due to its simplicity and good reproducibility.