Mário Gadanho

Polyphasic taxonomy of the basidiomycetous yeast genus Rhodosporidium: Rhodosporidium kratochvilovae and related anamorphic species

The phenotypic and genetic heterogeneity of the basidiomycetous yeast species Rhodosporidium kratochvilovae was investigated in a group of recent isolates and collection strains. A polyphasic taxonomic approach was followed which included micromorphological studies, nuclear staining, determination of sexual compatibility, physiological characterization, comparison of electrophoretic isoenzyme patterns, PCR fingerprinting, determination of mol% G+C, DNA-DNA reassociation experiments and 26S and ITS rDNA sequence analysis. The results allowed a more natural circumscription of the species, both from the genetic and phenotypic perspectives. The relationships with anamorphic species of the genus Rhodotorula were studied and isolates previously identified as Rhodotorula glutinis were found to belong to Rhodosporidium kratochvilovae. Other isolates included in the study were found to represent members of Rhodotorula glutinis var. dairenensis. Rhodosporidium kratochvilovae was found to include heterothallic strains, besides those already known to be self-sporulating. A total of 17 isolates, which were found to belong to this species, were heterothallic, self-sporulating and anamorphic strains. It is anticipated that integrated polyphasic studies of basidiomycetous yeasts will provide a more coherent classification system and the basis for accurate identification schemes, which in turn are essential for detailed ecological studies.

Molecular characterization of carotenogenic yeasts from aquatic environments in Patagonia, Argentina.

Fifteen aquatic environments (lakes, lagoons and rivers) of glacial origin in the northern Andean Patagonia (Argentina) were surveyed for the occurrence of red yeasts. Subsurface water samples were filtered and used for colony counting and yeast isolation. A preliminary quantitative analysis indicated that total yeast counts ranged between 0 and 250 cells l−1. A polyphasic approach including physiological and molecular methods was used for the identification of 64 carotenogenic yeast strains. The molecular characterisation of the isolates was based on the mini/microsatellite-primed PCR technique (MSP-PCR) employing the (GTG)5 and the M13 primers. Comparison of representative fingerprints of each group with those of the type strains of pigmented yeasts allowed the expeditious identification of 87.5% isolates. The sequence analysis of the D1/D2 domains of the 26S rDNA was employed to confirm identifications and in the characterization of the unidentified MSP-PCR groups. Teleomorphic yeast species were detected by performing sexual compatibility assays. The isolates corresponded to 6 genera and 15 yeast species, including four new yeast species of the genera Cryptococcus (1), Rhodotorula (1) and Sporobolomyces (2). Rhodotorula mucilaginosa was found in the majority of the samples and represented ca. 50% of the total number of isolates. However, this yeast was not detected in aquatic environments with very low anthropic influence. Other frequent yeast isolates were teleomorphic yeast species of Rhodosporidium babjevae, R. kratochvilovae and Sporidiobolus salmonicolor. This study represents the first report on red yeast occurrence and biodiversity in northwestern Patagonia.

Occurrence and diversity of yeasts in the mid-atlantic ridge hydrothermal fields near the azores archipelago

The yeast community associated with deep-sea hydrothermal systems of the Mid-Atlantic Rift was surveyed for the first time. This study relied on a culture-based approach using two different growth media: a conventional culture medium for yeasts supplemented with sea salts (MYPss) and the same medium additionally supplemented with sulfur (MYPssS). For the evaluation of species diversity, a molecular approach involving minisatellite-primed polymerase chain reaction (MSP-PCR) strain typing and sequence analysis of the D1/D2 domains of the 26S rDNA was followed. In the seven water samples that were studied, the number of colony-forming units per liter (cfu/L) ranged from 0 to 5940. The nonpigmented yeasts were much more abundant than the pink-pigmented ones. This disproportion was not observed in studies of other marine systems and may be due to the unique conditions of hydrothermal vents, characterized by a rich animal and microbial diversity and therefore by the availability of organic compounds utilizable by yeasts. Higher counts of nonpigmented yeast were obtained using MYPss, whereas for pink yeasts, higher counts were obtained using MYPssS. Moreover, among pink yeasts, some of the MSP-PCR classes obtained were composed of isolates obtained only on MYPssS, which might be an indication that these isolates are adapted to the ecosystems of the hydrothermal vents. Twelve phylotypes belonged to the Ascomycota and seven phylotypes belonged to the Basidiomycota. The nonpigmented yeasts were identified as Candida atlantica, C. atmosphaerica, C. lodderae, C. parapsilosis, Exophiala dermatitidis, Pichia guilliermondii, and Trichosporon dermatis, whereas the pigmented yeasts were identified as Rhodosporidium diobovatum, R. sphaerocarpum, R. toruloides, and Rhodotorula mucilaginosa. Some of the yeasts that were found belong to phylogenetic groups that include species reported from other marine environments, and eight phylotypes represent undescribed species. The new phylotypes found at Mid-Atlantic Ridge hydrothermal fields represent 33% of the total number of yeast taxa that were found.

Assessment of yeast diversity in a marine environment in the south of Portugal by microsatellite-primed PCR

The occurrence and diversity of yeasts in seawater was investigated in a study site located 20 Km off Faro, Portugal, above the Álvares 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.

Taxonomic studies in the Microbotryomycetidae: Leucosporidium golubevii sp. nov., Leucosporidiella gen. nov. and the new orders Leucosporidiales and Sporidiobolales

The subclass Microbotryomycetidae (Basidiomycota, Urediniomycetes) comprises a remarkably diverse assemblage of fungi. This group includes phytoparasites, mycoparasites and probably also saprobes that show a wide range of ecological preferences. In order to study the phylogenetic relationships within the Microbotryomycetidae, and to develop a more natural classification system, mitosporic and meiosporic taxa were investigated using an integrated approach. Sequence data of 26S rDNA D1/D2 domains were analyzed using several procedures, including the Bayesian Markov chain Monte Carlo method of phylogenetic inference. Ultrastructural markers such as the type of septal pore and presence / absence of colacosomes were investigated and micromorphological and nutritional properties were compared. In this study the current concept of the genus Leucosporidium and its apparent polyphyletic nature were addressed, as well as the relationships of this genus with the Microbotryales and Mastigobasidium. The classification of the anamorphic species closely related to Leucosporidium, and the concepts of the order Sporidiales and family Sporidiobolaceae were also reviewed.

Yeast Diversity in the Extreme Acidic Environments of the Iberian Pyrite Belt

In the Iberian Pyrite Belt (IPB), acid rock drainage gives rise to aquatic habitats with low pH and high concentrations of heavy metals, a situation that causes important environmental problems. We investigated the occurrence and diversity of yeasts in two localities of the IPB: São Domingos (Portugal) and Rio Tinto (Spain). Yeast isolation was performed on conventional culture media (MYP), acidified (pH 3) media (MYP3), and on media prepared with water from the study sites (MYPw). The main goal of the study was to determine the structure of the yeast community; a combination of molecular methods was used for accurate species identifications. Our results showed that the largest fraction of the yeast community was recovered on MYPw rather than on MYP and MYP3. Twenty-seven yeast species were detected, 48% of which might represent undescribed taxa. Among these, an undescribed species of the genus Cryptococcus required low pH for growth, a property that has not been observed before in yeasts. The communities of S. Domingos and R. Tinto showed a considerable resemblance, and eight yeast species were simultaneously found in both localities. Taking into consideration the physicochemical parameters studied, we propose a hierarchic organization of the yeast community in terms of high-, intermediate-, or low-stress conditions of the environment. According to this ranking, the acidophile yeast Cryptococcus sp. 5 is considered the most tolerant species, followed by Cryptococcus sp. 3 and Lecytophora sp. Species occurring in situations of intermediate environmental stress were Candida fluviatilis, Rhodosporidium toruloides, Williopsis californica, and three unidentified yeasts belonging to Rhodotorula and Cryptococcus.

New taxa in the Tremellales: Bulleribasidium oberjochense gen. et sp. nov., Papiliotrema bandonii gen. et sp. nov. and Fibulobasidium murrhardtense sp. nov

Two new genera, Bulleribasidium and Papiliotrema, and three new species, B. oberjochense, P. bandonii and Fibulobasidium murrhardtense are described. An integrated analysis of morphological, ultrastructural, physiological, and molecular data indicates that the new taxa belong to the Tremellales (Basidiomycota). Relevant characteristics of the new genera and species are discussed and compared with those of closely related taxa.

Cuniculitrema polymorpha (Tremellales, gen. nov. and sp. nov.), a heterobasidiomycete vectored by bark beetles, which is the teleomorph of Sterigmatosporidium polymorphum.

In a study of the mycobiota associated with bark beetles, a dimorphic fungus producing longitudinally septate basidia of the Tremella-type and yeast cells budding off from stalks, was collected. Detailed morphological, physiological and molecular studies revealed that this fungus represents the teleomorph of Sterigmatosporidium polymorphum. Consequently, a new genus, Cuniculitrema gen. nov., and a new species, C. polymorpha sp. nov., are proposed. Comparative morphological and molecular studies indicated that the new taxon belongs to a group that also comprises species of the stalk-forming anamorphic genera Fellomyces and Kockovaella. The new family Cuniculitremaceae is proposed for this group.

Taxonomic studies on the genus Cystofilobasidium: description of Cystofilobasidium ferigula sp. nov. and clarification of the status of Cystofilobasidium lari-marini

A new species of the genus Cystofilobasidium is described as Cystofilobasidium ferigula sp. nov. The new taxon represents the teleomorphic stage of Cryptococcus ferigula and was obtained in mating experiments using three strains deposited in the Portuguese Yeast Culture Collection (mating types A1) and a recent isolate (mating type A2). Cystofilobasidium ferigula is characterized using an integrated approach encompassing morphological studies, investigation of the ultrastructure of the septal pore, a comparative study of physiological traits, determination of the DNA base composition, DNA reassociation experiments and PCR fingerprinting. During the course of this study, a close similarity of microsatellite-primed PCR fingerprints was detected between Cystofilobasidium lari-marini and Cystofilobasidium capitatum. DNA-DNA reassociation experiments gave high homology values, which indicates that Cystofilobasidium lari-marini must be regarded as a synonym of Cystofilobasidium capitatum.

Cystofilobasidium lacus-mascardii sp. nov., a basidiomycetous yeast species isolated from aquatic environments of the Patagonian Andes, and Cystofilobasidium macerans sp. nov., the sexual stage of Cryptococcus macerans

Here, we report on two novel sexual basidiomycetous red yeast species of the genus Cystofilobasidium. Cystofilobasidium lacus-mascardii sp. nov. is based on sexually compatible strains isolated from Lake Mascardi, an ultraoligotrophic lake in north-western Patagonia, Argentina. Following the discovery of the first isolate of this species, additional (sexually compatible) strains were isolated using a selective medium containing erythritol as the sole source of carbon. The second novel species corresponds to the sexual state of Cryptococcus macerans. In spite of accounts over the last 20 years of sexually compatible strains of this species, the complete life has never been observed. We provide evidence of a Cystofilobasidium-like basidial stage with teliospores and slender holobasidia, based on the study of self-fertile (homothallic) and self-sterile (heterothallic) isolates of Cryptococcus macerans. A revised molecular phylogeny of the genus Cystofilobasidium is presented and the most salient features of Cystofilobasidium lacus-mascardii sp. nov. (type strain CBS 10642(T) =PYCC 5819(T) =CRUB 1046(T)) and Cystofilobasidium macerans sp. nov. (type strain CBS 10757(T)) are discussed and compared with those of the remaining species in the genus. Information on additional Patagonian isolates belonging to the Cystofilobasidiales is also included in this report.