A genome-informed higher rank classification of the biotechnologically important fungal subphylum Saccharomycotina.

The subphylum Saccharomycotina is a lineage in the fungal phylum Ascomycota that exhibits levels of genomic diversity similar to those of plants and animals. The Saccharomycotina consist of more than 1 200 known species currently divided into 16 families, one order, and one class. Species in this subphylum are ecologically and metabolically diverse and include important opportunistic human pathogens, as well as species important in biotechnological applications. Many traits of biotechnological interest are found in closely related species and often restricted to single phylogenetic clades. However, the biotechnological potential of most yeast species remains unexplored. Although the subphylum Saccharomycotina has much higher rates of genome sequence evolution than its sister subphylum, Pezizomycotina, it contains only one class compared to the 16 classes in Pezizomycotina. The third subphylum of Ascomycota, the Taphrinomycotina, consists of six classes and has approximately 10 times fewer species than the Saccharomycotina. These data indicate that the current classification of all these yeasts into a single class and a single order is an underappreciation of their diversity. Our previous genome-scale phylogenetic analyses showed that the Saccharomycotina contains 12 major and robustly supported phylogenetic clades; seven of these are current families (Lipomycetaceae, Trigonopsidaceae, Alloascoideaceae, Pichiaceae, Phaffomycetaceae, Saccharomycodaceae, and Saccharomycetaceae), one comprises two current families (Dipodascaceae and Trichomonascaceae), one represents the genus Sporopachydermia, and three represent lineages that differ in their translation of the CUG codon (CUG-Ala, CUG-Ser1, and CUG-Ser2). Using these analyses in combination with relative evolutionary divergence and genome content analyses, we propose an updated classification for the Saccharomycotina, including seven classes and 12 orders that can be diagnosed by genome content. This updated classification is consistent with the high levels of genomic diversity within this subphylum and is necessary to make the higher rank classification of the Saccharomycotina more comparable to that of other fungi, as well as to communicate efficiently on lineages that are not yet formally named. Taxonomic novelties: New classes: Alloascoideomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Dipodascomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Lipomycetes M. Groenew., Hittinger, Opulente, A. Rokas, Pichiomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Sporopachydermiomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Trigonopsidomycetes M. Groenew., Hittinger, Opulente & A. Rokas. New orders: Alloascoideomycetes: Alloascoideales M. Groenew., Hittinger, Opulente & A. Rokas; Dipodascomycetes: Dipodascales M. Groenew., Hittinger, Opulente & A. Rokas; Lipomycetes: Lipomycetales M. Groenew., Hittinger, Opulente & A. Rokas; Pichiomycetes: Alaninales M. Groenew., Hittinger, Opulente & A. Rokas, Pichiales M. Groenew., Hittinger, Opulente & A. Rokas, Serinales M. Groenew., Hittinger, Opulente & A. Rokas; Saccharomycetes: Phaffomycetales M. Groenew., Hittinger, Opulente & A. Rokas, Saccharomycodales M. Groenew., Hittinger, Opulente & A. Rokas; Sporopachydermiomycetes: Sporopachydermiales M. Groenew., Hittinger, Opulente & A. Rokas; Trigonopsidomycetes: Trigonopsidales M. Groenew., Hittinger, Opulente & A. Rokas. New families: Alaninales: Pachysolenaceae M. Groenew., Hittinger, Opulente & A. Rokas; Pichiales: Pichiaceae M. Groenew., Hittinger, Opulente & A. Rokas; Sporopachydermiales: Sporopachydermiaceae M. Groenew., Hittinger, Opulente & A. Rokas. Citation: Groenewald M, Hittinger CT, Bensch K, Opulente DA, Shen X-X, Li Y, Liu C, LaBella AL, Zhou X, Limtong S, Jindamorakot S, Gonçalves P, Robert V, Wolfe KH, Rosa CA, Boekhout T, Cadez N, Péter G, Sampaio JP, Lachance M-A, Yurkov AM, Daniel H-M, Takashima M, Boundy-Mills K, Libkind D, Aoki K, Sugita T, Rokas A (2023). A genome-informed higher rank classification of the biotechnologically important fungal subphylum Saccharomycotina. Studies in Mycology 105: 1-22. doi: 10.3114/sim.2023.105.01 This study is dedicated to the memory of Cletus P. Kurtzman (1938-2017), a pioneer of yeast taxonomy.

Public Microbial Resource Centers: Key Hubs for Findable, Accessible, Interoperable, and Reusable (FAIR) Microorganisms and Genetic Materials.

In the context of open science, the availability of research materials is essential for knowledge accumulation and to maximize the impact of scientific research. In microbiology, microbial domain biological resource centers (mBRCs) have long-standing experience in preserving and distributing authenticated microbial strains and genetic materials (e.g., recombinant plasmids and DNA libraries) to support new discoveries and follow-on studies. These culture collections play a central role in the conservation of microbial biodiversity and have expertise in cultivation, characterization, and taxonomy of microorganisms. Information associated with preserved biological resources is recorded in databases and is accessible through online catalogues. Legal expertise developed by mBRCs guarantees end users the traceability and legality of the acquired material, notably with respect to the Nagoya Protocol. However, awareness of the advantages of depositing biological materials in professional repositories remains low, and the necessity of securing strains and genetic resources for future research must be emphasized. This review describes the unique position of mBRCs in microbiology and molecular biology through their history, evolving roles, expertise, services, challenges, and international collaborations. It also calls for an increased deposit of strains and genetic resources, a responsibility shared by scientists, funding agencies, and publishers. Journal policies requesting a deposit during submission of a manuscript represent one of the measures to make more biological materials available to the broader community, hence fully releasing their potential and improving openness and reproducibility in scientific research.

Two yeast species Cystobasidium psychroaquaticum f.a. sp. nov. and Cystobasidium rietchieii f.a. sp. nov. isolated from natural environments, and the transfer of Rhodotorula minuta clade members to the genus Cystobasidium.

Many species of dimorphic basidiomycetes are known only in their asexual phase and typically those pigmented in different hues of red have been classified in the large polyphyletic genus Rhodotorula. These yeasts are ubiquitous and include a few species of some clinical relevance. The phylogenetic distribution of Rhodotorula spans three classes: Microbotryomycetes, Cystobasidiomycetes and Exobasidiomycetes. Here, the presented multi-gene analyses resolved phylogenetic relationships between the second largest group of Rhodotorula and the mycoparasite Cystobasidium fimetarium (Cystobasidiales, Cystobasidiomycetes, Pucciniomycotina). Based on the results, we propose the transfer of nine species belonging to the Rhodotorula minuta clade into the genus Cystobasidium. As a result, the clinically relevant species R. minuta will be renamed Cystobasidium minutum. This proposal follows ongoing reassessments of the anamorphic genus Rhodotorula reducing the polyphyly of this genus. The delimitation of the R. minuta clade from Rhodotorula species comprised in Sporidiobolales including the type species Rhodotorula glutinis is an important step to overcome obsolete generic placements of asexual basidiomycetous yeasts. Our proposal will also help to distinguish most common red yeasts from clinical samples such as members of Sporidiobolales and Cystobasidiales. The diagnosis of the genus Cystobasidium is amended by including additional characteristics known for the related group of species. The taxonomic change enables us to classify two novel species with the phylogenetically related members of the R. minuta clade in Cystobasidium. The recently from natural environments isolated species are described here as Cystobasidium psychroaquaticum f.a. sp. nov. (K-833(T) = KBP 3881(T) = VKPM Y-3653(T) = CBS 11769(T) = MUCL 52875(T) = DSM 27713(T)) and Cystobasidium rietchiei f.a. sp. nov. (K-780(T) = KBP 4220(T) = VKPM Y-3658(T) = CBS 12324(T) = MUCL 53589(T) = DSM 27155(T)). The new species were registered in MycoBank under MB 809336 and MB 809337, respectively.

Microbial ecology of sourdough fermentations: diverse or uniform?

Sourdough is a specific and stressful ecosystem inhabited by yeasts and lactic acid bacteria (LAB), mainly heterofermentative lactobacilli. On the basis of their inocula, three types of sourdough fermentation processes can be distinguished, namely backslopped ones, those initiated with starter cultures, and those initiated with a starter culture followed by backslopping. Typical sourdough LAB species are Lactobacillus fermentum, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus sanfranciscensis. Typical sourdough yeast species are Candida humilis, Kazachstania exigua, and Saccharomyces cerevisiae. Whereas region specificity is claimed in the case of artisan backslopped sourdoughs, no clear-cut relationship between a typical sourdough and its associated microbiota can be found, as this is dependent on the sampling, isolation, and identification procedures. Both simple and very complex consortia may occur. Moreover, a series of intrinsic and extrinsic factors may influence the composition of the sourdough microbiota. For instance, an influence of the flour (type, quality status, etc.) and the process parameters (temperature, pH, dough yield, backslopping practices, etc.) occurs. In this way, the presence of Lb. sanfranciscensis during sourdough fermentation depends on specific environmental and technological factors. Also, Triticum durum seems to select for obligately heterofermentative LAB species. Finally, there are indications that the sourdough LAB are of intestinal origin.

A comparison of post-ruminal provision of Ca-gluconate and Ca-butyrate on growth performance, gastrointestinal barrier function, short-chain fatty acid absorption, intestinal histology, and brush-border enzyme activity in beef heifers.

The objective of this study was to compare the effects of post-ruminal provision of Ca-butyrate (CaB) when delivered via abomasal dosing, and Ca-gluconate (CaG) when provided ruminally using a rumen protected form or using an unprotected form via abomasal dosing on short-chain fatty acid (SCFA) concentration throughout the GIT, nutrient digestibility, GIT barrier function, ruminal SCFA absorption, ruminal morphometrics, intestinal brush border enzyme activity, and blood parameters for beef heifers. Thirty-two beef heifers fitted with ruminal cannulas were used in a randomized complete block design and assigned to one of four treatments: 1) negative control (ruminal infusion of double-distilled water; CON); 2) abomasal infusion of CaB (AB; 0.0029% of BW); 3) abomasal infusion of CaG (AG; 0.0077% of BW); and 4) ruminal infusion of a hydrogenated fat-embedded CaG (RG; 0.0192% of BW) to provide ruminal protection. Excluding CON, treatments were designed to deliver the same amount of butyrate in the small intestine. Heifers were housed in individual pens and DMI was limited to 95% of voluntary intake to minimize a potential confounding effect of DMI on treatment responses. Total GIT barrier function was assessed on day 17 and SCFA disappearance was evaluated on day 21 using the temporarily isolated and washed reticulo-rumen technique. On day 28, heifers were slaughtered, and ruminal and colonic digesta were collected to assess SCFA concentration. Additionally, ruminal, jejunal, and colonic tissues were collected to assess SCFA fluxes and regional barrier function ex vivo using the Ussing chamber technique. For colonic digesta, both AB and CaG treatments reduced the proportion of acetate (P < 0.05) and increased the proportion on propionate (P < 0.05) compared to CON. Relative to CON, AB but not CaG treatments increased in vivo ruminal disappearance of total SCFA (P = 0.01), acetate (P = 0.03), propionate (P = 0.01), and butyrate (P > 0.01). Treatments did not affect (P ≥ 0.10) acetate and butyrate fluxes in the ruminal and colonic tissues when measured ex vivo; however, when compared with CON, AB tended to decrease (P = 0.09) mannitol flux across ruminal tissue. In addition, mannitol flux was affected (P < 0.01) by region, with greater mannitol flux across the jejunum than rumen and colon. We conclude that while both abomasal infusion of CaB and CaG affect the molar proportion of acetate and propionate in the colon, only abomasal CaB stimulated ruminal SCFA absorption for growing beef heifers.

Butyrate, a short-chain fatty acid (SCFA), has received attention due to its ability to promote gastrointestinal (GIT) health and development. However, butyrate in its free form presents a strong odor, limiting its use in diet formulation. Supplementation of butyrate precursors, such as gluconate, have been studied to enhance butyrate production in the GIT. This study evaluated the effects of post-ruminal infusion of Ca-butyrate (AB; 0.0029% of BW) and Ca-gluconate (AG; 0.0077% of BW) and ruminal infusion of a hydrogenated fat-embedded Ca-gluconate (RG; 0.0192% of BW) relative to control (CON; ruminal infusion of double-distilled water). Thirty-two beef heifers fitted with ruminal cannulas were fed for 28 d and GIT barrier function and ruminal SCFA absorption were assessed. At slaughter, the rumen, jejunum, and colon tissues were collected and barrier function and SCFA fluxes were assessed ex vivo. Relative to CON, AB but not AG and RG increased in vivo ruminal SCFA absorption and tended to increase ex vivo barrier function. Thus, the data presented in this study shows that butyrate and gluconate do not function through the same mode of action in the GIT of beef heifers.

Effect of feeding calcium gluconate embedded in a hydrogenated fat matrix on feed intake, gastrointestinal fermentation and morphology, intestinal brush border enzyme activity and blood metabolites in growing lambs.

Gluconate salts have been identified as a butyrate precursor when fed to non-ruminant species and may increase the butyrate concentration in the large intestine supporting gastrointestinal health and development. The objective of this study was to evaluate the dose response of hydrogenated fat-embedded calcium gluconate (HFCG) on performance and gastrointestinal tract (GIT) development in growing lambs. Thirty-two wether lambs were used in a randomized complete block design and assigned to 1 of 4 treatments differing in the inclusion of HFCG: 0.0% (CON), 0.075% (LOW), 0.30% (MED), and 0.60% of the diet (HIGH). Lambs were allocated into individual pens and fed ad libitum with feed delivered twice daily. Feed intake was recorded daily, and body weight (BW) was assessed at the beginning and the end of the 29-d period. Blood was sampled on day 21, prior to feeding and 6 h post-feeding to evaluate changes in ß-hydroxybutyrate, glucose, and insulin concentrations. Total fecal collection was conducted during days 25 to 28 to assess apparent total tract digestibility. On day 29, lambs were slaughtered, and the entire GIT was separated by region to enable sampling of tissue and digesta. Data were analyzed to assess linear, quadratic, and cubic effects of HFCG dose. Final BW, average daily gain, and dry matter intake decreased linearly (P ≤ 0.02) with increasing HFCG. Increasing inclusion of HFCG linearly decreased (P = 0.01) the thickness of the stratum corneum in ruminal papillae but did not affect other strata (P ≥ 0.34). Omasal digesta weight linearly decreased (P = 0.01) as the concentration of HFCG increased and abomasal digesta weight was cubically affected (P = 0.03) the increasing dose of HFCG. Short-chain fatty acid concentration in the cecum was cubically affected (P < 0.01) with increasing dose of HFCG where low dose had the greatest concentration. Moreover, increasing the dietary supply of HFCG linearly increased the proportion of acetate (P = 0.04) in the cecum and linearly decreased the proportion of propionate in the digesta of both the cecum (P < 0.01) and colon (P = 0.01). Colon crypt depth was quadratically (P = 0.03) affected with the increasing dose of HFCG, where lambs fed MED had greatest crypt depth. We conclude that feeding HFCG to growing lambs did not increase butyrate concentration in the large intestine and consequently does not increase the absorptive surface area of the whole tract, the size of the GIT, or the functionality of the intestine.

Gluconate salts have been reported to be metabolized by microbes in the gastrointestinal tract to yield butyrate. Butyrate has shown potential to enhance functionality of the gastrointestinal tract by increasing the absorptive surface area, enzyme activity, and the barrier function. This study evaluated the inclusion of four levels of hydrogenated fat-embedded Ca-gluconate (HFCG; 0.0%, 0.075%, 0.30%, and 0.60% of the diet) designed to increase the production of butyrate in the large intestine. Thirty-two wether lambs were fed for 28 d, slaughtered, and eviscerated to allow complete evaluation of the gastrointestinal tract and its contents. Growth and dry matter intake decreased linearly with increasing dose of HFCG. Dose of HFCG cubically affected short-chain fatty acid concentration in the cecum with increased concentrations at the 0.075% dose. Moreover, increasing dose of HFCG linearly increased the proportion of acetate and linearly decreased the proportion of propionate in the cecum without altering the proportion of butyrate. Thus, the supplementation of HFCG did not increase butyrate concentration in the large intestine and did not enhance gastrointestinal tract function.

Metabolic and Anthropometric Influences on Nerve Conduction Parameters in Patients with Peripheral Neuropathy: A Retrospective Chart Analysis.

BACKGROUND AND AIMS: Nerve conduction study (NCS) measures how fast an electrical impulse moves through the nerve and is a standard technique for diagnosing and assessing neurological diseases. Despite diabetes and obesity being a common accompaniment of peripheral neuropathy, their effects on NCS patterns have not been elucidated conclusively. Our study aimed to assess several anthropometric and metabolic factors with NCS outcomes to address this gap. RESEARCH DESIGN AND METHODS: This retrospective chart analysis study was conducted on subjects who underwent NCS between 1 January 2009 and 31 December 2019 at a regional hospital. Metabolic, anthropometric, demographical and NCS data were collected from patients' health records. RESULTS: In total, 120 subjects presenting with sensorimotor peripheral neuropathy symptoms were included in the study. Age, HbA1c, urea and ESR variables were significantly negatively associated with nerve conduction outcomes (Spearman's correlation rho between -0.210 and -0.456, p < 0.038). HbA1c and age consistently had the most substantial contribution to velocity and amplitude in all regression models (beta coefficients between -0.157 and 0.516, p < 0.001). Urea also significantly account for a large amount of variance in amplitude and velocity in the lower limbs. CONCLUSION: This study suggests that the severity of sensorimotor neuropathy is influenced by glycaemic control, age and uraemia. The interpretation of NCS results must consider these factors suggesting that improved glycaemic and uraemic control may improve nerve conduction outcomes.

Influence of Cane Molasses Inclusion to Dairy Cow Diets during the Transition Period on Rumen Epithelial Development.

The objective of this study was to evaluate the addition of cane molasses during a 60 day dry period on performance and metabolism of Holstein cows during prepartum and postpartum periods. For experiment 1, 26 primiparous and 28 multiparous cows were used. Upon freshening, all cows were offered a common lactation diet. For experiment 2, six multiparous cows fitted with rumen cannulas were used to measure performance and metabolism, following the same protocol as experiment 1. Ruminal propionate increased by 10% during both prepartum and postpartum periods; however, papillae area was greater for cows not fed molasses, and volatile fatty acids (VFA) absorption from the rumen was not increased, resulting in similar glucagon-like-peptide-2 receptor (GLP-2R) density. The improved dry matter intake, when molasses was added into prepartum diets, translated into increased milk yield and energy-corrected milk (ECM) in Experiment 1 only for multiparous cows. For experiment 2, the improvement on milk performance was also observed, where cows fed molasses had 18.5% greater ECM production. Feeding molasses during a 60 day dry period positively influenced transition cow performance, and it was not accompanied by changes in rumen morphometrics; however, this indicates enhanced adaptation by the rumen epithelium based on similar capabilities for VFA absorption.

The environmental and intrinsic yeast diversity of Cuban cocoa bean heap fermentations.

The environmental yeast diversity of spontaneous cocoa bean fermentations in east Cuba was investigated. Seven fermentations, 25 equipment- and handling-related samples, and 115 environmental samples, such as flowers, leaf and cocoa pod surfaces, as well as drosophilid insects, were analysed. The basic fermentation parameters temperature and pH were recorded during five fermentations for at least six days. A total of 435 yeast isolates were identified by a combination of PCR-fingerprinting of genomic DNA with the M13 primer and sequence analysis of DNA from representative isolates, using the internal transcribed spacer region, the D1/D2 region of the large subunit rRNA gene, and an actin gene-encoding fragment, as required. Among 65 yeast species detected, Pichia manshurica and Hanseniaspora opuntiae were the most frequently isolated species, obtained from five and four fermentations, followed in frequency by Pichia kudriavzevii from two fermentations. Saccharomyces cerevisiae was isolated only occasionally. Cocoa fermentation yeast species were also present on processing equipment. The repeated isolation of a preliminarily as Yamadazyma sp. classified species, a group of strains similar to Saccharomycopsis crataegensis from fermentations and equipment, and the isolation of fifteen other potentially novel yeast species in low numbers provides material for further studies. Environmental samples showed higher yeast diversity compared to the fermentations, included the most frequent fermentation species, whereas the most frequently isolated environmental species were Candida carpophila, Candida conglobata, and Candida quercitrusa. Potential selective advantages of the most frequently isolated species were only partly explained by the physiological traits tested. For instance, tolerance to higher ethanol concentrations was more frequent in strains of Pichia spp. and S. cerevisiae compared to Hanseniaspora spp.; the ability to also assimilate ethanol might have conferred a selective advantage to Pichia spp. In contrast, high glucose tolerance was common among strains of Hanseniaspora spp., Torulaspora delbrueckii, and Candida tropicalis, among which only Hanseniaspora spp. were frequently isolated.

Evaluation of ribosomal RNA and actin gene sequences for the identification of ascomycetous yeasts.

Highly similar gene sequences of the 5' region of the large subunit (LSU) are commonly interpreted to predict the organism's identity. However, it was recognised that closely related taxa do not always show sufficiently diverged D1/D2 LSU sequences to differentiate between them. The effectiveness of species separation using D1/D2 LSU sequences, small subunit (SSU) sequences and actin gene sequences was determined by pair-wise comparisons. The LSU data showed coinciding similarities among and within species. The actin data resolved all investigated species. Examples strengthened the value of almost complete SSU sequences for species separation. The larger number of differences in the highly conserved actin gene, compared to the overall more variable LSU gene, is due to the tolerance of protein coding genes to synonymous nucleotide changes. In contrast, the pairing in secondary structures of the rRNA, ensuring the functionality of the molecule, relies on longer and uninterrupted sequence sections. In conclusion, D1/D2 LSU sequences are not specific enough to identify closely related taxa. The actin gene is a better marker in these cases. However, because of the availability of a large database of fungal D1/D2 LSU sequences, this gene region is currently still the preferred target for sequence-based identification.

Polyphasic re-examination of Debaryomyces hansenii strains and reinstatement of D. hansenii, D. fabryi and D. subglobosus.

The type species of the genus Debaryomyces, Debaryomyces hansenii, is a highly heterogeneous species. It has been isolated from a large diversity of natural sources including fruit, air, water, soil, but most frequently from processed food products. The species delineation of this yeast species urgently needs clarification. The heterogeneity in taxonomic characteristics has resulted in the description of varieties linked to D. hansenii. The aim of this study was to re-examine and classify D. hansenii using a polyphasic approach. A total of 65 D.hansenii isolates were examined, 57 representing the variety hansenii and nine the variety fabryi. The selection of strains for DNA reassociation and phylogenetic analysis was based on polymerase chain reaction mediated fingerprints using four mini- and microsatellite-specific primers. The discriminating power of growth at 35 degrees C and 37 degrees C was re-examined and ascospore production was observed. DNA reassociations and phylogenetic analyses were performed on selected isolates from each of the clusters obtained from the DNA fingerprint analyses. The data indicated the presence of three distinct species within the D. hansenii group, which were represented by type strains of former species and that are proposed to be reinstated: D. hansenii (CBS 767(T) = MUCL 49680(T)), D. fabryi (CBS 789(T) = MUCL 49731(T)) and D. subglobosus (CBS 792(T) = MUCL 49732(T)).

Nanoscale gamma-AlO(OH) hollow spheres: synthesis and container-type functionality.

AlO(OH) hollow spheres are realized via a water-in-oil (w/o) microemulsion, applying the liquid-to-liquid-phase boundary of the micellar system as a template. Scanning electron microscopy, transmission electron microscopy (TEM), and dynamic light scattering analyses show the presence of nonagglomerated hollow spheres exhibiting an outer diameter of about 30 nm and a wall thickness of 5-6 nm. High-resolution TEM images show highly ordered lattice fringes, indicating the crystallinity of the sphere wall and identifying the wall to consist of gamma-AlO(OH) (boehmite). The container functionality of as-prepared AlO(OH) hollow spheres is validated as a proof of concept by encapsulating the fluorescent dye rhodamine (R6G) inside the alumina shell. Subsequent to centrifugation and careful purification, R6G is evidenced via photoluminescence to be still present. Finally, release of R6G is initiated by acidic dissolution of the sphere wall.

Partial sequence analysis of the actin gene and its potential for studying the phylogeny of Candida species and their teleomorphs.

The actin gene has been studied as a potential phylogenetic marker for selected members of the anamorphic genus Candida and seven related teleomorphic genera (Debaryomyces, Issatchenkia, Kluyveromyces, Saccharomyces and Pichia from the Saccharomycetaceae; Clavispora and Metschnikowia from the Metschnikowiaceae). The nucleotide sequences of 36 fungal taxa were analysed with respect to their molecular evolution and phylogenetic relationships. A total of 460 bp (47%) of the coding 979 bp were variable and 396 bp (40%) of these were found to be phylogenetically informative. Further analysis of the sequences showed that the genic G+C contents were higher than the nuclear G+C contents for most of the taxa. A strong positive correlation was found between G+C content over all codon positions and third positions. First and second codon positions were considered to be independent of the genic G+C content. The expected transition/transversion bias was detected only for third positions. Pairwise comparisons of transitional and transversional changes (substitutions) with total percentage sequence divergences revealed that the third position transitions showed no saturation for ingroup comparisons. A specific weighting scheme was set up, combining codon-position weights with change-frequency weights to enable the inclusion of distant outgroup taxa. Parsimony analyses of the investigated taxa showed four groups, three of which corresponded to major clusters that had been established previously in Candida by rDNA analysis. Interrelationships among the species groups in this heterogeneous anamorphic genus were determined. The polyphyletic origin of the selected Candida species and their close associations with several ascomycete genera were verified and known anamorph/teleomorph pairs confirmed. The actin gene was established as a valuable phylogenetic marker with the particular advantage of an unambiguous alignment.

The D1/D2 domain of the large-subunit rDNA of the yeast species Clavispora lusitaniae is unusually polymorphic.

Ten different versions of the D1/D2 divergent domain of the large-subunit ribosomal DNA were identified among interbreeding members of the yeast species Clavispora lusitaniae. One major polymorphism, located in a 90-bp structural motif of the D2 domain, exists in two versions that differ by 32 base substitutions. Three other polymorphisms consist of a two-base substitution, a two-base deletion, and a single-base deletion, respectively. The polymorphisms are independent of one another and of the two mating types, indicating that the strains studied belong to a single, sexually active Mendelian population. Several strains were heterogeneous for one or more of the polymorphisms, and one strain was found to be automictic and capable of producing asci on its own by isogamous conjugation or by bud-parent autogamy. These observations suggest circumspection in the use of sequence divergence as the principal criterion for delimiting yeast species.

Comparison of use of phenotypic and genotypic characteristics for identification of species of the anamorph genus Candida and related teleomorph yeast species.

A total of 49 type and neotype isolates and 32 clinical isolates of the anamorph genus Candida and related teleomorph genera were obtained from different culture collections and clinical laboratories. Isolates were subjected to two phenotypic methods of identification, Vitek yeast biochemical card (YBC) and API ID 32C, both based on carbohydrate assimilation, and one genotypic method, PCR fingerprinting, based on the detection of DNA polymorphisms between minisatellite-specific sequences with the primer M13 (5' GAGGGTGGCGGTTCT 3'). The correct identification of a strain at the Centraalbureau voor Schimmelcultures was used as the gold standard for the identification of an isolate. When the study was restricted to species included in the respective biochemical databases, the Vitek YBC and API ID 32C systems performed adequately with positive identification rates of 87.3 and 76.8%, respectively. When uncommon species were added to the study, several of which are not included in the databases, the identification efficiencies were 76.5 and 77.5%, respectively. By comparison, all isolates were correctly identified by PCR fingerprinting, with 63 reference species profiles in the databank. Sufficient polymorphisms among the total set of banding patterns were observed, with adequate similarity in the major patterns obtained from a given species, to allow each isolate to be assigned unambiguously to a particular species. In addition, variations in minor bands allowed for differentiation to the strain level. PCR fingerprinting was found to be rapid, reproducible, and more cost-effective than either biochemical approach. Our results provide reference laboratories with an improved identification method for yeasts based on genotypic rather than phenotypic markers.

Identification of pathogenic yeasts of the imperfect genus Candida by polymerase chain reaction fingerprinting.

With the increase in the number of immunocompromised hosts, the number of fungal pathogens has increased markedly. Identification and classification, especially of yeast species and strains, is often difficult when based solely on phenotypic characteristics. Since it became clear that different fungal pathogens require specific treatment strategies, there is a need for simple, rapid and reliable methods to identify fungal isolates. Polymerase chain reaction (PCR) fingerprinting was successfully applied here to identify yeast isolates. Microsatellite [(GTG)5; (GACA)4] and minisatellite [(5'GAGGGTGGCGGTTCT 3'), derived from the core-sequence of the phage M13] specific primers were used as single primers in the PCR to amplify hypervariable interrepeat DNA sequences from over 200 European, American and Australian clinical isolates within the genus Candida. Each species, represented by its type strain, could be identified by a specific multilocus pattern, allowing for the assignment of all the isolates to the appropriate species. Intra-species variation in the multilocus profiles was about 20% compared to inter-species variation, which was up to 80%. Anamorph-teleomorph pairs could be identified by highly homologous PCR fingerprint patterns. PCR fingerprinting was more discriminatory when compared with routinely used biochemical tests (Vitek YBC and API ID 32C). PCR fingerprinting has proven to be a powerful tool for the identification of medically important yeasts. It is rapid, sensitive, reliable, highly reproducible, stable in vitro and in vivo, and applicable to large scale experiments. Potential applications include: yeast taxonomy, epidemiology, environmental surveys, and improvement of the diagnosis of mycotic diseases.