No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Partial sequence analysis of the actin gene and its potential for studying the phylogeny of Candida species and their teleomorphs
Abstract
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.
... A review of the literature highlighted genes worthy of detailed investigation in the PMs. This shortlist includes actin (McElroy et al. 1990;Reece et al. 1992;Baldauf et al. 2000;Wöstemeyer 2000, 2001;Daniel et al. 2001;Daniel and Meyer 2003;Yun et al. 2003;Opalski et al. 2005;Hunter et al. 2006), βtubulin (O'Donnell et al. 1998bde Jong et al. 2001;McKean et al. 2001;Einax and Voigt 2003;Juuti et al. 2005), calmodulin (Stevens 1983;O'Donnell et al. 2000;Mulè et al. 2004;Wang and Zhuang 2007;Madrid et al. 2009;Romeo et al. 2011;Samson et al. 2014), chitin synthase (Chs) (Roberts et al. 1986;Debono and Gordee 1994;Kano et al. 1997;Zhang et al. 2000;Kong et al. 2012), elongation factor 1 alpha (EF1-α) (O'Donnell et al. 1998a;Roger et al. 1999;Baldauf et al. 2000;Seifert and Lévesque 2004;Kristensen et al. 2005;Hunter et al. 2006;Maphosa et al. 2006;Matheny et al. 2007;Amatulli et al. 2010), Mcm7 (Moir et al. 1982Kearsey and Labib 1998;Aguileta et al. 2008;Schmitt et al. 2009;Leavitt et al. 2011;Raja et al. 2011;Divakar et al. 2012;Morgenstern et al. 2012;Minnis and Lindner 2013;Tretter et al. 2013Tretter et al. , 2014Prieto and Wedin 2016), and Tsr1 (Gelperin et al. 2001;Schmitt et al. 2009;Tretter et al. 2013;Sadowska-Deś et al. 2013). In the present study, the possibility of developing working molecular markers for these regions for PM samples collected through a citizen science survey in the UK was investigated. ...
... The utility of each novel gene has varied across previous studies. The actin gene in particular has received both positive (Baldauf et al. 2000;Wöstemeyer 2000, 2001;Daniel et al. 2001;Daniel and Meyer 2003) and negative (Weiland and Sundsbak 2000;Hunter et al. 2006) reviews. In contrast, the few studies to have used Tsr1 have been mostly positive (Schmitt et al. 2009;Sadowska-Deś et al. 2013;Tretter et al. 2013); however, as in the present study, it was not considered to offer sufficient resolution in the study of Morgenstern et al. (2012). ...
The internal transcribed spacer (ITS) DNA marker is routinely used for fungal identification but gives a clear result for only three out of four powdery mildew samples. A search for new markers indicates that some genes offer enhanced identification in comparison with ITS. Others fail due to amplification and sequencing difficulties and lack of informative variability. Powdery mildews (Ascomycota, Erysiphales) are biotrophic, fungal plant pathogens that commonly occur worldwide on a wide range of host plants. They are unsightly and greatly reduce the vigor of their hosts and have major impacts on crop and other cultivated plants. Species within this order are straightforward to spot, but difficult to identify. A citizen science scheme was run in 2013–2016 in the UK to gather a wide array of samples on which identification methods could be developed. Current techniques for identification and phylogenetic reconstruction show scope for improvement. In this paper, we review genes used in other fungal groups for discrimination at species level. Working protocols for amplification and sequencing of seven genes (actin, β-tubulin, calmodulin,Chs, elongation factor 1-α [EF1-α], Mcm7, and Tsr1) are developed with varying success; Mcm7 proves to be the most useful at differentiation between closely related, phylogenetically young powdery mildew species for phylogenetic reconstruction when used separately and in tandem with ITS. We therefore propose this as the most appropriate candidate gene to be used commonly in powdery mildew diagnostics alongside the ITS; furthermore, this could be transferred to similarly troublesome fungal clades.
... The estimation of the gene copy number using QPCR indicates that the SDH is a single copy gene in the Pichia genome (Fig. S1). The Ct values obtained for a known single copy actin gene [23] and SDH gene are same at all the concentrations of DNA template used (Table S1). ...
... pastoris GS115 and CBS 7435) and found only one gene of SDH on the Chromosome 1 of P. pastoris (GenBank accession no: FN392319.1 and FR839628.1). We also carried out a relative copy number estimation by QPCR in comparison with single copy actin gene [23] and found the identical Ct values for both actin and SDH gene at all concentrations of DNA indicating that the SDH is also a single copy gene (Fig. S1, Table S1) in P. pastoris. The SDH gene upstream sequence found to be completely different from S. cerevisiae. ...
Sorbitol is used as a non-repressive carbon source to develop fermentation process for Mut(s) recombinant clones obtained using the AOX1 promoter in Pichia pastoris. Sorbitol dehydrogenase is an enzyme in the carbohydrate metabolism that catalyzes reduction of D-fructose into D-sorbitol in the presence of NADH. The small stretch of 211 bps upstream region of Sorbitol dehydrogenase coding gene has all the promoter elements like CAAT box, GC box, etc. It is able to promote protein production under repressive as well as non-repressive carbon sources. In this study, the strength of the Sorbitol dehydrogenase promoter was evaluated by expression of two heterologous proteins: Human Serum Albumin and Erythrina Trypsin Inhibitor. Sorbitol dehydrogenase promoter allowed constitutive expression of recombinant proteins in all carbon sources that were tested to grow Pichia pastoris and showed activity similar to GAP promoter. The Sorbitol dehydrogenase promoter was active in all the growth phases of the Pichia pastoris.
... Various markers have been developed for use in phylogenetic reconstructions, including the RNA polymerase II gene (Liu et al., 1999), the mitochondrial cytochrome C oxidase gene (Belloch et al., 2000), and the actin gene (Daniel et al., 2001;Daniel & Meyer, 2003). However, genes do not evolve similarly and often provide incongruent phylogenies when used separately, and consequently, multigenic analyses are now the preferred choice for the establishment of robust phylogenies (Kurtzman & Robnett, 2003). ...
... The markers implemented in YeastIP were chosen on the basis of several taxonomic studies (Peterson & Kurtzman, 1991;Cai et al., 1996;James et al., 1996James et al., , 1997Belloch et al., 2000;Daniel et al., 2001;Kurtzman & Robnett, 2003;Diezmann et al., 2004). The markers widely used in multi-genic analysis are described in Table 1 and were retrieved from the NCBI for all type strains of the Saccharomycotina subphylum. ...
With the advances in sequencing techniques, identification of ascomycetous yeasts to the species level and phylogeny reconstruction increasingly require curated and updated taxonomic information. A specific database with nucleotide sequences of the most common markers used for yeast taxonomy and phylogeny and a user-friendly interface allowing identification, taxonomy and phylogeny of yeasts species was developed. By the 1st of September 2012, the YeastIP database contained all the described Saccharomycotina species for which sequences used for taxonomy and phylogeny, such as D1/D2 rDNA and ITS, are available. The database interface was developed to provide a maximum of relevant information and data mining tools, including the following features: (1) the Blastn program for the sequences of the YeastIP database; (2) easy retrieval of selected sequences; (3) display of the available markers for each selected group of species; and (4) a tool to concatenate marker sequences, including those provided by the user. The concatenation tool allows phylogeny reconstruction through a direct link to the Phylogeny.fr platform. YeastIP is thus a unique database in that it provides taxonomic information and guides users in their taxonomic analyses. YeastIP facilitates multigenic analysis to encourage good practice in ascomycetous yeast phylogeny. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
... Primers V9G (de Hoog & Gerrits van den Ende, 1998) and LR5 (Vilgalys & Hester, 1990) were used to amplify the region of the nrRNA gene operon that includes the 39 end of the small-subunit rRNA gene, the ITS regions (ITS 1, ITS 2 and the intervening 5S rRNA gene), and the D1/D2 domain of the large-subunit (LSU) rRNA gene, as described by Knutsen et al. (2007). Primers CA14 and CA5R (Daniel et al., 2001) were used to amplify part of the actin gene using the method described by Daniel et al. (2001). The PCR products were separated by electrophoresis at 80 V for 40 min on a 0.8 % (w/v) agarose gel containing 0.1 mg ethidium bromide ml 21 in 16 TAE buffer (0.4 M Tris, 0.05 M sodium acetate, and 0.01 M EDTA, pH 7.85) and examined under UV light. ...
... Primers V9G (de Hoog & Gerrits van den Ende, 1998) and LR5 (Vilgalys & Hester, 1990) were used to amplify the region of the nrRNA gene operon that includes the 39 end of the small-subunit rRNA gene, the ITS regions (ITS 1, ITS 2 and the intervening 5S rRNA gene), and the D1/D2 domain of the large-subunit (LSU) rRNA gene, as described by Knutsen et al. (2007). Primers CA14 and CA5R (Daniel et al., 2001) were used to amplify part of the actin gene using the method described by Daniel et al. (2001). The PCR products were separated by electrophoresis at 80 V for 40 min on a 0.8 % (w/v) agarose gel containing 0.1 mg ethidium bromide ml 21 in 16 TAE buffer (0.4 M Tris, 0.05 M sodium acetate, and 0.01 M EDTA, pH 7.85) and examined under UV light. ...
The present classification of Galactomyces and its anamorph, Geotrichum, is based on various studies that used morphology, ecology, biochemistry, DNA-DNA reassociation comparisons and gene sequencing. In this study, the identities of strains of the Centraalbureau voor Schimmelcultures yeast culture collection as well as seven strains from South Africa were examined by analyses of the nucleotide divergence in the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene (nrRNA) operon, the D1/D2 domains of the 26S rRNA gene and partial actin gene sequences as well as compatibility studies. The South African strains were assigned to species in the genus Galactomyces. The phylogenetic analyses and mating studies revealed that Geo. silvicola and Geo. bryndzae are synonyms of Gal. candidus and that Geo. vulgare is a synonym of Gal. pseudocandidus.
... The actin encoding gene (ACT1) is an essential, ubiquitous, and highly conserved gene in eukaryotic organisms. In yeasts, it is one of the most commonly used marker genes in taxonomic analyses (Daniel et al. 2001;Daniel and Meyer 2003;Stielow et al. 2015). Inspection of the assemblies of the two strains of K. barnettii revealed that their genomes contain two copies of the gene encoding actin. ...
Recent studies have suggested that species of the Kazachstania genus may be interesting models of yeast domestication. Among these, Kazachstania barnettii has been isolated from various microbially transformed foodstuffs such as sourdough bread and kefir. In the present work, we sequence, assemble and annotate the complete genomes of two K. barnettii strains: CLIB 433, being one of the two reference strains for K. barnettii that was isolated as a spoilage organism in soft drink, and CLIB 1767, recently isolated from artisan bread-making sourdough. Both assemblies are of high quality with N50 statistics greater than 1.3 Mb and BUSCO score greater than 99%. An extensive comparison of the two obtained genomes revealed very few differences between the two K. barnettii strains, considering both genome structure and gene content. The proposed genome assemblies will constitute valuable references for future comparative genomic, population genomic or transcriptomic studies of the K. barnettii species.
... 1: negative droplets, 2: droplets that are only positive for C. truncatum sequence, 3: droplets that are only positive for C. scovillei sequence, 2 ∩ 3: dual positive droplets: C. truncatum and C. scovillei sequences are detected. (A) In monoplex detection: droplets were detected either for C. truncatum (FAM channel) or C. scovillei (HEX channel); amplification process was shown schematically, (B) In the 1D view of duplex detection of C. truncatum and C. scovillei, the FAM channel showing 2 + 2 ∩ 3 droplets for C. truncatum and negative droplets, while the HEX channel showed 3 + 2 ∩ 3 droplets for C. scovillei and negative droplets; amplification process was shown schematically, and (C) 2D view of duplex detection of C. truncatum and C. scovillei showing negative droplets, droplets only positive for C. truncatum, C. scovillei droplets, and dual positive droplets 2 ∩ 3 relationships among eukaryotes including fungi (Daniel et al., 2001;Dethoup et al., 2016). The identification of the isolates used in this study based on morphological characterization (Osman Abdelrazig et al., 2020;Suwannarat et al., 2017). ...
Chili anthracnose, caused by Colletotrichum species is a major disease of chili leading to severe economic loss worldwide. Here, two new species-specific primer pairs were designed based on the actin gene sequence and used for the detection of Colletotrichum truncatum (Ct) and Colletotrichum scovillei (Cs), the main causal agents of chili anthracnose. The primers amplified short sequences of 96 and 126 bp for Ct and Cs, respectively. These two species-specific primer pairs were applied in a droplet digital PCR (ddPCR) for duplex detections of Ct and Cs. Highly specific and sensitive assays for the detection of Ct and Cs in infected chili fruit and seeds were presented. The absence of PCR product for template DNA from other Colletotrichum species and different fungal genera was used to inform about the specificity of these primers for Ct and Cs. The sensitivity of duplex ddPCR was 10,000-fold lower than the conventional PCR.
... Furthermore, D1/D2 domain of the 26S rRNA has approved as a helpful tool for yeast identification and detecting biodegradation activity. D1/D2 domain includes different marker genes like, actin-1 (Daniel et al., 2001;Kurtzman and Robnett 2003), RNA polymerase II (Kurtzman and Robnett 2003) and mitochondrial encoded genes Robnett 2003, Belloch et al. 2000;Kurtzman and Robnett 2003). ...
Twenty five yeasts isolated were isolated from Khurais oil field in Saudi Arabia and assayed to evaluate their biodegradability. Only five isolates (namely, A1, A2, A3, A4 and A5) showed potential use of oil as sole carbon source. During incubation period, highest growth rate were recorded for A1, A2 and A3 isolates. Low growth distinguished A4 isolate; A5 isolate could not degrade oil.
Spectrophotometrical analysis for four yeast isolates biodegradation activities indicated that, A1 isolate was superior for oil degradation (61%) comparing with A4 isolate which reflected lowest degradation % (33%). A2 and A3 isolates showed moderate biodegradation activity (56 and 51 % respectively).
D1/D2 domain of the 26S rRNA gene sequence was used as molecular marker to identify five yeast isolates. After comparing 26S rRNA gene sequences of five yeast isolates with highly similarity isolates, five yeast isolates (A1, A2, A3, A4 and A5)were submitted to database as Candida tropicalis (MW488263), Candida tropicalis (MW488264), Rhodotorula mucilaginosa (MW488265) and Rhodosporidium toruloides (MW488266) respectively. Using OXF1/ACR1 primer, specific lipase gene amplicon with 250 bp were detected with in all four yeast isolates
... Actin-1, nucleotides 783^1680. Initial primers were from the study of Daniel et al. [14] followed by development of group-speci¢c primers for the species in the present study. RNA polymerase II (RPB2), nucleotides 2447^3127 [15]. ...
Species of Saccharomyces, Arxiozyma, Eremothecium, Hanseniaspora (anamorph Kloeckera), Kazachstania, Kluyveromyces, Pachytichospora, Saccharomycodes, Tetrapisispora, Torulaspora, and Zygosaccharomyces, as well as three related anamorphic species assigned to Candida (C. castellii, C. glabrata, C. humilis), were phylogenetically analyzed from divergence in genes of the rDNA repeat (18S, 26S, ITS), single copy nuclear genes (translation elongation factor 1α, actin-1, RNA polymerase II) and mitochondrially encoded genes (small-subunit rDNA, cytochrome oxidase II). Single-gene phylogenies were congruent for well-supported terminal lineages but deeper branches were not well resolved. Analysis of combined gene sequences resolved the 75 species compared into 14 clades, many of which differ from currently circumscribed genera.
... Нуклеотидные последовательности ядерного гена ACT1 и митохондриального гена ATP9 оказались более вариабельными. Следует отметить, что оба гена высоко консервативны и имеют идентичные аминокислотные последовательности у семи видов рода Saccharomyces: S. cerevisiae, S. arboricola, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae и S. paradoxus (Groth et al., 1999;Daniel et al., 2001;Spirek et al., 2003;Naumova et al., 2005;Naumov et al., 2010). При этом нуклеотидные последовательности этих генов у видов Saccharomyces отличаются несколькими молчащими заменами. ...
... The sequences of the nuclear gene ACT1 and the mitochondrial gene ATP9 exhibited more variation. It should be noted that both genes are highly conserved and encode identical amino acid sequences in seven species of the genus Saccharomyces: S. cerevisiae, S. arboricola, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, and S. paradoxus (Groth et al., 1999;Daniel et al., 2001;Spirek et al., 2003;Naumova et al., 2005;Naumov et al., 2010). However, nucleotide sequences of these genes differ among Saccharomyces species in several silent substitutions. ...
Molecular genetic study of Saccharomyces yeasts of different species was carried out using molecular karyotyping and comparative analysis of a number of nuclear and mitochondrial genes. Multigene phy-logenetic analysis showed that the recently described species S. jurei was phylogenetically closest to S. mika-tae, while S. bayanus and S. arboricola were the most divergent species within the genus Saccharomyces. Comparative analysis of amino acid sequences of the SUC β-fructosidase genes also indicated a close genetic relationship between the species S. jurei and S. mikatae. The biological species S. cerevisiae, S. kudriavzevii, and S. paradoxus possessed collinear karyotypes. Two reciprocal translocations were found in the S. jurei karyotype, one of which was unique (between chromosomes I and XIII), and the second was common with S. mikatae: VI/VII. Reciprocal translocations in the karyotypes of S. arboricola, S. bayanus, and S. cariocanus were species-specific. It was established that only chromosome III carrying the mating type locus MAT was of approximately the same size in all eight species of the genus Saccharomyces. The adaptive role of reciprocal translocations of chromosomes is discussed.
... The region has also the disadvantage to carry relatively few informative characters. Although the amino acid sequence of actin proteins is highly conserved (Hightower & Meagher 1996), the third codon positions of the DNA sequences show substantial variation among closely related taxa (Daniel et al. 2001). Conserved location of introns and low evolution rates are observed in the genes encoding actin protein so they may be useful for taxonomic and identification studies especially for Ascomycota (Díez et al. 2001, Verkley et al. 2014) and Zygomycota (Voigt & Wöstemeyer 2000, Wöstemeyer 2001. ...
Identification of fungi to the species level has some challenges because of great diversity in morphology and ecology. Therefore, developing an efficient species recognition system which is applicable for all fungi seems difficult. Currently, DNA sequences of different regions have become powerful molecular data for rapid identification of specimens and overcoming the different sets of traditional criteria used for describing fungal species. However, deciding the potential region(s) as DNA barcode is a crucial step to identify biological specimens and to assign them to a given species. In the present review, usefulness of several DNA markers (ITS, LSU, SSU, COX1, RPB1, RPB2, β-tubulin, MCM7, TEF1-α, γ-Actin, atp6 and CaM) were investigated based on current studies. Even though numerous studies were completed to decide a marker as DNA barcode for a specific taxon it was observed that potential DNA barcodes vary from species to species. However, ITS was indicated by lots of studies as the potential barcode for most of fungi due to high degree of interspecific variability, conserved primer sites and multiple copy nature in the genome. Several protein-coding regions such as TEF1-α, RPB1, RPB2, β-tubulin and CaM genes may be accepted as secondary barcodes for the vast majority of Ascomycota genera such as Penicillium, Aspergillus and Neonectria. Therefore, concatenated alignment of ITS region with one or more protein-coding genes may be effective for finer-scale species-level identification of specific fungi.
... A similar number of newly described species can be observed for other yeast genera, mostly as an extensive database of barcode sequences of D1/D2 and internal transcribed spacer (ITS) regions provides data of all described species [7]. However, additional protein-coding genes have also been employed in yeast taxonomy to construct statistically well-supported phylogenetic trees that reflect the evolutionary relationships among species and genera [8][9][10]. With emergence of whole genome sequencing, reconstructions of more robust yeast phylogenies are now being recovered [11][12][13][14], from which a frame for new species definition is being built [15][16][17][18][19][20][21]. ...
A novel yeast species was isolated from the sugar-rich stromata of Cyttaria hariotii collected from two different Nothofagus tree species in the Andean forests of Patagonia, Argentina. Phylogenetic analyses of the concatenated sequence of the rRNA gene sequences and the protein-coding genes for actin and translational elongation factor-1α indicated that the novel species belongs to the genus Hanseniaspora. De novo genome assembly of the strain CRUB 1928T yielded a 10.2-Mbp genome assembly predicted to encode 4452 protein-coding genes. The genome sequence data were compared to the genomes of other Hanseniaspora species using three different methods, an alignment-free distance measure, Kr, and two model-based estimations of DNA-DNA homology values, of which all provided indicative values to delineate species of Hanseniaspora. Given its potential role in a rare indigenous alcoholic beverage in which yeasts ferment sugars extracted from the stromata of Cytarria sp., we searched for the genes that may suggest adaptation of novel Hanseniaspora species to fermenting communities. The SSU1-like gene encoding a sulfite efflux pump, which, among Hanseniaspora, is present only in close relatives to the new species, was detected and analyzed, suggesting that this gene might be one factor that characterizes this novel species. We also discuss several candidate genes that likely underlie the physiological traits used for traditional taxonomic identification. Based on these results, a novel yeast species with the name Hanseniaspora gamundiae sp. nov. is proposed with CRUB 1928T (ex-types: ZIM 2545T = NRRL Y-63793T = PYCC 7262T; MycoBank number MB 824091) as the type strain. Furthermore, we propose the transfer of the Kloeckera species, K. hatyaiensis, K. lindneri and K. taiwanica to the genus Hanseniaspora as Hanseniaspora hatyaiensis comb. nov. (MB 828569), Hanseniaspora lindneri comb. nov. (MB 828566) and Hanseniaspora taiwanica comb. nov. (MB 828567).
... De ce fait, malgré une résolution taxonomique plus faible que la région ITS, la région 26S est encore largement utilisée. Les relations phylogénétiques entre les levures Arxiozyma, Candida, Eremothecium, Hanseniaspora, Kazachstania, Kluyveromyces, Pachytichospora, Saccharomyces, Saccharomycodes, Tetrapisispora, Torulaspora et Zygosaccharomyces, ont été établies à partir de la divergence dans les différentes parties de l'ADNr (18S, 26S, ITS), des gènes nucléaires de copie unique (facteur élongation de la traduction de 1'actine-1, l'ARN polymérase II) et des gènes mitochondriaux (petite sous-unité d'ADNr, cytochrome oxydase II) (Daniel et al., 2001 ;Kurtzman et Robnett, 2003 ;Daniel et Meyer, 2003). ...
The effects of different anthropogenic activities (vineyard, winery) on fungal populations from grape to wine were studied. To characterize these effects, it was necessary to access to the overall diversity of populations (pyrosequencing and spectroscopy FT-IR) but also to intra-specific diversity (FT-IR). Spectroscopy FT-IR has been validated for their ability to characterize the global population and to discriminate the strains for three species of non-Saccharomyces yeasts (NS). For the first time, it is shown that the grape berry is a limited source for NS yeasts while the winery seems to be a significant source; the air is an important vector for dissemination of these yeasts. In addition, persistence of NS yeast strains from year to year in the winery has been demonstrated. The studied anthropogenic activities modify the fungal diversity. Thus, lower biodiversity of grapes from organic modality was measured for the three vintages considered. The pressing / clarification step revises strongly fungal populations and the influence of the winery flora is confirmed. The addition of SO2 changes the population dynamics and favors the dominance of the species S. cerevisiae. The non-targeted chemical analysis shows, for the first time, that these wines can be distinguished at the end of the alcoholic fermentation (with or without SO2) depending on plant protection. Thus, the existence in wines of chemical and microbiological signatures associated with vineyard protection mode is highlighted.
... Phylogenetic trees produced using the three conserved gene approach revealed three distinct clusters: Cluster I, grouping C. albicans, C. dubliniensis, C. tropicalis, C. viswanathii, and C. parapsilosis: Cluster II, grouping C. glabrata, C. sphaerica, and C. kefyr; and Cluster III, grouping C. krusei, C. inconspicua, and C. lambica. These phylogenetic clusters are consistent with previously reported phylogenetic analyses based on the use of chromosomal small and large subunit ribosomal RNA gene sequences (Schmalreck et al., 2014;Barns et al., 1991;Gilfillan et al., 1998;Kurtzman and Robnett, 1998), nucleotide sequences of the mitochondrial large subunit ribosomal RNA gene (Yamada et al., 2004) or partial sequences of the actin gene (Daniel et al., 2001). Phylogenetic trees created by Diezmann et al. (2004) based on six concatenated genes different from the 3 genes we used, further supports our data. ...
Successful treatment of a Candida infection relies on 1) an accurate identification of the pathogenic fungus and 2) on its susceptibility to antifungal drugs. In the present study we investigated the level of correlation between phylogenetical evolution and susceptibility of pathogenic Candida spp. to antifungal drugs. For this, we compared a phylogenetic tree, assembled with the concatenated sequences (2475-bp) of the ATP2, TEF1, and TUF1 genes from 20 representative Candida species, with published minimal inhibitory concentrations (MIC) of the four principal antifungal drug classes commonly used in the treatment of candidiasis: polyenes, triazoles, nucleoside analogues, and echinocandins. The phylogenetic tree revealed three distinct phylogenetic clusters among Candida species. Species within a given phylogenetic cluster have generally similar susceptibility profiles to antifungal drugs and species within Clusters II and III were less sensitive to antifungal drugs than Cluster I species. These results showed that phylogenetical relationship between clusters and susceptibility to several antifungal drugs could be used to guide therapy when only species identification is available prior to information pertaining to its resistance profile. An extended study comprising a large panel of clinical samples should be conducted to confirm the efficiency of this approach in the treatment of candidiasis.
... The act1 region encodes actin protein found in all eukaryotes and was suggested by Daniel et al. [10] as reliable phylogenetic marker. Later, Daniel and Meyer [9] extended the act1 sequence database of selected yeast species. ...
... These regions may be particularly useful in the identifi cation of species that are poorly resolved by the D1/D2 domain. Other genes that have been used in yeast species identifi cation include the translation elongation factor 1α, actin-1, RNA polymerase II, pyruvate decarboxylase, β-tubulin, and cytochrome oxidase II (Belloch et al. 2000, Daniel et al. 2001, Daniel and Meyer 2003, Kurtzman and Robnett 2003. Some of them are superior to rRNA genes for resolving relationships at various taxonomic levels, for instance in Ascomycota (Schoch et al. 2009). ...
... The act1 region encodes actin protein found in all eukaryotes and was suggested by Daniel et al. [10] as reliable phylogenetic marker. Later, Daniel and Meyer [9] extended the act1 sequence database of selected yeast species. ...
Kazachstania slooffiae is the dominating yeast in pig's gut. No methods others than cultivation were applied for enumeration of yeasts within this ecosystem. Therefore, the aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay to quantitate total yeasts and K. slooffiae in the porcine gut. This work demonstrated that the copy numbers in gDNA can be determined by qPCR using PCR amplicons as a calibrator and one-point calibration method. The gDNA were then used as a calibrator for further analysis. The values of quantitation cycle and PCR amplification efficiency of gDNA calibrator were highly reproducible. DNA was extracted from feces and from 10 different cultured yeasts found in pigs' intestine. The qPCR results using primers NL1/LS2 encoding 26S rDNA correlated (r = 0.984, P < 0.0001) with cultivation results. From two primer sets developed, one set encoding act1 gene was suitable for quantitation of K. slooffiae. The copy numbers of K. slooffiae could be determined by 40 % analyzed animals, amounting to about 70 % of total yeasts. The application of this method in next studies will help to get more information about K. slooffiae and total yeasts in the gut of pigs.
... An isolate of Neurospora crassa was included as an outgroup. 9,10,30 The MEGA version 5.1 program 29 was used to estimate the best-fitting evolutionary models for each data and the Maximum Likelihood analysis. Bootstrap support was estimated by 1000 replicates. ...
Background:
Candida species are the main cause of hospital acquired fungal bloodstream infections. The main risk factors for candidemia include parenteral nutrition, long-term intensive care, neutropenia, diabetes, abdominal surgery and the use of central venous catheters. The antifungal drugs used to treat candidemia are mainly the echinocandins, however some isolates may be resistant to these drugs.
Aims:
This work aims to evaluate the in vitro susceptibility patterns of various Candida species isolated from blood samples and provide their identification by molecular characterization.
Methods:
Antifungal susceptibility testing was performed using the broth microdilution method. The sequencing of the ITS and D1/D2 regions of rDNA was used for molecular characterization.
Results:
Seventy-four of the 80 isolates were susceptible to anidulafungin, 5 were intermediate, and 1 was resistant. For micafungin 67 were susceptible, 8 were intermediate and 5 were resistant. All isolates were susceptible to amphotericin B. Lastly, 65 isolates were susceptible to fluconazole, 8 were dose-dependent and 4 were resistant. The molecular identification corroborated the phenotypic data in 91.3% of the isolates.
Conclusions:
Antifungal susceptibility data has an important role in the treatment of candidemia episodes. It was also concluded that the molecular analysis of isolates provides an accurate identification and identifies genetic variability within Candida species isolated from patients with candidemia.
... It was hypothesized that pseudohomothallic (mat +,-) strains are aneuploids. [8]. ...
Yeast identification has a real importance for selection of technological strains and also for studying biodiversity and population dynamics in the fermentation systems. Important studies on yeast population dynamics showed that Saccharomyces genus is dominant in alcoholic fermentation, while other genera like: Kloeckera, Candida, Pichia, Hansenula, Hanseniaspora and Metschnikowia are growing in the first step of the process. The purpose of this review was to emphasize the importance of the methylotrophic yeasts diversity, with the most important morpho-physiological characteristics, the complexity of the life cycle, and also the main pathway of the methanol metabolism, with the enzymes involved in this process.
... Influencing this work is lack of consensus on the definition of a genus and whether apparently independent lineages are sufficiently diverged to be considered genera. At present, all known ascosporic genera and several currently unnamed non-ascosporic lineages have been circumscribed from analyses of two to five genes, but some of the larger genera will probably be subdivided as whole-genome sequences become available (Nakase et al., 1988;Daniel et al., 2001;Tomaszewski et al., 2003;Kurtzman & Robnett, 2003Suh et al., 2004Suh et al., , 2006Nguyen et al., 2006;Kurtzman et al., , 2008Kurtzman et al., , 2011Kurtzman & Suzuki, 2010;Péter et al., 2012;Lachance & Kurtzman, 2013). Gene sequence analyses have shown that many species assigned to anamorphic genera, such as Candida, are members of currently accepted ascosporic genera, but other species are members of independent lineages that have not been taxonomically recognized and which represent new genera. ...
Detection, identification and classification of yeasts have undergone a major transformation in the past decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined gene sequences from domains 1 and 2 (D1/D2) of large subunit rRNA and from the internal transcribed spacer (ITS) now permits many laboratories to identify species accurately and this has led to a doubling in the number of known species of yeasts over the past decade. Phylogenetic analysis of gene sequences has resulted in major revision of yeast systematics, resulting in redefinition of nearly all genera. Future work calls for application of genomics to refine our understanding of the species concept and to provide a better understanding of the boundaries of genera and higher levels of classification. This increased understanding of phylogeny is expected to allow prediction of the genetic potential of various clades and species for biotechnological applications and adaptation to environmental changes.
... Therefore contemporary yeast taxonomy is mainly based on the comparative analysis of conserved parts of the genomes such as the nuclear rRNA operon, genes coding for components of the transcriptionary and translationary machineries, their combinations (e.g. [1] and references therein) and genes encoding cytoskeletal components [2,3]. In phylogenetic analysis of higher taxonomic units, a multigenic approach is preferred (e.g. ...
Modern taxonomy of yeasts is mainly based on phylogenetic analysis of conserved DNA and protein sequences. By far the most frequently used sequences are those of the repeats of the chromosomal rDNA array. It is generally accepted that the rDNA repeats of a genome have identical sequences due to the phenomenon of sequence homogenisation and can thus be used for identification and barcoding of species. Here we show that the rDNA arrays of the type strains of Metschnikowia andauensis and M. fructicola are not homogenised. Both have arrays consisting of diverse repeats that differ from each other in the D1/D2 domains by up to 18 and 25 substitutions. The variable sites are concentrated in two regions that correspond to back-folding stretches of hairpin loops in the predicted secondary structure of the RNA molecules. The substitutions do not alter significantly the overall hairpin-loop structure due to wobble base pairing at sites of C-T transitions and compensatory mutations in the complementary strand of the hairpin stem. The phylogenetic and network analyses of the cloned sequences revealed that the repeats had not evolved in a vertical tree-like way but reticulation might have shaped the rDNA arrays of both strains. The neighbour-net analysis of all cloned sequences of the type strains and the database sequences of different strains further showed that these species share a continuous pool of diverse repeats that appear to evolve by reticulate evolution.
... In contrast, numerous other studies have examined much larger groups of species, but have used far fewer genes, again resulting in weak resolution of relationships among genera (e.g. Kurtzman & Robnett, 1994, 1998Yamada et al., 1994;James et al., 1997;Daniel et al., 2001;Suh et al., 2006;Kurtzman et al., , 2008Kurtzman & Suzuki, 2010). ...
Relationships among ascomycetous yeast genera (subphylum Saccharomycotina, phylum Ascomycota) have been uncertain. In the present study, type species of 70 currently recognized genera are compared from divergence in the nearly entire nuclear gene sequences for large subunit rRNA, small subunit rRNA, translation elongation factor-1α, and RNA polymerase II, subunits 1 (RPB1) and 2 (RPB2). The analysis substantiates earlier proposals that all known ascomycetous yeast genera now assigned to the Saccharomycotina represent a single clade. Maximum likelihood analysis resolved the taxa into eight large multi-genus clades and four one and two genus clades. Maximum parsimony and neighbor-joining analyses gave similar results. Genera of the family Saccharomycetaceae remain as one large clade as previously demonstrated to which the genus Cyniclomyces is now assigned. Pichia, Saturnispora, Kregervanrija, Dekkera, Ogataea and Ambrosiozyma are members of a single large clade, which is separate from the clade that includes Barnettozyma, Cyberlindnera, Phaffomyces, Starmera and Wickerhamomyces. Other clades include Kodamaea, Metschnikowia, Debaryomyces, Cephaloascus and related genera, which are separate from the clade that includes Zygoascus, Trichomonascus, Yarrowia and others. This study once again demonstrates that there is limited congruence between a system of classification based on phenotype and a system determined from DNA sequences. © 2012 Federation of European Microbiological Societies. Published by Blackwell PublishingLtd. All rights reserved.
... The overall average genomic divergence between the contributors of CBS 7064 is 15.3% [21], consistent with the value of 15% for the introns of ACT1 and RPL33. Introns are therefore good indicators of overall sequence divergence in yeast genomes, and are superior to sequences like the rDNA D1/D2 [8] or the ACT1coding gene [46]. In the M. farinosahybrids, the overall divergence may be reduced by LOH. ...
Among ascomycetous yeasts, the CTG clade is so-called because its constituent species translate CTG as serine instead of leucine. Though the biology of certain pathogenic species such as Candida albicans has been much studied, little is known about the life cycles of non-pathogen species of the CTG clade. Taking advantage of the recently obtained sequence of the biotechnological Millerozyma (Pichiasorbitophila) farinosa strain CBS 7064, we used MLST to better define phylogenic relationships between most of the Millerozyma farinosa strains available in public collections. This led to the constitution of four phylogenetic clades diverging from 8% to 15% at the DNA level and possibly constituting a species complex (M. farinosa) and to the proposal of two new species:Millerozyma miso sp. nov. CBS 2004(T) ( = CLIB 1230(T)) and Candida pseudofarinosa sp. nov.NCYC 386(T)( = CLIB 1231(T)). Further analysis showed that M. farinosa isolates exist as haploid and inter-clade hybrids. Despite the sequence divergence between the clades, secondary contacts after reproductive isolation were evidenced, as revealed by both introgression and mitochondria transfer between clades. We also showed that the inter-clade hybrids do sporulate to generate mainly viable vegetative diploid spores that are not the result of meiosis, and very rarely aneuploid spores possibly through the loss of heterozygosity during sporulation. Taken together, these results show that in this part of the CTG clade, non-Mendelian genetic exchanges occur at high rates through hybridization between divergent strains from distinct clades and subsequent massive loss of heterozygosity. This combination of mechanisms could constitute an alternative sexuality leading to an unsuspected biodiversity.
... Genes other than those from the rRNA repeat have been examined for their capability to separate species. Daniel and Meyer (2003) and Daniel et al. (2001) compared species resolution from divergence in the gene sequences of D1/D2 LSU rRNA and actin-1. Divergence in actin-1 was greater than in D1/D2, thereby potentially providing greater resolution among closely related taxa. ...
The issue of description of new yeast species on the basis of a single strain is discussed. Single gene sequences, such as those from D1/D2 LSU rRNA, or sequences from ITS1/ITS2 are commonly used as the basis for recognizing new yeast species. Evidence is presented that hybrids and species with polymorphic gene sequences may not be recognized from a single gene analysis, but with multigene sequence comparisons, single-strain species can be accurately determined. Further, description of single-strain species will add to an understanding of yeast phylogeny and species diversity, which would be unknown if new species descriptions were limited to those taxa for which multiple strains were available.
... The nuclear genes, ACTIN-1 and TEF, and mitochondrial genes, SSU and COXII, were amplified and sequenced according to Daniel et al. (2001). For the amplifications primers described by Kurtzman and Robnett (2003), with few modifications, were used (Table 1). ...
The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from "Primitivo" grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO₂ and temperature) useful for industrial application.
... Quantitative real-time RT-PCR Real-time PCR primers (Table S1) were designed based on the sequences around 500–600 bp of CmADH genes where they have high sequence diversity to confirm specific gene product formation. Actin1 gene was chosen as endogenous gene[44]. The amplicon lengths were all about 120 bp. ...
The alcohol dehydrogenase (ADH) system plays a critical role in sugar metabolism involving in not only ethanol formation and consumption but also the general "cofactor balance" mechanism. Candida maltosa is able to ferment glucose as well as xylose to produce a significant amount of ethanol. Here we report the ADH system in C. maltosa composed of three microbial group I ADH genes (CmADH1, CmADH2A and CmADH2B), mainly focusing on its metabolic regulation and physiological function.
Genetic analysis indicated that CmADH2A and CmADH2B tandemly located on the chromosome could be derived from tandem gene duplication. In vitro characterization of enzymatic properties revealed that all the three CmADHs had broad substrate specificities. Homo- and heterotetramers of CmADH1 and CmADH2A were demonstrated by zymogram analysis, and their expression profiles and physiological functions were different with respect to carbon sources and growth phases. Fermentation studies of ADH2A-deficient mutant showed that CmADH2A was directly related to NAD regeneration during xylose metabolism since CmADH2A deficiency resulted in a significant accumulation of glycerol.
Our results revealed that CmADH1 was responsible for ethanol formation during glucose metabolism, whereas CmADH2A was glucose-repressed and functioned to convert the accumulated ethanol to acetaldehyde. To our knowledge, this is the first demonstration of function separation and glucose repression of ADH genes in xylose-fermenting yeasts. On the other hand, CmADH1 and CmADH2A were both involved in ethanol formation with NAD regeneration to maintain NADH/NAD ratio in favor of producing xylitol from xylose. In contrast, CmADH2B was expressed at a much lower level than the other two CmADH genes, and its function is to be further confirmed.
One important ecological question regarding the use of plant resistance genes against fungal pathogens concerns whether and how such resistance genes may modify pathogenic or beneficial members of the plant-associated microbiota. We studied the impact of a plant resistance gene by analyzing the mycobiota associated with Brassica napus organs over two cropping seasons. Sampling dates coincided with key stages of the life cycle of the B. napus pathogen Leptosphaeria maculans. Leaf samples were collected at three time points in autumn and spring, and stem base samples were collected at two time points a few weeks before and at harvest. Stem residues, where L. maculans survives in the intercropping season and develops sexual reproduction, were also analyzed at four time points between the two cropping seasons. The sampling was performed on two plant genotypes, Darmor and Darmor- Rlm11, only differing by the effective resistance gene against L. maculans, Rlm11. Altogether, 419 samples were analyzed using two barcode: internal transcribed spacer (ITS) and Actin. The plant organ was shown to be the main mycobiota structuring factor, as clear-cut alternation of the species suggested that each plant organ represented a specific ecological niche. The cropping season and plant genotype also significantly influenced the community structure in lower proportions. The resistance gene contributed differently to the community structure depending on the year and the organ concerned. A significant but low impact of Rlm11 on other B. napus fungal pathogens was detected. The ITS and Actin barcodes showed similar results, but the species assignation was limited for the latter.
The massive parallel sequencing technology, applied to the taxonomy of microorganisms, has been affecting the traditional phenotypic and molecular phylogenies based on the sequence of a single gene or a small handful of genes. The exponential accumulation of new, entire genome sequences of microorganisms in public databases in recent years, especially in the fields of taxonomic and biotechnology, is driving a conceptual revolution in the way of understanding the concepts of species in microorganisms in general and fungi in particular. The problems of drawing species boundaries, reclassification of species, discovering new taxa and clades, recognizing synonyms, and new species for science can now be addressed with genomic approaches. Derived from all this, much more robust high-resolution phylogenies, based on core genomes or broad collections of genes and their deduced proteins, are currently being reconstructed. Although this effort is still far from being a canon in the taxonomy of yeasts, it will gradually turn into a change and challenge that researchers are taking into account due to the great power and reliability of these genomic approaches and bioinformatics tools. Likewise, the complete sequence of the genomes of the strains of microorganisms of industrial or biotechnological interest will allow limiting biopiracy, help protect patents, recognize the appellation of origin, discourage violations of intellectual property rights, and resolve conflicts over the rights of the commercial exploitation of microorganisms. In this chapter, an effort is made to compare conventional taxonomy techniques with the latest work involving genomic sciences as a key tool in yeast taxonomy.
The primer sets used in polymerase chain reaction (PCR) assays for DNA barcoding of plant pathogenic fungi are presented. The key gene targets/loci for fungi are the internal transcribed spacer (ITS), β-tubulin (BenA/Tub2), Calmodulin (CAM or CAL), RNA polymerase II subunit 1 (RPB1) and 2 (RPB2), actin (ACT), Translation elongation factor 1-alpha (tef1), mitochondrial cytochrome c oxidase I (COI) and II (CO2), Chitin synthase (CHS-1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Glutamine synthetase (GS), Manganese-superoxide dismutase (SOD2), histone (his3), Intergenic sequence between Apn2 DNA lyase and Mat1-2-1 (ApMAT), partial apurinic DNA lyase gene (APN2), partial mating-types locus gene (MAT1-2) genes and the Ras-related protein gene (Ypt1) genes. Key references are provided for fungal group DNA barcodes, PCR assay cocktail preparation, and cycling conditions. These primer sets have been used or are currently used by mycologists, plant pathologists, and researchers from specific organizations (such as those in the plant quarantine services). Readers are still advised to check the correctness and completeness of these sequences in the relevant reference.There are other primer sets, which are not in the list, that have been used in DNA barcoding of plant pathogenic fungi. Nevertheless, these primers sets still targets the gene/loci indicated in this article.
Magnusiomyces capitatus and Saprochaete clavata are members of the clade of arthroconidial yeasts that represent emerging opportunistic pulmonary pathogens in immunocompromised patients. Given that standard rDNA identification often provides confusing results, in this study, we analyzed 34 isolates with the goal of finding new genetic markers for classification using multilocus sequencing and amplified fragment length polymorphism (AFLP). Interspecific similarity using rDNA markers [internal transcribed spacer (ITS) and large subunit] was in the range of 96–99%, whereas that of protein-coding loci (Rbp2, Act, and Tef1α) was lower at 89.4– 95.2%. Ultimately, Rbp2 was selected as the best marker for species distinction. On the basis of cloned ITS data, some strains proved to be misidentified in comparison with phenotypic characters, protein sequences, and AFLP profiles, indicating that different copies of the ribosomal operon were present in a single species. Antifungal susceptibility testing revealed that voriconazole had the lowest MIC against M. capitatus, while amphotericin B had the lowest MIC against S. clavata. Both species exhibited in vitro resistance to fluconazole and micafungin.
Plant pathogenic fungi cause significant economic crop yield losses every year. Proper identification to the species level is a critical first step in any investigation of plant infection, whether it is research driven or compelled by the need for rapid and accurate diagnostics during disease outbreak. Further, it is also helpful in decision making with respect to monetary loss and investment for necessary disease management practices. The recent developments of DNA barcoding technology have drastically translated the epitome of species identification and show promise in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment, ecological studies, diagnostics and monitoring of fungal plant pathogens. This chapter provides a snapshot vision on the current use and impact of DNA barcoding approaches in diagnosis and monitoring of fungal plant pathogens. Moreover, an effort has been put forward to understand various marker genes associated with barcode process, their suitability, limitation and applicability in diagnostic and monitoring of fungal plant pathogens.
Commercial banana varieties (Musa spp.) are cultivated in over 100 countries,
assuming important social and economic roles, given that this fruit is the most widely
consumed globally. Brazil is the world's fifth largest producer, where yellow Sigatoka
is the most common banana disease in production areas. Mycosphaerella musicola (R.
Leach ex. J. L. Mulder 1979) (Anamorfo: Pseudocercospora musae Zimm.
Deighton), causal organism of the disease, is the most commonly occuring
Mycosphaerella species in the country. Limited analysis of genetic diversity of this
pathogen has been conducted to date. Similary, little is known regarding fungicide
sensitivity of M. musicola to DMI (demethylation inhibitor) fungicides. Analysis of
pathogen effector LysM genes and their influence on the pathogenesis of
Mycosphaerella members is also limited. In this context, the goals of this study were
to verify the occurrence of sexual recombination in M. musicola populations from 13
different agroecological zones across Brazil, based on analysis of mating type
idiomorphs; characterize population genetic diversity by comparison of 19
microsatellite loci; analyse isolate sensitivity to DMI fungicide and correlate the
results with the occurrence of mutations in the CYP51 gene and, as a additional
component of the work, characterize the virulence effector – LysM in Mycosphaerella
graminicola. With the exception of isolates collected in one area of the Distrito
Federal and Rio Grande do Norte, populations displayed 1:1 proportions of mating
type gene idiomorphs MAT1-1 and MAT1-2, indicating the occurrence of sexual
reproduction as an evident process in M. musicola populations. Greatest genetic
diversity occurred among individuals within populations, which may be the result of
sexual recombination. A considerable number of migrants between populations from
different states were observed. These results indicate that anthropic action such as
transport of contaminated germplasm may be contributing to gene flow and the
genetic homogenization of populations, when considering that ascospore dispersal is
uncommon over large distances. The high clonal fraction in populations suggests that
asexual reproduction plays an important role in the genetic structure, supporting
documented evidence of conidial dispersal within banana blocks. Five M. musicola
isolates from Distrito Federal exhibited low sensibility to tebuconazole, triadimenol
and cyproconazole fungicides, with one point mutation in an amino acid at codon 461
observed across all these isolates. For LysM effector characterization, the
pathogenicity of M. graminicola strain B3 expressing GFP (green fluorescent protein)
was evaluated on 18 wheat genotypes, shown that GFP had no influence on pathogen
colonization. Colonization of the B3-GFP in wheat leaves was followed via confocal
laser scanning microscopy (CLSM). The results confirmed that GFP is expressed
during the whole infection cycle of the pathogen on susceptible wheat leaves.
Agrobacterium tumefaciens was used to mediate transformation to generate knockout
constructs for 3LysM in B3-GFP. The 3LysM homologous DNA was not integrated
in the target genomic site, such that continued experiments are required to develop the
desired strain for further analysis of the influence of LysM on M. graminicola
pathogenesis. The results contribute to our understanding of pathogen epidemiology
and contribute to development of enhanced disease control measures.
Fully revised, updated and offered in a new two-volume format, The Yeasts: A Taxonomic Study, 5th Edition remains the most comprehensive presentation of yeast taxonomy and systematics available. Nearly 1500 species of ascomycete and basidiomycete yeasts are included, each description offering not only standard morphological and physiological characters, but also information on systematics, habitat, ecology, agricultural and biotechnological applications and clinical importance. Extensive introductory chapters discuss clinical aspects of yeasts, their role in biotechnology, food and beverage spoilage, agriculture and ecology, while other chapters include methodology for isolation of species from various habitats, phenotypic characterization, chemotaxonomy, gene sequence analysis and phylogenetics, including whole genome analysis. Additionally, easy-to-understand trees illustrate the phylogenetic placement of each species in its assigned genus as they have been determined from gene sequence analysis. This essential work, prepared by the leading experts in the field, is the most definitive treatment of taxonomy and systematics of yeasts on the market, and a necessary reference for any bookshelf or workbench. High quality photomicrographs and line drawings enhance world-class content Detailed trees clearly illustrate phylogenetic placement of each species in its assigned genus Discussion of clinical aspects of yeasts, including their role in biotechnology, agriculture and ecology help contextualize content for a wide range of researchers.
The phylum Ascomycota has been resolved into three major phylogenetic lineages: the subphyla Saccharomycotina (e.g., Saccharomyces, Pichia, Candida), Taphrinomycotina (e.g., Protomyces, Taphrina, Pneumocystis), and Pezizomycotina (e.g., Aspergillus, Neurospora, Peziza). We discuss the ecology, physiology, molecular biology, biotechnology, phylogeny, and systematics of Saccharomycotina and Taphrinomycotina, which represent the yeasts and yeastlike fungi of Ascomycota. Major changes in all aspects of our knowledge of these two subphyla have resulted from molecular studies, and the focus of the chapter is on these changes and their impact on present and future applications of the yeasts.
The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1– D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β-tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5 – 6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra-and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.
This review discusses recognition of yeast species from gene sequence comparisons, which have been responsible for doubling the number of known yeasts over the past decade. The resolution provided by various single gene sequences is examined for both ascomycetous and basidiomycetous species, and the greater resolution of species provided by multigene analyses is discussed. Various methods are presented for rapid species identification using gene sequences.
The diagnosis of yeast infections has been dominated by culture-based methods and this continues in the modern medical microbiology
laboratory. The germ tube test to distinguish Candida albicans and Candida dubliniensis from other yeasts remains one of the few useful morphological identification tests; otherwise, identification of yeasts depends
largely on panels of sugar assimilation assays. In recent years, chromogenic agars have become popular for mixture or full
identifications. Automated biochemical systems for yeast identification are gaining popularity in larger laboratories. Molecular
identification has become an important approach, with sequences in rRNA genes and spacers being used in DNA sequence analysis,
microarrays or peptide nucleic acid probe based fluorescent hybridisation assays. Non-culture based approaches to the diagnosis
of yeast infections have been suggested as ways to improve the sensitivity and speed of a diagnosis, though currently few
have been proved as sufficient and reliable alternatives to culture.
This review discusses DNA-based methods used for identification of yeasts. Nuclear DNA reassociation was the first quantitative
molecular method employed for recognition of yeast species and has provided a baseline for interpretation of other molecular
comparisons. Among these, gene sequencing is the most definitive method, with ribosomal RNA gene sequences providing the preponderance
of available data. Multigene analyses that include the sequences of protein encoding genes are being increasingly developed
to provide a more definitive resolution of species. A number of rapid identification methods, such as denaturing gradient
gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and flow cytometry, which are based on species-specific
gene sequences, are available for use in diagnostic laboratories.
Two antagonistic yeast strains Metschnikowia pulcherrima MACH1 and Rhodotorula sp. PW34 were tested for their efficacy against Botrytis cinerea in vitro and in vivo on apples. Metschnikowia pulcherrima strain MACH1 showed higher inhibition of B. cinerea compared to the strain PW34 in vitro on potato dextrose broth. Further, yeast strain MACH1 showed higher efficacy in reducing
grey mould on apples compared to PW34 and the untreated control. In addition, partially purified extracellular proteins from
strain MACH1 showed an inhibition to B. cinerea in vitro. The antagonistic yeast strains were tested for their efficacy to produce chitinases in different liquid media,
including apple juice, amended with or without cell wall preparations (CWP) of B. cinerea. The study showed a higher production of chitinases from M. pulcherrima strain MACH1 when compared to PW34. Interestingly, the strain MACH1 secreted higher chitinases in the presence of cell wall
fractions of B. cinerea. For this reason, the chitinase gene of strain MACH1 was amplified using PCR reactions and the nucleotide sequence data showed
high homology to chitinases of other yeast strains. The results of the current study show that M. pulcherrima strain MACH1 has the ability to secrete chitinases in different liquid media including apple juice, and the enzyme could
be involved in the post-harvest biological control of B. cinerea.
Yeasts are among the economically and scientifically most important eukaryotic microorganisms known. At present, there are
1,500 recognized species, which are distributed between the ascomycetes and the basidiomycetes, but only a small fraction
of these species have undergone extensive genetic analyses. In this chapter, we discuss application of molecular methods for
identification of species and for their classification from phylogenetic analysis of gene sequences. The resulting phylogeny
is considered in the context of comparative genomics and evolution, and provides a useful background for selection of additional
species for whole genome sequencing as well as for new biotechnological applications.
DNA sequencing has revolutionized yeast taxonomy. Although initially rDNA sequences proved to be universal and convenient for assigning phylogenetic relationships, it was eventually supplanted by multigene analysis, which provided more discriminating and robust results. This led to a new classification of the major yeast clades, which is still used as a reference today. More recently, the availability of a large number of complete genome sequences has given a new perspective on the molecular taxonomy of yeasts by providing a high number of genes to compare. It also highlighted an unexpected aspect of yeast genome evolution: the existence of interspecific hybrids outside of the industrial Saccharomyces clade. Together with the loss of heterozygosity in interspecific hybrids and a reduced sexuality leading to clonal propagation, this observation obliges us to reexamine the present concept of species. In parallel, the ongoing challenge is to find a universal molecular marker, to improve fast authentication and, if possible, phylogeny of yeasts. The future of yeast taxonomy will involve the sequencing of more genomes, thorough analysis of populations to obtain a good representation of the biodiversity and integration of these data into dedicated databases.
RESUMEN La importancia de las levaduras se conoce desde hace mucho tiempo y con el desarrollo de la industria, se utilizan no sólo para la elaboración de un gran número de productos fermentados (cerveza, vino, quesos y embutidos), sino también para la producción de antibióticos, vitaminas, enzimas, etc. Sin embargo, las levaduras tienen también un aspecto negativo, debido a su potencial como alterantes de alimentos, lo que implica pérdidas económicas importantes para las industrias. En el presente trabajo se abordan aspectos relacionados con la identificación y caracterización molecular de algunas de las especies típicamente alterantes, pertenecientes a los géneros Debaryomyces, Zygosaccharomyces, Dekkera, Pichia y Saccharomyces. El objetivo es proporcionar a las industrias sistemas nuevos de identificación rápida de levaduras así como mostrar, con ejemplos concretos, la utilidad de la aplicación de las técnicas moleculares para resolver problemas a nivel industrial. Así, se ha determinado que la comparación de secuencias de la región ribosómica 5,8S-ITS y del gen de la actina resulta el método más óptimo para identificar de forma rápida y precisa las especies del género Debaryomyces, que están implicadas en la alteración de alimentos procesados. Además, el presente trabajo muestra cómo la aplicación de técnicas moleculares es de gran utilidad para determinar las levaduras alterantes a lo largo de la cadena de producción alimentaria. En el presente trabajo, se muestran dos ejemplos de contaminación en la industria, en concreto en la producción de turrón de frutas confitadas y en la elaboración de vino. Mediante las técnicas de RFLPs del mtDNA y RAPD-PCR se identificó la especie Zygosaccharomyces bailii como la levadura responsable de la alteración de dichos turrones, siendo los jarabes usados para macerar las frutas el origen de contaminación en la cadena de producción. Además, la caracterización fisiológica de dichas cepas, permitió evidenciar el potencial alterante de estas levaduras, ya que presentan una elevada resistencia a conservantes alimentarios, adaptación a bajo pH y baja aw. Por otro lado, la utilización de medios selectivos, como el DBDM, así como la técnica de análisis de restricción de la región 5,8S-ITS, permitieron identificar las especies Dekkera bruxellensis y Pichia guilliermondii como las levaduras alterantes más peligrosas en el vino, por su gran capacidad para producir elevados niveles de 4-etilfenol, responsables de olores desagradables en el vino. Mediante las técnicas de RFLPs mtDNA con HinfI y RAPD-PCR se determinó que el origen de contaminación de D. bruxellensis se produce antes del envejecimiento en barrica, y que las levaduras de la especie P. guilliermondii están presentes en las uvas y raspones, por lo que proceden del exterior de la bodega. Finalmente, y dado que la determinación del número de levaduras resulta esencial para los programas de conservación de alimentos, se consideró de gran interés desarrollar una técnica que permitiera la identificación y simultánea cuantificación de levaduras directamente en el alimento. En el presente trabajo, se ha desarrollado un protocolo de PCR a tiempo real específico para S. cerevisiae utilizando SYBR-Green como agente fluorescente. Dicha técnica resultó ser muy sensible, permitiendo la detección de 3-5 UFC/mL en vino. El sistema de PCR a tiempo real desarrollado es también útil para cuantificar de forma precisa el número de células de S. cerevisiae presentes en el vino, permitiendo de esta manera estimar el riesgo de alteración del vino durante su almacenamiento y distribución. Además, el sistema se puede extender a otros alimentos y bebidas donde S. cerevisiae puede causar alteración. __________________________________________________________________________________________________ Yeasts have been shown to be involved in the spoilage of an extensive range of foods, causing enormous economic losses. Consequently, a rapid and accurate identification of common spoilage yeasts is essential to detect and prevent food spoilage. In the present study, we assessed the identification of the Debaryomyces species. We found that these species can be identified both quickly and correctly by direct sequence comparison of the ribosomal 5.8S-ITS region and Actin gene. Moreover, the present work have shown two examples of the usefulness of the molecular monitoring of spoilage yeasts to solve contamination problems in the industry, specifically in the production of candied fruit nougats and in winemaking. Using RFLPs of mtDNA and RAPD-PCR for strain characterization, we determine that a strain of Zygosaccharomyces bailii was the responsible of the nougats alteration. Furthermore, physiological characterization showed that this isolate displayed a particular resistance to weak-acid preservatives, extreme osmotolerance, ability to adapt to high glucose concentrations, ability to vigorously ferment glucose and growth at low pH. Therefore, this strain can be considerate as significant spoiler of sugary products. By other hand, we studied those yeasts wich are responsible of spoilage in wine. We conclude that D. bruxellensis and P. guilliermondii are capable to produce high amounts of 4- ethylphenol and, consequently, off flavors in wine. Using RFLPs of mtDNA with HinfI and RAPD-PCR we could determine that D. bruxellensis strains are present in the wine before the aging in the barriques, so the contamination is not originating from the barrique wood. Moreover, the origin of P.guilliermondii is the vineyard, and the insects are vectors for its propagation within the winery. Finally, in order to provide winemakers with a rapid method to detect and prevent wine spoilage caused by Saccharomyces cerevisiae, we have developed a real-time PCR protocol. The method is very sensitive, and it is useful to estimate between 3.8 and 5 CFU/ml of S. cerevisiae directly in sweet and red wines respectively. Real-time PCR designed is useful to accurately quantify S. cerevisiae cells present in wine, and it could be adapted to other kinds of food where S. cerevisiae could cause spoilage.
Strains of Pichia ohmeri that form either spherical or hat-shaped ascospores comprise a well-defined assemblage, distinct in deoxyribonucleic acid base sequence and composition from other phenotypically similar yeasts. Thus, spore morphology, usually a stable systematic character, is not invariably reliable as a major taxonomic criterion among yeasts.
A new species of the genus Pichia has been recovered 38 times in the Sonoran Desert from "rot pockets" of cereoid cacti and from Drosophila species which utilize the cacti. We have named the species Pichia amethionina due to its absolute requirement for methionine or cysteine. P. amethionina is heterothallic and demonstrates an agglutination reaction when opposite mating types are mixed. Two varieties are designated based on the combination of mannitol assimilation and ecological habitat. P. amethionina var. amethionina, the type variety, was recovered from cacti in the subtribe Stenocereinae and cannot assimilate mannitol, whereas P. amethionina var. pachycereana was found in cacti of the subtribe Pachycereinae and can assimilate mannitol. Results are given which demonstrate that the assimilation of mannitol is controlled by a single genetic locus. An evaluation of the interfertility and postmating viability among the two varieties and possibly identical organisms was made. The base composition of the nuclear deoxyribonucleic acid (average of 10 strains) is 33.05 k 0.19 mol% guanine plus cytosine. The type strain of P. amethionina and of the type variety, P. amethionina var. amethionina, is UCD-FST 76-401B (=ATCC 36080 = CBS 6940). The type strain of P. amethionina var. pachycereana is UCD-FST 76-384A (=ATCC 36079 = CBS 6943).
The yeasts represent a diverse assemblage of fungi having in common a vegetative stage that is predominantly unicellular. Although from the end of the last century investigators have attempted to classify yeasts based on morphological and physiological differences, none of the various systems currently in use can be considered satisfactory, and no consensus exists among mycologists regarding the natural relationships among these organisms. We have therefore determined the deoxyribonucleic acid (DNA) base sequence relatedness among a phenotypically similar group of yeasts to establish whether the existing taxonomic criteria in fact reflect the evolutionary affinities within the group. Our results provide evidence that the methodology generally used for delimiting yeast species is inadequate to define natural yeast taxa.
Codon usage In a sample of 28 genes from the pathogenic yeast Candida albicans has been analysed using multivariate statistical analysis. A major trend among genes, correlated with gene expression level,
was identified. We have focussed on the extent and nature of divergence between C.albicans and the closely related yeast Saccharomyces cerevisiae. It was recently suggested that significant differences exist between the subsets of preferred codons In these two species
[Brown et al. (1991) Nucleic Acids Res. 19, 4293]. Overall, the genes of C.albicans are more A + T-rich, reflecting the lower genomic G + C content of that species, and presumably resulting from a different
pattern of mutatlonal bias. However, In both species highly expressed genes preferentially use the same subset of ‘optimal’
codons. A suggestion that the low frequency of NCG codons in both yeast species results from selection against the presence
of codons that are potentially highly mutable is discounted. Codon usage In C.albicans, as in other unicellular species, can be interpreted as the result of a balance between the processes of mutational bias
and translational selection. Codon usage in two related Candida species, C.maltose and C.troplcalls, is briefly discussed.
DNA sequences and other molecular data compared among organisms may contain phylogenetic signal, or they may be randomized with respect to phylogenetic history. Some method is needed to distinguish phylogenetic signal from random noise to avoid analysis of data that have been randomized with respect to the historical relationships of the taxa being compared. We analyzed 8,000 random data matrices consisting of 10-500 binary or four-state characters and 5-25 taxa to study several options for detecting signal in systematic data bases. Analysis of random data often yields a single most-parsimonious tree, especially if the number of characters examined is large and the number of taxa examined is small (both often true in molecular studies). The most-parsimonious tree inferred from random data may also be considerably shorter than the second-best alternative. The distribution of tree lengths of all tree topologies (or a random sample thereof) provides a sensitive measure of phylogenetic signal: data matrices with phylogenetic signal produce tree-length distributions that are strongly skewed to the left, whereas those composed of random noise are closer to symmetrical. In simulations of phylogeny with varying rates of mutation (up to levels that produce random variation among taxa), the skewness of tree-length distributions is closely related to the success of parsimony in finding the true phylogeny. Tables of critical values of a skewness test statistic, g1, are provided for binary and four-state characters for 10-500 characters and 5-25 taxa. These tables can be used in a rapid and efficient test for significant structure in data matrices for phylogenetic analysis.
The actin (ACT) gene from the budding yeast Kluyveromyces lactis was cloned, and the nucleotide sequence was determined. The
gene had a single intron 778 nucleotides in length which possessed the highly conserved splicing signals found in Saccharomyces
cerevisiae introns. We demonstrated splicing of heterologous ACT transcripts in both K. lactis and S. cerevisiae.
Atypical oral Candida isolates were recovered from 60 HIV-infected and three HIV-negative individuals. These organisms were germ-tube-positive and produced abundant chlamydospores which were frequently arranged in triplets or in contiguous pairs. They belonged to C. albicans serotype A and had atypical carbohydrate assimilation profiles. Fingerprinting the genomic DNA of a selection of these organisms with the C. albicans-specific probe 27A and five separate oligonucleotides, homologous to eukaryotic microsatellite repeat sequences, demonstrated that they had a very distinct genomic organization compared to C. albicans and C. stellatoidea. This was further established by random amplified polymorphic DNA (RAPD) and karyotype analysis. Comparison of 500 bp of the V3 variable region of the large ribosomal subunit genes from nine atypical isolates and the corresponding sequences determined from C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. glabrata, C. kefyr and C. krusei showed that they atypical organisms formed a homogeneous cluster (100% similarity) that was significantly different from the other Candida species analysed, but was most closely related to C. albicans and C. stellatoidea. These genetic data combined with the phenotypic characteristics of these atypical organisms strongly suggest that they constitute a novel species within the genus Candida for which the name Candida dubliniensis is proposed.
The taxonomy of Kluyveromyces has been the object of intense study since van der Walt's (1970) monograph. This is an account of the major developments and the classification to be adopted in the 4th edition of The Yeasts, a Taxonomic Study. The guiding principles that will be followed in eventual revisions of the genus are presented.
Phylogenetic relationships of species assigned to the genus Metschnikowia were estimated from the extents of divergence among partial sequences of rRNA. The data suggest that the aquatic species (Metschnikowia australis, Metschnikowia bicuspidata, Metschnikowia krissii, and Metschnikowia zobellii) and the terrestrial species (Metschnikowia hawaiiensis, Metschnikowia lunata, Metschnikowia pulcherrima, and Metschnikowia reukaufii) form two groups within the genus. M. lunata and M. hawaiiensis are well separated from other members of the genus, and M. hawaiiensis may be sufficiently divergent that it could be placed in a new genus. Species of the genus Metschnikowia are unique compared with other ascomycetous yeasts because they have a deletion in the large-subunit rRNA sequence that includes nucleotides 434 to 483.
In an effort to establish a suitable alternative to the widely used 18S rRNA system for molecular systematics of fungi, we examined the nuclear gene RPB2, encoding the second largest subunit of RNA polymerase II. Because RPB2 is a single-copy gene of large size with a modest rate of evolutionary change, it provides good phylogenetic resolution of Ascomycota. While the RPB2 and 18S rDNA phylogenies were highly congruent, the RPB2 phylogeny did result in much higher bootstrap support for all the deeper branches within the orders and for several branches between orders of the Ascomycota. There are several strongly supported phylogenetic conclusions. The Ascomycota is composed of three major lineages: Archiascomycetes, Saccharomycetales, and Euascomycetes. Within the Euascomycetes, plectomycetes, and pyrenomycetes are monophyletic groups, and the Pleosporales and Dothideales are distinct sister groups within the Loculoascomycetes. We confirm the placement of Neolecta within the Archiascomycetes, suggesting that fruiting body formation and forcible discharge of ascospores were characters gained early in the evolution of the Ascomycota. These findings show that a slowly evolving protein-coding gene such as RPB2 is useful for diagnosing phylogenetic relationships among fungi.
This chapter focuses on the role played by ribosomal RNA/DNA sequence comparisons in assessing the phylogenetic relationships between the yeasts. The first extensive use of rRNA comparisons for yeast systematics was described by Bicknell and Douglas, who measured species divergence from the extent of reassociation between tritium-labeled 25S rRNA and complementary sites on filter-bound nuclear DNA. The rRNA/rDNA comparisons on the taxonomy of ascomycetous yeasts has led to several major findings, such as, yeasts and yeastlike species are phylogenetically separate from the euascomycetes; the fission yeast genus Schizosaccharomyces is phylogenetically distant from the “budding” yeast clade and from the euascomycetes; and that various phenotypic characters such as ascospore morphology are poor indicators of phylogeny. rRNA/rDNA sequence analyses of basidiomycetous yeasts, have also demonstrated that Rhodotorula, Sporobolomyces, Cryptococcus, and Bensingtonia are polyphyletic, which further confirmed that commonly used phenotypic characters are insufficient for defining anamorphic genera.
This chapter focuses on Saccharomycopsis species and its member species. This genus demonstrates abundant development of true mycelium, often with blastoconidia. Budding cells are present in this genus, which may be formed on a broad or a narrow base. Hyphal septa are usually multiperforate, but may be limited to a single central pore. The asci are usually attached to hyphae and produce 1–4, or rarely 8, ascospores. These ascospores may be hat-shaped, reniform with terminal appendages, spheroidal or ellipsoidal, and may have one or more ledges, with smooth of roughened surfaces. The member species of this genus include Saccharomycopsis capsularis, Saccharomycopsis crataegensis, Saccharomycopsis fermentans, Saccharomycopsis fibuligera, Saccharomycopsis javanensis, and Saccharomycopsis malanga. The budding cells of Saccharomycopsis capsularis, after undergoing growth on 5% malt extract agar for three days at 25° C, appear as spheroidal, ellipsoidal, or elongate, in singles or in pairs. Asci may form terminally on the tips of hyphae or they may be intercalary following conversion of a hyphal cell to an ascus. The budding cells of Saccharomycopsis crataegens, after undergoing growth on 5% malt extract agar at at 25° C for three days, appear as ellipsoidal to elongate and frequently tapered on one end. This species is heterothallic and all known strains have been isolated as haploid mating types. Conjugation begins within 12 hours following mixing of complementary mating types and ascospores may be observed as early as 24 hours after pairing.
The primary structure of the small ribosomal subunit RNA (srRNA) molecule of the type strains of the ascosporogenous yeasts Debaryomyces hansenii, Pichia anomala (synonym: Hansenula anomala), Pichia membranaefaciens, Schizosaccharomyces pombe, Zygosaccharomyces rouxii and Dekkera bruxellensis was determined. The srRNA sequences were aligned with previously published sequences from fungi, including those of 5 Candida species, and an evolutionary tree was inferred.
The srRNA results were compared with chemotaxonomic criteria, e. g. the coenzyme Q system. The heterogeneity of the genera Candida and Pichia is clearly reflected by the srRNA analysis.
Actin is the best-studied member of a family of ATPases that originated, presumably via gene duplications, in the common ancestor of all living things.The discovery of actin-related proteins (ARPs) and the resolution of the three-dimensional structure of actin has refueled interest in the phylogeny of this coding region.In comparison to vertebrates and land plants, relatively little is known, however, about actin and actin gene families in protists.In this paper, the origin of actin is reviewed and sequence analyses of this coding region are presented.Phylogenetic analyses of actins are used to probe the evolutionary relationships between actin isoforms in three lineages, the prymnesiophytes (Le.Emiliana huxleyi), the heterokonts and the green~,algae/land plants, and the relationships between these sequences with actins from diverse eukaryotes.
One hundred and thirty-one cultures of the genera Debaryomyces, Saccharomyces, Kluyveromyces, Endomycopsis, and Saccharomycopsis were examined for the Co-Q system. The genus Debaryomyces was divided into two groups; Q-6 in the intense fermentors of D. globosus, D. franciscae, and D. nilsonii, and Q-9 in the weak fermentors such as D. hansenii, D. cantarellii, and D. tamarii. The genera Saccharomyces and Kluyveromyces were characterized by the only system comprising Q-6. The genus Endomycopsis represented three types of Co-Q system; Q-7 in E. bispora, E. platypodis, and E. monospora, Q-8 in most of the species in this genus, E. capsularis, E. fibuligera, E. vini, E. burtonii, E. javanensis, and E. selenospora, and Q-9 in E. ovetensis and E. lipolytica. Saccharomycopsis synnaedendra gave the Q-8 system. Discussions are made from the taxonomic point of view, especially on the yeasts and yeast-like organisms possessing the same system of Co-Q as found in the genera Hansenula and Pichia (Q-7, Q-8, and Q-9).
The gene coding for actin from Phaffia rhodozyma was cloned and sequenced. The Phaffia actin gene contains four intervening sequences and the predicted protein consists of 375 amino acids. The structural features of the Phaffia actin introns were studied and compared with actin introns from seven fungi and yeasts with ascomycetous and basidiomycetous affinity. It was shown that the architecture of the Phaffia introns most resembles that of the basidiomycete Filobasidiella neoformans (perfect stage of Cryptococcus neoformans), whereas least resemblance occurs with the ascomycetous yeasts. Based on the intron structure, the ascomycetous yeasts can be accommodated in one group in that their splice site sequences are very similar and show less homology with the other fungi investigated, including Phaffia. It was demonstrated that the Phaffia actin introns cannot be spliced in Saccharomyces cerevisiae, which shows that the differences found in intron structure are significant. Alignment of the Phaffia actin gene with the actin sequences from the yeasts and fungi investigated showed a high level of homology both on the DNA level and on the protein level. Based on these alignments Phaffia showed highest homology with F. neoformans and both organisms were accommodated in the same cluster. In addition, the actin gene comparisons also supported the distant relationship of Phaffia with the ascomycetous yeasts. These results supported the usefulness of actin sequences for phylogenetic studies. The sequence presented here has been submitted to the EMBL data library under Accession Number X89898.
According to currently accepted theories, rapidly evolving nucleotide sites are phylogenetically less informative than more slowly evolving ones, especially for recognizing more ancient groupings. For this reason third codon positions are often regarded as less reliable than first and second positions as indicators of phylogeny. Analysis of the largest nucleotide matrix treated to date—2538 rbc L sequences covering all major lineages of green plants—shows the opposite: although rapidly evolving and highly homoplastic, third positions contain most of the phylogenetic structure in the data. Frequency of change should thus be used with caution as a criterion for weighting or selecting characters.
We have characterized the cDNA and genomic sequences that encode actin from the multicellular red alga Chondrus crispus. Southern-blot analysis indicates that the C. crispus actin gene (ChAc) is present as a single copy. Northern analysis shows that, like the GapA gene, the actin gene is well expressed in gametophytes but weakly in protoplasts. Compared to actin genes of animals, fungi, green plants and oomycetes, that of C. crispus displays a higher evolutionary rate and does not show any of the amino-acid signatures characteristic of the other lineages. As previously described for GapA, ChAc is interrupted by a single intron at the beginning of the coding region. The site of initiation of transcription was characterized by RNAse protection. The promoter region displays a CAAT box but lacks a canonical TATA motif. Other noticeable features, such as a high content of pyrimidines as well as a 14-nt motif found in both the 5-untranslated region and the intron, were observed.
The phylogenetic relationships between species of yeasts assigned to the Saccharomyces sensu stricto group, which includes Saccharomyces cerevisiae and Saccharomyces bayanus, were studied together with Saccharomyces pastorianus and Saccharomyces paradoxus. The experimental approaches used were RFLP analysis of the PCR-amplified rDNA internal transcribed spacer (ITS) and intergenic spacer, and total ITS sequence analysis. Both RFLP and sequence analyses gave fairly similar results. The gene trees generated with either of the two data sets showed the distribution of the yeasts into two major, well-separated, phylogenetic clusters called 'cerevisiae' and 'bayanus'. The 'cerevisiae' cluster included the S. cerevisiae type strain, together with most of the species (16 out of 23), whereas the 'bayanus' cluster included the remaining seven type strains. Therefore, analysis of rDNA sequences confirmed S. cerevisiae and S. bayanus as two well-defined taxa. However, S. pastorianus and S. paradoxus, the two other usually accepted taxa of the now-defined Saccharomyces sensu stricto complex, could not be clearly separated from S. bayanus and S. cerevisiae, respectively. However, in both PCR-RFLP and ITS sequence analyses, S. paradoxus had the outermost position in the 'cerevisiae' cluster. PCR-RFLP analysis of the ribosomal spacer sequences was also carried out on 26 Saccharomyces strains isolated in various wine-growing regions of France in an attempt to clarify their positions in the Saccharomyces phylogenetic tree. Compared to the diversity of the Saccharomyces type strains, less genetic diversity was detected among these yeasts and several of them exhibited identical RFLP patterns. Most of the wine yeast strains (16 out of 26) were closely related to each other and were found within the 'cerevisiae' cluster. The remaining 10 wine yeast strains branched within the 'bayanus' cluster. PCR-RFLP analysis of ribosomal spacer sequences thus appears to be a useful and appropriate method for the correct characterization of Saccharomyces yeast strains used in food processing.
For the true fungi, phylogenetic relationships inferred from 18S ribosomal DNA sequence data agree with morphology when (1) the fungi exhibit diagnostic morphological characters, (2) the sequence-based phylogenetic groups are statistically supported, and (3) the ribosomal DNA evolves at roughly the same rate in the lineages being compared. 18S ribosomal RNA gene sequence data and biochemical data provide a congruent definition of true fungi. Sequence data support the traditional fungal subdivisions Ascomycotina and Basidiomycotina. In conflict with morphology, some zygomycetes group with chytrid water molds rather than with other terrestrial fungi, possibly owing to unequal rates of nucleotide substitutions among zygomycete lineages. Within the ascomycetes, the taxonomic consequence of simple or reduced morphology has been a proliferation of mutually incongruent classification systems. Sequence data provide plausible resolution of relationships for some cases where reduced morphology has created confusion. For example, phylogenetic trees from rDNA indicate that those morphologically simple ascomycetes classified as yeasts are polyphyletic and that forcible spore discharge was lost convergently from three lineages of ascomycetes producing flask-like fruiting bodies.
Actin genic regions were isolated and characterized from the heterokont-flagellated protists, Achlya bisexualis (Oomycota) and Costaria costata (Chromophyta). Restriction enzyme and cloning experiments suggested that the genes are present in a single copy and sequence determinations revealed the existence of two introns in the C. costata actin genic region. Phylogenetic analyses of actin genic regions using distance matrix and maximum parsimony methods confirmed the close evolutionary relationship of A. bisexualis and C. costata suggested by ribosomal DNA (rDNA) sequence comparisons and reproductive cell ultrastructure. The higher fungi, green plants, and animals were seen as monophyletic groups; however, a precise order of branching for these assemblages could not be determined. Phylogentic frameworks inferred from comparisons of rRNAs were used to assess rates of evolution in actin genic regions of diverse eukaryotes. Actin genic regions had nonuniform rates of nucleotide substitution in different lineages. Comparison of rates of actin and rDNA sequence divergence indicated that actin genic regions evolve 2.0 and 5.3 times faster in higher fungi and flowering plants, respectively, than their rDNA sequences. Conversely, animal actins evolve at approximately one-fifth the rate of their rDNA sequences.
Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida.
The actin gene of the fission yeast Schizosaccharomyces pombe has been isolated by using as a hybridization probe cloned actin DNA from the budding yeast Saccharomyces cerevisiae. In contrast to most actin genes studied from diverse eukaryotic species, the S. pombe gene is not interrupted by introns. The protein sequence deduced from the nucleotide sequence of the gene shows that the
S. pombe actin is more closely related to the mammalian γ-actin than to the actin of S. cerevisiae. Three transcripts of 1240, 1650 and 1850 nucleotides having the same 5′ end but differing in the length of their 3′ untranslated
region are generated in the fission yeast. Only one messenger RNA of 1330 nucleotides is formed from the S. pombe actin gene in S. cerevisiae. Contrary to the observation made with other S. pombe genes transcribed in the budding yeast, the heterologous actin gene transcript is initiated 39 nucleotides upstream of the
initiation start site used in the homologous yeast The mRNA termination (or 3′ processing) mechanism in the two ascomycetes
also differs as the 3′ end of the S. pombe actir. gene transcript in S.cerevisiae does not coincide with either of the three 3′ ends mapped in the fission yeast.
The buoyant density of nuclear and mitochondrial deoxyribonucleic acid (DNA) from 14 species of fungi was determined by CsCl density gradient equilibrium centrifugation. The buoyant density of both types of DNA was the same for all three Mucorales analyzed. The buoyant density of mitochondrial DNA was significantly lower than that of the nuclear DNA for nine species of Ascomycetes and two species of Basidiomycetes. No simple correlation could be obtained from the comparison of the two types of DNA. Mitochondrial DNA represented a very small proportion of total DNA. Heat-denatured mitochondrial DNA renatured more readily than nuclear DNA.
Extent of divergence in partial nucleotide sequences from large and small subunit ribosomal RNAs was used to estimate the evolutionary relationship between the genera Wingea and Debaryomyces. These data showed the monotypic genus Wingea to be congeneric with Debaryomyces, and it is proposed to transfer W. robertsii to Debaryomyces.
We clarified the evolutionary position of Candida maltosa, an n-alkane-assimilating yeast, by sequencing the nucleotides of the small-subunit ribosomal RNA gene. Phylogenetic analyses showed the close evolutionary relationships of C. maltosa with C. tropicalis, C. viswanathii, C. albicans, C. parapsilosis, and C. guilliermondii, forming a sub-group within this genus.
The twenty-seven strains of the hat-shaped ascospore-forming, nitrate-assimilating species, formerly classified in the genus Hansenula, of the genus Pichia were examined for their 18S and 26S rRNA partial base sequencings. All the strains examined were separate phylogenetically from the type strain of P. membranaefaciens (type species of genus Pichia). Based on the sequence data obtained [by number of base differences (five or more) with P. anomala and base sequences on fingerprint segment] in the 18S rRNA partial base sequences, these species were divided into seven groups. Group I, including P. anomala (identical to H. anomala, type species of genus Hansenula), P. canadensis, P. muscicola, P. silvicola, P. subpelliculosa, P. americana, P. bimundalis, P. ciferrii, P. syndowiorum, P. bispora, and P. fabianii, corresponded to the genus Hansenula Sydow et Sydow. Groups II and III were comprised of P. capsulata and P. holstii, respectively. Group IV included P. angusta, P. minuta var. minuta, P. minuta var. nonfermentans, P. philodendra, P. glucozyma, and P. henricii. Groups V, VI, and VII included P. jadinii, P. petersonii, and P. dryadoides, respectively. The nitrate assimilation-negative species, P. wickerhamii was phylogenetically distant from P. membranaefaciens. The seven groupings are discussed phylogenetically and taxonomically. For Groups IV, II, and III, the three new genera were proposed as Ogataea, Kuraishia, and Nakazawaea, respectively, with the type species, O. minuta (identical to P. minuta), K. capsulata (identical to P. capsulata), and N. holstii (identical to P. holstii).
We analyzed 18S and 26S rRNA partial base sequences [positions 1451-1618 (168 bases) of 18S rRNA and positions 1611-1835 (225 bases) and 493-622 (130 bases) of 26S rRNA] of a total of three strains of Pichia jadinii and Candida utilis. The three strains had identical base sequences with the type strain of P. jadinii (IFO 0987) in the 18S rRNA partial base sequencings. In the 26S rRNA partial base sequencings, there were partial base sequences similar to each other (1-0 base difference and 87-95 percent similarities). The sequence data obtained are discussed taxonomically and phylogenetically, especially in connection with Williopsis saturnus, the type species of the genus Williopsis Zender.
A PCR assay was developed for the diagnosis of candidemia. Primers were selected to amplify a 158-bp segment from the Candida actin gene that was detected by hybridization with a 32P_Ia_ beled oligomer probe. The lower limit of detection was DNA extracted
from 10 Candida yeasts/ sample, or 25 fg of purified Candida albicans DNA. This PCR was specific for medically important Candida species. Circulating Candida DNA was detected by this PCR from plasma of mice with induced candidemia and from sera in 11 (79%) of 14 patients with blood
cultures positive for Candida species. This PCR may offer a sensitive method for diagnosis of candidemia.
The rDNAs of strains of the cactophilic Pichia species P. amethionina, P. antillensis, P. barkeri, P. cactophila, P. caribaea, P. deserticola, P. heedii, P. kluyveri, P. norvegenesis, P. opuntiae, P. pseudocactophila, P. thermotolerans and their varieties and anamorphs were mapped with 15 restriction endonucleases, and compared to P. membranaefaciens and P. salictaria as possible non-cactophilic relatives. The existence of species complexes among those taxa was confirmed. P. membranaefaciens was a plausible ancestral species, and its closest relative in the cactophilic group was P. deserticola. These two species appeared to be moderately related to P. heedii and to P. barkeri, but the latter was shown clearly to belong to the P. kluyveri complex, in spite of a 6 mol% G+C difference in their nuclear DNAs. P. cactophila and P. pseudocactophila ostensibly emerged from P. norvegensis, a facultatively cactophilic yeast. The P. amethionina, P. cactophila and P. opuntiae species complexes appeared independent from one another and from all other species studied. P. salictaria did not appear to be related to P. amethionina.
Using heterologous probing of a genomic library, we have cloned and sequenced the actin gene from the pathogenic yeast Cryptococcus neoformans. The actin gene is 1371 bp in length, and exists as a single copy, as is the case for all fungi studied to date. The locations
of the introns in the C. neoformans actin gene are unique among all other known actin genes, and the deduced coding sequence results in a 375 amino acid chain
with very high homology to other actins. A phylogenetic tree comprising 31 actin-coding sequences from a wide variety of organisms
shows that the C. neoformans actin gene is grouped on a distinct branch together with all other known fungal actin sequences. The availability of the
C. neoformans actin gene will aid future phylogenetic and molecular studies of this important human pathogen.
Analysis of the coenzyme Q system and the monosaccharide pattern of purified cell walls were used for species characterization in the genus Kluyveromyces. All the type strains of the genus possess coenzyme Q-6 and the mannose-glucose ('Saccharomyces type') cell wall sugar pattern. With the help of Random Amplified Polymorphic DNA-Polymerase Chain Reaction analysis 17 species were separated: K. aestuarii, K. africanus, K. Bacillisporus, K. blattae, K. delphensis, K. dobzhanski, K. lactis (anamorph Candida sphaerica), K. lodderae, K. marxianus (syn. K. fragilis, K. bulgaricus, K. cicerisporus, anamorphs Candida macedoniensis, C. pseudotropicalis, C. kefyr), K. phaffii, K. piceae, K. polysporus, K. sinensis, K. thermotolerans (syn. K. veronae, anamorph Candida dattila), K. waltii, K. wickerhamii, K. yarrowii (anamorph Candida tannotolerans). A strain of K. drosophilarum showed with the type strain of K. lactis only 63% similarity. The strain originally described as the type strain of K. cellobiovorus nom. nud. was excluded from the genus (Q-9), and found to be conspecific with the type strain of Candida intermedia.
Actin molecules are major cytoskeleton components of all eukaryotic cells. All conventional actins that have been identified so far are 374-376 amino acids in size and exhibit at least 70% amino acid sequence identity when compared with one another. In the yeast Saccharomyces cerevisiae, one conventional actin gene ACT1 and three so-called actin-related genes, ACT2, ACT3 and ACT5, have been identified. We report here the discovery of a new actin-related gene in this organism, which we have named ACT4. The deduced protein, Act4, of 449 amino acids, exhibits only 33.4%, 26.7%, 23.4% and 29.2% identity to Act1, Act2, Act3 and Act5, respectively. In contrast, it is 68.4% identical to the product of the Schizosaccharomyces pombe Act2 gene and has a similar level of identity to other Sch. pombe Act2 homologues. This places Act4 in the Arp3 family of actin-related proteins. ACT4 gene disruption and tetrad analysis demonstrate that this gene is essential for the vegetative growth of yeast cells. The act4 mutants exhibit heterogenous morphological phenotypes. We hypothesize that Act4 may have multiple roles in the cell cycle.
Clinically important species of Candida and related organisms were compared for extent of nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region is sufficiently variable to allow reliable separation of all known clinically significant yeast species. Of the 204 described species examined, 21 appeared to be synonyms of previously described organisms. Phylogenetic relationships among the species are presented.
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.
A PCR system that can quickly and accurately identify 14 species of human pathogenic yeasts was developed. The procedure distinguished between nine species of a closely related clade, Lodderomyces elongisporus, Candida parapsilosis, a new Candida sp., C. sojae, C. tropicalis, C. maltosa, C. viswanathii, C. albicans, and C. dubliniensis and between another five more divergent species, Pichia guilliermondii, C. glabrata, C. zeylanoides, C. haemulonii, and C. haemulonii type II. A rapid DNA extraction procedure that yields purified DNA in about 1 h is also described. The system uses uniform conditions with four primers for each reaction, two 40- to 50-mer universal primers that serve as a positive control and two 23- to 30-mer species-specific primers. Species-specific primers were derived from a 600-nucleotide variable region (D1/D2) at the 5' end of the large-subunit (26S) ribosomal DNA gene and were generally designed to use mismatches at the 3' end. Universal primers were developed from conserved nucleotide sequences in the small-subunit (18S) rRNA gene. In this system, a control 1,200- to 1,300-base DNA fragment was produced in all reactions and a smaller 114- to 336-base DNA fragment was produced if the chromosomal DNA from the target species was present. The PCR procedure is rapid and easy to interpret and may be used with mixed cultures.
Systematics of the genus Arvicanthis, the African unstriped grass rat, are somewhat controversial. Most recent taxonomic revisions list five to six species but the definition of some of these (Arvicanthis dembeensis, Arvicanthis nairobae, and Arvicanthis niloticus) is uncertain. The complete mitochondrial cytochrome b gene (1140 bp) was sequenced for 20 specimens from throughout the range of the genus to determine the intrageneric genetic structure, construct a molecular phylogeny, and evaluate classical taxonomies. Neighbor-joining and maximum parsimony analyses yielded identical phylogenetic trees that identify two major lineages: the first one (1) is composed of specimens usually referred to A. niloticus but representing several distinct species, and the other (2) is a complex including "true" A. niloticus from Egypt and northern West Africa as well as Arvicanthis abyssinicus, Arvicanthis dembeensis, and Arvicanthis somalicus. An analysis on a 357-bp fragment of the cytochrome b including published data on A. nairobae indicates that this taxon is part of clade (1). Calibration of the number of 3rd position transversion changes with the murid fossil record suggests that clades (1) and (2) diverged approximately 5 Myr ago. Arvicanthis niloticus as currently recognized is a paraphyletic association and this name should be restricted to the Egyptian and northern West African sample. We also suggest referring to A. dembeensis as A. niloticus, as our cytochrome b data do not support its recognition as a distinct species. Clade (1) is subdivided in three lineages, geographically corresponding to southern West, Central, and East Africa. The high genetic divergence detected between the Central African lineage and the other two lineages suggests that they probably represent separate species. Clade (2) experienced rapid cladogenetic events during the late Pliocene, with the A. somalicus lineage being the first to emerge, followed by the ancestor of A. abyssinicus and A. blicki. This period was characterized by significant climatic and environmental changes, such as the extension of open habitats, which might have provided a stimulus for speciation in this savanna-dwelling genus. Confrontation of our molecular results with chromosomal data shows a high degree of congruence between the two datasets.
Rates of sequence evolution were estimated for the cytochrome b (cyt b) and NADH dehydrogenase sub-unit 2 (ND2) genes using a phylogeny of the dabbling ducks (Tribe: Anatini) and outgroups. This speciose group was densely sampled, reducing the impact of undetected homoplasy on rate comparisons. Phylogenies based on sequences of the two gene regions and various weighting schemes differed, but most of the differences involved weakly supported nodes. In addition, partition homogeneity tests show that these differences were not due to statistically significant conflict between the data sets. Cyt b and ND2 also showed similar rates and types of both nucleotide and amino acid substitutions. For both genes, substitutions between isoleucine and valine and between alanine and threonine were most common; both of these substitution types are the result of A-G transitions at first positions of codons. Rates of sequence evolution varied substantially and significantly among nucleotide positions, and even within a given codon position (first, second, or third), rates were significantly heterogeneous among sites. Within Anatini, cyt b and ND2 show similar levels of variation and homoplasy, and are equally useful for reconstructing the species level phylogeny of this group.
Approximately 500 species of ascomycetous yeasts, including members of Candida and other anamorphic genera, were analyzed for extent of divergence in the variable D1/D2 domain of large subunit (26S) ribosomal DNA. Divergence in this domain is generally sufficient to resolve individual species, resulting in the prediction that 55 currently recognized taxa are synonyms of earlier described species. Phylogenetic relationships among the ascomycetous yeasts were analyzed from D1/D2 sequence divergence. For comparison, the phylogeny of selected members of the Saccharomyces clade was determined from 18S rDNA sequences. Species relationships were highly concordant between the D1/D2 and 18S trees when branches were statistically well supported.
Saccharomycopsis Schio$ nning In The Yeasts, a Taxonomic Study
- C P Kurtzman
- M T Smith
Kurtzman, C. P. & Smith, M. T. (1998). Saccharomycopsis
Schio$ nning. In The Yeasts, a Taxonomic Study, 4th edn, pp.
374–386. Edited by C. P. Kurtzman & J. W. Fell. Amsterdam :
Elsevier.
, Phylogenetic Analysis Using Parsimony, version 3.1.1Champaign
- D L Swofford
Swofford, D. L. (1993). , Phylogenetic Analysis Using
Parsimony, version 3.1.1Champaign, IL : Illinois Natural
History Survey.
- P Sheterline
- J C Sparrow
Sheterline, P. & Sparrow, J. C. (1994). Actin. Protein Profile 1,
1–121.