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Evaluation of ribosomal RNA and actin gene sequences for the identification of ascomycetous yeasts
Abstract
Highly similar gene sequences of the 5' region of the large subunit (LSU) are commonly interpreted to predict the organism's identity. However, it was recognised that closely related taxa do not always show sufficiently diverged D1/D2 LSU sequences to differentiate between them. The effectiveness of species separation using D1/D2 LSU sequences, small subunit (SSU) sequences and actin gene sequences was determined by pair-wise comparisons. The LSU data showed coinciding similarities among and within species. The actin data resolved all investigated species. Examples strengthened the value of almost complete SSU sequences for species separation. The larger number of differences in the highly conserved actin gene, compared to the overall more variable LSU gene, is due to the tolerance of protein coding genes to synonymous nucleotide changes. In contrast, the pairing in secondary structures of the rRNA, ensuring the functionality of the molecule, relies on longer and uninterrupted sequence sections. In conclusion, D1/D2 LSU sequences are not specific enough to identify closely related taxa. The actin gene is a better marker in these cases. However, because of the availability of a large database of fungal D1/D2 LSU sequences, this gene region is currently still the preferred target for sequence-based identification.
Supplementary resource (1)
Nucleotide Sequence
September 2002
... For the identification of actinobacterial genera, different chemotaxonomic markers play a very important role [31][32][33][34][35][36][37][38][39][40][41][42] . We have chosen the fatty acids as the fastest and highly reproducible chemotaxonomic marker for the determination of the genus of potential novel isolates. ...
... A species was then defined as a group of strains generally having less than 1% nucleotide differences in the D1/D2 region 39 . Data obtained from D1/D2 sequences were found to correlate fairly well with the DNA reassociation values [38][39][40][41][42] . In a multigene sequence study, using five gene regions, 75 well-known species that belong to the Saccharomyces complex were placed into 14 phylogenetic clades, many of which were separated from their originally described taxonomic group 43 . ...
... This study was the stepping-stone of many follow-up studies in which numerous known species were reassigned to different genera and groups as well as the description of a 48 . The actin gene region was also shown to be a good marker for species delimitation of various closely related medical taxa 42 . The taxonomy of medically important yeasts was also studied using the multigene sequencing approach and revealed that the three groups described in Candida metapsilosis were actually three separate species, C. metapsilosis, C. parapsilosis and C. orthopsilosis 49 . ...
The role of taxonomy in natural product research can be estimated in different ways. As the biological active secondary metabolite is in the focus, different companies have developed their own philosophy, as Sanofi-Aventis has done. Surveying the patent literature and journals for the description of natural products, for example, the Journal of Antibiotics, it is found that most actinomycetes reported to produce biological active compounds are described only to the genus level1 or with an invalid taxonomic name. Many of them belong to novel species, as shown in our studies of members of the genus Actinoplanes during a research program for new lantibiotics2 which resulted in the validation of Actinoplanes liguriensis and A. teichnomyceticus which have been invalidly published by Parenti and co-workers. The characterisation and description of the antibiotic-producing actinomycetes at Sanofi-Aventis or its predecessors has a long history going back to the publication of three novel species of the genus Streptomyces that produce moenomycin6, an antibiotic compound which is still in fermentative production today.
... A species was then defined as a group of strains generally having less than 1% nucleotide differences in the D1/D2 region 39 . Data obtained from D1/D2 sequences were found to correlate fairly well with the DNA reassociation values [38][39][40][41][42] . In a multigene sequence study, using five gene regions, 75 well-known species that belong to the Saccharomyces complex were placed into 14 phylogenetic clades, many of which were separated from their originally described taxonomic group 43 . ...
... As the D1/D2 and ITS region were not successful in resolving closely related species in the Debaryomyces hansenii complex the actin gene was found to be a valuable gene region for species delimitation within this complex 48 . The actin gene region was also shown to be a good marker for species delimitation of various closely related medical taxa 42 . The taxonomy of medically important yeasts was also studied using the multigene sequencing approach and revealed that the three groups described in Candida metapsilosis were actually three separate species, C. metapsilosis, C. parapsilosis and C. orthopsilosis 49 . ...
The species richness and diversity existing in microorganisms are unparalleled on our planet. In recent years, the use of molecular techniques for the detection and identification of microorganisms, especially through DNA sequencing, has revolutionised the way taxonomists look at the systematics of a species, especially when applying multilocus sequence data and concepts such as the genealogical concordance phylogenetic species recognition (GCPSR) concept1. Particularly problematic topics arising in the fungi are, for example, naming pleomorphic fungi (that is, fungi for which a teleomorph, meaning sexual form, and one or more anamorphs, meaning asexual forms, are formed), polyphyletic genera (that is, the same genus being presented in several distinct phylogenetic lineages or clades), morphospecies (a species which is differentiated from other species based on its distinctive morphological characters) that in fact represent distinct phylogenetic lineages and how to deal with species that are only known by their DNA sequence(s). Here we provide a brief overview of the effects and implications that the applications of molecular techniques such as DNA sequencing are having on the taxonomy of fungi (part 1) and yeasts (part 2), with specific reference to those which can be cultured. Although fungi and yeasts both belong to the kingdom Fungi, sometimes different approaches to the taxonomy of these two ?groups? were followed in the past and, therefore, they are treated individually below. For the purpose of this overview, genera of Oomycetes are excluded.
... 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.
... Elle a largement été utilisée pour l'identification d'espèces levuriennes (James et al., 1996 ;Naumov et al., 2000 ;Cadez et al., 2003) pastorianus. La région 18S est la plus conservée et ne permet généralement pas la différenciation de certaines espèces proches (James et al., 1997 ;Naumov et al., 2000 ;Daniel et Meyer, 2003). La région 26S est historiquement la plus utilisée et pour un large nombre de microorganismes notamment ceux non cultivables ou difficilement cultivables en conditions de laboratoire. ...
... 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.
... The actin-encoding housekeeping gene ACT1 was partially sequenced if species determination was uncertain by the ribosomal gene complex sequences. This gene shows more reliable species resolution compared to ribosomal genes (Daniel and Meyer, 2003). The primers CA1, CA5R, CA21, and CA22R were used for amplification (Daniel and Meyer, 2003). ...
... This gene shows more reliable species resolution compared to ribosomal genes (Daniel and Meyer, 2003). The primers CA1, CA5R, CA21, and CA22R were used for amplification (Daniel and Meyer, 2003). The amplification conditions were: initial denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, followed by a final extension step at 72°C for 10 min. ...
The environmental yeast diversity of spontaneous cocoa bean fermentations in east Cuba was investigated. Seven fermentations, 25 equipment- and handling-related samples, and 115 environmental samples, such as flowers, leaf and cocoa pod surfaces, as well as drosophilid insects, were analysed. The basic fermentation parameters temperature and pH were recorded during five fermentations for at least six days. A total of 435 yeast isolates were identified by a combination of PCR-fingerprinting of genomic DNA with the M13 primer and sequence analysis of DNA from representative isolates, using the internal transcribed spacer region, the D1/D2 region of the large subunit rRNA gene, and an actin gene-encoding fragment, as required. Among 65 yeast species detected, _Pichia manshurica_ and _Hanseniaspora opuntiae_ were the most frequently isolated species, obtained from five and four fermentations, followed in frequency by _Pichia kudriavzevii_ from two fermentations. _Saccharomyces cerevisiae_ was isolated only occasionally. Cocoa fermentation yeast species were also present on processing equipment. The repeated isolation of a preliminarily as _Yamadazyma_ sp. classified species, a group of strains similar to _Saccharomycopsis crataegensis_ from fermentations and equipment, and the isolation of fifteen other potentially novel yeast species in low numbers provides material for further studies. Environmental samples showed higher yeast diversity compared to the fermentations, included the most frequent fermentation species, whereas the most frequently isolated environmental species were _Candida carpophila_, _Candida conglobata_, and _Candida quercitrusa_. Potential selective advantages of the most frequently isolated species were only partly explained by the physiological traits tested. For instance, tolerance to higher ethanol concentrations was more frequent in strains of _Pichia_ spp. and _S. cerevisiae_ compared to _Hanseniaspora_ spp.; the ability to also assimilate ethanol might have conferred a selective advantage to _Pichia_ spp. In contrast, high glucose tolerance was common among strains of _Hanseniaspora_ spp., _Torulaspora delbrueckii_, and _Candida tropicalis_, among which only _Hanseniaspora_ spp. were frequently isolated.
... Therefore, another gene, i.e., the act1 was selected as an alternative. In contrast to the D1/D2 (*600 bp), sequences of act1 are longer (*1 kb) and more variable [9]. ...
... 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. Until now more than 1500 act1 nucleotide sequences of various fungal species are available in the GenBank Ò on the NCBI's website; among those are there the majority of yeasts previously found in porcine GIT. ...
... These sequences can be found in the public Internet [46,89]. Additional genes and DNA fragments such as histone promoter sequences as well as actin-1, beta-tubuline, cytochrome oxidase II, RNA polymerase II, pyruvate decarboxylase, EF1-α, YCL008c, URA3, MET10, LRE1, GRC3, SED1, STE2, BUL2 genes are discussed, which can aid in the identification of relationships among yeast species [13,26,29,90,103,104,129]. These fragments are amplified using PCR and then sequenced or determined using gel electrophoresis or real-time methods. ...
... Overview of the methods for identification and differentiation used for yeast (adapted from[29,68]) spp., Claviaspora spp., Geotrichum spp., H. spp., I. spp., M. spp., P. spp., R. spp., S. cerevisiae, S. bayanus, Saccharomycopsis spp., T. spp., W. spp./orange juice[3] Bulleromyces spp., C. spp., Clavispora spp., Cryptococcus spp., Filobasidium spp., Galactomyces spp., H. spp., I. spp., P. spp., R. spp., S. spp., Sporobolomyces spp., Trichosporon spp., W. spp., Williopsis spp. ...
During the production of beverages or in terms of quality control, the question often arises as to which yeast species occur in which beverage as starter cultures or as spoilers? The species name should direct beverage microbiologists and technologists to information that is available about species' specific spoilage and fermentation characteristics. This review provides an overview regarding the yeast flora present in beverage processes in general and provides guidance on how this yeast flora can be identified at the level of genus, species or strain. Additionally, beverage-specific technological and microbiological information about specific yeast species is included. This review also provides an overview of the literature and information on the methods currently available for species identification from the point of view of beverage production, designed to aid microbiologists or technologists in coping with the challenges they face. Descriptions of the yeast flora found in beer, wine, sparkling wine and the mash used for the production of distilled beverages, as well as indigenous fermented beverages are provided below. Yeasts are grouped as either inoculated or spontaneous starter cultures and as direct or indirect spoilage organisms. Spoilage yeasts present in carbonated soft drinks are classified according to their spoilage and fermentative potential in a specific matrix. Detailed information about beverage-relevant characteristics is summarized in alphabetical order according to yeast genus and species name. Fermentation characteristics, fermentation by-products, sources of spoilage, resistance to preservatives and ethanol, osmotolerance, growth conditions and temperatures, characteristics in culture media and other beverage-specific background information are described.
... Yeast species were identified on the basis of three molecular markers: the AluI digestion profile of the amplified rDNA Non-Transcribed Spacer, NTS2 (Nguyen and Gaillardin, 1997), the sequence of the rDNA D1/D2 region (Kurtzman and Robnett, 2003), and the sequence of the actin encoding gene, ACT1 (Daniel and Meyer, 2003;Kan, 1993). All the oligonucleotide PCR primers used were obtained from Invitrogen (Invitrogen, Cergy Pontoise, France) and are listed in Supplementary material S4. ...
... Species identification was completed for 55 strains by sequencing ACT1 gene (Daniel and Meyer, 2003;Kan, 1993). PCR amplifications were run in 25 ml containing 0.2 mM of each dNTP, 0.2 mM of each primer CA14 and CA5R (Kan, 1993), 1 unit of Takara Ex Taq DNA polymerase (Takara Bio Inc., Shiga, Japan), 1Â Ex Taq Takara buffer and 25 ng of template DNA from pure cells. ...
... Similarly, 600 nM (each) NL-1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL-4 (5'-TCCGTGTTTCAAGACGG-3') (Kurtzman and Robnett, 1998) was used to amplify the 64 D1/D2 variable domain of the 26S ribosomal DNA (rDNA) gene, Rpb2_6F (5'-TGGGGKWTGGTYTGYCCTGC-3') and Rpb2_7R (5'-CCCATWGCYTGCTTMCCCAT-3') 2400 nM (each) was used to amplified the RNA polymerase II gene (Liu et al., 1999). CA14 (5'-AACTGGGATGACATGGAGAAGATCTGGC-3') and CA5R (3'-GTGAACAATGGATGGACCAGATTCGTCG-5') 600 nM (each) were used to amplified the ACT1 gene exon designed by Daniel and Meyer (2003). ...
... geotrichum (data not shown). These figures are well below the threshold to delineate species using the ACT1 coding sequence defined by Daniel and Meyer (2003). Three of these strains showed an identical sequence to that of G. sylvicola type strain CBS 9194T. ...
Geotrichum candidum is a hemiascomycetous yeast frequently found in the environment and foodstuffs. It is one of the main yeasts in cheese and it is widely used as adjunct culture in the maturation of cheese. Within ANR project ALIA Food Microbiomes in partnership with industry, we characterized the species the species G. candidum by a multigene phylogenetic study. MLST analysis allowed us to separate the studied strains into two groups. The first contains mainly environmental strains while the second contains only strains isolated from cheese. This suggests a specialization or a selection of a group of strains within industry. We developed a typing method by inter LTR profiles, which can provide a robust tool for an industrial monitoring of strains. The genome of G. candidum CLIB 918 = ATCC 204307 was sequenced. Preliminary analyses revealed evolutionary discontinuities among genes. 6802 genes where identified in which 315 genes have orthologs in filamentous fungi and not in yeast. This suggests that during evolution, G. candidum has retained a large number of genes which have been lost in other yeasts or has received some by horizontal gene transfer. The existence of this other yeasts also having a basal position in hemiascomycetous tree suggests that G. candidum and these other yeasts have an intermediate position during the evolutionary transition fungus to yeast. It is noteworthy that some of them are involved in the metabolism and may play a role in the adaptation of the yeast to the cheese environment.
... Therefore, another gene, i.e., the act1 was selected as an alternative. In contrast to the D1/D2 (*600 bp), sequences of act1 are longer (*1 kb) and more variable [9]. ...
... 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. Until now more than 1500 act1 nucleotide sequences of various fungal species are available in the GenBank Ò on the NCBI's website; among those are there the majority of yeasts previously found in porcine GIT. ...
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.
... Therefore, the study of A. pullulans can greatly benefit from detecting new variants with unique genetic traits. However, the resolution power of genomic studies is highly dependent on the number of available genomes, and frequently neither universal molecular markers nor automatic annotation methods allow detection of intraspecific variation (Daniel & Meyer, 2003;Kuhnert et al., 2021;Libkind, Peris, et al., 2020). In recent years, the overall genome relatedness indices (OGRIs) have been recognized as a useful tool to complement or trigger the proposal of novel fungal species, and several studies have established thresholds and standards to apply them (Gostinčar, 2020;Libkind, Čadež, et al., 2020). ...
Aureobasidium pullulans is a yeast‐like fungus with remarkable phenotypic plasticity widely studied for its importance for the pharmaceutical and food industries. So far, genomic studies with strains from all over the world suggest they constitute a genetically unstructured population, with no association by habitat. However, the mechanisms by which this genome supports so many phenotypic permutations are still poorly understood. Recent works have shown the importance of sequencing yeast genomes from extreme environments to increase the repertoire of phenotypic diversity of unconventional yeasts. In this study, we present the genomic draft of A. pullulans strain from a Patagonian yeast diversity hotspot, re‐evaluate its taxonomic classification based on taxogenomic approaches, and annotate its genome with high‐depth transcriptomic data. Our analysis suggests this isolate could be considered a novel variant at an early stage of the speciation process. The discovery of divergent strains in a genomically homogeneous group, such as A. pullulans, can be valuable in understanding the evolution of the species. The identification and characterization of new variants will not only allow finding unique traits of biotechnological importance, but also optimize the choice of strains whose phenotypes will be characterized, providing new elements to explore questions about plasticity and adaptation.
... At the same time, all four genetic populations can be differentiated only on the basis of the ACT1 nucleotide sequences. Using various genera of ascomycetous yeasts as an example, it was shown that in many cases the ACT1 gene is a preferred marker compared to rDNA sequences (Daniel and Meyer, 2003). Taking into account that GenBank already has a rather extensive database of yeast ACT1 gene sequences, this marker can be recommended for identification of intraspecific K. lactis populations. ...
... At the same time, all four genetic populations can be differentiated only on the basis of the ACT1 nucleotide sequences. Using various genera of ascomycetous yeasts as an example, it was shown that in many cases the ACT1 gene is a preferred marker compared to rDNA sequences (Daniel and Meyer, 2003). Taking into account that GenBank already has a rather extensive database of yeast ACT1 gene sequences, this marker can be recommended for identification of intraspecific K. lactis populations. ...
According to the modern classification of yeasts, the species Kluyveromyces lactis includes two tax-onomic varieties: cultural dairy yeast K. lactis var. lactis and nonlactose-fermenting environmental strains of K. lactis var. drosophilarum. This subdivision of the species, which is based only on phenotypic and ecological criteria, is formal and does not reflect the existing heterogeneity of K. lactis. Using various molecular methods and genetic hybridization analysis, we studied the genetic relationship of 35 K. lactis strains isolated from dairy products and environmental sources in different regions of the world. The lactose-fermenting yeasts K. lactis, including dairy strains, clinical and soil isolates, had identical molecular karyotypes, did not differ in the nucleotide sequences of a number of molecular markers, and formed fertile hybrids with 84-99% asco-spore viability. On the other hand, nonlactose-fermenting yeasts split into three genetically isolated populations: "krassilnikovii," "drosophilarum," and "phaseolosporus," which differed in molecular karyotypes, had unique SNP substitutions in the ACT1 gene, and formed semisterile hybrids: 6-34% ascospore viability.
... 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.
... This gene encodes conserved actin protein found in all eukaryotic cells (Dominguez & Holmes, 2011) and is a single copy in most fungi tested (Suwannarat et al., 2017). Moreover, the gene sequences are easy to amplify and align (Daniel & Meyer, 2003;Reeb et al., 2004), allowing the study of the species' relationships and variation. ...
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.
... 13 On the other hand, actin is a protein coding gene used to reclassify the genus Colletotrichum and contains more phylogenetic information compared to the ITS region; hence, it is an ideal marker to differentiate closely related species. 14 Moreover, the sequences of this gene are easy to amplify and align, 15,16 allowing the study of relationships and variation in the species. ...
A microfluidic analytical device based on wax-patterned Fusion 5 paper was designed and fabricated to facilitate early detection and improve control of anthracnose disease. Here, a rapid, specific, on-site, and low operational cost nucleic acid biosensor (ACT-Ct-PAD) based on the actin gene (ACT) and wax-patterned Fusion 5 paper was used to detect the PCR products of Colletotrichum truncatum (Ct), the main causal agent of chili anthracnose in Asia. The sensor was developed by using DNA conjugated gold nanoparticles (AuNPs-DNA) as a detection probe, which will hybridize to a complementary target sequence. Avidin coated mesoporous silica particles were attached to biotin-tagged DNA sequences forming capture probes, which were immobilized on the test and control zones of the device. The hybridization complex (MSP-dsDNA-AuNPs) produces an intense red color, which provides a platform for colorimetric detection. By targeting an actin gene sequence, the ACT-Ct-PAD device allows the detection of Ct DNA within 15 min. The specificity of the sensor was confirmed by the absence of a positive signal for DNA from non-target Colletotrichum species and two different fungal genera. Our wax-patterned Fusion 5 sensor provides a simple tool for the rapid nucleic acid diagnosis with a detection limit down to 17.42 femtomoles. This method has the potential to be applied for protein assay as well; hence, it has a considerable impact on on-site diagnostics.
... In our study, there was no difference between Aspergillus species as a result of cutting the Actin region with Taq I, Eco RI, Hind III, and Pst I enzymes. This result disagreed with Daniela and Meyera (2003), when they used the Actin gene in Ascomycetes yeasts and reported large number of sequence diversity. ...
Pistachio (Pistacia vera L.) is a strategic crop plant and Turkey is one of the major pistachio producing county together with Iran, Syria and the United States of America. Pistachio trees are affected by many fungal diseases. The Aspergillus spp. are one of the most important agent that produce mycotoxins and also known as human and animal pathogens. This study was conducted to determine the genomic DNA polymorphisms from Aspergillus spp. which were isolated from pistachio fruits collected from different climatic zones in Turkey. Total of 27 Aspergillus spp. isolates were used and specific primers for ITS, Actin and β-tubulin genes were subjected to RFLP analysis by using Hind III, Pst I, Taq I and Eco RI restriction enzymes. Three different groups were defined with the enzyme Taq I in the β-tubulin region. The results revealed low polymorphism and genetic diversity in Aspergillus spp. with PCR-RFLP of the ITS and actin regions.
(PDF) PCR-RFLP Analyses of Aspergillus spp. Isolated from Pistachio in Turkey. Available from: https://www.researchgate.net/publication/348843928_PCR-RFLP_Analyses_of_Aspergillus_spp_Isolated_from_Pistachio_in_Turkey [accessed Jan 28 2021].
... Partial ACT1 gene sequences were determined for representatives of the Debaryomyces hansenii group. The primers CA1, CA5R, CA21, and CA22R were used for amplification and sequencing [32]. PCR was performed using an initial denaturation at 94 • C for 5 min, 30 cycles at 94 • C for 30 s, at 60 • C for 30 s, 72 • C for 1 min, followed by a final extension at 72 • C for 10 min. ...
Pollen stored by bees undergoes a fermentation marked by the presence of lactic acid bacteria and yeasts. It results in bee bread. Past studies have singled out Starmerella (Candida) magnoliae as the most common yeast species in honey bee-stored bee bread. Starmerella species are ecological specialists with potential biotechnological value. The rarity of recent studies on yeasts in honey bees prompted us to generate new information on yeast diversity during the conversion of bee-collected pollen to bee bread. Bees and stored pollen from two apiaries in Belgium were sampled, a yeast isolation protocol was developed, yeast isolates were grouped according to their macro-and micromorphology, and representative isolates were identified using DNA sequences. Most of the 252 identified isolates belonged to the genera Starmerella, Metschnikowia, and Zygosaccharomyces. The high abundance of yeasts in fresh bee bread decreased rapidly with the storage duration. Starmerella species dominated fresh bee bread, while mostly Zygosaccharomyces members were isolated from aged bee bread. Starmerella (Candida) apis, a rarely isolated species, was the most frequent and abundant species in fresh bee bread. Yeasts from the bee's honey stomach and from pollen pellets obtained from bees hind legs were dominated by Metschnikowia species. The distinctive communities from pollen pellets over fresh bee bread to aged bee bread indicate a non-random distribution of these yeasts.
... As this level of genetic divergence lies at the margin of what can be regarded as genetic isolation among populations (Kurtzman and Robnett 1998;Vu et al. 2016), we also sequenced the complete large subunit of the ribosomal RNA gene (LSU) and the gene for translation elongation factor 1 (TEF1) of the strains, along with those of six strains of the closest relative, S. lactis-condensi (Table 1). The TEF1 gene was used for its ability to better resolve at the strain level and between closely related species compared to the ribosomal gene datasets due to more relaxed functional constraints at the third codon positions (Daniel 2003;Č adež et al. 2006). We assembled these datasets together with the ITS sequences and constructed a minimum spanning tree (MST) that combined all strains in a single, most-parsimonious network (Bandelt et al. 1999) in which the distances between the strains reflect genetic divergence. ...
A novel yeast species of Starmerella vitis f.a. sp. nov. is proposed to accommodate five strains isolated from flowers, grapes and an insect in the Azores, Canada, Hungary, Palau and Taiwan. As the strains were genetically distinct, we used parsimony network analysis based on ITS-D1/D2 sequences to delineate the species in a statistically objective manner. According to sequence comparisons and phylogenetic analysis, the novel species is most closely related to Starmerella lactis-condensi. The two species cannot be distinguished by conventional physiological tests. The type strain of Starmerella vitis f.a., sp. nov. is CBS 16418T; Mycobank number MB 835251.
... www.nature.com/scientificreports/ cerevisiae, the latter phylogenetically related to Candida 30,31 . Cross-detection of S. cerevisiae by the pan-Candida probe is not of medical concern as invasive infections caused by S. cerevisiae are rare compared to those caused by Candida and standard antifungal therapy with fluconazole is efficient in controlling such infections 32,33 . ...
Invasive candidiasis is one of the most common nosocomial fungal infections worldwide. Delayed implementation of effective antifungal treatment caused by inefficient Candida diagnosis contributes to its notoriously high mortality rates. The availability of better Candida diagnostic tools would positively impact patient outcomes. Here, we report on the development of a single-tube, dual channel pentaplex molecular diagnostic assay based on Multiplex Probe Amplification (MPA) technology. It allows simultaneous identification of C. auris, C. glabrata and C. krusei, at species-level as well as of six additional albicans and non-albicans pathogenic Candida at genus level. The assay overcomes the one-channel one-biomarker limitation of qPCR-based assays. Assay specificities are conferred by unique biomarker probe pairs with characteristic melting temperatures; post-amplification melting curve analysis allows simple identification of the infectious agent. Alerting for the presence of C. auris, the well-characterised multi-drug resistant outbreak strain, will facilitate informed therapy decisions and aid antifungal stewardship. The MPA-Candida assay can also be coupled to a pan-Fungal assay when differentiation between fungal and bacterial infections might be desirable. Its multiplexing capacity, detection range, specificity and sensitivity suggest the potential use of this novel MPA-Candida assay in clinical diagnosis and in the control and management of hospital outbreaks.
... 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. Daniel & Meyer (2003) compared LSU and actin regions in regard to their ability to resolve closely related yeast species. They indicated infeasibility of LSU (D1/D2) and practicality of actin sequences to identify closely related taxa. ...
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.
... All the 519 yeast isolates were identified on the basis of their pattern of AluI digestion of the amplified rDNA Non-Transcribed Spacer 2 (NTS2) (Nguyen and Gaillardin, 1997). Species identification was confirmed by sequencing the D1/D2 rDNA region (Kurtzman and Robnett, 2003) for 60 strains representatives of the different AluI profiles and by sequencing the actin coding gene, ACT1 for 46 strains (Daniel and Meyer, 2003). All the oligonucleotide PCR primers used were obtained from Invitrogen (Invitrogen, Cergy Pontoise, France) and PCR products were Sanger sequenced on both strands by Eurofins (Supplementary Table S1). ...
Microbial communities are essential for the maintenance and functioning of ecosystems, including fermented food ecosystems. The analysis of food microbial communities is mainly focused on lactic acid bacteria (LAB), while yeast diversity is less understood. Here, we describe the fungal diversity of a typical food fermented product, sourdough bread. The species diversity of 14 sourdoughs collected from bakeries located all over France was analyzed. Bakeries were chosen to represent diverse bakery practices and included bakers and farmer-bakers. Both non-culture-based (pyrosequencing of Internal Transcribed Spacer 1 amplicons) and culture-based methods were used. While both identification methods were in agreement regarding the dominant yeast species of each sourdough, the ITS1 metabarcoding analysis identified an increased number of fungal species in sourdough communities. Two third of the identified sequences obtained from sourdoughs were Saccharomycetales, mostly in the Kazachstania genus. No Saccharomycetales species was shared by all the sourdoughs, whereas five other fungal species, mainly known plant pathogens, were found in all sourdoughs. Interestingly, Saccharomyces cerevisiae, known as “baker’s yeast,” was identified as the dominant species in only one sourdough. By contrast, five Kazachstania species were identified as the dominant sourdough species, including one recently described Kazachstania species, Kazachstania saulgeensis and an undescribed Kazachstania sp. Sourdoughs from farmer-bakers harbored Kazachstania bulderi, Kazachstania unispora and two newly described Kazachstania species, while sourdough from bakers mostly carried Kazachstania humilis as the dominant species. Such yeast diversity has not been found in sourdoughs before, highlighting the need to maintain different traditional food practices to conserve microbial diversity.
... Similar to the C. guilliermondii species complex, differentiation of C. famata species complex was clearer by using ACT1 sequencing than by ITS or 26S sequencing, since the first one offers a better separation of species showing a higher gap between intraspecies and interspecies variations (Table 3).This is in agreement with the previous study of others. 16,31 MALDI-TOF MS identification is a powerful technique that has demonstrated to be successful in yeast identification, including the discrimination of closely related species. 21,32,33 Other studies have already evaluated the performance of MALDI-TOF MS by using the VITEK MS and MALDI Biotyper platform 12,13,28 in the identification of C. guilliermondii, C. fermentati, C. famata, and D. fabryi. ...
The aim of this work was to reidentify strains previously identified as Candida guilliermondii and Candida famata by conventional phenotypic methods conserved in a culture collection from Argentina using ribosomal DNA sequencing, ACT1 gene sequencing, and matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF MS). In addition, we performed antifungal susceptibility tests of eight antifungal drugs commonly used in clinical treatment. We identified 68 isolates belonging to the Candida guilliermondii species complex (59 C. guilliermondii, 8 C. fermentati, and 1 Candida carpophila), 16 isolates belonging to the Candida famata species complex (8 C. famata, 6 Debaryomyces nepalensis, 1 Debaryomyces fabryi, and 1 Debaryomyces tyrocola). Although sequencing of ITS region was able to identify C. guilliermondii and D. nepalensis isolates, sequencing of ACT1 gene seems to be the most appropriate technique for differentiation between C. fermentati and C. carpophila and between members of the C. famata species complex others than D. nepalensis. MALDI-TOF MS has a good potential for the identification of these yeasts, particularly in clinical laboratories since is a rapid and easy to perform technique. Here, we report the first isolation of D. tyrocola from a human patient and the first isolation of D. nepalensis from lungs and blood of human patients. Finally, correct identification and determination of antifungal susceptibility of those closely related species could be a useful tool for clinicians to choose the most effective antifungal treatment.
... The intergenic spacer (IGS) is another rRNA gene that has been used for identification of some closely related species (Diaz et al. 2000;Sugita et al. 2002;Diaz and Fell 2005;Fell et al. 2007;Kurtzman et al. 2011b). When the sequence of the D1/D2 region does not provide a clear separation of closely related species, identification has been performed by sequence analyses of protein-coding genes (Kurtzman et al. 2011a, b), such as actin (ACT; Daniel and Meyer 2003), translation elongation factor-1α (TEF1α; Kurtzman et al. 2008) or cytochrome oxidase II (COX II; Belloch et al. 2000;Kurtzman and Robnett 2003). In some case, analyses of multiple gene sequences are required (Kurtzman and Robnett 2003;Kurtzman et al. 2008;Sugita et al. 2001;Yurkov et al. 2015a). ...
Phylloplane, which refers to the surfaces of aboveground parts of plants, includes mainly leaves and has been recognized as an important habitat for microorganisms. The growth of phylloplane microorganisms is dependent on organic and inorganic substances on the leaf surfaces that are either secreted by the plant or originate from external sources. Several factors that structure the microbial communities in phylloplane such as leaf age, plant species, growing conditions, environmental factors, geography and competing microorganisms have been reported. To date, the diversity of microorganisms in the phylloplane has been studied intensively. However, only a small number of articles have focused on yeasts. In this chapter, we review the methodologies, both culture-dependent and culture-independent methods, which have been utilized for assessment of leaf-associated yeasts. The diversity of phylloplane yeasts in the tropical regions assessed by those techniques is discussed. In the course of investigations, phylloplane yeasts in tropical countries have been shown to be highly diverse. Many novel yeast species have been discovered by both culture-dependent and culture-independent methods. These studies suggest that extensive studies of phylloplane yeasts from tropical regions will lead to recovering a so far underappreciated diversity.
... The gene has been used to study evolutionary relationships. In addition, it is easy to amplify and align the sequences (Daniel and Meyer 2003;Reeb et al. 2004). This is good for studying the relationships and variation in the species. ...
Colletotrichum spp. are causal agents of anthracnose disease in chili fruits and other tropical crops. The disease is increasing in chili fruits in Thailand and significantly reduces fruit quality and fruit production. Forty-eight isolates of Colletotrichum spp. associated with chili anthracnose were collected from different areas of Thailand during 2010–2015. Based on morphological characteristic identification, 10 isolates were shown to belong to the C. gloeosporioides species complex, 24 isolates belong to the C. acutatum species complex and 14 isolates to C. capsici. For molecular identification, two primer sets, ITS1/ITS4 and ACT528/ACT738, were used for amplification of the internal transcribed spacer of rRNA gene (ITS1–5.8S–ITS2) and partial region actin gene (ACT), respectively. The phylogenetic analysis of individual and combined ITS region and actin nucleotide sequences identified the collected isolates into 4 species: C. gloeosporioides, C. siamense, C. acutatum and C. capsici. The pathogenicity test demonstrated that all four species were pathogenic on intact unwounded and healthy fruits. These results indicated that C. capsici, C. acutatum, C. gloeosporioides and C. siamense were the causal agents of chili anthracnose disease.
... Protein coding housekeeping nuclear genes like largest subunit of RNA polymerase II (Stockinger et al. 2014 ) β-tubulin (Mostert et al. 2006 ;Aroca et al. 2008 ) translation elongation factor 1 alpha (TEF1α) (Voigt and Wostemeyer 2001 ;Geiser et al. 2004 ;Knutsen et al. 2004 ;Kristensen et al. 2005 ). calmodulin (Mule et al. 2004) avirulence genes (Lievens et al. 2009 ) actin (Voigt and Wostemeyer 2001 ;Daniel and Meyer 2003 ) mating type genes (Wallace and Covert 2000 ;Foster et al. 2002 ) and mitochondrial genes like multicopy cox I and cox II and their intergenic region (Martin and Tooley 2003 ;Seifert et al. 2007 ;Nguyen and Seifert 2008 ) are widely tested. These housekeeping gene sequence typing based markers are superior over that of ribosomal genes to differentiate inter-and intra-species and to resolve relationships at various taxonomic levels (Schoch et al. 2009 ). ...
Fungi are the second most diverse group of organism after insects on the earth. While, there is huge morphological and biological diversity between different fungal taxon, nevertheless it is difficult to distinguish them within a single lowest rank of taxon or below that, due to their high morphological and biological similarity. The recent advancement in molecular biology and genomics helps in generation of vast amount of genetic data using various molecular markers that could be highly correlated with various morphological and physiological divergences within same group of fungi. The physiological variability even within same ecological niche raises the importance of genic markers for exploring the various aspects of diverse fungal ecology. The molecular markers developed for fungal genera in recent past were based on the conserved gene sequences but they were inadequate to distinguish between closely related strains or isolates. Of late, molecular marker based on random primers (Restriction Fragment Length Polymorphism and Random Amplified Polymorphic DNA) have been implemented for the differentiation of closely related strains, but due to their complicated methodology and low reproducibility, these hybridization and amplification based markers ran out of the race. This is invariably significant to differentiate between closely related fungal isolates or strains based on their virulence or aggressiveness or other physiological traits. Therefore, development of genic markers for a specific fungal species is requisite for establishing the correct taxonomic classification. The huge repository of genome sequences made it possible to develop genic markers for fungi. These genic markers are located either in regulatory or in functional site and are directly correlated with the trait of interest. Accordingly, genic molecular markers can be employed for the development of fungal diagnostics and deciphering their functional diversity in order to achieve marker-assisted selection for trait improvement, comparative genome mapping, and to explore fungal adaptation to different environments, etc. Hence, in the era of functional genomics, it will stand as one of the potential tool for the characterization of fungal population.
... Yeast identification was based only conventional identification methods previously. But now advanced yeast identification techniques, fatty acids profiles, mean molar percentage guanine plus cytosine (mol% G+C) and the application of two universal and two speciesspecific primers derived from the D1/D2 region of the 26S rDNA is used for identification (Herzberg et al., 2002; Daniel and Meyer, 2003). For example, the common yeast Saccharomyces cerevisiae (Figure 3. ...
... 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). ...
... Molecular techniques are increasingly adopted for identification of fungi and yeasts over conventional techniques due to the fact that they are rapid, easy and more precise (Arroyo- Lopez et al., 2006). The 26S rDNA region has approximately 600 bases in size reported to be a strong tool which allows accurate identification (Daniel and Meyer, 2003) and has the advantage that yeast species can be identified from sequence divergence in this region (Kurtzman and Robnett 1998). Masoud et al. (2004) successfully identified yeasts involved in fermentation of coffee arabica using D1/D2 LSU of 26S rDNA. ...
Coffee pulp waste (CPW), an abundant agro-waste available in Tanzania was studied as a potential substrate for bioethanol production. Selected yeast Pichia anomala M4 previously isolated from coffee environment was tested against three fermentation parameters namely pH, temperature and soy flour supplementation. Maximum ethanol of 4.7% (v/v) and 4.07% was produced at an optimum pH 4.5 and temperature of 30 0 C respectively. There was a significant increase (ANOVA, P˂0.05) in ethanol production on addition of soy flour as a supplement with a maximum yield of 6.04% (v/v) ethanol at a soy flour concentration of 2.0% g/l. A slight increase on ethanol production (6.3% v/v) was recorded when all the three parameters were put together. Appreciable amount of sugar content (8.2 g/l) found in CWP and significant levels of ethanol produced by yeast from the agricultural waste, calls for more research on how best to utilize this untapped bioresource.
... Molecular techniques are increasingly adopted for identification of fungi and yeasts over conventional techniques due to the fact that they are rapid, easy and more precise (Arroyo- Lopez et al., 2006). The 26S rDNA region has approximately 600 bases in size reported to be a strong tool which allows accurate identification (Daniel and Meyer, 2003) and has the advantage that yeast species can be identified from sequence divergence in this region (Kurtzman and Robnett 1998). Masoud et al. (2004) successfully identified yeasts involved in fermentation of coffee arabica using D1/D2 LSU of 26S rDNA. ...
Coffee processing environment harbours different microbiota of fungi and bacteria, some of which are of great economic value. This study isolated, screened and identified indigenous yeast associated with ethanol production potential in coffee processing environments of Mbinga in Ruvuma region and Hai in Kilimanjaro region using standard procedures. Yeast identification was done by amplification and sequencing D1/D2 domain of the gene 26S rDNA. A total of 21 yeast isolates were obtained from both sites and only 8 of them were able to ferment glucose. All 9 isolates fermented coffee pulp waste to ethanol at varying levels. Highest ethanol production was observed by isolate M4, which produced 3% (v/v) ethanol, followed by PDA4MB (2.8% v/v), PDA3 (2.6% v/v) and the least was by M2 which produced only 1.1% (v/v).Three isolates showed good attribute to ethanol production (2.76 to 3.56% v/v) even after an addition of higher sugar concentrations to coffee pulp waste. The basic local alignment search tool (BLAST) and phylogenetic analysis revealed the identity of the yeasts as Pichia kudriavzevii, Issatchenkia orientalis, Clavispora lusitaniae, Pichia guilliermondii, and Pichia anomala, all of them known to be associated with ethanol fermentation. Their attributes towards ethanol fermentation are taken to be potential for further investigation for bioethanol production.
... These sequences also have the advantage of being easily available via the Internet. Specifically, they correspond to the ITS (James et al., 1996) and D1/D2 (Kurztman & Robnett, 1998) rDNA regions, the mitochondrial gene COX2 Kurztman & Robnett, 2003), and the nuclear gene actin (Daniel & Meyer, 2003). These have been applied in real-time PCR systems developed for the detection and quantification of total yeasts in wine (Hierro et al., 2006a) and for the monitoring of populations of Saccharomyces species and Hanseniaspora species during alcoholic fermentation (Hierro et al., 2007). ...
This chapter discusses molecular identification and characterization of wine yeasts. Yeast species can be identified by comparison of nucleotide sequences from rDNA regions. The two most commonly used regions are the D1 and D2 regions at the 5' end of the genes encoding the 26S and 18S ribosomal subunits. The availability of sequences in DNA databases, particularly for the D1/D2 region of the 26S gene, makes this technique particularly useful for assigning unknown yeast to a specific species when the homology of the sequences is greater than 99%. Molecular characterization of commercial yeast strains is necessary for two reasons. First, it is needed for quality-control purposes to confirm that the obtained yeast is the one that was originally selected and not a contaminant, and, second, to detect fraud. Wine is a highly appropriate culture medium for the growth of a large number of microorganisms, in part due to its richness in organic acids, amino acids, residual sugars, growth factors, and mineral salts. Techniques for the detection of spoilage yeasts are essential. These techniques must be very sensitive and allow quantification of the number of microorganisms present. They must also be rapid to allow the application of corrective measures on the production line prior to release of the products onto the market.
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.
Cocoa fermentation is the key and most relevant process in the synthesis of aroma and flavor precursor molecules in dry beans or raw material for producing chocolate. Because this process occurs in an uncontrolled manner, the chemical and sensory quality of beans can vary and be negatively affected. One of the strategies for the standardization and improvement of the sensory quality of chocolate is the introduction of microbial starter cultures. Among these, yeasts involved in fermentation have been studied because of their pectinolytic and metabolic potential in the production of volatile compounds. This study was aimed at isolating and characterizing, both sensory and chemically, yeasts involved in cocoa fermentation that could be used as starter cultures from two agro-ecological regions for the cultivation of cocoa in Colombia. The microbiological analyses identified 22 species represented mostly by Saccharomyces cerevisiae, Wickerhamomyces anomalus and Pichia sp. The preliminary sensory analysis of eight of these species showed that Hanseniaspora thailandica and Pichia kluyveri presented sensory profiles characterized by high intensity levels of fruity notes, which could be ascribed to the production of ethyl acetate, isoamyl acetate, and 2-phenylethyl acetate.
Six polymorphic yeast strains with strong antifungal activities isolated from dicot plants in an alkaline-lake desert region were subjected to taxonomic examination. The phylogenetic trees reconstructed by using neighbour-joining, maximum-likelihood and Bayesian methods from concatenated D1/D2 and ITS-5.8S-ITS2 sequences revealed phylogenetic affinity to Ustilaginaceae, but the large phylogenetic distance separating the isolates from the most closely related groups of species indicates that they represent a separate species. The sequences of the genes coding for the LSU rDNA, act1, rpb2 and a protein of unknown function corroborate this position. The isolates can easily be distinguished from their closest relatives by physiological tests (utilisation of carbon and nitrogen sources). Based on these results, a new species, Mycosarcoma aegyptiacum sp. nov., is proposed to accommodate the isolates. All isolates are polymorphic. Transitions between budding-yeast and pseudohyphal morphologies which take place during colony formation result in morphologically different colony sectors and invasive growth into the medium. Neither sexual mating nor sporulation was observed in cultures growing on laboratory media.
Yeast have a fundamental role in winemaking. They carry out alcoholic fermentation and they contribute to the quality of the wine, although they can also cause spoilage during grape must transformation and in the final product. To detect and identify wine yeast and control their activities, a plethora of different methods can be utilized. As reported in the present chapter, these methods have different degrees of complexity and vary in terms of cost, rapidity and sensitivity. Those based on yeast isolation, namely culture-dependent methods, are widely utilized to define the composition of the microflora associated with wine-related environments and for yeast identification at the strain level, besides providing a means for ex-situ preservation of wine yeast biodiversity. Culture-independent methods bypass microorganisms cultivation, thus avoiding any bias introduced by their isolation and uncovering cell populations undetected by culture-dependent methods. These methods can be utilized to evaluate the impact of all of the components of the wine microbiota on the quality of the final product, to implement a quality control system based on real-time detection and quantification of specific targets, such as the inoculated starter (s) or the spoilage yeast, or to provide further insights into the composition of the microbial communities involved in the grape must transformation.
Fungi, in particular some of the Penicillium and Aspergillus species, are the main cause of dairy products contamination, they threaten the general health of community and food safety due to the production of mycotoxins. The purpose of this study was to isolate and identify saprophytic fungi contaminating various dairy products in Shiraz in comparison to the National Iranian Standard (2406).
In this study, 85 samples (40 cheeses, and 45 milk samples) were collected from Shiraz city of Iran and analyzed for the presence of mold and yeast. After morphological investigation, molecular tests was done by PCR method. The results were compared to the National Iranian Standard and were analyzed by SPSS version 20 and chi-square statistical analysis with about 95% accuracy and significant level (p-value<0.00001). Among the total of 85 samples, 15 (17.6%) samples showed contamination more than allowed limit. After molecular tests and sequencing of ITS region and D1/D2; Saccharomyces cerevisiae, Saccharomyces recorded in NBCI center. According to the results of this study, it is necessary to take serious measures to control the contamination of fungal dairy products without the use of preservatives.
Removal of petroleum benzene, toluene, and xylene compounds from the environment is necessary to ensure quality life. In this research, 41 yeasts were isolated from oily soils. Among them, nine yeasts named KKUs (A5, A6, A12, A20, A23, A24, A26, A29, and A38) were selected based on their use of benzene, toluene, and xylene as a sole carbon and energy source. Based on their growth rates, all selected yeasts displayed a high efficiency for toluene degradation, but had no ability to degrade benzene and a low ability to degrade xylene, except A29 and A38, which could not degrade xylene. HPLC analysis for toluene removal indicated that A6, A12, A20, A23, A24, and A26 almost completely removed the toluene compound after 3 days of incubation (92.74, 94.61, 95.05, 91.74, 91.85, and 97.29%, respectively). In addition, strains A29 and A38 showed moderate degradation (88.29 and 85.30%, respectively), while the ability of A5 was low (39.00%). The isolates were identified based on amplifying and sequencing the D1/D2 domain of the 26S rRNA gene. Alignments and comparisons of the 26S rRNA gene sequences of the isolates with those available in GenBank, plus phylogenetic analysis, proved isolates as Rhodotorula lactose KKU-A5, Rhodotorula nymphaeae KKU-A6, Rhodotorula graminis KKU-A12, Rhodotorula minuta KKU-A20, Exophiala dermatitidis KKU-A23, Candida davisiana KKU-A24, Rhodotorula slooffiae KKU-A26, Rhodotorula mucilaginosa KKU-A29, and Rhodosporidium diobovatum KKU-A38. Random amplified polymorphic DNA-PCR fingerprinting was accomplished within seven toluene-degrading red yeasts (A5, A6, A12, A20, A26, A29, and A38). The results indicated no correlation between the random amplified polymorphic DNA profile and the geographic origin of the isolates.
Protease enzymes (proteases), particularly those produced by microorganisms, play very important roles in industry, due to their diverse applications. Considering the richness of microbial diversity in nature, a good chance always exists that proteases more suitable, with better properties for commercial application, may be discovered while screening novel microorganisms from local environments. In this study, 94 yeasts were isolated from different natural sources collected from the Abha region, Kingdom of Saudi Arabia, to determine extracellular protease production and activity. Among them, 23 isolates (24.46%) showed protease activity using a casein hydrolysis test. Of these, five isolates (21.74%) were selected and identified as the best protease producers by exhibiting the largest clearance zones around colonies. A 26S rRNA gene D1/D2 domain sequence alignment, comparison, and phylogenetic analysis of our study yeasts to published D1/D2 domain rRNA gene sequences from GenBank, identifies the isolates as Rhodotorula mucilaginosa KKU-M12c, Cryptococcus albidus KKU-M13c, Pichia membranifaciens KKU-M18c, Hanseniaspora uvarum KKU-M19c, and Candida californica KKU-M20c. The influence of varying pH (4.0–9.0) on the yield and activity of the proteases was investigated using 0.5% (w/v) casein as a substrate, to detect optimum pH values for yeast extracellular protease production. Enzyme activity was measured using qualitative and quantitative assays. Results show all of the study yeasts secreting protease enzyme at all tested pH levels, with the exception of pH 9.0. This indicates that none of the five yeasts are alkaline protease producers. Maximum protease activity (187 U/mL) was observed in strain H. uvarum KKU-M19c at pH 6.0 (only), indicating that strain KKU-M19c only produces neutral protease. The other four yeast isolates, R. mucilaginosa KKU-M12c, C. albidus KKU-M13c, P. membranifaciens KKU-M18c, and C. californica KKU-M20c, produced both acidic (at pH 4.0) and neutral (at pH 6.0 and 7.0) proteases. Strain C. californica KKU-M20c was found to be the best acidic and neutral protease producer (138 U/mL at pH 4.0, and 185 U/mL at pH 7.0). This is the first report of the discovery and isolation of local, powerful yeasts producing acidic and neutral protease enzymes from the Abha region, Kingdom of Saudi Arabia.
Postharvest diseases of fruits and vegetables result in critical losses of production in worldwide. The losses often are caused by fungi and nowadays, most fungal pathogens are controlled by several strategies such as the use of fungicides. However, most of the fungicides are chemical-based compounds and are dangerous to human health and the nature. Therefore, the discovery of healthy and reliable strategies is crucial to control of fungal pathogens. In the paper, it was aimed to evaluate and characterize yeast isolates previously isolated from dairy products for the production of killer toxin. A total of 18 yeasts have been found to produce antagonistic behavior against susceptible fungal species. All of the yeasts expressing killer character were characterized by using several molecular techniques, and isolates TEM8 and 17 identified as D. Hansenii have showed the strongest antifungal activities. Improvement of killer toxin production by the yeasts also has been studied, and the highest production was found in YMB medium containing NaCl (6%) and DMSO (1000 ppm) at pH 4.0 and 20oC. The killer characters of these yeasts have indicated the potential use of the yeasts as antagonists for the control of postharvest diseases in agricultural industries.
The yeast Saccharomyces cerevisiae is the most important fermentative yeast and plays a leading role in the production of food and beverages (such as bread, wine, beer and sake, among others). Indeed, S. cerevisiae has a significant diversity, which is an important factor for the sensorial properties of the final fermentation product. Interestingly, recent studies have highlighted the importance of other fermentative yeast species. In this chapter we focus on the metabolic impact of yeasts on wine production and will cover the existing methods for recovery and identification fermentative yeasts, with a special focus on S. cerevisiae trains. We provide an update in the phenotypic and genetic characterization methods, the existing approaches that link the genome to phenotype and selection of important S. cerevisiae strains.
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.
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 chapter describes the taxonomy and biodiversity of yeasts and lactic acid bacteria (LAB) isolated from sourdoughs collected around the world. The phylogenetic position of yeast and LAB species found in sourdoughs is discussed, and important problems in taxonomic nomenclature of typical sourdough-associated species are highlighted. An overview is given of factors that may influence the taxonomic composition of sourdough ecosystems, such as geographical location, type of cereals and other raw materials, and technological process parameters. A brief history is provided of the most commonly used approaches to isolate yeast and LAB strains from sourdough samples. Finally, the main techniques for identification of sourdough yeasts and LAB are discussed, emphasizing both on conventional culture-based methods as well as on culture-independent molecular approaches.
Bootstrapping is a common method for assessing confidence in phylogenetic analyses. Although bootstrapping was first applied in phylogenetics to assess the repeatability of a given result, bootstrap results are commonly interpreted as a measure of the probability that a phylogenetic estimate represents the true phylogeny. Here we use computer simulations and a laboratory-generated phylogeny to test bootstrapping results of parsimony analyses, both as measures of repeatability (i.e., the probability of repeating a result given a new sample of characters) and accuracy (i.e., the probability that a result represents the true phylogeny). Our results indicate that any given bootstrap proportion provides an unbiased but highly imprecise measure of repeatability, unless the actual probability of replicating the relevant result is nearly one. The imprecision of the estimate is great enough to render the estimate virtually useless as a measure of repeatability. Under conditions thought to be typical of most phylogenetic analyses, however, bootstrap proportions in majority-rule consensus trees provide biased but highly conservative estimates of the probability of correctly inferring the corresponding clades. Specifically, under conditions of equal rates of change, symmetric phylogenies, and internodal change of less-than-or-equal-to 20% of the characters, bootstrap proportions of greater-than-or-equal-to 70% usually correspond to a probability of greater-than-or-equal-to 95% that the corresponding clade is real. However, under conditions of very high rates of internodal change (approaching randomization of the characters among taxa) or highly unequal rates of change among taxa, bootstrap proportions >50% are overestimates of accuracy.
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.
Heterothallic strains belonging to the biologically distinct yeast species CEavispora opuntiae and Clavisporu Zusituniae were studied by three different methods. The type cultures of the two yeast species exhibited 8% relatedness as measured by reassociation of unique deoxyribonucleic acid. Ten strains of C. opuntiue and nine strains of C. Zusitaniae were compared on the basis of their physiological phenotypes and the restriction maps of their ribosomal deoxyribonucleic acids (rDNAs). Although the two species possessed many similarities as well as certain amounts of intraspecific variation by both approaches, they appeared to constitute well-defined entities. Unlike C. opuntiae, C. Zusitaniae always utilized L-rhamnose as the sole carbon source and was resistant to 10 mg of cycloheximide per liter. Strains of C. opuntiae did not utilize L-lysine as the sole nitrogen source or utilized it very weakly, whereas all strains of C. Zusitaniae grew rapidly on this compound. By contrast, the hydrolysis of Tween 80 ad the utilization of lactic acid, citric acid, and hexadecane tended to be more pronounced in C. opuntiae. The rDNA repeating Unit was 9.0 kilobases long in C. lusitaniae, as compared with 7.6 kilobases in C. opuntiae. The conserved region identified previously in the rDNA of C. opuntiae was found almost intact in the rDNA of C. Zusitaniae, but the variable regions differed substantially between the two species. The genus Clavispora was proposed by Rodrigues de Miranda (6) to accommodate the teleomorphic state of Candida lusitaniae and its synonym Candida obtusa. Clavispora spp. are characterized by the formation of clav- ate ascospores with indistinct warts. Clavispora lusitaniae comprises interfertile heterothallic strains isolated from var- ious sources, including humans and other warm-blooded animals, and materials of plant origin (7). Clavispora opuntiae is also represented in nature by interfertile heterothallic strains, but its habitat is restricted almost entirely to necrotic tissue of various species of the prickly pear cactus, Opuntia (4, 9). Mating does not take place between the two species, and thus Clavispora is a good example of a yeast genus in which biological species (3) are clearly defined. It is therefore a useful model with which the significance of different taxonomic approaches may be eval- uated. This paper presents a comparison of the two known species of Clavispora based on the variation among physio- logical phenotypes normally investigated in the course of yeast identification, the relatedness of their unique deoxyri- bonucleic acids (DNAs), and the patterns observed among the restriction maps of their ribosomal DNAs (rDNAs). The last method has been used previously to assess intraspecific variation within C. opuntiae (2). In that study, 10 strains of C. opuntiae (not all the same as those used here) were found Ib share conserved regions in their rDNAs, but they also exhibited certain differences in their variable regions. We now show that this last approach is a valuable method for the study of yeast species delineation and that its results are
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 classification of species belonging to the genus Candida Berkhout is problematic. Therefore, we have determined the small ribosomal subunit RNA (srRNA) sequences of the type strains of three human pathogenic Candida species; Candida krusei, C. lusitaniae and C. tropicalis. The srRNA sequences were aligned with published eukaryotic srRNA sequences and evolutionary trees were inferred using a matrix optimization method. An evolutionary tree comprising all available eukaryotic srRNA sequences, including two other pathogenic Candida species, C. albicans and C. glabrata, showed that the yeasts diverge rather late in the course of eukaryote evolution, namely at the same depth as green plants, ciliates and some smaller taxa. The cluster of the higher fungi consists of 10 ascomycetes and ascomycete-like species with the first branches leading to Neurospora crassa, Pneumocystis carinii, Candida lusitaniae and C. krusei, in that order. Next there is a dichotomous divergence leading to a group consisting of Torulaspora delbrueckii, Saccharomyces cerevisiae, C. glabrata and Kluyveromyces lactis and a smaller group comprising C. tropicalis and C. albicans. The divergence pattern obtained on the basis of srRNA sequence data is also compared to various other chemotaxonomic data.
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.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
https://deepblue.lib.umich.edu/bitstream/2027.42/137546/1/evo03389.pdf
This chapter focuses on Candida genus and its member species. The cells of this genus are globose, ellipsoidal, cylindroidal, or elongate, occasionally ogival, triangular or lunate, and the cell wall is ascomycetous and two-layered. Reproduction in this genus occurs through holoblastic budding. The member species of this genus is Candida aaseri, Candida albicans, Candida amapae, Candida anatomiae, Candida ancudensis, and Candida antillancae. The cells of Candida aaseri, after undergoing growth in glucose-yeast extract-peptone broth for three days at 25° C, appear as elongate, in chains and clusters. The cells of Candida albicans, after undergoing growth in glucose-yeast extract-peptone broth for three days at 25° C, appear as globose to ovoidal, as single, pairs, chains, and clusters. The cells of Candida amapae, after undergoing growth in malt extract for three days at 25° C, become large and globose to elongate.
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.
The genetic and implicit taxonomic resolution provided by ribosomal RNA sequence divergence was estimated from comparisons of sibling yeast species. Two regions of the large subunit (25S) rRNA and four regions of the small subunit (18S) rRNA comprising a total of 855 nucleotides were examined in all strains. Within these regions, no nucleotide differences were detected between strains of the same species. The sibling species pairs Pichia mississippiensis/P. amylophila, P. americana/P. bimundalis, and Issatchenkia scutulata and its variety exigua could be separated from each other by differences in a ca. 200-base region of the large subunit rRNA. Sequence differences within the sibling species complex composed of Saccharomyces bayanus, S. pastorianius (S. carlsbergensis), and S. cerevisiae were consistent with the prior proposal that S. pastorianus is a partial amphidiploid formed by a rare mating between the other two Saccharomyces species. From these data, the highly variable region identified in the 25S subunit rRNA appears suitable for rapid separation of nearly all species by oligonucleotide probe technology.
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.
Deoxyribonucleic acid reassociation studies of 24 different wine and beer-associated strains of Saccharomyces confirmed the presence of three separate species. S. cerevisiae and S. bayanus strains had only 22% of their genomes in common. S. pastorianus, with intermediate hybridization values between S. cerevisiae and S. bayanus (52 and 72%, respectively) could possibly be a natural hybrid of the two species. S. pastorianus replaces S. carlsbergensis with which it is homologous for 93% of its genome, since the former species was described first by Hansen in 1904. These data do not agree with the results of traditional physiological tests.
Deoxyribonucleic acid (DNA) base composition and DNA base sequence relatedness comparisons were used for species delineation in the genus Kluyveromyces. Base composition values separated the members of the genus into three groups. The groups were further subdivided by comparing base sequences by using DNAIDNA renaturation experiments. Two DNA homology groups were identified. The first group included Kluyveromyces marxianus, Kluyveromyces fragilis, Kluyveromyces bulgaricus, Kluyveromyces cicerisporus, Kluyveromyces wikenii, and three anamorphs (Candida kefyr, Candida pseudotropicalis, and Torula cremoris); the members of this group exhibited 290 % DNA base sequence complementarity. The second group consisted of Kluyveromyces lactis, Kluyveromyces vanudenii, Kluyveromyces drosophilarum, and Kluyveromyces phaseolosporus; various pairs of these yeasts shared 64 to 98% of their DNA sequences. The two groups were only distantly related to each other (115% DNA base sequence complementarity). The other Kluyveromyces species appear to be unique, not being closely related to either of the two homology groups or to one another. Relationships deduced from comparisons of DNAs agreed well with those deduced by other workers from immunological comparisons of exo-P-glucanases and from isoenzyme analysis but were only in partial agreement with a taxonomic arrangement made on the basis of mating studies. We propose recognition of the following species: Kluyveromyces aestuarii, Kluyveromyces africanus, Kluyveromyces blattue, Kluyveromyces delphensis, Kluyveromyces dobzhanskii, K. lactis (syn. K. drosophilarum, K. phaseolosporus, and K. vanudenii), Kluyveromyces lodderi, K. marxianus (syn. K. bulgaricus, K. cicerisporus, K. fragilis, and K. wikenifi, Kluyveromyces phafii, Kluyveromyces polysporus, Kluyveromyces thermotolerans, Kluyveromyces waltii, and Kluyveromyces wickerhamii.
The evolution of mitochondrial ribosomal DNA sequences was investigated to infer the phylogenetic position of the Asian viper Azemiops feae and to refine weighting methodology for phylogenetic analysis of DNA sequences. To compare characteristics of sequence evolution and weighting methodology in a different group of vertebrates, the published mitochondrial ribosomal DNA data included in the study of chiropteran monophyly by Mindell et al. (1991, Proc. Natl. Acad. Sci. USA 88:10322-10326) were also analyzed. Sequence evolution in all taxa was marked by extreme substitution bias. For the L-strand, both mono- and ditypic substitutions involving G were rare in comparison with expected occurrences based on sequence composition. Character state weights were assigned prior to phylogenetic analysis in proportion to the ratio of expected to observed nucleotide differences in pairwise comparisons of sequences. Application of expected to observed ratio (EOR) weights strengthened support for an Azemiops/pitviper clade and for the monophyly of Chiroptera. This method may help in resolving rapid radiations and other relationships obscured by homoplasy.
Taxonomic studies on Debaryomyces hansenii (ZOPF) Lodder et Kregervan Rij and related yeast species were carried out using chemotaxonomic methods including DNA base composition, DNA-DNA hybridization, ubiquinone system, proton magnetic resonance spectrum of mannan, and serological methods. The 50 strains employed were classified into 3 groups, I, II, and III, based on DNA-DNA hybridization experiments. These groups were considered to represent three distinct species. Group I consisted of two subgroups, Ia and Ib, which were considered to represent two distinct varieties. These groups were also characterized by proton magnetic resonance spectra of alkali-extracted mannans and cell surface antigens, however, they could not be discriminated by DNA base composition and ubiquinone systems, in addition to the taxonomic criteria commonly employed in yeast taxonomy. Group Ia comprised 34 strains including the type or authentic strains of D. hansenii, D. nicotianae, D. nicotianae var. minor, D. kloeckeri, D. kloeckeri var. major, D. tyrocola, D. gruetzii, D. matruchoti, D. matruchoti var. cesarii, D. hildegaardi, D. guilliermondii, D. miso, Torulopsis westerdijkii, T. famata, and T. minor. Group Ib comprised 6 strains including the type or authentic strains Of D. subglobosus, D. fukuyamaensis, Pichia adzetii, and Candida flareri. Group II comprised 4 strains including the type or authentic strains of D. nepalensis, D. cavensis, and D. japonicus. Group III comprised 6 strains including the type strain of T. candida.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
DNA base composition and electrophoretic karyotypes of eight strains of Candida guilliermondii (Castellani) Langeron et Guerra including the type strains of the species and its synonym Torula fermentati Saito were determined by using thermal denaturation profiles (Tm) and the CHEF (contour-clamped homogeneous electric field) system of pulsed field gel electrophoresis (PFGE), respectively. These strains are separated into two distinct groups by the data obtained. The four strains in group I, including the type strain of C. guilliermondii, have 44.1–44.6 mol% G+C and homogeneous electrophoretic karyotypes consisted of 6–8 chromosomal DNA bands ranging in size from 0.4 to 2.1 Mb, while the other four strains in group II, including the type strain of T. fermentati, have 49.4–49.8 mol% G+C and almost identical karyotypes composed of 6 bands with molecular sizes ranging from 0.9 to 1.8 Mb. The approximately 5.0% difference in G+C contents and completely different electrophoretic karyotypes between the two groups demonstrate that these two groups represent two separate species. As a result, a new combination Candida fermentati (Saito) Bai comb. nov. is proposed.
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.
Abstract Cladistic data are more decisive when the possible trees differ more in tree length. When all the possible dichotomous trees have the same length, no one tree is better supported than the others, and the data are completely undecisive. From a rule for recursively generating undecisive matrices for different numbers of taxa, formulas to calculate consistency, rescaled consistency and retention indices in undecisive matrices are derived. The least decisive matrices are not the matrices with the lowest possible consistency, rescaled consistency or retention indices (on the most parsimonious trees); those statistics do not directly vary with decisiveness. Decisiveness can be measured with a newly proposed statistic, DD=S̄−S)/(S̄−S) (where S= length of the most parsimonious cladogram, S̄= mean length of all the possible cladograms for the data set and M= observed variation). For any data set, S̄ can be calculated exactly with simple formulas; it depends on the types of characters present, and not on their congruence. Despite some recent assertions to the contrary, the consistency index is an appropriate measure of homoplasy (= deviation from hierarchy). The retention index seems more appropriate for comparing the fit of different trees for the same data set.
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 determined the nucleotide sequence for the DNA encoding the 5·8S RNAs and downstream internal transcribed spacer (ITS2) regions for Candida albicans and the taxonomically related species C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. Phylogenetic analysis of all known fungal 5·8S RNA sequences revealed a close relationship between C. tropicalis and C. parapsilosis, and to a lesser extent C. albicans within the yeast-like fungi. This group can itself be delineated from predominantly filamentous species. The more distal relationships between Candida (torulopsis) glabrata and C. krusei support previous findings based on small (18S) ribosomal RNA sequence analysis, suggesting a greater degree of evolutionary divergence of these species from the C. albicans group. Among strains of C. albicans we observed conservation of the ITS2 region of the nucleotide level. Conservation was also observed for a more limited number of C. parapsilosis strains. Although the 3′ region of the ITS spacer was species specific, sequence homology was observed in the 5′ end within the albicans/parapsilosis/tropicalis group. Our findings suggest a rapid approach to species identification through the use of non-conserved regions flanked by highly conserved, functional domains.
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 single-copy actin gene of Giardia lamblia lacks introns; it has an average of 58% amino acid identity with the actin of other species; and 49 of its amino acids can be aligned with the amino acids of a consensus sequence of heat shock protein 70. Analysis of the potential RNA secondary structure in the transcribed region of the G. lamblia actin gene and of the single-copy actin gene of nine other species did not reveal any conserved structures. The G. lamblia actin sequence was used to root the phylogenetic trees based on 65 actin protein sequences from 43 species. This tree is congruent with small-subunit rRNA trees in that it shows that oomycetes are not related to higher fungi; that kinetoplatid protozoans, green plants, fungi and animals are monophyletic groups; and that the animal and fungal lineages share a more recent common ancestor than either does with the plant lineage. In contrast to smalls-ubunit rRNA trees, this tree shows that slime molds diverged after the plant lineage. The slower rate of evolution of actin genes of slime molds relative to those of plants, fungi, and animals species might be responsible for this incongruent branching.
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.
The genus Issatchenkia Kudriavzev has been emended to include all nitrate-negative, multilateral budding yeast species that form unconjugated persistent asci with roughened spheroidal ascospores and have Q-7 ubiquinone in the electron transport system. Pichia kudriavzevii (Issatchenkia orientalis), Pichiaterricola, Pichia scutulata var. scutulata, and Pichia scutulata var. exigua are assigned to this genus as Issatchenkia orientalis Kudriavzev, Issatchenkia terricola (van der Walt) comb. nov., Issatchenkia scutulata var. scutulata (Phaff et al.) comb. nov., and Issatchenkia scutulata var. exigua (Phaff et al.) comb. nov., respectively. Additionally, one new species, Issatchenkia occidentalis, is described. The type strain of I. occidentalis is NRRL Y-7552 (=CBS 5459). Electron microscopy demonstrated that the ascospore walls of I. terricola have thick inner and outer layers and that the ascospores of the other species have walls with a thick inner layer and a thin, dense outer layer. With the exception of I. scutulata var. scutulata, ascospore surface ornamentation arises from the dense outer layer. Deoxyribonucleic acid reassociation studies and mating tests confirmed the recognition of four species in the genus Issatchenkia and showed Candida krusei to be the imperfect state of I. orientalis; Candida sorbosa was identified as the imperfect form of I. occidentalis. I. scutulata var. scutulata and I. scutulata var. exigua showed only 25% deoxyribonucleic acid complementarity, yet intervarietal matings formed viable ascospores. This is the lowest deoxyribonucleic acid relatedness ever shown between strains capable of genetic hybridization, and the implications of this finding are discussed.
Twenty-four species assigned to the genus Saccharomyces sensu stricto were examined for deoxyribonucleic acid relatedness. Results with type strains demonstrated the presence of four distinct species: S. cerevisiae, S. bayanus, S. carlsbergensis, and S. kluyveri. S. carlsbergensis NRRL Y-12693T showed intermediate relatedness between S. cerevisiae and S. bayanus and has a genome size approximately 1.5 times those of the last two species. These data suggest S. carlsbergensis to be a partial amphidiploid which may have arisen from natural hybridization between S. cerevisiae and S. bayanus. Numerous studies in recent years have shown that the criteria used in traditional yeast taxonomy are not always satisfactory for delimitation of species. This is partly be- cause many separations are based on the presence or ab- sence of assimilative abilities which are often controlled by a single mutable gene (12, 14, 18, 23, 25). The species of Saccharomyces sensu stricto (34) provide a good example of this problem because of strain variability in standard carbon assimilation and fermentation tests (5, 16, 28, 34, 35, 36). As a result of the uncertainty of these tests, a number of other approaches have been attempted, such as comparisons of cell wall antigens (2, 10, 11, 27), proton magnetic resonance spectra of extracted mannans (11, 32), and attempted matings between haploid mating types of similar species (3, 21). Requirements for vitamins (9) and amino compounds (7) have also been used for taxonomic separations, as have differences in deoxyribonucleic acid (DNA) base composi- tion (guanine plus cytosine (G + C) contents in moles percent) (36). Finally, numerical analyses (1, 8, 15, 17) by Adansonian principles have been used for species separa- tions. None of these methods, however, seems to have resolved the status of the species investigated (25). More significant information concerning taxonomic rela- tionships among yeasts is obtainable by comparison of their nucleic acids (18, 25). In this light, some progress has been made with regard to Saccharomyces spp. Bicknell and Douglas (6) determined DNA relatedness among several species, whereas Rosini et al. (26) concluded from their DNA studies that common wine yeasts comprised at least three Saccharomyces species. In the present study, we measured DNA relatedness among all taxa assigned to Saccharomyces sensu stricto (34, 35, 36) in an effort to clarify their relationships.
Berlin, Techn. University, Thesis (doctoral).
Selected yeast classified as Candida sake van Uden et Buckley were examined for their physiological, morphological and immunological properties and their DNA relatedness. Candida maltosa Komagata, Nakase et Katsuya is herein recognized as a species separate from C. sake, Candida maltosa was distinguished from C. sake and from C. tropicalis by insignificant DNA reassociation. In addition, C. maltosa was distinguished from C. sake by its higher maximal growth temperature and lower guanine plus cytosine content of its DNA and from C. tropicalis by its failure to utilize soluble starch for growth and its resistance to cycloheximide. The species C. cloacae and C. subtropicalis are placed in synonymy with C. maltosa.
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.
Random amplified polymorphic DNA (RAPD) was used to better characterize the genotypic relatedness among medically important Candida species. By using short oligomer primers (10-mers) with arbitrarily chosen sequences in the polymerase chain reaction, distinctive and reproducible sets of polymerase chain reaction products were observed for isolates of C. albicans, C. lusitaniae, C. tropicalis, and Torulopsis (Candida) glabrata. The RAPD analysis differentiated a physiologically homogeneous panel of C. parapsilosis into three distinct groups and showed genetic diversity within C. haemulonii. Intraspecies DNA-length polymorphisms were seen for RAPD profiles derived from different isolates of each species. Analysis of RAPDs from a panel of C. albicans, which included 16 laboratory derivatives of two reference strains, showed that the profiles of unrelated strains differed and that the derivatives of each reference strain were identifiable. Minor differences in the RAPD profiles, suggestive of mutations that had occurred during the long-term maintenance of the strains, were detected. Because of its ease and reliability, RAPD analysis should be useful in providing genotypic characters for taxonomic descriptions, for confirming the identities of stock isolates, for typing Candida species in epidemiologic investigations, and for use in the rapid identification of pathogenic fungi.
A yeast culture isolated in Japan from soil and invalidly described as Kluyveromyces cellobiovorus nom. nud. DBVPG 6286 (CBS 7153) was compared for its physiological and morphological properties and by nDNA-nDNA reassociation experiments with the type strains of several species of the genus Kluyveromyces as well as of various Candida species exhibiting similar phenotypic profiles. DBVPG 6286 was found to be conspecific with the type strain of Candida intermedia (Ciferri & Ashford) Langeron et Guerra (1938).
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.