FEMS Yeast Research, 15, 2015, fov050
`
`doi: 10.1093/femsyr/fov050
`Advance Access Publication Date: 1 July 2015
`Minireview
`
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`M I N I R EV I EW
`Advances in yeast systematics and phylogeny and
`their use as predictors of biotechnologically important
`metabolic pathways
`Cletus P. Kurtzman1,∗, Raquel Quintilla Mateo2,3,4, Anna Kolecka2,
`Bart Theelen2, Vincent Robert2 and Teun Boekhout2
`
`1Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization
`Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604, USA, 2CBS Fungal
`Biodiversity Centre (CBS-KNAW), 3584 CT Utrecht, the Netherlands, 3Laboratory of Molecular Cell Biology,
`Institute of Botany and Microbiology, KU Leuven, B-3001 Leuven, Belgium and 4 Department of Molecular
`Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium
`∗Corresponding author: Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Agricultural
`Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA. Tel: +309-681-6385; Fax: +309-681-6672; E-mail:
`cletus.kurtzman@ars.usda.gov
`One sentence summary: Next generation sequencing enables improved phylogenetic analysis of yeasts and enables prediction of metabolic properties
`of different yeast species.
`Editor: Jens Nielsen
`
`ABSTRACT
`
`Detection, identification and classification of yeasts have undergone a major transformation in the last decade and a half
`following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily
`determined DNA sequences from domains 1 and 2 (D1/D2) of the nuclear large subunit rRNA gene and from ITS now
`permits many laboratories to identify species quickly and accurately, thus replacing the laborious and often inaccurate
`phenotypic tests previously used. Phylogenetic analysis of gene sequences is leading to a major revision of yeast
`systematics that will result in redefinition of nearly all genera. This new understanding of species relationships has
`prompted a change of rules for naming and classifying yeasts and other fungi, and these new rules are presented in the
`recently implemented International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). The use of molecular
`methods for species identification and the impact of Code changes on classification will be discussed, as will use of
`phylogeny for prediction of biotechnological applications.
`
`Keywords: yeasts; taxonomy; systematics; biotechnology; ecology
`
`INTRODUCTION
`
`Prior to phylogenetic analyses of DNA sequences, placement of
`yeasts among the fungi was guided by interpretation of pheno-
`type, which suggested that yeasts are primitive fungi (e.g. Guil-
`liermond 1912) or, alternatively, that they may be reduced forms
`
`of more mycelial taxa (Cain 1972; von Arx and van der Walt 1987).
`Initially, it was thought that all yeasts are ascomycetes, but dis-
`covery that some yeasts are basidiomycetes that produce bal-
`listoconidia (Kluyver and van Niel 1924, 1927; Nyland 1949) and
`basidia with basidiospores (Banno 1963, 1967) markedly changed
`
`Received: 24 February 2015; Accepted: 29 May 2015
`Published by Oxford University Press on behalf of FEMS 2015. This work is written by (a) US Government employee(s) and is in the public
`domain in the US.
`
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`2
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`FEMS Yeast Research, 2015, Vol. 15, No. 6
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`our perspective of the yeasts. With the discovery of such phylo-
`genetic diversity, the working definition of a yeast has become
`that of an ascomycete or basidiomycete fungus which divides by
`budding or fission and the sexual state, if known, is not enclosed
`in a fruiting body (Kurtzman, Fell and Boekhout 2011).
`In this review, we will discuss the advances that have arisen
`from application of DNA sequence analysis for identification of
`yeast species and for determining phylogenetic relationships
`among species. These new approaches are showing that the
`number of currently known yeast species is likely to be a small
`fraction of the total to be found in nature and that determi-
`nation of phylogenetic relationships will result in a major revi-
`sion of yeast classification. Furthermore, correct identification of
`species will significantly revise our understanding of yeast ecol-
`ogy, clinical microbiology and other areas of science that require
`accurate species identification. We have noted that some species
`groups share unique physiological characters and from this ob-
`servation, we will present the hypothesis that certain biotech-
`nological properties can be predicted from knowledge of phylo-
`genetic relationships.
`
`Species recognition
`
`Rapid identification of individual yeasts species is now routinely
`determined from nucleotide sequence divergence in domains
`1 and 2 (D1/D2) of the large subunit (LSU) rRNA gene. In prac-
`tice, this is done by conducting a Blast Search of the newly de-
`termined sequence with deposited sequences that are main-
`tained by GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and
`associated databases. The ca. 450–600 nucleotide D1/D2 region
`is bounded by highly conserved flanking sequences and essen-
`tially all species can be PCR amplified by a single set of primers
`(Kurtzman and Robnett 1998; Fell et al. 2000). The importance of a
`single gene diagnostic system is that as new species are discov-
`ered, the continuously expanding database provides documen-
`tation of all described species as well as evidence of undescribed
`species through absence of their sequences. A second database
`that is widely used is the internal transcribed spacer (ITS), which
`is located between the SSU and LSU rRNA genes. The ITS se-
`quence is divided into two sections (ITS1, ITS2) by the 5.8S gene,
`which is highly conserved and should not be included when
`comparing substitutions in ITS. The D1/D2 and ITS sequences
`are often similar in length, but notable length differences have
`been reported for some ascomycetes (Kurtzman and Robnett
`1998, 2003) and some basidiomycetes (Fell et al. 2000; Scorzetti
`et al. 2002), resulting in a different nucleotide length for deter-
`mining percent substitutions. With appropriate PCR primers, ITS
`and D1/D2 may be PCR replicated as a single amplicon. When
`conducting a Blast Search, the comparison should be made with
`the sequence of the type strain of the species that gives the near-
`est match because deposits in the database include many mis-
`named strains.
`Species resolution for both ascomycetes and basidiomycetes
`is based on the prediction that strains of a species diverge
`in D1/D2 and ITS sequences by no more than 1% (Kurtzman
`and Robnett 1998; Sugita et al. 1999; Fell et al. 2000; Scorzetti
`et al. 2002). For ITS, sequence divergence between closely related
`species may be relatively small in clades of the Saccharomyc-
`etaceae (Kurtzman and Robnett 2003), or quite large as seen for
`Citeromyces (Kurtzman 2012). Not surprisingly, exceptions have
`been found to the prediction of 1% or greater nucleotide diver-
`gence between species for D1/D2 and ITS sequences and this
`may result from interspecific hybridization, different substitu-
`tion rates or other genetic changes. Among these exceptions
`
`are Saccharomyces bayanus and S. pastorianus, which share the
`same rRNA repeat (Peterson and Kurtzman 1991; Groth, Hansen
`and Piskur 1999), and Clavispora lusitaniae, in which some strains
`show greater than 1% divergence in D1/D2 (Lachance et al. 2003).
`When taken in perspective, the D1/D2 and ITS databases present
`a powerful tool for rapid species identification and for presump-
`tive detection of previously unknown lineages. Use of DNA se-
`quences for identification has more than doubled the number of
`known yeast species during the past decade (Kurtzman, Fell and
`Boekhout 2011). It should be noted that ITS has been selected as
`the universal DNA sequence for identification of fungi because
`D1/D2 was less resolving of certain non-yeast fungal lineages
`(Schoch et al. 2012).
`
`Non-sequencing applications for species identification
`
`For some applications, DNA sequencing may not be required.
`Molecular methods based on sequences of known species are
`available from GenBank and other databases and can be used to
`develop species-specific primer pairs and probes. Other appli-
`cations include randomly amplified polymorphic DNA (RAPD),
`amplified fragment length polymorphisms (AFLP) and restric-
`tion fragment length polymorphisms (RFLP). The following is a
`brief description of these methods.
`
`Species-specific primers
`The use of species-specific primer pairs is effective when
`used for PCR-based identifications involving a small number of
`known species or when a particular species is the subject of the
`search (Fell 1993; Mannarelli and Kurtzman 1998; Chapman et al.
`2003; Hulin and Wheals 2014). Following the PCR reaction, the
`mixture is separated by gel electrophoresis to visually detect the
`band that identifies the target species.
`
`PNA
`Peptide nucleic acid (PNA) probes offer a means for detection
`and quantification of species in clinical samples, food products
`and other substrates through fluorescence in situ hybridization.
`PNA probes have a peptide backbone to which is attached nu-
`cleotides complementary to a species-specific target sequence,
`and a fluorescent label is added for detection by fluorescence
`microscopy (Stender et al. 2001; Rigby et al. 2002). If probes are
`complementary to rRNA, the whole cell of the target species will
`‘glow’ when visualized, which will also allow quantification by
`cell counts.
`
`RAPD/AFLP
`Microsatellite-primed RAPDs (Gadanho, Almeida and Sampaio
`2003) and AFLP fingerprints (de Barrios Lopes et al. 1999; Illnait-
`Zaragoz´ı et al. 2012) have been effectively used for rapid prelimi-
`nary identification of large numbers of isolates, and the pattern-
`based identification is then often followed by gene sequencing
`of representative strains from each group that has a unique pat-
`tern. One concern in using pattern-based identification tech-
`niques is reproducibility between laboratories, because small
`differences in PCR conditions may impact the species-specific
`patterns that serve as reference.
`
`Real-time PCR
`The technique of real-time PCR has also been widely studied
`for applications in medical mycology, especially those aiming to
`detect and quantify loads of Candida albicans. In typical assays,
`5 cfu ml−1 could be detected. Commonly used primers have been
`based on sequences of the rDNA repeat, such as ITS 1 and 2, or
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`Figure 1. Number of yeast species being described from 1790–2000. Data are
`based on data given in Barnett, Payne and Yarrow (2000) and Kurtzman et al.
`(2011). Top: cumulative graph of species being described in time (decades);
`bottom: number of species described per decade.
`
`tion of rDNA sequencing technology has contributed to the re-
`cent steep increase in number of yeast species being described
`(Kurtzman and Robnett 1998; Fell et al. 2000). From an analysis
`of species described over the past two centuries, it can be seen
`that yeast species discovery rapidly increased at the beginning
`of the 20th century (Fig. 1) with the use of physiological growth
`profiles resulting in the description of 460 yeast species by the
`1930s. This was followed by a decrease in the 1940–1950s due
`to the Second World War and it economic aftermath. A decline
`observed in the 1990s may be due to an awareness of the lim-
`itation of the use of physiological growth patterns in species
`distinctions, and hence reluctance emerged in using such data
`for the distinction of new species. However, with the introduc-
`tion of D1/D2 and ITS databases for both ascomycete and ba-
`sidiomycete yeasts (Kurtzman and Robnett 1998; Fell et al. 2000;
`Scorzetti et al. 2002; and the many publications that have fol-
`lowed), the description of new species rapidly increased. A ma-
`jor result from these barcoding studies was that more than 900
`species were described in the first decade of the 21st century,
`and it is clear that this trend will continue in the future given
`that large parts of the earth’s biomes have not yet been sam-
`pled for yeasts (Fig. 2). The fifth edition of The Yeasts, a Taxo-
`nomic Study (Kurtzman, Fell and Boekhout 2011) accepted >1400
`yeast species belonging to 85 ascomycetous genera and 61 ba-
`sidiomycetous genera, but since the publication of this treat-
`ment >230 yeast species have been registered in Mycobank
`(http://www.mycobank.org/).
`
`the small subunit (SSU) rRNA gene (Loeffler et al. 2000; Klingspor
`and Jalal 2006; Bergman et al. 2007; Khlif et al. 2009; Welling-
`hausen et al. 2009). This technique is also becoming widely em-
`ployed in food and beverage analyses and has been used for
`detection and quantification of spoilage yeasts in orange juice
`(Casey and Dobson 2004) as well as in wine fermentations (Co-
`colin, Heisey and Mills 2001).
`
`DGGE
`Denaturing gradient gel electrophoresis (DGGE) is a method
`that has been used for species identification and quantifica-
`tion of yeast populations in foods and beverages. The tech-
`nique is based on separation of DNA fragments that differ in
`nucleotide sequences (e.g. species specific) through decreased
`electrophoretic mobility of partially melted double-stranded
`DNA amplicons in a polyacrylamide gel containing a linear gra-
`dient of DNA denaturants (i.e. a mixture of urea and formamide).
`A related technique is temperature gradient gel electrophoresis,
`in which the gel gradient of DGGE is replaced by a temperature
`gradient (Muyzer and Smalla 1998). Applications of DGGE have
`included identification and population dynamics of yeasts, e.g.
`sourdough bread (Meroth, Hammes and Hertel 2003), in coffee
`fermentations (Masoud et al. 2004) and on wine grapes (Prak-
`itchaiwattana, Fleet and Heard 2004). Levels of detection are of-
`ten around 103 cfu ml−1, but 102 cfu ml−1 have been reported,
`which compares favorably with standard plate count methods
`(Prakitchaiwattana, Fleet and Heard 2004).
`
`Flow cytometry
`High-throughput probe hybridization methods are available
`for detection of multiple species in multiple samples. One
`method that is effective for yeasts (Diaz and Fell 2004; Page
`and Kurtzman 2005) is an adaptation of the Luminex xMAP
`technology (Luminex Corp), which consists of a combination
`of 100 different sets of fluorescent beads covalently bound to
`species-specific DNA capture probes. Upon hybridization, the
`beads bearing the target amplicons are classified in a flow cy-
`tometer by their spectral addresses with a 635-nm laser. The
`hybridized biotinylated amplicon is quantified by fluorescent
`detection with a 532-nm laser. Strains that differ by one nu-
`cleotide often can be discriminated and the assay can be per-
`formed, after amplification, in less than 50 min in a 96-well for-
`mat with as many as 100 different species-specific probes per
`well.
`The molecular detection methods just described have pro-
`vided some remarkable capabilities for yeast identification, but a
`number of factors affect detection and quantification. These in-
`clude (1) cellular copy number of the gene to be used, (2) whether
`the gene is sufficiently conserved to be PCR amplified by ‘univer-
`sal’ primers that will detect all species of interest, (3) efficiency
`of DNA extraction from cells in the sample, (4) efficiency of DNA
`recovery from the sample, (5) sample components that may in-
`terfere with DNA recovery or PCR amplification and (6) level of
`cell population that is detectable.
`The impact on identification of yeasts through use of DNA
`sequences has been remarkable. Earlier, yeast identification was
`based on comparative physiology in which the capability to fer-
`ment certain mono-, di- and trisaccharides was determined, to-
`gether with the utilization patterns of sugars, organic acids, al-
`cohols, sugar alcohols, starch and some nitrogen compounds.
`Molecular identification methods began with determination of
`guanine + cytosine (G + C) ratios (e.g. Nakase and Komagata
`1968) and was soon followed by nuclear DNA reassociation tech-
`niques (e.g. Price, Fuson and Phaff 1978). However, the introduc-
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`Figure 2. Geographical sources of approximately 7000 yeast strains present in the publicly available collection of CBS.
`
`MALDI-TOF Mass Spectrometry, an alternative
`identification method for yeasts
`
`MALDI-TOF MS-based identification has revolutionized micro-
`bial identification, including yeasts, in many laboratories world-
`wide. In comparison with DNA-based identification methods,
`such as sequence analysis of the D1/D2 domains of the LSU
`rDNA and the ITS 1 and 2 regions of the rDNA, MALDI-TOF
`MS gives identifications in short turnaround times (Tan et al.
`2012; Cassagne et al. 2013). MALDI-TOF MS has been successfully
`applied to identify isolates of many clinically relevant yeasts,
`e.g. Cryptococcus neoformans/C. gattii species complex, Ca. albi-
`cans and non-albicans Candida species, arthroconidial yeasts of
`Geotrichum and Trichosporon spp. and Malassezia spp. (Marklein
`et al. 2009; McTaggart et al. 2011; Cendejas-Bueno et al. 2012;
`Firacative, Trilles and Meyer 2012; Kolecka et al. 2013, 2014; Ha-
`gen et al. 2015). With an increasing coverage of yeast species in
`the databases, the utility of the technique will further increase.
`
`Phylogenetic placement of the ascomycete yeasts
`
`Although it was seen from sexual states that yeasts may
`be ascomycetes or basidiomycetes, their placement within
`these two fungal phyla was largely unknown before molecu-
`lar comparisons. An early study employed 5S rRNA sequences
`
`(Walker 1985) to examine relationships among the fungi, and
`this comparison divided the Ascomycota into three groups:
`(1) Schizosaccharomyces and Protomyces (Taphrinomycotina), (2)
`budding yeasts (Saccharomycotina) and (3) ‘filamentous fungi’
`(Pezizomycotina). Berbee and Taylor (1993) analyzed represen-
`tative members of the Mycota from SSU rRNA gene sequences
`and showed the same three major ascomycete lineages. This
`analysis also revealed that yeasts and ‘filamentous fungi’ are
`sister taxa and that Schizosaccharomyces and relatives represent
`an early diverging lineage. Kurtzman and Robnett (1994) showed
`from partial LSU and SSU rRNA sequences that all then ac-
`cepted ascomycetous yeast genera were members of a single
`clade, which was separate from Schizosaccharomyces and mem-
`bers of the ‘filamentous fungi’. The findings from single gene
`analyses have been supported by multigene sequence analyses
`(Fitzpatrick et al. 2006; James et al. 2006; Kuramae et al. 2006a,b;
`Sugiyama, Hosaka and Suh 2006; Schoch et al. 2009; Rosling et al.
`2011; Kurtzman and Robnett 2013).
`Sequence analyses are now providing an understanding of
`relatedness between both species and genera (Kurtzman 2011).
`Figure 3 shows phylogenetic relationships among genera of the
`Saccharomycotina as determined from a five-gene sequence
`analysis of type species from most presently accepted gen-
`era (Kurtzman and Robnett 2013). In this analysis, members of
`the Lipomycetaceae (Fig. 3, Clade 11) are the earliest diverging
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`5
`
`Clade 1
`
`Clade 2
`
`Clade 3
`
`Clade 4
`
`Clade 5
`
`Clade 6
`
`Clade 7
`Clade 8
`
`Clade 9
`
`Clade 10
`
`Clade 11
`
`Clade 12
`
`96
`61
`74
`
`58
`
`60
`
`100
`
`88
`
`96
`
`100
`
`100
`
`93
`
`58
`
`91
`
`100
`55
`
`96
`
`92
`100
`
`58
`
`100
`
`99
`
`99
`
`94
`
`100
`
`100
`
`Zygotorulaspora mrakii Y-12654 [Q-6]
`Torulaspora delbrueckii Y-866 [Q-6]
`Zygosaccharomyces rouxii Y-229 [Q-6]
`Cyniclomyces guttulatus Y-17561 [Q-6]
`Nakaseomyces delphensis Y-2379 [Q-6]
`Saccharomyces cerevisiae Y-12632 [Q-6]
`Naumovozyma dairenensis Y-12639 [Q-6]
`Kazachstania viticola Y-27206 [Q-?]
`Tetrapisispora phaffii Y-8282 [Q-6]
`Vanderwaltozyma polyspora Y-8283 [Q-?]
`Kluyveromyces marxianus Y-8281 [Q-6]
`Lachancea thermotolerans Y-8284 [Q-6]
`Eremothecium cymbalariae Y-17582 [Q-7]
`Saccharomycodes ludwigii Y-12793 [Q-6]
`Hanseniaspora valbyensis Y-1626 [Q-6]
`Barnettozyma populi Y-12728 [Q-7]
`Cyberlindnera americana Y-2156 [Q-7]
`Wickerhamomyces canadensis Y-1888 [Q-7]
`Starmera amethionina Y-10978 [Q-7]
`Phaffomyces opuntiae Y-11707 [Q-7]
`Komagataella pastoris Y-1603 [Q-8]
`Saccharomycopsis capsularis Y-17639 [Q-8]
`Ascoidea rubescens Y-17699 [Q-?]
`Nakazawaea holstii Y-2155 [Q-8]
`Pachysolen tannophilus Y-2460 [Q-8]
`Peterozyma toletana YB-4247 [Q-7]
`Citeromyces matritensis Y-2407 [Q-8]
`Kuraishia capsulata Y-1842 [Q-8]
`Candida boidinii Y-2332 [Q-7]
`Ogataea angusta Y-2214 [Q-7]
`100
`99
`Ogataea minuta Y-411 [Q-7]
`Ogataea glucozyma YB-2185 [Q-7]
`Dekkera bruxellensis Y-12961 [Q-9]
`Ogataea methanolica Y-7685 [Q-7]
`Ambrosiozyma monospora Y-1484 [Q-7]
`Kregervanrija fluxuum YB-4273 [Q-7]
`Pichia membranifaciens Y-2026 [Q-7]
`Candida abiesophila Y-11514 [Q-?]
`Saturnispora dispora Y-1447 [Q-7]
`Babjeviella inositovora Y-12698 [Q-9]
`Cephaloascus fragrans Y-6742 [Q-9]
`Yamadazyma philogaea Y-7813 [Q-9]
`Yamadazyma scolyti Y-5512 [Q-9]
`Yamadazyma mexicana Y-11818 [Q-9]
`Yamadazyma triangularis Y-5714 [Q-9]
`Spathaspora passalidarum Y-27907 [Q-?]
`Scheffersomyces spartinae Y-7322 [Q-9]
`Hyphopichia burtonii Y-1933 [Q-8]
`Hyphopichia heimii Y-7502 [Q-?]
`Candida multigemmis Y-17659 [Q-?]
`Kurtzmaniella cleridarum Y-48386 [Q-?]
`Debaryomyces hansenii Y-7426 [Q-9]
`Schwanniomyces occidentalis Y-10 [Q-9]
`Priceomyces haplophilus Y-7860 [Q-9]
`Priceomyces castillae Y-7501 [Q-9]
`Schwanniomyces etchellsii Y-7121 [Q-9]
`Millerozyma farinosa Y-7553 [Q-9]
`Meyerozyma guilliermondii Y-2075 [Q-9]
`Scheffersomyces stipitis Y-7124 [Q-9]
`Wickerhamia fluorescens YB-4819 [Q-9]
`Lodderomyces elongisporus YB-4239 [Q-9]
`Kodamaea ohmeri Y-1932 [Q-9]
`Aciculoconidium aculeatum YB-4298 [Q-9]
`Clavispora lusitaniae Y-11827 [Q-8]
`Metschnikowia agaves Y-17915 [Q-?]
`Metschnikowia bicuspidata YB-4993 [Q-9]
`Alloascoidea africana Y-6762 [Q-?]
`Sporopachydermia lactativora Y-11591 [Q-9]
`Nadsonia fulvescens Y-12810 [Q-6]
`Dipodascus albidus Y-12859 [Q-?]
`100
`Galactomyces geotrichum Y-17569 [Q-?]
`Magnusiomyces magnusii Y-17563 [Q-9]
`Middelhovenomyces petrohuensis Y-17663 [Q-?]
`Middelhovenomyces tepae Y-17670 [Q-9]
`Spencermartinsiella europaea Y-48265 [Q-?]
`Sugiyamaella smithiae Y-17850 [Q-9]
`Zygoascus hellenicus Y-7136 [Q-9]
`Diddensiella santjacobensis Y-17667 [Q-?]
`Trichomonascus petasosporus YB-2092 [Q-?]
`Candida blankii Y-17068 [Q-?]
`Yarrowia lipolytica YB-423 [Q-9]
`Wickerhamiella domercqiae Y-6692 [Q-?]
`Starmerella bombicola Y-17069 [Q-9]
`Trigonopsis variabilis Y-1579 [Q-9]
`Botryozyma nematodophila Y-17705 [Q-?]
`Tortispora caseinolytica Y-17796 [Q-?]
`Dipodascopsis anomala Y-7931 [Q-9]
`Dipodascopsis uninucleata Y-17583 [Q-?]
`Lipomyces starkeyi Y-11557 [Q-9]
`Pneumocystis carinii [Q-?]
`Schizosaccharomyces pombe Y-12796 [Q-10]
`Saitoella complicata Y-17804 [Q-10]
`Taphrina wiesneri IFO 7776 [Q-10]
`Protomyces inouyei IAM 14512 [Q-10]
`Filobasidiella neoformans CBS 6885 [Q-10]
`
`100
`96
`
`98
`
`98
`
`100
`
`85
`
`50
`
`100
`
`83
`
`93
`
`80
`
`98
`
`100
`
`100
`
`100
`
`100
`
`84
`
`80
`
`59
`
`100
`
`100
`
`99
`
`95
`
`77
`
`100
`
`100
`
`96
`
`65
`
`86
`
`0.2
`
`Figure 3. Phylogenetic relationships among type species of ascomycete yeast genera and reference taxa determined from maximum likelihood analysis of concatenated
`gene sequences for LSU rRNA, SSU rRNA, translation elongation factor-1α, and RNA polymerase II, subunits B1 and B2. The basidiomycete Filobasidiella (Cryptococcus)
`neoformans was the designated outgroup species in the analysis. Bootstrap values (1000 replicates) >50% are given at branch nodes and the final dataset included 11 773
`positions. Strain accession numbers are NRRL unless otherwise indicated. Reproduced from Kurtzman and Robnett (2013).
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`Figure 4. Schematic representation of the three subphyla, Agaricomycotina (top) Ustilaginomycotina (middle) and Pucciniomycotina (bottom) of Basidiomycota. Fungi
`with a yeast state, either as part of the life cycle or the entire known life cycle, occur in all three lineages. Courtesy: Holtermannia mycelialis (hyphae), Tremella encephala
`(basidiocarp), Xanthophyllomyces dendrorhous (=Phaffia rhodozyma, basidium), Malassezia globosa (budding yeast cell), Moniliella fonsecae (yeast cells and arthroconidia),
`Meira geulakonigii (pseudohyphae on mite) were taken from The Yeasts, a Taxonomic Study, 5th ed. (Kurtzman et al. 2011). Cryptococcus albidus (yeast cells), Farysizyma
`acheniorum (yeast cells), Rh. glutinis, Rh. minuta and Rh. hordea (both colonies and yeast cells) and Leucosporidium scottii (germinating teliospore with basidia, and
`pseudohyphae) were taken from the CBS website (http://www.cbs.knaw.nl/Collections/Biolomics.aspx?Table=CBS%20strain%20database).
`
`members of the Saccharomycotina. Species of Lipomyces are
`commonly isolated from soil and the genus represents one of
`the few groups of yeasts for which soil is the primary habitat.
`Saccharomyces and related genera appear to be among the most
`diverged members of the Saccharomycotina (Fig. 3, Clade 1), and
`this clade includes the vigorous sugar fermenting yeasts often
`used for ethanol production. Phenotypic characters previously
`used to infer relatedness, such as ascospore morphology and ni-
`trate assimilation, are found in many clades.
`Support for many lineages in present phylogenetic trees is
`often weak and more robust analyses of relationships among
`the Saccharomycotina will require whole genome comparisons.
`At present, genome sequences are available for less than 100
`yeasts and these are primarily from species of genetic, med-
`ical or biotechnological interest. Analyses of whole genomes,
`sometimes based on ca. 400 orthologous genes, have presented
`species relationships much like those determined from far fewer
`genes but, in contrast, phylogenetic trees derived from these
`analyses usually have much greater branch support (e.g. Fitz-
`patrick et al. 2006; Kuramae et al. 2006a,b). As demonstrated by
`Rokas et al. (2003) from analysis of genomes from Saccharomyces
`species, a minimum of 20 concatenated genes were needed to
`provide strong support of species placement in resulting phy-
`
`logenetic trees. Because Saccharomyces is a small genus, strong
`resolution of species relationships in larger genera will require
`additional genes. Similarly, resolution of families from cur-
`rent datasets is often uncertain. Placement of Cyniclomyces, Ere-
`mothecium, Kazachstania, Kluyveromyces, Lachancea, Nakaseomyces,
`Naumovozyma, Saccharomyces, Tetrapisispora, Torulaspora, Vander-
`waltozyma, Zygosaccharomyces and Zygotorulaspora in the family
`Saccharomycetaceae (Fig. 3, Clade 1) seems strongly supported
`as do some other clades that represent families, but other clades
`may represent several families, such as the large Clade 6 (Fig. 3),
`which is presently identified as the Debaryomycetaceae.
`
`Phylogenetic placement of the basidiomycete yeasts
`
`Basidiomycetous yeasts are polyphyletic and occur in all three
`subphyla of Basidiomycota, namely Pucciniomycotina, Agari-
`comycotina and Ustilaginomycotina (James et al. 2006; Hibbett
`et al. 2007; Boekhout et al. 2011) (Fig. 4). Within Pucciniomy-
`cotina, a biologically highly diverse group that is mainly unified
`by molecular phylogeny data, nine classes are recognized, but
`only four have species with a yeast state. These are Microbotry-
`omycetes, Cystobasidiomycetes, Agaricostilbomycetes and Mix-
`iomycetes. The latter contains only one species Mixia osmundae,
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`which is a fern parasite that in culture grows with a yeast
`state (Nishida, Robert and Sugiyama 2011). Within Agaricostil-
`bomycetes two major clades occur, Agaricostilbales with the
`yeast genera Kondoa, Agaricostilbum, some Bensingtonia species,
`Kurtzmanomyces, some Sporobolomyces species, Sterigmatomyces
`and Spiculogloeales with asexual yeasts presently classified in
`Sporobolomyces, namely Sp. subbrunneus, Sp. coprosmicola and Sp.
`linderae (Aime et al. 2006; Bauer et al. 2006). The Cystobasid-
`iomycetes include mostly pink-colored asexual yeasts classi-
`fied in the genera Bannoa, Cyrenella, part of Rhodotorula species,
`part of Sporobolomyces species and Erythrobasidium, and a diverse
`group of sexual and dimorphic species classified in the genera
`Cystobasidium, Occultifur, Naohidea and Sakaguchia. The Cystoba-
`sidiomycetes have three orders: (1) Cystobasidiales with Cysto-
`basidium and Occultifur, and some Rhodotorula species, includ-
`ing Rhodotorula minuta and Rh. slooffiae; (2) Erythrobasidiales with
`Erythrobasidium and Bannoa, Rh. lactosa and some Sporobolomyces
`species such as Sporobolomyces ogasawarensis and (3) Naohide-
`ales with the genus Naohidea that forms cream-colored colonies
`in culture. The Microbotryomycetes include many species of
`so-called red yeasts belonging to Rhodotorula and Rhodosporid-
`ium. Yeast taxa mainly belong to two orders, Leucosporidi-
`ales and Sporidiobolales. The former maintains two sexual and
`teliospore-forming genera, i.e. most Leucosporidium species and
`Mastigobasidium, and their asexual counterparts. Sporidiobolales
`include the pink-colored species in the sexual and teliospore-
`forming genera Rhodosporidium and Sporidiobolus and their asex-
`ual equivalents, Rhodotorula and Sporobolomyces, respectively.
`The classification of these fungi, especially that of the anamor-
`phic genus Rhodotorula, is in great need of revision and we expect
`that multigene-based and whole genome-based phylogenies in
`the near future will contribute to this.
`Among subphylum Agaricomycotina, yeasts or dimorphic
`taxa with yeast states occur only in class Tremellomycetes
`that has four (or five, depending on the view of the tax-
`onomist) orders, i.e. Cystofilobasidiales, Filobasidiales, Holter-
`manniales Tremellales and Trichosporonales that all contain
`yeast and yeast-like taxa (Fell et al. 2001; Sampaio 2004;
`Boekhout et al. 2011; Wuczkowski et al. 2011). The most basal lin-
`eage is Cystofilobasidiales (Fell, Roeijmans and Boekhout 1999;
`Boekhout et al. 2011) with seven well-circumscribed lineages
`and genera, such as Phaffia/Xanthophyllomyces, Cystofilobasidium,
`Itersonilia, Udeniomyces, Guehomyces and Mrakia. Taxonomic is-
`sues to be solved are the relationships between Tausonia and
`Guehomyces, Udeniomyces pannonicus and Itersonilia and the posi-
`tion of Mrakia curviuscula. Some species of Cystofilobasidiales,
`namely Mrakia spp. and Phaffia sp. are able to ferment sug-
`ars, which is a very rare trait among basidiomycetous yeasts
`and further known from Filobasidium capsuligenum belonging to
`Filobasidiales and some Bandoniozyma spp. belonging to Tremel-
`lales (Valente et al. 2012). Filobasidiales comprise all Filobasidium
`species as well as many species currently classified as Crypto-
`coccus. Several clades seem well defined and may represent sep-
`arate genera, such as the aerius clade, albidus clade, gastricus
`clade and cylindricus clade (Boekhout et al. 2011). The floriforme
`clade comprises Filobasidium species together with some Cryp-
`tococci, i.e. C. magnus, C. oeirensis, C. chernovii, C. stepposus and
`C. wieringae. The taxonomic relationship of F. uniguttulatum with
`the other species of Filobasidium requires further investigation.
`The largest order in the Tremellomycetes is the Tremellales
`that contains many mushroom-forming taxa that are mycopar-
`asitic and dimorphic. Molecular phylogenetic data strongly sup-
`port the inclusion of anamorphic taxa (Fell et al. 2001; Scorzetti
`et al. 2002; Sampaio 2004; Millanes et al. 2011; Xinzhan Liu, Feng-
`
`Kurtzman et al.
`
`7
`
`Yan Bai & Teun Boekhout, pers. comm). The genus Tremella
`also turned out to be polyphyletic and needs revision (Millanes
`et al. 2011; Xinzhan Liu, Feng-Yan Bai & Teun Boekhout, pers.
`comm). Multigene-based phylogenies showed the presence of
`25 well-supported clades within Tremellales (Xinzhan Liu, Feng-
`Yan Bai & Teun Boekhout, pers. comm). This an