Identification and Phylogeny of Ascomycetous Yeasts from Analysis
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Antonie van Leeuwenhoek 73: 331–371, 1998. 331 © 1998 Kluwer Academic Publishers. Printed in the Netherlands. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences Cletus P. Kurtzman∗ & Christie J. Robnett Microbial Properties Research, National Center for Agricultural Utilization Research, Agricultural Research Ser- vice, U.S. Department of Agriculture, Peoria, Illinois 61604, USA (∗ author for correspondence) E-mail: [email protected] Received 19 June 1998; accepted 19 June 1998 Key words: Ascomycetous yeasts, phylogeny, ribosomal DNA, systematics Abstract Approximately 500 species of ascomycetous yeasts, including members of Candida and other anamorphic genera, were analyzed for extent of divergence in the variable D1/D2 domain of large subunit (26S) ribosomal DNA. Divergence in this domain is generally sufficient to resolve individual species, resulting in the prediction that 55 currently recognized taxa are synonyms of earlier described species. Phylogenetic relationships among the ascomycetous yeasts were analyzed from D1/D2 sequence divergence. For comparison, the phylogeny of selected members of the Saccharomyces clade was determined from 18S rDNA sequences. Species relationships were highly concordant between the D1/D2 and 18S trees when branches were statistically well supported. Introduction lesser ranges of nDNA relatedness than those found among heterothallic species. Disadvantages of nDNA reassociation studies in- Procedures commonly used for yeast identification clude the need for pairwise comparisons of all isolates rely on the appearance of cellular morphology and dis- under study and that resolution is limited to the ge- tinctive reactions on a standardized set of fermentation netic distance of sister species, i.e., closely related and assimilation tests. These tests are laborious and species that have only recently become genetically sometimes ambiguous because of strain variability. isolated from one another. As a result, interest has Given these difficulties and the impracticality of iden- turned to other molecular comparisons that include se- tifying most species from genetic crosses, molecular quencing, restriction fragment length polymorphisms comparisons are increasingly used for yeast identifica- (Bruns et al., 1991) and random amplified polymor- tion. Initial molecular work centered on determining phic DNA (Hadrys et al., 1992). Of these, sequencing the extent of nuclear DNA (nDNA) relatedness be- appears the most robust because strain comparisons tween isolates. Kurtzman (1987) and Kurtzman & are easily made and, with the selection of appropriate Phaff (1987) reviewed results from nDNA reassoci- genes, both close and distant relationships can be re- ation studies of various heterothallic ascomycetous solved. For example, Peterson & Kurtzman (1991) se- yeasts and noted that members of a biological species quenced the variable D2 domain (ca. 300 nucleotides) 0 generally exhibit 70% or greater nDNA complemen- near the 5 end of large subunit (26S) rRNA from tarity. Isolates with 40 to 70% nDNA relatedness are selected heterothallic sister species in the genera Is- often considered varieties or subspecies unless genetic satchenkia, Pichia and Saccharomyces to determine if crosses indicate otherwise. These criteria have been closely related species could be separated from substi- applied to homothallic as well as to anamorphic (asex- tutions in this region. Conspecific strains generally had ual) yeasts with the argument that strains of species fewer than 1% nucleotide substitutions in this domain, from these groups appear to have neither greater nor whereas biological species were separated by greater MENNEN/SCHRIKS:Disk/CP: Pips Nr.:183584; Ordernr.:233759-bb; sp.code: 523-98 (antokap:bio2fam) v.1.1 anto523.tex; 13/10/1998; 7:32; p.1 332 than this number of substitutions, thus providing an DNA isolation for PCR was performed by a mod- empirical means for recognizing species. ified version of the sodium dodecyl sulfate protocol Numerous studies have presented the phylogeny of Raeder & Broda (1985). The lyophilized cell mass of different yeast groups from rRNA and rDNA se- from a single 1.5 ml microcentrifuge tube was broken quence comparisons, but these studies have focused apart with a pipette tip, and ca. 0.5 ml of 0.5-mm- either on individual genera, which are usually circum- diameter glass beads was added to the microcentrifuge scribed from phenotypic criteria, or on a relatively tube. The tube was shaken for 20 min on a wrist few widely divergent species. In either case, relation- action shaker at maximum speed. This treatment visi- ships are often incompletely understood because the bly fractured about 25% of the cells. The cells were number of taxa sampled has been small. To bring an suspended in 1 ml of extraction buffer (200 mM overall perspective to species relationships, we com- Tris-HCl [pH 8.5], 250 mM NaCl, 25 mM EDTA pared sequences from the ca. 600-nucleotide D1/D2 [pH 8.0], 0.5% sodium dodecyl sulfate) and extracted domain (Guadet et al., 1989) at the 50 end of large sub- with phenol-chloroform and chloroform. As an al- unit (LSU) rDNA for essentially all of the nearly 500 ternative to the laboratory use of phenol, the broken currently accepted species of ascomycetous yeasts, ap- cells were suspended in 700 µl2XCTABbuffer proximately 200 of which were included in our earlier (100 mM Tris-HCl [pH 8.4], 1.4 M NaCl, 25 mM studies (Kurtzman & Robnett, 1995; 1997). The data EDTA, 2% hexadecyltrimethyl-ammonium bromide), indicate that most yeast species can be identified from vortex-mixed with an equal volume of chloroform and sequence divergence in the D1/D2 domain, and show centrifuged for 10 min (K. O’Donnell pers. comm.). that 55 currently accepted species are either synonyms Following either extraction procedure, DNA was pre- or sister species of earlier described species. In addi- cipitated from the aqueous phase by adding 0.54 vol- tion, a phylogenetic analysis of the dataset provides an ume of isopropanol and pelleted for ca. 3 min in an overview of close species relationships. Eppendorf model 5415 microcentrifuge at 14,000 rpm. The pellet was washed gently with 70% ethanol, re- suspended in 100 µl of TE buffer (10 mM Tris-HCl, 1 Materials and methods mM EDTA [pH 8.0]), and dissolved by incubation at 55 ◦ C for 1 to 2 h. Dilute DNA samples for PCR were Organisms prepared by adding 4 µl of the genomic stocks to 1 ml of 0.1X TE buffer. The strains studied are listed in Table 1, and all The divergent D1/D2 domain (nucleotides 63–642 are maintained in the Agricultural Research Service 0 for Saccharomyces cerevisiae)atthe5 end of the LSU Culture Collection (NRRL), National Center for Agri- rRNA gene was symmetrically amplified with primers cultural Utilization Research, Peoria, Illinois. Strains NL-1 (50-GCATATCAATAAGCGGAGGAAAAG) and designated by a genus name followed by sp. are pu- 0 NL-4 (5 -GGTCCGTGTTTCAAGACGG) (O’Donnell, tative new species that will be described in future 1993). Amplification was performed for 36 PCR cy- publications. ◦ ◦ cles with annealing at 52 C, extension at 72 C for 2 min, and denaturation at 94 ◦ C for 1 min. The Growth of cultures, DNA isolation, PCR, and amplified DNA was purified with Geneclean II (Bio sequencing reactions 101, La Jolla, Calif.) according to the manufacturer’s Cells used for DNA extraction were grown for approx- instructions. Visualization of the amplified DNA was imately 24 h at 25 ◦ C in 50 ml of Wickerham’s (1951) performed following Geneclean II treatment by elec- YM broth (3 g yeast extract, 3 g malt extract, 5 g trophoresis in 1.5% agarose in 1X TAE buffer (0.04 M peptone, and 10 g glucose per liter of distilled water) Tris-acetate, 0.00l M EDTA [pH 8.0]) and staining − on a rotary shaker at 200 rpm and harvested by cen- with ethidium bromide (8 × 10 5 µg/µl). trifugation. The cells were washed once with distilled Both strands of the rDNA regions compared water, resuspended in 2 ml of distilled water, and 1 were sequenced with the ABI TaqDyeDeoxy Ter- ml of the suspension was pipetted into each of two 1.5 minator Cycle sequencing kit (Applied Biosystems ml microcentrifuge tubes. After centrifugation, excess Inc., Foster City, Calif.). Four sequencing re- water was decanted from the microcentrifuge tubes, actions were performed for each DNA sample. and the packed cells were lyophilized for 1 to 2 days Primers for these reactions were the external primers and stored in a freezer (−20 ◦ C) until use. NL-1 and NL-4 and the internal primers NL-2A anto523.tex; 12/10/1998; 14:25; p.2 333 Table 1. Strains of ascomycetous yeasts and reference species compared a,b c Species Strain designation No. of differences in GenBank NRRL CBS domain D1/D2 (ca. 600 accession no. nucleotides) between type strains and conspecific isolates T Aciculoconidium aculeatum YB-4298 5578 U40087 YB-4297 5293 2 T Ambrosiozyma (Hormoascus) ambrosiae Y-7524 6003 U73605 T A. cicatricosa Y-17594 6157 U40128 T A. monospora Y-1484 2554 U40106 T A. (Hormoascus) philentoma Y-7523 6276 U40113 T A. (Hormoascus) platypodis Y-6732 4111 U40083 T Arxiozyma telluris YB-4302 2685 U72158 T Arxula adeninivorans Y-17692 8244 U40094 Y-17693 7370 2 Y-17851 6461 2 Y-17993 2 T A. terrestris Y-17704 7376 U40103 T Ascoidea africana Y-17632 377.68 U40131 T A. corymbosa Y-17576 457.69 A A. hylecoeti Y-17634 355.80 U76198 Y-17703 0 A A. rubescens Y-17699 116.35 U76195 Y-17700 111.48 0 Y-17702 0 T Blastobotrys arbuscula Y-17585 227.83 U40108 T B. aristata Y-17579 521.75 U40109 T B. capitulata Y-17573 287.82 U40104 T B. elegans Y-17572 530.83 U40095 T B. nivea Y-17581 163.67 U40110 T B.