Multigene Phylogenetic Analysis of Pathogenic Candida Species in The

097

Multigene Phylogenetic Analysis of Pathogenic Candida Species in the Kazachstania (Arxiozyma) telluris Complex and Description of Their Ascosporic States as Kazachstania bovina sp. nov., K. heterogenica sp. nov., K. pintolopesii sp. nov., and K. slooffiae sp. nov. Cletus P. Kurtzman/* Christie J. Robnett,! Jerrold M. Ward,2 Cory Brayton,3 Peter Gorelick,4 and Thomas J. Walsh2 Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, u.s. Department ofAgriculture, Peoria, Illinois!,' Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, Bethesda, Marylanr£2; Center for Comparative Medicine, 3 The Baylor College ofMedicine, Houston, Texas ; and Animal Health Diagnostic Laboratory, National Cancer Institute at Frederick, SAIC Frederick, Frederick, Maryland4

Received 2 July 2004/Returned for modification 3 September 2004/Accepted 13 September 2004

A yeast causing widespread infection of laboratory mice was identified from 26S rRNA gene sequences as Candida pintolopesii. To determine the relationship of C. pintolopesii with other members of the Kazachstania (Arxiozyma) telluris species complex, nucleotide sequences from domains 1 and 2 of the 26S rRNA gene, the mitochondrial small-subunit rRNA gene, and the RNA polymerase II gene were phylogenetically analyzed. That analysis resolved the 48 strains examined into five closely related species: K. telluris, Candida bovina, C. pintolopesii, Candida sloojfiae, and a previously unknown species. One or more strains of each of the last four species formed an ascosporic state much like that of K. telluris. To place these ascosporogenous strains taxonomically, it is proposed that they be assigned to the teleomorphic genus Kazachstania as K. bovina (type strain NRRL Y-7283, CBS 9732, from the nasal passage of a pigeon), K. heterogenica (type strain NRRL Y-27499, CBS 2675, from rodent feces), K. pintolopesii (type strain NRRL Y-27500, CBS 2985, from the peritoneal fluid of a dead guinea pig), and K. sloojfiae (type strain NRRL YB-4349, CBS 9733, from the cecum of a horse). On the basis of multigene sequence analyses, K. heterogenica appears to be a hybrid ofK. pintolopesii and a presently unknown species. With the exception of K. bovina, the phylogenetically defined species show a moderate degree of host specificity.

During 2002, laboratory mice received by the ational In represent the anamorphic states of this ascosporogenous spe stitutes of Health from several different suppliers were infected cies (4, 19, 20). Mendon~a-Hagler and Phaff (12) tested this with a yeast, which rendered the mice unsuitable for laboratory possibility by measuring the extent of nuclear DA comple use. Domains 1 and 2 (Dl and D2, respectively) of the large mentarity among the four taxa. All four were reported to share subunit (26S) rR A genes of isolates from seven mice were 80% or greater nuclear DA relatedness, leading Mendonc;a sequenced in our laboratory, and the sequences were identical Hagler and Phaff (12) to propose that the four taxa represent to one another and to that of the type strain of Candida the same species. Hurt (2) reexamined the nuclear DNA re pintolopesii. Strains of a species generally have the same Dl latedness among the four taxa using a free-solution reassocia and D2 rR A gene sequences, or the sequences may some tion method rather than the filter-binding technique of Men times vary by 1 to 3 nucleotides (10). Consequently, from these donc;a-Hagler and Phaff (12). In contrast to the earlier results, sequence comparisons, we concluded that the mice were in Hurt (2) found only 20 to 56% nuclear DNA relatedness fected with C. pintolopesii. among the various pairings, which would suggest that the taxa C. pintolopesii is a member of the Kazachstania (Arxiozyma) represent four separate, but closely related, species. James et telluris species complex. Arxiozyma telluris was first described as al. (3) compared the four taxa by phylogenetic analysis of the Saccharomyces tellustris by van der Walt (17). Because the nucleotide sequences of the 18S rR A gene, 26S Dl and D2 ascospore walls of this species are roughened as a result of rRNA genes, and the mitochondrially encoded cytochrome surface ornamentation (5), van der Walt and Yarrow (18) oxidase II gene. These results again suggested that the taxa concluded that S. tellustris was not typical of the smooth-spored represent four separate but closely related species, and in view Saccharomyces sensu stricto species and redescribed it as A. of this genetic divergence, James et al. (3) proposed reinstate telluris. Candida bovina, C. pintolopesii, and Candida slooffiae ment of pintolopesii and slooffiae as distinct species. are phenotypically much like A. telluris and were believed to C. C. More recently, Kurtzman and Robnett (11) examined genus boundaries in the Saccharomyces complex by multigene se *' Corresponding author. Mailing address: Microbial Genomics and quence analysis. The ca. 80 species resolved into 14 clades, Bioprocessing Research Unit, ational Center for Agricultural Utili which were interpreted as genera, resulting in redescriptions of zation Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 . University St., Peoria, IL 61604. Phone: (309) some previously accepted genera and the proposal of five new 681-6561. Fax: (309) 681-6672. E-mail: [email protected]. genera (8). Clade 2 from the analysis included several species

101 102 KURTZM ET AL. J. eLi . MICROBIOL. of Saccharomyces and Kluyveromyces that were phylogeneti rection was used for neighbor-joining analyses. Bootstrap support for the phy cally unrelated to the type species of their respective genera, as logenetic trees was determined from 1,000 replications. well as species of the monotypic genera Arxiozyma, Kazach Nucleotide sequence accession numbers. The GenBank accession numbers of the sequences determined from the analysis of each gene, except for those of stania, and Pachytichospora. Of the last three genera, Kazach the RA polymerase II gene (GenBank accession numbers AY552467 to stania has taxonomic priority, resulting in the transfer of A. AY552472) and the mitochondrial small-subunit rRNA gene (GenBank acces telluris and other clade 2 species to Kazachstania. sion numbers AF442281 to AF442286), which were used for comparison with In the present study, we compared the mouse isolates with species in the Saccharomyces cerevisiae clade, are given on the phylogenetic trees. other strains of the K telluris complex and resolved five clades by Dl and D2 26S rR A gene sequence analysis, consistent RESULTS with the report of James et al. (3). We further compared these isolates by analysis of the mitochondrial small-subunit rR A Initial identification of yeasts from laboratory animals. In gene and RA polymerase II gene sequences. Analyses of the addition to the mice at the ational Cancer Institute that were last two data sets gave trees congruent with the tree con observed to have yeasts within the gastrointestinal tract, a line structed from the Dl and D2 sequences, supporting the con of knockout mice with a B6:129 background from that facility cept that C. pintolopesii, C. bovina, and C. slooffiae are distinct were shipped to another large research facility in another state species. The analyses also detected an additional, previously for rederivation and eventual research. The mice were rou unknown species. Microscopic examination of the cultures re tinely maintained in a receiving and quarantine room. The vealed that one or more strains from each of these four clades gastrointestinal infection had not been seen at that research formed ascospores. Consequently, we propose placement of facility for many years. The transported infected mice were the ascosporic states of C. pintolopesii, C. bovina, C. slooffiae, found to have gastric infection with chronic gastritis. DBN and the newly detected species in the teleomorphic genus 2 Cr mice placed in the same cage as infected null mice for 4 Kazachstania. months also developed gastric infection. Histopathology. The histopathological gastric changes con sisted of numerous but variable numbers of yeast-like organ MATERIALS AND METHODS isms in gastric epithelial surface debris and along the surfaces Initial identification of yeasts from laboratory animals. The yeast-like organ of epithelial cells throughout all regions of the glandular stom isms were first found during necropsy and by histopathology in sentinel and research mice of various strains and stocks from a facility where caging, food, and ach. Hematoxylin-eosin staining revealed that the organisms bedding were not autoclaved. These organisms were not observed in an adjacent were small, approximately 3 j.Lm in diameter, spherical to ovoid barrier facility where caging, food, and bedding were autoclaved. Similar organ yeast-like cells with a dark blue outer wall and some internal isms were found frequently in hamsters and sporadically in rat sentinels from the dot-like structures. These organisms were densely positive by same facility and in nude and heterozygous nude mice in a facility at an unrelated periodic acid-Schiff staining. Chronic inflammation with lym institu tion. Histology. Multiple tissues from laboratory animals were fixed in 10% neutral phocytes or myeloid cells was observed in a minority of ani buffered formalin and embedded in paraffin blocks. The specimens were sec mals. However, no inflammation was observed in most ani tioned and stained with hematoxylin-eosin and periodic acid-Schiff. mals. Organisms and culture media. Organisms from the infected mice at the Gene sequence comparisons. Phylogenetic analysis of the Dl ational Institutes of Health were cultured from stool specimens, gastric con tents, and homogenized tissues onto Sabouraud glucose agar supplemented with and D2 26S rR A gene nucleotide sequences resolved strains gentamicin and chloramphenicol. All strains examined in this study, their sources of the K telluris complex into five clades, with the level of of isolation, and observations on ascospore formation are provided in Table 1. intraclade divergence ranging from 1 to 3 nucleotides (Fig. 1). The composition of the culture media and the procedures for morphological The clades correspond to K telluris, C. bovina, C. pintolopesii, examination and characterization from fermentation and assimilation tests were C. slooffiae, and a previously unrecognized species. Analysis of given previously (25). Representative strains of each species group were tested with the seven fermentable sugars and by the 44 growth tests often used in yeast mitochondrial small-subunit rR A gene sequences also gave taxonomy (7). The small group of compounds likely to give positive responses five clades (Fig. 2) which were congruent with those in the tree was then tested with all strains in this study. Because growth responses were derived from the Dl and D2 sequences. However, analysis of sometimes weak, the yeast nitrogen base basal medium was supplemented with RA polymerase II gene nucleotide sequences resolved just 0.1 % yeast extract, which resulted in stronger positive reactions but which gave no false-positive results. Assimilation reactions were determined in liquid me four clades (Fig. 3). Strains of C. pintolopesii and the previously dium with manual shaking of the tubes every 2 to 3 days. Incubation was at 37°C. undetected species formed a single clade, which suggests that Yeast extract-malt extract-peptone-glucose (YM) agar slants were used for the new species is a hybrid of C. pintolopesii and another growth tests at 40 and 42°C. species that is not among those sequenced in this study. In the Ascospore formation. Strains were examined for ascospore formation on YM C. C. and McClary's acetate agars. The cultures were initially incubated for 2 days at work of James et al. (3), K telluris, bovina, pintolopesii, 37°C to facilitate growth and were then further incubated at 25°C to induce and C. slooffiae were resolved into separate clades from anal ascospore formation. The cultures were examined for ascospores at approxi ysis of the 18S rR A gene and Dl and D2 of the 26S rRNA mately weekly intervals for 4 weeks. gene, as shown here. Additionally, James et al. (3) sequenced Gene sequencing and phylogenetic analysis. Three gene regions were se the mitochondrially encoded COX II gene; and the analysis quenced: 01 and 02 (nucleotides 63 to 642) of the large-subunit (26S) rR A gene, the mitochondrial small-subunit rRNA gene bounded by primers MS-l and resolved C. pintolopesii and C. slooffiae into distinct popula MS-2, and the RA polymerase II gene (nucleotides 2447 to 3127). Primers and tions, but C. bovina and K telluris were found to be members details of the sequencing reactions were given by Kurtzman and Robnett (11). of the same clade. These results suggest that either K telluris or Sequences were determined on an Applied Biosystems model 3730 capillary C. bovina represents a hybrid species, which further suggests o A sequencer. that additional species of the K telluris complex are to be Estimates of phylogenetic relatedness among species were determined by using the maximum parsimony (MP) and neighbor-joining programs of the discovered and that interspecific hybridization in the complex PAUP*4.063a software package (16). The Kimura two-parameter distance cor- may be common. VOL. 43, 2005 PATHOGE IC KAZACHSTANIA SPECIES 103

TABLE 1. Sources of strains and their taxonomic designations as proposed in this study

Strain designation Ascospore Species Source (description of straint b NRRL CBSc formation Kazachstania bovina Y-7283T 9732 asal passage, pigeon, USA Y-27501 5812 Human, France + Y-27502 6264 asal passage, pigeon, USA + Y-27511 2760 Cecum, cow, Portugal (strain of Candida bovina) -? Y-27536 2678 Crop, turkey, UK

Kazachstania heterogenica Y-27499T 2675 Feces, rodent, ew Zealand + Y-2306 Lungs, spleen, mouse, Portugal -? Y-27537 2778 Cecum, rat, Portugal Y-27541 3046 Peritoneal cavity, mouse, USA -?

Kazachstania pintolopesii Y-27500T 2985 Peritoneal fluid, dead guinea pig, Indonesia Y-2251 Mouse, Paraguay? Y-2260 1787 Liver, mouse, Portugal (type strain of Candida pintolopesii) -? Y-7492 Stomach, mouse, USA Y-27461 Stomach, mouse, IH, USA Y-27462 Stomach, mouse, IH, USA -? Y-27463 Stomach, mouse, IH, USA + Y-27464 Stomach, mouse, IH, USA -? Y-27465 Feces, mouse, IH, USA Y-27466 Feces, mouse, IH, USA + Y-27467 Feces, mouse, IH, USA -? Y-27510 2676 Udder, mouse, The etherlands -? Y-27539 3011 Feces, rat, UK Y-27540 3045 Mouse, Uruguay Y-27542 3047 Gut, pigeon, USA Y-27543 3084 Gut, chinchilla, Canada Y-27544 6261 Unknown YB-1785 Mouse, USA + YB-2574 Rodent?, USA + YB-2576 Rodent?, USA -? YB-2577 Rodent?, USA YB-2578 Rodent?, USA -? YB-2579 Rodent?, USA YB-2580 Rodent?, USA YB-2581 Rodent?, USA YB-2582 Rodent?, USA -? YB-3291 Lungs, spleen, mouse, USA YB-3357 Intestines, rat, USA YB-3360 Intestines, rat, USA -? YB-3359 Intestines, rat, USA

Kazachstania slooffiae YB-4349T 9733 Cecum, horse, Portugal Y-27498 2419 Cecum, horse, Portugal (type strain of Candida slooffiae) Y-27538 2783 Cecum, horse, Portugal Y-27546 4068 Cecum, pig, Portugal YB-4351 Cecum, horse, Portugal

Kazachstania (Arxiozyma) telluris YB-4302T 2685 Soil, South Africa ++ Y-7284 6258 asal passage, pigeon, USA +++ Y-7285 asal passage, pigeon, USA ++ Y-27545 6265 asal passage, pigeon, USA +

a Abbreviations: IH, ational Institutes of Health; UK, United Kingdom; USA, United States. b -, no ascospores; -?, possible poorly formed ascospores; ,infrequent ascospores; , occasional ascospores; + , ascospores relatively common. C CBS, Centraalbureau voor Schimmelcultures, Utrecht, The etherlands.

Do the clades seen in this study and that of James et al. (3) study, K telluris and C. bovina, differed from one another by ca. represent individual species or genetically unique populations 2% substitutions (13 of 575 positions). The divergence in the within a single species? On the basis of earlier comparisons, mitochondrial small-subunit rR A gene and the RA poly strains that differ from one another by 1% or greater nucleo merase II gene data sets is also consistent with the concept of tide substitutions in the D1 and D2 domains of the 26S rR A separate species. For comparison, there was greater divergence gene have proven to be separate species on the basis of the among populations of the K telluris clade than among species results from genetic crosses and from the extent of nuclear of the S. cerevisiae clade in the D1 and D2 26S rR A gene DNA complementarity, as measured from DA reassociation sequences (Fig. 4), as well as for divergence in the mitochon analysis (10). The most closely related species pair in this drial small-subunit rR A gene and the RNA polymerase II 104 KURTZM ET AL. J. ell . MICROBIaL.

T YB-4302 / U72158 56 2 1 Y-7285/ AY545568 K. tel/uris 86 Y-27545/ AY545569 4 I Y-7284/ AY545570 T Y-7283 / AY545571 I Y-27501 .....;1~OO=--- +=6~31 TA L...-__ __ Y_27511 / AY545572 K. bovina 1 8 1 Y-27536 Y-27502 T Y-27500 / AY545573 Y-2251 TA Y_2260 / AY545574 Y-7492 Y-27461 Y-27462 Y-27463 Y-27464 Y-27465 Y-27466 Y-27467 Y-27510 ~ Y-27540 1 Y-27542 98 Y-27543 K. pint%pesii Y-27544 YB-1785 YB-2574 57 YB-2576 YB-2577 YB-2578 YB-2579 YB-2580 YB-2581 YB-2582 Y-27539 / AY545575 62 YB-3357 9 1 YB·3359 ~1 YB-3360 YB-3291 / AY545576 T 1""" Y-27499 / AY545577 L...--.=...;91~ Y-2306 K. heterogenica 3 Y-27537 Y-27541/ AY545578 T YB-4349 / AY545579 TA j§ Y_27498 / AY545580 99 1 Y-27546 K. s/ooffiae 1------=.:13=------~ YB-4351 1 Y-27538/ AY545581 L-- N. castelli; Y-12630 / U68557 36 FIG. 1. One of four most parsimonious trees from MP analysis of the Dl and D2 26S r A gene sequences from species of the K telluris clade. Tree length = 95, consistency index = 0.853, retention index = 0.956, rescaled consistency index = 0.815, homoplasy index = 0.147. Bootstrap values ;::::50% are given above the branches; and branch lengths, indicated as the number of steps, are given below the branches. Naumovia castellii was the designated outgroup species. Strains are designated by RRL accession numbers followed by GenBank accession numbers for strains with unique DA sequences. T, type strain of the designated Kazachstania species; TA, type strain of the previously described Candida anamorph. VOL. 43, 2005 PATHOGE IC KAZACHSTANIA SPECIES 103

TABLE 1. Sources of strains and their taxonomic designations as proposed in this study

Strain designation Ascospore Species Source (description of straint formationb NRRL CBSc Kazachstania bovina Y-7283T 9732 asal passage, pigeon, USA + Y-27501 5812 Human, France + Y-27502 6264 asal passage, pigeon, USA + Y-27511 2760 Cecum, cow, Portugal (strain of Candida bovina) -? Y-27536 2678 Crop, turkey, UK

Kazachstania heterogenica Y-27499T 2675 Feces, rodent, ew Zealand + Y-2306 Lungs, spleen, mouse, Portugal -? Y-27537 2778 Cecum, rat, Portugal Y-27541 3046 Peritoneal cavity, mouse, USA -?

Kazachstania slooffiae YB-4349T 9733 Cecum, horse, Portugal + Y-27498 2419 Cecum, horse, Portugal (type strain of Candida slooffiae) Y-27538 2783 Cecum, horse, Portugal Y-27546 4068 Cecum, pig, Portugal YB-4351 Cecum, horse, Portugal

Kazachstania (Arxiozyma) telluris YB-4302T 2685 Soil, South Africa +++ Y-7284 6258 asal passage, pigeon, USA +++ Y-7285 asal passage, pigeon, USA ++ Y-27545 6265 asal passage, pigeon, USA +

a Abbreviations: IH, National Institutes of Health; UK, United Kingdom; USA, United States. b -, no ascospores; -?, possible poorly formed ascospores; +, infrequent ascospores; +, occasional ascospores; +++, ascospores relatively common. C CBS, Centraalbureau voor Schimmelcultures, Utrecht, The etherlands.

Do the clades seen in this study and that of James et al. (3) study, K telluris and C. bovina, differed from one another by ca. represent individual species or genetically unique populations 2% substitutions (13 of 575 positions). The divergence in the within a single species? On the basis of earlier comparisons, mitochondrial small-subunit rR A gene and the RA poly strains that differ from one another by 1% or greater nucleo merase II gene data sets is also consistent with the concept of tide substitutions in the Dl and D2 domains of the 265 rR A separate species. For comparison, there was greater divergence gene have proven to be separate species on the basis of the among populations of the K telluris clade than among species results from genetic crosses and from the extent of nuclear of the S. cerevisiae clade in the Dl and D2 265 rR A gene DA complementarity, as measured from DA reassociation sequences (Fig. 4), as well as for divergence in the mitochon analysis (10). The most closely related species pair in this drial small-subunit rRNA gene and the RNA polymerase II 104 KURTZ ET AL. J. eLi . MICROBIaL.

T YB-4302 / U72158 2 56 Y-7285/ AY545568 86 1 K. tel/uris Y-27545/ AY545569 4 I Y-7284/ AY545570 T Y-7283 / AY545571 4 31 Y-27501 100 TA 63 Y_27511 / AY545572 K. bovina 8 11 Y.27536 Y-27502 T Y-27500 / AY545573 Y-2251 TA Y_2260 / AY545574 Y-7492 Y-27461 4 Y-27462 Y-27463 Y-27464 Y-27465 Y-27466 Y-27467 Y-27510 ~ Y-27540 1 Y-27542 98 4 Y-27543 K. pint%pesii Y-27544 YB-1785 YB-2574 57 9 YB-2576 YB-2577 YB-2578 YB-2579 YB-2580 YB-2581 YB-2582 Y-27539 / AY545575 62 YB·3357 { YB-3359 1 YB-3360 YB-3291 / AY545576 T '"1 Y-27499 / AY545577 91 Y-2306 K. heterogenica 3 Y-27537 Y-27541 / AY545578 T YB-4349 / AY545579 TA ~ Y-27498 / AY545580 1 99 Y-27546 K. s/ooffiae 13 YB-4351 ""1 Y-27538 / AY545581 N. caste/Iii Y-12630 / U68557 36 FIG. 1. One of four most parsimonious trees from MP analysis of the 01 and 02 26S rR A gene sequences from species of the K telluris clade. Tree length = 95, consistency index = 0.853, retention index = 0.956, rescaled consistency index = 0.815, homoplasy index = 0.147. Bootstrap values ;::::50% are given above the branches; and branch lengths, indicated as the number of steps, are given below the branches. aumovia castellii was the designated outgroup species. Strains are designated by RRL accession numbers followed by GenBank accession numbers for strains with unique 0 A sequences. T, type strain of the designated Kazachstania species; TA, type strain of the previously described Candida anamorph. VOL. 43, 2005 PATHOGE IC KAZACHSTANIA SPECIES 105

T ""3 YB-4302 / AY548473 five population groups in the K telluris clade appear to repre Y-7284/ AY548474 sent separate species. K. tel/uris 76 Y-7285 Habitat and host specificity. Members of the K telluris spe ~3 66 Y-27545 cies complex are mainly known from infections in rodents and T ~ Y-7283 / AY548475 birds and to a lesser extent in humans, horses, pigs, and cows II Y-27501 (Table 1). The clades determined from sequence analysis allow 1 Y-27502 K. bovina analysis of whether there is host specificity among these spe TA Y-27511 cies. As shown in Table 1, strains of C. slooffiae were from Y-27536 horses and a pig, C. pintolopesii and the new species were T Y-27500 /AY548476 almost exclusively from rodents, and K telluris was isolated 1" Y-2251 / AY548477 from soil and the nasal passages of pigeons. The least host Y_2260 TA specificity was shown by C. bovina, which has been isolated Y-7492 from a cow, birds, and a human. On the basis of the hybrid Y-27461 nature of the RRL Y-27499 clade and the apparent hybrid Y-27462 detected by James et al. (3), it appears that there are additional closely related species yet to be discovered within the Y-27463 K telluris complex, some of which may be specific to other animal hosts. Y-27464 Description of new ascosporic species. On the basis of the Y-27465 molecular analyses described above, five distinct species have Y-27466 88 been resolved in the K telluris complex: K telluris, C. bovina, C. Y-27467 pintolopesii, C. slooffiae, and a previously unrecognized species. Y-27510 The last four species were found to form ascospores, and on 57 Y-27540 1 the basis of multigene phylogenetic analyses, their placement Y-27542 in the teleomorphic genus Kazachstania is proposed. Y-27543 K. pint%pes;; Latin diagnosis ofKazachstania bovina Kurtzman & Robnett Y-27544 sp. nov. In agaro YM post dies 2 ad 3rc cellulae vegetativae YB-H85 globosae (2.8 to 6.0 joLm) aut ellipsoideae (2.8-7.1 X 3.8-11.0 YB-2574 fJ..m), singulae, binae et catenas brevis. In agaro morphologico YB-2576 post dies 7 ad 3rC, incrementum fuscum pallidum, nitens aut YB-2577 hebes et butyrosum; centrum coloniae deprimere; margo glabro Jl. YB-2578 vel lobate. Pseudohyphae et hyphae verae non fiunt. Species ho 3 YB-2579 mothallica. Asci liberi, ascospora globosa singulara, non liberi. "1 YB-2580 / AY548478 Glucosum fermentatur. Galactosum, sucrosum, maltosum, lac YB-2581 tosum, raffinosum et trehalosum non fermentantur. Assimilantur YB-2582 glucosum, ethanolum, DL-acidum lacticum (varia bile) et acidum Y-27539/ AY548479 succinicum (variabile). Non assimilantur galactosum, L-sorbo YB-3291 sum, maltosum, sucrosum, cellobiosum, trehalosum, lactosum, YB-3357 melibiosum, raffinosum, melezitosum, inulinum, amylum solu YB-3359 bile, D-xylosum, L-arabinosum, D-arabinosum, D-ribosum, L-rham 64 YB-3360 nosum, D-glucosaminum, N-acetyl-D-glucosaminum, glycerolum, T ~ Y-27499 / AY548480 methanolum, erythritolum, ribitolum, galactitolum, mannitolum, 11 7 Y-2306/ AY548481 K. heterogenica glucitolum, methyl a-D-glucosidum, salicinum, D-gluconas, 2-ke 1 Y-27537 to-D-gluconas, 5-keto-D-gluconas, saccharatas, acidum citricum, Y-27541 inositolum, hexadecanum et potassii nitras. Augmentum non fi YB-4349T / AY548482 I unt in 10% sal-5% glucosum. Amylum non formatur. Vitaminae ~_1..:..:0;,.=.O_~I Y-27498TA K. s/ooffiae externae ad crescentiam necessaria sunt. Augmentum fiunt in 9 I YB-4351 temperatura 40 et 42°C (variabile). Species nova a speciebus aliis jt 45 N. castellii Y-12630 / AY548483 sequentibus nucleotiditis D1 et D2 26S rRNA gene, mitochondrial submonas parvus rRNA gene et RNA polymerase II distinguenda. FIG. 2. One of 100 most parsimonious trees from MP analysis of Typus: NRRL Y-7283 (CBS 9732) designat stirpem typicam. Iso mitochondrial small-subunit rR A gene sequences from species of the K. telluris clade. Tree length = 79, consistency index = 0.924, retention lata naris Columba livida, USA, depositata in Collectione Cul index = 0.947, rescaled consistency index = 0.875, homoplasy index = turarum ARS (NRRL), Peoria, Ill. 0.076. Other designations are as defined in the legend to Fig. 1. Be Description ofKazachstania bovina Kurtzman & Robnett, sp. cause of sequencing difficulties, RRL Y-27538 and RRL Y-27546 nov. Vegetative reproduction. After growth for 2 days on YM were excluded from the analysis. agar at 37°C, the cells are spherical (2.8 to 6.0 joLm) to ellip soidal (2.8 to 7.1 x 3.8 to 11.0 joLm) and occur singly, in pairs, gene sequences (data not shown). Members of the S. cerevisiae or in short chains (Fig. 5A). Budding is multilateral. Growth is clade have been recognized as separate species from nuclear white to tannish white, semiglistening, and butyrous. Aerobic DNA reassociation analyses (21, 22), gene sequence compar growth after 7 days at 37°C in Dalmau plate culture on yeast isons (10, 11, 14), and genetic crosses (13). By comparison, the morphology agar is tannish white, dull to semiglistening, and 106 KURTZMAN ET AL. J. CUN. MICROBIOL.

r------YB-4302 / AF527897 58 9 100 1 Y·7284/ AY548486 K. tel/uris L...----;.;:.14~----t 84 Y·7285/ AY548487 2 Y.27545 T ~ Y·7283 / AY548488 Y·27501 L..- ---'-1.;..;00'-- __t Y-27502 K. bovina 18 TA Y·27511 / AY548489 Y·27536 Y·27500T /AY548490 TA """2 Y·2260 / AY548491 Y·27461 Y·27462 Y·27464 - 2 Y.27543 YB·2574 YB·2579 YB·2582 86 YB·3291 15 Y·2251 Y·7492 Y-27463/ AY548485 Y-27465 J Y-27466/ AY548493 K. pint%pesii 1] YB-2577 "1 YB-3357 l' Y-27467 Y·27539 Y-27542 Y-27544 YB-2578 YB-3359 Y-27510 / AY548494 80 Y-27540 13 YB-1785 YB·2576 YB-2580 YB-2581 YB-3360 T Y-27499 / AY548495 ~~ Y-2306/ AY548496 K. heterogenica 2 Y-27537 Y-27541 T YB-4349 / AY548497 Y-27498 TA t-- .;..;10:"-0 -i Y-27538 16 K. s/ooffiae Y-27546 YB-4351

FIG. 3. One of 100 most parsimonious trees from MP analysis of RA polymerase II nucleotide sequences from species of the K. telluris clade. Tree length = 198, consistency index = 0.798, retention index = 0.926, rescaled consistency index = 0.739, homoplasy index = 0.202. Other designations are as defined in the legend to Fig. 1.

butyrous with a depressed center. Margins are entire to finely formed on YM and McClary's acetate agars after 10 to 20 days lobed. either hyphae nor pseudohyphae are formed under at 25°C; asci are persistent and unconjugated and contain a the cover glass. single, finely roughened ascospore (Fig. 5B). Although asci are Ascospore formation. Three of the five available strains unconjugated, the occasional conjugations observed between formed ascospores, albeit at a low frequency. Ascospores independent cells, as well as apparent conjugations between VOL. 43, 2005 PATHOGE IC KAZACHSTANIA SPECIES 107

K. tel/uris Latin diagnosis of Kazachstania heterogenica Kurtzman & YB-4302 Robnett sp. nov. In agaro YM post dies 2 ad 3rC, cellulae U72158 vegetativae globosae (4.0 to 8.0 ,un) aut ellipsoideae (2.1-7.0 X K. bovina 4.0-12.0 ,un), singulae, binae et catenas brevis. In agaro mor L-..__ Y-7283 55 phologico post dies 7 ad 3rC, incrementum fuscum pallidum, AY545571 5 nitens aut hebes et butyrosum; centrum coloniae planae; margo K. pintolopesii glabro vel lobate. Pseudohyphae et hyphae verae non fiunt. Spe 84 Y-27500 12 AY545573 cies homothallica. Asci liberi, ascospora globosa singulara, non K. heterogenica liberi. Glucosum fermentatur. Galactosum, sucrosum, maltosum, 100 Y-27499 lactosum, raffinosum et trehalosum non fermentantur. Assimilan 27 AY545577 tur glucosum, ethanolum (variabi/e) et DL-acidum lacticum (va K. slooffiae riabi/e). Non assimi/antur galactosum, L-sorbosum, maltosum, YB-4349 sucrosum, cellobiosum, trehalosum, lactosum, melibiosum, raffi AY545579 nosum, melezitosum, inulinum, amylum solubi/e, D-xylosum, L S. cerevisiae arabinosum, D-arabinosum, D-ribosum, L-rhamnosum, D-glucos Y-12632 aminum, N-acetyl-D-glucosaminum, glycerolum, methanolum, U44806 erythritolum, ribitolum, galactitolum, mannitolum, glucitolum, methyl a-D-glucosidum, salicinum, D-gluconas, 2-keto-D-gluconas, 5-keto-D-gluconas, saccharatas, acidum succinicum, acidum cit ricum, inositolum, hexadecanum et potassii nitras. Augmentum S. cariocanus non fiunt in 10% sal-5% glucosum. Amylum non formatur. Y-27337 AY046086 Vitaminae extemae ad crescentiam necessaria sunt. Augmentum fiunt in temperatura 40°C (variabile), non 42°C. Species nova a S. mikatae Y-27341 speciebus aliis sequentibus nucleotiditis D1 et D2 26S rRNA gene AY046087 et mitochondrial submonas parvus rRNA gene distinguenda. Typus: NRRL Y-27499 (CBS 2675) designat stirpem typicam. 99 S. kudriavzevii 10 Y-27339 Isolata ex feces, rodentia incognita, Nova Zealandia, depositata AY046088 in NRRL. S.bayanus Description of Kazachstania heterogenica Kurtzman & Rob Y-12624 nett, sp. nov. Reproduction is vegetative. Mter growth for U94931 2 days on YM agar at 37°C, the cells are spherical (4.0 to 8.0 N. castellii J-lm) to ellipsoidal (2.1 to 7.0 x 4.0 to 12.0 J-lm) and occur singly L-.._---- Y-12630 17 in pairs or in short chains (Fig. 5C). Budding is multilateral. U68557 Growth is tannish white, semiglistening, and butyrous. Aerobic FIG. 4. Comparison of divergence among type strains of species of growth after 7 days at 37°C in Dalmau plate culture on yeast Saccharomyces and Kazachstania from MP analysis of the D1and D2 domains of the 26S r A gene sequences. One of three most parsi morphology agar is tan in color, dull to semiglistening, and monious trees is shown. Tree length = 126, consistency index = 0.794, re butyrous in texture. Colonies are low and fiat and have finely tention index = 0.867, rescaled consistency index = 0.688, homoplasy lobed margins. either hyphae nor pseudohyphae are formed index = 0.206. Other designations are as defined in the legend to Fig. 1. under the cover glass. Ascospore formation. Of the four strains examined, only buds and their parent cells, suggest that the species is ho RRL Y-27499 formed ascospores. Ascosporulation is quite mothallic. sparse and occurs on YM and McClary's acetate agars after 10 Fermentation, assimilation, and other growth reactions. to 15 days at 25°C. Asci are persistent and unconjugated and Glucose is the only sugar fermented in standard tests; and only contain a single, finely roughened ascospore (Fig. 5D). Occa glucose, ethanol, lactic acid (variable), and succinic acid (vari sionally, conjugation between independent cells as well as con able) are assimilated (Table 2). All strains grow at 40°C, but jugation between a bud and its parent cell is observed, which only three of the five strains grow at 42°C (Table 2). suggests that the species is homothallic. Type strain. RRL Y-7283 (CBS 9732) is preserved in the Fermentation, assimilation, and other growth reactions. lyophilized state, as well as in liquid nitrogen storage, in the Glucose is the only sugar fermented. Growth occurs on glu Agricultural Research Service Culture Collection, RRL, Pe cose, ethanol (variable), and DL-lactic acid (variable). Three of oria, Ill. The strain was received in 1971 from H. F. Hasen the four known strains grow at 40°C, but none grow at 42°C clever, ational Institutes of Health, Bethesda, Md., as strain (Table 2). B-3197 and had been isolated from the nasal passage of a Type strain. RRL Y-27499 (CBS 2675) is preserved in the pigeon. This isolate was selected as the type strain because it lyophilized state, as well as in liquid nitrogen storage, at produced the greatest number of ascospores of the three as RRL. The strain was isolated from the feces of an unidenti cosporogenous strains examined. fied rodent in ew Zealand. Etymology. The species name bovina comes from that of the Etymology. The species name heterogenica refers to the pos anamorphic state, Candida bovina van Uden & do Carmo sibility that this species is a hybrid between C. pintolopesii and Sousa. a presently unknown species. 108 KURTZMAN ET AL. J. CLI . MICROBIaL.

FIG. 5. (A and B) K bovina RRL Y-7283: (A) budding cells; (B) asci with a single ascospore, YM agar, 15 days. (C and D) K heterogenica RRL Y-27499: (C) budding cells; (D) ascus with an ascospore, YM agar, 25 days. (E and F) K pintolopesii RRL Y-27500: (E) budding cells; (F) ascus with an ascospore, YM agar, 23 days. (G to J) K slooffiae RRL YB-4349: (G) budding cells; (H) pseudohyphae, Dalmau plate with yeast morphology agar, 7 days, 37°C ( RRL Y-27498); (I) asci with a single ascospore, acetate agar, 22 days; (J) conjugation between two cells, acetate agar, 22 days. (K and L) K lelluris RRL YB-4302: (K) budding cells; (L) asci with ascospores, acetate agar, 8 days. For all panels, budding cells, YM agar, 2 days, 3rC and ascospore formation, 25°C. Bar,S J.Lm.

Latin diagnosis of Kazachstania pintolopesii Kurtzman, Rob maltosum, lactosum, raffinosum et trehalosum non fermentantur. nett, J. . Ward & T. J. Walsh, sp. nov. In agaro YM post dies Assimilantur glucosum, ethanolum (varia bile), et DL-acidum 2 ad 3'rC, cellulae vegetativae globosae (2.1 to 6.0 J.Lm) aut lacticum (variabile). Non assimilantur galactosum, L-sorbosum, ellipsoideae (2.1-8.0 X 2.5-11.0 J.Lm), singulae et binae. In agaro maltosum, sucrosum, cellobiosum, trehalosum, lactosum, meli morphologico post dies 7 ad 3'rC, incrementum fuscum palli biosum, raffinosum, melezitosum, inuhnum, amylum solubile, dum, nitens aut hebes, butyrosum; centrum coloniae deprimere D-xylosum, L-arabinosum, D-arabinosum, D-ribosum, L-rhamno aut planae, margo lobate. Pseudohyphae et hyphae verae non sum, D-glucosaminum, N-acetyl-D-glucosaminum, glycerolum, fiunt. Species homothallica. Asci liberi, ascospora globosa singu methanolum, erythritolum, ribitolum, galactitolum, mannitolum, lara, non liberi. Glucosum fermentatur. Galactosum, sucrosum, glucitolum, methyl a-D-glucosidum, salicinum, D-gluconas, 2-ke- VOL. 43, 2005 PATHOGE IC KAZACHSTANIA SPECIES 109

TABLE 2. Physiological and morphological characteristics of Kazachstania species in the K telluris clade

Growth" Glucose Presence of Species DL-Lactic Succinic 100/£ aCI-5% fermentation Glucose Ethanol 40°C 42°C pseudohyphae acid acid glucose

K bovina 5/5 b 5/5 5/5 4/5 2/5 0/5 5/5 3/5 0/5 K heterogenica 4/4 4/4 3/4 3/4 0/4 0/4 3/4 0/4 0/4 K pintolopesii 29/29 29/29 4/29 13/29 0/29 0/29 29/29 1/29 0/29 K slooifiae 5/5 5/5 0/5 0/5 0/5 0/5 5/5 2/5 5/5 K telluris 4/4 4/4 4/4 4/4 0/4 2/4 4/4 4/4 0/4

a The full set of seven sugars and 44 growth tests typically used for strain characterization are given by Kurtzman (7) and are also listed in the Latin description of each new species. b Data represent number of strains positive/total number of strains tested.

to-D-gluconas, 5-keto-D-gluconas, saccharatas, acidum succinicum, 4.8-14.0 fJ-rn), singulae, binae et catenas brevis. In agaro mor acidum citricum, inositolum, hexadecanum et potassii nitras. phologico post dies 7 ad 37C, incrementum fuscum pallidum, Augmentum non fiunt in 10% sal-5% glucosum. Amylum non nitens aut hebes et butyrosum. Centrum coloniae planae, margo formatur. Vitaminae externae ad crescentiam necessaria sunt. lobate. Pseudohyphaefiunt, hyphae verae nonfiunt. Species homo Augmentum fiunt in temperatura 40 et 42°C (variabile). Species thallica. Asci liberi, ascospora globosa singulara, non liberi. Gluco nova a speciebus aliis sequentibus nucleotiditis D1 et D2 26S sum fermentatur. Galactosum, sucrosum, maltosum, lactosum, raf rRNA gene, mitochondrial submonas parvus rRNA gene et RNA finosum et trehalosum non fermentantur. Assimilanturglucosum. polymerase II distinguenda. Typus: NRRL Y-27500 (CBS 2985) Non assimilantur galactosum, L-sorbosum, maltosum, sucrosum, designat stirpem typicam. Isolata humor peritonealis ex Cavia cellobiosum, trehalosum, lactosum, melibiosum, raffinosum, me porcellus, Iabada, depositata in NRRL. lezitosum, inulinum, amylum solubile, D-xylosum, L-arabinosum, Description of Kazachstania pintolopesii Kurtzman, Robnett, D-arabinosum, D-ribosum, L-rhamnosum, D-glucosaminum, J. N. Ward & T. J. Walsh, sp. nov. Vegetative reproduction. N-acetyl-D-glucosaminum, ethanolum, glycerolum, methanolum, After growth for 2 days on YM agar at 37°C, the cells are erythritolum, ribitolum, galactitolum, mannitolum, glucitolum, spherical (2.1 to 6.0 fJ-m) to ellipsoidal (2.1 to 8.0 X 2.5 to 11.0 methyl a-D-glucosidum, salicinum, D-gluconas, 2-keto-D-gluconas, fJ-m) and occur singly or in pairs (Fig. 5E). Budding is multi 5-keto-D-gluconas, saccharatas, DL-acidum lacticum, acidum suc lateral. Growth is tannish white, semiglistening, and butyrous. cinicum, acidum citricum, inositolum, hexadecanum et potassii Aerobic growth after 7 days at 37°C in Dalmau plate culture on nitras. Augmentum non fiunt in 10% sal-5% glucosum. Amylum yeast morphology agar is tannish white, semiglistening, and non formatur. Vitaminae externae ad crescentiam necessaria butyrous. Colonies may be flat or have a depressed center and sunt. Augmentum fiunt in temperatura 40 et 42°C (variabile). have a finely lobed margin. either hyphae nor pseudohyphae Species nova a speciebus aliis sequentibus nucleotiditis Dl et D2 are formed under the cover glass. 26S rRNA gene, mitochondrial submonas parvus rRNA gene et Ascospore formation. Five of the 30 available strains formed RNA polymerase II distinguenda. Typus: NRRL YB-4349 (CBS ascospores. Ascosporulation occurs on YM and McClary's ac 9733) designat stirpem typicam. Isolata ex caecum, Equus cabal etate agars after 10 to 15 days at 25°C. Ascospore formation is lus, Portugallia, depositata in NRRL. generally sparse with one finely roughened ascospore in each Description of Kazachstania slooffiae Kurtzman & Robnett persistent ascus (Fig. 5F). Asci are unconjugated, but the pres sp. nov. Vegetative reproduction. After growth for 2 days on ence of apparent conjugations between individual cells and YM agar at 37°C, the cells are spherical (4.0 to 11.0 fJ-m) to between cells and their buds suggests that the species is ho ellipsoidal (3.0 to 11.5 X 4.8 to 14.0 fJ-m) and occur singly in mothallic. pairs or infrequently in short chains (Fig. 5G). Budding is Fermentation, assimilation, and other growth tests. Glucose multilateral. Growth is tannish white, semiglistening, and bu is the only sugar fermented; and only glucose, ethanol (vari tyrous. Aerobic growth after 7 days at 37°C in Dalmau plate able), and DL-lactic acid (variable) are assimilated. All strains culture on yeast morphology agar is white to tannish white, grow at 40°C, but just one strain grows at 42°C (Table 2). semiglistening, and butyrous in texture. Colonies are low and Type strain. RRL Y-27500 (CBS 2985) is preserved in flat and have finely lobed margins. Sparingly differentiated the lyophilized state, as well as in liquid nitrogen storage, pseudohyphae are formed under the cover glass (Fig. 5H), but at RRL. The strain was isolated from the peritoneal fluid true hyphae are not formed. of a dead guinea pig in Indonesia. This isolate was select Ascospore fonnation. Of the five strains examined, only ed as the type strain because it produced the greatest num NRRL YB-4349 formed ascospores. Ascosporulation is infre ber of ascospores of the five ascosporogenous strains exam quent and occurs on YM agar after 15 to 20 days at 25°C. Asci ined. are unconjugated and persistent and form a single roughened Etymology. The species name pintolopesii comes from that of ascospore (Fig. 51). Although asci are unconjugated, apparent the anamorphic state, Candida pintolopesii (van Uden) S. A. conjugations between individual cells (Fig. 51), as well as be Meyer & Yarrow. tween cells and their buds, are observed and are interpreted as Latin diagnosis of Kazachstania slooffiae Kurtzman & Rob evidence that the species is homothallic. nett, sp. nov. In agaro YM post dies 2 ad 37C, cellulae vegeta Fermentation, assimilation, and other growth tests. Glucose tivae globosae (4.0 to 11.0 fJ-m) aut ellipsoideae (3.0-11.5 X is the only sugar fermented and assimilated. All strains grow at 110 KURTZMAN ET AL. J. CLI . MICROBIOL.

40°C, but three of the five strains available fail to grow at 42°C ACKNOWLEDGMENTS (Table 2). Amy Morgan is gratefully acknowledged for skillful operation of the Type strain. RRL YB-4349 (CBS 9733) is preserved in nucleic acid sequencer and oligonucleotide synthesizer, Eleanor Base the lyophilized state, as well as in liquid nitrogen storage, at hoar-Powers is acknowledged for conducting fermentation and assim NRRL. The strain was received from . van Uden and had ilation tests, Grace Lidl is acknowledged for assistance in identifying infected animals, and Don Fraser is acknowledged for preparation of been isolated from the cecum of a horse in Portugal. the final figures. Etymology. The species name slooffiae comes from that of The mention of firm names or trade products does not imply that the anamorphic state, Candida slooffiae van Uden & do they are endorsed or recommended by the U.S. Department of Agri Carmo-Sousa. culture over other firms or similar products not mentioned. This study was supported, in part, with federal funds from the CI Comparisons with K. tel/uris. The vegetative morphology of under contract NOl-CO-56000 to SAlC Frederick. K telluris is indistinguishable from that of the four new species described here (Fig. 5K). The one exception is that strains of REFERE CES the C. slooffiae clade produce relatively undifferentiated 1. Groth, c., J. Hansen, and J. Piskur. 1999. A natural chimeric yeast contain pseudohyphae (Fig. 5H), whereas strains of the other species ing genetic material from three species. Int. J. Syst. Bacteriol. 49:1933-1938. 2. Hurt, R. A. 1997. A molecular analysis of the relatedness of anamorphic do not. Ascosporulation by strains of K telluris appears to be yeasts currently classified as Candida pintolopesii. Ph.D. thesis. University of identical to that of the new species (Fig. 5L), although asci Tennessee, Knoxville. from K telluris may form two ascospores rather than one. As 3. James, S. A., M. D. Collins, and I. N. Roberts. 2001. Phylogenetic analysis of the psychrophobic yeast Ar.xiozyma telluris and the reinstatement of Candida with the other species, K telluris ferments only glucose. AIl pintolopesii (van Uden) Meyer et Yarrow and Candida slooffii van Uden et do four strains of K telluris tested assimilate glucose, ethanol, and Carmo-Sousa. Int. J. Syst. Evol. Microbiol. 51:1917-1925. 4. Kreger-van Rij, . J. W. 1958. The relationship between Saccharomyces DL-Iactic acid; and two of the strains grow in a medium with tellustris and Candida bovina. Antonie Leeuwenhoek 24:137-144. 10% aCI and 5% glucose. The four strains grow at both 40 5. Kreger-van Rij, N. J. W. 1966. Taxonomy of the genus Pichia, p. 51-58. In A. and 42°C (Table 2). Kockova-Kratochvilova (ed.), Proc. 2nd Int. Symp. Yeasts. Slovak Academy of Sciences, Bratislava, Czecholslovakia. 6. Kurtzman, C. P. 1987. Prediction of biological relatedness among yeasts from comparisons of nuclear D. A complementarity, p. 459-468. In G. S. de Hoog, M. T. Smith, and A. C. M. Wiejman (ed.), The expanding realm of DISCUSSIO yeast-like fungi. Elsevier, Amsterdam, The etherlands. 7. Kurtzman, C. P. 1998. Ar.xiozyma van der Walt & Yarrow, p. 134-135. In For more than 20 years and from reviews of several thou C. P. Kurtzman and J. W. Fell (ed.), The yeasts, a taxonomic study, 4th ed. sand sentinel animals, gastric infections in mice have seldom Elsevier Science BY, Amsterdam, The etherlands. 8. Kurtzman, C. P. 2003. Phylogenetic circumscription of Saccharomyces, been observed (15); but within the past 2 years numerous Kluyveromyces and other members of the Saccharomycetaceae, and the pro animals with gastric yeast infections have been found. Whether posal of the new genera Lachancea, Nakaseomyces, Naumovia, Vanderwal tozyma and Zygotorulaspora. FEMS Yeast Res. 4:233-245. this increase in frequency of observed infections reflects the 9. Kurtzman, C. P., and H. J. Phaff. 1987. Molecular taxonomy, p. 63-94. In emergence of a more virulent yeast, decreased resistance to A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 1, 2nd ed. Academic infection among present-day mouse breeds, or pathogen intro Press, London, United Kingdom. 10. Kurtzman, C. P., and C. J. Robnett. 1998. Identification and phylogeny of duction through poor sanitation is uncertain. However, mouse ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal colonies that received sterilized food, bedding, and cages did DA partial sequences. Antonie Leeuwenhoek 73:331-371. 11. Kurtzman, C. P., and C. J. Robnett. 2003, Phylogenetic relationships among stay free of infection. Consequently, the microbiological iden yeasts of the I Saccharomyces complex' determined from multigene sequence tity and mode of transmission reported here for these yeasts analyses. FEMS Yeast Res. 3:417-432. may alert others to a serious source of infection in laboratory 12. Mendon"a-Hagler, L. C., and H. J. Phaff. 1975. Deoxyribonucleic acid base composition and deoxyribonucleic acid/deoxyribonucleic acid hybrid for mice. mation in psychrophobic and related yeasts. Int. J. Syst. Bacteriol. 25:222 Separation of yeast species by phenotype has been a long 229. 13. aumov, G. I., S. A. James, E. S. aumova, E. J. Louis, and I. . Roberts. standing problem, and the application of molecular compari 2000. Three new species in the Saccharomyces sensu stricto complex: Sac sons to yeast systematics has further emphasized that it is often charomyces cariocanus, Saccharomyces kudriavzevii and Saccharomyces mi katae. Int. J. Syst. Evol. Microbiol. 50:1931-1942. impossible to separate species by using cellular morphology 14. Peterson, S. W., and C. P. Kurtzman. 1991. Ribosomal RA sequence and physiological responses (9, 23). Watson et al. (24) reported divergence among sibling species of yeasts. Syst. Appl. Microbiol. 14:124 some differences in temperature optima among species of the 129. 15. Savage, D. C., and R. J. Dubos. 1967. Localization of indigenous yeast in the K telluris complex, but as with other physiological criteria, murine stomach. J. Bacteriol. 91:1811-1816. strain variation precluded use of this criterion for the reliable 16. Swofford, D. L. 1998. PAUP* 4.0: phylogenetic analysis using parsimony. Sinauer Associates, Sunderland, Mass. recognition of species. Consequently, with the possible excep 17. van der Walt, J. P. 1957. Three new sporogenous yeasts from soil. Antonie tion of K. slooffiae, the only known means for the reliable Leeuwenhoek 23:23-29. separation of species in the K telluris complex is from gene 18. van der Walt, J. P., and D. Yarrow. 1984. The genus Ar.xiozyma gen. nov. (Saccharomycetaceae). S. Afr. J. Bot. 3:340-342. sequence analysis. The sequences of domains D1 and P2.of the 19. van Uden, ., and H. R. Buckley. 1970. Candida Berkhout, p. 893-1087. In 26S rR A gene have been quite helpful for rapid's'pecies . ,'. '1. Ladder (ed.),. The yeasts, a taxonomic study, 2nd ed. orth-Holland, Amsferdam, The etherlands. identification (10), but as we have demonstrated here' 'lli-IEi: . 20. van Uden, N., and M. Vidal-Leiria. 1970. Torulopsis Berlese, p. 1235-1308. In earlier (8, 14), single gene sequences will not detect hybrids J. Ladder (ed.), The yeasts, a taxonomic study, 2nd ed. orth-Holland, nor provide a full understanding of strain diversity within spe Amsterdam, The etherlands. 21. Vaughan-Martini, 'A., and C. P. Kurtzman. 1985. Deoxyribonucleic acid cies. In addition to this study, interspecific hybrids have been relatedness among species of the genus Saccharomyces sensu stricto. Int. J. detected in Williopsis (6) and Saccharomyces (1), suggesting Syst. Bacteriol. 35:508--511. 22. Vaughan-Martini, A., and A. Martini. 1987. Three newly delimited species of that hybrids are widespread among species and common to the Saccharomyces sensu stricto. Antonie Leeuwenhoek 53:77--84. speciation process. 23. Vaughan-Martini, A., and A. Martini. 1998. Saccharomyces Meyen ex VOL. 43, 2005 PATHOGE IC KAZACHSTANIA SPECIES 111

Reess, p. 358-371. In C. P. Kurtzman and J. W. Fell (ed.), The yeasts, a Stewart and I. Russell (ed.), Current developments in yeast research. Per taxonomic study, 4th ed. Elsevier Science BY, Amsterdam, The ether gamon Press, Toronto, Ontario, Canada. lands. 25. Yarrow, D. 1998. Methods for the isolation, maintenance and identification 24. Watson, K., H. Arthur, and M. Blakey. 1981. Biochemical and morphological of yeasts, p. 77-100. In C. P. Kurtzman and J. W. Fell (ed.), The yeasts, correlations in the thermophilic enteric yeasts, Saccharomyces telluris, Can a taxonomic study, 4th ed. Elsevier Science BY, Amsterdam, The eth dida slooffii, Torulopsis bovina and T. pintolopesii, p. 449-503. In G. G. eriands.

SU~ by the U.S. Department of bIiN....·re. National Center forT:uib.raI"",IV Utilization• Research, Peoria, ."noIS