Genetic and Molecular Delineation of Three Sibling Species in the Hansenula polymorpha Complex

System. Appl. Microbiol. 20, 50-56 (1997) © Gustav Fischer Verlag

Genetic and Molecular Delineation of Three Sibling Species in the Hansenula polymorpha Complex GENNADI I. NAUMOVI, ELENA S. NAUMOVA l , VERA I. KONDRATIEVA l , SERGEI A. BULATZ, NINA V. MIRONENK0 3 , LED A C. MENDON<;:A-HAGLER 4 and ALLEN N. HAGLER 4 I 2

3 4

State Institute for Genetics and Selection of Industrial Microorganisms, Moscow, Russia Petersburg Nuclear Physics Institute, Russia All-Russian Plant Protection Institute, St-Petersburg-Pushkin, Russia Instituto de Microbiologia, Unversidad Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Received August 1, 1996

Summary Genetic hybridization, molecular karyotyping and UP-PCR analysis showed that the taxonomic complex Hansenula polymorpha DE MORAIS et MAlA consists of three biological sibling species. H. angusta TEUNISSON et al. (= Pichia angusta (TEUNISSON et al.) KURTZMAN) is not synonymous with H . polymorpha and must be reinstated as a separate species. The third sibling species is apparently a new taxon associated with Opuntia cacti. The sibling species are able to cross with each other but their interspecific hybrids are sterile. Key words: Genetic analysis - Molecular karyotyping - UP-PCR - Hansenula polymorpha complex

Introduction The thermo tolerant methylotrophic species Hansenula polymorpha DE MORAIS et MAlA isolated mostly from desert and tropical regions has been the focus of a number of genetic and molecular studies (GLEESON et aI., 1986; GLEESON and 5UDBERY, 1988; 5UDBERY and GLEESON, 1989). In addition to its ability to use methanol, H. polymorpha can grow at temperatures up to 49°C, and may be able even to survive pasterization (TEUNISSON et aI., 1960). This thermotolerance is a favorable characteristic for use in industrial fermentations, especially in the tropics. The taxonomic status of H. polymorpha is still not adequately resolved (KOMAGATA, 1991; NAUMOV et aI., 1992). The species was first described as Hansenula angusta, however this was done without latin diagnosis (WICKERHAM, 1951). A valid description of H. angusta was made by TEUNISSON et al. (1960) based on an isolate from Drosophila. Earlier, another taxon H. polymorpha was described validly by DE MORAIS and MAlA (1959). WICKERHAM (1970) considered H. angusta to be synonymous with H. polymorpha. The latter species was included in the revised genus Pichia (KURZTMAN, 1984a), but since the name Pichia polymorpha was used previously for another species (now Debaryomyces polymorphus), the name P. angusta was adopted. More recently, a new

revlSlon of the genus Pichia based on the partial sequences of 185 and 265 ribosomal RNAs has been made by YAMADA et al. (1994). A new genus Ogataea YAMADA et al. contains five species, including Ogataea polymorpha (DE MORAIS et MAlA) YAMADA et al. Until recently scientists involved in studies of the methylotrophic yeasts did not follow the nomenclature revision made by KURTZMAN (1984a). In addition the classification of the genus Ogatae is controversial. Even the authors (YAMADA et aI., 1994) considered the created genus not to be quite homogenous. In the present paper we followed the most commonly used name Hansenula polymorpha (WICKERHAM, 1970; KURTZMAN, 1984b). Molecular techniques, such as electrophoretic karyotyping (CARLE and OLSON, 1985) and random amplified polymorphic DNA polymerase chain reaction (BULAT and MIRONENKO, 1990, 1992; WELSH and MCCLELLAND, 1990; WILLIAMS et aI., 1990), have been successfully applied to yeast taxonomy. We used genetic hybridization analysis, electrophoretic karyotyping and UP-PCR analysis to show that Hansenula polymorpha is a heterogeneous taxon containing at least three sibling species. Definition of sibling species is important for studies of geographic distribution, ecology, evolution, comparative genetics and systematics of yeasts.

Hansenula polymorpha complex

Materials and Methods Strains, cultivation and genetic crosses: The Hansenula polymorpha strains used, and their origins are listed in Table 1. The monosporic cultures of the strains studied were marked by UVinduced auxotrophic mutations. Two mutants, 1976-met6 and 1976-arg3, induced by GLEF.SEN at a1. (1986) using strain CBS 1976 (= NCYC 495), were received from the National Collection of Yeast Cultures (NORWICH, U. K.) under the numbers NCYC 2297 and NCYC 2282, respectively (PAITIl'
51

morpha were estimated using commercial chromosome standards of Saccharomyces cerevisiae (YNN 295) and Schizosaccharomyces pombe (972 h-) (Bio-Rad). After electrophoresis the gels were stained with ethidium bromide to visualize the chromosomes. PCR and amplified DNA products analysis: UP-PCR technique (Universally Primed Polymerase Chain Reaction) (BULAT and MIRONF.NKO, 1990, 1992) is an analog of AP-PCR (Arbitrary Primed Polymerase Chain Reaction) (WELSH and MCCLELLAND, 1990) or RAPD-PCR (Random Amplified Polymorphic DNA Polymerase Chain Reaction) (WILLIAMS et aI., 1990) based on the amplification of genomic DNA with single short primers of random nucleotide sequence. The main difference consists in primer design, reaction conditions and polymerase used (BULAT et aI., 1992a). Cell Iysates were prepared by suspending one loop full of yeast cells in 200 pi of lysis buffer (50 mM Tris-HCL, pH 7.8, 50 mM EDTA, 100 mM NaCl, 2% N-lauroyl sarcosine, 500 mM 2mercaptoethanol, 1000 y/ml Proteinase K). Following incubation at 65°C for 1 h, NaCl was adjusted to 1M and equal volume of chloroform/octanol mixture (24:1) was added. After gentle mixing for 10 min and centrifugation at 12,000 g for 2 min, the DNA was precipitated from the supernatant by adding 0.6 volume of isopropanol. The pellet was rinsed once with 70% ethanol prior to redissolving in TE buffer (1 mM Tris-HCI, 0.1 mM EDTA). For PCR 0.1 pI of obtained DNA was used. The PCR was performed according to SAIKI et a1. (1988) using 2-3 units of thermostable DNA polymerase Tth in the presence of 4 mM MgCI1 , 0.6 mM each of dNTPs and 40 ng of a primer. Single universal (arbitrary chosen) primers of our design were used: 215' -G GAT C C GA G G GT G G C G GTT CT 455' -G T AAAA C GA C G G C CA GT Amplifications were performed in a DNA thermal cycler TC 1-1000 (IRLEN, S.-Petersburg, Russia) programmed for a denat-

Table 1. Strains of the Hansenula polymorpha complex studied. Species originial designation, strains 1

Source

Geographic origin

Reference

Hansenula angusta

spoiled orange juice

Florida, U.S.A.

WICKERHAM (1951)

Drosophila pseudobscura

California, U.S.A.

TEUNISSON et a1. (1960)

soil

Brazil

DE MORAIS and MAlA (1959)

Drosophila pseudobscura

California, U.S.A.

WICKERHAM (1970)

Drosophila pseudobscura

California, U.S.A.

WICKERHAM (1970)

Opuntia phaeacantha

Arizona, U.S.A.

STARMER et al. (1986)

Opuntia lindheimeri

Texas, U.S.A.

STARMER et a1. (1986)

CBS 1976 (VKM Y-1397)

Hansenula angusta CBS 7073 (W (NRRL Y-2214)

Hansenula polymorpha CBS 4732 (W (VKM Y-2559)

Hansenula polymorpha NRRL Y-2216

Hansenula polymorpha NRRL Y-2267

Pichia angusta 81-530A

Pichia angusta 81-607.3

CBS - Centraalbureau voor Schimmelcultures, Delft, Holland; VKM - All-Russian Collection of Microorganisms, Moscow, Russia; NRRL - Northern Region Research Center, Peoria, IlL, U.S.A. The collection from which the corresponding strain was received is indicated in parenthesis. 2 T - type culture.

1

52

G. 1. NAUMOV et al.

uration step at 94°C for 3 min followed by 30 cycles of 50 sec at 94°C, 80 sec at 55 °C, 60 sec at 70°C and a final extension of 3 min at 70°C. The transcribed spacer region (ITSl) of the nucelar rDNA was amplified using primers X (3' end of the 175 RNA gene) and Y (5.85 RNA gene) of our design. Their sequences are as follows:

X 5' - TGAA C CTG C G GAA G GA T CA TT Y 5' - G CAT T T C G C T G C G T T C T T CAT The PCR conditions were the same as for previous experiments with the exception that the primer annealing temperature was 58°C. The PCR products were analysed by electrophoresis in agarose (1.7%) or PAA (6%) gels according to SAMBROOK et al. (1989).

Table 2. Genetic analysis of sibling species of the Hansenula polymorpha complex. NN

Origin of hybrids

Intra-specific hybrids 1976 x 1976 met6 xarg3 1 met6 xargl 2 4732 x 4732 met3 x leul 3 7073 x 7073 cysS x arg6 4 cysS x adel 5 81-530A x 81-530A adel xade3 6 81-607.3 x 81.607.3 hisl x met2 7 81-607.3 x 81-530A met2 xade3 8 7073 x 2267 ade4 xarg6 9 met8 xadel 10 4732 x 1976 met3 xargl 11 ade2 xmet6 12 Inter-specific hybrids 7073 x 1976 adel xmet6 13 14 arg6 xmet6 adel xargl 15 cysS xargl 16 7073 x 81-607.3 arg6 x hisl 17 arg6 xmet2 1) 18 2) 19 7073 x 81-530A arg6 xadel 20 arg6 xade3 21 7073 x 4732 met3 xarg6 22 met3 xade1 23 4732 x 81-607.3 ade2 x hisl 24 25 ade2 xmet2 1976 x 81-607.3 26 argl xmet2 met6 x hisl 27 1976 x 81-530A met6 xadel 28 met6 xade3 29

No. of spores isolated

Proportion of viable ascospores of hybrids (%)

Segregation of control markers! aB

Ab

AB

ab

19

16

16

91 104

78.0 88.5

19

116

81.9

19

16

25

16

160 104

68.1 96.2

72

24

3 3P: 5N: 14T

1

92

97.8

OP: IN: lOT

85

94.1

IP: 4N: 9T

89

92.1

OP: 4N: 6T

153 92

77.1 54.7

11 14

14 8

12 19

9 9

144 157

82.6 88.5

27 33

24 35

35 35

18 33

80 81 88 143

1.3

4.9 6.8 16.1

0 0 0 0

0 0 0 0

1 4 6 23

0 0 0 0

95

35.8

0

0

34

0

94 82

33.0 7.3

3 0

0 0

28 6

0 0

88 81

25.0 13.6

4 2

3 2

14 5

0 0

114 103

6.7 16.5

0 2

0 0

5 12

0 0

89 131

4.5 9.2

0 0

0 0

4 10

0 0

85 84

7.1 9.5

2 4

2 2

2 0

0 0

84 86

2.4 0.0

0 0

0 0

2 0

0 0

3P: IN: 7T

! Data of random spore or tetrad analyses are presented. a, b - auxotrophy of first (in front of crossing sign) and second parents, respectively; A, B - prototrophy; P, N, T - tetrads of parent and nonparent ditypes, and tetra type, respectively.

Hansenula polymorpha complex

A

1 2 3 4 5 6 7 8

Kb

B

1 2 3 4 5

Kb

2200 1 600 2200 1600

1125 1020 945 850 800 770

1125

700 630 580 460

850 800 770

B

A Kb

1 1,5 5 l' ' ........... 2,8 ......... 2,6_ 2,1_ 2,0"""""" 1,7/ 1,16_ 1,09 ......

1 2

Fig. 1. Karyotypic analysis of chromosomal DNAs of Hansenula polymorpha and its sibling species. CHEF was run at 200 V under conditions of switching intervals and run times described below: (A) 60 s for 15 h, then 90 s for 9 h. Lane 1, S. cerevisiae YNN 295; lanes 2 and 3, H. polymorpha CBS 1976 and CBS 4732; lanes 4-6, H. angusta CBS 7073, NRRL Y-2216 and NRRL Y-2267; lanes 7 and 8, cactus strains 81-530A and 81-607.3. (B) 60 s for 20 h, then 90 s for 16 h. Lane 1, S. cerevisiae YNN 295, lanes 2 and 3, H. polymorpha CBS 1976 and CBS 4732; lane 4, H. angusta CBS 7073; lane 5, cactus strain 81-530A. The chromosome sizes of the strain YNN 295 are indicated to the left. All cultUfes used are non-monosporic. Fig. 2. UP-PCR patterns of polymorphic DNA from the yeasts of Hansenula polymorpha and its sibling species using the primers (A) 21 and (B) 45. Lane 1, Pstl digested phage lambda DNA (molecular weight marker); lanes 2 and 3, H. polymorpha CBS 4732 and CBS 1976; lanes 4-6, H. angusta CBS 7073, NRRL Y-2216 and NRRL Y-2267; lanes 7 and 8, cactus strains 81-530A and 81-607.3. Molecular weight markers are indicated in Kb to the left. All strains used are monosporic, except NRRL Y-2267.

1020 945

370 290 245

3

4 5

6 7

8

53

1 2

3

4 5

6

7 8

54

G. 1. NAUMOV et al.

Results Genetic hybridization analysis

Only fertile homozygous parent strains can be used for taxonomic genetic analysis. All strains studied showed high fertility with ascospore viability of 96-100%. For each parent strain 25-30 spores were analyzed. UV-induced auxotrophic mutants were produced for the monosporic cultures. All mutants were able to cross independent of the origin of initial strains and the type of auxotrophy. Hybrids obtained had different sporulation ability but all of them were suitable for meiotic monosporic analysis using a micromanipulator. In the cases when tetrad formation occured at low frequency, triads were also dissected. Intra-strain hybrids nos. 1-7 were all fertile with normal digenic segregation of control auxotrophic markers (Table 2). Only hybrid no. 4 showed lower frequency for the arg segregants that is probably due to lower viability of its spores. From the viability of hybrid ascospores and segregation of control markers, we concluded that hybrids nos. 8-12 were intraspecific. The other hybrids (nos. 13-29) having low ascospore viability and irregular meiotic segregation of control auxotrophic markers were classified as inter-specific. Their rare viable ascospores proved not to be products of a normal meiosis since double auxotrophic recombinants were absent among them and each segregant carried only one parental marker or was prototrophic. Moreover, segregants from complete tetrads or triads were prototrophic indicating that in such asci there was no reductive meiotic division and the ascospores were at least diploid. For example, the four survival tetrads and the three triads of hybrid no. 17, and the seven tetrads of hybrid no. 18 were prototrophic. The analysis of the hybridization data allowed classification of the parent strains into three inter-sterile groups: 1) CBS 7073 and NRRL Y-2267; 2) CBS 1976 and CBS 4732; 3) 81-530A and 81-607.3. Molecular karyotyping

The taxonomic species Hansenula polymorpha is karyotypically heterogeneous and the seven strains can be classified into 3 distinct groups (Fig. 1A). Strains CBS 1976 and the type culture of Hansenula polymorpha CBS 4732 showed similar karyotypes (Fig. lA, lanes 2 and 3, respectively). Chromosomal DNAs of these strains resolved into five distinct bands ranging in size from 2200 to 1040 Kb. The type strain of H. angusta CBS 7073, together with the other Drosophila isolates, NRRL Y-2216 and NRRL Y-2267, formed group 2. Three strains showed very similar karyotypes characterized by the presence of a chromosomal band of 860 Kb (Fig. lA, lanes 4, 5 and 6). Four other chromosomal bands of these strains ranged in size from 1280 to 2200 Kb. Group 3 included the two cactus strains, 81-530A and 81-607.3 (Fig. lA, lanes 7 and 8, respectively). The karyotype patterns of these strains differed from group 1 in the sizes of the chromosomal bands in the range

below 1480 Kb. MARRI et al. (1993) comparing karyotypes of some H. polymorpha strains, including CBS 4732, noted that their chromosomal DNAs could be separated into bands in the range of 650 to 2200 Kb in size. To estimate more exactly the chromosome number in H. polymorpha, several different karyotyping conditions were used. The best resolution of chromosomal DNA was achieved by running the gel under the following conditions: 60 s for 20 h at 200 V, then 90 s for 16 h at 200 V. The chromosomal DNAs of three strains, CBS 1976, CBS 4732 and 81-530A, resolved into six bands (Fig. 1B, lanes 2, 3 and 5, respectively). Chromosomal bands no. 1 and no. 6 from the bottom of the gel were probably double according to their stronger relative fluorescence intensities. Strain CBS 7073 showed seven chromosomal bands (Fig. lB, lane 4). Taking into account that some chromosomes migrated as doublets, we consider that the three sibling species possess the same number of chromosomes, which is at least seven.

Kb

1

2 3

4

5 6

789

1,7

1,16 1,09

0,8

,0,51 0,47

0,45

Fig. 3. PCR polymorphisms of the nuclear rDNA ITS1 region . from Hansenula polymorpha and its sibling species. Lanes 1 and 9, Pstl digested phage lambda DNA (molecular weight marker); lanes 2 and 3, H. polymorpha CBS 4732 and CBS 1976; lanes 4-6, H. angusta CBS 7073, NRRL Y-2216, NRRL Y-2267; lanes 7 and 8, cactus strains 81-530A and 81-607.3. Molecular weight markers are indicated in Kb to the left. All strains used are monosporic, except NRRL Y-2267.

Hansenula polymorpha complex

UP peR analysis Universal primers, 21 and 45 were used for comparison of the H. polymorpha strains (Fig. 2) . Both primers were able to amplify numerous products from the DNA of the seven strains studied but the amplification products were specific for each primer. On the basis of UPPCR patterns the seven strains fell into the same 3 distinct groups as for electrokaryotyping. Strain NRRL Y2267 was not included in the genetic hybridization analysis. However, on the basis of both UP-PCR and electrokaryotyping (Fig. 1A) it should be classified together with the type culture of H. angusta CBS 7073 . The dot hybridization analysis of UP-PCR products showed that amplified DNAs from the representatives of three groups were unique and did not share any cross homology (data not shown). Our previous studies of different fungal species showed that the UP-PCR products were homologous within a group of genetically related orgamsms (BULAT et aI., 1992a, b). This has been observed only for DNA amplified with the same universal primer, and the D~A amplified DNA was species- and primer-specifi~ hybridization. UP-PCR patterns were also speCific III size and fragment arrangement for the group of genetically related organisms (BULAT and MIRONENKO, 1990, 1992; BULAT et aI., 1992a, b). All seven Hansenula strains were also compared for electrophoretic mobility of the rDNA amplified ITS1 region (Fig. 3). Strains CBS 4732 and CBS 1976 were characterized by identical length of the ITS1 regIOn that differed from the ITS 1 amplification products of strains 81-530A and 81-607.3. However, some length variation of this spacer was found for three Drosophila strains, CBS 7073, NRRL Y-2216 and NRRL Y-226 7. This indicates that the length of the ITS1 amplification products cannot be used as a taxonomic criterion for differentiation of sibling species in the Hansenula polymorpha complex. Similar observations have been seen in some other fungi (KLASSEN and BUCHKO, 1990; CHEN et aI., 1992).

In

Discussion Genetic hybridization, molecular karyotyping and UPPCR analyses indicated that the seven strains studied belong to three distinct species. One is known as Hansenula polymorpha DE MORAIS et MAlA (1959) (the type culture CBS 4732). Strain CBS 1976 isolated from orange juice also belongs to this taxon. The second sibling species is Hansenula angusta TEUNISSON et al. (1960) (the type culture CBS 7073). All three H. angusta strains studied by us, CBS 7073, NRRL Y-2216 and NRRL Y-2267, were isolated from Drosophila (Table 1). Our data showed that H. polymorpha and H. angusta are not synonymous and the latter species must be reinstated as separate from H. polymor-

pha. The third sibling species is apparently a new taxon associated with the Opuntia cacti. The two cactus strains

55

studied here (81-530A and 81-607.3) were genetically separate and different from the other two sibling species both in karyotype and UP-PCR profiles. Isolates from Opuntia cacti from the southwestern deserts of the USA and deserts of Australia are known to have slower and weaker utilization of methanol than other strains including CBS 4732 (PHAFF, 1985; LACHANCE et aI., 1988; STARMER et aI., 1990). The cactus strains, including 81-530A and 81-607.3, shared 99-100% DNA/DNA homology independent of geographic origin. The DNA relatedness of strains 81-530A and 81-607.3 with the type culture of H. polymorpha CBS 4 732 was 64.3%. It was 72 % with another strain (DL-1) isolated from mud and water of a polluted river (PHAFF, 1985; LACHANCE et aI., 1988; PHAFF, personal communication). Comparative chromosome identification of the three sibling species by Southern hybridization with cloned genes of H . polymorpha would allow a better understanding of their relatedness. Such research could be complicated by only one linkage group being described for H. polymorpha (GLEESON et aI., 1988). Recently, one strain of the methanol assimilating yeast Pichia pinus (HOLST) PHAFF has been reidentified by electrophoretic karyotyping and genetic hybridization analysis as Pichia methanolica MAKIGUCHI (TOLSTORUKOV, 1994). In this context it is also interesting to study by genetic and molecular methods the relationship of the H. polymorpha sibling species with other methanol assimilating and non-assimilating species included in the genus Hansenula. Some of them, based on similarities in GCcontent and phenotypic characteristics, may include synonyms to H. polymorpha.

Acknowledgements We thank C. KURTZMAN, H. PHAFF and I. ROBERTS for kindly providing yeast strains and their descriptions, O. KABOEV for thermostable DNA polymerase, and E. J. LOUIS for his useful comments. This research was supported by a Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil) to G.LN. and E.S.N., the ISC Program of the European Commission to L.C.M.H. and A.N.H. Also, G.LN., E.S.N., V.LK. and S.A.B. were supported in part by the grant "Frontiers in Genetic" from Russian Academy of Sciences.

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G. I. NAUMOV et a!.

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DEMAIN, N. A. SOLOMON, eds.), California, The Benjamin/ Cummings, Inc. Menlo Park 1985 SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHART, S. J., HIGUCHI, R., HORN, G. T., MULLIS, K. B., ERLICH, H. A.: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491 (1988) SAMBROCK, J., FRITSCH, E. E, MANIATIS, T.: Molecular cloning, a laboratory manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory 1989 STARMER, W. T., GANTER P. E, PHAFF, H. J.: Quantum and continuous evolution of DNA base composition in the yeast genus Pichia. Evolution 40, 1263-1274 (1986) STARMER, W. T., LACHANCE, M.-A., PHAFF, H. J., HEED, W. B.: The biogeography of yeasts associated with decaying cactus tissue in North America, the Caribbean, and northern Venezuela. Evolutionary Biology 24, 253-296 (1990) SUDBERY, P. E., GLEESON, M. A.: Genetic manipulation of methylotrophic yeasts, pp. 304-329. In: Molecular and cell biology of yeasts (E. P. WALTON, G. I. YARRONTON, eds.) Blackie (Glasgow and London), Van Nostrand Reinhold, NY 1989 TEUNISSON, D. J., HALL, H. H., WICKERHAM, L. J.: Hansenula angusta, an excellent species for demonstration of the coexistence of haploid and diploid cells in a homothallic yeast. Mycologia 52,184-188 (1960) TOLSTORUKOV, 1. I.: Genome structure and reidentification of the taxonomic status of Pichia pinus MH4 genetic lines. Genetika 30, 635-640 (1994) (in Russian) WELSH, J., MCCLELLAND, M.: Fingerprinting genomes using PCR with arbitrary primers. Nucl. Acid. Res. 18,7213-7218 (1990) WICKERHAM, L. J.: Taxonomy of yeasts. U. S. Department of Agriculture Tech. Bulletin N 1029, 1-56, U. S. Department of Agriculture, Washington DC (1951) WICKERHAM, L. J.: Hansenula, pp. 226-315. In: The yeasts, a taxonomic study (J. LODDER, ed.) 2nd ed., Amsterdam, ElsevierlNorth-Holland 1970 WILLIAMS, J. G. K., KUBELIC, A. R., LIVAK, K. L., RAFALSKI, J. A., TINGEY, S. V.: DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acid. Res. 18, 6531-6535 (1990) YAMADA, Y., MAEDA, K., MIKATA, K.: The phylogenetic relationships of the hat-shaped ascospore-forming, nitrate-assimilating Pichia species, formely classified in the genus Hansenula SYDOW et SYDOW, based on the partial sequences of 18S and 26S ribosomal RNAs (Saccharomycetaceae): the proposals of three new genera, Ogatae, Kuraishia and Nakazawaea. Biosci. Biotech. Biochem. 58, 1245-1257 (1994) Corresponding author: G. 1. Naumov, State Institute for Genetic and Selection of Industrial Microorganisms, I Dorozhnyi 1, Moscow 113545, Russia; Fax: (7095) 315 05 01; Phone: (7095) 315 07 83; email: gennadi@vnigen. msk. suo