Metschnikowia gruessii sp. nov., the Teleomorph of Nectaromyces reukaufii but not of Candida reukaufii

System. Appl. Microbiol. 15, 432-438 (1992) © Gustav Fischer Verlag, StuttgartlNew York

Metschnikowia gruessii sp. nov., the Teleomorph of Nectaromyces reukaufii but not of Candida reukaufiP~ GABRIELLA GIMENEZ-JURADO The Portuguese Yeast Culture Collection, Laboratory of Microbiology, Gulbenkian Institute of Science, Oeiras, Portugal

Received April 6, 1992

Summary One strain identified by conventional phenetic methods as Metschnikowia reukaufii did not hybridize with a species-specific probe produced in our laboratory. Molar % G+C composition and a relative heteroduplex formation of 7% with the type strain of M. reukaufii revealed that they did not belong to the same taxon. Comparisons with the type strain of Candida reukaufii (CBS 1903) and the authentic strain of Nectaromyces reukaufii (CBS 611) showed no similarity with the former (22% reassociation) and conspecificity with the latter (97% reassociation). Low nuclear DNA reassociation values with all the type strains of other Metschnikowia species provided sufficient evidence for proposing a novel species, Metschnikowia gruessii that represents the teleomorph of Nectaromyces reukaufii but not of Candida reukaufii. It differs from the type strain of M. reukaufii in its cell and ascus morphology, assimilation of trehalose and maximum temperature for growth, and from the other described Metschnikowia species by additional characteristics. More strains of Metschnikowia gruessii were repeatedly isolated from flowers in the natural park of Amibida, Portugal.

Key words: Yeast - Molecular taxonomy - Metschnikowia gruessii

Introduction Gruss (1918) first named the species Anthomyces reukaufii, and described the presence of a group of cells arranged in a tridental form, otherwise known as tetrad, cross, or airplane-like configurations as a distinguishing property. This species, later renamed Nectaromyces reukaufii by Sydow and Sydow (1918), as the name Anthomyces had already been assigned to a genus of the Uredinales, also became known as Nectaromyces cruciatus by Schoellhorn (1919), who apparently was unaware of the previous publications. Diddens and Lodder (1942) subsequently transferred this species to the genus Candida basing their proposal on its ability to form pseudomycelium with blastospores, and not true mycelium with conidia as claimed by Nadson and Krassilnikov (1927). Later, Lodder and Kreger-van Rij (1952) designated as the type culture of C. reukaufii the strain CBS 1903 received by the CBS in 1928 from Nadson, who also studied this yeast, and not Gruss's strain (CBS 611). After inducing sporulation in dilute V8 medium and studying the complete life * To the memory of Prof. Nicolau van Uden (1921-1991)

cycle Metschnikowia reukaufii was described (Pitt and Miller, 1968) and a new type culture was designated (CBS 5834). All the preceeding strains, including strains of Chlamydozyma zygota (Wickerham, 1964), simply became synonyms of M. reukaufii. Recently, in our laboratory, a species-specific probe for M. reukaufii was produced from a non-transcribed spacer sequence of the ribosomal DNA unit obtained from the type strain of M. reukaufii (Henriques et al., 1991). It hybridized only with DNA from strains of M. reukaufii but not with DNA from other Metschnikowia species. However, DNA from one strain of M. reukaufii that produced the typical cross-formations of cells and had been identified based on conventional phenetic identification criteria (Kreger-van Rij, 1984; Barnett et al., 1990) did not hybridize with the probe. Subsequent determinations of molar % guanine + cytosine and DNA-DNA relative reassociations revealed that the non-hybridizing strain does not belong to the species M. reukaufii (Henriques et al., 1991). This raised the question whether M. reukaufii might be a heterogenous species. Results presented here

Metschnikowia gruessii sp. nov.

reveal that this is the case and that the original strain of Nectaromyces reukaufii is the anamorph of the species now described.

Materials and Methods Strains. The strains examined and their origin are listed in Table 1. The strains of M. gruessii were isolated by direct inoculation of flowers in YM, yeast extract-malt extract broth (van der

433

Walt and Yarrow, 1984), the pH of which had been adjusted to 3.5-4.0 to discourage bacterial growth. Isolated colonies were obtained and purified in glucose (2%), peptone (1 %), yeast extract (0.5%) agar plates. All the strains were maintained on slopes of YM agar. Characteristics of the strains. Morphological and physiological characters were determined using the methods described by van der Walt and Yarrow (1984). Sporulation was induced using the conditions recommended by Pitt and Miller (1968). For DNA isolation the strains were grown for 2-3 days at 25°C in shaken

Table 1. Origin and DNA base composition of the strains examined Mol % G+C based on Species

IGC'

CBS b

Tm c

Buoyantd density

Source

Metschnikowia australis (Fell and Hunter) Mendonfa-Hagler et a!.

4212T

5847

46.9 ± 004

47.0±0.3

Antarctic ocean (J. W. Fell)

Metschnikowia bicuspidata (Metschnikov) Kamienski

4206 T

5575

45.0±0.9

48.0±02

Larva of Diplostomum flexicaudum in U.S.A. (1. J. Wickerham) Unknown

4604±0.3

4440

(A. Fonseca)

4382 T

7657

39.2±0.6

4796

7658

n.d.

Flower of Hebe salicifolia in Portugal (G. Gimenez-Jurado) Flower of Cistus monspeliensis in Portugal

4800

7659

41.1 ± 0.1

Flower of Salvia sp. in Portugal

Metschnikowia hawaiiensis Lachance, Starmer and Phaff

4220T

7432

47.3 ±0.6

46.6

Flower of Ipomoea accuminata in Hawaii

Metschnikowia krissii (van Uden and Castelo-Branco) van Uden

2895 T

4823

43.6 ± 0.1

4504± 004

Sea-water off California in U.S.A.

Metschnikowia lunata Golubev

4209 T

5946

48.1±0.3

44.2±0.3

Flower of Vicia cracea in U.S.S.R. (A. I. Egorova)

Metschnikowia pulcherrima Pitt and Miller

4830

5833

n.d.

45.6±0.1

Vitis labrusca (M. W. Miller)

2726

2245

47.8 ±0.5

4446 T

5834

42.8±0.2

41.3±0.2

Metschnikowia gruessii Gimenez-Jurado

(G. Gimenez-Jurado) (G. Gimenez-Jurado)

(M.-A. Lachance) (N. van Uden)

Unknown

3276

4370

43.3 ±0.3

Flower of Epilobium angustifolium in Canada (M. W. Miller) Leaf of lemon tree in Portugal (c. Cabefa-Silva) Raspberry Rubus strigosus in U.S.A. (1. J. Wickerham) Unknown (A. Capriotti)

Candida reukaufii (Gruss) Diddens and Lodder

2729 T

1903

42.5 ± 0.1

Unknown (G. A. Nadson)

Nectaromyces reukaufii (Gruss) Sydow and Sydow

3275 T

611

39.7±0.5

Flower of Linaria vulgaris in Germany (J. Gruss)

Metschnikowia zobellii (van Uden and Castelo-Branco) van Uden

2892 T

4821

44.0±0.8

Sea-water off California in U.S.A.

Metschnikowia reukaufii Pitt and Miller

4401

43.8±0.6

3684

5553

43.8 ± 0.2

(N. van Uden)

• IGC, Instituto Gulbenkian de Ciencia, Portuguese Yeast Culture Collection; T, Type strain. b CBS, Centraalbureau voor Schimmelcultures, Netherlands. C Tm molting temperature curve; Mean ± s.d. based on at least 4 determinations. d Mendonfa-Hagler et a!., (1985).

434

G. Gimenez-Jurado

YM broth, the pH of which had been adjusted to 8.0 (Sugiyama et aI., 1985). Cells were ruptured using a cell press as described by Eaton (1962). The DNA was purified, based on the method of Britten et al. (1970), following a modified protocol (Golubev et al., 1988). Determinations of the guanine plus cytosine of the nuclear DNA were done following the method of Marmur and Doty (1962) with a Gilford Response UV-VIS spectrophotometer, and its Thermal Programming software, using nDNA from Candida parapsilosis CBS 604 (mol% G+C = 40.5) as reference. The extent of DNA-DNA reassociations were determined spectrophotometrically using the same instrument and following the procedures described by Seidler and Mandel (1971) as modified by Kurtzman et al. (1980) . Ubiquinone isoprenologues were extracted from stationary cells of M . gruessii and determined following the methods referenced earlier (Gimenez-Jurado et aI., 1990).

Results

Description Metschnikowia gruessii Gimenez-Jurado sp. nov. In medio liquido cum dextroso et peptono et extracto levedinis post 3 dies 25°C cellulae sunt ellipsoidae, ovoidae, longovoidae aut cylindratae 2.5-6.3 X 8.8-27.5 !-tm. Post 7 dies chlamydosporae praesentes plerumque, ovoidae refractivae multiguttatae 5.0-12.5 X 10.0-15.0 flm in diam, reproductio vegetativa per gemmarum enteroblasticarum. Neque annulus nec velum fomantur, sedimentum abundus, Cultura in striis in medio agaro cum dextroso et peptono et extracto levedinis post 7 dies ad

25°C, color creme us, pagina laevis et nitens vel papillatus exigue, butyrosa, coloniae elevatus et convexus, margo integer. Pseudo mycelium constans hyphis tenuis cum ovales conidia ad terminalis. Asci in agaro V8 dilutus ellipsoidopedunculati 6.37.5 X 31.3-60.0 !-tm, pedunculi cylindrici, 2-spori. Ascosporae aciculares vel filiformes. Fermentatio: glucosum. Assimilat glucosum, gal actosum (variabilis), L-sorbosum, D-glucosaminum, D-ribosum (variabilis), D-xylosum (variabilis), sucrosum, maltosum, cellobiosum, salicinum, arbutinum, melezitosum, ethanolum, glycerolum, D-mannitolum, D-glucono-a-lacton urn, xylitolum et acidum succinicum ad non L-arabinosum, D-arabinosum, L-rhamnosum, trehalosum, a-methyl-D-glucosidum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, methanolum, erythritholum, ribitol urn, D-glucitolum, galactitolum, inositolum, qcidum glucuronicum, acidum lacticum, acidum citricum, acidum tartaricum et acidum malicum. Assimilat L-Iysini, ethylamini, cadaverini ad non kalii nitratis, sodii nitrosi, creatini nec creatinini. Vitamina externa ad cresentiam necessaria sunt. Temperatura maxima crescentiae 31°C. Materia amyloidae non formantur. Ureum non finditur. Proportio molaris guanini plus cytosini in acido deoxyribonucleico 39.2 ± 0.6 per centum. Ubiquinonum majus Q-9. Typus. IGC 4382 ex flore Hebe salicifolia isolatus, exsiccatus et vivus in collectione zymotica lusitanica et vivus in collectione zymotic a Delphis Batovorum (CBS 7657) praeservatus.

Fig. 1. Vegetative cells of M. gruessii IGC 4382 showing "airplane" configurations after 3 days at 25°C in 20% honey solution. Phase contrast. Insert bar represents 10 ~m.

435

Metschnikowia gruessii sp. nov. Growth in glucose (2%) yeast extract (0.5%) peptone (1 %) broth. After 3 days at 25°C cells are variable in

shape and size, elliptical, ovoidal, obovate, and sometimes cylindrical, measuring 2.5-6.3 X 8.8-27.5 11m, usually in pairs or in groups of four giving rise to characteristic "airplane" or trident (Fig. 1) configurations 3.8-6.3 X 21.339.0 11m. Budding is holoblastic-multilateral on a broad base. After 1 week refractile ovoid chlamydospores measuring 5.0-12.5 X 10.0-15.0 11m with one or more lipid globules are present. No ring or pellicle is formed, but an abundant sediment is produced. Growth on glucose (2%) yeast extract (0.5%) peptone (1 %) agar. After 1 week at 25 °C most strains studied form a streak culture that is cream-coloured, smooth, often papillate and glistening. The texture is butyrous. Colonies are slightly raised and convex, and the margin is entire. In on'e strain dull, rugose colonies are formed. Pseudomycelium formation varies with the strain, but generally consists of slender hyphae with oval conidia at the terminals (Fig. 2). A highly branched pseudo mycelium was also observed in CBS 611, consisting of large cylindrical-like cells. Dalmau plate cultures on corn meal agar. After 7 days at 25 °C growth under the cover glass shows short chains of stalagmoid cells with oval conidia. This pseudomycelium often becomes extensive, forming long, slender hyphae with lateral branching. Chlamydospores and "airplane" configurations are also present. Formation of asci and ascospores. Sporulation occurs after 1 week on dilute V8 (1 : 14) agar at 17°C. Ascus formation may initiate from cells proceeding from "airplane" groups of cells as well as chlamydospores. Ascus mother cells or ovoid chlamydospores differentiate, elon,

,

gating at one end giving rise to a long cylindrical peduncle (Fig.3). Mature asci measure 6.3-7.5 x 31.3-60.0 11m. Ellipsoidopedunculate asci when mature consist of two acicular to acerose ascospores, pointed at one end, often converging to appear bifurcated. Some ascospores penetrate a neighbouring cell attached to the mother ascus cell. Spores are persistent, often appearing swollen near the sharp pointed end or curled inside the original mother cell in the form of a loop. Fermentation of sugars. Glucose: + (delayed), other sugars not fermented. Assimilation of carbon compounds Glucose Galactose L-Sorbose D-Glucosamine D-Ribose D-Xylose L-Arabinose D-Arabinose L-Rhamnose Sucrose Maltose a,a-Trehalose Methyl-a-glucoside Cellobiose Salicin Arbutin Melibiose Lactose Raffinose Melezitose

+

variable

+ +

variable variable

+ + + + +

+

Inulin Starch Methanol Ethanol Glycerol Erythritol Ribitol D-Glucitol D-Mannitol Galactitol Inositol Glucono-b-lactone D-Gluconic acid D-Glucuronic acid D,L-Lactic acid Succinic acid Citric acid L-Tartaric acid L-Malic acid Xylitol

+ (weak)

+

+ (weak) +

variable

+ (weak)

+

Fig. 2. Slender pseudomycelium of M. gruessii IGC 4800 showing oval conidia at the terminals after 1 week at 17°C in dilute V8 agar (1 : 14). Phase contrast. Insert bar represents 10 ~m. 29 SyStem, Appl. Microbiol. Vol. 1513

436

G. Gimenez-Jurado

Fig. 3. Ellipsoidopedunculate asci with 2 acerose ascospores of M. gruessii IGC 4796 after 1 week at 17°C in dilute V8 agar (1 : 14). Phase contrast. Insert bar represents 10 !-1m.

Assimilation of nitrogen compounds Potassium nitrate Sodium nitrite Ethylamine L-Lysine

+ +

+

Cadaverine Creatine Creatinine

Discussion

Other characteristics Mol % G+C of nuclear DNA: Growth in 10% NaCI plus 5% glucose: Growth at 31°C: Growth at 32 °C: Growth in vitamin-free medium: Formation of starch-like compounds: Hydrolysis of urea: Splitting of arbutin: Growth with 0.01 % cycloheximide: Growth with 0.1 % cycloheximide: Colour reaction with Diazonium Blue B: Growth on 50% (w/v) glucose: Lipolytic activity on Tween 80 agar:

tugal) and under number CBS 7657 in the Yeast Division of the Centraalbureau voor Schimmelcultures in Delft (Netherlands) .

39.2 ± 0.6 + (weak)

+

The formation of ellipsoidopedunculate asci with acerose spores, the presence of Q9 as the principal ubiquinone, together with physiological data characterize the new species as a member of the genus Metschnikowia KamienTable 2. DNA relatedness between M.gruessii and other Metschnikowia species % DNA reassociation with"

+

Coenzyme Q system. In addition to the principal ubiquinone Q9 (79%) ubiquinone Q8 (9.2%) was also present. Etymology. The specific epithet gruessii was chosen in honour of J. Gruss who first isolated and named the anamorph of the species. Origin and Deposits. The type strain IGC 4382 was isolated from the nectaries of Hebe salicifolia collected at Oeiras, Portugal. It has been deposited (living and dried) in the Portuguese Yeast Culture Collection in Oeiras (Por-

Species

IGC C

M.gruessii IGC 4382 T

M. australis M. bicuspidata M. hawaiiensis M. krissii

4212 4206 4220 2895 4209 2726 4446 2892

5±3.2 5±6.7 20±7.0 6±0.2 4± 1.6 16 ± 0.3 7±5.9 8±3.2

M.lunata

M. pulcherrima M. reukaufii

M.Zobellii

" Mean ± s.d. based on three determinations. b Strain numbers correspond to type strains except IGC 2726. C IGC, Instituto Gulbenkian de Ciencia, Portuguese Yeast Culture Collection (PYCC).

Metschnikowia gruessii sp. nov.

ski (Miller and Phaf!, 1984). Of the eight presently accepted species of Metschnikowia, four are parasitic in invertebrates or free-living in aquatic environments, and the others are associated with terrestrial environments, mainly fruits and flowers. On the basis of genome comparisons Mendon(a-Hagler et aI., (1985), determined the taxonomic position of the aquatic group of Metschnikowia and their varieties, reporting no close relationship between species belonging to the aquatic and terrestrial groups. Since all our isolates originate from flowers, produce chlamydospores and differ by over 2% in G+C content from all the type strains of the aquatic species (Table 1), no close relatedness with these would be expected. Indeed, DNA-DNA reassociation studies between M. gruessii (type strain, IGC 4382) and the type cultures of M. australis, M. bicuspidata, M. krissii, and M. zobellii gave relatively low reassociation values (5-8%) indicating that M. gruessii bears no significant genomic similarity with this group of aquatic yeasts (Table 2). Distinct vegetative cells, asci, and chlamydospore morphology allowed us to distinguish all the terrestrial species from one another and from M. gruessii. The occurrence of exceptionally large, elongate asci in M. hawaiiensis, globose chlamydospores and sphaeropedunculate asci in M. pulcherrima, broadly ellipsoidal chlamydospores, clavate asci and the absence of pigment in M. reukaufii, in contrast to "airplane" configurations, ellipsoidopedunculate asci and the absence of pigment in M. gruessii, were criteria used for differentiation. Based on phenetic characteristics, the species that most closely resembles the new taxon is M. reukaufii (Table 3). We found a low extent of reassociation with the type strains of M. reukaufii and C. reukaufii, 7% and 22% respectively, giving support to earlier investigations conducted with a species-specific DNA probe (Henriques et aI., 1991). However, one strain

Table 4. Salient differences between M.gruessii, M. reukaufii and M. pulcherrima

437

Table 3. Extent of DNA relatedness between M.gruessii and strains identified as M. reukaufiia % DNA reassociation with: b

Strain

IGC 4382 IGC 3275 IGC 4446

C

IGC 4446 IGC 3684 IGC 4401

7±5.9 8±6.0 1±0.2

7±5.3 10 ± 2.6

C. reukaufii

IGC 2729 IGC 3276

22±0.8 7±0.5

14 ± 0.9

N. reukaufii

IGC 3275

97±0.6

M.gruessii

IGC 4796 IGC 4800

91 ±2.3 92 ± 0.4

M. reukaufii

a

b C

n.d.

n.d.

5±4.8 86±0.7 91±3.1 88 ± 1.2 7±5.3

n.d. n.d.

n.d. n.d.

Strains were identified by conventional methods according to van der Walt and Yarrow (1984). Mean ± s.d. based on three determinations. The type strains of Candida reukaufii and Nectaromyces reukaufii were listed as anamorphs of M. reukaufii by Miller and van Uden (1970).

that was listed as an anamorph of M. reukaufii by Miller and van Uden (1970), Nectaromyces reukaufii (IGC 3275), shared 97% base sequences with the new taxon, suggesting that they belong to the same species (Table 4). This strain was studied by Gruss (1918), Wickerham (1964), and Pitt and Miller (1968), and the presence of cross-formations was depicted in the 1952 monograph of The Yeasts (Lodder and Kreger-van Rij), although reported to be an unstable character by some authors (Gruss, 1918; Hautmann, 1924). More recently, Eisikowitch et ai. (1990) reported the presence of two strains of M. reukaufii, an "airplane" morphotype and a second

Characteristic Fermentation Glucose Assimilation a-a Trehalose Maximum Temp (0G) of growth Presence of: "Cross formations" in 20% honey solution True mycelium with ovoid conidia

Metschnikowia gruessii

Metschnikowia pulcherrima

Metschnikowia reukaufii

+D

+

+

+

+

35-36

34-35

31-32

+ +

Chlamydospore shape:

subglobose

globose

cylindroical

Asci shape:

ellipsoidopedunculate

sphaeropedunculate

clavate

39.2± 0.6

47.9±0.5

Pulcherrimin pigment: Mol % (G+C): 3a a

+

Mol % (G+C) represents the mean ± s.d. of the type strains.

42.8 ±0.2

438

G. Gimenez-Jurado

strain with large ovoid chlamydospores, affecting pollen germination in the common milkweed Asclepias syriaca. This report reinforces what we observed in the process of isolating our strains from different flowers, i. e. that M. gruessii ("airplane" morphotype) and M. reukaufii occur side by side in nature, although the latter is far more frequent. Whether these species are native to the nectars of flowers or carried by the insects that pollinate them remains to be seen. Numerous isolates of both species have been recovered from a variety of flowers, often from the same samples, suggesting that these two species of Metschnikowia share a common habitat. Attempts to induce cross-formations in other species, including the type strain of M. reukaufii, by growing them in 20% honey solution or media with high sugar concentration failed. Cross-forming isolates gave DNA reassociation values of 90% and higher, indicating that this morphological trait has taxonomic relevance at the species level. Physiologically, M. gruessii can be distinguished from M. reukaufii by the inability to assimilate trehalose, delayed fermentation of glucose and a lower maximum temperature for growth. Salient differences are summarized in Table 3. Acknowledgments. The author is very grateful to Drs. Isabel Spencer-Martins and Marc-Andre Lachance for critical reading of the manuscript, to Marilia Henriques for unveiling this new species with the ribosomal DNA probe, to Ema Fonseca, Gulbenkian Institute of Science, for the HPLC analysis of the extracted ubiquinones of M. gruessii, and to David Yarrow, CBS, Delft, for the type strains of Metschnikowia species. I will ever be deeply indebted to Prof. Nicolau van Uden, without whose support and commitment to Yeast Taxonomy the Portuguese Yeast Culture Collection would not exist today, and none of this work would have been possible.

References Barnett,]. A., Payne, R. W., Yarrow, D.: Yeasts, Characteristics and Identification. London, Cambridge University Press, 1990 Britten, R. ]., Pavich, M., Smith, J.: A new method of DNApurification. Carnegie Institute of Washington Year Book 68, 400-402 (1970) Diddens, H. A., Lodder, J. (eds.): Die anaskosporogenen Hefen. Zweite Hiilfte. Amsterdam, North Holland Publishing Co. 1942 Eaton, N. R.: New press for disruption of microorganisms. J. Bact. 83, 1359-1360 (1962) Eisikowitch, D., Lachance, M. A., Kevans, P. G., Willis, S., Collins-Thompson, D. L.: The effect of the natural assemblage of microorganisms and selected strains of the yeast Metschnikowia reukaufii in controlling the germination of pollen of the common milkweed Asclepias syriaca. Can. J. Bot. 68,1163-1165 (1990) Gimenez-Jurado, G., Placido, T., Cidadiio, A. ]., Cabe(a-Silva, c., Fonseca, E., Roeijmans, H. J., Eijk, G. W., van Uden, N.: Kurtzmanomyces tardus sp. nov., a new anamorphic yeast species of basidiomycetous affinity. Antonie v. Leeuwenhoek 58, 129-135 (1990)

Golubev, W. 1., Smith, M. Th., Poot, G. A., Kock, J. L. F.: Species delineation in the genus Nadsonia. Antonie v. Leeuwenhoek 55, 369-382 (1989) Griiss, J.: Die Anpassung eines Pilzes (Anthomyces reukaufii) an den Bliitenbau und den Bienenriissel. Ber. Dtsch. Botan. Gesellsch. 35, 746-761 (1918) Hautmann, F.: Dber die Nektarhefe Anthomyces reukaufii. Arch. Protistenk. 48, 213-244 (1924) Henriques, M., Sa-Nogueira, I., Gimenez-Jurado, G., van Uden, N.: Ribosomal DNA spacer probes for yeast identification: Studies in the yeast genus Metschnikowia. Yeast 7, 167-172 (1991) Kreger-van Rij, N. J. W. (ed.): The Yeasts: A Taxonomic Study. Amsterdam, Elsevier Science Publ. 1984 Kurtzman, C. P., Smiley, M. J., Johnson, C. J., Wickerham, L. J., Fuson, G. B.: Two closely related heterothallic species, Pichia amylophila and Pichia mississipiensis: Characterization by hybridization and deoxyribonucleic acid reassociation. Int. J. System. Bact. 30, 208-216 (1980) Lachance, M. A., Starmer, W. T., Phaff, H. J.: Metschnikowia hawaiiensis sp. nov., a heterothallic haploid yeast from hawaiian morning glory and associated drosophilids. Int. J. System. Bact. 40, 415-420 (1990) Lodder, J., Kreger-van Rij, N. J. W.: Candida, pp.553. In: The Yeasts: A Taxonomic Study. Amsterdam, North Holland Publishing Co., 1952 Marmur,]., Doty, P.: Determination of the base composition of DNA from its thermal denaturation temperature. J. Mol. BioI. 5, 109-118 (1962) Mendon(a-Hagler, L., Hagler, A., Phaff, H. J., Tredick, J.: DNA relatedness among aquatic yeasts of the genus Metschnikowia and proposal of the species Metschnikowia australis comb. nov. Can. J. Microbiol. 31, 905-909 (1985) Miller, M. W., van Uden, N.: Metschnikowia, pp.408-429. In: The Yeasts: A Taxonomic Study 1]. Lodder, ed.). Amsterdam, North Holland Publishing Co., 1970 Miller, M. W., Phaff, H. J.: Metschnikowia, pp. 266-278. In: The Yeasts: A Taxonomic Study (N. J. W. Kreger-van Rij, ed.). Amsterdam, Elsevier Science Publ., 1984 Nadson, G. A., Krassilnikov, N. A.: Bull. Soc. myco!. France 43, 232 (1927) Pitt, J. I., Miller, M. W.: Sporulation in Candida pulcherrima, Candida reukaufii and Chlamydozyma species: Their relationships with Metschnikowia Mycologia 60, 663-685 (1968) Schoellhorn, K.: Sur la fermentation de quelques levures des nectars des plantes d'hiver. Bull. Soc. Botan. de Geneve 11, 154-190 (1919) Seidler, R.]., Mandel, M.: Quantitative aspects of deoxyribonucleic acid renaturation: Base composition, site of chromosome replication and polynucleotide homologies. J. Bact. 106, 608-614 (1971) Sugiyama, J., Fukagawa, M., Ehiu, S., Komagata, K.: Cellular carbohydrate compOSitIOn, ubiquinone systems, and diazonium blue B colour test in the genera Rhodosporidium, Leucosporidium, Rhodotorula and related basidiomycetous yeasts. J. Gen. App!. Microbiol. 31, 519-550 (1985) Sydow, H., Sydow, P.: Mykologische Mitteilungen. Ann. Myco!. 16,240-248 (1918) van der Walt, J. P., Yarrow, D.: Methods for the isolation, maintenance, classification, and identification of yeasts, pp. 45-104. In: The Yeasts: A Taxonomic Study. Amsterdam, Elsevier Science Pub!., 1984 Wickerham, L. J.: A preliminary report on a perfect family of exclusively protosexual yeasts. Mycologia 56, 253-266 (1964)

Gabriella Gimenez-Jurado, Laboratory of Microbiology, Gulbenkian Institute of Science, Apartado 14,2781 Oeiras Codex, Portugal