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Int J Syst Evol Microbiol 55 (2005), 1353-1363; DOI  10.1099/ijs.0.63277-0
© 2005 International Union of Microbiological Societies

Taxonomy and phylogeny of the ascomycetous yeast genus Zygoascus, with proposal of Zygoascus meyerae sp. nov. and related anamorphic varieties

Maudy Th. Smith, V. Robert, G. A. Poot, Wendy Epping and A. W. A. M. de Cock

Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

Correspondence
Maudy Th. Smith
smith{at}cbs.knaw.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Physiological characters, mating compatibility, PCR-RAPD fingerprints, mol% G+C content, DNA–DNA relatedness, and large-subunit and internal transcribed spacer rRNA gene sequences of strains assigned to the genus Zygoascus were re-examined. On the basis of those data, and after phylogenetic analyses, an emendation of Zygoascus hellenicus (type material is a cross of CBS 6736TxCBS 5839T) is proposed, comprising two novel anamorphic varieties, Candida steatolytica var. steatolytica (CBS 6736T) and C. steatolytica var. inositophila (CBS 5839T). A novel teleomorphic species, Zygoascus meyerae sp. nov. (type material is a cross of CBS 4099TxCBS 7521T) is described, together with two novel anamorphic varieties corresponding to it, Candida hellenica var. hellenica (CBS 4099T) and C. hellenica var. acidophila (CBS 7115T).

Abbreviations: ITS, internal transcribed spacer; LSU, large subunit

Published online ahead of print on 3 December 2004 as DOI 10.1099/ijs.0.63277-0.

The GenBank/EMBL/DDBJ accession numbers for the 26S and ITS rRNA gene sequences determined in this study are given in Table 1Go.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The monotypic genus Zygoascus was introduced in 1986 by Smith to accommodate the teleomorph state of three Candida species: Candida hellenica (Verona & Picci) D. S. King & S.-C. Jong, Candida inositophila Nakase and Candida steatolytica Yarrow. These species were all found to represent mating types of a single species, with C. hellenica and C. inositophila representing one mating type, designated a, and C. steatolytica representing the opposite type, designated {alpha}. C. inositophila and C. steatolytica were placed into synonymy with C. hellenica, since that name has priority, and the nomenclaturally independent teleomorph name Zygoascus hellenicus was modelled after the accepted anamorph name.

In earlier studies, Meyer et al. (1984)Go reported that, of four strains previously identified as being representatives of C. steatolytica [Centraalbureau voor Schimmelcultures (CBS catalogues; CBS, 1972Go)], only one showed a DNA–DNA relatedness of 98 % with the type strain. Therefore, the remaining strains were excluded from this species and remained unclassified. Furthermore, Meyer et al. (1984)Go kept C. steatolytica and C. inositophila as separate taxa, because the DNA–DNA reassociation value between the type strains of these species was around 57 %, as determined by the DNA filter reassociation technique (S. A. Meyer, personal communication), a value considered by Meyer to be low enough to keep the species apart. However, the observation of mating among the three unclassified isolates and the type strains of the three aforementioned species, resulting in asci with ascospores, was considered sufficient to conclude that they all belonged to the species Z. hellenicus (Smith, 1986Go). Accepting the presence of mating as an indicator of conspecificity, Meyer et al. (1998)Go excluded these three species from the list of purely anamorphic Candida species in a later revision of this genus.

As these species concepts based on sexual compatibility and DNA–DNA relatedness values were discrepant and, as we had obtained some new and difficult-to-place Z. hellenicus-like isolates, we undertook a revision of this group. In this study, physiological characters and mating behaviour were studied, and genome-level differences were examined by means of PCR-randomly amplified polymorphic DNA (RAPD) analyses, DNA–DNA reassociation studies and sequencing of the D1/D2 domain of the large-subunit (LSU) rRNA gene, as well as the internal transcribed spacer (ITS) regions ITS1 and ITS2 and the intervening 5·8S rRNA gene.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Cultures.
In Table 1Go, 14 Zygoascus strains as well as the type strain of Pichia hangzhouana (Lu & Li, 1989Go) are listed, together with their respective origins and original identifications. P. hangzhouana was included because, from the original physiological description and the sequence data of Kurtzman & Robnett (1998)Go, this species resembled Z. hellenicus. Strain CBS 8813T belonging to Candida bituminiphila (Robert et al., 2001Go) was used as an outgroup for phylogenetic analyses.


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  		Table 1. List of strains of the genus Zygoascus examined

 
Phenotypic characterization.
Fermentation ability and utilization of carbon and nitrogen compounds were tested according to the methods described by Yarrow (1998)Go. Physiological relationships between the strains of the Zygoascus clade were analysed by using the BioloMICS software (Robert & Szoke, 2003Go). Mating type behaviour was determined by mixing strains in various combinations, as shown in Table 2Go. Actively growing 2–7 day-old cultures were used. Ascosporulation medium was composed of 5 % powdered malt extract (Difco) and 2 % agar (Yarrow, 1998Go), with or without supplemented vitamins (van der Walt & Yarrow, 1984Go). Mating type assignment was in compliance with the designations of Smith (1986)Go. Mating experiments were repeated twice, and microscopic observations were done at regular intervals from 2 weeks to 3 months of growth.


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  		Table 2. Matrix of mating combinations and DNA–DNA reassociation values among strains of the genus Zygoascus

Lower triangle, mating combinations; upper triangle, DNA–DNA reassociation values. –, No ascus production; (+), ascus production without or with immature ascospores; +, ascus production with immature and mature ascospores; mt, mating type.

 
DNA isolation.
For PCR, DNA from 3–7 day-old yeast cultures grown on 4 % glucose/0·5 % peptone/0·5 % yeast autolysate/2 % agar (Yarrow, 1998Go) was isolated according to the method of Möller et al. (1992)Go. To determine the mol% G+C content and DNA–DNA reassociation values, DNA was isolated from cells grown in 2 l yeast malt broth (Wickerham, 1951Go) for 2 days, by using hydroxyapatite column chromatography, as described by Smith et al. (1995)Go.

PCR-RAPD analysis.
For fingerprinting analyses, a decamer primer, OPA13 (Operon Technologies) and the microsatellite primers M13 (Vassart et al., 1987Go) and (ATG)5 (Garbelotto et al., 1993Go) were used in PCRs. The final reaction mixtures of 50 µl containing 50 ng genomic DNA, 10 mM Tris/HCl (pH 9·5), 50 mM KCl, 1·5 mM MgCl2, 0·01 % gelatin, 200 µM of each of the dNTPs (Roche), 10 pM of primer and 0·5 U of Supertherm polymerase (ITK Diagnostics) were processed in a Biomed thermocycler (model 60; Theres). The programmes of 40 cycles of the thermocycler were: 94 °C for 60 s, 34 °C for 60 s and 72 °C for 120 s for OPA13; 94 °C for 20 s, 60 °C for 60 s and 72 °C for 120 s for M13; and 94 °C for 60 s, 48 °C for 60 s and 72 °C for 120 s for (ATG)5. The PCR products were separated on 1·7 % agarose gels in 1x Tris/acetate/EDTA buffer (40 mM Tris/HCl and 2 mM EDTA; pH 8·0), chilled to 14 °C. The PCR-RAPD profiles for each strain obtained with the three primers were combined in a composite fingerprint by using GelCompar 3.1 (Applied Maths). Similarities between combined fingerprints were calculated by using the Pearson product-moment correlation coefficient. A dendrogram was generated by using the UPGMA method.

Mol% G+C content and DNA–DNA relatedness.
Mol% G+C content and DNA–DNA relatedness values were calculated according to previously described procedures (Smith et al., 1995Go).

rRNA gene sequencing and sequence analysis.
The primers NL-1 and NL-4 (O'Donnell, 1993Go) were used to amplify the D1/D2 region of the LSU rRNA gene, and the primers ITS1 and ITS4 (White et al., 1990Go) were used to amplify the whole ITS region, including ITS1, ITS2 and the intervening 5·8S rRNA gene. The compositions and final volumes of the reaction mixtures were as for the PCR-RAPD analysis. An initial denaturation step at 94 °C for 5 min was followed by 35 cycles of 94 °C for 45 s, 52 °C for 30 s and 72 °C for 2 min for the D1/D2 amplification, or 35 cycles of 94 °C for 60 s, 55 °C for 60 s and 72 °C for 2 min for the ITS amplification, followed in both cases by final extension at 72 °C for 6 min. The amplified DNA was stored at –20 °C and was purified before use by using GFX columns (27-9602-01; Amersham Biosciences), according to the manufacturer's instructions. For the sequencing of both strands of the D1/D2 and ITS regions, the primers used were the same as those for the PCRs. The final sequence reaction mixture of 10 µl contained 2 µl Dyenamic (Amersham Biosciences), 1 µl primer (final concentration 0·4 pmol µl–1), 1 µl buffer, 5 µl Milli-Q-water (Millipore) and 1 µl PCR product. Purified sequencing reactions were loaded on an ABI 3700 capillary sequencer (PE Biosystems). Sequences were edited and assembled by using the SeqMan package (DNAStar) and aligned using CLUSTAL W software (Thompson et al., 1994Go). No manual adjustments were done, except that the ends were trimmed. Phylogenetic relationships were inferred by a combined analysis of the D1/D2 and the ITS regions, by using the maximum-likelihood optimality criterion and the general heuristic search option of PAUP* software, version 4.0 beta 10 (Swofford, 1993Go). Bootstrap values of less than 50 % were not reported.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Positive mating reactions merely resulting in asci with ascospores are generally accepted as a guideline for conspecificity. However, the ultimate criterion is to confirm the presence of recombinants of parental strains. When such genetic analyses are difficult to perform, alternative approaches must be applied to determine the conspecificity and the taxonomic status of the strains. Initially, DNA–DNA relatedness, by measuring reassociation kinetics of denatured DNA mixtures, was applied; later on, other molecular parameters were introduced. A revision of the genus Zygoascus was started by using various approaches to classify recently obtained Zygoascus-like isolates.

Molecular analysis
PCR-RAPD results.
Individual OPA13, M13 and (ATG)5 patterns and the dendrogram derived from the combined fingerprints are shown in Fig. 1Go. At a similarity level of 40 %, the 15 strains could be separated into two major groups, Group I and Group II. At a similarity level of 80 %, however, each group could be further subdivided. Group Ia was represented by a single strain (CBS 6736T), Group Ib contained six strains (CBS 4028, CBS 5839T CBS 6726, CBS 6779, CBS 7652, CBS 8424), Group IIa included five strains (CBS 4075, CBS 4099T, CBS 6360, CBS 7521T, CBS 8426) and Group IIb comprised three strains (CBS 6173, CBS 7115T, CBS 8425).



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  		Fig. 1. PCR-RAPD fingerprints of strains of Z. hellenicus and Z. meyerae obtained with primers OPA13, (ATG)5 and M13, and the dendrogram resulting from the combined analysis of the fingerprints using the Pearson product-moment correlation coefficient and the UPGMA clustering method. Ia, Ib, IIa and IIb refer to clusters recognized on the basis of the fingerprints.

 
DNA base composition and DNA–DNA reassociation.
Analysis of the DNA base composition, expressed as mol% G+C content, showed that the range of values obtained was very narrow, spanning only 1·5 mol%. CBS 5839T was at the lower end of the spectrum with 43·6 mol%, whereas CBS 4028 was at the upper end, with 45·1 mol% (Table 1Go).

The separation into groups as observed by PCR-RAPD fingerprinting was further examined by determining DNA–DNA relatedness values calculated from reassociation rates (Table 2Go, upper triangle). Two main reassociation groups could be distinguished, which were identical to the major groups observed following PCR-RAPD fingerprint analysis (Fig. 1Go). The reassociation values obtained when Group I isolates were paired with Group II isolates ranged from 19 to 38 % (Table 2Go). Detailed examination revealed a further subdivision similar to that obtained in the fingerprint analyses (Fig. 1Go). Reassociation values of strains of Group Ia, represented by CBS 6736T (the type strain of C. inositophila), with strains of Group Ib ranged from 66 to 76 %. The values obtained in pairings among the six strains of Group Ib (including the type strain of C. steatolytica) ranged from 91 to 100 %. Within Group IIa, reassociation values ranging from 93 to 100 % were observed. This group included the type strains of C. hellenica (CBS 4099T) and P. hangzhouana (CBS 7521T), which by this way of measuring appeared to share 100 % of their DNA. The name P. hangzhouana, though pertaining to the teleomorphic genus Pichia, was originally given to material that did not contain true teleomorphic elements; ascospores that were ostensibly seen appear to have been oil droplets or artefacts. According to article 59.3 of the International Code of Botanical Nomenclature (Greuter et al., 2000Go), this name is therefore considered to be that of an anamorph. On the basis of the reassociation results obtained, P. hangzhouana can be considered to be conspecific with C. hellenica. Reassociation values among the three strains of Group IIb, CBS 6173, CBS 7115T and CBS 8425, originally identified as Z. hellenicus, ranged from 99 to 100 %. The DNA–DNA reassociation values between strains of groups IIa and IIb ranged from 65 to 79 %.

Sequence analyses.
Analysis of the D1/D2 and ITS regions showed the separation of the strains into two well-supported main clades (100 % bootstrap), which could be further divided into two subclades (Fig. 2Go) The groupings based on the phylogenetic analysis were identical to those obtained from the PCR-RAPD fingerprints (Fig. 1Go) and DNA–DNA reassociation analyses (Table 2Go and Fig. 3Go), except for strain CBS 5839T, which was closer to CBS 6736T than to the other members of clade Ib. The latter topology was also found in the phylogenetic analysis of the actin gene (Daniel, 2002Go).



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  		Fig. 2. Phylogenetic tree based on rRNA gene sequences of D1/D2 and ITS (ITS1, 5·8S and ITS2) regions. The tree was constructed using the maximum-likelihood method as optimality criterion and the general heuristic search option of PAUP* software version 4.0 beta 10. The numerals represent 100 replicated bootstrap samplings (values less than 50 % are not reported). Ia, Ib, IIa and IIb refer to clusters recognized on the basis of the PCR-RAPD fingerprints.

 


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  		Fig. 3. Relationships between groups of strains of Z. hellenicus and Z. meyerae based on average DNA–DNA reassociation values and distribution of mating types (mt a and mt {alpha}). Distances are not proportional to levels of relatedness. SD values for levels of reassociation within groups were <=5 % and are means of at least two determinations. SD values for levels of reassociation between groups were <=7 %. Ia, Ib, IIa and IIb refer to clusters recognized on the basis of the PCR-RAPD fingerprints.

 
Morphology and physiology
The physiological profiles were compared by using the BioloMICS software based on pairwise similarity (simple matching coefficient), as listed in the CBS yeast database (http://www.cbs.knaw.nl). Isolates belonging to the Zygoascus clade were very similar, with only a few strains being slightly different (see Table 3Go). The differences seen mainly involved the assimilation of D-ribose, D-arabinose, methyl {alpha}-D-glucoside, L-arabinitol, DL-lactate and 1,2-propanediol. The two teleomorphic species newly distinguished below, Zygoascus meyerae sp. nov. and Z. hellenicus, could not be distinguished physiologically. Among the four anamorphic varieties newly distinguished below, only Candida hellenica var. hellenica and Candida steatolytica var. steatolytica showed different assimilation properties for DL-lactate, whereas Candida steatolytica var. inositophila differed from Candida hellenica var. acidophila in the assimilation of methyl {alpha}-D-glucoside and 2-keto-D-gluconate.


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  		Table 3. Physiological results for the strains belonging to the Zygoascus clade

Comp., compiled; d, delayed reaction; V, variable reaction; W, weak reaction; ND, not determined. All tested strains were positive for growth in the presence of D-glucose, D-galactose, L-sorbose, D-glucosamine, D-xylose, L-rhamnose, sucrose, trehalose, cellobiose, salicin, glycerol, xylitol, D-glucitol, D-mannitol, myo-inositol, 5-keto-D-gluconate, D-glucuronate, D-galacturonate and ethanol as carbon source; growth on L-lysine, cadaverine and glucosamine as nitrogen source; growth without myo-inositol, pantothenate, pyridoxine, niacin, para-aminobenzoic acid and cycloheximide (0·01 and 0·1 %); and growth at 25, 30, 35 and 37 °C. All tested strains were negative for growth on melibiose, inulin, erythritol, methanol, 2,3-butanediol, quinic acid, D-glucarate and D-galactonate as carbon source; growth on nitrate, creatine, creatinine and imidazole as nitrogen source; growth without vitamins, thiamine, biotin plus thiamine, and pyridoxine plus thiamine as nitrogen source; growth on 1 % acetic acid; starch formation; acetic acid production; urea hydrolysis; Diazonium blue B reaction; and growth at 42 and 45 °C. Strain designations given are CBS numbers.

 
Results of crosses among strains of the four groups obtained by PCR-RAPD fingerprints and DNA–DNA reassociations are presented in the lower triangle of Table 2Go. None of the studied strains was self-fertile. Asci with or without (im-) mature, hat-shaped ascospores were usually observed after 2–4 weeks of incubation (Fig. 4Go), when mating was compatible (Table 2Go). In Group I, ascus formation was not observed for four crosses (CBS 4028xCBS 6726T; CBS 7652xCBS 6726; CBS 7652xCBS 6779 and CBS 6736TxCBS 8424). The same occurred in Group II for the combination of CBS 4099T and CBS 8425. Asci without or with (im-) mature ascospores were produced when compatible mating types of Group I and Group II were mixed. From the mating tests, it can be concluded that eight strains, including the type strains of C. hellenica and C. inositophila, represent mating type a, and seven strains, including the type strains of C. steatolytica and P. hangzhouana, represent mating type {alpha}. The predicted relatedness of P. hangzhouana with Z. hellenicus on the basis of the D1/D2 rRNA gene sequences (Kurtzman & Robnett, 1998Go) was confirmed by the observation of ascospore production after mixing of the type strains (Table 2Go). A summary of the mean reassociation values and the distribution of the mating types between the various groups are given in Fig. 3Go.



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  		Fig. 4. Ascus and one hat-shaped ascospore of Z. meyerae CBS 4099TxCBS 7521T (arrow). Bar, 5 µm.

 
The occurrence of mating between Group I and Group II isolates with low DNA–DNA relatedness indicates that they possess the same mating type system and are closely related (belonging to the same genus). Whether recombination can occur between these two groups should be investigated by genetic analysis, but this would be an onerous and difficult procedure because of the extreme hyphal state of the teleomorph. However, the latter genetic analysis has been performed for the yeast genus Arthroascus (Naumova et al., 2003Go), where mating occurred between taxa with low (19–31 %), as well as intermediate (65 %), nuclear DNA–DNA relatedness. The progeny of crosses of taxa with low DNA–DNA relatedness showed an absence of recombinants, indicating that the crosses represented a poorly compatible type of interspecies hybridization (Naumov et al., 2003Go). Analyses of crosses between taxa sharing intermediate DNA–DNA reassociation values of 65–70 % has revealed some genetic exchange (G. I. Naumov & M. Th. Smith, unpublished data). The progeny of crosses between strains within the same group with high reassociation values gave fertile recombinants (Naumov et al., 2003Go). A similar example was demonstrated for the teleomorphic genus Stephanoascus. In this genus, mating occurred among taxa with low DNA–DNA relatedness, as well as differences in 18S rRNA gene sequences (Ueda-Nishimura & Mikata, 2002Go). These authors therefore concluded that mating indicates close relatedness, not conspecificity.

In addition, the division of the Zygoascus isolates into four clusters is supported by the actin gene-sequencing study of Daniel (2002Go; and unpublished data). Each of the four groups could be distinguished by differences of more than 20 nucleotides. H.-M. Daniel (personal communication) considered this value to be high enough to support the hypothesis that these groups represented separate taxa.

On the basis of the results summarized in Fig. 3Go and the above cited data, it seems warranted to propose two novel teleomorphic species within the genus Zygoascus, namely, Z. hellenicus and Z. meyerae sp. nov. These species correspond to Group I and Group II, respectively. To deal with the two molecular genetic subgroups found within each of these Zygoascus species, we propose C. steatolytica var. steatolytica and C. steatolytica var. inositophila as two novel anamorphic varieties connected to Z. hellenicus, and C. hellenica var. hellenica and C. hellenica var. acidophila as two novel anamorphic varieties linked to Z. meyerae. The varieties are proposed only at the anamorphic level, because the level of DNA–DNA relatedness (71 %) between varietal subgroups within each species appears sufficient to predict fully compatible mating; thus, the organisms at the teleomorphic level represent (at least potentially) unified biological species. This matter needs to be further investigated with studies on actual introgression levels among the groups and subgroups within the genus Zygoascus. In the meantime, because strong consistency was observed among all the molecular characters studied, we feel confident to propose the two teleomorphic species. Because all the observations (DNA–DNA reassociation, D1/D2, ITS and actin gene sequences, and PCR-RAPD fingerprints) consistently suggest that two subgroups can be distinguished within each teleomorphic species, but not to the point where we could describe them as separate species, we propose to consider them as four anamorphic varieties.

Taxonomy
The following species and varieties within the Zygoascus clade are proposed.

I. Zygoascus hellenicus M. Th. Smith.

Antonie van Leeuwenhoek (1986) 52, 27.

Teleomorph of C. steatolytica var. steatolytica and C. steatolytica var. inositophila.

Type specimen: a dried specimen of mixed sporulating culture of CBS 5839TxCBS 6736T is preserved at the CBS.

Ia. Candida steatolytica Yarrow var. inositophila (Nakase) M. Th. Smith & V. Robert comb. nov., stat. nov.

Basionym: C. inositophila Nakase

Antonie van Leeuwenhoek (1975) 41, 206.

Anamorph of Z. hellenicus.

Type strain: ex-type isolate CBS 6736T (=IFO 1575T) is a living strain in the CBS yeast collection.

Ib. Candida steatolytica Yarrow var. steatolytica.

Antonie van Leeuwenhoek (1969) 35, 24.

Anamorph of Z. hellenicus.

Type strain: ex-type isolate CBS 5839T (=IFO 10184T) is a living strain in the CBS yeast collection.

II. Zygoascus meyerae M. Th. Smith & V. Robert sp. nov.

Etymology: the epithet meyerae is chosen in honour of S. A. Meyer, who played a great role in the establishment of DNA–DNA reassociation methods in yeast taxonomy.

Teleomorph of C. hellenica var. hellenica and C. hellenica var. acidophila.

Type specimen: a dried specimen of a mating culture of CBS 4099TxCBS 7521T with ascospores is preserved at the CBS as Herb. CBS 6599T.

Latin diagnosis of Zygoascus meyerae M. Th. Smith et V. Robert sp. nov.
Species heterothallica. Coloniae in agaro maltoso post 7 dies 25 °C 7–10 mm diametro, elevatae, restrictae, tenaces, rugosae vel crispatae, pubescentes vel floccosae, albae vel cremeae, margine fimbriate circumdatae. Cellulae gemmantes ovoideae, elongatae vel cylindricae, (2·2–5·4)x(5·4–10·2) µm, saepe catenulatae et pseudohyphae formantes. Hyphae septatae, ramosae, 2·0–3·5 µm latae. Microsporus singulus in centro septum. Blastosporae ellipsoidae, ovoideae vel elongatae, cylindricae. Asci ovoidei vel globosi, parietibus persitentibus, plerumque unispori, sed etiam 4-spori (4·1–6·2)x(5·2–7·4) µm. Ascosporaehemisphericae ad galeatae, ad basim margine prominente praeditae, (1·5–3·5)x(2·5–4·5) µm. Glucosum, galactosum, maltosum, sucrosum, trehalosum, cellobiosum, raffinosum (variabile) fermentur, at non methyl {alpha}-D-glucosidum, melibiosum, lactosum, melezitosum, inulinum, amylum nec xylosum. Glucosum, galactosum, sorbosum, glucosaminum, xylosum, L-arabinosum, rhamnosum, sucrosum, trehalosum, cellobiosum, salicinum, glycerolum, ribitolum, xylitolum, glucitolum, mannitolum, galactitolum, inositolum, 5-ketogluconatum, glucuronatum, galacturonatum et ethanolum assimilantur, at non melibiosum, lactosum, inulinum, erythritolum, methanolum, butane-2,3-diolum, acidum quinicum, glucaratum nec galactonatum. Ribosum, D-arabinosum, maltosum, methyl {alpha}-D-glucosidum, arbutinum, raffinosum, melezitosum, amylum, L-arabinitolum, glucono-1,5-lactonum, 2-ketogluconatum, gluconatum, acidum DL-lacticum, acidum succinicum, acidum citricum, propane-1,2-diolum variabile assimilantur. Lysinum, cadaverinum, glucosaminum assimilantur at non kalium nitricum, kalium nitrosum, creatinum, ceatininum nec imidazolum. Ad crescentiam vitaminae necessariae sunt. Diazonium caeruleum B negativum. Augmentum ad 37 °C. Holotypus cultura conjugate ascigera CBS 4099TxCBS 7521T, exsiccate in collectione CBS, Utrecht, praeservatur.

IIa. Candida hellenica (Verona & Picci) D. S. King & S.-C. Jong var. hellenica.

Mycotaxon (1977) 6, 413.

Basionym: Trichosporon hellenicum Verona & Picci, Ann Microbiol Enzimol (1958) 8, 106.

Synonym: Pichia hangzhouana X.-H. Lu & M.-X. Li, Acta Mycol Sin (1989) 8, 253.

Anamorph of Z. meyerae.

Type strain: ex-type isolate CBS 4099T (=IFO 10246T) is a living strain in the CBS yeast collection.

IIb. Candida hellenica var. acidophila M. Th. Smith & V. Robert nov. var.

Etymology: the epithet acidophilus is chosen because some strains were isolated from acidic source material.

Anamorph of Z. meyerae.

Type strain: ex-type isolate CBS 7115T (=NCYC 2544T) is a living strain in the CBS yeast collection.

Latin diagnosis of Candida hellenica var. acidophila M. Th. Smith et V. Robert nov. var.
Caractera morfologica similes Candida hellenica var. hellenica. Glucosum fermentur et galactosum, maltosum, sucrosum, trehalosum, cellobiosum, raffinosum variabile fermentur, at non methyl {alpha}-D-glucosidum, melibiosum, lactosum, melezitosum, inulinum, amylum nec xylosum. Glucosum, galactosum, sorbosum, glucosaminum, amylum, glucono-1,5-lactonum, acidum succinicum, acidum citricum, xylosum, L-arabinosum, rhamnosum, sucrosum, trehalosum, cellobiosum, salicinum, glycerolum, ribitolum, xylitolum, glucitolum, mannitolum, galactitolum, inositolum, 5-ketogluconatum, glucuronatum, galacturonatum et ethanolum assimilantur, at non melibiosum, D-arabinosum, methyl {alpha}-D-glucosidum, 2-ketogluconatum, lactosum, inulinum, erythritolum, methanolum, butane-2,3-diolum, acidum quinicum, glucaratum nec galactonatum. Ribosum, maltosum, arbutinum, raffinosum, melezitosum, L-arabinitolum, gluconatum, acidum DL-lacticum, propane-1,2-diolum variabile assimilantur. Lysinum, cadaverinum, glucosaminum assimilantur at non kalium nitricum, kalium nitrosum, creatinum, ceatininum nec imidazolum. Diazonium caeruleum B negativum. Ad crescentiam vitaminae necessariae sunt. Augmentum ad 37 °C. Typus CBS 7115T lyophilus depositus in collectione zymotica CBS, Utrecht.

As mentioned above, a dried, sporulated specimen from a cross between the two mating strains, C. steatolytica CBS 5839TxC. inositophila CBS 6736T was originally deposited by Smith (1986)Go as the holotype of Z. hellenicus. This specimen was selected because asci with ascospores were abundantly present. In crossings with the ex-type isolate of C. hellenica, reduced ascospore formation was seen, but Smith considered this isolate to be conspecific with the isolates used in making the holotype. This then made the basionym T. hellenicum the oldest available name for the anamorphic species involved, and Smith (1986)Go selected ‘hellenica’ as the epithet for the species as revised. The epithet of the teleomorph name Z. hellenicus was adopted as an echo, at another taxonomic level, of the established anamorph epithet, following examples such as the echoing of the anamorph name Cryptococcus neoformans in the teleomorph name Filobasidiella neoformans. Our current application of molecular criteria has somewhat inconveniently shown that the type strain of Candida hellenica differs at the species level from both allotype isolates of Z. hellenicus. Therefore, in the present study, we were obliged to redispose C. hellenica as the anamorph of a novel teleomorph species, which in this case bears the quite different name Z. meyerae.

It should be noted that the practice of echoic designation of teleomorph epithets automatically carries with it the potential disadvantage that the anamorph and teleomorph epithets may later become separated. Though superficially it may seem sensible to apply a ‘one yeast-one epithet’ policy in order to simplify nomenclature when a new teleomorph species is being described, the separate rooting of each epithet to its own type material needs to be borne in mind. The potential for later separation of the two identical epithets is, of course, minimized when the ex-type isolate of the anamorph name is a vigorously mating isolate used in preparing the holotype of the teleomorph name. It seems sensible to suggest that reuse of currently valid anamorph epithets for newly obtained teleomorphs should be restricted to cases where this condition holds.


   ACKNOWLEDGEMENTS
 
The authors would like to thank R. Summerbell, W. Gams and A. Aptroot for valuable discussions, and M.-A. Lachance, Elma Pretorius and J. P. van der Walt for providing cultures. Raimon Zoetemelk and Lisette Smit are thanked for technical assistance.


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