Antonie van Leeuwenhoek 55:36%382 (1989) �9 Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands

Species delineation in the genus Nadsonia Sydow

W.I. GOLUBEV 1, MAUDY T. SMITH 2., G.A. POOT 2 & J.L.F. KOCK 3 l All Union Collection of Microorganisms, Institute of Biochemistry and Physiology of Microorgan- isms, Pu~'hchino 142292, USSR; 2 Centraalbureau voor Schimmelcultures, Yeast Division, Juliana- laan 67, BC2628 Delft, The Netherlands; 3 Department of Microbiology, University of Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa (*requests for offprints)

Received 24 June 1988; revised and accepted 19 October 1988

Key words: taxonomy, Nadsonia. DNA reassociation, amino acids, fatty acids

Abstract. The genus Nadsonia Sydow is revised on the basis of morphology, physiology, amino acid and fatty acid composition, electrophoretic patterns of some enzymes and DNA relatedness. Two species, N. commutata (type CBS 6640) and N. fulvescens, with two varieties, N. fulvescens var. fulvescens (type CBS 2596) and N. fulvescens var. elongata (type CBS 2594) nov. comb. are recognized. A modified diagnosis of the genus and a key are given.

Parts of this work were presented at the IXth International Specialized Symposium on Yeasts (Smolenice, CSSR, April 18-22, 1983); Vlth General Symposium on Yeasts (Montpellier, France, July 9-13, 1984) and International Symposium 'The Expanding Realm of Yeast-like Fungi' (Amersfoort, The Netherlands, August 3-7, 1987).

Introduction

The genus Nadsonia represents a distinct taxon comprising yeast organisms characterized by bipolar bud fission, conjugation between the mother cell and a bud(pedogamy), brown spherical ascospores with warty walls and inability to grow at 30 ~ C. The diagnosis of this genus, fully based on the description of N. fulvescens (Nadson & Konokotina 1911) Sydow 1912, is a rare example of stability in the yeast taxonomy. It was not amended until 1984 when some modifications were made by Miller and Phaff who took into account the features of N. commutata Golubev 1973.

On the other hand, the number of accepted species in this small genus is unstable. The species N. slovaca Kockov~i-Kratochvfiov~i & Svobod~i-PoNko- v~i 1959 and N. ukrainica Nagornaya 1973 nom. nud. are not included in this study. The strains of the former species were identified as Candida humicola by Phaff in 1970 and the authentic strain of the latter as Kloeckera corticis by Golubev et al. in 1987. For instance, Stelling-Dekker (1931) maintained N.

370

richteri Kostka 1925 as a separate species, but Lodder & Kreger-van Rij (1952) placed this name into synonymy with N. elongata Konokotina 1913. Kudryavt- sev (1954) combined N. fulvescens and N. elongata because their habitats are similar (slime fluxes of deciduous trees), but Phaff (1970) and Miller & Phaff (1984) retained both species. It is necessary to emphasize that the criteria used for species delineation in early works (Nadson & Konokotina 1911; Konokoti- na 19t3; Kostka 1925; Couch 1944) and in subsequent taxonomic treatments (Stelling-Dekker 1931; Lodder & Kreger-van Rij 1952; Phaff 1970; Barnett et al. 1983; Miller & Phaff 1984). are different, namely primarily cellular mor- phology and cultural characteristics in the former and almost exclusively physiological properties in the latter. In addition, it should be noted that species delimitation in the genus Nadsonia is especially complicated due to mother-daughter cell conjugation, so interfertility between strains cannot be applied as a guideline to their conspecificity.

To clarify the species delineation in this genus, we have examined the cultural, morphological and physiological properties, the amino acid and fatty acid composition, the electrophoretic patterns of some enzymes, and the DNA relatedness of the three taxa recognized so far.

Material and methods

Organisms

The strains studied are listed in Table 1.

Morphology and physiology

The morphological and physiological properties were tested by methods cur- rently employed in yeast taxonomy (van der Walt & Yarrow 1984). The length and width of 200 cells and diameter of 200 ascospores were measured in the type strains and of 100 cells and 100 ascospores in other strains. The growth was examined also on glucose-peptone gelatin (Nadson & Konokotina 1911). In all experiments the growth temperature was 18 ~ C.

Determination of amino acids and fatty acids

Cultures from the late exponential phase of growth were used for the study of the cell composition. They were grown in Yeast Nitrogen Base (6.7 g/l) with 1 or 8% glucose (see Table 4 and 5) on a shaker (200 r.p.m.). Quantitative total

371

Table 1. List of strains.

Species and Strain number Source

Nadsonia commutata VKM Y-1573, CBS 6640 VKM Y-1941 VKM Y-1942 VKM Y-2610

Nadsonia elongata l l x l 17N VKM Y-268 VKM Y-269 VKM Y-270 VKM Y-1653, CBS 6008 VKM Y-2531, CBS 2594 VKM Y-2578

VKM Y-2579, CBS 2593

CBS 2595

Nadsonia fulvescens NCYC 46 VKM Y-2532, CBS 2596 VKM Y-2618

VKM Y-2619

Type strain, isolated from soil, East Falkland. From soil, East Falkland. From soil, East Falkland. From soil, Carpatians, USSR.

From birch sap, Moscow region, USSR. From birch sap, Moscow region, USSR. From birch sap, Moscow region, USSR. From birch sap, Moscow region, USSR. From elm sap, Alma Ata, USSR. From birch sap, Moscow region, USSR. Type strain, from birch sap, Smolensk Region, USSR. Received from C.P. Kurtzman as N. fulvescens NRRL Y-991. Type strain of N. richteri, from hornbeam sap, Brno, CSSR. Obtained from G.A. Nadson.

Received from Carlsberg Laboratory as strain 1146 Type strain, from oak sap, Leningrad, USSR. Obtained from All-Union Institute of Agricultural Micro- biology as N. elongata 433 Obtained from All-Union Institute of Agricultural Micro- biology as strain 445

CBS: Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. NCYC: National Collection of Yeast Cultures, Food Research Institute, Norwich, United King- dom. VKM: All-Union Collection of Micro-organisms, Moscow, USSR.

amino acid analysis was pe r fo rmed on a AAA - 881 model A u t oma t i c A m i n o

Acid Ana lyze r (Micro techna Praha, CSSR) after cell hydrolysis in 6 N HC1 for

24h at 110~ Ext rac t ion and analysis of fatty acids were per formed as de-

scribed by Kock et al. (1985).

Electrophoresis o f enzymes

For e lect rophoret ic analysis of enzymes the cells were disrupted by a single

passage through a French press at a pressure of 1400 atm. Cell debris was

372

removed by centrifugation at 120.000 g for 1 h. The supernatant obtained was stored at -8~ till use. Protein content was determined by the method of Lowry et al. (1951). Enzyme electrophoresis was performed in 8.8% acryla- mide gels as described by Davis (1964). Tris (hydroxymethyl)-aminomethane (6.0 g/l) -glycine (28.8 g/l) of pH 8.3 was used as electrode buffer, and bromophenol blue as the tracking dye. The relative mobilities (Rm) of the enzyme bands were calculated as the ratio of the distance that the enzyme moved from the origin to the distance that the bromophenol blue moved. Alcohol dehydrogenase (EC 1.1.1.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and alkaline phosphatase (EC 3.1.3.1) were stained by methods of Harris and Hopkinson (1976). All samples were studied at least twice for each enzyme. Glucose-6-phosphate dehydrogenase was measured spectro- photometrically.

Purification ofD NA , determination of D NA base composition and D NA- D NA homology

The deoxyribonucleic acid (DNA) was prepared as follows. Cultures were grown in 500 ml 2% glucose-0.5% peptone- 1% yeast extract medium, washed twice with saline EDTA (0.15 M sodium chloride and 0.01 M sodium ethylene- diamine tetra-acetate, pH 8.0), resuspended in 5-10 ml 0.27 M sodium phos- phate buffer (pH 6.8) containing 9 M urea and 0.9% (w/v) sodium dodecyl- sulphate, and passed 3 times through a French press at 1150 atm. The disrupted cells were then centrifuged for 20 min at 9.000 g. The DNA was purified from the supernatant by hydroxylapatite (Biorad Bio-Gel HTP) column chroma- tography as described by Britten et al. (1970). The fraction was finally dialysed against 0.1 SSC (1 SSC buffer pH 7.0 is 0.15 M NaC1 and 0.015 M trisodium- citrate). If necessary, the DNA was precipitated with cold ethanol and redis- solved in the minimal volume 0.1 SSC. Ratios of A260/A280 = 1.86 and A230/ A260 = 0.5 were used to determine the quality of the DNA prepared. The G + C percentage was determined in 0.1 SSC from the thermal denaturation profile of the DNA by the method of Marmur & Doty (1962) and calculated from the formula mol % G + C = 2.08 - Tm 106.4 of Owen et al. (1969). A standard preparation of Candida parapsilosis CBS 604 DNA (TM = 70.6 ~ C in 0.1 SSC) was included in every determination as control. The extent of DNA-DNA reassociation was determined spectrophotometrically using the procedures described by Seidler and Mandel (1971) and modified by Kurtzman et al. (1980).

373

R e s u l t s

Morphology

The strains under study are distinguished by growth in liquid media. In contrast to N. commutata strains, the cultures of N. elongata and N. fulvescens (except VKM Y-2618) form a thin, dull, creeping pellicle. The growth of all the strains on malt extract agar is rather similar: greyish- or cream-white (in sporulating cultures the colour ranges from yellowish to brown depending on the abundance of ascospores), pasty, dull, smooth (finely wrinkled in the type strain of N. elongata), somewhat raised; the margin is entire, sinuated. How- ever, the giant colonies look different on the glucose peptone gelatin used in works by Nadson & Konokotina. The following colony types were observed:

- Greyish white with yellowish centre, smooth, slightly convex (strains of N. commutata).

- Greyish white, concentrically wrinkled, flat (type strain of N. elongata). - Greyish white, smooth; slightly raised (strains of N. fulvescens). - White, radially plicate, raised with a cone shaped centre (strains of

N. elongata). - Chalky-pallid, squamulose, raised (strains of N. elongata).

The diameters of colonies of the fourth and fifth types (about 2 cm) after one month of growth were twice as large as those of the others. In addition, the colonies of N. elongata and N. fulvescens strains had often sectors or spots which differed from the mother colony in tint of colour or elevation. The sporulating cultures liquified gelatin weakly and slowly. Both width and length of vegetative cells vary rather widely (Table 2). In all the strains investigated the sizes of 90% of the cells were 4--8 x 7-14/zm and the width/length ratios were 1.3-2.5. Only some strains of N. elongata (VKM Y-268, 270, 2531 and 2578) formed a rudimentary pseudomycelium. Sporulation was observed in all strains of N. commutata and N. elongata, with the exception of strain 11 x 1. The mode of conjugation and ascospore morphology corresponded with the descriptions given by the authors of these species. Conjugation was observed in strain 11 x 1 during isolation, but ascospores were not formed (Golubev et al. 1977). In N. commutata strains the mother cell becomes an ascus, while in N. elongata (and in N. fulvescens according to the original description) the ascus is a special third (meiotic) cell and the presence of cell triads in sporulat- ing cultures is specific for N. elongata. The ascospores are smaller in the strains with narrower cells which have an average width of less than 5/zm (Table 2). Neither ascospore formation, nor conjugation was observed in the strains of N. fulvescens.

374

Table 2. Morphological characteristics of Nadsonia strains.

Strain Size of vegetative cells, Mean number t~m(morphology agar, 3

days)

Length: width ratios

Mean Diameter of Mean ascospores

N. commutata 1573 4.3- 9.4 x 6.0-14.2 6.4 x 9.0 1941 3.4-11.0 x 6.0-26.4 4.9 x 12.6 1942 3.4- 8.5 x 5.1-19.6 4.8 x 10.2 2610 4.3- 9.4 x 6.0-14.5 7.0 x 9.9

N. elongata 11 x I 2.6- 8.5 x 3.4-19.6 6.2 x 11.3 17N 3.4- 9.4 x 5.1-21.3 5.9 x 12.8 268 4.3- 9.4 x 6.0-17.9 6.1 x 11.0 269 3.4- 8.5 x 6.0-17.9 6.5 x 11.7 270 3.4- 9.4 x 5.1-19.6 6.2 x 11.9 1653 3.4- 8.5 x 6.0-16.2 5.5 x 10.8 2531 2.6- 8.5 x 8.5-21.3 4.9 x 13.6 2578 3.4-15.3 x 6.0-23.0 6.6 x 12.9 2579 4.3- 8.5 x 7.7-17.0 6.3 x 12.3

N. fulvescens 2532 3.4- 8.5 x 4.3-21.3 5.7 x 10.9 2618 4.3- 7.7 x 6.8-17.0 5.2 x 9.2 2619 3,4- 6.8 x 5,1-12.8 4.7 x 8.3

1.1-2.0 1.4 3.4--6.0 4.6 1.4--4.8 2.1 2.6-4.3 3.9 1.4-3.4 2.1 2.6--4.3 3.7 1.1-2.3 1.4 2.6--6.8 4.7

1.1-3.0 1.8 -i 1.2--3.3 2.0 3.4-4.3 -2 1.2-3.0 1.8 3.4-6.8 5.5 1.2-3,3 1.8 3.4-6.0 -2 1.3-2.9 1.9 3.4-6.8 5.1 1.3-3.2 2.0 3.4-6.8 4.9 1.8-5.0 2.8 2.6--5.1 3.7 1.1-5.4 2.1 '3.4-5.1 4.7 1.3-3.0 2.0 3.4-6.0 5.6

1.0--3.0 1.9 -1 1.3-2.5 1.8 -1 1.3-2.5 1.8 -1

1 No formation of ascospores. 2 Ascospore formation is vely poor.

Physiology

All Nadsonia strains assimilated glucose, e thanol , glycerol, succinic acid,

g lucono- f - l ac tone but no t g lucosamine, cellobiose, t rehalose, lactose, meli-

ziose, raff inose, melezi tose, inul in , soluble starch, xylose, L- and D-arabi -

nose , r ibose, rhamnose , erythri tol , galactitol, salicin, a rbu t in , citric acid,

inosi tol , g lucuronic acid, 2- and 5-keto-gluconate . They were unab le to utilize

po tass ium ni t ra te and sodium nitr i te and methy lamine . They failed to hydro-

lyze u rea and to grow on media con ta in ing more than 4--5% NaC1 or 0.01%

cycloheximide. The physiological differences be tween the strains are shown in

Tab le 3.

Amino acid and fatty acid composition

The quant i t a t ive amino acid composi t ion of cell hydrolyzates of the type

strains and V K M Y-1653 are p resen ted in Table 4. W h e n compar ing the results

Table 3. Physiological differences between Nadsonia strains.

375

N. commutata N. elongata N. fulvescens

1 1 1 2 1 1 2 2 2 1 2 2 2 2 2 2 5 9 9 6 1 7 6 6 7 6 5 5 5 5 6 6

7 4 4 1 x N 8 9 0 5 3 7 7 3 1 1

3 1 2 0 1 3 1 8 9 2 8 9

Fermentation Glucose Galactose Sucrose Maltose a - M - g l u c .

Assimilation Galactose L-Sorbose Maltose Sucrose Ribitol M a n n i t o l

G l u c i t o l

a - M - g l u c .

Lactic acid Growth in vitamin free medium Max. Temp. of growth, ~ C

+ + S + + + + + + + + +

S w +

V W W W

+ S +

W W - -

S S +

+ + + S vw S + S + + + + + + + + + +

V W W W

S vw w + + +

vw w w + w + + +

+ S S

vw vw vw + S S S + S S + S w S + +

+ + + + + + - + + - + +

27 27 27 27 26 26 27 27 27 27 27 27 22 22 22 22

+ Positive reaction, S S low, w w e a k , vw v e r y w e a k , - negative.

an allowance of 5% should be made for experimental error. These data reveal that the strain VKM Y-1573 differs from the others in the amounts of 7 or 8 amino (alanine, arginine, glycine, leucine, lysine, phenylalanine, serine and valine) while the strains of N. elongata and N. fulvescens differ from one another by contents of 1-3 amino acids only (arginine, histidine, lysine and threonine). There are also marked differences in cellular long-chain fatty acid composition (Table 5). The statistical interpretation of the results was per- formed by Student's t-test (Schefler 1969).

The strains of N. commutata contain a statistically significant higher mean percentage linoleic (C~8:2) and linolenic (C18:3) and a statistically significant lower mean percentage palmitoleic (C16:~) and oleic acid (C18:~) compared to N. elongata and N. fulvescens (P < 0.05 - in all comparisons). The N. elongata

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Table 4. Amino-acid compositions of Nadsonia species at the late exponential phase of growth (Yeast Nitrogen Base with 1% of glucose).

Amino acids N. commutata N. elongata N. fulvescens 1573 1653 2531 2532

Percentage of total amino acids Alanine 9.87 7.08 7.43 7.17 Arginine 5.71 7.11 8.46 7.48 Aspartic acid 11.09 11.21 10.34 11.06 Glutamic acid 13.25 13.02 14.04 13.14 Glycine 5.30 4.70 4.73 4.75 Histidine 2.16 1.99 2.38 2.26 lsoleucine 4.53 4.68 4.30 4.54 Leucine 7.95 9.25 8.85 9.74 Lysine 7.83 9.51 9.85 8.80 Phenylalanine 5.18 5.12 4.66 4.93 Proline 4.08 4.19 4.16 3.82 Serine 5.71 6.77 6.47 6.83 Threonine 5.50 5.61 5.05 5.69 Tyrosine 3.91 4.03 3.80 4.15 Valine 7.95 5.74 4.57 5.63

Protein (g per 100 g of dry yeast) 24.53 39.36 28.57 39.12

J

I573

i

I

I

I ' I 9 4 I 1942 I I x ~ I?N

" 11 . I I I , I I J

_ I I 268 269 2'70 I653 253I

1 0 , 4

0,2

0 2578 25"79 2 5 3 2

J I 1

I i

-0,4

-0,2

Fig. 1. Electrophoretic patterns of Nadsonia strains for alkaline phosphatase (above) and glu- cose-6-phosphate dehydrogenase (below).

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Table 5. Long-chain fatty acid composition (%) of Nadsonia species (grown in Yeast Nitrogen Base with 8% of glucose, 2 days) 1.

Strain numbers Fatty acids 2

14:0 14:1 16:0 16:1 18:0 18:l 18:2 18:3

N. commutata VKM Y-1573 0.2 0.3 ll .8 4,4 1.7 17.9 56.9 6.7 VKM Y-1941 0.4 0.1 15.1 2,3 5.5 25.4 41.(t 10.1 VKM Y-2610 0.4 0.4 12.8 3.3 3.3 12.5 61.1 6.0

J~ 0.33 0.27 13.23 3.33 3.5 18.6 53.0 7.6 S 0.08 0.11 1.19 0.74 1.35 4.58 7.5 1.55

N. elongata 11 x 1 2.1 1.1 27.0 3.8 6.7 26.5 32.1 0.6 VKM Y-268 0.4 0.2 12.2 17.1 1.6 36.0 30.4 2.1 VKM Y-269 0.5 0.I 13.1 14.8 2.1 31.4 35.0 3.1 VKM Y-270 0.5 0.2 12.8 16.3 1.6 34.0 32.8 1.8 VKM Y-1653 2.6 1.1 20.6 5.6 4.2 25.6 40.0 0.4 VKM Y-2531 0.3 0.2 11.4 20.2 0.9 40.0 26.7 0.2 VKM Y-2578 0.0 (1.3 11.9 12.0 1.3 25.0 46.6 3.0

0.91 0.46 15.57 12.83 2.63 31.21 34.8 1.6 S 0.87 0.38 5.15 5.28 1.81 5.0 5.73 1.05

N. fulvescens VKM Y-2618 0.5 0.2 16.9 11.8 2.4 41.1 26.5 0.6 VKM Y-2619 0.4 0.1 14.1 14.9 1.4 42.1 26./I 0.8

0.45 0.15 15.5 13.35 1.9 41.6 26.25 0.7 S 0.04 0.04 1.14 1.27 0.41 0.41 0.2 0.08

Values are the means of three and more repetitions. The standard deviation is about 8% of the mean value (range 3% to 10%). 2 Fatty acids designated as a number of carbon atoms: number of double bonds.

a n d N. fu lvescens s t ra ins a re c h a r a c t e r i z e d by s imi la r fa t ty ac id c o m p o s i t i o n s

a l t h o u g h the s t ra ins o f t he l a t t e r c o n t a i n a s ta t i s t ica l ly s ign i f ican t h i g h e r m e a n

p e r c e n t a g e o le ic ac id and l o w e r m e a n p e r c e n t a g e l ino le ic ac id (P < 0.05 in

b o t h cases) .

Electrophoret ic e n z y m e patterns

F i g u r e 1 shows the e l e c t r o p h o r e t i c p a t t e r n s o f Nadsonia s t ra ins fo r a lka l ine

p h o s p h a t a s e ( A P ) and g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e ( G 6 P D ) . A l c o h o l

d e h y d r o g e n a s e d id n o t p r o v i d e r e p r o d u c i b l e resu l t s due to p o o r ac t iv i ty and

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Table 6. D N A base composit ion and D N A reassociation between Nadsonia strains.

Species and

Strain number

mol% G + C + sd ~ D N A relatedness (% + sd 1)

V K M Y-2531 V K M Y-2596

N. commutata V K M Y-1573 39.5 z 15.8 +_ 4.7

40.03 0.00 + 1.8

N. elongata V K M Y-1653 40,9 __+_ 0.18 97.7 + 2.0 60.8 + 4.0

V K M Y-2531 41.83 - 70.1 + 4.7

V K M u 40.1 + 0.00 86.9 + 2.0 70.5 + 2.1

CBS 2595 - 96.4 +_ 0.4 68.7 + 2.9

N. fulvescens N C Y C 46 39.3 _+ 0.10 - 96.6 + 2.0

VKM Y-2532 41.53 70,1 + 4.7 -

Standard deviation calculated from three determinations. 2 Golubev & Vagabova (1977).

3 Miller & Phaff (1984).

stability and were not considered. The strains of N. elongata and N. fulvescens produced similar patterns of AP which had a single band (Rm 0.34-0.37). In N. commutata strains AP gave the main band (Rm 0.26--0.28) and I or 2 minor bands. The thicker major band in strain VKM Y-1942 apparently resulted from irresolution of a slow minor band which had a Rm close to (0.21-0.23). The strain VKM Y-1941 differed from other N. commutata strains by the presence of a fast minor band which had Rm (0.38), very close to that of AP in N. elongata and N. fulvescens. G6PD gave the sharp and well-stained main band and 2 to 3 slow minor bands in the strains of N. elongata and N. fulvescens. With the exceptionn of the strain 11 x 1, having the main band with Rm 0.43, all other strains had the main band of G6PD with Rm 0.37 + 0.03. Most of them had the minor bands with Rm from 0.20 to 0.27 and about 0.34, only the strains 17N and VKM Y-270 did not have the latter but had a slow minor band with Rm about 0.11. The electrophoretic image for G6PD was quite different in the strains of N. commutata. The main enzyme activity (specific activity of samples was about 20 nmoles/min/mg protein in both N. elongata and N. commutata strains) was revealed as a very broad zone which stained somewhat lighter in the strains VKM Y-1941 and 1942. In these strains darker bands (Rm 0.02, 0.09, 0.13 and 0.33) were on the background of the stained zones. The strains VKM Y-1573 and 1942 had 2 or 3 distinct slow minor bands with Rm 0.02, 0.04 and 0.07.

379

DNA base composition and DNA-DNA homology

The G + C values of the strains and results of the DNA-DNA reassociation experiments are presented in Table 6. G + C contents of all Nadsonia spp. are about 40 mol %. As expected, high reassociation values (96, 97%) were found between the type strain of N. fulvescens VKM Y-2532 and NCYC 46, and between the type strain of N. elongata VKM Y-2531 and CBS 2595. Taking into account the history of the strains NCYC 46 and CBS 2595, they most likely originate from the type strain of either species. The DNA-DNA reassociation experiments between the N. elongata strains (including the type strains of N. elongata and N. richteri) of independent origin (Table 1) also resulted in high values of 87 and 98%. The type species N. fulvescens shared 61 to 71 base sequences with N. elongata but is not related to N. commutata. The level of reassociation between the latter and N. elongata was only 16%.

Discussion

The results obtained clearly prove the specifically distinct status of N. commu- tata which differs from N. fulvescens and N. elongata in many aspects, in- cluding cultural properties, ascus formation, none or low DNA relatedness, different amino acid and fatty acid compositions, electrophoretic enzyme patterns and physiological characters. Besides that, N. commutata is dissimilar with respect to ascospore surface ornamentation and sources of isolation (Orlova et al. 1978; Golubev et al. 1987).

The question of the taxonomic status of N. fulvescens and N. elongata (the descriptions of which were based on one single strain) is more complicated. Although our data confirm the morphological and physiological distinctness of their type strains, the study of other strains demonstrated that these differ- ences are mainly strain features or unstable. Extensive spontaneous variability in N. elongata (N. richteri) was reported by Skovsted (1943) with respect to colony morphology, shape and size of cells, ability to sporulate and in N. fulvescens as well by Nadson (1920), Nadson and Philippov (1928) after X-ray irradiation. The physiological properties are also unstable as is shown by the discrepancies among the data of various authors who studied the same strains (Kostka 1925; Stelling-Dekker 1931; Lodder & Kreger-van Rij 1952; Miller & Phaff 1958; Phaff 1970). In particular conflicting results were obtained con- cerning the ability to ferment sucrose and maltose. Probably, these contradic- tions are a consequence of long-term maintenance of the cultures under different conditions. It appears that neither morphological nor physiological characteristics can provide a reliable basis for species distinction in the genus Nadsonia. Our chemotaxonomic study reveals close relationship between N.

380

fulvescens and N. elongata. In comparison with N. commutata they are more similar in their cellular composition and the electrophoretic patterns of AP and G6PD. The type species of N. fulvescens has 61-71% DNA complementarity with N. elongata. These values are lower than among N. elongata strains, and they are close to the low limit of DNA-DNA homology values (75-85%) which are acceptable within the same species (Kurtzman 1985). On the basis of all above data we propose the following varietal distinction as:

- N. fulvescens (Nadson & Konokotina) Sydow var. elongata (Konokotina) Golubev et M.Th. Smith nov. comb.

- Basionym: N. elongata Konokotina in Bull. Jard. Imp. Botan. St.Peters- bourgh, 13: 32, 1913.

- Synonyms: Guilliermondia elongata Konokotina 1913; Nadsonia richteri Kostka 1925.

This species delineation in the genus Nadsonia allows a choice from the following morphological and physiological properties as species character- istics: the mode of ascus formation, ascospore surface ornamentation, the ability to ferment glucose and to assimilate L-sorbose, maximum temperature of growth and, for fresh isolates, pellicle formation and the ability to grow in vitamin free medium. The natural habitat of N. fulvescens is spring fluxes of deciduous trees, whereas all strains of N. commutata have been isolated from soils. Characteristics which can be used to differentiate varieties of N. fulves- cens are the ability to ferment galactose, sucrose, maltose and the utilization of these sugars as well as of ribitol, mannitol, glucitol and a-methyl-D-glucoside (Table 3). Taking into consideration only the most clearly distinctive and stable characters we suggest the following key for identification of species and varieties of the genus Nadsonia:

la. No growth at 24~ b. Growth at 24~

2a. Mannitol assimilated b. Mannitol not assimilated

N. commutata 2 N. fulvescens var. fulvescens N. fulvescens var. elongata

In order to eliminate negative and variable characteristics depending on the species, variety or strain we propose to accomodate the diagnosis of the genus Nadsonia as follows:

'Cells spheroidal to elongate, usually 4-8/xm wide. Apiculate cells acquiring their shape as a result of vegetative reproduction by bud-fission at both poles.

381

As a role a conjugation between the mother cell and a bud precedes ascospore formation. Ascospores are spherical, warty-walled, brownish with a promi- nent lipid globule. Maximum temperatures of growth are below 30 ~ C.'

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