Biochemical Genetics, Vol. 26, Nos. 5/6, 1988

Isozyme Polymorphism of fl-Glucosidase in Aspergillus nidulans

Sunita Sharma, 1 Dhanwant K. Sandhu, 1'2 and Parmjit S. Bagga I

Received 19 Aug. 1987--Final 16 Feb. 1988

Electrophoretic analysis of the distribution of various electromorphs at different fl-glucosidase zones was carried out in natural populations of A. nidulans, the A. nidulans group, and various species belonging to the genus Aspergillus from diverse geographical areas of India. The data show the existence of three segregating zones for fl-glucosidase, designated fl-GluL fl-GlulI, and fl-GlulII. All three zones are present in wild isolates of A. nidulans, and only two, i.e., fl-Glul and fl-GlulII, in the A. nidulans group and fl-GlulI and fl-GlulII in different species of Aspergillus except A. terreus, A. flavus, and A. brevipes, where only fl-GlulII is present. Overall nine electro- morphs are observed at fl-GluL three at fl-GlulI, and six at fl-GlulII zones, respectively, It can be concluded that there may be three structural genes for fl-glucosidase coding the three polymorphic zones in A. nidulans.

KEY WORDS: fl-glucosidase; Aspergillus nidulans; isozyme; electromorph; natural variation.

INTRODUCTION

Over the last few decades, evidence has accumulated that a considerable amount of genetic variability, which can easily be detected by gel electropho- resis, exists in natural populations. Since then, there has been an immense exploration of genetic polymorphism in natural populations of organisms varying from Escherichia coli to Homo sapiens (Singh et al., 1976). All natural populations of organisms are highly polymorphic for gefies specifying

This research work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi.

Department of Biology, Guru Nanak Dev University, Amritsar-143005, Punjab, India.

331 0006-2928/88/0600-0331506.00/0 © 1988 Plenum Publishing Corporation

332 Sharma, Sandhu, and Bagga

the amino acid sequences of enzymes. Among the fungi, numerous studies of experimental taxonomy have made use of protein analysis by gel electrophore- sis to distinguish related species and to establish possible reciprocal relation- ships (Catherine and Dubayle, 1983; Yamazaki et al., 1985). However, an experimental sampling of the genome by electrophoretic analysis of isozymes is practically nonexistent. Fungi as a whole are very suitable organisms for the study of natural variation because of some peculiarities of fungal biology, viz., a prolonged haploid phase permitting direct observation of genotype, hetero- karyosis, and more varied parasexual as well as sexual recombination. These characteristics can help to give a suitable explanation to the enormous data on population genetics (Spieth, 1975).

~-Glucosidase is one of the three components of the cellulase enzyme complex. It catalyzes the hydrolysis of cellobiose to glucose. In addition to the fungi, this enzyme is also of wide occurrence in both plant and animal tissues. So far very little information on the isozymes of/3-glucosidase has been published. Multiplicity of this enzyme has been detected with the help of fractionation studies in the case of some fungi, e.g., Trichoderma spp. (Labudova and Farkas, 1983), Sporotrichum pulverulentum (Deshpande et al., 1978), and Monilia spp. (Dekker, 1981). There is not a single report on the intraspecific or interspecific variability of isozymes of ~3-glucosidase in any fungus. In the present study, an attempt has been made to evaluate the intraspecific variability of /3-glucosidase in wild isolates of Aspergillus nidulans and species belonging to the A. nidulans group. Other species of Aspergillus have also been included for a comparative study.

MATERIALS AND METHODS

A total of 96 wild isolates of A. nidulans was used in the present study, of which 73 were isolated from soil samples collected from different geographical

Table I. List of Isolates ofA. nidulans of Indian Origin

Number of isolates Site of collection Year of collection

6 Assam 1984 6 Chandigarh 1984

27 Delhi 1984 4 Goa 1984 5 Gujrat 1984 3 Himachal Pradesh 1984 2 Jammu 1984 2 Meghalaya 1984 7 Punjab 1984 3 Rajasthan 1984 2 Trivandrum 1984 6 Uttar Pradesh 1984

Isozyme Polymorphism of 13-Glucosidase in A. nidulans 333

2 Chandigarh Jo I 3 Delhi

"~ q L~Goa 5 OuI rat

,~ 6 Himachal _n 7 Jammu

8 Meghalaya 9 Punlab 10 Ralastnan 11 Trivanclrum 12 Uttar Prade~h

Fig. 1. Geographic locations of wild-type isolates of Aspergillus species.

regions of India (Table I, Fig. 1) and 23 were from the genetic stock collection of A. nidulans from the United Kingdom (Table II). Among the Indian isolates, the majority was from Delhi (Table I). From other places, the number of isolates studied varied from one to seven (Table I). Eighteen species and varieties of the A. nidulans group were isolated mainly from the Punjab (Table III). For the interspecific comparison, 13 species of the genus Aspergillus were selected (Table IV). These species were also from the Punjab except A. fumigatus, for which different isolates were collected from different parts of India (Table IV). A. fumigatus, A. niger, A. Terreus var. aureus, and A. terreus comprised 14, 4, 3, and 2 isolates, respectively, whereas all the other species had only one representative. Delineation and classifica- tion of various species within the genus were done on the basis of differences in cultural and morphological characters (Raper and Fennel, 1965). After single spore isolation, these species were preserved in soil at 4°C. Subculturing was done on Vogel's complete medium whenever required.

Enzyme Production. Vogel's (1956) complete medium supplemented with 1% pectin as the sole source of carbon was employed for culturing, as pectin was found to bring about the best production of t3-glucosidase in A. nidulans from some preliminary studies. Each 250-ml Erlenmeyer flask

334

Strain

Sharma, Sandhu, and Bagga

Table II. List of Isolates of A. nidulans of U.K. Origin

Site of collection Year of collection

1 Birmingham 26 Birmingham 28 Birmingham 33 Birmingham 36 Birmingham 37 Birmingham 74 Birmingham 38 Durham (North) England 48 Durham

178 Isle of Wight 124 Somerset 56 Dudley 84 Pembroke, Wales

101 Essex 0035 Glasgow 0043 Glasgow

00136 Glasgow 0051 Glasgow

00109 Glasgow 0066 Glasgow 0067 Glasgow 0068 Glasgow 0080 Glasgow

1954 1961 1962 1962 1962 1962 1962 1962 1962 1963 1962 1962 1962 1962

containing 50 ml of the above medium was inoculated with seven mycelial disks (7-mm diameter) cut from the periphery of actively growing colonies. The flasks were incubated for 8 days at 37°C, after which the contents of each flask were centrifuged at 22,000g for 30 rain at 4°C. The supernatant was used as the extracellular crude enzyme extract. Protein from these extracts was concentrated using a 100% ammonium sulfate precipitation.

Electrophoresis. Electrophoresis was performed following the method of Davis (1964) with some modifications. A horizontal polyacrylamide gel

Table I l l . List of Isolates of Varieties and Species of the A. nidulans Group i

Number Site of Year of Species of isolates collection collection

A. nidulans var. acristatus 2 Punjab 11984 A. aureolatus 3 Punjab 1984 A. nidulans var. echinulatus 1 Punjab 1984 ~. heterothallicus 1 Punjab 1984 A. nidulans var. latus 2 Punjab 1984 A. rugu!osus 4 Punjab, Delhi 1984 A. speluneus 4 Punjab 1984 A. nidulans var. violaceous 1 Punjab 1984

Isozyme Polymorphism of ~-Glucosidase in A. nidulans

Table IV. List of Isolates of Species of the Genus Aspergillus

Number Site of Species of isolates collection

335

Year of collection

A. aculeatus 1 Punjab 1984 A. brevipes 1 Punjab 1984 A. carneus 1 Punjab 1984 A.flavus 1 Punjab 1984 A. flavus var. colurnnaris 1 Punjab 1984 A. fumigatus 14 Assam 1984

Bengal 1984 Uttar Pradesh 1984 Nepal 1984 Gujrat 1984 Punjab 1984

A. furnigatus var. ellipticus 1 Punjab 1984 A. niger 5 Trivandrum, 1984

Punjab A. oryzae 1 Punjab 1984 A. parasiticus 1 Punjab 1984 A. spectabilis 1 Punjab 1984 A. terreus 2 Punjab 1984 A. terreus var. aureus 3 Punjab, 1984

Uttar Pradesh

prepared with 6% cyanogum (Sigma) in Tris-glycine buffer, p H 8.3, was employed for electrophoresis. The enzyme extract adsorbed on rectangular wicks (10 × 3 mm) of Whatman No. 3 filter paper was inserted into slots made in the gel slab. Electrophoresis was performed at 250 V until the marker dye bromophenol reached the end of the gel. A continuous buffer system was used for electrophoresis. After running, the gel was incubated in a 1 mM solution of p-nitrophenyl-/3-D-glucopyranoside (PNPG) (CSIR Centre), p H 5, at 37°C for 30 min, and afterward it was dipped in 0.2 M NaOH/g lyc ine buffer, p H 10.6. This resulted in the appearance of yellow-colored bands of released p-nitrophenol.

RESULTS

Varia t ion in the W i l d - T y p e I so la tes o f A. nidulans. Each isolate from A. n idu lans yielded a regular electrophoretic pattern of/3-glucosidase depicting the existence of its isozymes. Three zones of/3-glucosidase activity were resolved according to their migration toward the anode. In order to differen- tiate different zones, they have been given numbers after the abbreviated name of the enzyme (/3-Glu):/3-GlulII,/3-GlulI, and/3-GluI, starting from the least anodal-migrating zone to the most anodal/3-glucosidase zones (Rider and Taylor, 1980). The patterns of variation at each such zone are indepen- dent of one another. Various electromorph types are assigned numbers

336 Sharma, Sandhu, and Bagga

Z

m

r i J I i I I i l ~ I r - I I - I I - I i I~

d r I ~ r I I I J I I r I I I I i I - - ~

I ~ l l l ~ r i l l

I J l ~ f l ~ l l r

I I I I F l i l - I I i -

~ I - J F I I - I I

J I I I I PI

I - , I - I I I P I I r I I

] - I - I I - I I~ I I I ]

I I I i - I r I I I - I J I I I I I ~

I I l 1 ~ I I I 1 1 1 I I I F I I I ~

~ o

N ~

Isozyme Polymorphism of/%Glucosidase in A. n idu lans 337

according to their electrophoretic mobility in millimeters from the point of origin (Table V).

The/3-GluI zone was represented by eight different segregating electro- morphs, labeled 30, 32, 35, 36, 38, 40, 42, and 45. Single-band variants are mutually exclusive in their appearance. In Indian populations, ET30 was a rare form restricted to only a few isolates from Delhi. ET32 was present in some isolates from the populations of Punjab and Delhi origin. ET38 was widespread in local populations of Chandigarh, whereas ET40 was frequently found in almost all the populations of different regions studied in the present investigation (Table V). The frequency (percentage of each electromorph is given in Table VIII. In the case of the U.K. isolates, three electromorphs were present at this zone, i.e., 35, 40, and 42. Of these, ET42 was observed exclusively in local U.K. populations. Moreover, a null form was also present at this zone in one isolate (0043) from the U.K.

The ~3-GluII zone was observed in only some isolates of Punjab, Goa, and Rajasthan from Indian populations and it was also present in one isolate from U.K. Two segregating electromorphs, ET23 and ET25, were present at this zone (Table V).

The ~-GluIII zone was of wide occurrence. Overall four different electromorphs, viz., 10, 12, 15 and 20, were present at this zone (Table V). ET15 was the most frequent form present in all the isolates from the U.K. except two isolates in which ET12 was common. In the Indian populations also, ET15 was the most common form, followed by ET10, observed in 16 isolates, ET20 in 5 isolates, and ET12 in 4 isolates only.

Table VI. Distribution of Electromorphs at/3-Glucosidase Zones in Natura l Isolates of the A. n idu lans Group

Zone and electromorph

~-GluI /3-GlulII Number

Species of isolates 33 35 38 45 11 12 15 20

A. n idu lans var. aer i s ta tus 2 - - 2 A. aureo la tus 3 1 - -

A. n idu lans var. ech inu la tus 1 1 A. he tero tha l l i cus 1 - - 1 A. n idu lans var. la tus 2 1 1 A. rugu losus 2 - - 2

1 - - 1 1

A. spe luneus 3 - - 3 1

A. n idu lans vat. vio laceous 1 - - 1 Total 18 3 11

1 1

1 -- 1 1

2 1 2 14

1

338 Sharma, Sandhu, and Bagga

Variation in Different Species and Varieties of the A. nidulans Group. Two zones,/%GluI and B-GIulII, were present in different species of the A. nidulans group. Four electrophoretic variants were observed at ~-GluI, i.e., ET33, 35, 38, and 45, and similarly at ~-GlulII also four variants, i.e., ET11, 12, 15, and 20, were detected (Table VI). ET35 at the ~-GluI zone was the most common form in almost all the species except A. aureolatus and A. nidulans var. echinulatus, whereas ET33 was observed in A. aureolatus, A. nidulans var. echinulatus, and A. nidulans var. latus. ET38 was restricted to isolates of A. aureolatus only, while ET45 was present in strains of A. rugulosus and A. speluneus. At the B-GlulII zone, ET15 was present in all the species except A. nidulans var. violaceous, whereas ET11 and ET20 were restricted to A. rugulosus only. ET12 was found to be present in A. speluneus and A. nidulans var. violaceous (Table VI).

Variation in Different Isolates of A. fumigatus. In A. fumigatus and A. fumigatus var. ellipticus, two zones, /~-GlulI and /~-GlulII, were present (Table VII). Both zones were found to be monomorphic in A. fumigatus. However, different isolates showed variations with respect to the staining intensity of different bands at the two zones.

Variation Among Different Species of Aspergillus. Various electro- morph types in different species of the genus Aspergillus are listed in Table VII. In A. terreus, only ~-GlulII was present, whereas in A. terreus var.

Table VII. Dis t r ibut ion of Elec t romorphs at 13-Glucosidase Zones in Species of the Genus A s p e r g i l l u s

Zone and e lec t romorph

/~-GlulI j3-GluIII N u m b e r ~-GluI ,

Species of isolates 35 25 28 13 15 20

A . a c u l e a t u s 1 - - - - 1 1 - - - -

A . b r e v i p e s 1 . . . . . 1

A . c a r n e u s 1 - - 1 - - - - 1 - -

A . f l a v u s 1 . . . . 1 - -

A . f l a v u s var. c o l u m n a r i s 1 . . . . 1 - -

A . f u m i g a t u s 14 - - 14 - - - - 14 - - A . f u m i g a t u s var. e l l i p t i c u s 1 - - 1 - - - - 1 - -

A . n i g e r 4 - - 4 - - - - 4 - -

A . n i g e r 1 . . . . 1 - -

A . o r y z a e 1 - - 1 - - 1 - - - -

A . p a r a s i t i c u s 1 - - - - - - 1 - - - -

A . s p e c t a b i l i s 1 1 1 - - - - 1 - -

A . t e r r e u s 2 - - - - - - 2 - - - -

A . t e r r e u s var. a u r e u s 3 - - 3 - - - - 3 - -

Total 33 1 25 1 5 27 1

Isozyme Polymorphism of ~-Glucosidase in A. nidulans 339

aureus/3-GluII as well as /3-GlulII was present. Both the species can be differentiated on the basis of the differences in electrophoretic mobility of the /3-GluIII band. In A. flavus and A. flavus var. collumnaris, only/3-GluIII was present. In A. carneus and A. oryzae, both/3-GluII and/3-GluIII of the same electrophoretic mobility were present, while A. brevipes and A. aculea- tus showed species specificity for various zones of/3-glucosidase. A. parasiti- cus shared a common zone,/3-GluIII, with A. flavus, whereas A. spectabilis showed an affinity with the A. nidulans group by exhibiting /3-GluI and /3-GluIII zones.

DISCUSSION

Electrophoretic analysis of species populations has shown that natural popula- tions are heterogeneous mixtures of largely heterozygous individuals and evolution of a population is related to the amount and nature of genetic variation present in it (Dobzhanskey et al., 1977). Both these aspects result from the presence of allelic variability and the frequency of different alleles at different loci. Thus isozyme analysis constitutes a useful tool to reveal genetic variation in natural populations. In any population, a locus is considered polymorphic only when the frequency of the most common allele does not exceed 95% (Ayala et al., 1972). Thus according to this criterion, A. nidulans populations can be considered as polymorphic at all the ~-glucosidase zones. Two zones,/3-GluI and/3-GluIII, are highly polymorphic, with eight and four variants each. Of eight electromorph types at/3-GluI, seven are present in Indian populations and one is restricted to isolates from the U.K. It has been observed that the large population of the region (Table V) shows almost all the different electromorphs at various zones. ET42 is specific for U.K. popula- tions, which may be due to climatic and geographical variations, because of which this electromorph is selected in those populations. The occurrence of large numbers of electromorphs at/3-GluI and/3-GluIII is in accordance with the earlier reports, where 20 electrophoretic variants have been observed at /3-GluI in maize, which is a diploid organism (Stuber and Goodman, 1976). This finding can be explained on the basis of studies suggesting that a high mutation rate at /3-GluI may be responsible for such a large number of electrophoretic variants at this zone (Pryor, 1976), or it may be due to a high rate of crossing over-during sexual reproduction in diploid organisms as compared to haploid organisms. It has been observed that the frequency of different electromorphs at variable /3-glucosidase zones is quite different. Frequencies of certain eleetromorphs (Table VIII) are high, while those of others are low. It could be presumed that the electromorphs showing low frequencies either are of recent origin or do not confer any selective advantage. The high frequency of other electromorphs may be the result of

340 Sharma, Sandhu, and Bagga

Table VIII. Frequency (Percentage) of Electromorph Types in Total Number of Isolates of Each Group

II

A. nidulans A. nidulans Aspergillus Zones Electromorph (96)" group (18) spp. (33)

~-GluI 30 2.1 - - - - 32 2.1 - - - - 33 - - 1.6 - - 35 39.6 61.1 3.3 36 5.2 - - - - 38 5.2 11.1 - - 40 29.4 - - - - 42 9.3 - - - - 45 5.2 11.1 - -

B-GIulI 23 1.0 - - - - 25 6.2 - - 75.7 28 - - - - 3.3

/~-GlulIl 10 16.6 - - - - 11 - - 5.5 - - 12 4.1 11.1 - - 13 - - - - 15.1 15 74.0 77.7 81.8 20 5.2 5.5 3.3

"Number of isolates tested in parentheses.

high adapt ive values of these forms in different par ts of a heterogeneous environment (Rockwood-Sluss et al., 1973). Both these aspects resul t if the envi ronmenta l he terogenei ty is large as compared to the average selection intensi ty (Gil lespie and Langley, 1974). In common with all the other species which have been extensively examined (Lewontin, 1973), much var ia t ion and po lymorphism are found in na tu ra l populat ions o fA . nidulans for a number of different morphological , physiological , and b iochemical proper t ies (Grindle , 1963a,b; Hol t and MacDona ld , 1968; Kurze ja and Garbe r , 1973; M e r r i c k and Caten, 1975). The pa t t e rn of var iab i l i ty for different charac te rs in wild populat ions of A. nidulans suggests tha t na tu ra l populat ions of this o rgan ism can be divided into a number of independent subpopula t ions in which evolution may be proceeding independent ly with low frequencies of recombi- nat ion between them (Crof t and Jinks, 1977).

The occurrence of null, i.e., nonfunct ional e lec t romorphs at t h e / ~ - G l u l I zone suggests tha t this enzyme appears to serve ei ther some dispensable metabol ic role or a physiological role tha t is not well unders tood (Bewley and Lucchesi , 1975). Thus, gene si lencing could arise f requent ly and spread in organisms with dup l ica te loci (Al lendorf , 1979; Li, 1980).

Inspect ion of the zymograms of other species of Aspergi l lus indicates tha t the e lec t rophoret ic pa t t e rn of this enzyme m a y be used as a potent ia l

Isozyme Polymorphism of/%Glucosidase in A. nidulans 341

taxonomic tool for the differentiation of various Aspergillus spp. From Table VIII, it is clear that the A. nidulans group is phylogenetically closer to A. nidulans itself, as they share most of the forms at various zones, although the frequency of some electromorphs is different in the two groups and some forms are exclusive to each group. On the other hand, various species of Aspergillus share only one electrophoretic variant at t3-GluI, two at/3-GluII, and three at /3-GluIII. Most of the other species show specificity for the isozyme of 13-glucosidase. However, 13-GluIII is found to be present in all the species studied so far and this zone can be treated as the ancestral form. The similarity of various bands at different/3-glucosidase zones can be used as a criterion for their genetic similitude (Hubby and Throckmorton, 1965; Iglesias, 1984). The present studies, therefore, suggest that the three polymor- phic zones of ~-glucosidase represent true isozymes. It can be inferred that all the three forms have separate structural genes. This is supported by some other studies where the three forms have been found to be expressed differentially during the development of A. nidulans and show heat-sensitive and urea denaturation polymorphism (data to be published elsewhere). Since A. terreus, A. brevipes, and A.flavus have only one electromorph at/3-GluIII, there is only one structural gene for/3-glucosidase in these species.

REFERENCES

Allendorf, F. W. (1972). Rapid loss of duplication gene expression by natural selection. Heredity 43:247.

Ayala, F. J., Powell, J. R., Tracey, H. L., Mourao, C. A., and Perezsalos, S. (1972). Enzyme variability in the Drosophilia willistoni group. IV. Genetic variation in natural populations of Drosophila willistoni. Genetics 70:113.

Bewley, C. G., and Lucchesi, J. C. (1975). Lethal effects of low and null activities alleles of 5-phosphogluconate dehydrogenase in Drosophilia melanogaster. Genetics 79:451.

Catherine, M., and Dubayle, B. (1983). Taxonomic significance of enzyme polymorphism among isolates of Pleurotus from Umbellifers. Trans. Br. MycoL Soc. 81:121.

Croft, J. H., and Jinks, J.L. (1977). Aspects of the population genetics of Aspergillus nidulans. In Smith, J. E., and Pateman, J. A. (ed.), Genetics and Physiology of Aspergillus, Academic Press, New York and San Francisco, pp. 340-359.

Davis, B. J. (1964). Disc electrophoresis. II. Method and applications to human serum protein. Ann. N.Y. Acad. Sci. 121:404.

Dekker, R. F. H. (1981). Induction, localization and characterization of t3-D-glucosidases produced by a species of Monilia. J. Gen. MicrobioL 127:177.

Deshpande, V., Eriksson, K. E., and Pettersson, B. (1978). Production, purification and partial characterization of 1,4-/3-glucosidase enzymes from Sporotrichurn pulverulentum. Eur. J. Biochem. 90:191.

Dobzhansky, T., Ayala, A. J., Stebbins, G. L., and Valentine, J. W. (1977). The genetic structure of populations. In Evolution, W. H. Freeman, San Francisco, pp. 20-52.

Gillespie, J. H., and Lamgley, G. H. (1974). A general model to account for enzyme variation in natural populations. Genetics 76:837.

Grindle, M. (196-3a). Heterokaryon incompatability in unrelated strains in the Aspergillus nidulans group. Heredity 18:191.

Grindle, M. (1963b). Heterokaryon compatability of closely related wild isolates of Aspergillus nidulans. Heredity 81:397.

342 Sharma, Sandhu, and Bagga

Holt, G., and MacDonald, K. B. (1968). Penicillin production and its mode of inheritance in A. nidulans. Ant. Van. Leeuwen. 34:409.

Hubby, J. L., and Throckmorton, L. H. (1965). Protein differences in Drosophila. II. Compara- tive species genetic and evolutionary problems. Genetics 52"203.

Iglesias, L. (1984). Study on the origin and phylogenetic relationship of bananas. Cent. Agr. 11:45.

Kurzeja, K. C., and Garber, E. D. (1973). A genetic study of electrophoretically variant extracellular amylolytic enzymes of wild type strains of A. nidulans. Can. J. Genet. Cytol. 15:275.

Labudova, I., and Farkas, V. (1983). Multiple enzyme forms in the cellulase system of Trichoderma reesei during its growth on cellulose. Biochim. Biophys. Acta 744:135.

Lewontin, R. C. (1973). Population genetics. Annu. Rev. Genet. 7:1. Li, W. H. (1980). Rate of gene silencing a duplication loci: A theoretical study and interpretation

of data from tetraploid fishes. Genetics 95:237. Merrick, M. J., and Catch, C. E. (1975). The inheritance of penicillin titre in wild type isolates of

A. nidulans. J. Gen. Microbiol. 86:283. Pryor, T. (1976). Glucosidase in maize seedlings. Maize Genet. Coop. News Lett. 50:15. Raper, K. B., and Fennel, D. I. (1965). The Genus Aspergillus, Robert E. Krieger, New York. Rider, C. C., and Taylor, C. B. (1980). Structural aspects of isoenzymes. In Isoenzymes,

Chapman and Hall, New York, p. 8. Rockwood-Sluss, E. S., Johnson, J. S., and Heed, W. B. (1973). Allozyme genotype environment

relationships. I. Genetics 73:135. Singh, R. S., Lewontin, R. C., and Felton, A. A. (1976). Genetic heterogeneity within

electrophoretic alleles of xanthine dehydrogenase in D. pseudoobscura. Genetics 84:606. Spieth, P. T. (1975). Population genetics of allozyme variation in Neurospora intermedia.

Genetics 80:785. Stuber, C. W., and Goodman, M. M. (1976). B-Glucosidase isozyme variation in maize races.

Isozyme Bull. 9:59. Vogel, H. J. (1956). A convenient growth medium for Neurospora (Medium N). Microbial.

Genet. Bull. 13:42. Yamazaki, M., Gao, S., and Komagata, E. (1985). Taxonomic analysis of the genus Tilletiopsis

or physiological properties and electrophoretic comparison of enzymes. Trans. Mycol. Soc. Jpn. 26:13.