Nuclear Cycle Analyses of
mellea AND ROBERT
Department of Biological Sciences, Bridgewater State College, Bridgewater, Massachusetts 02324, and Department of Biology, Stonehill College, North Easton, Massachusetts 02356 Accepted for publication PEABODY,
December 27, 1983
Armillaria mellea. Experimental Mycology 8, 161-169. Absorption microspectrophotometry and fluorescence microspectrophotometry have been compared for their relative abilities to measure Feulgen-stained DNA in post fusion basidial nuclei of the basidiomycete Armillaria mellea. The variance of the fluorescence method is significantly lower than that of the absorption method (P < O.OOl), and it is concluded that fluorescence is superior for analysis of small nuclei where data interpretation relies heavily on measuring levels of variability about the mean. F-DNA (Feulgen-stained nuclear DNA) was measured by fluorescence microspectrophotometry in four stages of the nuclear cycle of A. mellea. The nuclei of spores were haploid and unreplicated [mean F-DNA = 53.8, SE = 1.9 arbitrary units (a.u.)], nuclei of unmated, monosporous hyphae were haploid and replicated z = 84.1, SE = 1.6 a.u.), nuclei of prefusion basidiai nuclei were haploid and replicated (x = 80.0, SE = 2.0 a.u.), and the nuclei of postfusion basidia were diploid and replicated (%?= 166.4, SE = 1.7 a.u.). The basidial data indicated that DNA synthesis occurred during the nuclear cycle prior to fusion of basidial nuclei. There was no evidence of polyploidy in any of the nuclear cycle stages. INDEX DESCRIPTORS: Armillaria mellea; microspectrophotometry; nuclear cycle; ploidy. of
Since the early work of Kniep (1928) it has been accepted that the typical hymenomycetous basidiomycete has a haploid-dikaryotic nuclear cycle (Kniep, 1928; Raper, 1966; Burnett, 1975). However, mating experiments, light microscopy, and absorption photometry indicate that the agaric Armillaria mellea (Vahl ex Fr.) Kummer has a nuclear cycle more appropriately described as diploid (Hintikka, 1973; Korhonen and Hintikka, 1974; Korhonen, 1978, 1980; Peabody et al., 1978; Ullrich and Anderson, 1978; Anderson and Ullrich, 1982). Anderson’s work (1982) reveals that a closely related species, Armillaria tabescent (Stop. ex Fr.) Dennis, Orton and Hora, also has an extended vegetative diploid phase in nature. Therefore some members of the A. mellea complex and at least one of their closely related species, A. tai To whom correspondence
should be addressed.
bescens, are at variance with the esta lished nuclear cycle pattern for this group of basidiomycetes. Traditionally rmclear cycle studies of most fungi have been hindered by loid chromosome numbers an nuclear and chromosomal sizes, maki chromosomal counts difficult at best. circumvent these problems, absorption photometry with the two~wave~en~tb method of Patau (1952) has been used in studying ploidy levels of some fungaj nuclei. Measurements of fungal nuclear DNA using this approach were first obtained by Therrien (1966) and Rossen and Westergaard (1966) in studies of nuclear cycle nomena in two myxomycetes and an ascomycete, respectively. Quantification clear DNA employing this method w used to determine the site of meios oomycete, Saprolegnia terrestris and Howard, 1969), and to ~~afft~f~ 161 0147-5975184 $3.00 Copyright Q 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
Feulgen-stained DNA and azure bluestained RNA at different stages of ascus development in Sordariu fimicola (Bell and Therrien, 1977). Extensive absorption photometric analyses of myxomycetes have provided valuable insights into the nuclear dynamics and life histories of members of this group (Yemma and Therrien, 1972; Therrien and Yemma, 1974; Collins and Therrien, 1976; Therrien and Collins, 1976; Haskins and Therrien, 1978; Therrien and Haskins, 1981; Clark and Mulleavy, 1982). As indicated earlier this quantitative approach has been used to study the nuclear cycle of one isolate (76-54, Sullivan County, N.Y.) of the A. mellea complex (Peabody et al., 1978). Data from isolate 76-54 indicated that basidiospores were haploid Gl (lC), monosporous hyphae (hyphae derived from a single spore) were probably predominantly diploid G 1 (2C) and G2 (4C), the dikaryotic clamped subhymenium of basidiocarp lamellae were predominantly haploid G2 (2C), and the trama of lamellae were predominantly diploid G2 (4C). Polyploidy in some postfusion, premeiotic basidial nuclei was suggested because of the high DNA values noted in the distribution pattern of these nuclei. Although it was proposed that the variability of measured DNA contents at other stages in the nuclear cycle were indications of possible polyploidy in those stages, the reported levels of variability cast doubt on this interpretation. Additional nuclear cycle variability has been found in the A. melleu complex in that some members have a dikaryotic, clamped subhymenium and young basidia while others have a monokaryotic subhymenium and basidia lacking clamps (Kniep, 1911; Singer, 1956; Romagnesi, 1970, 1973; Korhonen, 1978; Peabody and Motta, 1979; Korhonen, 1980). Although the two-wavelength method of Patau (1952) shows a high degree of accuracy in absorption photometry, alternate methods are desirable as a means of verifying data reliability in cases where new
phenomena such as polyploidy in nuclear stages of A. melleu are reported. Fluorescence photometry is another method which has been used to obtain quantitative measurements of fungal nuclear DNA (Mortimer and Shaw, 1975; Williams and Mendgen, 1975; Laffler and Dove, 1977; Coleman et al., 1981). Because the absorption photometric study of a single isolate of the A. melleu complex indicated that its nuclear cycle is unusual among Hymenomycetes, the present study was undertaken to (1) compare absorption and fluorescence photometry of Feulgen-stained A. melleu material, and (2) use the less variable method to determine whether the same nuclear cycle patterns would emerge in another isolate of the same species complex. MATERIALS
Material for study was taken from basidiocarps of A. melleu collected on Syringu sp. at 34 Oak Street, Bridgewater, Massachusetts, and designated 82-3. All tissues were fixed, hydrolyzed, stained, and prepared for absorption and fluorescence photometry according to the Feulgen method (Bryant and Howard, 1969) with modifications that follow. Lamellae of A. melleu basidiocarps were gently macerated with a mortar and pestle in 100 mM phosphate buffer, pH 7.0; this supernatant was mixed with spores from fresh spore falls of the same basidiocarps and with erythrocytes of the salamander, Plethodon cinereus, suspended in amphibian Ringer’s solution. P. cinereus erythrocytes were included as an internal standard on all slides in the study to provide a constant basis for comparison among slides. Since monosporous hyphae were not available when slides of other stages were prepared, spores that had been stored at 18°C were included as an additional internal standard. Slides of monosporous hyphae were prepared by removing tufts of hyphae growing from small pieces of malt extract agar suspended in liquid glucose asparagine medium (Weinhold, 1963)
CYCLE OF Avmillnn’a mellea
and placing them in a drop of 100 mM phosphate buffer, pH 7.0, along with spores (823) suspended from a spore fall in the same buffer and a drop of P. cinereus erythrocytes in amphibian Ringer’s solution. After air drying, smeared slides were fixed in freshly prepared 3: 1 ethanol:acetic acid 30 min at 25°C. After a washing in distilled water, some slides were treated with DNase I (Sigma, crystallized and lyophilized, 0.25 mg/ml in 100 mM phosphate buffer plus 10 mM MgSO,, pH 7.7, for 2 h at 37°C); some slides were treated with RNase A (Sigma, 5 x crystallized, 0.5 mgl ml in 100 mM phosphate buffer plus 10 mM MgSO,, pH 7.7, for 30 min at 37°C followed by a 1.5-h rinse in distilled water); some slides, not treated with DNase or RNase, were designated as controls and remained in distilled water at 25°C during this 2-h period. All slides were run in parallel in all other steps of the Feulgen procedure (except for monosporous hyphae); monosporous hyphae with spores and P. cinereus erythrocytes were carried through the same steps as the control slides but at a different time. Optimum hydolysis time, 30 min at 25°C in 5 N HCl, was determined by a standard hydolysis curve (Jordanov, 1963). Slides were stained in Schiff reagent (Bryant and Howard, 1969) for 2 h in the dark at 25°C. DNA, expressed in arbitrary units (a.u.),2 was determined for individual Feulgen-stained nuclei (F-DNA) with a Zeiss universal microscope fitted with an MPM OlK photometer attached to a Zeiss photomultiplier indicator. For absorption photometry, relative F-DNA values were determined by the Rasch and Rasch (1970) modification of the two-wavelength method of Patau (1952). The maximum absorption wavelength, 540 nm, and the half-maximum wavelength, 483 nm, were determined from an absorption curve. A Zeiss motorized 2 Abbreviations used: F-DNA, Feulgen-stained clear DNA; a.u., arbitrary units of F-DNA.
continuous filter monochromator b was used to isolate the desired maximum an’ half-maximum wavelengths for abso~ti~~ readings. For fluorescence photometry an epi-illumination system with a 100-W mercury lamp and a 50/60-Hz light modulator was utilized. In conjunction with the photometer, an electronic shutter control functioned to allow controlled short-term fluorometric measurements. The Zeiss filter combination 48 77 15 was used for Schiff reagent (pararosaniline) fluorescence. Each reading of a nucleus was corrected for background by subtracting the amount of fluorescence observed after moving stage until the measuring pinhole was external to the cell. A fluorescent mineral standard available from Zeiss was fit to the 40X objective by a magnetic ho1 and its fluorescence was monitored after every 50 nuclear readings to check instrument stability; variation was generally less than 1%. To accomplish objective ( 1)) comparison of fluorescence and absorption photometry, Schiff reagent was used since it can be measured by both methods. Forty postfusion basidial nuclei were read with fluorescence photometry; stage coordinates were noted and sketches were made so that nuclei could be reliably relocated. All 40 nuclei were relocated and read with abs~r~t~o~ photometry. To eliminate the p~ss~b~~~ty that the process of taking fluorescence measurements affected subsequent absorption measurements, 10 additional nuclei were measured with the absorption method both prior to and following exposure to the controlled pulse of uv light used when making fluorescence readings. This test confirmed that prior measurement of nuclei with fluorescence did not influence subsequent absorption measurements ~~wo-wad ANOVA, paired comparison, P 3 0.75). Examination of stained lamellar tissue with phase contrast microscopy cQnfi~~ed the similarity of the 82-3 isolate to the 76
FIG. 1. A comparison of the variability of the fluorescence and absorption photometric methods. The same 40 nuclei were read by both methods and the resulting frequency distributions plotted on scales of arbitrary units of F-DNA per nucleus. The mean value for each distribution is indicated by the position of the arrow. For ease of comparison, the scale for the fluorescence readings is positioned on the page so that its mean (z = 166.4 au.) is directly above the mean value for absorption readings (y = 739.8 a.u.).
54 isolate studied by,Peabody et al. (1978) with respect to the presence of clamped, dikaryotic subhymenium and young basidia. RESULTS
Comparison of the control slides and the RNase-treated slides revealed no significant difference (one-way ANOVA, P > 0.75). Both the control and the RNasetreated slides were highly significantly different from the DNase-treated slides (oneway ANOVA, P < 0.0001). Figure 1 illustrates the frequency distribution of F-DNA content per nucleus for 40 postfusion basidial nuclei measured with fluorescence and absorption photometry. The null hypothesis that the two methods
FIG. 2. Histograms of the F-DNA content in spores, monosporous hyphae, prefusion and postfusion basidia. F-DNA values are in units. The mean value for each distribution is by the position of the arrow.
of nuclei basidia, arbitrary indicated
have equal variances (absorption variance = 259, 986.0 and fluorescence variance = 387.8) was tested by calculating the variance ratio and testing its significance (Sokol and Rohlf, 1969, p, 186). As the variance ratio was highly significant [F&39, 39) = 670.4, P < 0.0011, the null hypothesis was rejected and it was concluded that the fluorescence method was significantly less variable than the absorption method. Figure 2 represents the frequency distributions of F-DNA content per nucleus for spores, monosporous hyphae, prefusion basidia, and postfusion basidia; Table 1 lists means, relative C values, and estimates of variability for the same four nuclear stages. Mean F-DNA values for monosporous hy-
CYCLE OF Armillaria
phae and prefusion basidia were not significantly different from one another (one-way ANOVA, 0.50 > P > 0.25); spores and postfusion basidia were each highly significantly different from all other nuclear stages (one-way ANOVA, P < 0.001). The ratio of mean F-DNA contents for spores: prefusion basidia:postfusion basidia was 53.8:80.0:166.4 a.u. DISCUSSION
Over the years there have been various studies concerning the use of the two-wavelength method of Patau (1952) to quantify relative amounts of a chromophore (Garcia and Iorio, 1966; Rasch, 1970; Rasch and Rasch, 1970; Noeske, 1971). In 1976, Van Oostveldt and Boeken evaluated the twowavelength method and determined that it shows high performance in estimating the amount of dye in microscope preparations. There are two sources of error inherent in the absorption method, however. The most important source of error in absorption measurements is the distributional error due to the nature of the relationship between transmission and concentration of the substances being quantified. The twowavelength method minimizes or eliminates
this type of error (Pearse, 1972). In contrast, fluorescence measurements are not affected by uneven distribution of material. In fluorescence photometry, the light emitted by a substance within an object is proportional only to its amount an dependent of distribution. A second source of error, that due to light scattering, is smaller in fluorescence than in absorption photometry (Ruth, 1966). Because fluorescence photometry detects substances that are present in very low concentrations, this approach is ideal for measuring small fungal nuclei. The smallest fungal genomes are smaller than the largest bacterial genomes (Ulhich and Raper, 1977). Not only is orescence photometry highly sensitive its optical specificity is high, as both excitation and emission wavelengths can lected. According to Pearse (1972) th sical Schiff reagent (pararosaniline) is among the fluorochromes of choice for quantification of DNA. In this study fluorescence measurements were significantly less variable than abs tion measurements. In addition to tistieally significant difference strated by the variance ratio, Fig. 1 illustrates that on the absorption histogram, two
TABLE 1 Mean F-DNA, Relative C Values, 99% Confidence Intervals about Means, and Frequencies (Observed and Expected) of Nuclei Falling within the 50, 95, and 99% Ranges for Spores, Monosporous Hyphae, Prefusion Basidia, and Postfusion Basidia
Relative C value
99% Confidence intervals about means
Spore (n = 30)
Monosporous hyphae (n = 30) Prefusion basidia (n = 30) Postfusion basidia (n = 100)
Observed Expected Observed Expected Observed Expected Observed Expected
17 15.0 13 15.0 16 15.0 60 50.0
28 28.5 28 28.5 28 28.5 95 95.0
29 29.7 29 29.7 30 29.7 98 99.0
a Means are expressed in arbitrary units. b 50% range = x 2 0.674 SD (i.e., the range expected to contain 50% of the measured values); 95% range = x 5 1.960 SD; 99% range = x t 2.576 SD.
readings actually exceed 2 times the mean value of 739.8 a.u. These values might be interpreted as evidence for polyploidy if it were not for two factors that argue against such a conclusion. First, with a mean of 739.8 a.u. and a standard deviation of 509.9 a.u., 5% of the 40 readings (i.e., approximately 2 readings) would be expected to lie outside the range specified by the mean + 1.960 SD (i.e., O-1739 a.u.). Since two values are outside of these limits, it is reasonable to conclude that the observed distribution of values represents expected statistical fluctuation about the mean and is not evidence of polyploidy. Second, if the high values obtained with absorption are the result of high variability of the method rather than real indications of polyploidy, they should not be obtained if a less variable method of measurement is used. This is precisely the result obtained when the same 40 nuclei are measured with the less variable fluorescence technique (Fig. 1). Because fluorescence photometry is less variable than absorption photometry, and because the ability to detect real variability is essential for the analysis of polyploidy, fluorescence was used for all subsequent measurements of spores, monosporous hyphae, prefusion basidia, and postfusion basidia. Spores appear to be 1C and differ significantly from all other stages measured, suggesting a haploid Gl nuclear condition. This is consistent with the findings of Peabody et al. (1978) and accepted views of hymenomycetous basidiomycete nuclear cycles in general. In the single monosporous hyphal isolate of A. mellea studied in 1978 (Peabody et al.) the frequency distribution of F-DNA in hyphal nuclei was interpreted as having values in both the 2C and 4C ranges. It was concluded that monosporous hyphae were probably diploid with some Gl (2C) and G2 (4C) nuclei. In A. mellea 82-3 where the hyphal nuclei of three monosporous iso-
lates were analyzed with fluorescence photometry, the frequency distribution shows all 30 nuclei within the 2C range. The highest values from the monosporous hyphal nuclei values falls below the range of 4C postfusion basidial values. This suggests that the monosporous hyphal nuclei of isolate 82-3 are actually haploid G2 (2C). When 30 nuclei from actively growing monosporous hyphal cultures were selected for measurement, nuclei that appeared to have recently divided were avoided, thus accounting for the lack of 1C values. Division figures were also avoided because of the difficulty of enclosing them within the measuring aperture. An alternate hypothesis consistent with the 2C value is that these nuclei are diploid Gl (2C). This interpretation seems unlikely because, if so, the 30 readings from the cultures of actively growing hyphae should have included at least some 4C values. An interesting result obtained with subhymenial nuclei of isolate 76-54 (Peabody et al., 1978) was their apparent haploid, G2 (2C) condition. Since subhymenial cells of this isolate have unfused nuclei (i.e., are dikaryotic), this result was unexpected; prefusion DNA synthesis has been reported in only one basidiomycete, .Coprinus cinereus (Lu and Jeng, 1975; Ld, 1982), and three ascomycetes, Neotiella rutilans (Rossen and Westergaard, 1966), Sordaria fimicola (Bell and Therrien, 1977), and Neurospora crassa (Iyengar et al., 1977). While this finding supported the hypothesis of an S phase during the meiotic cycle prior to fusion of basidial nuclei, it was not possible to eliminate the alternative that subhymenial nuclei underwent an additional mitotic division and entered the basidial stage of the life cycle in the haploid 1C condition. In the present study of isolate 82-3 (also with dikaryotic subhymenium and young basidia), this problem was resolved by measuring young, prefusion basidial nuclei rather than subhymenial nuclei.
CYCLE OF Armillaria
The prefusion basidial nuclei were 2C and the hypothesis of prefusion DNA synthesis during the meiotic cycle was supported. In the 1978 study of isolate 76-54 it was concluded that postfusion basidial nuclei are predominantly diploid G2 (4C), but with a definite tendency to produce some polyploid nuclei. A major objective of the present study was to measure a large number of postfusion basidial nuclei with fluorescence photometry to detect polyploidy and to estimate the frequency of its occurrence. Surprisingly, the present data offer no support for polyploidy in any of the 100 nuclei measured in isolate 82-3. The frequency distribution clusters tightly about the mean value of 166.4 a.u. (SE = 1.7), and the frequencies of nuclei falling within the 50, 95, and 99% ranges (i.e., z ? 0.674 SD, x r 1.960 SD, and x + 2.576 SD, respectively) are close to the expected frequencies in every case. It would be interesting to reexamine isolate 76-54 as well as other new geographic isolates with fluorescence photometry to determine whether there is any variation in nuclear cycles among members of the A. melEeacomplex. Despite the fact that data on polyploidy in fungi are somewhat sketchy, polyploidy has been documented in all major groups (Ogur et al., 1952; Wilson, 1952; Olive, 1953; Lu, 1964; Fincham and Day, 1971; Rogers, 1973; Collins and Therrien, 1976; Win-Tin and Dick, 1975; Ullrich and Raper, 1977; Maniotis, 1980). Although the question of polyploidy in other isolates of the A. m&a complex is unresolved, none of the nuclear stages examined in isolate 82-3 supports its existence. The present study indicates that dikaryotic isolate 82-3 of A. mellea may have a nuclear cycle similar to the cycle described for A. ostoyyae (Korhonen, 1980) in which it was proposed that there may be two diploidizations and two haploidizations. If this is true of isolate 82-3 of A. mellea, the first diploidization would probably occur
sometime after compatible mating types of monosporous hyphae have anastomosed and the first haploidization would probably occur prior to or during the formation of the dikaryotic subhymenium. Nuclei woul again become diploid when prefusion basidial nuclei fuse to form postfusion basidial nuclei. A final haploidization would then take place meiotically as basidiospores form on the mature basidium. An tive to the hypothesized first diploi and haploidization must be conside cause nuclear stages intermediate between the monosporous hyphal nuclei and the prefusion basidial nuclei were not measured in this study, it is possible that ~a~l~idy sisted throughout these stages. If so, first proposed diploidization and subsequent haploidization suggested above would not be required to account for the relative F-DNA values measured in this isolate. A study of these intermed stages is currently being undertaken to tinguish between these alternatives. ACKNOWLEDGMENTS This investigation was supported by National Science Foundation Grants CDP-7927124 and PRM8025 174 (D.C.P. and R&P.), a grant from Sigma Xi (D.C.P.), and Faculty Development Grants from Bridgewater State College (D.C.P.) and Stonehill College (R.B.P.). Thanks are given to James R. Brennan and to an anonymous reviewer for reviewing the manuscript and for making helpful suggestions and to Jerome 9. Motta for providing the program for Patau’s two-wavelength method. An earlier version of this paper was presented at the 6th International Meeting IUFRO S2.06.01 on Root and Butt Rots in Melbourne, Australia, August 25, 1983. REFERENCES J. B. 1982. Bifactorial heterothallism and vegetative dipioidy in Glitocybe tabescens. Mycologia 74: 911-916. ANDERSON, J. B., AND ULLRICH, R. C. 1982. Diploids of Armillaria mellea: Synthesis, stability, and mating behavior. Canad. 4. Bot. 40: 432-439. BELL, W. R., AND THERRIEN, C. D. 1917. A cytophotometric investigation of the relationship of DNA ANDERSON,
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