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Department of Biological Sciences, Stanford University, Stanford, California 94305-5020
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Neurospora pannonica is homothallic, with 8-spored asci. Immature asci are usually swollen and noncylindrical while the mature asci are narrow and cylindrical. The two meiotic divisions resemble those of other Neurospora species. However, the orientation of third-division mitotic spindles and the distribution of nuclei in the swollen asci are irregular. Ascospores are arranged irregularly at first, but as the ascospores enlarge and mature the asci gradually become cylindrical, with the ascospores aligned in single file. The asci cannot be considered ordered tetrads, because ascospore order does not reliably reflect the assortment of chromosomes at the first and second meiotic divisions. Contrary to the original species description, ascospores require heat shock for germination and hyphae are sent out at both ends of germinating ascospores.
Key words: ascospore dormancy, ascus-shape, homothallic Neurospora, nonlinear asci, unordered tetrads
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The species is nonconidiating, like other homothallic species of Neurospora. The asci and ascospores are much larger, however. Ascospore ornamentation and the linear alignment of spores in the mature asci are typical of the genus Neurospora (Figs. 1, 2
).
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, Raju and Perkins 1994
). Asci of these three species are cylindrical. The original description of N. pannonica by Krug and Khan (1991)
showed mature asci were cylindrical, with the ascospores arranged linearly. I report here that the developing immature asci are usually noncylindrical. At early stages the asci are often swollen and the nuclei and young ascospores are not usually aligned in single file. However, as the maturing ascus elongates and becomes cylindrical, pressure from the ascus walls appears to force the 8 ascospores into a linear alignment. A similar sequence of events, with noncylindrical young asci becoming linear as they mature, has been described in several large-spored homothallic isolates of Gelasinospora (Glass et al 1990
). Certain mutants of N. crassa (e.g., peak) also produce swollen, noncylindrical asci but the mature ascospores are not linearly ordered (Pincheira and Srb 1969
, Raju 1988
). I describe here the nuclear behavior in immature swollen asci and the subsequent linear alignment of ascospores in the mature cylindrical asci of N. pannonica. These observations, together with those made previously on Gelasinospora species, show clearly that linearity of ascospores in mature asci cannot be assumed to reflect the true order of segregating alleles at the first or second division of meiosis. New observations on ascospore dormancy and germination are also reported.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), which was further modified by replacing half the maltose in the original formula by glucose to give the following recipe: Maltose 3.12 g, Glucose 3.12 g, Malt extract 6.25 g, KH2PO4 1.25 g, Yeast extract 1.0 g, MgSO4·7H2O 0.625 g, Peptone 0.625 g, Agar 20 g, Distilled water 1000 mL. Neurospora pannonica grows vigorously on this medium and perithecia are formed within 6 to 8 d at 25 C in a 12 h light/dark regimen. Neurospora pannonica also fruits adequately on Neurospora synthetic crossing medium but not on standard minimal or complete media (see Davis and de Serres 1970
).
Strips of agar medium bearing developing perithecia were fixed at 12 h intervals from 8 to12 d, and the perithecial contents were stained using an iron-hematoxylin procedure (Raju and Newmeyer 1977
, Raju 1978
). Acriflavin staining and fluorescence microscopy were used for obtaining chromosome counts during meiosis (Raju 1986
). For photographing rosettes of mature asci, unfixed perithecia were opened and the asci were lightly stained using a 10-fold dilution (in 50% propionic acid) of ferric acetate mordant and hematoxylin staining solutions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Soon after karyogamy, the resulting diploid zygote nucleus undergoes meiosis and a postmeiotic mitosis in the common cytoplasm of the enlarging ascus. The chromosome number is 7, and the pachytene karyotype resembles that of other Neurospora species (Figs. 3, 4
).
The developing asci of N. pannonica are generally broader than those of other Neurospora species. Immature asci at or shortly after spore delimitation measure 40 x 150 µm as compared to 20 x 200 µm for N. crassa. The asci are narrow at the base but much broader at the apical two-thirds of the ascus. Apical pore formation in asci of N. pannonica is delayed until after meiosis I, and the pore is less conspicuous than in the cylindrical asci of other Neurospora species. Following an apparently normal meiotic prophase, the first-division spindle orients along the long axis of the ascus, at approximately half-way between the distal and proximal ends. At the second division, the two spindles are well-spaced and are often aligned in tandem, oriented longitudinally in the ascus. However, the resulting sister nuclei of each pair are neither spaced equidistant along the ascus nor aligned in single file as in N. crassa. The 4 interphase II nuclei are often aligned irregularly or biseriately (Fig. 5
). At the subsequent postmeiotic mitosis, the 4 spindles in the swollen asci are oriented irregularly (Fig. 6
). The resulting 8 nuclei are not lined up in single file, nor do all 8 spindle pole bodies face the same side of the ascus (Figs. 7, 8 ), as occurs in the cylindrical asci of other Neurospora species (see photos in Raju 1978, 1980
). Eight uninucleate ascospores are then cut out (Fig. 9
). A second postmeiotic mitosis in the just-delimited ascospores makes them binucleate (Fig. 10
). The positions of nuclei and ascospores in the swollen immature asci usually overlap (Figs. 5–10
).
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).
The young binucleate ascospores, typically 20 x 27 µm, enlarge and mature, with gradual thickening and pigmentation of ascospore walls. The nearly full grown but just pigmenting ascospores show characteristic striations (Fig. 2
) and measure 25 x 35 µm, in contrast to 16 x 30 µm for N. crassa and 18 x 32 µm for N. tetrasperma. The ascospores of N. pannonica are egg-shaped, like those of N. terricola, rather than spindle-shaped as in most other Neurospora species (Frederick et al 1969
, Raju 1978
).
Linear alignment of ascospores in mature asci –
As the ascospores mature, the swollen asci (40 x 150 µm) become narrow and cylindrical (30 x 275 µm). Of the 4 heterothallic and 6 homothallic species of Neurospora that I have examined cytologically over the years, only N. pannonica shows this developmentally programmed change of ascus-shape. Asci of other Neurospora species are normally cylindrical throughout meiosis and ascospore development (Raju 1978, 1980
).
Several large-spored homothallic species of Gelasinospora resemble N. pannonica in ascus development (Glass et al 1990
). For example, a large-spored isolate (No.115-3) from Yucatan Peninsula, Mexico, produces large, ovoid ascospores (26–29 µm x 33–37 µm). The young ascospores (15–17 µm x 21–24 µm) are arranged nonlinearly in the immature noncylindrical asci, but the mature ascospores become linearly arranged in the older asci that become cylindrical (see Figs. 8 and 9 in Glass et al 1990
). I have also witnessed a similar rearrangement of mature ascospores in the asci of the corn pathogen Gibberella fujikuroi (= Fusarium moniliforme). In this heterothallic species, unlike in N. pannonica, the developing asci are nearly cylindrical. However, the second and third-division spindles usually overlap and the resulting nuclei and ascospores are not linearly ordered in the immature asci. All 8 ascospores later become more or less linearly arranged as they enlarge and mature, but the asci cannot be considered as linearly ordered, at least in the genetic sense (Raju unpubl).
In both N. pannonica and in large-spored Gelasinospora, the transition from immature swollen asci to mature cylindrical asci has important consequences for the linear order of alleles in the ascus. For tetrads to be truly ordered in a genetic sense, as in the heterothallic N. crassa, the ascospore order in the cylindrical asci must reflect crossover events and the orientation of segregating alleles at the first and second division of meiosis (see Fig. 29 in Raju 1980
). Although mature asci of N. pannonica are physically linear, they cannot be considered to be genetically ordered tetrads because the primary order of nuclei and ascospores in the immature noncylindrical asci is irregular. The linear order of alleles becomes significant when self-sterile mutations and other marker differences are segregating in heterozygous asci.
Ascospore dormancy –
In the original description, Krug and Khan (1991)
reported that the freshly discharged mature ascospores of N. pannonica are not dormant and that they readily germinate without the need for heat shock or other artificial conditions. They also noted that the ascospores germinate from only one end of the spore. My observations are inconsistent with both of their observations. I find that while a few ascospores germinate spontaneously without heat shock, a vast majority of ascospores germinate only after being subjected to heat-shock treatment, and germinating ascospores usually send out hyphae from both ends. In one experiment, freshly ejected ascospores from a 2-wk-old culture plate were spread on 4 Leonian agar plates and on two 4% water-agar plates. The plates, each with several hundred ascospores, were incubated at 25 C for 24 h. Only 1 to 2% of ascospores germinated spontaneously and all others remained ungerminated even after 48 h. In another experiment, ascospores from the lid of a 1-mo-old culture plate were spread and rehydrated on a Leonian agar plate and left for 24 h at 25 C without heat shock. Only two ascospores, among several hundred, germinated and started growing into colonies. When the plate was then heat shocked at 60 C for 30 min, most of the previously ungerminated ascospores germinated within 24 h. Another batch of rehydrated ascospores were heat shocked and left overnight at room temperature. Among 89 ascospores scored, 43 germinated from both ends, 6 germinated from one end and 40 spores remained ungerminated. Thus, N. pannonica resembled other Neurospora species in requiring heat activation for ascospore germination.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). I suggest that the change in ascus shape is genetically programmed, and that linearity is adaptively important for ascospore dispersal.
A vast majority of fungi in the Sordariaceae eject ascospores from cylindrical asci. Apparently, the cylindrical ascus shape is required for the formation of an apical pore through which mature ascospores are forcefully ejected. These observations were borne out from the behavior of noncylindrical asci in the Prf (perforated ascus apex) and peak mutants of N. crassa (Raju 1987, 1988
). The asci of peak (a colonial mutant having vegetative aerial growth resembling mountain peaks) are typically swollen and do not form apical pores, nor do they eject their mature ascospores. However, a few exceptional cylindrical asci in the peak mutant do differentiate apical pores, and these asci are able to eject their spores forcefully.
Ascospore dormancy and germination –
Dodge (1912)
first discovered that heat-shock induces ascospore germination in species of Ascobolus, and subsequently showed the same requirement for ascospores of Neurospora spp. (Shear and Dodge 1927
). Typically heat activation is applied at 60 C for 30–40 min in a water bath. However, Krug and Khan (1991)
reported that freshly discharged ascospores of N. pannonica readily germinate without the need for heat shock or other artificial conditions. My own observations show that only 1 to 2% of ascospores germinate spontaneously, and that heat shock increases germination frequency to 60% or more. Ascospores of exceptional genotypes of N. crassa germinate spontaneously, however. For example, mature ascospores of per-1 mutant (lacks pigment in perithecial wall) are unpigmented and do not become dormant when mature. They germinate spontaneously and are killed if heat shocked (Howe 1976
). The per-1 ascospores are unique in that they lack certain wall components as well as the pigment, allowing the stained nuclei to be readily seen through the transparent ascospore walls (see Fig. 27 in Raju 1980
). In another N. crassa mutant, fluffy (aconidial), mature ascospores are black but a high proportion of them germinate spontaneously, without heat shock (Perkins et al 2001
).
Both N. pannonica and another homothallic species, N. terricola, were isolated from soils in a temperate climate. The ascospores of both species are ovoid, rather than spindle-shaped, but apparently only the ascospores of N. terricola germinate from one end (Gochenaur and Backus 1962
). In this respect, N. pannonica differs from N. terricola and the dominant Round-spore mutant of N. crassa, both of which differentiate a single germ pore (Novak 1971
, Srb et al 1973
, Raju unpubl).
Species relationships –
Glass et al (1990)
have shown that the homothallic species N. africana, N. dodgei, N. galapagosensis and N. lineolata carry a single mating-type sequence similar to the mat A sequence of N. crassa. In contrast, N. terricola and N. pannonica, like many homothallic ascomycetes, contain both mat A and mat a sequences (Glass et al 1990
, Vellani 1998
, Pöggeler 1999
, Yun et al 1999
). In addition, N. pannonica and N. terricola are more similar in ascospore shape and in geographical distribution to some homothallic Gelasinospora and Sordaria species than they are to other homothallic Neurospora species. Dettman et al (2001)
have further examined the relationships between species of Neurospora and Gelasinospora using the DNA sequences of four nuclear genes. Their results indicate that some species of the two genera are polyphyletic in origin, and that ascospore ornamentation (ribbed vs pitted) is not a reliable indicator of phylogenetic relationships.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication June 22, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Dettman JR, Harbinski FM, Taylor JW., 2001 Ascospore morphology is a poor predictor of the phylogenetic relationships of Neurospora and Gelasinospora Fungal Genet Biol 34:49–61
Dodge BO., 1912 Methods of culture and the morphology of the archicarp in certain species of the Ascobolaceae Bull Torrey Bot Club 39:139-197
Frederick L, Uecker FA, Benjamin CR., 1969 A new species of Neurospora from the soil of West Pakistan Mycologia 61:1077-1084
Glass NL, Metzenberg RL, Raju NB., 1990 Homothallic Sordariaceae from nature: the absence of strains containing only the a mating type sequence Exp Mycol 14:274-289
Gochenaur SE, Backus MP., 1962 A new species of Neurospora from Wisconsin lowland soil Mycologia 54:555-562
Howe HB., 1976 Phenotypic diversity among alleles at the per-1 locus of Neurospora crassa Genetics 82:595-603
Krug JC, Khan RS., 1991 A new homothallic species of Neurospora from Hungary Mycologia 83:829-832
Malloch D., 1981 Moulds: their isolation, cultivation, and identification Toronto: University of Toronto Press. 99 p
Novak DR., 1971 A study of developmental mutants of Neurospora [PhD Thesis] Ithaca, New York: Cornell University. 139 p
Perkins DD, Radford A, Sachs MS., 2001 The Neurospora compendium: chromosomal loci San Diego: Academic Press. 325 p
Pincheira G, Srb AM., 1969 Genetic variation in the orientation of nuclear spindles during the development of asci in Neurospora Amer J Bot 56:846-852
Pöggeler S., 1999 Phylogenetic relationships between mating-type sequences from homothallic and heterothallic ascomycetes Curr Genet 36:222-231[Medline]
Raju NB., 1978 Meiotic nuclear behavior and ascospore formation in five homothallic species of Neurospora Can J Bot 56:754-763
Raju NB., 1980 Meiosis and ascospore genesis in Neurospora Eur J Cell Biol 23:208-223[Medline]
Raju NB., 1986 A simple fluorescent staining method for meiotic chromosomes of Neurospora Mycologia 78:901-906
Raju NB., 1987 A mutant of Neurospora crassa with abnormal croziers, giant ascospores and asci having multiple apical pores Mycologia 79:696-706
Raju NB., 1988 Nonlinear asci without apical pores in the peak mutant of Neurospora Mycologia 80:825-831
Raju NB., 1992a Functional heterothallism resulting from homokaryotic conidia and ascospores in the pseudohomothallic Neurospora tetrasperma Mycol Res 96:103-116
Raju NB., 1992b Genetic control of the sexual cycle in Neurospora Mycol Res 96:241-262
Raju NB., Newmeyer D., 1977 Giant ascospores and abnormal croziers in a mutant of Neurospora crassa Exp Mycol 1:152-165
Raju NB., Perkins DD., 1994 Diverse programs of ascus development in pseudohomothallic species of Neurospora, Gelasinospora, and Podospora Dev Genet 15:104-118[Medline]
Shear CL, Dodge BO., 1927 Life histories and heterothallism of the red bread-molds of the Monilia sitophila group J Agri Res 34:1019-1042
Srb AM, Basl M, Bobst M, Leary JV., 1973 Mutations in Neurospora crassa affecting ascus and ascospore development J Hered 64:242-246
Vellani TS., 1998 Positional regulation and evolution of mating-type genes in heterothallic and homothallic species of Neurospora [PhD Thesis] Vancouver: University of British Columbia. 148 p
Yun SH, Berbee ML, Yoder OC, Turgeon BG., 1999 Evolution of the fungal self-fertile reproductive life style from self-sterile ancestors Pros Natl Acad Sci USA 96:5592-5597
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