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Basidiosporogenesis, meiosis, and post-meiotic mitosis in the ectomycorrhizal fungus Pisolithus microcarpus
Fungal Genetics and Biology 47 (2010) 477–483
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Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi
Basidiosporogenesis, meiosis, and post-meiotic mitosis in the ectomycorrhizal fungus Pisolithus microcarpus André Narvaes da Rocha Campos *, Maurício Dutra Costa Departamento de Microbiologia, Campus Universitário, Universidade Federal de Viçosa, Viçosa 36570-000, Minas Gerais, Brazil
a r t i c l e
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Article history: Received 23 December 2009 Accepted 24 February 2010 Available online 3 March 2010 Keywords: Basidiospores Nuclear migration Fluorescence microscopy Calcofluor SYBR Green IÒ Ectomycorrhiza
a b s t r a c t Pisolithus microcarpus (Cooke and Massee) G. Cunn. is a model organism for the studies on the ecology, physiology, and genetics of the ectomycorrhizal associations. However, little is known about the basidiosporogenesis in this species and, in particular, the nuclear behavior after karyogamy. In this work, the events involved in basidiosporogenesis and meiosis in P. microcarpus were analyzed using fluorescence and scanning electron microscopy. The basidia are formed inside peridioles by the differentiation of the cells along the whole hyphae. Basidial cells measure 12–18 lm in length and 6–7 lm in diameter. P. microcarpus produces eight basidiospores per basidium imbibed in a gelatinous matrix in the basidiocarp. The basidiospores are globose, equinate, with blunt spines, and measure 6–8 lm. Karyogamy can take place inside basidia as well as in undifferentiated hyphal cells followed by nuclear migration to a newly developed basidium where meiosis takes place. After the formation of the meiotic tetrad, one round of post-meiotic mitosis occurs, resulting in the production of eight nuclei per basidium. The newly-formed nuclei migrate into the basidiospores asynchronously, resulting in the production of eight uninucleate spores. This corresponds to pattern A of post-meiotic mitosis. This work is the first report on meiosis and post-meiotic mitosis during basidiosporogenesis in P. microcarpus and contributes to clarify some aspects of the biology and genetics of this ectomycorrhizal species. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The ectomycorrhizal fungus Pisolithus microcarpus associates with plants of economic interest, such as eucalypts, and has been used as a model organism for studies on several ecological, physiological, and genetic aspects of the ectomycorrhizal associations (Chambers and Cairney, 1999). The Pisolithus life cycle encompasses the release of basidiospores, produced inside closed basidiocarps, after the weathering of the basidiocarp apical layers, allowing basidiospore dispersion by the wind, rain water, or animals (Brundrett et al., 1996; Chambers and Cairney, 1999). Upon reaching the soil, basidiospore germination takes place in the favorable environment of the host plant rhizosphere, where appropriate chemical signals released by the roots trigger the process (Fries, 1987; Martin, 2007). Once germinated, basidiospores give rise to monokaryotic mycelia containing one nucleus per cell. Pisolithus is heterothallic and the fusion of two sexually compatible monokaryons, i.e., plasmogamy, leads to the production of a dikaryotic mycelium, characterized by the presence of two haploid nuclei per cell and clamp connections along the hyphae (Kope, 1992; Carvalho et al., 1997). Once formed, the dikaryon associates * Corresponding author. Fax: +55 31 3899 2573. E-mail address: [email protected] (A.N. da R. Campos). 1087-1845/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2010.02.008
symbiotically with the roots of compatible tree species and is subsequently stimulated to produce basidiocarps upon receiving unknown signals from the host plant and the environment (Chambers and Cairney, 1999). Important nuclear events, such as karyogamy, meiosis, mitosis, and nuclear migration, have been reported to occur during the life cycle of basidiomycetes and are essential for basidiosporogenesis (Hasebe et al., 1991; Mueller et al., 1993; Hibbett et al., 1994; Obatake et al., 2003; Kamzolkina et al., 2006; Shimomura et al., 2008). However, for Pisolithus, detailed descriptions of these processes as well as information on the nuclear events involved are still lacking. The understanding of the synthesis and distribution of organelles during this stage of the fungal life cycle could clarify some of the factors that are likely to influence spore germination in Pisolithus, reported to be as low as 0.38% (Bulmer, 1964; Kope and Fortin, 1990; Nara, 2009). Interestingly, the percentage of nucleate basidiospores in P. microcarpus has been shown to correspond to 57% and this could partially explain the low germination rates in this species (Costa, personal communication). Knowledge on basidiospore formation, such as the concurrent deposition of spore cell wall layers and storage of reserve compounds have been described for a few Pisolithus species, indicating provision for future spore germination (Mims, 1980; Campos et al., 2008; Campos and Costa, 2010). The early steps of basidiospore
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formation in Pisolithus tinctorius (Pers.) Coker and Couch occur in the lower portion of the basidiocarp, containing dikaryotic vegetative hyphae with clamp connections. Basidium production is reported to occur at the apical cells of the hyphae present in the peridioles of the upper portions of the basidiocarp. The basidia are immersed in a fibrilar matrix and are uninucleate, what is attributed to the occurrence of karyogamy (Mims, 1980). The basidiospore cell wall is composed of four layers (Mims, 1980) and its external surface is highly water-repellent due to the deposition of hydrophobic proteins, making the basidiospores almost impermeable to hydrophilic solutes. Also, sterigmata remnants can be easily seen in the free basidiospores (Mims, 1980). The synthesis of new organelles and their correct migration to developing spores are necessary for the production of viable spores. Basidiomycetes usually produce four basidiospores per basidium; however, this can vary for some basidiomycetal species, reflecting complex patterns of meiosis, post-meiotic mitosis, and nuclear migration events during basidiosporogenesis (Duncan and Galbraith, 1972; Arita, 1979; Mueller et al., 1993). Six distinct patterns of nuclear behavior during basidiosporogenesis have been described for basidiomycetes and are characterized by the occurrence or absence of post-meiotic mitosis, the fungal structure in which it takes place and the fate of nuclei after mitotic division (Duncan and Galbraith, 1972; Arita, 1979; Mueller et al., 1993). In pattern A, post-meiotic mitosis occurs inside the basidium and each nucleus migrates to a single spore, resulting in the production of eight uninucleate basidiospores. In pattern B, post-meiotic mitosis occurs inside the sterigmata, followed by the migration of the distal nucleus into the basidiospore, while the proximal one migrates back to the basidium, originating four uninucleate basidiospores. The pattern C differs from pattern B in that post-meiotic mitosis occurs in the basidiospores. In pattern D, after the occurrence of post-meiotic mitosis in the basidiospores, the nuclei do not migrate back to the basidia, giving rise to four binucleate basidiospores (Duncan and Galbraith, 1972). In pattern E, post-meiotic mitosis is absent, originating four uninucleate basidiospores (Arita, 1979). Finally, in pattern F, post-meiotic mitosis occurs inside the basidium with the subsequent migration of two nuclei to each basidiospore, resulting in the production of four binucleate basidiospores (Mueller et al., 1993). To our knowledge, the patterns of nuclear division during basidiosporogenesis in Pisolithus have not been described. Knowledge on these patterns could clarify many aspects of the dispersion strategies, biology, and genetics of this ectomycorrhizal genus, throwing light on the factors that may be involved in spore viability and germination. The objective of this work was to characterize the basidiosporogenesis and the events of meiosis and post-meiotic mitosis in P. microcarpus. 2. Materials and methods 2.1. Biological material This study was conducted at the Mycorrhizal Associations Laboratory, Microbiology Department, located at the Instituto de Biotecnologia Aplicada à Agropecuária (BIOAGRO), Federal University of Viçosa (UFV), Viçosa, MG, Brazil. Twenty fresh basidiocarps of P. microcarpus were collected under Eucalyptus urophylla S.T. Blake and Eucalyptus grandis Hill Ex Maiden forest sites at the Sylviculture Section, UFV, from August, 2003, to December, 2004. The collected basidiocarps were immediately transported to the laboratory and processed for microscopical analyses. Three samples (fragments of 3 3 3 mm) of each region of the basidiocarp gleba, characterized by distinct developmental stages of the peridioles, namely, unconsolidated peridioles, young peridioles, mature peridioles, internal basidiospores, and mature basidiospores, were
collected and processed as described below. Additional basidiocarps specimens were air-dried and deposited at Vic Herbarium under the code VIC 26495. 2.2. Fluorescence microscopy The gleba samples were fixed in ethanol:acetic acid (3:1 v/v) for 5 h at 5 °C and then treated with 35% ethanol for 15 min. The fixed samples were mounted in Jung Tissue Freezing MediumÒ (Leica Microsystems, Germany) and sectioned in a Leica CM 1850 cryomicrotome at 20 °C, producing 25-lm thick sections. For the observation of basidium and basidiospore development, the sections were collected in 100 mmol L 1 phosphate buffer, pH 7.0, and stained on the microscope slide for 5 min in 20 lL of calcofluor at 1 lg mL 1. The preparation was observed under a Nikon E600 light microscope, using an excitation wavelength range of 450– 520 nm, and photographed with a Fujix HC-300Z digital camera. The images were processed with Photograb and Eztouch (Fujifilm). For the observation of meiosis, post-meiotic mitosis, and nuclear migration, the sections were collected in 100 mmol L 1 phosphate buffer, pH 7, stained on the microscope slide in 20 lL of 0.001% SYBR Green IÒ, prepared in 10 mmol L 1 KH2PO4 containing 18% glycerol and 1 lg mL 1 calcofluor, and observed as described above. 2.3. Scanning electron microscopy Gleba samples collected as described above were fixed in 5% glutaraldehyde prepared in 100 mmol L 1 phosphate buffer, pH 7.0, for 12 h at 5 °C. After fixation, the samples were washed three times in the same buffer for 5 min and, then, post-fixed for 2 h in 1% OsO4, prepared in 100 mmol L 1 phosphate buffer, pH 7.0. The post-fixed peridioles were dehydrated by immersion in an acetone series (50%, 70%, 80%, and 90%) for 10 min for each concentration, and finally for 20 min in 100% acetone. The final drying was performed with 1% hexamethyldisilazane (HMDS) for 10 min, followed by air drying for 24 h. The dried peridioles thus obtained were sectioned with a pair of tweezers to expose the basidia and basidiospores. The sections were metalized with a 150-Å thick gold layer and observed under a Jeol JSM-5600 pv scanning electron microscope. 3. Results and discussion 3.1. Basidiosporogenesis During basidiosporogenesis, predominant stages of basidium and basidiospore development were observed at different portions of the basidiocarps. The unconsolidated peridiole region is found at the base of the basidiocarp and is characterized by the presence of white, flattened hyphae densely arranged and with larger diameters, surrounded by a pigmented, dark brown, gelatinous matrix containing loosely arranged hyphae. The young peridioles show reduced size, are surrounded by the same pigmented matrix, and contain only young basidia without spore primordia. Mature peridioles are fully developed and contain basidia with spores at different developmental stages; no gelatinous matrix is noticed in this region. Internal basidiospores corresponded to those inside partially ruptured peridioles in the upper portion of a closed basidiocarp. Finally, free basidiospores corresponded to mature spores collected at the upper portion of the basidiocarp from fully ruptured peridioles. The differentiation of hyphal cells into basidia is fully completed when peridioles become clearly distinguishable in the basidiocarps. Several newly-differentiated basidia occur along the
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whole length of the hypha inside the young peridioles and, from each hyphal cell in this region, one or several basidia can be produced (Fig. 1a and b). Basidial development initiates at the apical cell and progresses backwards to the sub-apical cells. The apical cell of the hypha may undergo further differentiation, originating up to two additional basidia (Fig. 1b). Basidium production is concurrent with an intense branching of the hyphae inside the peridioles (Fig. 1a and b). The basidia develop into a typically clavate shape, with 12–18 lm in length and 6–8 lm in diameter. The differentiation of basidia from each hyphal cell is described here for the first time and probably contributes to the production of the abundant masses of basidiospores typical of P. microcarpus. This characteristic contrasts with other basidiomycete species, such as Laccaria spp. and Agaricus bisporus, in which a single basidium is usually originated from the terminal cell of the hypha in the lamellae of the gilled basidiocarp (Mueller et al., 1993; Kamzolkina et al., 2006). In the gasteromycete fungi, Phallus impudicus L. and Rhizopogon roseolus (Corda) Th. M. Fr., the whole gleba differentiates into a mass of spores (Gull, 1981; Shimomura et al., 2008); however, no information is available on hyphal branching and distribution of basidia along the length of the hyphae. In Scleroderma verrucosum (Bull.) Pers., the basidiospores are produced from metamorphosed basidia derived from hyphal knots which are a common feature of development in tissues where a great number of new cells must be formed rapidly (Reijnders, 1999). Basidiospore formation begins after the differentiation of a hyphal cell into a basidium (Fig. 1c), with the appearance of basidiospore primordia asymmetrically distributed on the upper portion of the basidial cell (Fig. 1d and e). This characteristic is shared among different fungal species, such as Coprinus lagopus (Fries) Redhead, Vilgalys, and Moncalvo, Boletus rubinellus Peck., and Exobasidium spp., and seems to occur regardless of the number of basidiospores produced per basidium (Lu, 1967; Yoon and McLaughlin, 1986; Mims et al., 1987). Basidiospore development is asynchronous in P. microcarpus (Fig. 1e), and spore size becomes homogeneous later on, when basidiospores acquire similar diameters (Fig. 1f–h). After staining with calcofluor, the fluorescence emission patterns were shown to change according to the developmental stage of the basidiospores (Fig. 1j–l). Thin-walled spores show a pale blue1 fluorescence (Fig. 1j) that turns to reddish orange as new cell wall material is deposited (Fig. 1k). At the end of development, when basidiospores mature, fluorescence emission turned back to pale blue (Fig.1l). Such changes in fluorescence emission patterns during basidiospore development has already been reported for Flammulina velutipes (Curtis) Singer and has been explained as reflecting changes in cell wall composition and spore shape during development (Yoon and McLaughlin, 1980). For P. tinctorius, the deposition of four layers of cell wall material has been reported (Mims, 1980). The presence of layers with contrasting patterns of electron transmission suggests that their chemical compositions are distinct. Our results indicate that the young basidiospores of P. microcarpus present a cell wall composed predominantly of b-glucans, which can be stained with calcofluor leading to a blue fluorescence emission. Further deposition of b-glucans in the cell wall can also be inferred as fluorescence emission turns back to blue as basidiospores reach maturity. P. microcarpus produces eight basidiospores per basidium, with diameters of 6–8 lm (Fig. 1h). Nevertheless, the simultaneous observation of all the basidiospores is sometimes difficult, since some can be hidden behind the basidium, while others can be detached during sample preparation. Also, although the basidiocarps
1 For interpretation of color in Figs. 1 and 3, the reader is referred to the web version of this article.
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used in this study were collected from the same site, variation in basidiospore numbers due to environmental factors cannot be excluded (Mims et al., 1987). At the end of development, the basidiospores become free after detaching from fragments of collapsed basidial cells (Fig. 1i). Calcofluor staining allowed the visualization of the sterigmata (Fig. 1i), where polysaccharide deposition takes place to seal the mature basidiospores (Fig. 1i and l). Similar events were also observed during the maturation of P. impudicus and R. roseolus basidiospores, both also gasteromycete fungi (Gull, 1981; Shimomura et al., 2008). The basidia and basidiospores are immersed in a gelatinous matrix during the development inside the peridioles. (Fig. 2a). The abundance of the gelatinous matrix decreases as the basidiospores go through more advanced developmental stages and has been shown to be composed mainly by polysaccharides (Campos and Costa, 2010). The gelatinous matrix is also commonly found in the basidiocarps of other gasteromycetal species and is probably derived from the lyses of hyphae inside the peridioles (Mims, 1980; Moore-Landecker, 1996). It has been suggested that the products of hyphal lysis are subsequently absorbed by the basidiospores to constitute their carbon reserves (Moore-Landecker, 1996). In the final stage of basidiosporogenesis, the basidiospores become densely packed inside the mature peridioles (Fig. 2b). The cavities formed in the dried matrix by the detachment of the basidiospores during sample preparation highlight the complete imbibition of these propagules on the course of their development (Fig. 2b). P. microcarpus basidiospores have blunt spines measuring, approximately, 1 lm (Fig. 2b), and correspond more closely to B1 type described by Burgess et al. (1995). 3.2. Meiosis, post-meiotic mitosis, and nuclear migration During basidiospore development in P. microcarpus, conjugated mitotic division, karyogamy, meiosis, post-meiotic mitosis, and nuclear migration events were observed. After the occurrence of mitosis at the tips of the hyphae inside the young peridioles, the apical cell becomes clavate, indicating the differentiation into a basidium (Fig. 3a). After the migration of one haploid nucleus to the sub-basidial cell through a clamp connection, the two haploid nuclei left in the basidium are positioned at the longitudinal axis (Fig. 3a). They subsequently approach each other, and, then, fuse to give rise to a diploid nucleus (Fig. 3b–d). After karyogamy, distinct degrees of chromosome condensation can be observed (Fig. 3c and d). At the beginning of prophase I, the diploid nucleus is weakly condensed (Fig. 3c). At the end of prophase I and during metaphase I, the diploid nucleus becomes highly condensed and migrates to the basidial apex (Fig. 3d) where anaphase I takes place subsequently (Fig. 3e). During interphase I, the two diploid nuclei are positioned in opposite sides of the basidial apex (Fig. 3f). Anaphase II takes place leading to the formation of the meiotic tetrad (Fig. 3g). During interphase II, the four haploid nuclei become disorganized and are located at the base of the basidium (Fig. 3h). After the formation and development of the basidiospores, the haploid nuclei are reorganized at the apical portion of the basidium and then go through post-meiotic mitosis (Fig. 3i). Once reorganized, the four haploid nuclei become fusiform (Fig. 3j). As a result of post-meiotic mitosis, eight haploid nuclei are formed and migrate to the respective basidiospore (Fig. 3k and l). Thus, P. microcarpus presents the pattern A of post-meiotic mitosis, corresponding to the occurrence of post-meiotic mitosis within the basidium, followed by the migration of each haploid nucleus into a basidiospore, giving rise to eight uninucleate basidiospores. To our knowledge, this work represents the first complete report on the nuclear division and migration events during basidio-
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Fig. 1. Developmental stages of basidia and basidiospores of Pisolithus microcarpus observed by fluorescence microscopy with calcofluor. (a) Abundant production of basidia along the whole length of the hypha. (b) Typical differentiation of the hyphal apex, producing three basidia (arrow). (c) Newly differentiated basidium with clamp connection (arrow). (d and e) Basidium with asymmetrically distributed basidiospore primordia. (e) Asynchronous development of the basidiospores. Notice primordia with distinct sizes (arrows). (f–h) Development of the basidiospores. (h) Basidium with eight mature basidiospores at the end of development. (i) Basidiospores attached to fragments of a collapsed basidium (arrow, a). Notice the sterigma scar linking the basidiospore to the collapsed basidium (arrow, b). (j–l) Basidium and basidiospore development inside the peridiole. Notice the variation in the fluorescence emission pattern of the basidiospores stained with calcofluor along the development and the number of spores per basidia. Notice the sterigma scars at the sites of basidiospore attachment (l; arrows). Bars: a and b = 20 lm; c–i = 5 lm; j–l = 20 lm.
spore formation in P. microcarpus. The occurrence of pattern A is a rare event and also occurs in a few fungal species, such as Cantharellus cibarius Fr. and Pholiota nameko (T. Itô) S. Ito & S. Imai (Duncan and Galbraith, 1972; Arita, 1979). Although the patterns of post-meiotic mitosis is not considered appropriate for the estab-
lishment of phylogenetic relationships between different fungal species (Mueller and Ammirati, 1993), this characteristic has been used for fungal identification at higher taxonomical levels, for example, to differentiate the genus Neolentinus from the genera Panus and Lentinus (Hibbett et al., 1994). So far, no information is
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Fig. 2. Scanning electron microscopy of Pisolithus microcarpus basidiospores. (a) Basidia and basidiospores imbibed in a gelatinous matrix inside the peridioles. (b) Mature basidiospores. Notice the ruptured dried matrix involving the basidiospores at the end of development (arrows). Bars: a and b = 10 lm.
Fig. 3. Meiosis and post-meiotic mitosis during basidiosporogenesis in Pisolithus microcarpus observed by fluorescence microscopy with calcofluor and SYBRÒ Green I. (a) Dikaryotic basidium. (b) Beginning of karyogamy. (c) Prophase I. (d) Preparation for anaphase I. (e) Anaphase I. (f) Interphase I. (g) Anaphase II, beginning of formation of the meiotic tetrad. (h) Basidia with four haploid nuclei. (i) Nuclear reorganization in basidial apex. (j) Beginning of post-meiotic mitosis. (k) End of post-meiotic mitosis. Notice the presence of eight haploid nuclei and the asynchronous nuclear migration. (l) Uninucleate basidiospores after nuclear migration. (m) Behavior of nuclei in a basidium with less than eight basidiospores. Notice that two nuclei in excess of the number of basidiospores remain in the basidium. (n) Binucleate basidiospore (arrow). (o) Haploid nuclei in a hyphal cell that has produced two basidia. (p) Diploid nucleus in a sub-apical cell. (q) Migration of a diploid nucleus (arrow) from a sub-apical cell of the hypha to a newly formed lateral basidium.
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available on the occurrence of pattern A of post-meiotic mitosis among different species within the genus Pisolithus, but is noteworthy that Pisolithus species from Australia and Japan produce more than four spores per basidia (Brundrett et al., 1996; Kasuya et al., 2008), suggesting that this pattern must be prevalent within the genus. After mitosis, asynchronous nuclear migration from the basidium to the basidiospores takes place (Fig. 3k). The nuclei become fusiform during migration and each basidiospore receives a single haploid nucleus (Fig. 3l). The fusiform morphology of the nuclei during migration has also been observed for other basidiomycetes, (Mueller et al., 1993; Obatake et al., 2003), indicating the participation of microtubules and the cell molecular motors in the process (Xiang and Fisher, 2004). Our results have shown that the occurrence of enucleate basidiospores is negligible. In fact, basidiospores without a nucleus were not observed. Previous reports suggested that a great proportion of Pisolithus spp. basidiospores were enucleate (Mims, 1980; Costa, personal communication), but this might have been due to limitations of the microscopy techniques used, since nuclei are generally pressed against the spore cell wall due to the accumulation of lipid bodies inside the basidiospores, making their visualization very difficult (Mims, 1980; Campos et al., 2008). Also, the low permeability of the basidiospore cell wall due to the deposition of different cell wall layers and hydrophobic proteins usually prevent stains from diffusing into the spore cytoplasm, hindering the observation of nuclei, especially in mature basidiospores (McLaughlin, 1982). Visualization of nuclei at this stage is only possible after the chemical scarification of the cell wall before staining procedures (Verrinder-Gibbins and Lu, 1982), but homogeneity by this chemical treatment is difficult to obtain.
Basidia containing less than eight basidiospores were also observed (Fig. 3m). In such cases, the nuclei in excess of the number of basidiospores remained in the basidium during nuclear migration (Fig. 3n). Similar events were reported for C. cibarius (Duncan and Galbraith, 1972), a species that shows the same pattern of post-meiotic mitosis as P. microcarpus. The nuclei remaining in the basidium are reported to degenerate (Duncan and Galbraith, 1972). Though rarely observed, binucleate basidiospores were also observed (Fig. 3n) and could possibly result from the aberrant migration of two haploid nuclei to a single basidiospore or, alternatively, from the absence of nuclear disjunction at the end of post-meiotic mitosis. Production of binucleate basidiospores, having two genetically different nuclei, is suggested as a strategy to long distance dispersion in some basidiomycetes (Horton, 2006). Since post-meiotic mitosis in P. microcarpus occurs inside basidia, two genetically different nuclei might potentially migrate to single spore originating a binucleate basidiospore that could give rise to a dikaryotic mycelium without requiring plasmogamy. Although tempting, this hypothesis needs experimental confirmation. In P. microcarpus, karyogamy occurs not only in the basidium, but also in undifferentiated sub-apical or sub-basidial cells (Fig. 3o and p). When this is the case, once formed, the diploid nuclei migrate from the sub-apical or sub-basidial cell to a newly developed lateral basidium (Fig. 3q) and, then, undergo the usual steps of meiosis and post-meiotic mitosis. This seems to be a particular characteristic of P. microcarpus, since no reports of sub-apical and sub-basidial karyogamy could be found for other fungi, including other gasteromycetes. Besides, in holobasidiomycetes, karyogamy is known to occur only inside the basidia, while in heterobasidiomycetes, it is reported to take place in the probasidia (Mims et al., 1987; Reijnders, 2000).
Fig. 4. Schematic representation of meiosis and post-meiotic mitosis during basidiosporogenesis in Pisolithus microcarpus, showing the approximate location of the nuclear events inside the fungal basidiocarp. Bars: a and e = 200 lm; b = 1 mm; c and d = 500 lm.
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Our results allowed the completion of the life cycle of P. microcarpus, bringing novel information on the nuclear events that take place during basidiosporogenesis (Fig. 4). The fact that all spores are nucleate and contain large quantities of storage compounds (Mims, 1980; Campos et al., 2008; Campos and Costa, 2010) indicates the provision of carbon and organelles to direct basidiospore germination and initial vegetative growth in the host plant rhizosphere. This is the first report on meiosis and post-meiotic mitosis in a fungal species within the genus Pisolithus and contributes to a better understanding of fungal biology and reproduction. Acknowledgments The authors are grateful to Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG; Project CAG 775/03), Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial support. References Arita, I., 1979. Cytological studies on Pholiota. Rep. Tottori Mycol. Inst. 17, 1–118. Brundrett, M., Bougher, N., Dell, B., Grove, T., Malajczuk, N., 1996. Working with Mycorrhizas in Forestry and Agriculture – ACIAR Monograph 32. Pirie Printers, Canberra. Bulmer, G.S., 1964. Spore germination of forty-two species of puffballs. Mycologia 56, 630–632. Burgess, T., Malajczuk, N., Dell, B., 1995. Variation in Pisolithus based on basidiome and basidiospore morphology, culture characteristics and analysis of polypeptides using 1D SDS-PAGE. Mycol. Res. 99, 1–13. Campos, A.N.R., Costa, M.D., Tótola, M.R., Borges, A.C., 2008. Total lipid and fatty acid accumulation during basidiospore formation in the ectomycorrhizal fungus Pisolithus sp. Braz. J. Soil. Sci. 32, 1531–1540. Campos, A.N.R., Costa, M.D., 2010. Histochemistry and storage of organic compounds during basidiosporogenesis in the ectomycorrhizal fungus Pisolithus microcarpus. World J. Microbiol. Biotechnol., doi:10.1007/s11274010-0353-3. Carvalho, D., Rosado, S.C.S., Souza, A.M., Oliveira, A.F., 1997. Produção de culturas monocarióticas e compatibilidade sexual intra- e interpopulacional para o fungo ectomicorrízico Pisolithus tinctorius. Cerne 3, 143–160. Chambers, S.M., Cairney, J.W.G., 1999. Pisolithus. In: Cairney, J.W.G., Chambers, S.M. (Eds.), Ectomycorrizal Fungi: Key Genera in Profile. Springer-Verlag, Berlin, pp. 1–31. Duncan, E.G., Galbraith, M.H., 1972. Post-meiotic events in the homobasidiomycetidae. Trans. Brit. Mycol. Soc. 58, 378–392. Fries, N., 1987. Ecological and evolutionary aspects of spore germination in the higher basidiomycetes. Trans. Brit. Mycol. Soc. 88, 1–7. Gull, K., 1981. Meiosis, basidiospore development and post-meiotic mitosis in the basidiomycete, Phallus impudicus. Arch. Microbiol. 128, 403–406. Hasebe, K., Murakami, S., Tsuneda, A., 1991. Cytology and genetics of a sporeless mutant of Lentinus edodes. Mycologia 83, 354–359. Hibbett, D.S., Murakami, S., Tsuneda, A., 1994. Postmeiotic nuclear behavior in Lentinus, Panus, and Neolentinus. Mycologia 79, 331–333.
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Horton, T.R., 2006. The number of nuclei in basidiospores of 63 species of ectomycorrhizal homobasidiomycetes. Mycologia 98, 233–238. Kamzolkina, O.V., Volkova, V.N., Kozlova, M.V., Pancheva, E.V., Dyakov, Y.T., Callac, P., 2006. Karyological evidence for meiosis in the three different types of life cycles existing in Agaricus bisporus. Mycologia 98, 763–770. Kasuya, M.C.M., Coelho, I.S., Tamai, Y., Miyamoto, T., Yajima, T., 2008. Morphological and molecular characterization of Pisolithus occurring in Hokkaido Island, Northern Japan. Mycoscience 49, 334–338. Kope, H.H., 1992. Interactions of heterokaryotic and homokaryotic mycelium of sibling isolates of the ectomycorrhizal fungus Pisolithus arhizus. Mycologia 84, 659–667. Kope, H.H., Fortin, J.A., 1990. Germination and comparative morphology of basdiospores of Pisolithus arhizus. Mycologia 82, 350–357. Lu, B.C., 1967. Meiosis in Coprinus lagopus: a comparative study with light and electron microscopy. J. Cell Sci. 2, 529–536. Martin, F., 2007. Fair trade in the underworld: the ectomycorrhizal symbiosis. In: Howard, R.J., Gow, N.A.R. (Eds.), Biology of the Fungal Cell, second ed., The Mycota, vol. VIII Springer-Verlag, Berlin Heidelberg. McLaughlin, D.J., 1982. Ultrastructure and cytochemistry of basidial and basidiospore development. In: Wells, K., Wells, E.K. (Eds.), Basidium and Basidiocarp. Evolution, Cytology, Function and Development. Springer-Verlag, New York, pp. 37–74. Mims, C.W., 1980. Ultrastructure of basidiospores of the mycorrhizal fungus Pisolithus tinctorius. Can. J. Botany 58, 1525–1533. Mims, C.W., Richardson, E.A., Roberson, R.W., 1987. Ultrastructure of basidium and basidiospore development in three species of the fungus Exobasidium. Can. J. Botany 65, 1236–1244. Moore-Landecker, E., 1996. Fundamentals of the Fungi, fourth ed. Prentice Hall, Upper Saddle River, New Jersey. Mueller, G.J., Mueller, G.M., Shih, L., Ammirati, J.F., 1993. Cytological studies in Laccaria (Agaricales). I. Meiosis and post-meiotic mitosis. Am. J. Botany 80, 316– 321. Mueller, G.M., Ammirati, J.F., 1993. Cytological studies in Laccaria (Agaricales) II. Assessing phylogenetic relationships among Laccaria, Hydnangium, and other Agaricales. Am. J. Botany 80, 322–329. Nara, K., 2009. Spores of ectomycorrhizal fungi: ecological strategies for germination and dormancy. New Phytol. 181, 245–248. Obatake, Y., Murakami, S., Matsumoto, T., Fukumasa-Nakai, Y., 2003. Isolation and characterization of a sporeless mutant in Pleurotus eryngii. Mycosciense 44, 33– 40. Reijnders, A.F.M., 1999. The formation of spores by metamorphosed basidia in Mycocalia and Scleroderma. Mycol. Res. 103, 521–526. Reijnders, A.F.M., 2000. A morphogenetic analysis of the basic characters of the gasteromycetes and their relation to other basidiomycetes. Mycol. Res. 104, 900–910. Shimomura, N., Aimi, T., Matsumoto, T., Maekawa, N., Otani, H., 2008. Ultrastructure of developing basidiospore in Rhizopogon roseolus (=R. rubescens. Mycoscience 49, 35–41. Verrinder-Gibbins, A.M., Lu, B.C., 1982. An ameiotic mutant of Coprinus cinereus halted prior to pre-meiotic S-phase. Curr. Genet. 5, 119–126. Yoon, K.S., McLaughlin, D.J., 1980. Fluorescence microscopic examination of basidiosporogenesis in Boletus rubinellus and Coprinus cinereus. Mycologia 72, 1150–1158. Yoon, K.S., McLaughlin, D.J., 1986. Basidiosporogenesis in Boletus rubinellus. II. Late spore development. Mycologia 78, 185–197. Xiang, X., Fisher, R., 2004. Nuclear migration and positioning in filamentous fungi. Fungal Genet. Biol. 41, 411–419.
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Basidiosporogenesis, meiosis, and post-meiotic mitosis in the ectomycorrhizal fungus Pisolithus microcarpus André Narvaes da Rocha Campos *, Maurício Dutra Costa Departamento de Microbiologia, Campus Universitário, Universidade Federal de Viçosa, Viçosa 36570-000, Minas Gerais, Brazil
a r t i c l e
i n f o
Article history: Received 23 December 2009 Accepted 24 February 2010 Available online 3 March 2010 Keywords: Basidiospores Nuclear migration Fluorescence microscopy Calcofluor SYBR Green IÒ Ectomycorrhiza
a b s t r a c t Pisolithus microcarpus (Cooke and Massee) G. Cunn. is a model organism for the studies on the ecology, physiology, and genetics of the ectomycorrhizal associations. However, little is known about the basidiosporogenesis in this species and, in particular, the nuclear behavior after karyogamy. In this work, the events involved in basidiosporogenesis and meiosis in P. microcarpus were analyzed using fluorescence and scanning electron microscopy. The basidia are formed inside peridioles by the differentiation of the cells along the whole hyphae. Basidial cells measure 12–18 lm in length and 6–7 lm in diameter. P. microcarpus produces eight basidiospores per basidium imbibed in a gelatinous matrix in the basidiocarp. The basidiospores are globose, equinate, with blunt spines, and measure 6–8 lm. Karyogamy can take place inside basidia as well as in undifferentiated hyphal cells followed by nuclear migration to a newly developed basidium where meiosis takes place. After the formation of the meiotic tetrad, one round of post-meiotic mitosis occurs, resulting in the production of eight nuclei per basidium. The newly-formed nuclei migrate into the basidiospores asynchronously, resulting in the production of eight uninucleate spores. This corresponds to pattern A of post-meiotic mitosis. This work is the first report on meiosis and post-meiotic mitosis during basidiosporogenesis in P. microcarpus and contributes to clarify some aspects of the biology and genetics of this ectomycorrhizal species. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The ectomycorrhizal fungus Pisolithus microcarpus associates with plants of economic interest, such as eucalypts, and has been used as a model organism for studies on several ecological, physiological, and genetic aspects of the ectomycorrhizal associations (Chambers and Cairney, 1999). The Pisolithus life cycle encompasses the release of basidiospores, produced inside closed basidiocarps, after the weathering of the basidiocarp apical layers, allowing basidiospore dispersion by the wind, rain water, or animals (Brundrett et al., 1996; Chambers and Cairney, 1999). Upon reaching the soil, basidiospore germination takes place in the favorable environment of the host plant rhizosphere, where appropriate chemical signals released by the roots trigger the process (Fries, 1987; Martin, 2007). Once germinated, basidiospores give rise to monokaryotic mycelia containing one nucleus per cell. Pisolithus is heterothallic and the fusion of two sexually compatible monokaryons, i.e., plasmogamy, leads to the production of a dikaryotic mycelium, characterized by the presence of two haploid nuclei per cell and clamp connections along the hyphae (Kope, 1992; Carvalho et al., 1997). Once formed, the dikaryon associates * Corresponding author. Fax: +55 31 3899 2573. E-mail address: [email protected] (A.N. da R. Campos). 1087-1845/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2010.02.008
symbiotically with the roots of compatible tree species and is subsequently stimulated to produce basidiocarps upon receiving unknown signals from the host plant and the environment (Chambers and Cairney, 1999). Important nuclear events, such as karyogamy, meiosis, mitosis, and nuclear migration, have been reported to occur during the life cycle of basidiomycetes and are essential for basidiosporogenesis (Hasebe et al., 1991; Mueller et al., 1993; Hibbett et al., 1994; Obatake et al., 2003; Kamzolkina et al., 2006; Shimomura et al., 2008). However, for Pisolithus, detailed descriptions of these processes as well as information on the nuclear events involved are still lacking. The understanding of the synthesis and distribution of organelles during this stage of the fungal life cycle could clarify some of the factors that are likely to influence spore germination in Pisolithus, reported to be as low as 0.38% (Bulmer, 1964; Kope and Fortin, 1990; Nara, 2009). Interestingly, the percentage of nucleate basidiospores in P. microcarpus has been shown to correspond to 57% and this could partially explain the low germination rates in this species (Costa, personal communication). Knowledge on basidiospore formation, such as the concurrent deposition of spore cell wall layers and storage of reserve compounds have been described for a few Pisolithus species, indicating provision for future spore germination (Mims, 1980; Campos et al., 2008; Campos and Costa, 2010). The early steps of basidiospore
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formation in Pisolithus tinctorius (Pers.) Coker and Couch occur in the lower portion of the basidiocarp, containing dikaryotic vegetative hyphae with clamp connections. Basidium production is reported to occur at the apical cells of the hyphae present in the peridioles of the upper portions of the basidiocarp. The basidia are immersed in a fibrilar matrix and are uninucleate, what is attributed to the occurrence of karyogamy (Mims, 1980). The basidiospore cell wall is composed of four layers (Mims, 1980) and its external surface is highly water-repellent due to the deposition of hydrophobic proteins, making the basidiospores almost impermeable to hydrophilic solutes. Also, sterigmata remnants can be easily seen in the free basidiospores (Mims, 1980). The synthesis of new organelles and their correct migration to developing spores are necessary for the production of viable spores. Basidiomycetes usually produce four basidiospores per basidium; however, this can vary for some basidiomycetal species, reflecting complex patterns of meiosis, post-meiotic mitosis, and nuclear migration events during basidiosporogenesis (Duncan and Galbraith, 1972; Arita, 1979; Mueller et al., 1993). Six distinct patterns of nuclear behavior during basidiosporogenesis have been described for basidiomycetes and are characterized by the occurrence or absence of post-meiotic mitosis, the fungal structure in which it takes place and the fate of nuclei after mitotic division (Duncan and Galbraith, 1972; Arita, 1979; Mueller et al., 1993). In pattern A, post-meiotic mitosis occurs inside the basidium and each nucleus migrates to a single spore, resulting in the production of eight uninucleate basidiospores. In pattern B, post-meiotic mitosis occurs inside the sterigmata, followed by the migration of the distal nucleus into the basidiospore, while the proximal one migrates back to the basidium, originating four uninucleate basidiospores. The pattern C differs from pattern B in that post-meiotic mitosis occurs in the basidiospores. In pattern D, after the occurrence of post-meiotic mitosis in the basidiospores, the nuclei do not migrate back to the basidia, giving rise to four binucleate basidiospores (Duncan and Galbraith, 1972). In pattern E, post-meiotic mitosis is absent, originating four uninucleate basidiospores (Arita, 1979). Finally, in pattern F, post-meiotic mitosis occurs inside the basidium with the subsequent migration of two nuclei to each basidiospore, resulting in the production of four binucleate basidiospores (Mueller et al., 1993). To our knowledge, the patterns of nuclear division during basidiosporogenesis in Pisolithus have not been described. Knowledge on these patterns could clarify many aspects of the dispersion strategies, biology, and genetics of this ectomycorrhizal genus, throwing light on the factors that may be involved in spore viability and germination. The objective of this work was to characterize the basidiosporogenesis and the events of meiosis and post-meiotic mitosis in P. microcarpus. 2. Materials and methods 2.1. Biological material This study was conducted at the Mycorrhizal Associations Laboratory, Microbiology Department, located at the Instituto de Biotecnologia Aplicada à Agropecuária (BIOAGRO), Federal University of Viçosa (UFV), Viçosa, MG, Brazil. Twenty fresh basidiocarps of P. microcarpus were collected under Eucalyptus urophylla S.T. Blake and Eucalyptus grandis Hill Ex Maiden forest sites at the Sylviculture Section, UFV, from August, 2003, to December, 2004. The collected basidiocarps were immediately transported to the laboratory and processed for microscopical analyses. Three samples (fragments of 3 3 3 mm) of each region of the basidiocarp gleba, characterized by distinct developmental stages of the peridioles, namely, unconsolidated peridioles, young peridioles, mature peridioles, internal basidiospores, and mature basidiospores, were
collected and processed as described below. Additional basidiocarps specimens were air-dried and deposited at Vic Herbarium under the code VIC 26495. 2.2. Fluorescence microscopy The gleba samples were fixed in ethanol:acetic acid (3:1 v/v) for 5 h at 5 °C and then treated with 35% ethanol for 15 min. The fixed samples were mounted in Jung Tissue Freezing MediumÒ (Leica Microsystems, Germany) and sectioned in a Leica CM 1850 cryomicrotome at 20 °C, producing 25-lm thick sections. For the observation of basidium and basidiospore development, the sections were collected in 100 mmol L 1 phosphate buffer, pH 7.0, and stained on the microscope slide for 5 min in 20 lL of calcofluor at 1 lg mL 1. The preparation was observed under a Nikon E600 light microscope, using an excitation wavelength range of 450– 520 nm, and photographed with a Fujix HC-300Z digital camera. The images were processed with Photograb and Eztouch (Fujifilm). For the observation of meiosis, post-meiotic mitosis, and nuclear migration, the sections were collected in 100 mmol L 1 phosphate buffer, pH 7, stained on the microscope slide in 20 lL of 0.001% SYBR Green IÒ, prepared in 10 mmol L 1 KH2PO4 containing 18% glycerol and 1 lg mL 1 calcofluor, and observed as described above. 2.3. Scanning electron microscopy Gleba samples collected as described above were fixed in 5% glutaraldehyde prepared in 100 mmol L 1 phosphate buffer, pH 7.0, for 12 h at 5 °C. After fixation, the samples were washed three times in the same buffer for 5 min and, then, post-fixed for 2 h in 1% OsO4, prepared in 100 mmol L 1 phosphate buffer, pH 7.0. The post-fixed peridioles were dehydrated by immersion in an acetone series (50%, 70%, 80%, and 90%) for 10 min for each concentration, and finally for 20 min in 100% acetone. The final drying was performed with 1% hexamethyldisilazane (HMDS) for 10 min, followed by air drying for 24 h. The dried peridioles thus obtained were sectioned with a pair of tweezers to expose the basidia and basidiospores. The sections were metalized with a 150-Å thick gold layer and observed under a Jeol JSM-5600 pv scanning electron microscope. 3. Results and discussion 3.1. Basidiosporogenesis During basidiosporogenesis, predominant stages of basidium and basidiospore development were observed at different portions of the basidiocarps. The unconsolidated peridiole region is found at the base of the basidiocarp and is characterized by the presence of white, flattened hyphae densely arranged and with larger diameters, surrounded by a pigmented, dark brown, gelatinous matrix containing loosely arranged hyphae. The young peridioles show reduced size, are surrounded by the same pigmented matrix, and contain only young basidia without spore primordia. Mature peridioles are fully developed and contain basidia with spores at different developmental stages; no gelatinous matrix is noticed in this region. Internal basidiospores corresponded to those inside partially ruptured peridioles in the upper portion of a closed basidiocarp. Finally, free basidiospores corresponded to mature spores collected at the upper portion of the basidiocarp from fully ruptured peridioles. The differentiation of hyphal cells into basidia is fully completed when peridioles become clearly distinguishable in the basidiocarps. Several newly-differentiated basidia occur along the
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whole length of the hypha inside the young peridioles and, from each hyphal cell in this region, one or several basidia can be produced (Fig. 1a and b). Basidial development initiates at the apical cell and progresses backwards to the sub-apical cells. The apical cell of the hypha may undergo further differentiation, originating up to two additional basidia (Fig. 1b). Basidium production is concurrent with an intense branching of the hyphae inside the peridioles (Fig. 1a and b). The basidia develop into a typically clavate shape, with 12–18 lm in length and 6–8 lm in diameter. The differentiation of basidia from each hyphal cell is described here for the first time and probably contributes to the production of the abundant masses of basidiospores typical of P. microcarpus. This characteristic contrasts with other basidiomycete species, such as Laccaria spp. and Agaricus bisporus, in which a single basidium is usually originated from the terminal cell of the hypha in the lamellae of the gilled basidiocarp (Mueller et al., 1993; Kamzolkina et al., 2006). In the gasteromycete fungi, Phallus impudicus L. and Rhizopogon roseolus (Corda) Th. M. Fr., the whole gleba differentiates into a mass of spores (Gull, 1981; Shimomura et al., 2008); however, no information is available on hyphal branching and distribution of basidia along the length of the hyphae. In Scleroderma verrucosum (Bull.) Pers., the basidiospores are produced from metamorphosed basidia derived from hyphal knots which are a common feature of development in tissues where a great number of new cells must be formed rapidly (Reijnders, 1999). Basidiospore formation begins after the differentiation of a hyphal cell into a basidium (Fig. 1c), with the appearance of basidiospore primordia asymmetrically distributed on the upper portion of the basidial cell (Fig. 1d and e). This characteristic is shared among different fungal species, such as Coprinus lagopus (Fries) Redhead, Vilgalys, and Moncalvo, Boletus rubinellus Peck., and Exobasidium spp., and seems to occur regardless of the number of basidiospores produced per basidium (Lu, 1967; Yoon and McLaughlin, 1986; Mims et al., 1987). Basidiospore development is asynchronous in P. microcarpus (Fig. 1e), and spore size becomes homogeneous later on, when basidiospores acquire similar diameters (Fig. 1f–h). After staining with calcofluor, the fluorescence emission patterns were shown to change according to the developmental stage of the basidiospores (Fig. 1j–l). Thin-walled spores show a pale blue1 fluorescence (Fig. 1j) that turns to reddish orange as new cell wall material is deposited (Fig. 1k). At the end of development, when basidiospores mature, fluorescence emission turned back to pale blue (Fig.1l). Such changes in fluorescence emission patterns during basidiospore development has already been reported for Flammulina velutipes (Curtis) Singer and has been explained as reflecting changes in cell wall composition and spore shape during development (Yoon and McLaughlin, 1980). For P. tinctorius, the deposition of four layers of cell wall material has been reported (Mims, 1980). The presence of layers with contrasting patterns of electron transmission suggests that their chemical compositions are distinct. Our results indicate that the young basidiospores of P. microcarpus present a cell wall composed predominantly of b-glucans, which can be stained with calcofluor leading to a blue fluorescence emission. Further deposition of b-glucans in the cell wall can also be inferred as fluorescence emission turns back to blue as basidiospores reach maturity. P. microcarpus produces eight basidiospores per basidium, with diameters of 6–8 lm (Fig. 1h). Nevertheless, the simultaneous observation of all the basidiospores is sometimes difficult, since some can be hidden behind the basidium, while others can be detached during sample preparation. Also, although the basidiocarps
1 For interpretation of color in Figs. 1 and 3, the reader is referred to the web version of this article.
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used in this study were collected from the same site, variation in basidiospore numbers due to environmental factors cannot be excluded (Mims et al., 1987). At the end of development, the basidiospores become free after detaching from fragments of collapsed basidial cells (Fig. 1i). Calcofluor staining allowed the visualization of the sterigmata (Fig. 1i), where polysaccharide deposition takes place to seal the mature basidiospores (Fig. 1i and l). Similar events were also observed during the maturation of P. impudicus and R. roseolus basidiospores, both also gasteromycete fungi (Gull, 1981; Shimomura et al., 2008). The basidia and basidiospores are immersed in a gelatinous matrix during the development inside the peridioles. (Fig. 2a). The abundance of the gelatinous matrix decreases as the basidiospores go through more advanced developmental stages and has been shown to be composed mainly by polysaccharides (Campos and Costa, 2010). The gelatinous matrix is also commonly found in the basidiocarps of other gasteromycetal species and is probably derived from the lyses of hyphae inside the peridioles (Mims, 1980; Moore-Landecker, 1996). It has been suggested that the products of hyphal lysis are subsequently absorbed by the basidiospores to constitute their carbon reserves (Moore-Landecker, 1996). In the final stage of basidiosporogenesis, the basidiospores become densely packed inside the mature peridioles (Fig. 2b). The cavities formed in the dried matrix by the detachment of the basidiospores during sample preparation highlight the complete imbibition of these propagules on the course of their development (Fig. 2b). P. microcarpus basidiospores have blunt spines measuring, approximately, 1 lm (Fig. 2b), and correspond more closely to B1 type described by Burgess et al. (1995). 3.2. Meiosis, post-meiotic mitosis, and nuclear migration During basidiospore development in P. microcarpus, conjugated mitotic division, karyogamy, meiosis, post-meiotic mitosis, and nuclear migration events were observed. After the occurrence of mitosis at the tips of the hyphae inside the young peridioles, the apical cell becomes clavate, indicating the differentiation into a basidium (Fig. 3a). After the migration of one haploid nucleus to the sub-basidial cell through a clamp connection, the two haploid nuclei left in the basidium are positioned at the longitudinal axis (Fig. 3a). They subsequently approach each other, and, then, fuse to give rise to a diploid nucleus (Fig. 3b–d). After karyogamy, distinct degrees of chromosome condensation can be observed (Fig. 3c and d). At the beginning of prophase I, the diploid nucleus is weakly condensed (Fig. 3c). At the end of prophase I and during metaphase I, the diploid nucleus becomes highly condensed and migrates to the basidial apex (Fig. 3d) where anaphase I takes place subsequently (Fig. 3e). During interphase I, the two diploid nuclei are positioned in opposite sides of the basidial apex (Fig. 3f). Anaphase II takes place leading to the formation of the meiotic tetrad (Fig. 3g). During interphase II, the four haploid nuclei become disorganized and are located at the base of the basidium (Fig. 3h). After the formation and development of the basidiospores, the haploid nuclei are reorganized at the apical portion of the basidium and then go through post-meiotic mitosis (Fig. 3i). Once reorganized, the four haploid nuclei become fusiform (Fig. 3j). As a result of post-meiotic mitosis, eight haploid nuclei are formed and migrate to the respective basidiospore (Fig. 3k and l). Thus, P. microcarpus presents the pattern A of post-meiotic mitosis, corresponding to the occurrence of post-meiotic mitosis within the basidium, followed by the migration of each haploid nucleus into a basidiospore, giving rise to eight uninucleate basidiospores. To our knowledge, this work represents the first complete report on the nuclear division and migration events during basidio-
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Fig. 1. Developmental stages of basidia and basidiospores of Pisolithus microcarpus observed by fluorescence microscopy with calcofluor. (a) Abundant production of basidia along the whole length of the hypha. (b) Typical differentiation of the hyphal apex, producing three basidia (arrow). (c) Newly differentiated basidium with clamp connection (arrow). (d and e) Basidium with asymmetrically distributed basidiospore primordia. (e) Asynchronous development of the basidiospores. Notice primordia with distinct sizes (arrows). (f–h) Development of the basidiospores. (h) Basidium with eight mature basidiospores at the end of development. (i) Basidiospores attached to fragments of a collapsed basidium (arrow, a). Notice the sterigma scar linking the basidiospore to the collapsed basidium (arrow, b). (j–l) Basidium and basidiospore development inside the peridiole. Notice the variation in the fluorescence emission pattern of the basidiospores stained with calcofluor along the development and the number of spores per basidia. Notice the sterigma scars at the sites of basidiospore attachment (l; arrows). Bars: a and b = 20 lm; c–i = 5 lm; j–l = 20 lm.
spore formation in P. microcarpus. The occurrence of pattern A is a rare event and also occurs in a few fungal species, such as Cantharellus cibarius Fr. and Pholiota nameko (T. Itô) S. Ito & S. Imai (Duncan and Galbraith, 1972; Arita, 1979). Although the patterns of post-meiotic mitosis is not considered appropriate for the estab-
lishment of phylogenetic relationships between different fungal species (Mueller and Ammirati, 1993), this characteristic has been used for fungal identification at higher taxonomical levels, for example, to differentiate the genus Neolentinus from the genera Panus and Lentinus (Hibbett et al., 1994). So far, no information is
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Fig. 2. Scanning electron microscopy of Pisolithus microcarpus basidiospores. (a) Basidia and basidiospores imbibed in a gelatinous matrix inside the peridioles. (b) Mature basidiospores. Notice the ruptured dried matrix involving the basidiospores at the end of development (arrows). Bars: a and b = 10 lm.
Fig. 3. Meiosis and post-meiotic mitosis during basidiosporogenesis in Pisolithus microcarpus observed by fluorescence microscopy with calcofluor and SYBRÒ Green I. (a) Dikaryotic basidium. (b) Beginning of karyogamy. (c) Prophase I. (d) Preparation for anaphase I. (e) Anaphase I. (f) Interphase I. (g) Anaphase II, beginning of formation of the meiotic tetrad. (h) Basidia with four haploid nuclei. (i) Nuclear reorganization in basidial apex. (j) Beginning of post-meiotic mitosis. (k) End of post-meiotic mitosis. Notice the presence of eight haploid nuclei and the asynchronous nuclear migration. (l) Uninucleate basidiospores after nuclear migration. (m) Behavior of nuclei in a basidium with less than eight basidiospores. Notice that two nuclei in excess of the number of basidiospores remain in the basidium. (n) Binucleate basidiospore (arrow). (o) Haploid nuclei in a hyphal cell that has produced two basidia. (p) Diploid nucleus in a sub-apical cell. (q) Migration of a diploid nucleus (arrow) from a sub-apical cell of the hypha to a newly formed lateral basidium.
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available on the occurrence of pattern A of post-meiotic mitosis among different species within the genus Pisolithus, but is noteworthy that Pisolithus species from Australia and Japan produce more than four spores per basidia (Brundrett et al., 1996; Kasuya et al., 2008), suggesting that this pattern must be prevalent within the genus. After mitosis, asynchronous nuclear migration from the basidium to the basidiospores takes place (Fig. 3k). The nuclei become fusiform during migration and each basidiospore receives a single haploid nucleus (Fig. 3l). The fusiform morphology of the nuclei during migration has also been observed for other basidiomycetes, (Mueller et al., 1993; Obatake et al., 2003), indicating the participation of microtubules and the cell molecular motors in the process (Xiang and Fisher, 2004). Our results have shown that the occurrence of enucleate basidiospores is negligible. In fact, basidiospores without a nucleus were not observed. Previous reports suggested that a great proportion of Pisolithus spp. basidiospores were enucleate (Mims, 1980; Costa, personal communication), but this might have been due to limitations of the microscopy techniques used, since nuclei are generally pressed against the spore cell wall due to the accumulation of lipid bodies inside the basidiospores, making their visualization very difficult (Mims, 1980; Campos et al., 2008). Also, the low permeability of the basidiospore cell wall due to the deposition of different cell wall layers and hydrophobic proteins usually prevent stains from diffusing into the spore cytoplasm, hindering the observation of nuclei, especially in mature basidiospores (McLaughlin, 1982). Visualization of nuclei at this stage is only possible after the chemical scarification of the cell wall before staining procedures (Verrinder-Gibbins and Lu, 1982), but homogeneity by this chemical treatment is difficult to obtain.
Basidia containing less than eight basidiospores were also observed (Fig. 3m). In such cases, the nuclei in excess of the number of basidiospores remained in the basidium during nuclear migration (Fig. 3n). Similar events were reported for C. cibarius (Duncan and Galbraith, 1972), a species that shows the same pattern of post-meiotic mitosis as P. microcarpus. The nuclei remaining in the basidium are reported to degenerate (Duncan and Galbraith, 1972). Though rarely observed, binucleate basidiospores were also observed (Fig. 3n) and could possibly result from the aberrant migration of two haploid nuclei to a single basidiospore or, alternatively, from the absence of nuclear disjunction at the end of post-meiotic mitosis. Production of binucleate basidiospores, having two genetically different nuclei, is suggested as a strategy to long distance dispersion in some basidiomycetes (Horton, 2006). Since post-meiotic mitosis in P. microcarpus occurs inside basidia, two genetically different nuclei might potentially migrate to single spore originating a binucleate basidiospore that could give rise to a dikaryotic mycelium without requiring plasmogamy. Although tempting, this hypothesis needs experimental confirmation. In P. microcarpus, karyogamy occurs not only in the basidium, but also in undifferentiated sub-apical or sub-basidial cells (Fig. 3o and p). When this is the case, once formed, the diploid nuclei migrate from the sub-apical or sub-basidial cell to a newly developed lateral basidium (Fig. 3q) and, then, undergo the usual steps of meiosis and post-meiotic mitosis. This seems to be a particular characteristic of P. microcarpus, since no reports of sub-apical and sub-basidial karyogamy could be found for other fungi, including other gasteromycetes. Besides, in holobasidiomycetes, karyogamy is known to occur only inside the basidia, while in heterobasidiomycetes, it is reported to take place in the probasidia (Mims et al., 1987; Reijnders, 2000).
Fig. 4. Schematic representation of meiosis and post-meiotic mitosis during basidiosporogenesis in Pisolithus microcarpus, showing the approximate location of the nuclear events inside the fungal basidiocarp. Bars: a and e = 200 lm; b = 1 mm; c and d = 500 lm.
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