Mycologia, 95(2), 2003, pp. 232–238. q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897
Stereum sanguinolentum: Is it an amphithallic basidiomycete?
M. Calderoni T. N. Sieber1 O. Holdenrieder
Key words: meiosis, parasexuality, vegetative compatibility groups (VCGs)
Swiss Federal Institute of Technology, Department of Forest Sciences, Forest Pathology and Dendrology, ETH- Zentrum, CH-8092 Zurich, Switzerland
INTRODUCTION
Our knowledge of the sexuality of the basidiomycete Stereum sanguinolentum (Alb. & Schwein. : Fr.) Fr. (Stereaceae) is incomplete (Piazzi et al 1998, Rayner and Turton 1982, Robak 1942, Vasiliauskas et al 1996). There exists no mycelial dimorphism between supposed homo- and heterokaryons, i.e., mycelial morphology of monospore, polyspore and trama isolates is identical on the micro- and macroscopic level (Robak 1942). Hyphal cells are multinucleate, and clamp connections are produced sporadically. Monospore isolates from the same basidiocarp are compatible with each other in all possible combinations (Piazzi et al 1998, Rayner and Turton 1982), i.e., either the mating type genes are absent or non functioning or the basidiospores contain both complementary sets of mating type genes. Rayner and Turton (1982) concluded that S. sanguinolentum was best regarded as amictic or apomictic. Robak (1942) came to a similar conclusion based on his studies of nuclear behavior in the basidia. He considered spores to form parthenogenetically without any preceding karyogamic process because young basidia were mostly monokaryotic. Robak (1942) observed only a few cases of karyogamy and regarded them as fusions between sister nuclei and, consequently, the resulting spores as products of homothallism. The conception of S. sanguinolentum to be either apomictic (5 parthenogenetic) or homothallic, however, is hardly coherent with the high number of sympatric and allopatric vegetative compatibility groups (VCGs) (Piazzi et al 1998, Rayner and Turton 1982, Vasiliauskas et al 1996). We postulate, therefore, that sexual or parasexual recombination must occur (or at least must have occurred in the past) to generate this high diversity. In this study, we tried to find clues to whether meiosis really is the exception in S. sanguinolentum, as suggested by Robak (1942). The nuclear behavior in basidia of a series of basidiocarps originating from different hosts and places were examined. The frequency and distribution of various meiotic stages and of the number of nuclei per basidiospore were given special attention.
Abstract: Monobasidiospore isolates were prepared from basidiocarps of Stereum sanguinolentum. Five isolates per basidiome were paired with each other and with isolates from the trama. Interbasidiome pairings of the trama isolates and of a selection of single-spore isolates also were performed. Thin sections of the hymenium were stained with DAPI and examined by fluorescence microscopy to study the nuclei in the basidia. Spore prints were stained with DAPI to count the number of nuclei per spore. SEM was used to determine the number of basidiospores per basidium. All intrabasidiome pairings were compatible. In contrast, interbasidiome pairings, except one, were incompatible, independent of whether single-spore or trama isolates were paired. Fertile basidiomes were formed in single-basidiospore cultures. Basidia were regularly four-spored. On average, 5% of the basidiospores possessed one nucleus, 82% two, 2% three and 1% four nuclei. Ten percent of the spores appeared to be empty. Karyogamy, meiosis and postmeiotic mitosis were observed in the basidia. Nuclei resulting directly from meiosis, i.e., without having undergone postmeiotic mitosis, sometimes were observed in the sterigmata or spore primordia. The high number of vegetative compatibility groups (VCG) of S. sanguinolentum observed in this study and earlier studies is difficult to explain without sexual or parasexual recombination. We suppose that the majority of spores with $2 nuclei are amphithallic, possessing at least one nucleus of each mating type. Recombination could occur by exchange of nuclei among VCGs via anastomoses between homothallic compartments. Transfer of nuclei from heterothallic to homothallic mycelia or matings between homothallic mycelia, which originate from monokaryotic spores, might be other paths for gene exchange. Accepted for publication August 30, 2002. 1 Corresponding author. E-mail:
[email protected]
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MATERIALS AND METHODS
Basidiocarps of S. sanguinolentum were collected from logs of Abies alba, Picea abies, Larix decidua and Pinus sylvestris in Nov and Dec 2000 in forests at the eastern shore of Lake Zurich within an area of approximately 35 km2. Two or three basidiocarps (n 5 21) were excised from each of two logs per host species. Minimal distance between the logs was 400 m. Basidiocarps were stored in sterile Petri dishes at 4 C and processed within 24 h. Monospore and trama isolates. Basidiomes were mounted 10 cm above the surface of cornmeal agar (CMA) (17 g L21 CMA, Difco Laboratories, Detroit, MI) in 9-cm-diam Petri dishes for 5–10 min under humid conditions. The dishes were examined daily under a dissecting microscope with transmission light at a magnification of up to 1003. Most basidiospores germinated within two days. At least 10 germinated spores per basidiome were transferred to thiabendazol agar {TA; 2% (w/v) malt extract agar (MEA; 20 g L21 malt extract, 15 g L21 agar) containing 230 mg L21 thiabendazol [2-(1,3 thiazol-4-yl)-benzimidazol] (Sieber 1995)} to inhibit nonbasidiomycetes. Mycelium was subcultured on MEA as soon as isolates showed macroscopically visible growth to prepare stock cultures. Similarly, small pieces of trama also were excised from each basidiocarp and incubated first on TA and subcultured later on MEA. Monospore pairings and vegetative compatibility (vc) tests. At least five monospore isolates per basidiocarp were paired in all possible combinations. Interbasidiome pairings of monospore X monospore isolates and intrabasidiome monospore X trama isolate pairings also were performed. In addition, all trama isolates were paired with each other. All pairing experiments were performed on 2% (w/v) MEA in 9-cm-diam Petri dishes. The inoculi consisted in 4 3 4 3 4 mm cubes taken from the margins of actively growing colonies on MEA. The distance between the inoculi was 2 cm for monospore pairings or pairings between monospore and trama isolates and 3 cm for pairings of trama isolates (vc-tests). Incubation occurred at room temperature (22 6 1 C) in the dark, and the pairings were evaluated after 20 d. A comparatively long incubation period was chosen because the demarcation line between incompatible isolates becomes more pronounced the longer the incubation lasts. Presence of a distinct demarcation line (‘‘barrage’’) between the colonies of two isolates was indicative of incompatibility, and absence of the same indicated compatibility. Compatible and incompatible reactions also were studied microscopically. Pairings were performed on glass slides in humid chambers at 20 C in the dark, using inoculi that consisted in cylinders of 6 mm diam and 4 mm height extracted from the margin of actively growing colonies on MEA. The inoculi were placed approximately 4 mm apart. Colonies were allowed to grow for several days after initial contact between the colonies was established to allow time for maturation of possible differentiated contact zone morphology (Petersen and Halling 1993). Basidiocarp development in monospore cultures. Two glass beakers (1 L) were filled with wood chips of Norway spruce, covered with aluminum foil and autoclaved twice at a time
interval of 48 h. Three hundred ml of hot, autoclaved MEA was poured uniformly over the chips in each beaker under aseptic conditions. The next day, four 5 3 5 3 5 mm cubes, taken from the margin of an actively growing colony (MEA) of a monospore isolate of a basidiocarp collected from Norway spruce, were placed in each of the two beakers. Incubation was at 22 6 1 C under uncontrolled light in the laboratory for the first five months and then at 4 C in the dark. Colonies were checked monthly for the formation of basidiocarps. Number of basidiospores per basidium and nuclear behavior in the basidia. Thin sections were prepared and examined under epifluorescence to study the meiotic and/or mitotic stages in the basidia. Scanning electron microscopy (SEM) of the hymenial surface served to study the average number of mature basidia per unit area and the average number of basidiospores per basidium. Several pieces of maximally 7 3 4 3 2 mm (length 3 width 3 thickness) were cut from each basidiocarp, dehydrated in a graded ethanol series (50%, 70%, 90% and 96% ethanol, $4 h per concentration) and stored in 96% ethanol at 4 C until further preparation for thin-sectioning or SEM. The samples for thin-sectioning were embedded in Technovit 7100, a water-soluble glycol-methacrylate-based resin, according to the manufacturers description (Kulzer Heraeus GmbH, Germany). The resin blocks were sectioned at a thickness of 4–6 mm using a rotation microtome ( Jung 2065 Supercut, Leica Instruments GmbH, Germany) equipped with a metal knife (cut D) fixed at an angle of 208 and examined under epifluorescence as described below. Tissues for SEM were transferred to 100% acetone after dehydration, stored for at least 24 h at 4 C and critical-point dried (CPD 030, Bal-tec, Liechtenstein). Specimens were mounted, gold coated at 25 mA and 160 V with the vacuum adjusted to 0.1 mbar (MED 020, Bal-tec, Liechtenstein) and examined with a Leo 435 VP SEM (Leo Elektronenmikroskopie GmbH, Germany). Number of nuclei per basidiospore. Basidiomes were mounted 1 cm above microscopic slides for up to 8 h under humid conditions to collect basidiospores. No microscopically visible basidiome or basidial aberrations were observed even after 8 h of spore collection. A 0.5% (w/v) solution of DAPI (4,6-diamidino-2-phenylindole) in distilled water was mixed at a ratio of 4:6 with McIlvaine buffer of pH 7 (1:1 mixture of 0.2 M Na2HPO4 and 0.1 M citric acid) and used to stain the nuclei (Campbell and Duffus 1988). One drop of this staining solution was applied to the spore print, which then was covered with a coverslip and examined immediately with a Zeiss Axiophot microscope equipped for epifluorescence with an HBO 50W/AC mercury lamp. Filters and reflectors were a 365-nm excitation filter, a 395-nm chromatic beam splitter and a 420-nm barrier filter.
RESULTS
Monospore pairings and vegetative compatibility (vc) tests. Interbasidiome pairings of monospore X mon-
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ospore and trama X trama isolates were incompatible, except for the pairings between isolates from two basidiomes, which grew adjacent to each other on the same log. This contrasts with intrabasidiome pairings of monospore X monospore and monospore X trama isolates which always were compatible. Differentiation between compatible and incompatible interactions on the macroscopic level was always clear-cut without ambiguity. Distinct demarcation lines formed in all incompatible interactions. These lines were characterized by an up to 3 mm wide zone without any aerial mycelium and ‘‘walls’’ of dense white to cream-colored aerial mycelium on both sides of that zone. The agar in this zone was dark brown and contained an accumulation of intensively interwoven substrate hyphae. Compatibility was inferred when the crosses did not exhibit a differentiated contact zone morphology. The contact zone of compatible crosses appeared identical to all other areas of individual monospore colonies. Differentiation between compatible and incompatible interactions on the microscopic level was difficult because the mycelium of both monospore and trama isolates was composed of four to nine nucleate cells (DAPI stain), and clamp connections were present, though rarely, on mycelia of either origin. The mycelium in the interaction zone on MEA was too dense to allow interpretation of microscopic mounts. However, anastomoses could be observed with a microscope between mycelia of compatible isolates when pairings were performed directly on glass slides. Basidiocarp development in monospore cultures. Fertile basidiocarps were observed after one year of incubation at 4 C. The hymenial structures formed in culture corresponded to those formed in nature. Sterile elements, such as acanthohyphidia, conducting elements and pseudocystidia, were abundant, whereas basidia and consequently spores were observed rarely. Number of basidiospores per basidium and nuclear behavior in basidia. No basidia were formed at the basidiocarp margin and within the first few mm from it (FIG. 1). The frequency of basidia in the more central parts of the hymenium was variable. In some areas, up to 35 mature basidia were counted within a square of 0.01 mm2 (100 3 100 mm) (FIG. 2), in others only a few or no basidia were detected. Basidia regularly produced four sterigmata on which four basidiospores were formed (FIG. 3). In addition to the basidia, sterile elements, such as cells with a central mammiform protuberance, acanthohyphidia (30–45 3 4 mm) (FIG. 4) and pseudocystidia (90–150 3 10 mm), were present. The observed nuclear stages in the basidia clearly
could be interpreted as karyogamy and meiosis. The basidium is a chiastobasidium with both meiotic divisions occurring at the tip of the basidium and the nuclear spindle apparatus of at least the first meiotic division forming across the basidium (see below). In active hymenia, approximately 30% of the basidia were in prekaryogamy and contained constantly two nuclei, which were more or less longitudinally aligned in the middle of the basidium (FIGS. 5 and 6). More than one third (36%) of the basidia were in Prophase I (FIGS. 5 and 7). Fewer were in another meiotic stage. Basidia initially were cylindrical to subclavate but became wider and more markedly clavate during karyogamy and Prophase I and reached their final size at the end of Prophase I (though without sterigmata). The two prekaryogamy nuclei fused to form the zygote. Prophase I started immediately after karyogamy. The volume of Prophase I nuclei was up to 20 times greater than that of prekaryogamy nuclei and the fluorescence was strongly reduced (FIG. 7). Nuclei in meta-Anaphase I were located in the apex of the basidium, and in Telophase I two daughter nuclei lay separately on opposite sides of the basidium, indicating that the microtubules attached to the spindle poles separated the chromosomes perpendicularly to the long axis of the basidium (FIG. 8). During Interphase I the nuclei remained in the apical portion of the basidium. In early metaphase II, nuclei were in the same position as in telo- or Interphase I but often appeared falcate, an effect of the chromatin being condensed in the middle of the spindles. Later, the spindles elongated parallel to the long axis of the basidium to divide the nuclei (Anaphase II). Telophase II nuclei were highly condensed initially but relaxed somewhat and descended a little bit toward the center of the basidium (FIG. 9). Sterigmata and basidiospore initials formed simultaneously with the meiotic tetrad. Meiosis was followed by a mitosis (FIG. 10). Usually, two nuclei migrated into each basidiospore. It was impossible, however, to establish whether the two nuclei originated from the same or one each of the two progenitor nuclei. Occasionally, nuclei resulting from the second meiotic division, i. e., nuclei that had not undergone a postmeiotic mitosis, were observed in the sterigmata or spore primordia (FIGS. 11 and 12). Number of nuclei per basidiospore. Most basidiospores possessed two nuclei (FIG. 13, TABLE I). Depending on the basidiocarp between 1% and 10% had only one nucleus, between 0% and 4% three nuclei and between 0% and 3% four nuclei. The proportion of empty or apparently empty spores was considerable in some spore prints. In addition, spores containing
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FIGS. 1–4. Scanning electron micrographs of the basidiocarp structures of Stereum sanguinolentum. 1. Sterile hyphae at the margin of a basidiocarp. Bar 5 10 mm. 2. Central part (approx. 100 3 100 mm) of a mature hymenium showing a high number of regularly four-spored basidia. Bar 5 10 mm. 3. Close-up of two basidia with four basidiospores each. Bar 5 3 mm. 4. Acanthohyphidia (arrowheads) and developing basidia and basidiospores. Bar 5 3 mm.
amorphous DNA instead of clearly delimited nuclei also were observed frequently in some spore prints. DISCUSSION
FIG. 5. Frequency distribution of meiotic stages in the basidia. PK, prekaryogamy (dikaryon); K, karyogamy; PI, Prophase I; MTI, meta-, ana-, Telophase I; INI, Interphase I; MTII, meta-, ana-, Telophase II; MIT, eight nuclei in the basidia; B, two nuclei in each basidiospore, basidia empty.
The inter- and intrabasidiome pairings confirmed earlier findings (Piazzi et al 1998, Rayner and Turton 1982, Vasiliauskas et al 1996). Monospore intrabasidiome pairings always were compatible. In contrast, interbasidiome monospore X monospore and trama X trama crosses were incompatible, with the exception of crosses between isolates from two basidiocarps growing adjacently on the same log. Our observations of the behavior of the nuclei in the basidia of S. sanguinolentum deviate in some respects from those made by Robak (1942). Young basidia usually contained only one nucleus in Robak’s (1942) study, whereas they were constantly dikaryotic in this study. Correspondingly, Robak (1942) detect-
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FIGS. 6–12. Epifluorescence microscopy of DAPI-stained sections (FIG. 12 differential interference contrast, DIC) depicting various meiotic stages in the basidia. 6. Dikaryon (prekaryogamy); 7. Prophase I; 8. Telophase I. 9. Telophase II. 10. Basidium with eight nuclei after postmeiotic mitosis. 11. Migration of nuclei through the sterigmata. Postmeiotic mitosis did not take place in this basidium. Only three of the four nuclei are visible, two of which are migrating through the sterigmata. 12. Same basidium as in FIG. 11 but picture taken with DIC optics. 6–12. Bar (in FIG. 6) 5 10 mm.
ed only a few ‘‘great fusion nuclei’’. These probably correspond to Prophase I nuclei, which frequently were found in this study (FIGS. 5 and 6). Moreover, Robak (1942) never observed an eight-nucleate stage in the basidia. The basidia in his study had maximally four nuclei, which migrated directly into the spores where an additional mitosis occurred. We found postmeiotic mitosis to occur primarily before migration. Consequently, Robak (1942) interpreted what we consider to be meiosis as two subsequent mitoses. The differences in regard to the behavior of the nu-
clei in the basidia between our and Robak’s (1942) observations might be due to the fact that we did not examine the collections studied by Robak. It is possible that the nuclear behavior in Swiss collections deviates from that in Norwegian collections. Karyogamy and meiosis seem to be the rule, not the exception, although definite proof could not be produced, i.e., the halving of the zygotic chromosome number during meiotic division was not demonstrated. Presence of karyogamy and meiosis is further supported by the frequency distribution of the
FIG. 13. Epifluorescence micrograph of DAPI-stained spores of Stereum sanguinolentum depicting spores with one, two, three or four nuclei. Bar 5 10 mm.
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Frequency distribution (%) of the number of nuclei per basidiospore Basidiocarp (Host and no.)
Number of nuclei
Spruce 1 n 5 92a
Spruce 2 n 5 220
Larch 1 n 5 384
Larch 2 n 5 258
Larch 3 n 5 326
Pine 1 n 5 75
Pine 2 n 5 105
Pine 3 n 5 167
Mean n 5 1627
Empty 1 2 3 4
4 10 81 4 2
5 5 89 2 0
5 7 84 3 1
6 7 81 3 3
23 1 75 1 0
40 7 53 0 0
2 10 82 4 3
2 1 96 1 0
10 5 82 2 1
a
Number of spores examined.
different meiotic stages. The frequencies correspond surprisingly well with those observed by Berbee and Wells (1989) for meiosis in the basidiomycete Clavicorona pyxidata. The formation of four basidiospores on each basidium represents another piece of evidence for the occurrence of meiosis. Conjugation of sexually complementary thalli (‘‘heterothallism’’ in the sense of Blakeslee (1904)) is obviously not necessary for the development of fertile basidiomes, because these were formed in single-basidiospore cultures. Based on our results regarding the nuclear behavior in the basidia, S. sanguinolentum cannot be considered apomictic or amictic, as suggested by Rayner and Turton (1982). S. sanguinolentum is either a homothallic or amphithallic species. The high diversity of sympatric VCGs and, according to Petersen and Halling (1993), the mostly binucleate basidiospores make homothallism improbable. It might be best to regard S. sanguinolentum as amphithallic for several reasons. Monobasidiospore and trama isolates are plurinucleate and bear clamp connections, though these are rare, independent of the origin of the isolate, and basidiospores most frequently are dikaryotic. Moreover, monospore isolates from the same basidiocarp are compatible with each other in every possible combination. This suggests that basidiospores are heterodikaryotic, which would indicate a form of directed amphithallism (Petersen 1995). Likewise, the basidiomycete Oudemansiella canarii with a very similar behavior was considered amphithallic (Petersen and Halling 1993). Mycelia growing from heterodikaryotic spores consequently are heterokaryotic. Recombination occurs either by matings between homokaryons originating from monokaryotic basidiospores (TABLE I) or by parasexual processes (Anderson and Kohn 1998). A very interesting possibility for nuclear reassortment could be the fusion between homokaryotic hyphae, which might be formed in gaps between incompatible heterokaryons. This hypothesis was forwarded by Hansen et al (1993) who found single hyphal tip isolates from the gap between incompatible heterokaryons of Heterobasidion annosum to be somatically in-
compatible with either progenitor. A similar phenomenon also was observed in a Picea-abies stump naturally colonized by various genets of H. annosum (Swedjemark and Stenlid 2001). ‘‘New’’ genets were detected inside the stump in the interaction zone between two other genets. S. sanguinolentum is a very effective and fast colonizer of newly dead or wounded conifer sapwood. Amphithallism probably confers a selective advantage upon such organisms by enhancing their survival and dispersal (Petersen and Halling 1993). Dispersal occurs solely by basidiospores. Spores of various VCG groups are deposited on fresh wounds, where they germinate and establish small thalli. The dynamics of competition and turnover among adjacent individuals, which modify the initial pattern of colonization, are not well known, but we suggest that nuclear reassortment and, thus, the formation of new VCGs occur exactly during these processes. It will be a challenge to study the mating behavior of this fungus in more detail. ACKNOWLEDGMENTS
We are grateful to Mrs. Grazyna-Maria Gantenbein, ETH Zu¨rich, for her help and support with the SEM study and to Ms. Karin Langenegger, ETH Zu¨rich, for excellent technical assistance.
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