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INRA, Unité de Recherches sur les Champignons BP 81, 33883 Villenave d'Ornon cedex, France
Christophe Desmerger
Centre Technique du Champignon, Munet, 49400 Distré, France
Ioanna Theochari 2
NAGREF, Laboratory of Edible Fungi, 41110 Larissa, Greece
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Among 400 wild specimens of A. bisporus collected in Europe, only three were tetrasporic. In the case of two of them from France, a previous study showed that one was homokaryotic and hypothetically belonged to a homothallic entity while the other was heterokaryotic and possibly resulted from hybridization between a member of this entity and a classical bisporic strain. A third tetrasporic specimen recently was discovered in Greece. Morphological and genetic comparisons, using alloenzymatic markers, molecular markers and ITS polymorphisms, reveal that this third specimen is homokaryotic and belongs, with the homokaryotic specimen from France, to the same entity. Dissimilarity analysis confirms the hybrid origin of the heterokaryotic specimen. Varietal status is proposed for this homothallic, highly homogeneous entity, and A. bisporus var. eurotetrasporus is described. This novel variety clearly differs from var. bisporus by its tetrasporic basidia and from var. burnettii by its longer spores. It has a complex story because it can interbreed with var. bisporus and shares the same habitat; however, because of its homothallic life cycle and its partial intersterility, it is probably in the process of speciation.
Key words: Agaricus bisporus var. eurotetrasporus, basidial spore number variation, cultivated mushroom, evolution, homothallism, ITS polymorphism
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INTRODUCTION |
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). Ploidy level refers here to the number of sorts of genetically different nuclei per cell. This number, which can be one or two respectively in homokaryotic and heterokaryotic cells, might not equal the number of nuclei per cell. In Agaricus bisporus var. burnettii Kerrigan and Callac (Callac et al 1993), described for specimens from the Sonoran Desert of California, the life cycle is predominantly heterothallic (Kerrigan et al 1994); most of the basidia are tetrasporic and produce homokaryotic spores that give rise to homokaryotic mycelia. Plasmogamy between two sexually compatible and generally infertile homokaryons restores a fertile heterokaryon. In contrast, the other known wild populations, as well as the cultivars, belong to the predominantly pseudohomothallic (secondary homothallic; Raper et al 1972) A. bisporus var. bisporus; most of the basidia are bisporic and produce spores that usually receive two non-sister postmeiotic nuclei carrying different mating type alleles that give rise to fertile heterokaryons.
The collection at INRA-Bordeaux comprises isolates from about 400 wild European specimens of A. bisporus. Among about 250 isolates from France, only two, Bs 261 and Bs 423, exhibited a tetrasporic phenotype with less than 5% bisporic basidia (Callac et al 1994, 1996, Guinberteau et al 1998). For these strains and the tetrasporic strains of var. burnettii, we have shown that this trait is the result of a dominant Bsn-t allele at the BSN (basidial spore number) locus (Imbernon et al 1996, Callac et al 1998). We also found that Bs 423 was homokaryotic, while Bs 261 was heterokaryotic, with one of its nuclei having the same haploid genotype as Bs 423 and bearing a Bsn-t allele, while the second nucleus had a different genotype and harbored a bisporic recessive allele Bsn-b. These results suggested that Bs 423 could belong to a homokaryotic, tetrasporic, homothallic entity, while Bs 261 could result from a natural hybridization between a member of this genet and a homokaryon from A. bisporus var. bisporus. The homokaryotic status of Bs 423, deduced from its homoallelic genotype, and the fertility of its spores were consistent with a homothallic (or primarily homothallic, Hawksworth et al 1995) life cycle. "Homothallic" is used in a broad sense because we do not know if a homomictic process (meiosis in haploid thalli) occurs in the basidia or if it is an amictic (mitotic) process conferring, in fact, an asexual life cycle.
Among about 100 isolates from Greece, a third tetrasporic strain, Bs 514, was found. Preliminary studies using only two allozyme markers suggested that Bs 514 might be homokaryotic, with the same genotype as Bs 423 (Callac et al 2000). In this paper, we report the comprehensive characterization of European tetrasporic strains based on their basidial spore number, spore size, gross morphology, mycelial growth rate and 21 genotypic loci, including five ITS (internal transcribed spacer) polymorphisms. These studies confirm the existence of a novel homothallic variety that we described below as A. bisporus var. eurotetrasporus.
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MATERIALS AND METHODS |
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, 2000). To determine the genotype of the nuclei (A and B) within the Bs 261 heterokaryon, we previously tried to isolate homokaryons of each type by using the protoplast method described by Kerrigan et al (1994). Unfortunately, only one type was recovered; therefore a single homokaryon, Bs 261–150, was studied and its haploid genotype was designated the genotype of the nucleus A of Bs 261 (Callac et al 1998).
The homokaryons JB 3–83, U 1–7 and PS-1, were used as reference strains for genotypic analysis. JB 3–83 is a single-spore isolate from the wild isolate JB 3 belonging to A. bisporus var. burnettii. U 1–7 and PS-1 respectively were recovered by protoplasting the cultivar U 1 and a wild isolate PS from France, both belonging to A. bisporus var. bisporus. JB 3–83 and U 1–7 have been used previously (Kerrigan et al 1994). JB 3, U 1–7, Bs 261, Bs 423 and Bs 514 have been deposited at ATCC (respective accession numbers ATCC 200853, ATCC 96326, MY-MYA-2385, MY-MYA-2386 and MY-MYA-2389).
Fructification – Fruiting was performed in plastic trays containing 500 g of commercial compost that was inoculated with cultures from four Petri dishes of compost-extract agar. Fruiting was conducted at 16 C in a controlled environment. Cultivated specimens were used for micro- and macromorphological observations.
Basidial spore number and spore size –
Counts of two-, three-, and four-spored basidia were conducted with light microscopy (Callac et al 1993). At least 400 basidia were observed for each strain (100 on each of two distinct lamellae for two different sporocarps). The average spore number per basidium (ASN), which can vary from two to four, was calculated. Spore size was determined by light microscopy; spores from recent spore prints were measured in 3% KOH with an ocular micrometer and 100x oil-immersion objective.
Mycelial growth rate test –
In an earlier study, 128 SSIs (single spore isolates) of Bs 261 and 136 SSIs of Bs 423 were obtained from spore germination rates of about 12% and 10% respectively (Callac et al 1998). Two random samples of 40 SSIs of each offspring were used for the mycelial growth-rate test. Among those from Bs 261, 39 were homokaryotic and the remaining one was heterokaryotic. Colony diameters were determined after 14 d growth on a malt medium (2% agar, 1.5% Cristomalt from Société Difal) with two replicates.
Allozyme markers –
Three allozyme markers, EST1, PGM and ßGLU, were characterized with PAGE or IEF, according to the respective methods of Roux and Labarère (1990), Cameleyre and Olivier (1993) and Kerrigan and Ross (1989). Allelic nomenclature and relative mobilities of the allelic products, or their isoelectric points, have been reported by Kerrigan et al (1996). EST1 and PGM were characterized at INRA with a PhastSystem (Pharmacia) apparatus (Callac et al 1993).
SCAR markers –
Twelve sequence-characterized amplified-region (SCAR; Paran and Michelmore 1993) markers were analyzed. DNA extraction was made with the RPN8510 Nucleon Phytopure plant DNA extraction kit (Amersham Pharmacia Biotech) and PCR reactions using two primers were performed as previously described (Imbernon et al 1996). After digestion by restriction endonuclease, restriction fragments were separated by electrophoresis in 1.2% w/v agarose. The PR3 marker was derived from the sequence of an RFLP probe and detected using the primer sequences (1)5'-GGAGCATCATCAGGACTTGG and (2)5'-CGCCACATGTTTCCCTTCAAT. After digestion by HaeIII, we identified alleles Pr3–1 and Pr3–2 giving bands at approximately 770 bp and 380 bp, and at approximately 1150 bp, respectively. For PR19, the pair of primer sequences are (1)5'-GTGTGCTCATACCTGCCAAC and (2) 5'-CGAACTTTCTTCAACCAGTG. After digestion by HaeIII, we identified the alleles Pr19–1 and Pr19–2 giving bands at approximately 900 bp, 400 bp and 200 bp, and at approximately 1100 bp and 400 bp, respectively. For the ten remaining SCARs, the primer sequences, the restriction endonucleases used, and the identification of the codominants alleles 1 and 2 have been described by Callac et al (1997) or Moquet et al (1999); the Pr5–5, Pr5–7, and Pr6–4 alleles have been described by Callac et al (1998).
Sequencing –
To characterize the tetrasporic strains using ITS1 and ITS2 regions of the nuclear rDNA, a single product was amplified using the PN3 (5'-CCGTTGGTGAACCAGCGGAGGGATC) and PN34 (5'-TTGCCGCTTCACTCGCCGTT) primers (Rafin et al 1995). PCR products were directly sequenced with big dye-terminator chemistry on ABI Prism (Applied Biosystems) DNA sequencers. Alignments were manually performed and revealed only five polymorphisms in ITS1 and ITS2 regions. The region used in comparisons started 5'-TTGAATTATG, finished 5’-AAAGAAACTA, and spanned a maximum length of 719 bases that included the ITS1 (1 to 290), the 5.8S rDNA gene (291 to 444), the ITS2 (445 to 652), and a part of the 28S rDNA gene (653 to 719). For several strains the beginning of the ITS1 sequence (19 bases maximum) was missing. For each strain, the GenBank accession number, the sequenced region and the region confirmed by double-stranded sequence (in brackets) follow: PS-1 (AF465402): 16–719 [87–719]; JB 3–83 (AF465401): 1–719 [100–683]; Bs 423 (AF465400): 11–719 [79–678]; Bs 514 (AF465399): 1–719 [25–700]; Bs 261–150 (AF465403): 16–719 [24–678]; Bs 261(AF465404): 19–719 [95–114]. ITS1 sequences for PS-1, JB3–83, Bs 423 and Bs 514 were confirmed against earlier data (Challen et al 2002) for the convarietal isolate RWK1737 (var. bisporus), the parental isolate JB 3, Bs 423 and Bs 514.
Dissimilarity analysis –
To confirm the origin of the two Bs 261 nuclear components, a dissimilarity analysis using Nei's distance (Nei 1972) was performed on polymorphisms for 31 loci from eight haploid genotypes comprising the two nuclei A and B of Bs 261 separately considered, the two European tetrasporic isolates Bs 423 and Bs 514, the three reference strains (JB3–83, U1–7, PS-1), and, as presumed outgroup, the homothallic isolate RWK 1441 belonging to A. subfloccosus (see Kerrigan et al 1999). The ITS sequence of RWK 1441 is available in GenBank (AF432887, R.W. Kerrigan) and ten ITS polymorphisms discriminated RWK 1441 from all the A. bisporus genotypes analyzed. The remaining 21 loci comprised three allozyme loci, 12 SCAR loci, the BSN locus, and five ITS polymorphisms within the A. bisporus sample. The matrix of dissimilarity values was evaluated with the Fitch-Margoliash algorithm as implemented in the FITCH program, and the phenogram was produced by the DRAWGRAM program of the PHYLIP software package (Felsenstein 1993).
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RESULTS |
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). Because the Bsn-t allele exists in the two first tetrasporic isolates found in Europe and because the third tetrasporic isolate recently found, Bs 514, is genetically related to the two others (see below), we infer that the Bs 514 isolate probably also has a Bsn-t allele.
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, which was based on the ITS1 sequence. The polymorphisms and the corresponding allele nomenclature are given in Table III. The single ITS(102)-2 allele corresponds to a T deletion within a tract of five Ts. All of the genotypes at these ITS loci were confirmed by second-strand data. However, in the case of the Bs 261 heterokaryon, the sequence of each strand was affected at the position of the heterogeneous insertion/deletion; each strand clearly could be read to the indel at Position 102, beyond which a double sequence could be deciphered, comprising two identical sequences with one shifted -1 position (due to a deleted T). This interpretation clearly agreed with an ITS(102)-1/2 heteroallelic ITS genotype. Because Bs 261-150 (nucleus A) lacked this deletion, we concluded that the B nucleus had it. Moreover, Bs 261 was heteroallelic at Position 628, with a 5'-taatcRtctaa sequence.
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TAXONOMY |
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. Therefore, and because we have found a natural intervarietal hybrid, the homothallic entity is not so divergent as to represent a new species.
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Pileus: 4–7 (8.5) cm broad at maturity, generally greater that the total height of the sporocarp, at first broadly convex with inrolled margin, then expanding to plane or subplane at maturity with exceeding margin, sometimes peripherally flexuose and generally slightly depressed at the disc. Surface dry, lustrous, appressed-fibrillose, then, at maturity, with distinct and darker appressed squamae (2 x 5 mm). Suprapellis cream to light brown (hazel), generally paler to whitish in the peripheral zone (
to 
of the radius) giving a bicolored general aspect to the pileus; context 6–13 mm, whitish, quickly turning orange-sooty, then vinaceous when cut, odor pleasant, like the type, or slightly stronger, evoking a Scleroderma.
Lamellae: free, close (ca 16/cm at 1 cm from the stipe), to 3–5 mm broad, dull pinkish in youth, becoming dark brown chocolate, turning vinaceous when creased.
Stipe: 3–6 cm long x 7–12 mm broad, cylindrical, slightly bulbous at the base with a diameter abruptly increasing of 1 to 3 mm. Surface pale gray-rosy and covered, mainly under the ring, by appressed whitish and fibrillose scales arranged in overlapping transverse rows.
Veils: universal veil persisting as appressed whitish patches on the pilei-pellis; small, delicate submedian annulus, poorly joined to the stem, sometimes remaining attached to the pileus margin.
Spores: dark brown, broadly ellipsoid to ellipsoid [Q = (1.2–) 1.36–1.41–1.45 (–1.7)], (5.7–) 6.3–7.5 (–8.5) x (4–) 4.5–5.3 (–6) µm, the mean 6.9 x 4.9 µm, two collections, N = 120 for each. Basidia cylindro-clavate, 18–27 x 6–7 µm, predominately tetrasterigmate, the sterigmata acute, ca 3 µm long. Cheilocystidia 27–37 x 7–12 µm, polymorphic, cylindro-clavate, sublageniform; lamellar margin sterile.
Culture: Behaves comparably to var. bisporus. On commercially prepared compost under standard conditions, timing of fructification is normal and yield is similar to certain wild isolates of var. bisporus but is 20% to 40% lower than commercial strains.
Sexuality: Homothallic. Mycelia issued from single spores are homokaryotic, fertile and produce sporocarps morphologically similar to the parental ones (Fig. 7).
Characteristic internal transcriber spacer polymorphisms –
‘tctgatgt’ (positions 607–614 in the present study) characterizes the species A. bisporus within the section Duploannulatae Wasser ex Wasser emend. Kerrigan, Challen, & Callac (Challen et al 2002; corresponding polymorphism ‘tctg[-2-]atgt @637–638’); ‘tcttt-tcagg’ or ITS(102)-2 allele characterizes A. bisporus var. eurotetrasporus (see also Challen et al 2002; corresponding polymorphism ‘cttt[-3-]tcag @118’); ‘taatcAtctaa’ or ITS(628)-2 allele characterizes the tetrasporic isolates Bs 423 and Bs 261 (nucleus B, inferred, only) from France.
Habitat, distribution, occurrence – Under C. macrocarpa or C. sempervirens, habitat shared with var. bisporus, Europe (France and Greece), extent of range unknown, rare.
Specimens examined.—Material cultivated at INRA, Villenave d'Ornon, France, 28 May 2001, from a tissue culture of the wild specimen collected at Olonne-sur-mer, Vendée, FRANCE, Atlantic coast, under Cupressus macrocarpa, 22 Nov 1994, P. Callac and J. Guinberteau, Bs 423 (HOLOTYPE. LIP. ATCC MY-MYA-2386); material cultivated from single spore isolates from Bs 423: Bs 423–2, Bs 423–8, Bs 423–13. Material cultivated at INRA, Villenave d'Ornon, France, 27 Jun 2001, from a tissue culture of the wild specimen collected at Larissa, Thessaly, GREECE, under Cupressus. sempervirens, Jan 1997, I. Theochari, Bs 514 (LIP. ATCC MY-MYA-2389).
Interfertility and intervarietal hybrid examined – Variety eurotetrasporus is partly interfertile with the other varieties of A. bisporus. Bs 261 (Fig. 8) appears to be a natural hybrid between var. bisporus and var. eurotetrasporus. Material was cultivated at INRA, Villenave d'Ornon, France, 28 Jun 2001, from a tissue culture of the wild specimen collected at Dinard, Ille-et-Vilaine FRANCE, Channel coast, under Cupressus macrocarpa, 14 Oct 1992, P. Callac, Bs 261 (ATCC MY-MYA-2385). It is tetrasporic but mainly differs by a wider annulus better joined to the stipe and a more uniform and brown color of the pilei-pellis.
Etymology – The ‘tetrasporus’ epithet refers to the tetrasporic trait of the variety; the ‘euro’ indicates the known range.
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DISCUSSION |
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). Genetically, heterozygosity, which is absent in var. eurotetrasporus, and genotypes at the two ITS polymorphisms, ITS(102) and ITS(240), might be the best criteria for discriminating among varieties (see Table IV; see also Challen et al 2002: corresponding positions are respectively 118 and 261). Biologically, the three varieties have different life cycles; var. bisporus is amphithallic and predominantly pseudohomothallic, var. burnettii is amphithallic and predominantly heterothallic, and var. eurotetrasporus is homothallic. Considerations about homothallism – The fact that the two homokaryotic isolates belong to a highly homogeneous entity is consistent with a homothallic life cycle. Homothallism is the primary diagnostic feature of the novel variety eurotetrasporus; however, three important aspects of this reproductive syndrome remain to be elucidated.
First, we do not know if homomictic or amictic process occurs in basidia of var. eurotetrasporus. Cytogenetic studies will be necessary to elucidate the basidial events, but we already can note that the spores have variable size and that the spore germination rate was only about 10% while the spores are theoretically haploid and genetically identical.
Second, does homothallism exist in the other varieties? Haploid fruiting was observed in culture of homokaryons from var. bisporus (Dickart 1985) and from var. burnettii (Callac et al 1993), but fructification was generally poor and late. There is no solid evidence that homothallism occurs in field conditions, although for certain wild strains heterozygozity was not detected (Xu et al 1997); such strains, mainly of var. burnettii, could be homokaryotic. In var. bisporus, homothallism would be less expected than in var. burnettii because pseudohomothallism maintains a high level of heterozygosity and inbreeding depression is known (Xu 1995).
Finally, what is the genetic basis of homothallism in A. bisporus? The analysis of a small homokaryotic progeny of Bs 261 revealed that about half was able to fruit (Callac et al 1998). This suggested a relatively simple genetic determinism. A hybrid between var. eurotetrasporus and var. bisporus, Bs 423 x U 1–2, previously has been obtained and genetically confirmed (Callac et al 1998). To elucidate the genetic determinism of the haploid fruiting, the analysis of a larger offspring of this hybrid is in progress.
Origin of the three European tetrasporic isolates –
Except for one marker, the two homokaryotic isolates, Bs 423 and Bs 514, and the nucleus B of Bs 261 had the same haploid genotype. Knowing that high genetic diversity exists among the European populations (Xu et al 1997, Callac et al 2000), such a particular haploid genotype would be unlikely to appear independently three times by chance alone (i.e., by recombination and outcrossing). We think instead that, in agreement with their homothallic life cycle, the genotypic resemblance of the two homokaryons Bs 423 and Bs 514 results from their belonging to a single, highly homogeneous lineage. They must have inherited the same Bsn-t allele responsible for their tetrasporic phenotype from a common ancestor through many homothallic generations. The dissimilarity analysis and the confirmed existence of a homothallic, tetrasporic entity reinforce our previous hypothesis that Bs 261 is a natural hybrid that has received its nucleus bearing the tetrasporic allele Bsn-t from a member of this entity and its nucleus bearing the bisporic allele Bsn-b from a bisporic parent belonging to var. bisporus. In other respects, it appears that the three varieties of A. bisporus are related more closely to each other than they are to the A. subfloccosus isolate, in agreement with the phylogenetic analysis of Challen et al (2002).
The origin of the intervarietal hybrid Bs 261 must be relatively recent because the alleles inherited from var. eurotetrasporus remain grouped in the apparently intact parental nucleus. An earlier analysis of Bs 261 homokaryotic offspring showed that recombination occurred between four markers linked on chromosome I (Pr2, Pr5, Pr6, and BSN) and that EST1 segregated in a 1:1 manner (Callac et al 1998). Therefore, since meiosis occurs in the basidia of Bs 261, the latter isolate itself must be a first generation hybrid between the homothallic var. eurotetrasporus and the predominantly pseudohomothallic var. bisporus. Among basidiomycetes, we do not know of any report of such an intervarietal cross under field conditions; however, restricted hybridization between homothallic and heterothallic strains of Sistotrema brinkmani, an aggregate of biological species, has been forced with nutritional marker (Ullrich and Raper 1975).
In contrast, var. eurotetrasporus must be relatively ancient with a complex history. First, it is characterized not only by the ITS(102)-2 allele but also by different and probably independent biological traits, such as partial intersterility, homothallism and tetrasporic basidia, which are unlikely to have been acquired at one time. Second, Bs 423 and the nucleus B of Bs 261 bear the unique ITS(632)-2 allele, while Bs 514, collected 2000 km apart, bears the frequent allele ITS(632)-1. This intravarietal polymorphism could result from a mutation that occurred since the origin of the homothallic entity.
Allelic frequencies known among different A. bisporus populations could help to elucidate the origin of the homothallic variety. While they are not homogeneous and would have to be completed, data from earlier analyses merit discussion. For example, for alloenzymatic loci (see Callac et al 2000 and Kerrigan et al 1996), the two alleles Pgm-3 and ßGlu-3 of var. eurotetrasporus are rare among the bisporic French isolates (f < 0.05) while we know that Pgm-3 is abundant in Greece (f = 0.29), as is ßGlu-3 in North America (f = 0.47). Finally, at the BSN locus, both tetrasporic varieties bear what might be the same Bsn-t allele (Callac et al 1998), but this does not necessary imply an historical relationship between the two varieties because we do not know the precise history of the tetrasporic trait. In conclusion, further studies will be necessary to elucidate the origin of var. eurotetrasporus, but its unexpected genotype at least in comparison with the bisporic French population suggests that var. eurotetrasporus does not simply derive from this population.
Consequences of the interfertility between the varieties –
To understand the complex history and the genesis of the homothallic lineage, the possible role played by intervarietal hybrids such as Bs 261 must be examined. The life cycle of such a hybrid is amphithallic and predominantly heterothallic, and most of the produced spores are homokaryotic. However, not only can it be pseudohomothallic, because few spores are heterokaryotic and fertile, but also it is potentially homothallic, because some of its homokaryotic spores give rise to fertile homokaryons (Callac et al 1998). In the latter case, new homothallic lineages could be generated. Such scenarios could have important implications: First, var. eurotetrasporus itself could derive from a more ancient homothallic entity. The combination of dispersal, reproductive isolation, and rare hybridizations producing new homothallic lineages could have generated the unexpected genotype of var. eurotetrasporus. Second, the existence of daughter homothallic entities is not excluded. However, in areas where both var. euroterasporus and var. bisporus coexist, we have not found any other homothallic lineages. On the other hand, the haploid sporocarps we observed in single-spore isolates from the natural intervarietal hybrid Bs 261 were poor and late (Callac et al 1998), possibly because of unfavorable recessive alleles inherited from the parent of var. bisporus. In the absence of gametic selection for haploid fruiting in this highly heterozygous and predominantly pseudohomothallic variety, such alleles would be accumulated. This suggests that apparition of new homothallic lineages resulting from intravarietal hybridization could be rare in the known areas where both varieties coexist.
The coexistence of the two varieties could have another consequence: While the homothallic lineage is reproductively isolated, intervarietal hybrids such as Bs 261, which has a predominantly heterothallic life cycle, can produce gene flow from the homothallic lineage toward var. bisporus. In other terms, var. tetrasporus is potentially a spatio-temporal carrier of alleles through the amphithallic populations, but its impact probably is limited because it is poorly represented and is partly intersterile with the other varieties. In western France, the introduction of C. macrocarpa a century and a half ago (Camus 1914) on the coast could have helped the proliferation of the species and possibly favored the conjunction between the two varieties. For example, the two rare alleles Pgm-3 or ßGlu-3 borne by the two tetrasporic isolates Bs 423 and Bs 261 were found only in three bisporic isolates collected at less than 100 km from Bs 423 in the same cypress coastal habitat and in three other isolates collected at the same site as Bs 261. The latter site was included in a recent genetic analysis of local populations that strongly suggested that out-crossing and recombination was common although the populations were mainly bisporic (Xu et al 2002). These data suggest that the alleles of intervarietal hybrids such as Bs 261 can spread, at least locally, into the bisporic population.
Evolutionary considerations –
Current and previous data suggest that var. eurotetrasporus is in the speciation process: First, it is partly intersterile with the other varieties; second, it is relatively ancient and is accumulating private or semispecific alleles; third, its homothallic life cycle maintains its biological isolation; finally, there are numerous similarities among the homothallic entities of A. subfloccosus. That complex of species which is closely related to A. bisporus (Challen et al 2002) comprises two sister entities, which are tetrasporic, homothallic and intersterile (Kerrigan et al 1999). One of the two, the lowland entity, is highly homogeneous, such as A. bisporus var. eurotetrasporus. It can share the same cypress habitat, where it is equally rare in France. This comparison suggests that a process, similar to the one by which A. subfloccosus would have derived from A. bisporus or from their common ancestor, would be in progress for var. eurotetrasporus. However, this process is not complete; var. bisporus and var. eurotetrasporus appear to be two entities that have progressed far into speciation but which are still capable of hybridizing and which have the potential to continue to interact in ways that could have several outcomes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Dr. Theochari died while this paper was in review. We dedicate this work to her memory
Accepted for publication August 28, 2002.
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LITERATURE CITED |
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