National Agricultural Research Foundation, Institute of Kalamata, Lakonikis 85, 24100 Kalamata, Greece1
Dipartimento di Scienze Botaniche, Via Archirafi 38, I-90123 Palermo, Italy2
Author for correspondence: Georgios I. Zervakis. Tel: +30 721 91984. Fax: +30 721 27133. e-mail: zervakis{at}kal.forthnet.gr
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ABSTRACT |
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Keywords: Pleurotus nebrodensis, Apiaceae, mushroom systematics, fungal speciation and evolution, host-specificity
Abbreviations: ET, electrophoretic type; RAPD, random amplified polymorphic DNA
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INTRODUCTION |
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There is much controversy on the proper assignment of host-specialized populations within the P. eryngii complex. For example, some authors consider most or all ecotypes as distinct species (Boisselier-Dubayle, 1983 ; Joly et al., 1990
), some view them as varieties of P. eryngii (Bresinsky et al., 1987
; Hilber, 1982
), and others express intermediate arguments (Venturella, 2000
; Zervakis & Venturella, 1998
). In addition, there are several other names that have been proposed to accommodate taxa of dubious validity, like P. fossulatus (Cooke) Sacc. or P. hadamardii Constantin, growing also on umbellifers (Joly et al., 1990
; Pegler, 1977
). This confused situation presents a major challenge for fungal taxonomy, speciation and co-evolution studies. Its clarification would also be of substantial benefit for applied research, since the use of Pleurotus fungi is linked to several agro-industrial activities of great economic importance, e.g. conversion of lignocellulosic residues to food and feed, biocontrol of plant diseases, degradation of noxious pollutants, production of enzymes and medicinal compounds, etc. (Heinfling et al., 1998
; Philippoussis et al., 2001
; Ruiz-Duenas & Martinez, 1996
; Wasser & Weis, 1999
; Zervakis et al., 1996
).
There are significant problems in classifying Pleurotus isolates using only morphological characters (which are often unreliable or inconclusive mainly due to the large influence exerted by environmental factors) or compatibility experiments (which are based on the application of the controversial biological species concept). Therefore, the application of molecular criteria is essential for providing a thorough insight into the taxonomic relationships between Pleurotus populations and in pertinent speciation processes (Iraçabal et al., 1995 ; Petersen & Hughes, 1999
; Vilgalys et al., 1996
). Isozyme analysis has been successfully applied to several taxonomic studies in mycology (Micales et al., 1986
). Interpretation of zymograms has been useful in identifying genetic variability within and between fungal species (Gottlieb et al., 1998
; Urbanelli et al., 1998
; Zervakis et al., 1994
), and for revealing the extent of variation in diverse populations from numerous hosts (Harrington et al., 1996
; Surve-Iyer et al., 1995
; Yoon et al., 1990
) and geographical origins (Bonde et al., 1993
; Stanosz et al., 1999
; Zervakis & Labarère, 1992
). On the other hand, the random amplified polymorphic DNA-PCR technique (RAPD-PCR) has permitted the study of the population structure of many fungi that are difficult to characterize with other markers. This technique allows rapid generation of reliable and reproducible DNA fingerprints and has been used to investigate the genetic variation within several fungal groups (Bryan et al., 1999
; Paavanen-Huhtala et al., 1999
; Raina et al., 1997
), or to clarify systematics based on traditional criteria (Assigbetse et al., 1994
; Holmes et al., 1994
).
The aim of this study was to elucidate the systematics and assess the diversity of European Pleurotus taxa associated with umbellifers, and determine speciation processes under way. Forty-six dikaryotic strains belonging to the P. eryngii species-complex isolated from five different host-plant genera were examined by the use of RAPD-PCR and isozyme techniques, and evaluated in conjunction with ecological observations and morphology of basidiomata.
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METHODS |
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Dikaryotic and homokaryotic fungal cultures.
For the establishment of dikaryons in pure culture, small pieces from the basidioma context were taken and placed on Petri dishes with water agar. After a few days, hyphal tips were transferred to fresh medium. For the assessment of the loci and alleles responsible for the enzyme activities tested, Pleurotus homokaryotic (monokaryotic) strains were produced from at least three dikaryons per host-associated population. Single-spore isolates were obtained either from naturally occurring basidiomata or from basidiomata grown in vitro (Zervakis & Balis, 1995 ).
Enzyme extraction, starch-gel electrophoresis and analysis of isozyme data.
For the production of mycelial cultures, dikaryotic or homokaryotic strains were grown in 250 ml Erlenmeyer flasks containing 100 ml CYM. Culture conditions, mycelium harvest and enzyme extraction were as described by Zervakis et al. (1994) .
Enzymes were separated by horizontal starch-gel electrophoresis. The five different tray and gel buffer systems used for optimal resolution of isozyme patterns, and preparation of gels, were as described by Zervakis et al. (1994) . Conditions for electrophoresis and staining protocols for the 23 enzyme activities assayed were as follows (enzyme abbreviations for data-informative loci are defined in Table 2
): ADH, GDH, LDH, SOD, XDH (Allendorf et al., 1977
), APH, ALP, GADH, ME (Loukas & Krimbas, 1980
), PHE-DRE, PHE-TRE (Kerrigan & Ross, 1989
), DIA, EST, LAP, PGM (Zervakis & Labarère, 1992
), and G6PD, HK, PHI, IDH, MDH, PEP-LT, PEP-PHE-PRO, 6PGD (Zervakis et al., 1994
). The gels were read just after the end of the incubation period, and then preserved for future reference in a water/methanol/acetic acid (50:40:10) solution. All protein extracts were electrophoretically examined at least three times and produced consistent zymograms, which only occasionally showed some fluctuations in their staining intensity (band staining intensity was not taken into account when the zymograms were interpreted).
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Isozyme data were partitioned by locus and population for further analysis. For all loci, the allele frequencies, the genetic diversity per locus and the mean diversity were calculated using the formulae of Selander et al. (1986) and Nei (1978)
with a correction for bias in small samples. In addition, for each population (either host-associated or geographical) examined, the percentage of polymorphic loci (0·95 criterion), the mean number of alleles per locus and the mean diversity were determined to provide an estimate of the intrapopulation variability.
DNA extraction and RAPD-PCR analysis.
Mycelia, grown on solidified CYM, were frozen in liquid nitrogen in Eppendorf tubes and ground into powder with a micropestle (Kontes Pellet Pestle, Fisher cat. no. K749520-0-000). DNA extraction followed Rogers & Bendich (1988) . DNA concentration and dilution were estimated by gel electrophoresis and spectrophotometry. Amplification reactions were performed in a final volume of 25 µl containing 10 ng genomic DNA. The reaction solution consisted of 200 µM each of dATP, dCTP, dGTP and dTTP, 50 µmol oligonucleotide primer (Operon Technologies, Kit OPB) and 2 units Taq polymerase (Boehringer Mannheim) in 10 mM Tris pH 8·3, 2 mM MgCl2, 0·001% gelatin, 0·05% Tween 20, 50 mM KCl. Amplification was performed in a Techne Progene thermalcycler: one cycle at 94 °C for 3 min, 37 °C for 1 min and 72 °C for 1 min, and 44 cycles at 94 °C for 30 s, 37 °C for 1 min and 72 °C for 1 min. Amplified fragments were resolved on a 1·1% agarose gel, run under standardized conditions, and stained by ethidium bromide. A 100 bp ladder DNA marker (Pharmacia) was used as a size standard.
Each amplification run included a negative control reaction without the addition of DNA and each reaction was performed at least twice. Initially, a subset of the isolates was used to perform a preliminary screening of 20 decamer oligonucleotide primers to identify those that gave reproducible marker profiles and to exclude those producing very low proportion of polymorphic bands. Five primers were selected: OPB01 (5'-GTTTCGCTCC-3'), OPB02 (5'-TGATCCCTGG-3'), OPB10 (5'-CTGCTGGGAC-3'), OPB14 (5'-TCCGCTCTGG-3') and OPB18 (5'-CCACAGCAGT-3'). Some variations in RAPD patterns were detected in the duplicate experiments (i.e. approximately 10% of the total amplified bands were not consistently amplified). However, only distinct, clearly resolved and reproducibly amplified fragments were selected for RAPD analysis and scored as present (1) or absent (0). Comparisons of RAPD profiles were only made between samples that were included in the same run, and which had been separated on the same agarose gel. There was no differential weighting for band intensity. The assumption was made that amplification products of the same size which were present in the profiles generated by different isolates represented products from equivalent loci.
Statistical analysis.
Cladistic analysis was performed with the tester version 4.0b4a of PAUP* (Phylogenetic Analysis Using Parsimony) written for PowerPC (Swofford, 2000 ). All characters (26 from the isozyme data, and 48 from the RAPD data) were unordered and of equal weight. Parsimony settings were as follows: ACCTRAN (accelerated transformation) character-state optimization, stepmatrix option allowed assignment of states not observed in terminal taxa to internal nodes (all states in stepmatrix), and multiple states of taxa were interpreted as polymorphism (in the case of isozymes only). Genetic distances were calculated by the PAUP* software on the basis of mean genetic differences.
Branch robustness of the derived cladograms was evaluated in PAUP* using two different methods: (a) bootstrapping, and (b) jacknife-resampling. Both methods used maximum-parsimony as the optimality criterion and performed an heuristic search with 500 replicates and the following settings: simple addition sequence (reference taxon: P. pulmonarius), starting trees obtained via stepwise addition, 10 trees held at each step during stepwise addition, tree-bisection-reconnection (TBR) as branch-swapping algorithm, MULPARS option not in effect, steepest descent option not in effect, MAXTREES setting 500, branches having maximum length zero allowed to collapse to yield polytomies, topological constraints not enforced. In addition, the jacknife-resampling method was set to 50% of characters deleted in each replicate. The respective cladograms shown in Results derived from the enforcement of the 50% majority-rule consensus.
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RESULTS |
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Allele frequencies, genetic diversity and mean diversity for all 13 isozyme loci provided an indication of the overall variation that existed among the ETs produced by this study (Table 2). The genetic diversity varied significantly among the different loci, ranging from 0·117 (Sod-1) to 0·663 (Ldh). The mean diversity for all strains was 0·470. Partition of isozyme bands into loci and grouping of strains by host allowed estimation of the mean number of alleles per locus, the percentage of polymorphic loci, and the mean diversity within each host-associated population (Table 3
). Increased percentages of polymorphic loci and high mean diversity values were detected for Eryngium- and Ferula-associated strains (mean diversity 0·261 and 0·288 respectively), whereas isolates growing on Cachrys, Elaeoselinum and Thapsia plants yielded significantly lower values (0·122, 0·123 and 0·099 respectively). Furthermore, a similar type of evaluation performed within groups of strains of identical geographical origin (Italy and Greece, only for Eryngium- and Ferula-associated specimens) indicated higher diversity for Greek isolates irrespective of the host plant they were collected from: e.g. mean no. of alleles per locus 2·231 vs 1·769 when data from both host plants are compared, or 2·000 vs 1·462 for Ferula only. However, these values were consistently lower than those previously obtained when strain partitioning was performed on the basis of the host plant.
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Evaluation of RAPD-PCR data produced infrahost genetic distance values ranging from 0·178 (strains growing on T. garganica) to 0·262 (strains growing on Eryngium spp.) (Table 4). In contrast, significantly higher values were obtained by interhost pairwise comparisons. As in the case of the isozyme analysis, Pleurotus strains growing in association with C. ferulacea were the most distant within the P. eryngii species-complex. Specimens isolated from Eryngium hosts were relatively closer to Ferula and Thapsia; in general, T. garganica revealed consistently lower distance values with all other hosts (0·309<D<0·387). Noteworthy also was the relative affinity between strains from Ferula and Elaeoselinum hosts.
The cladograms produced by the 42 RAPD phenotypes were evaluated by the bootstrap and jacknife-resampling methods, and are presented as one (Fig. 1b). All Pleurotus strains were arranged in five major clusters in accordance with their associated host-plant. As previously, isolates collected from Eryngium spp. were linked together with low support, with the exception of IKP63 and IKP64 from eastern Crete (>44%). In contrast, clades within the other clusters were of high robustness: Ferula (between the isolates of Italian origin), and between all Sicilian specimens collected from Elaeoselinum, Thapsia and Cachrys. Especially in the case of C. ferulacea, high statistical support was observed both within the group and externally with the larger cluster formed by all other groups. As was noted in the case of the cladograms produced by the isozyme analysis, individual clusters belonging in P. eryngii sensu stricto (i.e. including all host groups except the C. ferulacea one) showed poor statistical support with one another (<10%).
Combination of isozyme and RAPD-PCR data
When the two isozyme and the RAPD-PCR datasets were combined into one, clusters of the resulting tree showed higher statistical support than those of the individual datasets (Fig. 1c). Grouping of populations was again in accordance with the associated plant-hosts. All populations, with the exception of the isolates from Eryngium spp., demonstrated high intragroup bootstrap and jacknife values. Furthermore, the majority of the strains within the Eryngium and Ferula clusters were positioned with respect to their geographical origin. The statistical support within isolates growing on C. ferulacea was high, whereas the coherence within Ferula, Elaeoselinum and Thapsia related strains was satisfactory. Of interest was again the fact that the relative positioning of the host-associated groups within P. eryngii showed weak support, which is indicative of their close affinity.
In an attempt to evaluate the correlation between the genetic distances obtained from isozyme and RAPD-PCR datasets, a logarithmic curve was produced demonstrating high correlation (r2=0·73) between the values which derived from the application of the two approaches (Fig. 2). This graph indicates the lower distances obtained within populations, and illustrates a relatively faster saturation of RAPD data at high genetic distances.
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DISCUSSION |
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Levels of isozyme variation found within the P. eryngii species-complex are high whether measured in terms of percentage of polymorphic loci, alleles per locus, or genetic diversity. They are indicative of high genetic differentiation among populations, with the largest proportion of diversity resulting from differences among individuals from different hosts rather than from different locations. Since most allelic variation at isozyme loci is unlikely to be subject to strong selection (Nei & Graur, 1984 ), the levels of isozyme variability within populations are primarily determined by effective population size (Crow & Kimura, 1970
). In the Mediterranean region, Pleurotus populations are unlikely to have passed through severe bottlenecks over the past. Thus, the history of populations, the reproductive system of P. eryngii, allochrony, and co-evolution phenomena with the host plant favour high intraspecific heterogeneity. Similar isozyme diversity has been reported in populations of Puccinia graminis (Burdon & Roelfs, 1983
), Morchella esculenta (Yoon et al., 1990
), Crumenulopsis sororia (Ennos & Swales, 1991
), Agaricus bitorquis (Roux & Labarère, 1990
) and Pleurotus spp. (Zervakis et al., 1994
).
While isozyme analysis provides an indication of variation in the products of certain genes, RAPD-PCR is a means of assessing polymorphisms at a wide range of loci (Williams et al., 1990 ). In this study, the resolution of the RAPD-PCR analysis was better than that of isozyme analysis. Every examined strain of the P. eryngii species-complex showed a unique genotype. Previous studies have shown comparable high levels of intraspecific genetic diversities for the tree endophytes Rhabdocline parkeri (McCutcheon et al., 1993
) and Gnomonia setacea (Lappalainen & Yli-Mattila, 1999
), and for plant pathogens like Claviceps purpurea (Jungehülsing & Tudzynski, 1997
) and Stagonospora nodorum (McDonald et al., 1994
). Such phenomena were mainly attributed to the predominance of sexual reproduction, whereas in biotrophic fungi adaptation to different hosts causes accumulation of genetic differences within the same species due to isolation phenomena. For example, RAPD-PCR permitted the differentiation of pathogenic races of Fusarium oxysporum f. sp. vasinfectum (Assigbetse et al., 1994
), F. solani f. sp. cucurbitae (Crowhurst et al., 1991
), Gremmeniella abietina (Hamelin et al., 1993
), and isolates of A. alternata f. sp. citri (Weir et al., 1998
) based on host specialization, without any apparent correlation with geographical origin. Such types of cases might be broadened to include some facultative parasites like Sphaeropsis sapinea (Stanosz et al., 1999
), which have a restricted group of long-lived hosts belonging in a single gymnosperm family, or the Pleurotus populations growing on umbellifers.
The use of parsimony analysis for RAPD-PCR data has been criticized in the past, especially above the species level (Adams & Demeke, 1993 ; Backeljau et al., 1995
). This could be justified because none of the commonly used parsimony analyses provide an appropriate model for RAPD character state change, while a few non-homologous characters can more drastically affect the topology of trees produced than in phenetic analyses based on similarity (Adams & Demeke, 1993
). On the other hand, trees obtained with UPGMA and other phenetic clustering methods do not always accurately represent the phylogeny of closely related organisms (Hillis et al., 1992
), they offer no optimality criteria for choosing between different topologies, and they reduce the rich character-based matrix to abstract distance values offering no possibility of ancestral state reconstruction (Paavanen-Huhtala et al., 1999
). Hence, phenetic clustering methods should be seen merely as a means of constructing an initial tree for more thorough analysis, not as a method for choosing the final tree (Swofford et al., 1996
). Along this line of approach, we started our data analysis from NJ and UPGMA trees (data not shown), before proceeding with parsimony analysis. The phenetic trees of RAPD-PCR and isozyme data do not differ substantially from one another, and both are congruent with parsimonious trees (minor differences detected between them could be attributed to the smaller size of the isozyme data matrix). In general, the parsimonious trees deriving from isozyme or RAPD-PCR and from combined isozyme and RAPD-PCR data were very similar to each other, confirming the agreement of the approaches. In contrast, previous phylogenetic studies demonstrated that the use of additional molecular characters such as the mitochondrial small-subunit rRNA and/or of the nuclear rRNA ITS sequences were of particular value only above the species level (Gonzalez & Labarère, 2000
; Wu et al., 2000
; Vilgalys & Sun, 1994
).
Systematics and speciation
The use of RAPD-PCR and isozyme analyses permitted grouping of the P. eryngii complex isolates into five main clusters in accordance with the separation of individual populations on host specialization; pairwise genetic distances within host isolates were lower than those between host populations. Pleurotus isolates growing on C. ferulacea formed a distinct group with relatively high statistical support. Therefore their separation from the rest of the populations examined and their classification within a distinct taxonomic entity at the species level, i.e. P. nebrodensis (Inzenga) Quél., seems well justified and confirms reports based on morphology (Venturella, 2000 ). In previous studies, P. nebrodensis showed intercompatibility values as low as 618% in crosses with P. eryngii var. eryngii and var. ferulae (Cailleux et al., 1981
; Hilber, 1982
; Zervakis & Balis, 1996
). In addition, Cailleux et al. (1981)
demonstrated that among the successful inter-ecotype matings, a large percentage of hybrid-dikaryons showed disturbed morphogenesis and abnormal reproductive physiology. Moreover, the pleuroti from C. ferulacea, which appear in Sicily from late spring to early summer at altitudes exceeding 1200 m, are in the process of morphological differentiation, already showing distinct characters in pileus colour and texture, cuticle, spore size, cheilocystidia and chromosome number (Table 5
).
All other strains were positioned within the larger P. eryngii group, which was further divided into four main clusters corresponding to Eryngium, Ferula, Elaeoselinum and Thapsia hosts. Pleurotus strains growing on T. garganica show a very narrow distribution range (Madonie Mt., Sicily) and hence their intrapopulation genetic distances and diversities are relatively low. They are characterized by their distinct pileus size and colour, cuticle, habit and habitat (Table 5). Although this population seems to constitute a new variety, additional specimens need to be examined before any definite conclusions are drawn. In contrast, Pleurotus isolates from Eryngium spp. and F. communis plants (originating from various geographical areas) are very heterogeneous, showing high infrahost genetic distances and diversities. These populations could be discriminated on the basis of ecomorphological characters from the rest of the taxa examined (Table 5
). However, the results from previous mating studies which provided intercompatibility percentages exceeding 40% (Hilber, 1982
; Zervakis & Balis, 1996
), in conjunction with the molecular evidence furnished by this work, support their currently accepted status: P. eryngii (DC.: Fr.) Quél. var. eryngii, and P. eryngii (DC: Fr.) Quél. var. ferulae Lanzi.
Pleurotus growing on E. asclepium subsp. asclepium seems to hold an intermediate position, demonstrating relatively high variability, which could be probably explained by the restricted gene exchange with the other P. eryngii taxa. In fact, isolates from these populations give higher percentages of positive results when mated with P. eryngii var. eryngii and var. ferulae (4570% of successful matings; G. Zervakis & G. Venturella, unpublished results). Isozyme and RAPD data confirm the relative affinity of this group to P. eryngii sensu stricto, and especially to var. ferulae; co-evaluation of ecomorphological characters such as pileus size, colour, surface and cuticle as well as spores and cheilocystidia size support its taxonomic assignment as a new variety, P. eryngii var. elaeoselini Venturella et al. (Venturella et al., 2000 ). As it is the case with all other Pleurotus growing on umbellifers, morphological differentiation seems to follow genetic isolation for this taxon as well; this is in accordance with previous reports on synnematoid Pleurotus taxa (Zervakis, 1998
), or other Basidiomycetes (Kemp, 1975
).
As regards taxa of ambiguous validity, P. hadamardii Constantin should be considered as a nomen nudum, since the original description by Constantin referred to a fungus isolated from Eryngium alpinum, but it proved to be erroneous (Heim, 1960 ). Another plant associated with P. hadamardii is Laserpitium latifolium (Joly et al., 1990
), which has also been regarded as principal host for P. nebrodensis (Heim, 1960
; Hilber, 1982
). However, Pleurotus strains growing on Laserpitium spp. in central Europe and Northern Italy show identical microscopical characteristics to P. eryngii var. elaeoselini (G. Venturella & G. Zervakis, unpublished data), and differ substantially from the original descriptions of P. nebrodensis based on biological material collected from Sicily (Inzenga, 1863
). Therefore, P. nebrodensis should only include strains isolated from C. ferulacea, at least until isolates from Laserpitium can be thoroughly investigated. In addition, the taxonomic position of Pleurotus isolates growing in association with Diplotaenia cachrydifolia at high elevations in Iran (Heim, 1960
; Saber, 1990
) remains dubious, as does that of strains collected from wood in Afghanistan and classified under the name P. fossulatus (Pegler, 1977
). Both of them apparently belong to the P. eryngii complex and morphologically resemble the descriptions of P. eryngii var. elaeoselini and P. nebrodensis.
The level of genetic variation is generally considered adaptive and related to the breadth of geographical ranges and/or to the ecological heterogeneity within the ranges (Lewinsohn et al., 2000 ; Nevo, 1988
). Speciation and the development of species richness appear to be facilitated by restricted gene flow and isolation of small populations (Lande, 1984
). Hence, the high diversity in many infraspecific taxa that are trophically highly specialized (e.g. the P. eryngii species-complex) suggests that ecologically specialized populations are particularly prone to speciation (Futuyma, 1986a
). However, if those populations are brought into contact, much of the divergence they have accomplished will be lost by interbreeding. On the other hand, if they have become different species they can retain their diverse adaptations, and refine them even while sympatric (Futuyma, 1986b
). In many cases sympatric populations are in an intermediate stage of speciation (i.e. partially reproductively isolated), and they usually interbreed along a hybrid zone that can persist for long periods (Futuyma, 1986a
). This explains the mating behaviour between ecotypes of P. eryngii, where ecological or seasonal barriers can readily break down, unless some type of isolation factor(s) reduces gene flow to a very low level.
We believe therefore that the P. eryngii species-complex includes many host-associated taxa, which maintain distinct gene pools through an efficient mechanism of premating barriers. These populations have recently diverged through a sympatric speciation process based on both intrinsic reproductive barriers (i.e. partial compatibility in inter-ecotypes matings) and extrinsic factors (host specialization). In addition, ecogeographical parameters such as allochrony in the appearance of basidiomata (and hence discharge of basidiospores), elevation and host plant distribution hinder, to a certain extent, gene exchange among P. eryngii ecotypes. This hypothesis is clearly supported in the present study by the distinct clustering of the P. eryngii taxa when subjected to isozyme and RAPD-PCR analysis. Similar phenomena of sympatric speciation based on habitat specialization within a common area, resembling the microevolutionary units (Duncan, 1972 ), were noted in the past for other homobasidiomycetes such as Hirschioporus abietinus (Macrae, 1967
), Heterobasidion annosum (Worrall et al., 1983
), Paxillus involutus (Fries, 1985
) and Peniophora cinerea (Chamuris, 1991
). In these cases, population divergence was promoted by locally strong ecological discontinuities (e.g. habitat or temporal isolation), and was accompanied by partial or total reproductive isolation.
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ACKNOWLEDGEMENTS |
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Received 23 March 2001;
revised 4 July 2001;
accepted 12 July 2001.