Genetic polymorphism and taxonomic infrastructure of the Pleurotus eryngii species-complex as determined by RAPD analysis, isozyme profiles and ecomorphological characters

Georgios I. Zervakis1, Giuseppe Venturella2 and Kalliopi Papadopoulou1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Pleurotus eryngii species-complex includes populations of choice edible mushrooms, growing in the greater Mediterranean area in close association with different genera of plants of the family Apiaceae. Their distinct host-specialization served as the principal criterion for the discrimination of several taxa; however, the genetic relationships among the various P. eryngii ecotypes remain ambiguous. In the present study, 46 Pleurotus strains with a wide range of geographical origins were isolated from Eryngium spp., Ferula communis, Cachrys ferulacea, Thapsia garganica and Elaeoselinum asclepium subsp. asclepium, and were subjected to isozyme and random amplified polymorphic DNA-PCR (RAPD) analysis. The 16 enzyme activities tested were controlled by 28 loci, 11 of which were monomorphic. Host-exclusive zymograms for the Aph (acid phosphatase) and Phe-1 (dopa-phenoloxidase) loci were obtained from Pleurotus strains associated with C. ferulacea. Allele frequencies, genetic diversity and mean diversity were high for isolates from Eryngium spp. and Ferula communis. In RAPD analysis, the use of five primers allowed the production of 45 (out of 48) polymorphic bands, while four molecular markers specific for the identification of Pleurotus strains growing on E. asclepium subsp. asclepium and C. ferulacea were obtained. The Pleurotus strains produced 35 distinct electrophoretic types and 42 RAPD patterns, which independently permitted the separation of the fungal populations into five clusters in accordance with their host-specificity. In addition, the evaluation of the principal ecological and morphological characters provided further evidence for discriminating between P. nebrodensis growing on C. ferulacea and the rest of the host-associated populations. The latter represent taxa at the varietal level: P. eryngii var. eryngii, P. eryngii var. ferulae and P. eryngii var. elaeoselini. The position of taxa of dubious validity, such as P. hadamardii and P. fossulatus, is discussed in relation to the new findings. All Mediterranean Pleurotus populations growing on umbellifers seem to have recently diverged through a sympatric speciation process, that is based on both intrinsic reproductive barriers and extrinsic ecogeographical factors.

Keywords: Pleurotus nebrodensis, Apiaceae, mushroom systematics, fungal speciation and evolution, host-specificity

Abbreviations: ET, electrophoretic type; RAPD, random amplified polymorphic DNA


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fungi of the genus Pleurotus (Basidiomycotina, Poriales) are tetrapolar heterothallic; dikaryons produce edible reproductive structures (i.e. basidiomata) on a large array of lignocellulosic substrates. The only group of this genus growing in association with living plants is P. eryngii sensu lato. This species-complex includes populations which are weak parasites on the roots and stems of umbellifers (family Apiaceae, genera Eryngium, Ferula, Ferulago, Cachrys, Laserpitium, Diplotaenia and Elaeoselinum), appearing mostly in groups from autumn until late spring. Their distribution is located within a rather well-defined area of the northern hemisphere, extending westwards to the Atlantic coasts of France and Morocco, and along a zone lying within 30–50° N from central Europe to the Mediterranean coast of Africa, and eastwards to Kazakhstan and India.

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.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Biological material.
Details of the 46 Pleurotus dikaryons used in this study are listed in Table 1. Strains were maintained on complete yeast medium (CYM) solidified with agar (Raper et al., 1972 ); cultures were stored at 4 °C in vials with sterile distilled water, and are kept in the fungal culture collection of the Institute of Kalamata (NAGREF-IK). All voucher specimens are deposited in the Herbarium Mediterraneum (PAL) and in the Herbarium of the Institute of Kalamata (NAGREF-IK).


View this table:
[in this window]
[in a new window]
 
Table 1. Details of the 46 Pleurotus dikaryons used in this study

 
Morphological and ecological data.
Field surveys and periodical observations on Pleurotus taxa growing on umbellifers were carried out in Sicily and Greece from autumn 1995 to autumn 2000. Each taxon was evaluated as regards morphological, anatomical, distributional and ecological characters (Venturella et al., 2000 ; Zervakis & Balis, 1996 ).

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).


View this table:
[in this window]
[in a new window]
 
Table 2. Summarized results of isozyme electrophoresis

 
The gene nomenclature adopted in this paper follows that of May et al. (1979) . Bands (and hence their genetic basis) were interpreted using the outcome of the electrophoretic runs of the homokaryotic isolates. Encoding of every distinct area of enzymic activity (locus), assignment of numbers to alleles of each locus, and encoding of electrophoretic data for input into the computer software followed Zervakis et al. (1994) . Pleurotus isolates which shared identical multilocus phenotypes were grouped into the same electrophoretic type (ET) (Table 1).

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.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isozyme analysis
For the purposes of this study 23 enzyme activities were examined; for seven of them (ALP, DIA, GADH, GDH, ME, PHE-TRE, XDH) no activity was detected or they were poorly resolved and therefore abandoned. For the rest of the enzymes tested, interpretation of electrophoretic patterns was based on the zymograms produced by the homokaryotic progenies (Table 2). Hence, the production of the other 16 activities was governed by 28 loci, 11 of which were monomorphic (HK, PHI, PEP-LT and PEP-PHE-PRO were monomorphic at all loci). Among the rest of the activities examined, the production of EST and SOD was controlled by three loci in each case; however, only one locus per activity (Est-1 and Sod-1) was clearly resolved for all strains tested. In total, 13 clearly interpretable and polymorphic loci were obtained which permitted the classification of the 40 strains into 35 distinct electrophoretic types (data not shown). Only five pairs of isolates produced identical zymograms for all loci: IKP63 and IKP64 from eastern Crete on Eryngium sp. (ET1), UPA10 and UPA12 from Apulia on E. campestre (ET6), UPA7 and UPA8 from Madonie Mt. on C. ferulacea (ET26), UPA30 and UPA31 from Mussomeli on E. asclepium (ET32), and UPA32 and UPA35 from Madonie Mt. on T. garganica (ET34). Host-exclusive zymograms were produced only from Pleurotus strains growing in association with C. ferulacea, for the Aph and Phe-1 loci.

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.


View this table:
[in this window]
[in a new window]
 
Table 3. Number of strains per population, mean number of alleles per locus, percentage of polymorphic loci and mean diversity for all loci examined in the five populations of the P. eryngii species-complex based on host preference

 
A matrix of genetic distances among all Pleurotus strains examined was generated by the PAUP* software on the basis of mean character differences. Evaluation of isozyme data produced infrahost distance values ranging from 0·109 (strains growing on T. garganica) to 0·365 (strains growing on Ferula spp.) (Table 4). As expected, pairwise comparisons between strains associated with different hosts produced higher genetic distance values, with the exception of isolates from Elaeoselinum and Thapsia. In almost all cases, particularly high values were obtained among strains growing on C. ferulacea and the rest of the isolates growing on other hosts (D>0·514; only Cachrys vs Thapsia resulted in lower values, D=0·398). Specimens collected from Eryngium hosts were relatively closer to Thapsia (D<0·491), and the same was observed between Ferula and Elaeoselinum strains (D=0·451).


View this table:
[in this window]
[in a new window]
 
Table 4. Genetic distances resulting from isozyme (above the diagonal) and RAPD-PCR (below the diagonal) analysis among five different populations of Pleurotus isolated from different plant hosts

 
Cladograms resulting from the isozyme data and evaluated by the bootstrap and jacknife-resampling methods are presented as one in Fig. 1(a). In general, Pleurotus strains were clustered according to their associated hosts and formed five major groups. Robustness for clades corresponding to isolates growing on Eryngium and Ferula plants was low, and only strains IKP65 and IKP66 from NE Greece, and ATCC 36047 and CBS 10082 from the former Czechoslovakia showed relatively good bootstrap support (>40%). In contrast, higher robustness was observed within groups associated with the rest of the hosts: isolates UPA24, UPA4 and UPA29 from Elaeoselinum, and UPA32, UPA35 and UPA36 from Thapsia, were supported by values exceeding 54%. Furthermore, three clusters included more than four strains of the same geographical origin: UPA1 and UPA17–20 (Ferula, Sicily), UPA26, UPA32, UPA35 and UPA36 (Thapsia, Madonie Mt.), as well as the entire Cachrys group from Sicily. High values were also obtained for strains AMRU244 and AMRU245 isolated from Ferula sp. in Western Crete, which clustered outside the respective host group. Of interest was the poor statistical support among host-based clusters; thus the relative position of the groups of strains associated with Eryngium, Ferula, Elaeoselinum and Thapsia was not particularly solid. In contrast, higher robustness was apparent in the comparisons between the Cachrys cluster with the rest of the groups.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 1. Cladograms based on the results of (a) the isozyme analysis of 40 dikaryons, (b) the RAPD-PCR analysis of 42 dikaryons and (c) the combination of the isozyme and RAPD-PCR datasets depicting 36 dikaryons, assigned in the P. eryngii species-complex originating from five distinct plant-hosts: Eryngium spp., Ferula communis, Elaeoselinum asclepium subsp. asclepium, Thapsia garganica and Cachrys ferulacea. The numbers at branch points represent bootstrap and jacknife values (they are shown only when they exceed 40%). The numbers at the ends of the branches correspond to Pleurotus strain numbers (Table 1). One strain of P. pulmonarius (IKP26) was used for rooting of the P. eryngii species-complex clade.

 
RAPD analysis
Five primers were selected to survey the genetic diversity within a collection of 42 P. eryngii species-complex isolates. A minimum of 6 (OPB10) and a maximum of 14 (OPB01) unambiguously amplified bands were generated, furnishing a total of 48 bands ranging in size from 150 to 1900 bp. Forty-five of these 48 bands (93·75%) were polymorphic. Only two bands with sizes of 750 and 1050 bp produced by primer OPB1, and one band with a size of 950 bp produced by OPB10 were monomorphic. All banding patterns were unique for each strain studied; in addition, a number of bands could be used as molecular markers for the identification of host-specific Pleurotus strains. Thus, all Pleurotus strains growing in association with E. asclepium were distinguished by the presence of one DNA fragment with an approximate size of 1600 bp produced by OPB1. Accordingly, all specimens isolated from C. ferulacea produced three distinct bands of 150 bp (OPB2), 500 bp (OPB10) and 1200 bp (OPB14).

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.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2. Correlation between the genetic distances obtained from isozyme and RAPD-PCR datasets. Filled and open circles indicate within-host and between-host distances respectively.

 
Morphological and ecological features
All specimens used for the purposes of this work were studied as regards their morphological characters and ecological preferences. The main discriminating features of strains originating from different hosts are presented in Table 5.


View this table:
[in this window]
[in a new window]
 
Table 5. Summary of the most distinctive discriminating morphological characters and ecological features of various Pleurotus taxa associated with umbellifers

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Evaluation of the biochemical and molecular data
This work forms part of a research project on the systematics and phylogeny of Mediterranean Pleurotus taxa. Forty-six dikaryotic Pleurotus strains growing on umbellifers were selected to reflect the geographical and host range of the populations under examination.

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 6–18% 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 (45–70% 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.


   ACKNOWLEDGEMENTS
 
This paper was prepared with the financial support of the Italian Council of National Research (CNR), and it was carried out in the frame of a bilateral project between Italy and Greece (project no. 96.00168.CT06). Additional funding was obtained through the National Agricultural Research Foundation of Greece (Programme DIMITRA). The authors wish to thank two anonymous reviewers for their helpful comments and suggestions.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Adams, R. P. & Demeke, T. (1993). Systematic relationships of Juniperus based on random amplified polymorphic DNAs (RAPDs). Taxon 42, 553-571.

Allendorf, F. W., Mitchell, N., Ryman, N. & Stahl, G. (1977). Isozyme loci in brown trout (Salmo trutta L.): detection and interpretation from population data. Hereditas 86, 179-190.[Medline]

Assigbetse, K. B., Fernandez, D., Dubois, M. P. & Geiger, J. P. (1994). Differentiation of Fusarium oxysporum f. sp. vasinfectum races on cotton by random amplified polymorphic DNA (RAPD) analysis. Phytopathology 84, 622-626.

Backeljau, T., de Bruyn, L., de Wolf, H., Jordaens, K., van Dongen, S., Verhagen, R. & Winnepennickx, B. (1995). Random amplified polymorphic DNA (RAPD) and parsimony methods. Cladistics 11, 119-130.

Boisselier-Dubayle, M.-C. (1983). Taxonomic significance of enzyme polymorphism among isolates of Pleurotus (Basidiomycetes) from Umbellifers. Trans Br Mycol Soc 81, 121-127.

Bonde, M. R., Micales, J. A. & Peterson, G. L. (1993). The use of isozyme analysis for identification of plant-pathogenic fungi. New Phytol 128, 135-143.

Bresinsky, A., Fischer, M., Meixner, B. & Paulus, W. (1987). Speciation in Pleurotus. Mycologia 79, 234-245.

Bryan, G. T., Labourdette, E., Melton, R. E., Nicholson, P., Daniels, M. J. & Osbourn, A. E. (1999). DNA polymorphism and host range in the take-all fungus, Gaeumannomyces graminis. Mycol Res 103, 319-327.

Burdon, J. J. & Roelfs, A. P. (1983). The effect of sexual and asexual reproduction on the isozyme structure of populations of Puccinia graminis. Phytopathology 75, 1068-1073.

Cailleux, R., Diop, A. & Joly, P. (1981). Relations d’interfertilité entre quelques représentents des Pleurotes des Ombellifères. Bull Soc Mycol Fr 97, 97-124.

Chamuris, G. P. (1991). Speciation in the Peniophora cinerea complex. Mycologia 83, 736-742.

Crow, J. F. & Kimura, M. (1970). An Introduction to Population Genetics Theory. New York: Harper & Row.

Crowhurst, R. N., Hawthorne, B. T., Rilkkerink, E. H. A. & Templeton, M. D. (1991). Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplified polymorphic DNA. Curr Genet 20, 391-396.[Medline]

Duncan, E. G. (1972). Microevolution in Auricularia polytricha. Mycologia 64, 394-404.

Ennos, R. A. & Swales, K. W. (1991). Genetic variability and population structure in the canker pathogen Crumenulopsis sororia. Mycol Res 95, 521-525.

Fries, N. (1985). Intersterility groups in Paxillus involutus. Mycotaxon 24, 403-409.

Futuyma, D. J. (1986a). Evolutionary Biology. Sunderland, MA: Sinauer Associates.

Futuyma, D. J. (1986b). Patterns and Processes in the Evolution of Life. Edited by D. Raup & D. Jablonski. Berlin & New York: Springer.

Gonzalez, P. & Labarère, J. (2000). Phylogenetic relationships of Pleurotus species according to the sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6 and V9 domains. Microbiology 146, 209-221.[Abstract/Free Full Text]

Gottlieb, A. M., Saidman, B. O. & Wright, J. E. (1998). Isoenzymes of Ganoderma species from southern South America. Mycol Res 102, 415-426.

Hamelin, R. C., Ouellette, G. B. & Bernier, L. (1993). Identification of Gremmeniella abietina races with random amplified polymorphic DNA markers. Mol Plant–Microbe Interact 5, 479-483.

Harrington, T. C., Steimel, J. P., Wingfield, M. J. & Kile, G. A. (1996). Isozyme variation and species delimitation in the Ceratocystis coerulescens complex. Mycologia 88, 104-113.

Heim, R. (1960). Le Pleurote des Ombellifères en Iran. Rev Mycol 25, 242-247.

Heinfling, A., Martinez, M. J., Martinez, A. T., Bergbauer, M. & Szewzyk, U. (1998). Transformation of industrial dyes by manganese peroxidases from Bjerkandera adusta and Pleurotus eryngii in a manganese-independent reaction. Appl Environ Microbiol 64, 2788-2793.[Abstract/Free Full Text]

Hilber, O. (1982). Die Gattung Pleurotus (Fr.) Kummer unter besonderer Berücksichtigung des Pleurotus eryngii-Formenkomplexes. Bibliotheca Mycologica 87. Vaduz: J. Cramer.

Hillis, D. M., Bull, J. J., White, M. E., Badgett, M. R. & Molineux, I. J. (1992). Experimental phylogenetics: generation of a known phylogeny. Science 255, 589-592.[Medline]

Holmes, G. J., Eckert, J. W. & Pitt, J. I. (1994). Revised description of Penicillium ulaiense and its role as a pathogen of citrus fruits. Phytopathology 85, 522-527.

Inzenga, G. (1863). Nuova specie di agarico del Prof. Giuseppe Inzenga. Giorn Reale Ist Incoragg Agric Sicil Palermo 1, 161-164.

Iraçabal, B., Zervakis, G. & Labarère, J. (1995). Molecular systematics of the genus Pleurotus: analysis of restriction polymorphisms in ribosomal DNA. Microbiology 141, 1479-1490.

Joly, P., Cailleux, R. & Cerceau, M.-T. (1990). La stérilité male pathologique, élément de la co-adaptation entre populations de champignons et de plantes-hotes: modèle des Pleurotes des Ombellifères. Bull Soc Bot Fr 137, 71-85.

Jungehülsing, U. & Tudzynski, P. (1997). Analysis of genetic diversity in Claviceps purpurea by RAPD markers. Mycol Res 101, 1-6.

Kemp, R. F. O. (1975). Breeding biology of Coprinus species of the section Lanatuli. Trans Br Mycol Soc 65, 375-388.

Kerrigan, R. W & Ross, I. K. (1989). Allozymes of a wild Agaricus bisporus population: new alleles, new genotypes. Mycologia 81, 433-440.

Lande, R. (1984). The expected fixation rate of chromosomal inversions. Evolution 38, 743-752.

Lappalainen, J. H. & Yli-Mattila, T. (1999). Genetic diversity in Finland of the birch endophyte Gnomonia setacea as determined by RAPD-PCR markers. Mycol Res 103, 328-332.

Lewinsohn, D., Nevo, E., Hadar, Y., Wasser, S. P. & Beharav, A. (2000). Ecogeographical variation in the Pleurotus eryngii complex (higher Basidiomycetes) in Israel. Mycol Res 104, 1184-1190.

Loukas, M. & Krimbas, C. (1980). Isozyme techniques in Drosophila subobscura. Drosophila Inf Serv 55, 157-158.

Macrae, R. (1967). Pairing incompatibility and other distinctions among Hirschioporus (Polyporus) abietinus, H. fusco-violaceous and H. laricinus. Can J Bot 45, 1371-1398.

McCutcheon, T. L., Caroll, G. C. & Schwab, S. (1993). Genotypic diversity in populations of a fungal endophyte from Douglas fir. Mycologia 85, 180-186.

McDonald, B. A, Miles, J., Nelson, L. R. & Pettway, R. E. (1994). Genetic variability in nuclear DNA in field populations of Stagonospora nodorum. Phytopathology 84, 250-255.

May, B., Wright, J. E. & Stoneking, M. (1979). Joint segregation of biochemical loci in Salmonidae: results from experiments with Salvelinus and review of the literature on other species. J Fish Res Board Can 36, 1114-1128.

Micales, J. A., Bonde, M. R. & Peterson, G. L. (1986). The use of isozyme analysis in fungal taxonomy and genetics. Mycotaxon 27, 405-449.

Nei, M. (1978). Estimation of average heterozygosity and genetic distance from a small sample of individuals. Genetics 89, 583-590.[Abstract/Free Full Text]

Nei, M. & Graur, D. (1984). Extent of protein polymorphism and the neutral mutation theory. Evol Biol 17, 73-118.

Nevo, E. (1988). Genetic diversity in nature. Patterns and theory. Evol Biol 23, 216-246.

Paavanen-Huhtala, S., Hyvönen, J., Bulat, S. M. & Yli-Mattila, T. (1999). RAPD-PCR, isozyme, rDNA RFLP and rDNA sequence analyses in identification of Finnish Fusarium oxysporum isolates. Mycol Res 102, 625-634.

Pegler, D. N. (1977). Pleurotus (Agaricales) in India, Nepal and Pakistan. Kew Bull 31, 501-510.

Petersen, R. H. & Hughes, K. W. (1999). Species and speciation in mushrooms. BioScience 49, 440-452.

Philippoussis, A., Zervakis, G. & Diamantopoulou, P. (2001). Bioconversion of agricultural lignocellulosic wastes through the cultivation of the edible mushrooms Agrocybe aegerita, Volvariella volvacea and Pleurotus spp. World J Microbiol Biotechnol 17, 191-200.

Raina, K., Jackson, N. & Chandlee, J. M. (1997). Detection of genetic variation in Sclerotinia homoeocarpa isolates using RAPD analysis. Mycol Res 101, 585-590.

Raper, C. A., Raper, J. R. & Miller, R. E. (1972). Genetic analysis of the life cycle of Agaricus bisporus. Mycologia 64, 1088-1117.

Rogers, S. O. & Bendich, A. J. (1988). Extraction of DNA from plant tissues. In Plant Molecular Biology Manual , pp. 1-11. Edited by S. B. Gevin, R. A. Schilperoort & D. P. S. Verma. Dordrecht:Kluwer Academic Publishers.

Roux, P. & Labarère, J. (1990). Isozyme characterization of dikaryotic strains of the edible basidiomycete Agaricus bitorquis (Quél.) Sacc. (syn. Agaricus edulis). Exp Mycol 14, 101-112.

Ruiz-Duenas, F. J. & Martinez, M. J. (1996). Enzymatic activities of Trametes versicolor and Pleurotus eryngii implicated in biocontrol of Fusarium oxysporum f. sp. lycopersici. Curr Microbiol 32, 151-155.

Saber, M. (1990). Contribution to the knowledge of Agaricales, pleurotoid in habit in Iran. Iran J Plant Pathol 26, 29-40.

Selander, R. K., Caugant, D. A., Ochman, H., Musser, J. M., Gilmour, M. N. & Whittman, T. S. (1986). Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl Environ Microbiol 51, 873-884.[Medline]

Slézec, A.-M. (1984). Variabilité du nombre chromosomique chez les pleurotes des ombellifères. Can J Bot 62, 2610-2617.

Stanosz, G. R., Swart, W. J. & Smith, D. R. (1999). RAPD marker and isozyme characterization of Sphaeropsis sapinea from diverse coniferous hosts and locations. Mycol Res 103, 1193-1202.

Surve-Iyer, R. S., Adams, G. C., Iezzoni, A. F. & Jones, A. L. (1995). Isozyme detection and variation in Leucostoma species from Prunus and Malus. Mycologia 87, 471-482.

Swofford, D. L. (2000). PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4.0b4a. Sunderland, MA: Sinauer Associates.

Swofford, D. L., Olsen, G. J., Waddell, P. J. & Hillis, D. M. (1996). Phylogenetic inference. In Molecular Systematics , pp. 406-514. Edited by D. M. Hillis, C. Moritz & B. K. Mable. Sunderland, MA:Sinauer Associates.

Urbanelli, S., Sallicandro, P., De Vito, E., Bullini, L. & Biocca, E. (1998). Biochemical systematics of some species in the genus Tuber. Mycologia 90, 537-546.

Venturella, G. (2000). Typification of Pleurotus nebrodensis. Mycotaxon 75, 229-231.

Venturella, G., Zervakis, G. & La Rocca, S. (2000). Pleurotus eryngii var. elaeoselini var. nov. from Sicily. Mycotaxon 76, 419-427.

Vilgalys, R. & Sun, B. L. (1994). Ancient and recent patterns of geographic speciation in the oyster mushroom Pleurotus revealed by phylogenetic analysis of ribosomal DNA sequences. Proc Natl Acad Sci USA 91, 4599-4603.[Abstract]

Vilgalys, R., Moncalvo, J.-M., Liou, S.-R. & Volovcek, M. (1996). Recent advances in molecular systematics of the genus Pleurotus. In Mushroom Biology and Mushroom Products , pp. 91-102. Edited by D. J. Royse. Pennsylvania:PennState University Press.

Wasser, S. P. & Weis, A. L. (1999). Medicinal properties of substances occurring in higher Basidiomycetes mushrooms: current perspectives (review). Int J Med Mushr 1, 31-62.

Weir, T. L., Huff, D. R., Christ, B. J. & Romaine, C. P. (1998). RAPD-PCR analysis of genetic variation among isolates of Alternaria solani and Alternaria alternata from potato and tomato. Mycologia 90, 813-821.

Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. & Tingey, S. V. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18, 6531-6535.[Abstract]

Worrall, J. J., Parmeter, J. R.Jr & Cobb, F. W.Jr (1983). Host specialization of Heterobasidion annosum. Phytopathology 73, 304-307.

Wu, O. X., Mueller, G. M., Lutzoni, F. M., Huang, Y. Q. & Guo, S. Y. (2000). Phylogenetic and biogeographic relationships of eastern Asian and eastern North American disjunct Suillus species (fungi) as inferred from nuclear ribosomal RNA ITS sequences. Mol Phylogenet Evol 17, 37-47.[Medline]

Yoon, C., Gessner, R. & Romano, M. (1990). Population genetics and systematics of the Morchella esculenta complex. Mycologia 82, 227-235.

Zervakis, G. (1998). Mating competence and biological species within the subgenus Coremiopleurotus. Mycologia 90, 1063-1074.

Zervakis, G. & Balis, C. (1995). Incompatibility alleles and mating behaviour between and within Pleurotus species. In Science and Cultivation of Edible Fungi , pp. 53-62. Edited by T. Elliott. Rotterdam:A. Balkema.

Zervakis, G. & Balis, C. (1996). A pluralistic approach on the study of Pleurotus species, with emphasis on compatibility and physiology of the European morphotaxa. Mycol Res 100, 717-731.

Zervakis, G. & Labarère, J. (1992). Taxonomic relationships within the fungal genus Pleurotus as determined by isoelectric focusing analysis of enzyme patterns. J Gen Microbiol 138, 635-645.

Zervakis, G. & Venturella, G. (1998). Towards the elucidation of the systematics of the Pleurotus taxa growing on Umbellifers. In Proceedings of the Sixth International Mycological Congress, p. 7 (abstract). Jerusalem: IMI.

Zervakis, G., Sourdis, J. & Balis, C. (1994). Genetic variability and systematics of eleven Pleurotus species based on isozyme analysis. Mycol Res 98, 329-341.

Zervakis, G., Yiatras, P. & Balis, C. (1996). Edible mushrooms from olive mill wastes. Int Biodeterior Biodegrad 38, 237-243.

Received 23 March 2001; revised 4 July 2001; accepted 12 July 2001.