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Population genetics and spatial structure of the fairy ring fungus Marasmius oreades in a Norwegian sand dune ecosystem

     Division of Molecular Biology, Department of Biology, University of Oslo, P.O. Box 1066, Blindern, 0316 Oslo, Norway

Klaus Høiland ²

     Division of Botany and Plant Physiology, Department of Biology, University of Oslo, P.O. Box 1066, Blindern, 0316 Oslo, Norway

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

The population genetics and spatial structure of the fairy ring fungus Marasmius oreades (Bolt. : Fr.) Fr. was studied by DNA amplification fingerprinting (DAF). Basidiocarp samples were collected from fairy rings from two separate sand dune systems of about 560 m² and 1750 m², respectively, on the Lista Peninsula in southwestern Norway in 1996. Samples were collected after a careful mapping of fairy rings and a vegetation survey of the composition and spatial structure of vascular plants, bryophytes and lichens. DAF with standard arbitrary oligonucleotide primers was used to examine the genetic relationship between basidiocarp samples. The study showed that the fungal population contained a high number of genotypes and that about 90% of the fairy rings represented a separate genet. Both cluster and phylogenetic analyses of DAF amplification products established relationships between fairy rings and showed that genetically similar basidiocarps were found close to each other. Overall results showed a weak correspondence between genotype and spatial distribution and no correspondence between genotype and composition of the surrounding vegetation. Furthermore, the occurrence of the four dominant sand dune grass species was randomly distributed among the localities housing the various fungal genotypes, indicating that the fungus did not exhibit genotypic specialization to the various grass species that could host it as a pathogen. Results show that establishment of new individuals generally was mediated by basidiospore dispersal and not by fragmenting dikaryotic, vegetative mycelium, as previously proposed.

Key words: basidiomycetes, basidiocarps, cladograms, DNA amplification fingerprinting, genetic dissimilarity

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Organismal units must be identified when studying populations at the genetic level to establish patterns of propagation, inheritance and evolution. In fungi, genetic clones resulting from asexual reproduction can be characterized by recurrent multilocus genotypes (Milgroom 1996, Anderson and Kohn 1998). Fungal clones generally are of recent origin (Guidot et al 1999, Gryta et al 2000). However, in some cases, such as those that establish symbiosis with fungus-farming ants (Mueller et al 1998), they can be ancient. Clones can separate from their origin for dispersal, while in other cases they can be physically connected and can be highly territorial. Territoriality can occur at different spatial scales, ranging from centimeters in Coriolus versicolor growing on birch stumps (Rayner and Todd 1978) to kilometers in Phellinus weirii growing in a mountain hemlock forest (Dickman and Cook 1989), and local populations can be almost as diverse as the entire meta-population (Guidot et al 1999). Spatial connections between mycelia can be favored or broken by somatic incompatibility reactions between neighbors, sometimes resulting in few and large (e.g., Armillaria bulbosa, see Smith et al 1996) or many and small individuals, respectively. The study of these spatially connected fungal clones offers a unique opportunity to address important phenomena in biology, such as the existence of barriers to gene flow, the role of somatic incompatibility, the evaluation of the cost and benefits of sex in populations of varying size, and the role of deleterious mutations in evolution (e.g., Carbone et al 1999).

Marasmius oreades (Bolt. : Fr.) Fr., commonly known as the fairy ring mushroom, belongs to the family Tricholomataceae, order Agaricales, division Basidiomycota. It can form fairy rings, distinct ring or arch-like structures generally recognized by the stimulation or suppression of the surrounding plant growth and the seasonal production of basidiocarps. Marasmius oreades can be destructive to lawns, parks, golf courses and pastures (Lebeau and Hawn 1961, Couch 1973) and has been reported to be a pathogen of grasses such as Poa pratensis and Festuca rubra (Blenis et al 1997). The mycelium, which is found in the soil beneath the ring, interferes with plant-water relationships and produces hydrogen cyanide, polyacetylene and sesquiterpene metabolites capable of damaging grass roots (Traquair and McKeen 1986, Ayer and Craw 1989). The mycelium from each fairy ring is a genetically homogenous entity that can be considered a discrete fungal individual (Burnett and Evans 1966). Furthermore, these fairy ring individuals can be as old as 100–150 yr and possibly 500 yr (Bayliss-Elliott 1911, Schantz and Piemeisel 1917, Burnett and Evans 1966). The fungus is heterothallic and has a unifactorial mating system controlled by a multiallelic locus (Mallett and Harrison 1988). In southern Norwegian sand dune landscapes, M. oreades is common in the dry dune pastures behind outer dune ridges (Høiland 1977). A significant positive correlation was found between the diameter of the rings and their distance from the outermost dune ridge in this dynamic ecosystem (Høiland 1993). The age of the oldest rings was estimated to be less than 15 yr (Høiland 1993). However, the fungus has not been studied with contemporary molecular tools that would better define its genetics and population structure.

DNA amplification fingerprinting (DAF) is a nucleic acid scanning technique (Caetano-Anollés et al 1991) capable of resolving taxa efficiently at the subspecies level (Caetano-Anollés 1996). Short oligonucleotides of arbitrary sequence, generally 5–8 nucleotides in length, are used to amplify a collection of anonymous nucleic acid segments in a genome. These oligonucleotide primers bind to naturally occurring sets of short, complementary and closely spaced inverted repeats, driving the DNA polymerase-based amplification of the spanning sequences. The very high primer-to-template mass ratios provide both a highly stringent and stable amplification reaction and result in relatively complex and highly reproducible DNA profiles. These profiles are visualized by silver staining and prove far more reliable and robust than those generated using other techniques (e.g., random amplified polymorphic DNA [RAPD] analysis). DAF can characterize closely related organisms and has been used successfully to study fungal populations that genetically were highly homogeneous (Trigiano et al 1995, Caetano-Anollés et al 1996, 2001, Bentley et al 1998).

In this study, we explored the population structure of M. oreades at the molecular level. DAF was used to examine the genetic relationships between basidiocarp samples from fairy rings collected from sand dunes in southern Norway. Experimental objectives included mapping the distribution pattern of each fairy ring in two separate dune systems, determining genetic variation among these rings and comparing this variation with the spatial structure and composition of the surrounding vegetation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Study area – The study area was in the sand dune systems on the Lista Peninsula in the Farsund community, Vest-Agder County, southwestern Norway. Dune vegetation has been described in detail (Høiland 1978). The dominating grasses in the dune pastures on Lista were Festuca arenaria (a segregate from F. rubra) and Corynephorus canescens (Høiland 1978). In less established pastures, Marram Grass Ammophila arenaria was frequent. Poa pratensis ssp. subcaerulea was common in vegetation under weak cultural influence. Outside Poaceae, numerous lowland or coastal plant species occurred in the pastures, some more frequent on pioneering sites, other more dominant on the more established areas (Høiland 1978). Bryophytes and lichens also were common. The species of grasses, herbs, mosses and lichens usually occurred in mosaic patterns (Høiland 1978). In the dune ecosystem, M. oreades appeared to be confined to the dry dune pastures (Høiland 1977). The fungus avoided the outer dunes and was absent in the wetter dune slacks and Salix repens-dominated vegetation types (Høiland and Elven 1980).

Field work – Two separate dune systems, areas A and B, of about 560 and 1750 m², respectively, were chosen on the small Einarsneset Peninsula (Universal Transverse Mercator Grid: UTM_ED50 LK 69 38), SE on Lista. Area A was a sand plain sheltered from the prevailing western winds by rocks (on the west and north) and otherwise delimited by dune ridges and blowouts with naked sand. Area B was part of a moving sand dune system containing building Ammophila dunes in the west. Going eastward, there was a succession gradient of nonestablished to fixed Ammophila dunes, dune pastures and dune heaths dominated by Empetrum nigrum, in that order. The delimitation of Area B was set arbitrarily to cover a representative part of the vegetation types where M. oreades could be found. Area B was 175 m N and 150 m E of Area A.

All data presented in this paper were collected the first week of Sep 1996, with some additional replicates from the same rings in Sep 1997 and 1998. All fairy rings in areas A and B were identified, mapped and permanently marked for future investigation. Size and form of the rings were depicted on the map. In the part of the ring with the highest density of basidiocarps, a plot of 1 m² was laid in the S-N direction. A full analysis of percentage cover of vascular plants, bryophytes and lichens were performed for each plot. A basidiocarp representing the actual fairy ring was collected for DNA extraction from the middle of the 1 m² plot. The westernmost ring (A-011) was defined as the "zero" ring for areas A and B together, with value 0 for both geographical coordinates. With this "zero" ring as base, the geographical coordinates for the position of each basidiocarp collected were determined and reported as meters in E and N directions, respectively. These coordinates defined the location of each ring and were used to calculate Euclidean metric distances between rings.

The vegetation data from the 1 m² plots were read by the program BDP/PC (Pedersen 1988). The Sørensen's (1948) similarity index was calculated for pairs of all plots:

where S_i,j is Sørensen's similarity index between plots i and j, p_i,j is the number of species of vascular plants, bryophytes and lichens common for i and j, p_i is the number of species found in i, and p_j is the number of species found in j.

DNA preparation – Basidiocarps were collected, dried and kept in paper bags at ambient room temperature. DNA was extracted from a 20 mg dried basidiocarp sample with a commercial kit (Puregene, Gentra Systems, Minneapolis, Minnesota), by grinding the samples to a fine powder with a presterilized mortar and pestle under liquid nitrogen and following manufacturer specifications. No attempt was made to dedikaryotize the dikaryotic hyphae in the basidiocarps, a technique found invaluable to unambiguously identify isolates of the same genotype of a dikaryotic basidiomycete (Gryta et al 2000). Our investigation, therefore, relies on the sum of DNA from both nuclei. Of all samples studied, only one (Ring B-111) was refractory to repeated DNA extraction. DNA concentrations were measured by fluorescent enhancement of the fluorescent dye Hoechst H33285 (1 mg/µL dye stock solution), using a Hoefer DyNA Quant 200 fluorometer (Amersham Pharmacia Biotech, San Francisco, California) and DNA sample stocks diluted to 2 ng/µL for amplification.

DNA amplification – DAF cocktails were assembled in a final volume of 10 µL, adding the components in this order: 4.2 µL double distilled water, 1 µL of 10 x Stoffel buffer, 1 µL of deoxyribonulceotides of each deoxynucleoside triphosphate (200 µM), 1.2 µL MgCl₂ (25 mM/1.5 mL), 0.3 µl oligonucleotide primer (300 µM), 0.3 µl AmphiTaq Stoffel fragment DNA polymerase (0.3 U/µL), and 2 µL of template DNA (2 ng/µL). Enzyme and buffers were obtained from Perkin-Elmer (Norwalk, Connecticut). Master mixes were prepared with common components. Reaction mixtures were amplified for 35 cycles of 2.1 min at 96 C, 1 min at 48 C, and 1 min at 74 C using a Robocycler Gradient 96 thermocycler with a hot lid assembly (Strategene, La Jolla, California).

Eight standard arbitrary oligonucleotide primers were used to generate fingerprints from the isolates of M. oreades. The 5'–3' sequences of the primers were: GACGTAGG, GAAACGCC, GTATCGCC, GCAGGTGG, GCTGGTCG, GCAGGTGC, GGACCCGC, and AACCTGCGG. All primers generated fingerprints with varying degree of polymorphism. Repeated amplification of the same DNA sample generated consistent and reproducible fingerprints.

Separation and visualization of amplified products – Amplified products were diluted 1:5 and separated by electrophoresis on 0.45 mm-thick 10 % T:0.2 % C polyacrylamide-1.7 M urea-5% glycerol gels backed on Gelbond PAG films (FMC Bioproducts, Rockland, Maryland, U.S.A.). Wells were loaded with 3 µL of the diluted amplified products mixed with 3 µL of loading buffer (12 g urea, 2 mL xylene cyanol [4 mg/mL], and 8 mL H₂O). Electrophoresis was run at 180 V for 15–20 min and, after the samples were loaded, run at 250 V for 30–45 min. The polyester-backed gels were stained silver (Bassam and Caetano-Anollés 1993), treated with an anticracking solution (10% glacial acetic acid, 35% ethanol, and 1% glycerol) for 5 min and preserved by drying at room temperature.

Fingerprint analysis – Bands (700 bp in length) were scored by direct visual observation as present (1), absent (0), or uncertain or missing (u), then encoded in a data matrix. The data initially were encoded as unordered, nondirected and unweighted characters. Unweighted pair group cluster analysis using arithmetic means (UPGMA) (Sneath and Sokal 1973) with Dice similarity index (Dice 1945, Nei and Li 1979) was used to examine the hierarchy of genotypes. The analysis was run with the program NTSYSpc 2.02h (Applied Biostatistics Inc., Setauket, New York). Phylogenetic relationships were estimated with the maximum-parsimony optimality criteria (Farris 1970) in PAUP* v. 4.0b8a (Swofford 2001). Bands were treated as: (1) unordered character types or (2) ordered types with weighting of 1:5 for absence and presence of band. The weighting reflects the asymmetric probabilities of gaining and losing an amplification product because it is easier to loose a primer annealing site (e.g., by a single mutation complementary to a nucleotide close to the 3' terminus of the primer) than to gain one (requiring homology to the first five or six nucleotides from the 3' terminus; Caetano-Anollés et al 1992). It also reflects the existence of primer mismatching. This "generalized" parsimony approach was defined by an asymmetric step matrix of character transformation costs with the USERTYPE command in PAUP and represents a statement of character polarization and a more realistic model of character-state transformation. Asymmetric step matrices constrain free reversibility of character change by the differential cost of reversing a given transformation. Because the length of trees depends upon the position of the root, these stepmatrices produce inherently rooted phylogenies that help optimize the interplay between phylogenetic analysis and the evolutionary model. To diminish phylogenetic noise, 33 phylogenetically uninformative characters representing monomorphic products and autapomorphies were excluded from the analysis. Most-parsimonious trees (MPTs) were obtained using the heuristic search option with tree-bisection-reconnection (TBR) branch-swapping, random stepwise addition sequence, and 100 replicates to avoid tree islands (Maddison 1991). The save all most-parsimonious trees (MULPARS) option was chosen to obtain all MPTs, and the collapse zero-length-branches option was switched on to eliminate excessive unsupported branching topologies. In the absence of a specified outgroup, trees were rooted by the midpoint when using unordered types. When using ordered characters with weighted transformations, trees were rooted automatically at the point where the hypothetical ancestor connects to the tree. Phylogenetic reliability was assessed with 10³ bootstrap (BS) replicates (Felsenstein 1985). The consistency index (CI), retention index (RI) and rescaled consistency index (RC) were used as measures of homoplasy and character fit. In a preliminary parsimony search, homoplastic characters were identified by their low RC index values, and this information was used to reweight characters for further study. The structure of the phylogenetic signal in the data was tested by Hillis' skewness (g₁) of the length distribution of 10⁴ random trees (Huelsenbeck 1991) and permutation-tail probability (PTP) tests of cladistic character co-variation using 10³ replicates (Archie 1989, Faith and Cranston 1991). The homogeneity of data partition resulting from the amplification with different primers was analyzed with a modified Michevich-Farris index of incongruence among datasets and 10³ heuristic replicates (Farris et al 1995).

To make direct comparison between samples, or between sample pairs and their corresponding Euclidean metric distance and Sørensen's similarity index, the genetic distance between the samples was calculated by this equation:

where G_i,j is the genetic distance between sample i and j, n is the total number of DAF character loci, q_i,j(1,0/0,1) the number of polymorphic loci shared by i and j (i.e., where one band is present and the other absent), and q_i,j(u) the number of loci with uncertain or missing bands in either i or j.

Genetic diversity was estimated with the Shannon information index (Lewontin 1972) and was calculated according to this equation:

where p_i is the frequency of DAF fragment at the ith locus and n is the total number of DAF character loci.

Analysis of molecular variance (AMOVA) (Weir and Cockerham 1984, Weir 1996, Excoffier et al 1992) between and within populations (areas A and B) were performed by the program ARLEQUIN (Schneider et al 2000) using 2 x 10⁴ permutations.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Techniques that generate arbitrarily amplified DNA depend on many amplification factors (reviewed by Tyler et al 1997) and become robust and transportable only when the many variables are carefully and laboriously optimized. We applied the Taguchi method of robust experimental design to DAF analysis (Caetano-Anollés 1998) and optimized amplification conditions necessary for the reliable characterization of fungi (Caetano-Anollés et al 2001). The interaction of reaction components and thermal-cycling parameters was studied using L₉(3⁴) and L₁₈(3⁸) orthogonal arrays; analysis of variance (ANOVA), which decomposed the contribution of individual amplification factors to the responses of amplification yield and product number; and verification experiments, which established that optimum conditions were predictable and reproducible. The optimized DAF protocol was based primarily on high annealing temperatures (48 C) and primer concentrations (9 µM), and produced reproducible and relatively complex fungal DNA profiles (Fig. 1). In at least two experiments, profiles were reproducibly generated from DNA obtained from extractions of two replicate basidiocarps from the same fairy ring (Fig. 1A). Even basidiocarps collected during three years (1996–1998) from the same fairy ring produced identical or nearly identical fingerprints (Fig. 1B). The small differences of fingerprints might be due to either the fact that the fairy ring is a composite of mycelia from different but genetically similar individuals or the result of somatic mutations within the same individual.

View larger version (118K): [in this window] [in a new window]   FIG. 1. DAF analysis of Marasmius oreades. DNA fragments were separated using denaturing polyacrylamide gels and visualized using silver staining. The sizes of molecular weight markers are given in kbp. A. DNA extracted from two replicate basidiocarps collected from the same fairy ring (A-031 and A-042, respectively) in 1996 was amplified using the oligonucleotide primer GAAACGCC in separate experiments (1 and 2) but separated in a same gel. B. DNA from basidiocarps appearing in a same fairy ring (A-081 and A-161, respectively) in three consecutive years (1996–1998) were amplified with primer GTATCGCC.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Map of Marasmius oreades fairy rings in areas A and B. All rings—viz. genotypes—expressing genetic dissimilarity (Gi,j) values of less than 0.2 for representative basidiocarps are connected with lines. Basidiocarp B-111 failed to produce reliable DNA and was omitted from the study. All fairy rings were arch shaped. Area B was situated 175 m N and 150 m E of area A. On this map a line separates these areas and distances are scaled in meters.

View larger version (14K): [in this window] [in a new window]   FIG. 3. Genetic relationships between Marasmius oreades fairy rings revealed by cluster analysis using the unweighted pair group method of averages (UPGMA). DAF profiles were produced with eight arbitrary primers. DNA fingerprints were scored and similarities determined using the Dice coefficient. The dendrogram shows genotypes together with the occurrence of the four dominant sand dune grasses Ammophila arenaria (A,a), Corynephorus canescens (C,c), Festuca arenaria (F,f) and Poa pratensis ssp. subcaerulea (P,p). Uppercase and lowercase letters represent coverage above or similar to 20%, or below 20%, respectively. Hyphens indicate no occurrence.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Phylogenetic analysis of Marasmius oreades fairy rings. A total 117 informative out of 150 DAF characters were analyzed using maximum parsimony as the optimality criterion in PAUP*. A. Cladogram showing a single most-parsimonious tree (68 steps; CI = 0.444, RI = 0.665, RC = 0.295; g1 = -3.040; PTP test, P = 0.001) retained after a heuristic search with 100 replicates of TBR branch swapping. Characters were considered unordered and were reweighted by maximum RC values in three iterations. Bootstrap support indices are given for nodes with values more than 50%. B. Cladogram showing a single most-parsimonious tree (604 steps, g1 = -0.187; PTP test, P = 0.001) obtained after a heuristic search with 100 replicates of TBR branch swapping. Characters were treated as ordered types with weighting of 1:5 for absence and presence of bands. Weighting reflects the asymmetric probabilities of gaining and losing markers and the existence of primer-template mismatching. The number of genetic links to other fairy rings at Gi,j values less than 0.2 (see Fig. 3) was treated as an ordered and polarized character that could be traced in the dichotomous and inherently rooted tree. These values are indicated for each terminal branch of the tree and are coded in the scale.

Figure 5 shows the relationship between the genetic dissimilarity of all pairs of basidiocarps, G_i,j, in areas A and B, respectively, and the logarithm of the Euclidean metric distance between the basidiocarps constituting those pairs. Genetic similarity weakly increased with decreasing metric distance between the rings. The samples from pairs with G_i,j = 0 always were found less than 2 m apart. However, the regressions have low r² values—0.027 and 0.011—both not significant, for areas A and B, respectively. No relationship was found between the genetic dissimilarity of all pairs of basidiocarps, G_i,j, and the similarity, S_i,j, of the vegetation between the actual plots analyzed (data not shown).

View larger version (16K): [in this window] [in a new window]   FIG. 5. The relationship between the genetic dissimilarity (Gi,j) of all pairs of Marasmius oreades basidiocarps in areas A and B and the logarithm of the Euclidean metric distance (log[Distij]) between basidiocarps constituting those pairs. The regression lines, r2 = 0.027 for area A and r2 = 0.011, both not significant, for area B, are indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Fungi can establish extensive mycelial networks in the soil, sometimes occupying a continuous territory encompassing many adjacent root systems. One example is that of the fairy ring fungi. However, the establishment of M. oreades is poorly understood. Couch (1973) suggested that fragments of dikaryotic mycelia are probably the main sources of inoculum. On the other hand, Mallett and Harrison (1988) suggested that most fairy rings were started by spores because either their mating types differed or they shared the same mating type genotype but were genetically distinct, based on vegetative interactions between dikaryotic isolates. In this study, we show that the M. oreades population contained a high number of genotypes (Table I), varying on a fine spatial scale. Our investigation indicates that each ring generally constitutes a separate genet. Only three of the rings (A-031, A-041 and A-042), situated close to each other, were genetically indistinguishable and must have originated by patching of a single genet. The establishment of new individuals in these populations therefore is mediated by basidiospore dispersal and not by fragments of dikaryotic, vegetative mycelium, as stated by Couch (1973). Similar conclusions were reached in several studies of saprotrophic or ectomycorrhizal basidiomycete populations (Worrall 1994, Gryta et al 1997, Chiu et al 1999, Gherbi et al 1999, Dettman and van der Kamp 2001).

A weak correspondence between genotype and spatial distribution was seen (Fig. 5), but no correspondence between genotype and composition of the surrounding vegetation was noted. Populations of the wood-inhabiting basidiomycete Lentinula edodes in China, Chiu et al (1999) demonstrated a similar pattern; more heterogeneity was encountered between isolates the greater the distance separating the genets. On the other hand, neither Saville et al (1996) nor Dettman and van der Kamp (2001) could prove any relationship between geographic distance and genetic similarity of genets for two species of the root pathogen basidiomycete Armillaria in North America. The same was seen in a population of the ectomycorrhizal Hebeloma cylindrosporum in coastal sand dune areas in France (Guidot et al 1999, Gryta et al 2000).

Because the sexual behavior of M. oreades is heterothallic and unifactoral (Mallett and Harrison 1988), monokaryotic mycelia from basidiospores from two individuals belonging to two mating types have to dikaryotize to make a new individual fungus. Consequently, these individuals will be closely related genetically to their two parental progenitors. Looking at Fig. 2, where all rings with G_i,j < 0.2 are connected with lines, every ring is linked to two or more other rings, with very few exceptions. Theoretically it could be possible to trace the two parents giving rise to a particular individual. This possibility, however, is confounded by the fact that: (i) an individual produces many basidiospores, and these give rise to several individuals with different genetic constitution arising from the contributions of the other parent and meiotic crossing over; (ii) not every fungal individual fructifies in the sampling season; and (iii) somatic mutations can occur in a growing, dikaryotic mycelium. Despite caveats, phylogenetic analysis showed that one particular group of genotypes, A-022 and A-101, was very different from the rest. These genotypes were unlinked genetically from the rest of genotypes (Fig. 2) and formed a well-supported and separate group (Fig. 4). The common genetic origin of these genotypes was confirmed by cluster analysis (Fig. 3). These genotypes might represent derivatives of the original colonization event that gave rise to the bulk of the genotypes analyzed in this study. In contrast, rings A-023, A-051, A-061, A-071, A-081, A-091, A-141, B-021 and B-091 were linked with more than five other rings and were derived phylogenetically. One can speculate that these rings represent young individuals that are participating in the production of a long series of new generations. Assuming ancestors share characteristic with its descendants, the reconstructions of genetic links here suggest that ancestral genotypes in M. oreades are those that become genetically isolated.

	ACKNOWLEDGMENTS

 
We wish to thank Håvard Kauserud for advice and valuable comments; the county governor of Vest-Agder for granting permission to collect samples in the Husebysanden landscape protected area; and the International Atomic Energy Agency in Vienna, Austria, (FAO/IAEA RCP5808151) for financial support.

	FOOTNOTES

 
¹ Present address: Department of Crop Sciences, University of Illinois, 332 NSRC, 1101 West Peabody Drive, Urbana, IL 61801. E-mail: gca{at}uiuc.edu

² Corresponding author. E-mail: klaus.hoiland{at}bio.uio.no

Accepted for publication April 15, 2003.

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